Download Compartmentalization of Cyclic GMP

Compartmentalization of Cyclic GMP-Dependent Protein Kinase in Formyl-Peptide
Stimulated Neutrophils
By Katherine B. Pryzwansky, Todd A. Wyatt, Harriette Nichols, and Thomas M. Lincoln
The presence and physiologic role of cyclic GMP-dependent protein kinase (G-kinase) in human neutrophils was
investigated by Western blot analysis and immunocytochemistry. Small quantities of G-kinase were found in the
cytoskeletal-enriched fraction of neutrophil lysates as detected by Western blots using a polyclonal antibody raised
against bovine aorta G-kinase. Immunofluorescencemicroscopy demonstrated in adherent neutrophils that G-kinase
was localized diffusely within the cytoplasm, at the microtubule organizing center, and in the euchromatin of the
nucleus. Because cyclic GMP is implicated as a modulator
of neutrophil chemotaxis. G-kinase localizationwas investigated in neutrophils activated with N-formyl-methionylleucyl-phenylalanine (fMLP). fMLP stimulated transient
focal changes in G-kinase localization that coincided with
transient changes in cell shape. G-kinase translocated over
a period of 5 minutes from diffuse staining of the cytosol to
filaments within the uropod of polarizedcells (1 minute), to
bundles of filaments associated with loss of cell polarity
(2.5 minutes), and finally to more intense staining of the
nuclear euchromatin (5 minutes). Optical sectioning of
neutrophils by confocal laser scanning microscopy confirmed that G-kinase was restricted to specific sub-cellular
compartments during cell activation. This transient localization of G-kinase was disrupted by cytoskeletal inhibitors
and was augmented by 8-Br-cyclic GMP. These data
provide evidence for the first time that G-kinase plays a
physiologic role in human neutrophils, and support the
concept of compartmentalization of cyclic nucleotides during neutrophil activation.
0 1990by The American Society of Hematology.
C
ubiquitous nor abundant, and the physiologic role of cyclic
G M P is not understood. While G-kinase and its counterpart
cyclic AMP-dependent protein kinase (A-kinase) are homologous proteins, the more restrictive distribution and substrate specificity of G-kinase suggest a specialized function
for this
At this time, G-kinase has not been isolated, characterized, or localized in neutrophils. In this report we demonstrate the presence of G-kinase in human neutrophils, and
describe a transient redistribution of G-kinase to specific
targeting proteins or structures in cells stimulated with the
chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP).
YCLIC G M P has been implicated as a modulator of
neutrophil chemotaxis because agents that elevate
cyclic G M P levels enhance chemotaxis.’ Nevertheless, the
physiologic role of cyclic G M P has remained an enigma for
the past 15 years, since elevation of cyclic G M P levels by
chemoattractants has led to conflicting
A new
interest in cyclic G M P has recently evolved, in that nitric
oxide (NO), a potent stimulant of guanylate cyclase, is
generated by neutrophils that are stimulated with chemoattractants4 Furthermore, chemotaxis is inhibited by blocking N O synthesis and is restored with either di-butryl cyclic
G M P or with L-arginine, the precursor for NO prod~ction.~
The mechanism by which the rise in cyclic G M P after
activation of guanylate cyclase mediates its effects in neutrophils remains to be established. An important effector for
transducing cyclic G M P signals into biologic responses is
cyclic GMP-dependent protein kinase (G-kinase). Although
G-kinase has been characterized biochemically, its physiologic role is only now emerging, particularly in vascular
smooth muscle function.6 Information on the role of G-kinase
in other cells is minimal since this enzyme is neither
From the Department of Pathology, University of North Carolina, Chapel Hill; and Department of Pharmacology, University of
South Alabama, Mobile.
Submitted February 2,1990; accepted April 13.1990.
Supported by the American Cancer Society (ACS-401),National
Institutes of Health (CA-07017 and HL34646). and USPHS General Research Support Award (5-501-FR-05406). T.M.L. is an
Established Investigator for the American Heart Association.
A portion of this report was presented at the FASEB Meetings.
3:A1086.1989.
