Download Neuronal Control of Mucus Secretion by Leeches: Toward a General

Neuronal Control of Mucus Secretion by Leeches:
Toward a General Theory for Serotonin
CHARLES M. LENT
Department of Cellular and Comparative Biology, State University of New York,
Stony Brook, New York 11790
SYNOPSIS. The large Rctzius cells are serotonin-containing neurons whose impulse
activity controls the secretion of mucus from the skin of leeches. Serotonin elicits the
secretion of mucus without any apparent synaptic transfers in either the central or
peripheral nervous systems. Such a secretogogue function may be more general as
serotonin controls the secretion of mucus from the gastrointestinal tract of mammals
and from the ciliated gills of bivalved molluscs. Furthermore, the qualitative and
quantitative distribution of serotonin in molluscs, annelids, arthropods, and vertebrates
corresponds approximately with mucosecretory structures. Serotonin appears also to
control other secretory functions in some of these animals. It is proposed therefore
that serotonin might often function in controlling secretion.
INTRODUCTION
Each of the 21 segmental ganglia comprising the ventral nerve cord in leeches
contains approximately 175 bilaterally symmetrical pairs of neurons. The "kolossal"
cells of Retzius (1891) are the largest pair
in each of these neural assemblages (Fig. I).
These large neurons can be identified not
only in ganglia of various species of leeches
(Lent, 1973a), but also in their "brains"
which consist of fused segmental ganglia
(Wilson and Lent, 1973). An elegant utilization of autoradiography, chromatography,
and microspectrofluorometry (Rude et al.,
1969) demonstrated conclusively that
Retzius cells contain serotonin (5-hydroxytryptamine, 5-HT) in high concentrations.
In addition, these neurons contain up to 70
times more aromatic acid clecarboxylase, the
enzyme catalyzing the synthesis of 5-HT
from 5-HTP (5-hydroxytryptophan), than
other large neurons in the leech ganglion
(Coggeshall et al., 1972). Thus, the Retzius
cell might be a model serotonergic neuron.
And given the nature of a symposium, I
shall treat it as such a neuron and proceed
I thank Gunther S. Stem for helpful discussions
and critical ideas for the research reported herein.
I am also grateful for the technical assistance of
Ellis Story, Ginna Davidson, Sharon Swift, and Trey
Parrish. Supported in part by PHS Grant CM
17866 (to G.S.S.) and in part by N'SK Grant GB39614.
931
FIG. 1. The large paired Retzius cells in a segmental ganglion of the leech. (From Retzius, 1891.)
from its function in the Jeech to suggest
that serotonin might function similarly in
other animals.
THE FUNCTION OF RETZIUS CELLS IN LEECHES
As their large size (50 to 80 ^m) renders
them experimentally accessible, Retzius
cells have been subjected to histochemical
932
CHARLES M. LENT
(Ehinger et al., 1968), electron-microscopic
(Gray and Guillery, 1963), neuropharmacological (Kerkut and Walker, 1967), microchemical (Rude et al., 1969), and electrophysiological studies
(Hagiwara and
Morita, 1962). Despite these studies however, the functional role of these large neurons has remained unknown until recently
(Lent, 19736). It had been postulated on the
bases of the studies cited above that Retzius
cells function as inhibitory motor neurons
since (i) their function must be intra- rather
than inter-segmental as their processes do
not enter the longitudinal connectives; (ii)
their axonal branches project to the body
wall via all the major ipsilateral ganglionic
roots; (iii) action potentials travel centrifugally along these axons; and (iv) the
perfusion of leech muscle fibers with 5-HT
reduces the amplitude of their excitatory
junction potentials. I have executed a
variety of experiments designed to reveal
effects of Retzius cell impulse activity on
the musculature of the leech as a test of the
validity of this suggestion (Lent, 19736).
