Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells

1993 ◽  
Vol 264 (3) ◽  
pp. G470-G477 ◽  
Author(s):  
J. G. Jin ◽  
K. S. Murthy ◽  
J. R. Grider ◽  
G. M. Makhlouf
Keyword(s):  
Protein Kinase ◽  
Muscle Cells ◽  
Cell Types ◽  
No Production ◽  
Taenia Coli ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Cgmp Production ◽  
Gastric Muscle ◽  
Dependent Protein

The mechanism of action of vasoactive intestinal peptide (VIP) was examined in isolated gastric and taenia coli muscle cells and compared with that of nitric oxide (NO), sodium nitroprusside (SNP), and isoproterenol. In gastric muscle cells, VIP stimulated NO production, increased adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels, and induced relaxation in a concentration-dependent fashion. The NO synthase inhibitor NG-nitro-L-arginine abolished NO and cGMP production and partly inhibited relaxation. The soluble guanylate cyclase inhibitor LY 83583 abolished cGMP production and partly inhibited relaxation. (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], a preferential inhibitor of cAMP-dependent protein kinase (cAK), and KT5823, a preferential inhibitor of cGMP-dependent protein kinase (cGK), partly inhibited relaxation separately and abolished relaxation in combination. The pattern implied that VIP induced relaxation by activation of cAK and by NO-mediated stimulation of cGMP and activation of cGK. In taenia coli muscle cells, VIP did not increase NO production or cGMP levels: relaxation was accompanied by an increase in cAMP and was partly inhibited by (R)-p-cAMPS and KT5823 and abolished by a combination of both inhibitors. Isoproterenol increased only cAMP levels in both cell types, which induced relaxation by activating cAK at low concentrations of agonist and both cAK and cGK at high concentrations in a pattern identical to that observed with VIP in taenia coli muscle cells. SNP and NO increased only cGMP levels in both cell types, which induced relaxation by activating cGK only. We conclude that cAK and cGK can be activated separately and mediate relaxation independently.(ABSTRACT TRUNCATED AT 250 WORDS)

cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells

1990 ◽  
Vol 258 (3) ◽  
pp. C399-C407 ◽  
Author(s):  
T. M. Lincoln ◽  
T. L. Cornwell ◽  
A. E. Taylor
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dependent Protein Kinase ◽  
Aortic Smooth Muscle ◽  
Cyclic Monophosphate ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The major action of forskolin, the diterpine activator of adenylate cyclase, in primary (unpassaged) rat aortic smooth muscle cells is to reduce vasopressin-stimulated Ca2+ concentrations. In repetitively passaged cells, however, forskolin by itself increased Ca2+ levels by apparently stimulating Ca2+ uptake into the cell and had much smaller effects on inhibiting vasopressin-stimulated Ca2+ elevations. Both primary and passaged smooth muscle cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase was greatly reduced or absent in passaged smooth muscle cells. The introduction of purified cGMP-dependent protein kinase into the cytoplasm of passaged cells prevented forskolin from elevating intracellular Ca2+ and restored the capacity of forskolin to reduce vasopressin-stimulated Ca2+ mobilization. Similar effects were observed for isoproterenol in passaged smooth muscle cells. When introduced into cells, the active catalytic subunit of the cAMP-dependent protein kinase did not lead to reductions in Ca2+ levels. These results suggest that cAMP elevations lead to profound changes in Ca2+ metabolism through activation of both cAMP- and cGMP-dependent protein kinases. Activation of cGMP-dependent protein kinase by cAMP leads to the reduction in intracellular Ca2+, whereas activation of cAMP-dependent protein kinase may only mediate the uptake of Ca2+ from extracellular sources.

Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells

1995 ◽  
Vol 268 (1) ◽  
pp. C171-C180 ◽  
Author(s):  
K. S. Murthy ◽  
G. M. Makhlouf
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Rabbit Muscle ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.

Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors

1994 ◽  
Vol 267 (1) ◽  
pp. C236-C244 ◽  
Author(s):  
J. Geiger ◽  
C. Nolte ◽  
U. Walter
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Rapid Phase ◽  
No Donor ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp ◽  
Stimulation Of

Stimulation of Ca2+ mobilization and entry by agonists such as ADP, thrombin, and thromboxane is an early step of platelet activation. Here, we compared the effects of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating prostaglandins, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating nitrovasodilators, membrane-permeant selective activators of cAMP- or cGMP-dependent protein kinases, and physiological endothelium-derived factors on the agonist-evoked Ca2+ mobilization and entry in human platelets. Prostaglandin E1, the prostacyclin analogue Iloprost, the nitric oxide (NO) donor 3-morpholinosydnonimine hydrochloride, and selective activators of cGMP- or cAMP-dependent protein kinase strongly inhibited the agonist-evoked Ca2+ mobilization from intracellular stores and associated late Ca2+ entry but had little effects on the rapid (1st) phase of ADP-evoked Ca2+ entry. During coincubation of platelets with endothelial cells, endothelium-derived factors that were released strongly inhibited platelet agonist-evoked Ca2+ mobilization and only moderately affected the rapid phase of ADP-evoked Ca2+ entry. These effects were partially prevented when endothelial cells were preincubated with cyclooxygenase and/or NO synthase inhibitors. Endothelial cells therefore produce sufficient quantities of labile platelet inhibitors whose effects on the platelet Ca2+ response resemble those observed with selective cAMP- and cGMP-dependent protein kinase activators.