Address reprint requests to Katherine B. Pryrwansky, PhD.
Department of Pathology, Brinkhous-Bullitt Building 7525. University of North Carolina, Chapel Hill. NC 27599-7525.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7603-0019$3.00/0
612
MATERIALS AND METHODS
Neutrophil isolation. Neutrophils were isolated from human
peripheral blood with Polyresolving media (Flow Lab, McLean,
VA). The cells were resuspended at 3 x lo6 cells/mL in Gey’s
Balanced Salts buffered with 10 mmol/L HEPES, pH 7.2, and
supplemented with 1.5 mmol/L CaCl,, 1 mmol/L MgCl,, 0.3
mmol/L MgSO, (GBS), and 10% human type AB serum. Neutrophils were allowed to adhere to glass coverslips for 15 minutes at
37OC. Unattached cells were removed by washing with GBS. Cells
consisted of greater than 95% neutrophils, and were greater than
98% viable by trypan blue exclusion. All reagents were checked for
endotoxin by the limulus test and contained less than 0.07 ng/mL of
endotoxin.
Neutrophil stimulation. Neutrophil monolayers were stimulated with lo-’ mol/L fMLP (Sigma, St Louis, MO) in GBS plus
10% human type AB serum from 30 seconds to 5 minutes at 37OC.
The culture media was removed and the cells were quickly fixed in
1% paraformaldehyde for 10 minutes, followed by 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 minutes, -20°C
methanol for 4 minutes, and -2OOC acetone for 1 minute. Cells were
washed in PBS after formaldehyde and acetone.* In some instances
cells were pre-incubated with 1 pmol/L 8-Br-cyclic GMP (Sigma)
for 15 minutes, followed by incubation with lo-’ mol/L fMLP in the
presence of 8-Br-cyclic GMP. Cells were also pre-incubated with 5
pg/mL cytochalasin B (Sigma) for 5 minutes or 2.5 pg/mL
nocodazol (Janssen, Piscataway, NJ) for 10 minutes to inhibit
microfilaments or microtubules, respectively.
Immunofluorescence microscopy. Fixed monolayers were incubated at 4°C overnight with rabbit anti-bovine aorta G-kinase,
B l ~ d VOI
, 76,
NO
3 (August 1). 1990: pp 6 12-6 18
OKINASE COMPARTMENTALIZATION IN NEUTROPHLS
washed in PBS. and stained for 30 minutes with F I X goat
anti-rabbit immunoglobulin G (1s)(Cappel-Worthington. West
Chester. PA). The antibody to bovine aorta G-kinase has been
characterized for specificity in smooth muscle cells.' Cells were
mounted in polyvinyl-alcohol and viewed on a Leitr fluorescence
microscope or a Zciss Confocal Dual Laser Scanning Microscope
(CLSM). Immunofluorescence photomicrographs were taken on a
Leitz Orthomat Camera with Kodak Tri-X Pan film, ASA 400.
ConfMol-laser-sconning immunofluorescence micrarcopy. To
tesolve differential staining patterns at various levels within a cell,
neutrophils were optically Kctioned at 0.4 pm using a Zciss CLSM.
An argon laser at 488 nm was used for excitation with a 10118p a s
filter cutoff at 520 nm. using a 63X Zciss planapochromat objective
lens with a numerical aperture of 1.4. This technology allows for
immunofluorescence detection of specific regions of
a
cell without
fluorescence interference from areas removed from the selccted
plane of focus. The digital image was recorded on Kodak Technical
Pan film 2415 using a Polaroid Freeze Frame Video Recorder.
Wesfern blot unalysis. A neutrophil pellet consisting of 5 x IO'
cells was lysad for 5 minutes at 25OC in 3 mL of 0.5%-Triton X-100
in 0.1 mol/L NaCI. 0.3 mol/L suctose. 3 mmol/L MgCI,. 0.01
mol/L piperazine aham sulfonic acid (PIPES). 1.2 mmol/L
paminokntamidine. and 1 pg/mL Apmtinin.'" The DNA and
RNA were digested with 2 mg/mL DNase I and IO mg/mL RNase
A (Sigma) for I5 minutes at 25%. and (NH,),SO, was added to a
final concentration of 0.1 mol/L to the lysate for 5 minutes at 25%.