The results of these experiments were uniformly negative. For example, simultaneous
intracellular recordings from Retzius cells
and individual muscle fibers failed to show
any correlation between neuronal action
potentials and either inhibitory or excitatory postjunctional potentials in the fibers
of longitudinal, circular, oblique, or dorso-
ventral muscles. Moreover, bursts of impulses induced in the Retzius cells by
current injection caused neither visible relaxations nor contractions. When the longitudinal or circular muscles were made to
contract by stimulating the longitudinal
nerve connective, alterations of the impulse
activity of the Retzius cells did not affect
the rate of tension increase, the maintenance of tonus, or the rate of relexation of
either muscle layer.
In view of the intrinsic likelihood of
Retzius cells having a peripheral function,
and the failure to demonstrate any effects
on the musculature, I examined the possibility that Retzius cells might govern the
secretion of mucus from the large glands
which lie between the skin and muscle layers (Lent, 19736). The 30- to 80-ju.m glands
are either tubular or globose (Fig. 2) and
many axonal processes terminate near them.
Leeches secrete large amounts of mucus as
do many aquatic animals and, although a
precise role for this slime has not been
formulated, it is thought to function in
sucker adhesion and skin cleansing. Two
preliminary experiments demonstrated that
the secretion of mucus from the skin of the
leech is under neural control. Firstly, one
or two segments of body wall in an otherwise intact leech were severed from the
ventral nerve cord by cutting ganglionic
roots. Visual inspection indicated that the
Dermis"
Globose
Glands-
Circular Muscle
Tubular Glands
Longitudinal Muscle
Dorso-ventral Muscle
Botryoidal Tissue
FIG. 2. A cross section of leech body wall illus-
trating the position of the mucous glands. (After
Bhatia, 1941.)
LEECHES, SEROTONIN, AND SECRETION
Body wall
Dorsal mid line
Innervated segment
Mucus field
933
Ganglion
L
Microelectrode
Retzius cells
Suction electrode
Connective
FIG. 3. Diagrammatic representation of the preparation and experimental apparatus. Retzius cell
impulse activity is generated by passing current
through the microelectrode and the efferent impulses are monitored by the suction electrode. The
ganglionic and body wall compartments are separated by a petroleum jelly dam. The vertical
calibration bar represents 25 mV for the intracellular trace A and 100 ^V for the extracellular trace B.
The time calibration bar is 1 sec. During stimulation, 8 to 9 annuli secrete mucus and therefore the
effector field of the Retzius cell overlaps adjacent
segments (each with y annuli) about 2 in each
direction. (After Lent, 19736.)
denervated, and only the denervated, segmental skin remained free of slime. Secondly, the longitudinal connective in another group of leeches was cut near its
midpoint and either the anterior or posterior cut end was stimulated by means of a
suction electrode. The stimulated half animals—whether anterior or posterior—invariably secreted more mucus than the
unstimulated control halves. The results
presented below show that the neurons
which control the secretion of mucus are
the Retzius cells.
Quantitative experiments on the relation
of mucus secretion to Retzius cell activity
were conducted on a preparation from the
medicinal leech, Himdo medicinalis. This
preparation consists of a single ganglion
connected unilaterally by its roots to a corresponding half body wall extending
slightly over three segments (15 annuli) in
length (Fig. 3). The ganglion, pinned to
a layer of transparent Sylgard resin (Dow
Chemical) and viewed with transmitted
light, is separated from the body wall by a
dam of petroleum jelly, in order that the
ganglion and wall can be bathed in salines
of different composition. The body wall is
usually bathed in leech physiological saline
(Nicholls and Baylor, 1968), whereas the
ganglion is usually bathed in saline whose
Mg-+ concentration is raised to 20-mM.