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

2003 ◽  
Vol 284 (6) ◽  
pp. H2311-H2319 ◽  
Author(s):  
Jean-Christophe Schneider ◽  
Driss El Kebir ◽  
Christiane Chéreau ◽  
Sophie Lanone ◽  
Xiao-Lin Huang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
No Production ◽  
Ionophore A23187 ◽  
Dependent Protein Kinase ◽  
Aortic Endothelial Cells ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Endothelium Dependent Relaxation ◽  
Aortic Endothelial

Nitric oxide (NO) is synthesized froml-arginine by the Ca2+/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca2+ ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion ofl-[3H]arginine tol-[3H]citrulline. Three CaMK II inhibitors, polypeptide 281–302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca2+-dependent activation of eNOS.

cAMP-dependent protein kinase differentially regulates prestalk and prespore differentiation during Dictyostelium development

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 135-146 ◽  
Author(s):  
S.K. Mann ◽  
R.A. Firtel
Keyword(s):  
Protein Kinase ◽  
Spatial Localization ◽  
Cell Types ◽  
Basal Region ◽  
Cell Type ◽  
Dependent Protein Kinase ◽  
Multicellular Development ◽  
Prespore Cell ◽  
Dependent Protein ◽  
Cell Type Specific

We and others have previously shown that cAMP-dependent protein kinase (PKA) activity is essential for aggregation, induction of prespore gene expression and multicellular development in Dictyostelium. In this manuscript, we further examine this regulatory role. We have overexpressed the Dictyostelium PKA catalytic subunit (PKAcat) in specific cell types during the multicellular stages, using prestalk and prespore cell-type-specific promoters to make PKA activity constitutive in these cells (independent of cAMP concentration). To examine the effects on cell-type differentiation, we cotransformed the PKAcat-expressing vectors with reporter constructs expressing lacZ from four cell-type-specific promoters: ecmA (specific for prestalk A cells); ecmB (specific for prestalk B and anterior-like cells in the slug); ecmB delta 89 (specific for stalk cells); and SP60 (prespore-cell-specific). By staining for beta-galactosidase expression histologically at various stages of development in individual strains, we were able to dissect the morphological changes in these strains, examine the spatial localization of the individual cell types, and understand the possible roles of PKA during multicellular development. Expression of PKAcat from either the ecmA or ecmB prestalk promoters resulted in abnormal development that arrested shortly after the mound stage, producing a mound with a round apical protrusion at the time of tip formation. Prestalk A and prestalk B cells were localized in the central region and the apical mound in the terminal differentiated aggregate, while prespore cells showed an aberrant spatial localization. Consistent with a developmental arrest, these mounds did not form either mature spores or stalk cells and very few cells expressed a stalk-cell-specific marker. Expression of PKAcat from the prespore promoter resulted in abnormal morphogenesis and accelerated spore cell differentiation. When cells were plated on agar, a fruiting body was formed with a very large basal region, containing predominantly spores, and a small, abnormal sorocarp. Mature spore cells were first detected by 14 hours, with maximal levels reached by 18–20 hours, in contrast to 24–26 hours in wild-type strains. When cells were plated on filters, they produced an elongated tip from a large basal region, which continued to elongate as a tubular structure and produce a ‘slug-like’ structure at the end. The slug was composed predominantly of prestalk cells with a few prespore cells restricted to the junction between the ‘slug’ and tube. As the slug migrated, these prespore cells were found in the tube, while new prespore cells appeared at the slug/tube junction, suggesting a continual differentiation of new prespore cells at the slug's posterior.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells

1997 ◽  
Vol 272 (6) ◽  
pp. G1391-G1399 ◽  
Author(s):  
K. S. Murthy ◽  
J. G. Jin ◽  
J. R. Grider ◽  
G. M. Makhlouf
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Muscle Cells ◽  
Cyclic Monophosphate ◽  
Pituitary Adenylate Cyclase ◽  
Gastric Muscle ◽  
Pacap Receptors ◽  
G Protein Coupled ◽  
Camp Formation ◽  
Cgmp Formation

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.

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