The lysate was centrifuged at lO.OOOg for IO minutes at 4%. and the
pellet dissolved with 1% Triton X-100 in PBS. The protein was
precipitated at 4% with 10% trichloroacetic acid as described.'
Immunoprecipitation was performed on recovered protein ( 9 mg)
using 5 pL affinity-purified rabbit anti4-kinase for 4 days at 4%
followed by I O pL Protein A Sephanac (Sigma) for 4 hours at 4%.
The precipitate was washed and applied to a 12% polyacrylamide
vertical mini-gel and transferred to a nylon transfer membrane
(ICN. Irvine. CA) as described." Transfers w m probed with rabbit
anti-G-kinase followed by '"I Protein A (ICN). and exposed to
X-ray film for 24 hours at -70%:
RESULTS
Demonsrrarion of Gkinase expression by neutrophils.
Western blot analysis of neutrophil extracts showed the
presence of G-kinase in human neutrophils (Fig I ) . The
neutrophil antigen was a single polypeptide that comigrated
with bovine aorta G-kinase on sodium dodecyl sulfatepolyacrylamide reducing gels at 77 Kd. Based on the number
of cells required to detect the enzyme ( 5 x IO' cells), it is
estimated that the level of G-kinase in neutrophils is approx-
1
2
Fb1. wmrnbbtuw)yo b o f ~ i n h u r w n
philr. I
.
"1. bovirw lung M
i
0
GMP-d.p.nd.nt protoin kino00
nandord I6 ng a( .nynwl. MC
nor bando or. known protootytk f r a g n " Ot G - k i M d
Ian. 2. noutrophll protoin
o x t r m a0 doocribod in M o t r i alr and Mothod..
613
imately 100-fold lower than that in vascular smooth muscle?
Nevertheless. the kinase was enriched in the cytoskeleton
fraction, suggesting that most of the G-kinase in neutrophils
is localized in this compartment. In addition, cross-reactivity
of the antibody, which was raised against bovine aorta
G-kinase and characterized for specificity in smooth muscle
cells. suggests homology (if not identity) with neutrophil
G-kinase.'
halizarion of Gkinase in jMLP-srimulared neurrcr
phils. Immunofluorescence microscopy showed that
G-kinase is transiently compartmentalized in fMLP-stimulated neutrophils. In untreated cells. G-kinase was diffusely
localized throughout the cytoplasm. Staining of the microtubule organizing center (MTOC) and euchromatin of the
nucleus was also observed (Fig 2A). In unstimulated cells
that were polarized (30% to 50%. depending on the donor).
some diffuse staining was also observed at the trailing end. or
uropod.
FMLP stimulated transient focal changes in G-kinase
localitation that coincided with transient changes in cell
shape. Within 30 seconds intense focal staining of G-kinase
was observed along the cell margin and at the trailing end of
polarized cells (Fig 2B). After I minute, when most of the
neutrophils were polarized. G-kinase was focally localized on
filaments in the uropod, and at the MTOC.No staining was
observed on filaments in the uropod of polarized neutrophils
that were not stimulated with M L P (Fig 2A). suggesting
that fMLPstimulation is required for translocating G-kinase
to specialized structures or targeting proteins in the uropod.
After I minute, some staining of the nucleus was also
observed (Fig 2C). By 2.5 minutes, bundles of filaments
stained for G-kinase within one region of the cell. as cells
displayed less polarity and became round (Fig 2D). These
filament bundles were strongly reactive for G-kinase and
were frequently localized adjacent to the nucleus. After 5
minutes cells were round, and cytoplasmic staining for
G-kinase was. in general, less intense and confined primarily
to the MTOC. However, more pronounced staining of the
nuclear euchromatin was alsoobserved at this time (Fig 2E).
Insignificant staining was observed in unstimulated and
fMLP-stimulated neutrophils that were stained with pnimmune serum (Fig 2F).