Thus, while the high Mg-+ in the ganglion
compartment abolishes chemical synaptic
transmission in the central nervous system
of this preparation (Stuart, 1970)—presumably by inhibiting the release of neurotransmitters from axon terminals—the
saline in the body wall compartment permits release of transmitters in the periphery. This procedure has the advantage that,
being deprived of synaptic inputs, the
Retzius cells lose most of their spontaneous
impulse activity until depolarized experimentally. Furthermore, any increase in the
rate of mucus secretion can be attributed to
a direct effector action of Retzius cells on
the periphery rather than any synaptic activation of other central neurons. Since
leeches often secrete copious amounts of
mucus during their dissection, the preparation is removed from the animal in a high
Mg2+ saline. This dissection procedure reduces neural activity which in turn restricts
somewhat the loss of glandular stores of
mucus prior to an experiment.
The Retzius cell innervating the body
wall is impaled with a micropipette con-
934
CHARLES M. LENT
taining 4-M potassium acetate. Depolarizing
current pulses are passed into the cell by
means of a balanced bridge circuit which
allows the same electrode simultaneously to
detect the impulse activity, which in turn is
monitored on an oscilloscope, recorded on
a penwriter, and summed automatically on
an electronic counter. The efferent impulse
activity from the ganglion was also monitored by means of a suction electrode
attached to the dorsal branch of the posterior root, usually at the cut end of the
contralateral root, but occasionally en
passant on the intact ipsilateral root. Since
the pair of Retzius cells is so strongly
coupled by an electrotonic junction that
their impulses are nearly synchronous
(Hagiwara and Morita, 1962), either side
provides a good measure of the impulses
traveling from the central cell body to the
innervated half body wall.
The secreted mucus is assayed by taking
advantage of its adsorption of carmine red.
For this purpose, the skin is inundated with
an excess of carmine suspended in saline
at an approximate concentration of 75 mg/
ml. After 5 min, the unadsorbed carmine is
removed by gentle washing with saline. The
mucus together with its adsorbed carmine is
then collected with a suction pipette, transferred into a test tube, and sedimented into
a pellet in a clinical centrifuge. After removing the supernatant fluid, the pellet is
resuspended in a fixed volume of diluted
sulfuric acid which dissolves the carmine.
The concentration of carmine is assayed
colorimetrically by measuring intensity of
red color in solution and this is taken to
be proportional to the amount of mucus. As
the rate of mucus secretion by different
preparations is variable (about twofold), results are reported as percentages of the
maximum amount of mucus secreted by a
particular preparation.
The Retzius cell was depolarized by current pulses sufficient to produce different
numbers of impulses during successive 15min periods. The rate of mucus secretion is
roughly proportional to the number of impulses (Fig. 4) rising at least eightfold from
the control period (when the 500 impulses
were spontaneous) to the period of maxi-
~
'00
-
£60 r
0
2
4
6
3
Retzius cell impulses (10 per 15 min)
FIG. 4. Percent mucus secreted as a function of
Retzius cell impulses during 15-min stimulation
periods. The depolarizing pulses were delivered at
0.1 Hz and each lasted 4.5 sec. The current injected
determined the total impulse number. (After Lent,
197S/>.)
mum stimulus depolarizations (when 6000
impulses were generated). The maximum
amount of mucus secreted by this preparation corresponded to a weight of about 10
mg. In this experiment, the number of impulses was increased in successive stimulation periods in order to insure that any
exhaustion of mucus reserves during the
experiment would result in an underestimate of the dependence of the rate of mucus
secretion on impulse activity. As it takes a
few minutes for the accelerated rate of
mucus secretion to return to its low spontaneous level, successive stimulation periods
were separated by intervals of at least 10
min. In other experiments, the number of
impulses was decreased in successive stimulation periods. Since here, too, the rate of
mucus secretion corresponded to the total
impulse number, one must conclude that
these results (Fig. 4) cannot be attributed
to any cumulative effect of successive collections of mucus in the assay procedure.
Thus, these results demonstrate that the
impulse activity of Retzius cells controls the
rate of mucus secretion by the dermal
glands of leeches.
