Download Membrane receptors in the gastrointestinal tract

Bioscience Reports, Vol. 8, No. 3, 1988
REVIEW
Membrane Receptors in the Gastrointestinal
Tract
Christian Gespach, 1 Shahin Emami and Eric Chastre
Received March 4, 188
This review focusses on the roles that membrane receptors and their transducers play in the
physiology and pathology of the gastrointestinal tract. The multifactorial:factorial regulation of
mucosal growth and function is discussed in relation to the heterogeneity of exocrine and endocrine
populations that originate from progenitor cells in stomach and intestine.
INTRODUCTION
We shall describe here the plasma membrane receptors of cells originating from
the gastrointestinal tract that are--as are such receptors in other tissues-integrated components of one or other of three different types of communication
pathway. Each pathway consists of extracellular receptor agonists, the receptor,
and a cellular amplifing mechanism, producing intramembrane, intracytoplasmic
or nuclear messengers.
One pathway makes use of adenylate cyclase, cyclic A M P and cAMPdependent kinase A, guanylate cyclase, cyclic GMP, cGMP-dependent kinase G
and phosphodiesterase. Adenylate cyclase is composed of a catalytic subunit C
and two regulatory subunits Gs (activating) and Gi (inhibiting). The subunits Gi
and Gs are heterotrimers composed of a r-protein (Mr = 35 KDa), a )'-protein
(lvlr= 5 KDa) and an a~ or an o:~ protein (1). The re-proteins are different:
M r = 4 0 K D a for oq and 4 2 - 5 2 K D a for a's. These two latter structures are
GTPases (2, 3). The addition of sodium fluoride, magnesium ions and GTP or
certain of its analogues dissociates the as, aq-/3-7-protein complex. This complex
thus separates into two parts: /3-y and o:~-GTP or oq-GTP which activate or
inhibit the catalytic subunit C. A third GTP-binding protein Go closely related to
G~ in structure and serving as substrate for pertussis toxin has been identified in
several tissues (4). The function of this novel type of transducer has not yet been
clarified. A number of proteins, drugs and bacterial toxins are able to act upon
INSERM U.55, Unit6 de Recherche sur les peptides neurodigestifs et le diab~te, HOpital
Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris, France.
1To whom all correspondence and reprint requests should be addressed.
199
0144-8463/88/0600-0199506.00/09 1988 Plenum Publishing Corporation
200
Gespach, Emamiand Chastre
the different subunits and components of the adenylate cyclase enzymatic system.
Calmodulin, a calcium-binding, a calcium-modulated protein stimulates adenylate
cyclase by acting directly upon the catalytic subunit of the enzyme; cholera toxin
produces the same effect on the o:s moiety of the subunit Gs by ADP-ribosylating
it. Forskolin stimulates adenylate cyclase by acting directly on the catalytic
cyclase unit and on the tr~/a~i moieties of GJGi (5). By binding to the tri moiety,
pertussis toxin dissociates the fl-o:i complex, thus preventing stimulation of GTP
hydrolysis by the inhibitory hormones acting on the Ri cyclase receptors (i.e.
opiates, somatostatin, angiotensin, acetylcholine: M1 receptorsand adrenergic
a~2) and performs an ADP ribosylation of the o~i moiety of Gi. For instance,
somatostatin inhibits cAMP production in the $49 lymphoma by stimulating the
aq protein-associated GTPases (6). Cyclic AMP was thus recognized to be one of
the second messengers of the hormones stimulating the secretions of the gastric
and intestinal epithelium (7). Finally, cAMP might play a role in the proliferation
of the crypt epithelium of the rat colon and jejunum (8) and in the transcription
of the mRNA encoding prepro-VIP and PHM 27 in a human neuroblastoma.
Guanylate cyclase is stimulated by sodium nitroprussiate, sodium azide,
hydroxylamine, sodium nitrite and insulin in various cell systems (9).
The second pathway uses phospholipase C which transforms
phosphatidylinositol-4,5-bisphosphate (PIP2) into two messengers: inositol 1,4,5trisphosphate (IP3) and 1,2-diacylglycerol (10-13). This transmembrane signalling
system regulates the concentration of Ca 2+ in the cytosol and controls the activity
of kinases of type C (PKC). Diacylglycerol diffuses into the membrane lipid layer
and activates PKC in the presence of phosphatidylserine and calcium ions.
Phosphatidylserine is a component of the membrane acting as a cofactor of kinase
C activation by diacylglycerol or by mutagenic agents, such as the tumor
promotors phorbol esters (14). Further, cAMP analogues and diacylglycerol
mediate the translocation of protein kinase C from the cytosol to the nucleus (15)
or to the plasma membrane. Thus PKC may function in the regulation of gene
expression (15) and cell differentiation (16). Of the several inositol phosphates
formed, IP3 acts as the intracellular messenger mobilizing Ca 2§ from the
endocytoplasmic reticulum. Changes in free cytosolic Ca 2§ were monitored using
the fluorescent probe Quin-2 in permeabilized cells (17) or Fura-2 and Aequorin
in cell layers with normal integrity (18, 19). Membrane receptor activation of
phospholipase C involves a guanine nucleotide binding protein G of type p (Gp)
structurally related to the other G proteins (20). This contention is supported by
the knowledge that both Gi and Go can reconstitute phospholipase C activation
by formylMet-Leu-Phe when native Gp is inactivated by pertussis toxin in HL-60
cell membranes (21). The action of acetylcholine and gastrin on acid secretion is
influenced by extracellular calcium; carbachol and gastrin respectively induce
persistent and transient increases in free calcium in the cytosol of parietal ceils of
rabbit and dog (22). In gastric chief cells isolated from the guinea pig, CCK and
carbachol increase free intracellular calcium concentrations, independently of a
Ca 2§ entry mechanism (23). Further, the minimum effective dose of CCK or
carbachol to elicit an increase in Quin-2 fluorescence is similar to that for
pepsinogen secretion, suggesting that enzyme release by the chief cell might be
G.I. Tract Membrane Receptors
201
mediated by intracellular C a 2+ levels. These biochemical events are consequent
to inositol phospholipid hydrolysis in guinea pig gastric glands (24).
Membrane receptors coupled to G proteins possess an extracellular domain
with a consensus site for N-linked glycosylation at the amino terminus, seven
membrane spanning domains and cytoplasmic regions involved in recognition and
coupling to regulatory G proteins, and phosphorylation domains for regulatory
desensitization by receptor kinases (25). Recent cloning of the genes and cDNAs
for the /32-adrenergic receptor reveals that an intronless gene G-21 encodes for
members of the class of receptors regulating intracytoplasmic GTPases, including
muscarinic receptors and the visual light receptor rhodopsin (26).
The third communication pathway is illustrated by, e.g., the receptortyrosine kinase complex of insulin and epidermal growth factor (EGF). This
complex, inserted in the membrane, comprises a receptor component which binds
the hormone, and a transduction system which phosphorylates the tyrosine
residues of certain proteins (27, 28). Certain oncogene products display structural
and functional analogies with the membrane-bound transduction systems. For
example, the erb gene encodes a protein analogous to the EGF receptor and the
ras gene encodes a GTPase. The three functional ras genes of the human genome
(c-H-ras-1, c-K-ras-2 and N-ras) encode a p21 protein associated with the
cytosolic surface of the plasma membrane. The c-ras genes are expressed during
normal human development in fetuses at 12 to 18 weeks gestation (29). This time
subsequent to embryogenesis coincides with a general situation of rapid cell
proliferation and differentiation. The p21 ras protein expresses an intrinsic GTPase
activity which might be involved in the increase in inositol phosphate production
in response to growth factors including bombesin, gastrin releasing peptide and
bradykinin (30). Insulin was found to stimulate the phosphorylation level of the
p21 protein encoded by the ras oncogene of Harvey murine sarcoma virus
v-Ha-ras (31). These systems play a very important part in the differentiation and
cell growth processes in eukaryotes. Thus, PDGF (platelet-derived growth factor)
is a 30KDa protein made up of 2 polypeptidic chains: A, encoded on
chromosome 7 and B, encoded on chromosome 22. It activates a receptor
belonging to the same "family" as those of insulin. The PDGF B chain is virtually
identical to the transforming protein of the simian sarcoma virus (SSV) encoded
by the v-cis gene of SSV. Only cells with the PDGF receptor are transformed by
~his virus, which synthesizes an agonist analogue to PDGF. There is thus a close
relationship between the mitogenic action of growth factors and retroviral
transforming proteins.
These three communication pathways are probably interdependent in the
multifactorial integration of cell functions. By activating the PIP2-IP3-Ca 2.+
pathway, certain hormones may thus modulate the activity of the guanylate and
adenylate cyclases that react on the variations in intracytoplasmic free calcium.
Activation of these transduction systems (ion channels, enzymes, exocytosis)
results in phosphorylation of serine, threonine or tyrosine residues in the
especially mobilized protein structures: the receptor itself or the regulating
protein. Other still unknown messenger systems might also participate in these
regulations.