To confirm that G-kinase is focally localized at I minute,
neutrophils were optically sectioned at 0.4-pm increments by
C U M . The three optical sections shown in Fig 3 demonstrate that G-kinase is confined to the uropod and indentation
of the nucleus, or hof.
When cells were allowed to adhere to glass in the presence
of 10% human serum. washed free of serum. and stimulated
with MLP. similar translocation of G-kinase was observed.
However. if cells were adhered in the absence of 10% human
serum. cells avidly adhered to the coverslips. remained
~
well-spread throughout the time course of M L P stimulation
( 5 minutes), and did not undergo shape changes or display
changes in G-kinase localitation. Staining of these cells was
confined to the MTOC and euchromatin. with minimal
staining observed in the cytoplasm (not shown). When serum
was replaced with 1% human serum albumin (lipid-fm) or
10% heat-inactivated human serum (serum heated at 56%
614
b
I
for 30 minutes). cells remained spherical throughout the time
course of fMLP treatment. and staining of G-kinase was
difTuse (not shown). These results suggest that a heat labile
serum factor(s) is required during cell adhesion to observe
translocation of G-kinase and the changes in cell shape
required for motility (eg. establishing polarity) after M L P
stimulation.
Eflecr of cyroskclcrol inhibirors. Because Western blots
and immunofluorescence microscopy suggested an association of G-kinase with the cytoskeleton. cells were exposed to
cytoskeletal inhibitors to determine if G-kinase localization
was dependent on an intact cytoskeleton. Cytochalasin B
inhibited both cell polaritation and transient changes in
G-kinase localization. suggesting a relationship between
microfilaments and the putative translocating or targeting
protein (Fig 4A). Nocodazol. a microtubule inhibitor, did not
inhibit polarization, but did inhibit the selective staining of
filaments in the uropod (Fig 4B). Thus, agents that disrupt
microfilaments and microtubules alter the focal staining of
G-kinase on filaments.
Eflect of exogenous cyc/ic GMP. G-kinase exists in an
inactive state when cyclic GMP levels are not elevated.
Elevation of cyclic GMP via M L P may enhance G-kinase
-2.
-
~ l i Z O t h o t ~ k , r m C
trophih .tkmJ.1.d with fMLP
tor various timos (A through D).
Shown h c)-kiMSO kulbtlor,
in unnknu)n.d neutrophils (A)
and noutrophilr stimuktd with
fMLP for 30 soconds (E): 1
minuto (C): 2.6 minuns (0):
and 6 &uta (E). T r r t d c d b
stminod with pro-immuno sorum ar. shown in (F) (origkul
"tion
x 1.600).
localization in neutrophils. To test whether cyclic GMP
augments the transient localization of G-kinase, cells were
pre-incubated with 8-Br-cyclic GMP and stimulated with
fMLP. After incubation with 8-Br-cyclicGMPalone,CUM
demonstrated intense staining of the cytoplasm and some
nuclear staining. However, G-kinase was also focally localized within one optical section at the MTOC (Fig SD).
Enhanced changes in staining intensity and localization of
G-kinase were observed in cells that were pre-incubated with
8-Br-cyclic GMP and stimulated with fMLP. Within 30
seconds. compartmentalization of G-kinase was observed at
the MTOC and uropod (Fig 6). and by 2.5 minutes C U M
showed that G-kinase was predominantly localized within
the euchromatin of the nucleus and MTOC (Fig 7). Thus,
increased levels of cyclic GMP within specific compartments
may augment the transient localitation of G-kinase after
chemotactic stimulation.