No increases in the rates of mucus secretion with impulse activity were observed
when the body wall was bathed in a high
Mg2+ saline, which suggests that the Retzius
cell exerts its effect by the release of 5-HT
from its axon terminals in the body wall.
To test this possibility, I examined the
LEECHES, SEROTONIN, AND SECRETION
935
these large neurons. Nor do the data allow
any decision as to whether the control of
mucus secretion is mediated by a synaptic
or indirect route such as neurosecretion or
diffusion through tissue spaces. An indirect
route appears more likely as very few 5-HTcontaining axons terminate directly upon
the mucous glands (Yaksta-Sauerland and
Coggeshall, 1973).
O "
10~
5
10"
4
10"3
10"
2
5-Hydroxytryptamine (M)
FIG. 5. Per-cent mucus secreted by deganglionated
body wall of leech as a function of 5-HT concentration. The data are from a single experiment on
5 half sections of body wall each 1.5 segments long
and bathed in 20 mM Mg2+. (After Lent. 19736.)
A GENERAL FUNCTION FOR SEROTONIN?
The literature on 5-HT (Garattini and
Shore, 1968; Page, 1968; Barchas and Usdin,
1973) contains several lines of evidence
which suggest that the mediation of mucus
secretion by serotonin may not be restricted
to leeches, but may be more widespread.
The parallel to the neuron-serotonin-secretion process _n leeches is especially evident
in preparations from two diverse groups of
animals: the gastro-intestinal tract of mammals and the ciliated gills of bivalve molluscs.
effect that the direct application of 5-HT
onto the body wall has on mucus secretion.
Preliminary experiments showed that a
drop of concentrated 5-HT applied to the
skin results in the immediate formation of
a glob of mucus in the treated area. For a
more quantitative study of this effect, equalsized deganglionated sections of body wall
(lateral halves, three segments long) were The mammalian gastro-intestinal tract
each bathed in high Mg2+ saline to which
different amounts of 5-HT had been added.
The abundant serotonin of the gastroAfter 45 min, the mucus was collected and intestinal (G-I) tract is localized within its
3
assayed. Exposure of the body wall to 10~ enterochromaffin cells at concentrations up
M and 10~2 M 5-HT increased the rate of to 10 jw,g/g—nearly 100 times more concenmucus secretion more than threefold over trated than in neural tissue (Feldberg and
the control with no added 5-HT, while in- Toh, 1953; Pletscher et al., 1955). The G-I
termediate concentration produced sub- mucous membranes both synthesize and
maximal effects (Fig. 5).
release this serotonin (Blaschko, 1958). The
The finding that the direct application of addition of serotonin to the stomach of
5-HT to the body wall causes mucus secre- either dogs (Smith, 1958; Menguy, 1967) or
tion offers further support for the inference guinea pigs (Wilson, 1958) stimulates the
that the Retzius cell controls the secretory secretion of copious mucus. Intravenous adprocess without synaptic transfers within ministration of serotonin to dogs stimulates
the central nervous system, because the only the secretion of so large a volume of G-I
axons in the ganglionic roots containing mucus (up to a 700% increase) as to induce
this neurochemical are those of the Retzius vomiting and defecation (White and Magee,
cells (Ehinger et al., 1968; Marsden and 1958). Furthermore, the serotonergic secreKerkut, 1969). Since the applied 5-HT tion of G-I mucus is probably under neural
mediates the secretion of mucus in the pres- control, as stimulation of the vagus nerve
ence of Mg2+, the 5-HT from the Retzius results in the release of 5-HT from the
cells most likely activates the mucous glands stomach (Bulbring and Gershon, 1968) and
directly without any synaptic transfers gastric mucus secretion follows hypothalathrough a peripheral nerve plexus. These mic stimulation (Leonard et al., 1967).
data do not demonstrate that the control of
Serotonin has often been implicated in
mucus secretion is the only effector role for increasing G-I tract motility; however, in
936
CHARLES M. LENT
the experiments reported above, either
atropine or hexamethonium blocked the
motility induced by the 5-HT without altering its secretory effects. This implies, of
course, that the serotonin is activating
either other neurons or different receptor
sites which in turn control the gut musculature. Thus, the mammalian gut is rich in
serotonin which induces its epithelium to
secrete mucus. Here too then, as in the body
wall of the leech, serotonin appears to mediate mucus secretion.