2112
Gespach, Emami and Chastre
RECEPTORS INVOLVED IN THE REGULATION OF GASTRIC
SECRETION
Acidification by the proton pump of the intracellular compartments of the
parietal cell gives an indirect measure of the in vitro H + ion production. Capture
of aminopyrine, a weak base with a pKa of 5, demonstrates an equilibrium
between the formation and secretion of H + ions during H+/K + ATPase
activation (32). This technique has been widely used in various laboratories to
evaluate the effects on gastric acid secretion of different agents including
histamine and its H E antagonists, gastrin, acetylcholine, dibutyryl cAMP,
isobutylmethylxanthine, forskolin, CCK, cerulein, somatostatin, glucagon, prostaglandins E2 and prostacyclins I2 (32-34). The activity of the parietal cell can be
visualized in electron microscopy by the ultrastructural transformations it
undergoes during secretory processes (32, 35). Omeprazole inhibits basal acid
secretion stimulated by dibutyryl cAMP, histamine, pentagastrin, but does not
inhibit the development of secretory channels or the disappearance of the parietal
cell's tubulovesicular system (36-42). Thiocyanate ions have a comparable action.
Omeprazole seems to be an irreversible inhibitor of the proton pump (39).
However, this inhibition and irreversibility can be counteracted by the addition of
fl-mercaptoethanol, dithiothreitol, cysteine or glutathione, or by pH Values close
to neutrality (43). These observations suggest that this benzimidazole derivative
reacts with the sulfhydryl groups controlling the proton pump's activity after
being decomposed into a derivative which is active at pH 6 (43). When acid
secretion is stimulated, the half-life of omeprazole is 3 m in, and when it is
inhibited, 73 min. In addition to omeprazole and thiocyanate and nitrite ions,
which inhibit the proton translocation by the H+/K § pump (44), other molecules
with a functional tertiary amino group can inhibit the K § site of the proton
ATPase (45). Thus, there are drugs able to control gastric acid secretion by acting
either upon the basal pole (antagonists of the histaminergic, cholinergic and
gastrinic receptors), or upon the apical pole of the parietal cell (omeprazole,
trifluoroperazine, verapamil, TMB).
The secretory activity of the parietal cell is regulated by gastrin, acetylcholine
and histamine. Gastrin controls acid secretion and the growth of the gastrointestinal epithelium and has certain sequence analogy with CCK, since these two
peptides have the same C-terminal extremity GWMDF-amide which is their
active site on the receptors. Gastrin has specific receptors in fundic and duodenal
mucosa of the rat (46, 47). The binding capacity of the gastrin receptor decreases
after weaning and is restored after refeeding the animal, suggesting a receptor
self-regulation by serum gastrin (47). These results were confirmed later by Speir,
who also demonstrated, in the rat, a homologous desensitization of the gastrinic
receptor in oxyntic glands after a gastrin injection (48). Weaning reduces the
number of gastrin receptors without altering their affinity (48). In the guinea pig,
gastrin and CCK seem to share the same receptor site in fundic glands (49). This
can be explained by the common GWMDF-amide sequence of the C-terminus of
both peptides, and by the important role of this pentapeptide in the biological
activity of gastrin and CCK. Replacement of the terminal phenylalanine residue
G.I. Tract Membrane Receptors
203
by aspartic acid produces competitive antagonists of the CCK and gastrin
receptors 20 and 200 times more potent than proglumide, a nonselective
CCK/gastrin antireceptor (50). The new CCK antagonist asperlicin isolated from
the fungus Aspergillus alliaceus has 300 to 400 times higher potency than
proglumide on pancreatic, ileal and gallbladder CCK receptors. This nonpeptide
antagonist is highly selective for peripheral CCK receptors, relative to brain CCK
and gastrin receptors, since asperlicin has no effect on the CCK-induced acid
secretion mediated by gastrin receptors in the isolated mouse stomach. A recent
observation shows that in a human gastric adenocarcinoma (SC-6-JCK cell line)
CCK inhibited cAMP production and cell proliferation induced by pentagastrin
(51).
Acetyleholine is secreted by neurons of the central and peripheral nervous
system. In the vagus nerve these neurons are preganglionic autonomous neurons
and fibers, and in the gastrointestinal wall, postganglionic neurons. These
parasympathetic fibers have endings in the region of the epithelium and smooth
muscle effector cells. The muscarinic receptors stimulate gastrin, histamine,
pepsin and acid secretion by the stomach (52). The nicotinic and muscarinic
receptors stimulate gastric muscle contraction and acid secretion. The cholinergic
receptors are classified as nicotinic receptors (subclasses N1 and N2) and
muscarinic receptors (subclasses M1, M2 and M3).
The nicotinic receptor comprises 5 subunits (2 o~ and one/~, 7 and c5) located
in the postsynaptic membrane of cholinergic neurons (53). This complex is
arranged in the form of a rosette which integrates sodium transport, two binding
sites for acetylcholine and o~-bungarotoxin on the o~-subunits, and allosteric sites
where the ion channel is blocked by histrionicotoxin, chlorpromazine and
phencycline(54). Phosphorylation of the 7 and 6 subunits increases the rate of
receptor desensitization induced by acetylcholine in myotubules. Lobeline,
nicotine and dimethyl phenyl piperazinium iodine are agonists at nicotinic
cholinergic receptors (55). An autoimmune disease, myasthenia gravis is associated in most cases with hyperplasia of the thymus, whose lymphocytes
synthesize antibodies against the acetylcholine receptor (54).
Muscarinic receptors are coupled via the guanine nucleotide binding proteins
m, adenylate cyclase, phospholipase C and potassium channels. In the gastrointestinal tract, they inhibit the release of somatostatin in canine oxyntic mucosa (56)
and regulate muscle contraction or electrolyte plus water secretions in intestine
(57). Electron microscopic autoradiographic studies using the potent muscarinic
agonist [3H]quinuclidinyl benzilate, showed that the plasma membranes in
acid-secreting parietal cells and the basal plasma membrane of the capillary
endothelium possess muscarinic acetylcholine receptors (58). Acid secretion
stimulated by bethanechol seems to be mediated by the M2-receptor subtype
which is located on parietal cells and displays low affinity for pirenzepine
(Ko = 500 nM). In contrast, vagally-induced acid secretion appears to be mediated
by neural Ml-receptors with high affinity for pirenzepine (Ka = 20 riM). Accordingly carbachol and oxotremorine both inhibit cAMP formation via M2-receptors,
but only carbachol stimulates phosphoinositide hydrolysis via Ml-receptors (59).
Scopolamine and atropine are non-specific antagonists of the muscarinic receptors
204
Gespach, Emami and Chastre
M1 and M2. However, it has been demonstrated recently that a single type of
muscarinic receptor, subtype M2, might be coupled with adenylate cyclase and
phosphoinositides in Chinese hamster ovary cells after transfection with a vector
directing the expression of the porcine atrial M2-type receptor (60). Finally,
N-ethylmaleimide distinguishes between M1- and M2-receptors and the two states
of the M2-receptors (61). The purified muscarinic receptor has a relative
molecular mass Mr of 70,000 (62-66).
Histamine is a very potent stimulant of gastric acid secretion, but this
stimulation is not sensitive to mepyramine or diphenhydramine, which are drugs
that act on the histamine H1 receptors located in the smooth muscles of bronchi
and intestinal wall. The synthesis by Black and associates (67) of a series of
imidazole derivatives analogous to histamine (e.g. burimamide) allowed the
demonstration of the existence of a second class of histamine receptors (H2),
which stimulate gastric acid secretion and the heart rate, and inhibit uterine
contraction in the rat. These investigators completed their work by creating a
series of agonists that act selectively on histamine H1 and H2 receptors (68). The
H2 receptor agonists impromidine and 4-methylhistamine (4-MH) have a stronger
effect on the biological functions controlled by the histamine Hz receptors than
the H~ receptor agonists aminoethylthiazole (AET) and pyridylethylamine
(PEA). A third class of histamine receptors (Hs) has been identified in central
histaminergic neurons and perivascular nerve endings (69-71). In brain, these Hs
receptors are mobilized during inhibition of histamine secretion and biosynthesis.
A new class of histamine receptors, H2h (h = highly sensitive to the H2 antagonists
tiotidine, famotidine, ranitidine, metiamide and cimetidine) has just been
demonstrated and characterized by pharmacological and biochemical studies.
These HEh receptors control the uptake of serotonin by human platelets (9).