DISCUSSION
This study provides evidence for a physiologic role for
G-kinase in human neutrophils. The data suggest that
regulation of neutrophil activation via cyclic GMP and
G-kinase is a localized intracellular event. fMLP induces
G-KINASE COMPARTMENTALIZATION IN NEUTROPHILS
615
Fig 3. Localization of G-kinase in neutrophils
stimulated for 1 minute with fMLP and viewed by
CLSM. Neutrophils were optically sectioned a t
0.4-pm increments (B through D). Differential interference microscopy is shown in (A). G-kinase is
focally localized within the nuclear hof and uropod
(original magnification x 1.100).
remarkable changes in G-kinase localization that are transient, and apparently cytoskeleton-dependent. Because fMLP
may also elevate cyclic G M P levels through NO production:
it may be predicted that 8-Br-cyclic G M P would augment
the localization produced by fMLP. This is, in fact, what was
observed (Figs 5 through 7). The focal localization of
G-kinase during neutrophil activation together with the
observed low levels of G-kinase, suggest that compartmentalized levels of this enzyme are important for the physiologic
regulation of neutrophil function. Thus, the overall levels of
cyclic G M P or G-kinase activity may not be as important in
contributing to a biologic effect as the placement of G-kinase
via targeting proteins in the vicinity of substrate protein(s).
Unraveling the regulatory role of cyclic nucleotides in
neutrophils is particularly difficult to resolve, because cell
activation is transient, and the sites of activation are probably confined to specialized intracellular compartments or
structures. Immunocytochemistry combined with CLSM
was used to localize G-kinase in neutrophils that were
stimulated with the chemotactic peptide fMLP. Optically
sectioning cells by CLSM was particularly useful in visualizing targeting structures and/or compartments of G-kinase.
With this approach, changes in localization of G-kinase
during neutrophil activation suggest targeting proteins that
may be important in the actions of G-kinase, and offer clues
that may eventually determine the regulatory role of this
enzyme within single cells.
G-kinase was localized at all time points at the MTOC and
nuclear euchromatin. fMLP stimulated transient focal
changes in G-kinase localization that coincided with the
transient changes in cell shape. A filamentous staining
pattern was observed near the cell margin and within the
uropod of polarized cells. When the cells began to lose
polarity and become round, strong staining of filament
Fig 4. Localization of G-kinase in neutrophils that were pre-incubated with cytochalasin B (A) or nocodazol (B) and stimulated with
fMLP for 2.5 minutes. CytochalasinB inhibited polarization of neutrophils and focal staining of G-kinase. Diffuse staining of the uropod was
observed in nocodazol treated cells (original magnification x 1.600).
616
PRYZWANSKY ET Al
Fig 6. Suios of throo C U M hugon (B through
DI of nwtrophih incubotod w+th 1 pnolll. RBrq d i c GMP for 16 minutn ond noinod for Gkinoao.
Difforontiol intorformco m r o n is shown in (A).
Cytopbomk and n w l u r noining h obsowod in
most of tho sconrwd inug... At o w optic01 @on0
(D), &kill000 is diotinctfy h l h d O t tho MTOC
(origin01mognitlcotion x 1,OOO).
bundles was observed within one region of the cell near the
nucleus. The staining intensity of G-kinase and change in cell
shape were enhanced when cells were pre-incubated with
8-Br-cyclicGMP. These results suggest that increased levels
of cyclic GMP augment the transient localization of G-kinase
after chemotactic stimulation.
It is important to note that in the absence of 10% human
serum. neutrophils were nonmotile. They did not polarize
and remained well-spread throughout the time course of
fMLP treatment. Under these conditions, G-kinase was
intensely localized at all time points at the MTOC and
nuclear euchromatin, and minimally locali7A within the
cytoplasm. In addition, when serum was replaced with
albumin or heat-inactivated serum, cells were nonmotile
after M L P treatment. as demonstrated by no changes in cell
shape (polarization). Also. under these conditions no translocation of G-kinase was observed. These observations suggest
that the motile activity and/or degree of adhesion to the
Fig 6. L o a ( h n k n of Gkhwoo In m m o p h b thn -0
proincubmod with R B r y d i c GMP for 16 hutn ond nfmu(n.d
whh fMtP for 30 ..cond..cOmp.rtnwnnliL.tbn of Gkinoao io
o b d at tho MTOC ond uropod (original nugnitlcotion x 1,200).
substratum may influence cyclic GMP and G-kinase activation. Keller et all?." have shown that chemokinesis is controlled by mechanisms that influence motility and adhesion,
and chemotactic factors for neutrophils in human serum
have been described." Studies are in progress to determine if
a serum factor (eg, C5a) is required for priming G-kinase
activation in neutrophils, and/or if an adhesive protein is
important.