Additional inferential support for serotonin subserving the secretion of a protective coat of mucus is provided by the experimental application of reserpine to the
gastric mucosa of rats (Rasanen, 1968).
Reserpine is effective both in depleting
gastric enterochromaffin cells of their serotonin and in producing ulcer-like lesions
in the treated areas. These studies suggest
that G-I serotonin directly stimulates secretion of a lubricating mucus and indirectly
stimulates the movements of the bowels;
therefore, the answer to the query of Collier
(1958), "Is HT nature's remedy for constipation?", is very likely—yes!
The ciliary cpithclia of molluscs
Bivalved molluscs trap their food in a sheel
of mucus which is continually secreted,
moved across the surface of large, paired
gills by ciliary activity, and then ingested
together with entrapped food particles. The
frequency of the ciliary metachronal waves
on each of these gills is controlled by the
impulse activity of the branchial nerve,
which when cut reduces ciliary activity
(Aeillo, 1960) and when stimulated increases the ciliary activity (Aeillo, 1970) of
nearly all species examined. Serotonin is
suggested to be the transmitter responsible
for this cilio-excitation, not only because it
can be measured in the gills (1.5 /xg/g in
Mytilus), but also because the addition of
serotonin to these gills increases the frequency of their ciliary movements two to
three times (Gosselin et al., 1962). Furthermore, the gills of these bivalves enzymatically decarboxylate 5-HTP to 5-HT,
which in turn stimulates cilio-excitation,
from which it follows that cilio-excitation
by 5-HTP has a slower onset and a longer
duration than excitation by serotonin.
Treatment of the gills with BOL (bromolysergic acid diethylamide, an inhibitor believed to block serotonin receptor sites)
interferes with cilio-excitation produced
either by neural stimulation or by addition
of 5-HT (Aeillo, 1970)—implying that both
these types of excitation possess a common
receptor.
The central ganglia of bivalves contain
higher concentrations of 5-HT (up to 60
jttg/g) than neural tissues of the many other
species examined (Welsh and Moorhead,
1960). The innervation of the bivalve gill
has been investigated by fluorescence histochemistry—a technique which utilizes a reaction between the serotonin in freeze-dried
tissues and formaldehyde producing a
fluorophore which emits a characteristic
yellow color unlike the greener colors from
other monoamines (Falck et al., 1962).
Pharmacological agents can either enhance
or deplete particular monoamines, and
these techniques yield evidence suggesting
that the yellow fluorescent axons in the bivalve branchial nerve contain serotonin,
enter the gill filaments, and innervate the
ciliated epithelial cells (Paparo, 1972).
The evidence presented above strongly
suggests that the neuronal release of serotonin excites the gill cilia of bivalves.
Remembering that a major function of
these motile organelles is the propulsion of
a mucous sheet, I have conducted some
qualitative experiments in order to ascertain whether neuronally-released serotonin
also controls the secretion of mucus from
the gills of bivalved molluscs. The neural
control of secretion was investigated by
transection and stimulation of the branchial
nerve in the ribbed mussel, Modiolus dcmissus. The distal end of the cut nerve was
stimulated through a suction electrode and
the amount of mucus secreted was visually
compared (by the application of powdered
carmine) to that of the control gills on the
unstimulated half of the mussel. In all cases,
branchial nerve stimulation produced obvious increases in the rates of mucus secretion (and ciliary activity) by the gills of this
LEECHES, SEROTONIN, AND SECRETION
bivalved mussel. Experiments measuring
the effects of 5-HT on mucus secretion were
conducted by exposing isolated pairs of gills
from Modiolus and Mytilus edulis to 5-HT
(10~3 M) in artificial sea water (Instant
Ocean, Eastlake, Ohio). The gills of both
species secreted more mucus than control
gills without added 5-HT. Therefore, the
serotonin in the neural tissues of bivalves
stimulates not only cilio-excitation, but also
the secretion of the large volumes of mucus
for which they are so well known. Such a
concomitant function for serotonin presumably has the advantage to the mollusc of
maintaining a uniformly thick layer of
mucus which is propelled across the gill
surfaces at various velocities dictated by
neural activity.