In addition to mastocytes and endocrine cells of the gastric epithelium,
histamine has been localized in the ganglions and nervous fibers adjacent to the
rat ileum submucosa (72). It was shown in a carcinoma originating from a gastric
metastasis that pentagastrin induces histamine secretion in vivo, whereas
somatostatin decreases its concentration in plasma (73). The vasodilating effect of
histamine has been shown in the central and peripheral nervous systems, the
skeletal and intestinal muscle, the mesentery and the circulation in the gastric
epithelium (74). It was also observed that intragastric histamine perfusion
increases the permeability of the microvascular system to macromolecules via
histamine H1 receptors, but only when/3-adrenergic receptors are blocked. In the
treatment of Zollinger-Ellison syndrome, gastric acid secretion might be selectively controlled by different drugs (Fig. 1), such as H 2 receptor antagonists:
cimetidine, ranitidine, famotidine; suicidal inhibitors of the luminal proton pump:
omeprazole, or of histidine decarboxylase: c~-fluoromethylhistidine; anticholinergic agents: pirenzepine; and non-competitive inhibitors of the histamine 1-12receptor: somatostatin, its long-acting analog SMS 201-995 and prostaglandins
(75). Histidine decarboxylase is the histamine-forming enzyme in nerve fibers and
the other endocrine or paracrine APUD cells containing the bioamine. Compared
to the classical HE antihistamine cimetidine, the new drugs ranitidine and
famotidine exhibit the following pharmacological differences in vivo and in vitro:
G.I. Tract Membrane Receptors
205
cimetidine
HN~
N-C~N
ranitidine
famotidine
s•/C\CHzSCH2CHzCNH2
NSO~NNj
N
J
H~N NHI
OCH3
H3Cn~r
CH3 0
N-~/~{O
CH3
omeprazole
H
H O
i .
p, enzep,oe
~CH2/N~jN-CH3
I
!
(D) Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Th9
SMS 201-995
Prostaglandins
Fig. 1. Antiacid drugs in the treatment of the ZoUinger-Ellison syndrome.
(1) increased inhibitory potency (famotidine >ranitidine >cimetidine), (2) more
prolonged action, (3) non-competitive antagonism and (4) apparently irreversible
blockade (76, 77). As shown in Fig. 2, large differences were observed for the
pharmacological properties of the histamine H2 receptors in gastric glands
isolated from the human and guinea pig stomach. In contrast, the relative
potencies and inhibition constants for the H1/H2 receptor agonists and
antagonists are remarkably similar in normal and cancerous gastric cells in man
(Table 1). The HGT-1 human gastric cancer cell line is therefore a suitable model
for studying the pharmacological properties of histamine H2 receptor antagonists
under clinical investigation in ulcer therapy.
The anti-acid role of prostaglandins is due to their ability to induce the
biosynthesis and secretion of the cytoprotecting mucus in the fundic mucous ceils,
as well as the secretion of bicarbonate ions by the gastric surface epithelium. A
spectacular illustration of this was published by Fanning and Tyler, who used as a
model the Australian frog Rheobatrachus silus (78). The female swallows the
2116
Gespach, Emami and Chastre
=100 ~.~HGT-1
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Fig. 2. Pharmacology of histamine H2 receptor in normal or cancerous human gastric cells and
guinea pig fundic glands. The activity of the histamine H 2 receptor has been analyzed: Upper panels:
after stimulation by histamine and its selective agonists for H 1 receptors (AET, PEA) or H2 receptors
(Impromidine: I, 4-MH); Lower panels: after blockade by its selective antagonists at H 1 receptors
(diphenhydramine: DPH) or H2 receptors (ranitidine: RA, oxmetidine: OX, cimetidine: C).
Table 1. Relative potencies of histamine H 1 and H 2 respectively agonists and antagonists on
histamine H 2 receptor activity in fundic glands isolated from man and guinea pig. Comparison with
the HGT-1 human gastric cancer cell line
Agonists
Antagonists
Models
I
Guinea pig
Human
HGT-1 cells
200
30
50
H 4-MH AET PEA
1
1
1
0 . 0 9 0 . 1 8 0.18
0.21 0.1
0.01
0.16 0.2
0.01
RA
OX
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5
7
9
0.8
6.8
9
1
1
1
0.01
0.02
0.05
The relative potency of each drug was established as the ratio: ECso for histamine/ECso for agonist;
ICso for cimetidine/ICso for antagonist. H 2 receptor agonists: impromidine (I), histamine (H), 4-MH
and antagonists: ranitidine (RA) and oxmetidine (OX). H 1 receptor agonists: histamine (H), AET,
PEA and antagonist: dephenhydramine (DPH). The data revealed that differences were observed
between the guinea pig and the human models concerning the relative potencies of the agonists PEA
and impromidine (I) as well as for the antagonist oxmetidine (OX). AET: 2-(2-aminoethyt) thiazole;
PEA: 2-(2-pyridyl) ethylamine.
G.I. Tract Membrane Receptors
207
fertilized eggs. The stomach is then converted into a uterus until the expulsion of
the mature frogs (gestation time 8 weeks). The parietal cells then exhibit an
expanded tubulo-vesicular system characteristic of non-secreting cells. The
substances responsible for this hospitable activity of the stomach seem to be the
prostaglandins secreted by the larva during its development. However, the
cytoprotecting mechanism of prostaglandins E2 against necrosis of the gastric
mucosa caused by the ingestion of ethanol and its penetration into the digestive
tissues is still poorly understood (79). The possibility that the PGE2 controlled by
the central nervous system inhibits acid secretion cannot be excluded. Prostaglandins synthesized by the parietal and surface mucus cells of the epithelium control
gastric acid secretion by inhibiting the activity of the histamine He receptor
coupled to the parietal cell adenylate cyclase (80, 81). These biological actions of
prostaglandins are controlled by the activation of binding sites in the fundic
mucosa, as shown in pig and rat (82). Secretion of the intrinsic factor seems to be
one of the parietal cell functions in man, and of the pepsin chief cells in rat (83).
Tile histamine H2 receptor is involved in that stimulation (83, 84). The existence
of autoantibodies against the parietal cell has been demonstrated in the plasma of
patients with pernicious anemia and Biermer disease (atrophic gastritis) and in
the BB rat, which develops severe insulin-dependent diabetes at the age of
60-120 days (85). The immunoglobulins G extracted from the serum of
pernicious anemia patients contain autoantibodies against the gastrin receptor
(8:5). These antibodies inhibit the binding of gastrin-17 and the stimulation of
aminopyrine capture by the parietal cell.
Many other hormones, neuromediators and agents control the digestive
secretions and activity, either directly, by acting on the gastrointestinal epithelium, or indirectly through relays between the central nervous system and the
target tissues. We will first consider the agents opposing the aggressive acid and
pepsin secretions of the stomach, and formation of ulcers, i.e. prolactin,
dopamine, a~2-adrenergics, neurotensin (86) and PYY (87); then, the agents able
to stimuate these secretions and induce ulcer formation, i.e. leukotrienes and
porcine ileal peptide (88, 89). The gastrin releasing peptide (GRP) isolated from
the dog intestinal muscle and brain has 27 amino acids (90, 91). An analogous
peptide bombesin, was previously identified in amphibian skin (92). GRP is
present in nervous fibers of the human gastrointestinal tract (90). GRP and
bombesin stimulate gastrin release by the antral mucosa and act directly on acid
secretion of the fundic mucosa kept in an Ussing chamber (93). These two
peptides have an identical C-terminal sequence of 7 amino acids which determines their biological action on gastric acid secretion. When GRP is injected into
the intracisternal cavity of the rat, it inhibits the volume and activity of basal or
histamine-stimulated gastric secretion, despite an increase in plasma gastrin. In
the rat, intracerebral injection of GRP stimulates gastric mucus secretion (94). In
man, perfusion with GRP stimulates the secretion of numerous hormones acting
on the gastroenteropancreatic axis, including gastrin, pancreatic polypeptide,
insulin, glucagon and GIP. The natural ligands of the opiate receptors,
Met-enkephalin and Leu-enkephalin, are pentapeptides which were first isolated
from sheep hypophysis and hypothalamus (95). Related endogenous substances
208
Gespach, Emami and Chastre
with analgesic and morphinic properties were also called "endorphins", such as
fl-endorphin, 31 residues (96) and dynorphin, 17 or 32 residues (97, 98). Peptides
of the type to which enkephalins, endorphins and dynorphins belong are
nowadays referred to as "opioid peptides" (99).
Enkephalins are located in endocrine cells (enterochromaffine cells) and in
nervous fibers of the gastric antrum and myenteric plexus. Met- and Leuenkephalins increase basal or pentagastrin- and histamine-stimulated acid secretion and the blood flow of gastric mucosa in a dog with a Heidenhain pouch or
gastric fistulae. Morphine- or Met-enkephalin-induced stimulation is inhibited by
naloxone (an antagonist of opiate receptors), and also by atropine and metiamide, which also suggests cooperativity between opiates and cholinergic and
histaminergic receptors (100). Morphiceptin is a small peptide composed of 4
amino acids: NH4-Tyr-Pro-Phe-Pro-CONH2. This tetrapeptide, which is the
amide derivative of a tetrapeptide that is released on the digestion of bovine
fl-casein (101), is a potent and selective agonist of the opiate #-receptors and has
analgesic properties (102). The ealeitonin gene-related peptide (CGRP) is present
in the central nervous system and in the innervations of the gastrointestinal tract.
This neuromediator, composed of 37 residues, inhibits acid secretion stimulated
by injection into the intracisternal cavity of pentagastrin, histamine, bethanechol
and TRH (103).