The filamentous staining patterns were disrupted with
cytochalasin B and nocodaml, suggesting that one or more
cytoskeletal proteins are targeting proteins for G-kinase.
Possible targeting proteins include cytoskeletal-associated
proteins that contribute to the contractile. metabolic, and
structural activities associated with cell motility. Once localized to the appropriate substrate. G-kinase might regulate
the number of microtubule nucleating sites during neutrophil
activation, since increased levels of cyclic GMP are associated with an increase in microtubule numbers." and INLP
stimulates an increase in microtubule numbers and length.I6
Alternatively, G-kinase may mediate its effects by lowering
Ca" concentrations once localized within a specific cellular
compartment(s). For example, in vascular smooth muscle,
cyclic G M P lowers Ca?' concentrations resulting in
relaxation." G-kinase apparently mediates this efTcct. since
introduction of the enzyme into cells deficient in G-kinase
results in the lowering of Ca" and dephosphorylation of the
light chain of smooth muscle myosin?'" The significance of
G-kinase localization within the nucleus is unknown, although it is known that G-kinase phosphorylates nuclear
chromatin and nuclear histones?'
Recently, Schmidt et al' have demonstrated that fMLP
produces a transient increase in neutrophil NO, a compound
long known to stimulate guanylate cyclase," and which has
recently been identified as the endothelial-derived relaxing
factor or EDRF." Because the rise in NO is transient (due to
GKINASE COMPARTMENTMUAWN IN NEUTROeHlLS
-7.
617
8wk.oft)lmcLsM
imogom (0through D) of nMl0philo pucncuatod with &Brcydk O W for 16 minuton and
nimulotod for 2.6 minutoswith
MLP. O-khu- io fodb k a C
hod within tho nuckum and
MTOC. D)t(rWW i n ”
Contro.1 is ohown in (A) (origiMI nugnbtion x 2.100).
the lability of the radical), it is likely that cyclic G M P
increases arc also transient. Kaplan et at’ have shown that
chemotaxis is inhibited by blocking NO synthesis with the
competitive inhibitor of L-arginine. N”-monomethyl-Larginine. Inhibition of chemotaxis could be abrogated by
L-arginine or dibutryl-cyclic GMP. These data support the
involvement of cyclic G M P and G-kinase in regulating
chemotaxis. Thus, the greater effectiveness of 8-Br-cyclic
GMP in augmenting translocation may be due to its longer
duration of action compared with cyclic GMP.
Compartmentalitation of cyclic nucleotides in neutrophils
is also supported by our observations that cyclic AMP and RI
of A-kinase are compartmentalized within 30 seconds at the
forming phagosome during phagocytosis? The concept of
functional compartments in regulating cyclic nucleotide
effectors has been described and/or postulated in other
cellular systems.”u Further work is required to identify the
targeting proteins for these effectors and determine the
physiologic consequence. Immunocytochemistry. combined
with biochemical analysis, offers an approach to correlate
physiologic functions of intact cells with kinase modulation.
A C K N M E D G M EN f
We thank Dn M.Caplow. S.Earp. and K. Harden for review of
the manuscript; Dr Bill Reed for his technical advice and revim of
the manuscript: Dr Alton Steiner for amsultation; Dr Robert
Bagncll for assistance with the confocal dual laser scanning miscope; and Elizabeth Parker for photographic assistance. Informed
consent regarding the nature and possible consequences of the study
was obtained from human subjects before donating blood. This
investigation was approved by an Institutional Review Board and in
accord with an assurance filed with. and approved by, the Department of Health and Human Services.