PHYLOGENETIC DISTRIBUTION OF SEROTONIN
The experiments cited above demonstrate that neuronally released serotonin
mediates the secretion of mucus by preparations from three phylogenetically distant
species: leech skin, mammalian gastric mucosa, and clam gill. Such observations lead
to the inference that serotonin may often
function as a mediator of secretion. If this
suggestion is valid, then the qualitative and
quantitative phylogenetic distribution patterns of serotonin ought to correspond with
muco-secretory structures. Indeed, it does,
as the quantities of serotonin within the
neural tissues of the major taxa correlate
approximately with their relative mucussecreting abilities. Thus, molluscs and annelids—animals whose outer surfaces are
composed of active mucous epithelia—have
the highest amounts of serotonin within
their nervous systems, while the arthropods
with their cuticular exoskeletons have the
lowest amounts. The vertebrates, which are
intermediate in their mucus-secreting abilities, have intermediate amounts of 5-HT
in their nervous systems (Welsh, 1968).
Further monoamine oxidase, the enzyme
which inactivates 5-HT can be detected
in annelids, molluscs, echinoderms, and
lampreys—all known slime producers
(Blaschko, 1958). A detailed examination
937
of the available data on molluscs, annelids,
arthropods, and vertebrates follows.
Mollusca
The concentration of serotonin within
the nervous systems of the three major
classes nf molluscs correlates approximately
with their relative mucus-secreting ability.
Thus, bivalves with 10 to 60 jxg 5-HT/g
of nerve tissue produce more mucus than
cephalopods (1 to 4 jxg/g) and the gastropods are intermediate between them with 2
to 10 ng/g (Welsh and Moorhead, 1960).
Fluorescence histochemistry indicates that
in bivalves both gills (see above) and some
kidney cells contain serotonin (Sweeney,
1968). The escargot, Helix, has a pair of
large cerebral serotonergic neurons (Bardessono et al., 1972) whose axons terminate in
the body wall near the lip (Penreath and
Cottrell, 1972)—a structure known to secrete much of the mucus lubricating the
foot (Bullough, 1958). Large amounts of
serotonin are found also in the muricid
gastropod hypobranchial glands—organs
which produce large amounts of a shelldissolving secretion when these marine
snails "drill" their bivalve prey (Carriker,
1961; Welsh, 1970). Very high amounts of
serotonin are associated also with the
salivary glands of Octopus; however, it cannot now be decided whether this serotonin
stimulates release of a secretion used to drill
through the shells of molluscan prey
(Arnold and Arnold, 1969; Wodinsky,
1969) or whether the serotonin is used as a
toxin (a role which is further discussed
below). Thus, in molluscs, the amounts and
sites of serotonin seemingly correspond with
mucus or with secretory structures.
Annelida
All three classes of annelids have similar
quantities of serotonin within their neural
tissues (3 to 10 /*g/g). The central neurosomata contain serotonin, fluoresce a brilliant yellow-gold, and send their axons
peripherally in polychaetes (Clark, 1966),
earthworms (Rude, 1966), and leeches
(Ehinger et al., 1968; Marsden and Kerkut,
938
CHARLES M. LENT
be stimulated also by 5-HT at 10~7 M
(House, 1973) even though its actual transmitter appears to be dopamine (House et
al., 1973).