In earlier studies, we established the tissue distribution and the pharmacological properties of the receptor-cAMP systems sensitive to secretin and its
structurally related peptides in gastric cells isolated from normal and transformed
epithelia in man and laboratory animals (104-111). These peptides include the
glucagon-related pancreatic peptide (GRPP), glicentin, pancreatic glucagon
(G-29), oxyntomodulin (G-37), the intact and the truncated glucagon-like
peptides 1 (GLP-1 and TGLP-1), GLP-2, vasoactive intestinal peptide (VIP), the
peptides with N-terminal histidine and C-terminal isoleucine amide (PHI) or with
C-terminal methionine amide (PHM), the growth hormone releasing factors
(GRF), secretin, helodermin, helospectrin, gastric inhibitory peptide (GIP) and
somatostatins (Fig. 3). Most of these peptides are known to exert an inhibitory
action on gastric acid secretion (glicentin, G-29, G-37, GLP-1, TGLP-1, VIP,
secretin, GIP and somatostatin) beside the stimulation of exocrine and endocrine
secretions by the stomach and intestine. These regulatory agents are located in
brain and endocrine cells or nerve fibers in the gastrointestinal wall and mucosa.
DEVELOPMENT OF RECEPTORS IN THE GASTROINTESTINAL
TRACT
Numerous hormones and mediators are synthesized and stored in the
digestive tract during fetal life in man and laboratory animal. They include
gastrin, secretin, motilin, GIP, VIP, enteroglucagon and somatostatin (112-114).
In 15-21 week-old human fetuses, immunoreactive VIP is present in the fundus
at 16pmol/g and in the antrum at 25pmol/g (114). This biosynthesis is
accompanied by the formation of nervous structures ensuring the release of
G.I Tract Membrane Receptors
209
t
porcine GRPP
1O
20
3O
R SL ONT E EKS RS F PA POT DP L DDR DOMTED(KR)
porcine G-29
catfish G-34
HSQGTF T S D Y S K Y L D S R R A Q D F V Q W L M N T
H A D G T Y T S D V S S Y L Q D Q A A K D F I TWLKSGQPKPE
human G-37
H SQGTF T S D Y S K Y L D S R R A Q D F V Q W L M N T K RNRNN I A
porcine G-37
HSQGTF TSDYS KYL DSRRAQDFVQWLMNTK RNKNN I A
human rat GLP-1 HDEFERHAEGTFTSDVSSYLEGQAAKE F I A W L V K G R
human GLP-2
H A D G S F S D E M N T I L D N L A A R D F I N W L I QTK I T DR
chicken VlP
H S D A V FTDNYSR F RKQMAVKKYL N S V L T
dog, rat,human,porcine,bovine VlP H SDAV FTDNYTR L RKQMAVKK YL NS I LN
guinea pig VlP H S D A L FTDTYTR L RKQMAMKKYL N SVL N
orcine PHI
H A D G V F T S DFSR L L G Q L S A K K Y L E S L I
~uman PHM
bovine PHI
rat PHI
human pancreatic GRF
rat hypothalamic GRF
porcine hypot halamic GRF
~orcine bovine Secretin
uman Secretin
chicken Secretin
Helodermin
Helospectrin
orcine GIP
~uman GtP
Somatostatin
H A D G V F T S DFS K LLGQ L S A K K Y LES
H A D G V F T S DYSR L L G Q L S A K K Y LES
H A D G V F T S DYSR LLGQ I S A K K Y L E S
YADA I FTNSYRKVLGQLSARKLLQD
HADA I F TSSYRR I L G Q L Y A R K L L H E
YADA I F TNSYRKVLGQLSARKLLQD
LM
LI
LI
IMSRQQGESNQERGA
IMNRQQGERNQEQRSRFN
IMSRQQGERNQEQGARVRL
HSDGTFTS ELSRLRDSARLQR LLQGLV
H S D G T F T S E L S R L REGARLQ R L L Q G L V
HS DG LF TS EYS KMRGNAQVQK F I QN LM
H S D A I F T Q Q Y S K L L A K L A L Q K Y L A S I LGSRTSPPP
H S D A T F T A Q Y S K L L A K L A L Q K Y L E S I LGSSTSPRPPSS
Y A E G T F I SDYS I AMDK I RQQDF VNWLLAQKGKKSDWKHN I TQ
Y A E G T F I SDYS I AMDK I HQQDFVNWLLAQKGKKNDWKHN I TQ
AGCKNF FWKT F TS C
1
10
20
30
40
Fig. 3. Sequence comparison of secretin and its structurally related peptides. A: alanine; D: aspartic
acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine;
M: methionine; N: asparagine; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W:
tryptophan; Y: tyrosine. According to: A one letter notation for amino acid sequence. Eur. J.
Biochem. 5:151 (1968),
neuromediators close to their target tissues (112). The enzymatic systems
responsible for neuromediator metabolism become operative. They consist of
histidine decarboxylase (HDC) and histamine methyl-transferase (HMT) for
histamine, choline acetyl-transferase and acetylcholine esterase for the cholinergic
agents, and tyrosine hydroxylase and monoamine oxidase for the adrenergics and
histamine (115).
The gastric mucosa differentiates very late in the rat (116). Pepsinogen
becomes detectable in fetal stomach as from the 17th day of gestation (117,118).
Maturation of the pepsin chief cells in the fundus starts on the 15th day
postpartum and is only completed 25-30 days after birth. Administration of
hydrocortisone and thyroxin to 2-9 day-old rats or 5-10 day-old mice induces
early pepsin activity, maturation of the pepsinogen molecular forms and
differentiation of the gastric mucosa cells containing pepsinogen (118). Similarly,
injection of another glucocorticoid, corticosterone, to 8 day-old rats causes
premature induction (at 12 days) of acid secretion stimulation by gastrin,
histamine and carbachol, and of pepsin secretion by carbachol. The secretin
receptors, preferentially located in rat antrum (107,109, 119), can thus play a role
in the differentiation and function in rat stomach, inasmuch the secretin-sensitive
adenylate cyclase has been identified during the neonatal period in rats (120). The
parietal cells begin to differentiate on the 19th day of gestation (117, 121). Basal
acid secretion appears 15 days after birth; it is stimulated by carbachol, not by
210
Gespach, Emami and Chastre
histamine nor gastrin (122). According to Ackerman, impromidine does not
stimulate H § secretion in 11 to 18 day-old rats. Acid secretion and the H2
receptor are stimulated by impromidine and histamine between the 19th and the
22nd day after birth (123). By contrast, pentagastrin and the cholinergic agonist
bethanechol stimulate acid secretion 3 to 5-fold in the 14-day-old rat. Gastrin
receptors in the gastric mucosa are detectable 20 days after birth in rat, i.e. 2 days
after weaning (122). Their number increases considerably until adulthood (60
days). Acid secretion induced by gastrin and the histamine H2 receptor seems to
be lacking during fetal and postnatal life in rat, but cholinergic receptors are
detected and functionally coupled to acidification of the stomach contents during
fetal life. This apparent insensitivity of the gastric mucosa can be explained by the
fact that the binding sites for radioiodinated gastrin are undetectable during the
period from 5 to 20 days postpartum (122) and that the H+-K+-ATPase activity
was altered in rat gastric mucosa on day 12 after birth (124).
In man, parietal cells differentiate earlier, at 10 weeks of gestation. They are
present in the fundus of 11-12 week-old fetuses (125). In agreement, it has been
observed that histamine activates adenylate cyclase in gastric glands isolated from
15 and 23-week-old human fetuses (126,127). In the rat, gastric receptors for
histamine, secretin, somatostatin and glucagon are functional during fetal life
(120). However, large differences in the activity of these receptors appear during
the postnatal period and weaning (21 days). These two developmental stages
respectively coincide with milk taking and the transition from milk feeding to
adult-type diet. There thus exists a transition period, before weaning, during
which the suckling rat gradually ingests the adult diet and abandons its milk
feeding. Advanced weaning at 14 days induced early maturation of gastric
receptor systems in rats at 21 days after birth. Coupling of the histamine 1-12
receptors to adenylate cyclase is also lacking between the 10th and 21st day after
birth (120). It is therefore likely that milk feeding uncouples the histamine 1-12
receptor at the basal pole and the proton pump at the apical pole of the
acid-secreting cell (124).