REFERENCES
1. Estenm RD. Hill HR. Quie PG. Hogan N. Goldberg N D
CyclicGMPand cell mavement. Nature 249458,1973
2. Simchovitt L, Fischbein LC. Spilberg 1. Atkinson J P Induction of a transient elevation in intracellular levels of adenosine-3’-5’cyclic monophosphate by chemotactic factors: An early event in
human neutrophil activation. J lmmunol 124:1482.1980
3. Smolen JE. Korchak HM. Weissmann G: Increased lmls of
cyclic adenosine-3’-5’-monophosphatein human polymorphonuclear
leukocytes after surface stimulation. J Clin lnvat 65:1077.1980
4. Schmidt HH. Seifert R. Bohme E Formation and release of
nitric oxide from human neutrophils and HL-60 cells induccd by a
chemotactic peptide. platelet activating factor, and leukotriene 84.
FEBS Lett 24437.1989
5. Kaplan SS.Billiar T. Curran RD,Zdziarski UE.Simmons RL.
Basford R E Inhibition of chemotaxis with N“-monomethyl-Iarginine: A role for cyclicGMP. Blood 741885.1989
6. Lincoln TM. Corbin J D Characterization and biological role
of the cGMPdepcndent protein kinase. Adv Cyclic Nucleotide Res
15:139. 1983
7. Walter U: Cyclic GMP-regulated enzymes and their possible
physiological functions. Adv Cyclic Nucleotide Protein Phosphor
R a 17:249.1984
8. Prymansky KB. Steimr AL. Spitznagel JK. Kapaar C L
Compartmentalization of cyclic AMP during phagocytosis by human neutrophilicgranulocytes. Science 21 1 :407. 198 I
9. Comwell TL. Lincoln TM: Regulation of intracellular Ca**
levels in cultured vascular smooth muscle cells. J Biol Chem
2W1146.1989
IO. Papadopoula V. Hall PF: Isolation and characterization of
protein kinase C from Y-l adrenal cell cytoskeleton. J Cell Biol
108553, 1989
I I. Towbin H. Staehelin T, Gordon J: Electrophomic transfer of
proleins from polyacrylamidegelsto nitrocellulose sheets: Rocedure
and some applications. Proc Natl Acad Sci USA 764350.1979
12. Keller HU. Zimmermann A. Cottier H: Crawling- I‘k
I embva
menu. adhesion to solid substrata and chemokinesis of neutrophil
granulocytes.J Cell Sci M89.1983
13. Keller HU, Wider JH. D a m u B Diverging ctTccts of
chemotacticserum peptides and synthetic f-Mct-Lcu-Phcon ~ u t r o phi1 locomotion and adhesion. Immunology 42379,198 I
14. Jungi TW: Monocyte and neutrophil chemotactic activity of
normal and diluted human mrum and plasma. Int A r c h Allergy
Appl lmmun 53:18.1977
618
15. Hoffstein ST: Intra- and extra-cellular secretion from polymorphonuclear leukocytes, in Weissmann G (ed): Cell Biology of
Inflammation. New York, NY, North-Holland, Elsevier, 1980, p
387
16. Schliwa M, Pryzwansky KB, Euteneuer U: Centrosome
splitting in neutrophils: An unusual phenomenon related to cell
activation and motility. Cell 31:705, 1982
17. Lincoln TM: Cyclic GMP and mechanisms of vasodilation.
Pharmacol Ther 41:479,1989
18. Lincoln TM, Cornwell TL, Rashatwar SS, Johnson RM:
Mechanism of cyclic GMP-dependent relaxation on vascular smooth
muscle. Biochem Soc Trans 16:497, 1988
PRYZWANSKY ET AL
19. Arnold WP, Mittal CK, Katsuki S, Murad F Nitric oxide
activates guanylate cyclase and increases guanosine 3’:s’-cyclic
monophosphate levels in various tissue preparations. Proc Natl Acad
Sci USA 74:3203,1977
20. Palmer RMJ, Ferrige AG, Moncada S: Nitric oxide release
accounts for the biological activity of endothelium-derived relaxing
factor. Nature 327524, 1987
21. Earp HS, Steiner AL: Compartmentalization of cyclic nucleotide-mediated hormone action. Ann Rev Pharmacol Toxicol 18:
431,1978
22. Hayes JS, Brunton LL: Functional compartments in cyclic
nucleotide action. J Cyclic Nucleotide Res 8:1, 1982