Many crustaceans are bioluminescent,
and in the euphausiid Meganyctiphanes
serotonin is able to both directly stimulate
and increase the sensitivity of their light
organs (photophores) to other forms of
stimulation (Kay, 1965). Many photophores
are glandular structures only slightly modified from mucous glands and other crustaceans emit their extra-corporeal bioluminescent secretions cojointly with mucus
from unmodified glands—apparently lending cohesiveness to the luminescent chemicals (Nicol, 1962).
The venom glands of many arthropods—
scorpions, spiders, wasps, and hornets, but
not bees—contrast with most of their tissues
by containing massive amounts of 5-HT (up
to 19 mg/g dry weight; Welsh, 1970). A
role for serotonin in venoms is widespread,
however, and it can be found highly conArthropoda
centrated in the salivary glands of Octopus,
Arthropods usually have less than 0.2 jxg the labial poisons of the Gila monster, and
of serotonin per gram of neural tissue—a the poisonous skin secretions of the toad
fact which correlates with the low secretory Bnfo (Garattini and Valzelli, 1965). Many
activity of their hypodermis. Thus, the of these venoms produce intense pain as
crayfish Astacus has but a single pair of does serotonin, and the venom of the Gila
yellow fluorescent neurons (Osborne and monster produces symptoms similar to the
(Elofsson et al., 1966). However, the lobster effects of injected sertonin, vomiting, salivastomatogastric ganglion contains several tion, and defecation (Minton and Minton,
yellow fluorescent axons (Osborne and 1969); however, no inference can be drawn
Dando, 1970) whose axons presumably in- regarding the function of serotonin in toxnervate the muco-secretory intestine. The ins. At the very least there is some assoneurosecretory pericardial organs (Cooke, ciation of the serotonin in arthopods with
1964) of decapod crustaceans contain 100 to secretory activity.
200 times more serotonin than their ordiVertebrata
nary neural ganglia (Welsh, 1970).
Serotonin can be detected within the
High concentrations of serotonin are
brain of blowflies and their salivary glands
are sensitive to it—more so in fact than any present within these vertebrate structures:
related chemical species tested by Berridge G-I tract, pulmonary epithelium, pancreas,
(1972). These glands respond to 5-HT in thyroid, pineal gland, spleen, mast cells,
concentrations as low as 10~10 M with in- and platelets. Furthermore, chromaffin cells
creases in their rates of fluid secretion of can be identified by histochemistry in the
20 to 40 times (Berridge and Patel, 1968). biliary tract, pancreatic duct, prostate
This serotonergic salivation is mediated by gland, and vaginal epithelium of mammals
a Ca2+-dependent, cyclic AMP mechanism (Hakanson, 1970). Many of these structures
(Berridge and Prince, 1972). The secretion are secretory.
of saliva by the glands of the cockroach can
A secretagogue function for serotonin in
1969). These yellow axons terminate most
often in the body wall, and in the polychaete Glycera large yellow terminals can
be seen within the skin (Manaranche and
L'Hermite, 1973). A yellow fluorescence is
often noted also within the tissues of the
nephridia, pharynx, and intestinal tract.
Thus, the amounts and distribution of
serotonin within the bodies of annelid
worms correlate somewhat with mucosecretory structures, and further, for each class
there are experimental findings which support the general hypothesis. The data reported above on leeches form the nucleus
for this hypothesis. In polychaetes, Lawry
(1967) demonstrated that stimulation of the
parapodial nerves in Harmathoe causes the
discharge of its mucous glands. Additionally, the topical application of drops of
concentrated 5-HT to the deganglionated
skin of earthworms elicits a localized secretion of mucus (Lent, unpublished).
939
LEECHES, SEROTONIN, AND SECRETION
the G-I tract was detailed above, and there
are data suggesting a similar function for
the serotonin within the pulmonary tree.