Milk has been recognized as efficacious in preventing stress-induced gastrointestinal bleeding in hospitalized children (128). Milk phospholipids constitute a
hydrophobic barrier on the surface of gastric epithelium and might thus be one of
the physicochemical elements responsible for the ulcer-protecting function of
milk. The phospholipid components phosphatidylcholine and phosphatidylethanolamine are present in milk at rather high concentrations (129). During
digestion of milk caseins, particular peptides are formed, e.g. caseinomacropeptide and phosphopeptides, whose physiological role is still unknown. For
example, caseinomacropeptide (CMP) is formed by the action of chymosin or
rennin and of pepsin on casein K, which is cleaved between phenylalanine 105
and methionine 106. This 6.8 KDa CMP might be absorbed as such and might act
as an antagonist to gastrin. Another peptide resulting from the digestion of
fl-casein is remarkably identical to a specific ligand of the #-opiate receptors,
morphiceptin NHa-Tyr-Pro-Phe-Pro-CONH~ (102). It is thus possible that the
release of this peptide during milk digestion might modulate pain perception and
stomach secretions due to its resistance to gastric proteases. The presence of
G.L Tract Membrane Receptors
211
phosphopeptides in milk might favor the absorption of CMP, which controls vagal
electric activity by inhibiting histamine secretion by gastric mucosa. Other human
milk components might also participate in this anti-ulcer effect. For example:
calcium, prostaglandins, insulin, EGF-urogastrone, bombesin/GRP, the transforming growth factors oL-TGF and fl-TGF, pepsin-resistant mammary growth
factor MDGF1, growth factor HMGF III, opiates, VIP, neurotensin, somatostatin, fl-casomorphin and 8-prolyl-fi-casomorphin (129-138).
The peptides and other components of milk may thus act against ulceration
of the gastric and duodenal mucosa through the following different mechanisms:
(1) a direct effect on gastric receptors controlling the secretion of acid, pepsin,
mucus and bicarbonate via the regulation of information-transducing systems by
the cell basal pole; (2) an indirect effect via the receptors, or via effector systems
involved in the cephalic phase of gastric digestion; (3) a direct effect on the
biosynthesis and secretion of endogenous agents stored either in the gastric and
intestinal mucosa such as prostaglandins, histamine, polyamines, gastrin, GIP,
somatostatin, glucagon and secretin, or in the wall of the digestive tube, such as
VIP, neurotensin, bombesin, etc., via the regulation of cell metabolism and
endocrine, paracrine and neural secretions; (4) a direct effect on the renewal and
selective differentiation of the protective digestive cells from precursor cells via
control of epithelial growth and maturation. The fi-TGF, for instance, interacts
with specific binding sites in human carcinoma cells and induces the expression of
fibronectin and collagen, both components of the cellular matrix involved in the
differentiation processes in malignant and normal epithelial cells (139); (5) a
direct or indirect effect of the milk components inhibiting the proton pump via
control of enzymatic activity by the apical pole of the parietal cell. For example,
po]tyunsaturated fatty acids irreversibly inhibit H+/K + ATPase in pig and rat
gastric membranes. EGF acts synergistically with this direct effect of milk
prostaglandins to stimulate prostaglandin secretion in the isolated rat stomach
(140). Milk itself is able to stimulate prostaglandin biosynthesis in fibroblasts
obl:ained from cultured human skin biopsies.
In agreement with these results, the prostaglandin E2 receptors are mobilized
in the gastric epithelium of the adult rat on a milk diet (141). The opposite
regulation was observed, in the same model, for the histamine H2 receptors,
which were not mobilized in vivo during the 4 day-milk feeding. In man,
EGF-urogastrone inhibits acid secretion stimulated by pentagastrin, histamine
and insulin and the secretion of intrinsic factor. EGF-urogastrone secreted by the
submaxillary glands inhibits histamine-stimulated acid secretion in perfused rat
stomach and in dogs with Heidenhain pouches (142). The anti-acid effect of EGF
is probably controlled by the receptors identified and characterized in gastric
glands (143). EGF is atrophic peptide on the gastric mucosa in 10-day-old and in
adult rats. It stimulates DNA, RNA and protein synthesis (144). It can also
stimulate stomach and duodenum ornithine decarboxylase activity in 8-day-old
mic.e (145). Ornithine decarboxylase is a key enzyme in the biosynthesis of the
polyamines that may inhibit the parietal cell proton pump (146). Polyamines such
as putrescine, spermidine and spermine are present in most tissues and play a
vital role in cell growth and differentiation. A mutant line of hamster ovary cells,
212
Gespach, Emami and Chastre
deficient in polyamine biosynthesis, exhibits absolute dependence on polyamines
for growth (147).
In rat, the intestinal epithelium also maturates very late and displays an
antero-posterior differentiation gradient (148). In the 15-16 day fetus, the
epithelium is multilayered, as a result of cell proliferation. During postnatal
development, there is a spectacular rise in the activity of numerous digestive
enzymes of the intestinal epithelium, including disaccharidases, alkaline phosphatase, sucrase-isomaltase, lactase, phospholipase A2 and fructosediphosphatase (149). Weaning, glucocorticoids or food deprivation may influence
their activities and colon growth (150). In man, the intestinal epithelium is
multilayered in the 7-8 week-old fetus. Villi and crypts structures start appearing
very early between the 9th and 12th weeks of gestation (125). Villi start to appear
from the 8th week in the duodenum and this coincides with the maturation of
lactase and aminopeptidase of the small intestine brush border (151). The anlagen
of the villus structures and immunoreactive VIP appear at the 8th week
(112, 114). VIP receptors are functional from the 10th week on (Christian
Gespach and coworkers, unpublished). At this stage of development the
duodenal lumen is manifest. Villi and crypts start appearing in the terminal part
of the small intestine (125). Villi isolated from 18-23-week human fetuses possess
specific *25I-VIP receptors coupled to adenylate cyclase. This transduction system
is not activated by other hormones or regulation agents of the gastro-enteropancreatic system (152). At that stage of development (18-23 weeks), the
meconium is present in intestinal lumen, the activity of dipeptidases and
disaccharidases increases and Paneth cells appear (125).
Cystic fibrosis (CF) can be detected during fetal life in man by measuring the
activity of alkaline phosphatase of intestinal origin in the amniotic fluid (153).
Already, discovery of DNA markers closely linked to a CF locus, have provided
early pre-natal diagnosis in families with an affected child (154). Cystic fibrosis is
a genetic disease characterized by general anomalous muciparous and hydroelectrolytic secretion/reabsorption of the exocrine glands. While a mutant gene of
cystic fibrosis has been localized on chromosome 7, near 4 already known g e n e s
(oncogene met, a T cell receptor component, parooxonase and collagen), the
biochemical bases of the disease are still unknown (154, 155). The intestine is not
functional (obstructive meconium). This disease affects 1 child in 2000 and is
lethal, generally before the age of 30. Cystic fibrosis patients secrete substances
into serum, sweat, saliva and urine. These factors are inhibitory on the
Na+/K+-ATPase activity in membranes of rat submandibular glands (156). Sweat
collected by micropuncture of the sweat glands of cystic fibrosis patients contains
very high concentrations of C1- and Na § Sweat ducts and airway epithelia have a
decreased C1- permeability, intact (157) or accelerated Na § absorption (158),
coupled with fluid movements that may contribute to the relative dehydration of
the airway secretions. When excised from the apical membrane, cystic fibrosis
epithelial cells contain C1- channels that have the same conductive properties as
those from normal cells (159). In contrast, normal regulation of the Ca 2§
activated K § channels occurred at the basolateral membrane. Thus, the disease
might result from the presence of an inhibitor of the C1- channel, an adnormal
G.L Tract Membrane Receptors
213
regulatory site on the channel itself, or a faulty insertion at the apical membrane
(1:59,160).
In agreement, (1) the catalytic subunit C of a cAMP-dependent protein
kinase opens the apical C1- channel in cell-free patches from normal cells, but
fails to open C1- channels in CF cells (161,162); (2) the VIPergic innervation of
the acini and ducts of the eccrine glands is deficient, which suggests a causal
relationship between this abnormality and the ability of VIP to promote water
and chloride exchanges through the transporting epithelia (163). It seems that a
Na+-K+-ATPase might act in concert with a Na+-Cl--cotransferring system
located in the basolateral membranes of colon cells, such as those of line T-84
(164). The entry of chloride "pumped" from the extracellular medium might lead
to chloride secretion on the apical side of the cell. The common denominator of
these abnormalities could thus be the result of a dysfunctioning of the
receptor-transductor system of VIP or its biosynthesis and secretion (Chastre
et al., unpublished observation); (3) naturally-occurring antibodies to VIP were
increased 3.3-fold in plasma samples from patients with cystic fibrosis (165).
These antibodies might therefore interfere on the prejunctional regulation of VIP
release by inhibitory autoreceptors in intestine and stomach (166), or on the
stimulus-secretion coupling associated with the regulation of enterocyte ions
transport by VIP, including protein phosphorylation (167); (4) a serum factor has
been identified at elevated levels in patients and obligate heterozygotes (168). For
CF patients, the plasma levels of the antigen MRP-14 were in the range of
250-5,000ng. m1-1. For control individuals, values ranged between 2-50 and
55-135ng. m1-1 (169). The gene for this serum protein has been mapped to
chromosome 1 (170) and the use of the anti-MRP-14 serum will be important on
sceening plasmas of carriers of the CF gene and their relatives (169). Together
with molecular genetic approaches, analysis of the biochemical steps involved in
the stimulus-secretion coupling in secretory epithelia are therefore crucial in
determination of the basic defect in cystic fibrosis.
