Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

1997 ◽  
Vol 272 (5) ◽  
pp. L865-L871 ◽  
Author(s):  
B. Tolloczko ◽  
Y. L. Jia ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells

1995 ◽  
Vol 268 (6) ◽  
pp. L1047-L1051 ◽  
Author(s):  
K. Maruno ◽  
A. Absood ◽  
S. I. Said
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Thymidine Incorporation ◽  
Inhibitory Effect ◽  
Airway Smooth Muscle Cells ◽  
Dependent Manner ◽  
Cyclic Monophosphate ◽  
Cell Counts ◽  
Dependent Protein ◽  
Camp Levels

Airway smooth muscle (ASM) cell proliferation contributes to increased airway resistance in bronchial asthma. We have examined the modulation of ASM proliferation by vasoactive intestinal peptide (VIP), a cotransmitter of airway relaxation. Human ASM cells were grown in culture as a monolayer. VIP (1.0 nM-1.0 microM) inhibited proliferation in a dose-dependent manner by up to 82% on day 2, but the related peptide glucagon had no effect. Histamine (100 nM-100 microM) increased cell counts by 66%, but in the presence of VIP, cell counts and [3H]thymidine incorporation were reduced by up to 55%. Adenosine 3',5'-cyclic monophosphate (cAMP)-promoting agents, including 3-isobutyl-1-methylxanthine, forskolin, and 8-bromo-adenosine 3',5'-cyclic monophosphate, alone and especially combined with VIP, reduced cell counts and [3H]thymidine incorporation, in correlation with cAMP levels. KT-5720 (1.0 nM-1.0 microM), a selective inhibitor of cAMP-dependent protein kinase A (PKA), abolished the inhibitory effect of VIP. The results show that VIP selectively and potently inhibits human ASM cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA-mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens.

Intracellular mechanisms involved in the regulation of vascular smooth muscle tone

1995 ◽  
Vol 73 (5) ◽  
pp. 565-573 ◽  
Author(s):  
Michael P. Walsh ◽  
Gary J. Kargacin ◽  
John Kendrick-Jones ◽  
Thomas M. Lincoln
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Cyclic Gmp ◽  
Vascular Tone ◽  
Light Chains ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regulation Of Vascular Tone

Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling – sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation–dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca2+-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure–function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca2+–calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as β-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.Key words: vascular smooth muscle, regulation of contraction, smooth muscle structure, calcium, cyclic GMP, myosin.

An inhibitor of Ca2+/calmodulin-dependent protein kinase II, KN-62, inhibits cholinergic-stimulated parietal cell secretion

1992 ◽  
Vol 262 (1) ◽  
pp. G118-G122 ◽  
Author(s):  
Y. Tsunoda ◽  
M. Funasaka ◽  
I. M. Modlin ◽  
H. Hidaka ◽  
L. M. Fox ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Parietal Cell ◽  
Parietal Cells ◽  
Cell Secretion ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
High Concentrations ◽  
Total Membrane

Cholinergic stimulation of parietal cell secretion is mediated by an increase in intracellular calcium. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), has recently been synthesized (Tokomitsu et al. J. Biol. Chem. 265: 4315-4320, 1990). To define the role of CaMK II in parietal cell secretion, we determined the effects of KN-62 on secretagogue-stimulated acid secretion in isolated rabbit parietal cells. Pretreatment of parietal cells with KN-62 resulted in the inhibition of carbachol-stimulated [14C]aminopyrine uptake over a concentration range of 3 to 60 microM (IC50 of 20 microM). KN-62 (60 microM) reduced carbachol-stimulated aminopyrine uptake to unstimulated levels. KN-62 did not alter carbachol-stimulated increases in cytoplasmic free Ca2+ concentration. High concentrations of KN-62 (60 microM) elicited a small decrease in aminopyrine uptake stimulated by forskolin, but did not significantly inhibit histamine stimulation. A potent CaMK II activity was identified in total membrane from parietal cells. These results suggest that CaMK II may mediate cholinergic-stimulated parietal cell secretion.

Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP

1994 ◽  
Vol 267 (5) ◽  
pp. C1405-C1413 ◽  
Author(s):  
T. L. Cornwell ◽  
E. Arnold ◽  
N. J. Boerth ◽  
T. M. Lincoln
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Activity Ratio ◽  
Dependent Protein Kinase ◽  
Inhibition Of Proliferation ◽  
Cyclic Monophosphate ◽  
Dependent Inhibition ◽  
Dependent Protein

Recent studies indicate that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may inhibit the proliferation of vascular smooth muscle cells (SMC) in vitro. The purpose of this study was to investigate the mechanism of NO- and cGMP-dependent inhibition of cultured rat aortic SMC. The cytokine interleukin-1 beta (IL-1 beta) inhibited serum- and platelet-derived growth factor-stimulated [3H]thymidine incorporation into DNA in subcultured rat aortic SMC. Incubation with IL-1 beta for 24 h markedly increased cGMP levels but not adenosine 3',5'-cyclic monophosphate (cAMP) levels. However, the IL-1 beta-induced increase in cGMP was correlated with an activation of the cAMP-dependent protein kinase (cAMP kinase) activity ratio. The activation of the cAMP kinase was prevented by treatments that blocked NO and cGMP production. The NO-generating vasodilator, S-nitroso-N-acetylpenicillamine (SNAP) also inhibited DNA synthesis and elevated cGMP levels. The inhibition of DNA synthesis by both IL-1 beta and SNAP was observed only when cGMP levels were elevated to high levels (10-fold or more). As was the case for IL-1 beta, SNAP increased the activity ratio of cAMP kinase. Selective inhibition of cAMP kinase using (R)-p-bromoadenosine 3',5'-cyclic monophosphorothioate prevented the inhibition of proliferation by IL-1 beta. By contrast, the inhibitor of the cGMP-dependent protein kinase, (R)-p-bromoguanosine 3',5'-cyclic monophosphorothioate, had no effect on IL-1 beta-induced inhibition of cellular proliferation. These studies suggest that cGMP-dependent activation of the cAMP kinase may be responsible in part at least for the NO-dependent inhibition of proliferation of subcultured rat aortic SMC.

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

1998 ◽  
Vol 274 (5) ◽  
pp. C1199-C1205 ◽  
Author(s):  
Karnam S. Murthy ◽  
Gabriel M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Muscle Cells ◽  
Ruthenium Red ◽  
Dependent Protein Kinase ◽  
Cell Content ◽  
Gastric Smooth Muscle ◽  
Dependent Protein ◽  
Permeabilized Muscle

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca2+-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 ( Gastrointest. Liver Physiol. 28): G660–G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 μM), NO (1 μM), and VIP (1 μM) stimulated45Ca2+release (21 ± 3 to 30 ± 1% decrease in45Ca2+cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 ± 0.1 μM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]iby stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.

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.

scholarly journals Regulation of jun-B expression by a cyclic AMP (cAMP)-dependent mechanism in human myeloid cells

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 83-88 ◽  
Author(s):  
R Datta ◽  
T Nakamura ◽  
ML Sherman ◽  
D Kufe
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Jun B

Abstract The present studies have examined the regulation of the jun-B early response gene by cyclic AMP (cAMP)-dependent signaling pathways. The 2.0-kb jun-B transcript was at low but detectable levels in uninduced human HL-60 myeloid leukemia cells. In contrast, treatment with 1 mmol/L8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) in the presence of isobutylmethylxanthine, an inhibitor of cAMP-dependent phosphodiesterase, was associated with increases in jun-B transcripts that were maximal by 1 hour and then decreased to near pretreatment levels by 6 hours. Similar findings were obtained with 8–(4- chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (8-CPT-cAMP) and N6,2′–0-dibutyryladenosine 3′,5′-cyclic monophosphate (dBt-cAMP). jun-B transcripts were also increased with other agents that increase intracellular cAMP levels, such as prostaglandin E2 (PGE2) and forskolin. Moreover, inhibition of cAMP-dependent protein kinase by the isoquinolinesulfonamide H-8 blocked 8-Br-cAMP-induced increases in jun- B expression. The results of nuclear run-on assays demonstrate that treatment of HL-60 cells with PGE2, forskolin, 8-Br-cAMP, and dBt-cAMP is associated with increases in the rate of jun-B transcription. The present findings also demonstrate that the related jun-D gene is similarly regulated by a cAMP-dependent pathway. Taken together, these findings suggest that stimulation of cAMP-dependent protein kinase is involved in the induction of jun gene expression in myeloid leukemia cells.

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