Foreign particles are removed from the
respiratory epithelium on a sheet of mucus
propelled by ciliary activity. The epithelial
mucous glands are under neural control
(Schlesinger, 1973) and ciliary activity of
isolated rabbit iiddiea is modestly accelerated by 10~9 M 5-HT. Even lower concentrations of 5-HT are cilio-excitatory if the
trachea is pretreated with reserpine
(Tsuchiya and Kensler, 1959). The fine
structural organization of pulmonary epithelium in rabbit occasioned the proposal
that neuro-epithelial bodies (serotonincontaining cells) might modulate mucus
secretion (Layweryns et al., 1973). Thus,
this muco-ciliary epithelium in mammals
may be excited by serotonin as is the mucociliary epithelium of the molluscan gill (see
above).
Serotonin is implicated in a variety of
other secretory functions in mammals including the following:
1) the initiation of milk secretion from
the mammary glands of virgin rats (Meites
et al., 1959).
2) an increase in the amylase and protein
secreted with saliva from rabbit parotid
glands (Kojima et al., 1973).
3) stimulation of excess salivation by
goats (Andersson et al., 1966).
4) being essential in the secretion of insulin from the pancreatic islet cells (Lernmark, 1971).
5) secretion of both ACTH (Verdesca et
al., 1961) and a substance which reduces
tetrazolium blue (Rosenkranz and LaFerte,
1960) from adrenal glands, organs where the
secretagogue function of serotonin is Ca2+~
dependent (Douglas, 1965).
CONCLUSION
Many data are consonant with the proposal that serotonin often functions by
controlling secretion. This thesis in no way
argues against other serotonergic functions,
such as relaxing the "catch" of molluscan
tonic muscles (Twarog, 1967) or as a neuro-
ACh
E3=5-HT
ED'SECRETION
PERIPHERY
B
FIG. 6. Anatomical relationships between serotonergic cells and mucous glands. A, The unicellular
mucous glands in the brain of Nepthys. (After
Clark, 1!)56.) B, A central serotonergic neuron controlling peripheral mucous glands. C, Extrinsic
serotonergic control of mucous glands. The peripheral seiotoneigic cells are controlled by central
neurons.
transmitter within the central nervous system (e.g., Greschenfeld, 1971).
The neuronal control of mucus secretion
has been a common theme throughout this
paper; however, there are diverse morphological relationships of the serotonergic cell
and mucous gland to the central nervous
system and the periphery. In some animals,
such as the polychaete worm, Nepthys, cell
bodies of unicellular mucous glands reside
within the brain where they are presumably
excited and their secretions are then carried
outside the body by long tubular lumina
(Fig. 6/4). The mucous glands are situated
in the periphery in mollusca and most annelids. Serotonergic neurons within the
central nervous system control these glands
through peripheral axons (Fig. 6B). The
leech Retzius cell is such a neuron and it is
excited by cholinergic neurons (Kerkut and
Walker, 1967). In the G-I tract and pulmonary epithelium of mammals, the serotonergic cells are themselves in the periphery and closely associated with the mucous
glands (Fig. 6C). The activity of other
central neurons (which may be cholinergic,
e.g., the Vagus nerve and gastric secretion,
940
CHARLES M.
see above) modulate the activity of the
serotonergic cells, indirectly controlling the
secretion of mucus.
This thesis for serotonin functioning
often as a secretagogue should provide an
impetus for definitive research designed to
answer questions such as these:
1) By what mechanisms does serotonin
control mucous glands?
2) Do these mechanisms depend upon
Ca2+ and cyclic AMP?
3) What biophysical mechanisms underlie the secretory process?
This thesis should be easily testable. If it
is not found coherent, we can discard or
modify it. However, if it is even moderately
accurate, we can begin formulating answers
to Irwing Page's (1968) question on serotonin: "What does it do?"
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