RECEPTORS IN NORMAL, TRANSFORMED AND IMMORTALIZED
GASTROINTESTINAL CELLS IN CULTURE
Studies on membrane receptors in the gastrointestinal tract have been mainly
performed on epithelial and muscle cells isolated at various stages of human and
animal development after chemical and/or enzymic treatment to dissociate the
tissue. Previous in vitro studies on the function and metabolism of freshly isolated
cells and tissues were limited by the rapid cell alteration and necrosis after 2 or 3
hours of incubation. Moreover, analysis of the active proliferation and
differentiation processes associated with the active renewal of the gastrointestinal
mucosa was not possible in these short-term experiments. Thus, only the ability to
culture gastric and intestinal ceils made it possible to investigate the synthetic,
secretory and proliferative functions of digestive cells in vitro. These models
included organ culture from normal specimens and epithelial cells in primary
cultures (171-174), cancer cell lines established from spontaneous or chemically
214
Gespach, Emamiand Chastre
induced tumors (175-177) and immortalized epithelial cells obtained after
transfection by viral or cellular oncogenes (178,179). Different investigations on
the growth, differentiation (180, 181), ultrastructure (149), specific antigenic
determinants (149), membrane receptors (173, 178, 182, 183), and function in
digestive epithelial cells were then performed on these models (172, 174).
The transformed human colonic cell line HT-29, clone 18, has the ability to
differentiate into enterocyte-like cells when glucose is replaced by galactose in the
culture medium (180,182). This differentiation is accompanied by the inhibition
of cell proliferation (doubling time: 19 hr for undifferentiated cancerous cells and
52 hr for the enterocytes HT29-18), a ten-fold increase in villin biosynthesis and
storage, the appearance and increase of brush border enzymes such as alkaline
phosphatase,
disaccharidases and aminophosphatase.
The enterocytic
differentiation of these cells was accompanied by a spectacular loss in VIPergic
receptor activity when they were incubated at 37~ in the absence of IBMX
(182). In agreement with these biochemical and morphological data, replacement
of galactose by glucose led to a partial return of cyclase activation by VIP and
forskolin towards the values previously observed in undifferentiated cells (182).
The same phenomenon was observed in rat intestinal villi isolated during fetal
and postfetal life (183). In rat, another difference appears in the VIPergic
activation of the intestinal epithelial cell during fetal life and the first days after
birth. In the 17-19-day fetus the intestinal epithelial cells are thus 50 times more
sensitive than the cells isolated between the postnatal period and adult age. These
differences might be due to a series of nutritional, biochemical and physicochemical mechanisms analyzed in (183). The variations in sensitivity to VIP were also
observed in human intestinal mucosa during the period of fetal development
under consideration (152). The functional characteristics of the VIPergic receptor
is a good marker for the differentiation state of the cancerous or normal intestinal
cell in both man and rat. In the human colonic adenocarcinoma HT-29 cell line,
nuclear receptors for VIP have been identified with apparent molecular sizes
similar to those present in plasma membranes of the cancer cells (184). This is
consistent with the active internalization processes of VIP together with its
surface receptors in HT-29 cells, leading to cell desensitization (vide infra), and
possibly to intracellular/intranuclear regulations by the neuropeptide and/or its
receptor (183,184). The presence of nuclear VIP receptors in normal intestinal
cells and other tissues remains to be explored.
VIP receptors are also retained in immortalized intestinal cell lines derived
from rat fetuses at 19 days of gestation after electroporation in the presence of
different recombinant DNAs containing the viral oncogenes E1A and large T
(178). These immortalized cell lines, designated SLC, possess several properties
observed in the parent cells of this tissue, including the expression of cytoplasmic
villin, enkephalinase and retention of membrane receptors. The effects of VIP
has been compared in the SLC-11 cell line (178), in normal rat intestinal cells
freshly isolated from fetuses at 19 days of gestation (183) or in primary culture for
9 days (173), in the rat fetal intestinal cell line FT33 (174), in the rat colon
carcinoma cell line PROb induced in vivo after trbatment by 1,2-
G.I. Tract Membrane Receptors
215
Table 2. Effects of vasoactive intestinal peptide VIP on membrane receptors in normal, cancerous
and the E1A-immortalized intestinal SLC-11 cells. Comparison with VIP receptor agonists and other
adenylate cyclase activators
Tissues
Intestine
Animal Species
Fibroblasts
Rat
Human
Human
Hamster
Cell culture
Primary
SLC-11
FF33
PROb
HT29-18
Fr, N1
DC3F
Phenotypes
N
I
C
C
C
N
C
+
-
-
+
+
+
+
+
Membrane Receptors
VII?
PHI
G1LF
GIP
Glucagon
Isoproterenol
Forskolin
Prostaglandins
+
.
.
.
.
+
+
+
+
-
.
.
-
.
.
.
.
.
.
.
.
.
+
+
+
.
.
.
+
+
+
.
.
+
+
+
.
.
+
+
+
Nolnnal (N) rat intestinal cells were freshly isolated from rat fetuses at 19 days of gestation (183) or
maintained in primary cultures for 9 days (173). Immortalized (I) rat fetal intestinal SLC-11 cells (178)
were maintained 16 months in culture (54 passages). The fetal rat intestinal cells FT33 (178) are
spontaneously transformed towards the malignant phenotype (C). The PROb cell line originate from
a poorly differentiated rat colon carcinoma induced by 1,2-dimethylhydrazine (a generous gift from Dr
F. Martin, INSERM U. 252, Groupe de recherches sur les cancers digestifs humains et
exp6rimentaux, Facult6 de M6decine, 21033 Dijon, France). The HT29-18 cells (181) originate from a
huraan colonic cancer (C). The human skin fibroblast Fr cells and the foreskin N1 cells established
from a newborn child were respectively donated by Dr V. Barbu (Laboratoire de Biochimie, CHU,
H6pital St-Antoine, Paris, France) and by Dr J. Cornicelli (Columbia University, Atherosclerosis
Center, New York, USA). The DC3F cells are spontaneously transformed from the hamster lung
(178). Cyclic AMP generation induced by PHI, GRF, secretin and helodermin in SLC-11 cells was
probably related to the ability of these peptides to interact with and activate VIP receptors in these
EIA-immortalized rat intestinal cells (Emami et al., manuscript in preparation).
dimethylhydrazine and in the HT29-18 human colonic cell line (182). VIP
inc.reased cellular cAMP generation with a potency ECs0 of 1 0 - 9 M VIP in
E1A-immortalized SLC-11 cells, 3.16 x 1 0 - 1 ~ M in normal rat fetal intestinal cells
and 10-1~
in HT29-18 cells (Table 2). Other systems, including the rat
intestinal cell lines FT33 and PROb, normal fibroblasts from the human skin (Fr
and N1 cells) and spontaneously transformed fibroblasts from the hamster lung
(DC3F cells) are insensitive to VIP with respect to cAMP generation. The rat
fetal intestinal cell line FT33 has some morphological and electrophysiological
properties associated with the smooth muscle (174). The SLC-11 cells were highly
contact-inhibited, formed only monolayers and grew at low starting density, as
judged by their capacity to be cloned. The SLC-11 cells are not tumorigenic in the
athymic nude mice. In contrast, human colonic epithelial cells treated in vitro by
SV 40 were transformed toward the malignant phenotype (179). The SLC cell
lines have been established as models to investigate membrane receptors, gene
expression and cell maturation in the intestine. In that way, these E1A- and large
T-immortalized cells might constitute a reference in the analysis of cancer
progression in the gastrointestinal tract.
216
Gespach, Emamiand Chastre
RECEPTOR DESENSITIZATION AND UP-REGULATION
The insensitivity of a receptor-transducer system to a hormone might be the
consequence of previous exposure of the target tissue to this hormone. The
importance of this insensitivity, variously called desensitization, tachyphylaxis,
refractoriness or down-regulation, is undeniable and applies to numerous
hormones, neuromediators, paracrine agents and drugs (185), including insulin
(186), VIP (106, 187, 188), glucagon (189), opiates (190), carbamylcholine, PTH,
prostaglandins (191), histamine H1 and H2-type receptors (76, 192-195), serotonin, dopamine, isoproterenol (196), EGF (197), LH, ACTH and TSH. Desensitization of the receptors might reflect the cessation of hormonal action when it
is too persistent and massive, which is the case of hormone-secreting tumors,
endocrine, paracrine and neurocrine dysfunctioning and stress. Conversely,
desensitization of a receptor can be related to pathologies resulting from excess of
hormone secretion, as is the case of insulin receptors in diabetes, of adrenergic
receptors in Alzheimer's disease (198), drugs used for asthma treatment,
autoimmune diseases in diabetics (anti-insulin and anti-insulin receptor effects) or
asthmatics (anti-r2 adrenergic receptor effect). Receptor desensitization can thus
be regulated by an antibody against that receptor. A monoclonal antibody specific
for the insulin receptor competes with iodinated insulin for binding to its receptor
in human IM-9 lymphocytes when it is preincubated prior to 125I-insulin addition
(199). This antibody, like insulin, can enhance the degradation of its receptor, but
does not mimick the metabolic effects of the insulin-receptor complex which, in
this cell line, possesses intrinsic tyrosine kinase activity.
Desensitization can be homologous or heterologous. In the first case, an
agent desensitizes the cell to a second exposure to the same regulatory agent; in
the second, other transduction systems are affected by the desensitizing agent.
Chronic treatment of HGT-1 cells with histamine for 6 days specifically
desensitizes the activity of the histamine H2 receptor, while peptidergic receptors
present are not affected, as shown by the ECs0 values of the VIP-glucagon- and
GIP-induced cAMP synthesis (76). Similarly, exposure of the cells to VIP
specifically desensitizes the activity of the VIPergic receptor, while the histamine
H2, glucagon and GIP receptors are not affected (106). Similar results are
obtained when HGT-1 cells are incubated with histamine for shorter periods of
20min to 3 h (192, 193). In this model desensitization of the histamine H2
receptors is regulated by the action of histamine on these receptors and is not due
to a loss of binding activity of the H2 receptor, but to uncoupling between this
receptor and the catalytic subunit C of cyclase, as shown by the unaltered cyclase
activation by sodium fluoride and peptides. This desensitization of the H2
receptors in the HGT-1 cell line might explain the transient effect of histamine
when these cells were incubated at 37~ This transient effect was not observed
when the cells were incubated at 20~ (200). The characteristics of histamine H1
receptor desensitization are different from those described above for H2 receptors
of the human gastric cell line HGT-1 (192, 193), the promyelocytic cells HL-60
and T lymphocytes. A first exposure to histamine significantly reduces the
number of H1 receptors, as shown by tritiated mepyramine binding, and alters
G.L Tract MembraneReceptors
217
hi~tamine potency without changing biochemical and biological responses such as
guanylate cyclase stimulation, glycogen hydrolysis and smooth muscle
contraction.
In the case of the VIP receptors in the HT-29 cell line, the pharmacological,
biochemical and functional characteristics of the process of desensitization by VIP
can be summarized as follows and compared with the histaminergic desensitization described above (187,188). In this cell line, desensitization of the VIPergic
receptor was observed both at 37~ and 10~ Contrarily to what was described
for histaminergic desensitization in HGT-1 cells, VIPergic desensitization in
HT-29 cells was directly correlated with a considerable loss of VIP receptor sites,
e.g. 3,000 to 20,000 sites per cell, while the affinity of the residual binding sites
was not modified. This disappearance of the binding sites in HT-29 cells exposed
to VIP is explained by the internalization of VIP together with its receptor.
Recovery of the sensitivity to VIP is only partial (50-70%). It is possible that the
biochemical and functional characteristics of the desensitization processes are
different in normal and malignant cells. However, the experiments on the human
gastric HGT-1 and colonic HT-29 cell lines show that homologous desensitization
can be mobilized during the digestive processes through a series of biochemical
and pharmacological properties which characterize the biologically competent
systems. These properties include sensitivity, time dependence, temperature
dependence, homology, pharmacological specificity, reversibility, alteration of
the Gs/Gi coupling with cyclase unit, ligand binding and receptor internalization
and recycling.
Desensitization of a receptor-transducer system is heterologous when exposure of a tissue to a hormone induces partial or complete loss of its cellular
response to other hormones or agents. This has already been observed in many
tissues, for instance, in the desensitizing action of isoproterenol in ACTH in
adipocytes (201), of glucagon on PGE1 in the renal MDCK cell line (189), or
PGE1 on isoproterenol in cultured human fibroblasts and on glucagon in rat
hepatocyte membranes (202). Various agents and techniques have been utilized
and developed in order to determine the molecular level at which the receptortransducer system is modified and becomes non-functional. This modification may
be caused by (a) the binding of the ligand to the receptor, (b) the action of
sodium fluoride, GppNHp and cholera toxin on the bipolar coupling system of
receptors-subunit Gs-catalytic unit, and (c) the action of pertussis toxin, somatostatin and carbachol on the bipolar coupling system receptoq-subunit Gi-catalytic
unit.
The following mechanisms are worth mentioning. (1) Binding between the
hormone and the receptor induces receptor internalization into cytosol fractions
or into internalization vesicles. The receptor "disappears" from the cell surface
and is internalized, degraded or recycled. This process is called "receptor
down-regulation" and takes place with the receptors for insulin, VIP, glucagon,
cz.techolamines and prostaglandins (188, 191, 199, 203). (2) During hormonereceptor interaction, the receptor is modified and loses its functional activity of
coupling to the transducer system, e.g. by its phosphorylation, as observed for the
fl.-adrenergic receptors (196,203). Phosphorylation of these receptors depends on
218
Gespach, Emamiand Chastre
kinases activated by cAMP produced by these receptors activation. This is
homologous desensitization (204, 205), because activation of the PGE2 receptor
in erythrocytes is not connected with phosphorylation of the fl-adrenergic
receptor (185,206). In the latter case, the receptor is still able to bind the ligand,
but can no longer transmit this information to the biological systems which are
later mobilized in cells not exposed to the ligand. This is an example of
uncoupling. (3) There is uncoupling between the receptor and the subunits Gi and
Gs of adenylate cyclase or between these regulatory subunits and the catalytic
unit (189, 192, 202). (4) Regulation distal to the receptor-transducer system is
brought about by stimulating cAMP-degrading phosphodiesterases in the case of
transduction systems connected with the cyclic nucleotide metabolism (207). (5)
Modification of the receptor and of the subsequent transduction mechanisms has
also been observed in the case of the/3-adrenergic and VIPergic receptors (188,
203,204).
Other kinases may play a role in the phosphorylation/desensitization of the
/3-adrenergic receptor, for instance protein kinase C and kinases dependent on
the calmodulin-Ca ++ complex (204). For instance, phosphorylation/desensitization of that receptor is mimicked by phorbol esters and calmodulin, but is
reversed by EGTA. In the mutant cell fines UNC, H21a, and cyc- and kin- of
the $49 mouse lymphoma, internalization of the/3-adrenergic receptors seems to
be independent of the cyclase Gs subunit and of the cAMP-dependent kinases
(208). In that case, internalization results in the loss of surface receptors
measured at 4~ At 37~ the down-regulation of the /3-adrenergic receptors in
these systems is influenced by the Gs subunit and leads to the total loss of
functional /3-adrenergic receptors. This is a case of internalization + phosphorylation + uncoupling. The mutants used were UNC, a line in which the
electrophoretic mobility of Gs is modified and the Gs-receptor interaction
defective; H21a, a line with defective Gs-cyclase coupling; cyc-, a line lacking the
45 KDa protein of Gs, and kin-, a line lacking cAMP-dependent kinases. The
/3-adrenergic receptors internalized in $49 cells desensitized at 37~ for 20 min
can be functionally recovered (209) by treatment with polyethylene glycol (PEG).
PEG recouples the /3 receptor with the Gs subunit by releasing it from the
intramembranous compartments in which it was sequestered.
Recovery of the activity of desensitized receptors will be the last mechanism
of receptor regulation considered in this article. This process could also be called
desensitization reversibility. This aspect is especially bound up with the fate of the
internalized receptor, with its possible recycling/insertion in the plasma membrane or with its degradation in specialized intracellular structures. Recovery of
cell sensitivity to a regulatory agent also depends on the receptor biosynthesis and
turn-over, whose regulation is still poorly known. The characteristics of the
processes of cell response recovery depend on time, temperature, ligand
concentration during the desensitization period, and the mechanisms involved in
desensitization such as internalization, receptor phosphorylation, modification of
the receptor transduction systems. After a brief exposure, the receptor can be
rapidly recycled: 20 min is necessary for the insulin receptors, and 10 min for the
VIP receptors (188). After a first prolonged or even chronic exposure, the time
G.I. Tract Membrane Receptors
219
necessary to recover 50% of the initial sensitivity becomes much longer: 40 min
for the histamine HI receptors (195), 3 h or more for the H2 receptors (192), 6h
for the fl-adrenergic and PTH receptors, 12 h for the serotonin receptors, 16 h for
the insulin receptors (199), and 24-36 h for the fl-adrenergic receptors (210). In
the last case, which concerned cultured 1321 N1 human astrocytes, /3 receptor
biosynthesis and recovery depend on the state of confluency of the cells.
Biosynthesis of newly formed proteins is necessary, since the recovery of the
12:5I-hydroxybenzylpindolol binding sites was blocked by cycloheximide. Reinsertion of preformed reserve receptors becomes necessary for the expression of
possible cell sensitivity to a second exposure to the regulatory agent.
The mechanisms involved in the recovery of cell sensitivity after a desensitizing step call for a brief mention of the converse processes of receptor sensitization
of "up-regulation". The sensitization processes have been described for the
gastrin receptors in rat oxyntic membranes (48), the insulin receptors in cultured
3T3-L1 preadipocytes (186), prolactin receptors in lung and liver, the LH
receptors in ovaries and the prostaglandin receptors in liver after indomethacin
treatment (211). In the case of the histamine 1-12receptors in the gastric mucosa
of the adult rat on a milk diet, receptor sensitization may be connected with a
defect in histamine secretion/biosynthesis in the gastric mucosa, and with an
increase in the number of histamine receptor-transducer units or their efficiency.
Similarly, sensitization of the gastrin, LH and prolactin receptors has been
observed in vivo (48) or in cultures of differentiable cells. In the case of insulin
receptor autoregulation, it has been indicated that the receptor recycled on the
cell surface may have a different structure from that of the newly synthesized one
(186).
CONCLUSIONS
We have presented evidence that membrane receptors and their related
transducers play important roles for the physiological as well as for various
pathological states of the gastrointestinal tract. The multifactorial regulation of
the mucosal growth and function coincide with the heterogeneity of the exocrine
and endocrine populations originating from the progenitor cells in the stomach
and intestine.
These regulatory systems are operative before the morphological and
functional differentiation of the mucosa and are retained in cancerous and
immortalized epithelial cells. Membrane receptor activity is modified: (1) during
the fetal and postnatal life; (2) according to nutritional events such as milk and
food intake, the weaning; (3) during the differentiation and maturation of
transformed intestinal cells; (4) following desensitization to the ligand; (5) by a
wLriety of agents secreted from the central nervous system, the periphery or
locally; (6) by pharmacological drugs acting as competitive or non-competitive
inhibitors of the receptor-ligand interaction (36, 67, 75) or as regulator of its
transduction system (81,104); (7) by receptor and ligand autoantibodies.
The same ligand may activate sequentially two different transduction systems
220
Gespach, Emami and Chastre
(213,214) and, accordingly, induce heterologous receptor desensitization to
regulatory signals. Conversion and maturation of the ligand precursor by specific
enzymes occur in neurocrine-/endocrine-/paracrine-/secreting cells and by the
target tissue (215). From the cleavage of active peptides by proteolytic enzymes
(arginine vasopressin, pancreatic glucagon and glucagon-like peptide-1) arises
truncated derivatives with potent and new biological activities (216,217).
Peptides and bioamines are the natural ligands activating membrane receptors
coupled with the Gs/Gi subunits of adenylate cyclase, the IP3/CaZ+/protein
kinase C cascade and tyrosine kinases (Tables 3 and 4).
The molecular components of the receptors coupled with the GTPases of
adenylate cyclase and phospholipase C are similar in apparent molecular weight
but are different from those coupled with tyrosine kinase activation (Tables 3 and
4). Membrane receptors and their transduction systems or ligands possess some
structural and functional homologies with viral and cellular oncoproteins: EGF,
NGF, PDGF, the CSF-1 macrophage colony-stimulating factor, gastrin,
Table 3, Transduction systems and molecular identification of membrane receptors for histamine,
secretin and its related peptides in digestive organs and other tissues
Receptors
Transducers
Tissues
Species
Cross-linking,
solubilization
agents
Mr,
KDa
Refs.
(109)
Secretin
Gs
Stomach
Rat
DSP
62-33
Glucagon
Gs
ICK
Liver
Rat
HSAB
63
Intestine
HT-29 cells
Rat
Man
Man
Man
Rat
VIP
Gs
Stomach
Liver
Brain
Rat
DSP
DSP
DSP (CSR)
DSS (NR)
DSP
Triton
X-100
DSS
Somatostatin
Gi
Pancreatic
Acini
Rat
HSAB
88
GIP
Gs
Pancreatic
fl cell
Hamster
DSP
59-41
GRF
Gs
Pituitary
Rat, Ox
DSS
Histamine
(H1)
(Ha)
ICK
Brain
Thymocytes
Thymocytes
Man
Calf
Calf
U.V.
Nonidet
Nonidet
Gs
73-33
63-30
120-64
49
69-34
200
77-19
72-47
(214,218)
(219,220)
(221)
(222)
(184)
(108)
(223)
(224)
(6)
(225)
(106,110)
(226)
72
(227)
160
50
40
(228)
(229)
Transduction systems: GTPases of adenylate cyclase (GJGi subunits) or phospholipase C
(IP3Ca2+/protein kinase C: ICK). The lzSI-labelled ligands have been immobilized on their specific
cell surface receptors (CSR), using the cross-linking agents DSP (dithiobis-succinimidyl propionate),
HSAB (hydroxysuccinimidyl-4-azidobenzoate), EGS (ethylene glycol bis succinimidyl succinate), DSS
(disuccinimidyl suberate), SANAH (N-succinimidyl 6-4'-azido-2'-nitrophenylamino-hexanoate) or
U.V. irradiation: U.V. The complex between the ligand and its receptor has been solubilized using
the following detergents: SDS, Triton X-100, Lubrol, Nonidet or CHAPS. In HT-29 cells, the
apparent molecular sizes for 125I-labeledVIP cross-linked with cell surface receptors CSR and nuclei
(nuclear receptors NR) are quite similar in apparent molecular mass (184).
G.I. Tract Membrane Receptors
221
Table 4. Tranduction systems and molecular identification of membrane receptors for gastrin/CCK,
neurotensin, opiates, nicotinic, muscarinic and adrenergic receptors in digestive organs and other
tissues. Comparison with cell surface receptors for growth factors
Receptors
Transducers Tissues
Species
Cross-linking,
solubilization
agents
Mr,
KDa
Refs.
Ga~,;trin
CCK
ICK
ICK
Stomach
Pancreatic
acini
Hog
Mouse
DSS
DSS
EGS
DSP
78
120
76-46
(230)
(231)
Neurotensin
ICK
Brain
Intestinal
epithelium
Rat
DSS
CHAPS
U.V.
100-120
50
(232)
(233)
OpJLates
Gi
Brain
Rat
CHAPS
#: 94-44-35
6:53
(234)
Nicotinic
Gs/G i
Electrocytes
Fish
20:: 38-44
13: 48-53
7:57-60
6:64-67
72
(53)
(235)
Muscarinic
Adlrenergic
Gi/ICK
I ICK
Gi
Gs
Gs
Brain
Calf
Triton
X-100
Digitonin
Erythrocytes Frog
Reticulocytes Turkey
Spleen
Rat
Rabbit
Digitonin
U.V.
o:1:78-85
0/2:64
/31: 39-45
f12:52-65
(236)
(237)
(238)
(239)
DSS
0/: 130
t : 95
(240)
Lubrol
160-180
185
250-68
(241)
(242)
(243)
Insulin
TK
Liver
EGFurogastrone
PDGF
IGF-II
TK
TK
TK
Placenta
Man
Fibroblasts
Man
Chondrocytes Rat
Rat
Triton
X 100
Transduction systems (1-5, 10-21): GTPases of phospholipase C (IP3/Ca2+/protein kinase C: ICK)
or adenylate cyclase (Gs/G ~subunits), tyrosine kinases (TK).
bombesin/GRP, bradykinin, insulin and somatomedin C/insulin-like growth
factor I (27-31,244-247). In intestine, the acquired immune deficiency syndrome
(AIDS syndrome) is associated with the secretory diarrhoea which might be
related to the co-occurrence of VIP and/or its receptors in effector intestinal cells
(enterocytes, T lymphocytes in Peyer' patches or lamina propria). In this view,
VIP or its N-terminal 7-11 fragment TDNYT may be an endogenous ligand for
the CD4 receptor in human monocytes (248). The human immunodeficiencyvirus
HIV, the etiologic agent of AIDS, has been shown to bind to the CD4 receptor
present on T lymphocytes and macrophages. The TDNYT sequence is analogous
wit.h potent chemotactic peptides for human monocytes, inhibiting the binding of
the. external glycoprotein molecule gp 120 of the HIV virus. The expression of
insulin receptors and the function of the Gi proteins of adenylate cyclase is also
altered in auto-immune disease and in streptozotocin-diabetic animals (249).
Thus, membrane receptors and transducers may contribute to the expression of
several dysfunctions in the gastrointestinal tract, including cancer transformation,
222
Gespach, Emami and Chastre
ulcer, type I diabetes, cystic fibrosis (250), V e r n e r - M o r r i s o n syndrome, Hirschsprung disease, myasthenia gravis, pernicious anemia and B i e r m e r disease.
Although this review has dealt primarily with the regulation of m e m b r a n e
receptors, little is k n o w n about their chemical structure and biosynthetic
pathways f r o m the gene to their functional domains. Thus, a better understanding
of receptor gene expression, (pro)receptor m a t u r a t i o n and m e m b r a n e insertion,
conformational changes of the receptor during ligand and transducer activations
will require further investigations in the future.
ACKNOWLEDGEMENTS
W e t h a n k Miss M. Le H e i n for secretarial assistance in preparing this
manuscript. A i d e d by Research Grants f r o m Le Centre Interprofessionnel de
D o c u m e n t a t i o n et d ' I n f o r m a t i o n Laiti~res ( C I D I L No. 1150/86), l'Association
Fran~aise de Lutte contre la Mucoviscidose ( A F L M , 1986-1987) and M e r c k Sharp
and D o h m e Chibret (1987) to C . G . , and by a G r a n t f r o m Le Minist~re de la
Recherche et de l ' E n s e i g n e m e n t Sup6rieur No. 87 T 0424 (178).
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