scholarly journals Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways

1994 ◽  
Vol 297 (1) ◽  
pp. 233-239 ◽  
Author(s):  
P A Stevens ◽  
S Pyne ◽  
M Grady ◽  
N J Pyne
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Phospholipase D ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Tracheal Smooth Muscle ◽  
Constitutive Activation ◽  
Cyclic Amp Accumulation

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

1989 ◽  
Vol 66 (3) ◽  
pp. 1397-1407 ◽  
Author(s):  
J. M. Madison ◽  
C. A. Jones ◽  
R. M. Sankary ◽  
J. K. Brown
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Prostaglandin E2 ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Tracheal Smooth Muscle ◽  
Inhibitory Effects ◽  
Camp Content

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3′,5′-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.

scholarly journals Structural specificity for prostaglandin effects on hepatocyte glycogenolysis

1990 ◽  
Vol 267 (1) ◽  
pp. 59-62 ◽  
Author(s):  
E P Brass ◽  
M J Garrity
Keyword(s):  
Glucose Metabolism ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Sprague Dawley ◽  
Hepatic Glucose Metabolism ◽  
Sprague Dawley Rats ◽  
Cyclic Amp Accumulation

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.

Acetylcholine inhibits positive inotropic effect of cholera toxin in ventricular muscle

1982 ◽  
Vol 243 (3) ◽  
pp. H434-H441
Author(s):  
A. J. Pappano ◽  
P. M. Hartigan ◽  
M. D. Coutu
Keyword(s):  
Chick Embryo ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Positive Inotropic Effect ◽  
Phosphodiesterase Inhibitor ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Inotropic Effect ◽  
Cyclic Monophosphate

Acetylcholine (ACh, 10(-6) M) had no effect on basal adenylate cyclase activity (3.4 +/- 0.56 pmol cyclic AMP . min-1 . mg wet wt-1), adenosine 3',5'-cyclic monophosphate (cyclic AMP) content (0.88 +/- 0.09 pmol/mg wet wt), or the force of contraction in paced (2.5 Hz) chick embryo right ventricles superfused with Tyrode solution. After 60-180 min of superfusion in the presence of cholera toxin (5 x 10(-6) g/ml), adenylate cyclase activity (1.7 times), cyclic AMP content (2.4 times), and contractility (2.4 times) had increased significantly above basal levels. ACh reversed the positive inotropic effect of cholera toxin but did not change the increased activity of adenylate cyclase and content of cyclic AMP obtained in cholera toxin. Stimulation of adenylate cyclase by isoproterenol (ISO) was inhibited by ACh in the absence and presence of cholera toxin. ACh did not change guanosine 3',5'-cyclic monophosphate (cyclic GMP) content in the absence or presence of cholera toxin. Cholera toxin has actions on chick embryo ventricle similar to those of the beta-adrenergic agonist, ISO, and the phosphodiesterase inhibitor, isobutylmethylxanthine. The ability of ACh to reverse the positive inotropic effect of cholera toxin without preventing the accumulation of cyclic AMP may involve the prevention or reversal of cyclic AMP-dependent phosphorylation. In this regard, reduction of Ca2+ influx through voltage-sensitive membrane channels may be an essential component of muscarinic inhibition.

Muscarinic cholinergic inhibition of adenylate cyclase in airway smooth muscle

1987 ◽  
Vol 253 (1) ◽  
pp. C97-C104 ◽  
Author(s):  
C. A. Jones ◽  
J. M. Madison ◽  
M. Tom-Moy ◽  
J. K. Brown
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Inhibitory Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adrenergic Stimulation ◽  
Biochemical Effect ◽  
Cholinergic Agents ◽  
Muscarinic Cholinergic

The goal of our study was to test for an inhibitory effect of acetylcholine on adenylate cyclase activity in canine trachealis muscle. Therefore, cells were dispersed from the muscle enzymatically and lysed, and adenylate cyclase activity was assayed in a membrane suspension isolated from the lysates. Maximal beta-adrenergic stimulation, in the presence of GTP (10(-4) M), increased the activity of adenylate cyclase twofold above the activity induced by GTP alone. In the presence of GTP, acetylcholine (10(-4) M) decreased activity from 97 +/- 21 to 55 +/- 13 pmol cyclic AMP X min-1 X mg protein-1 (means +/- SE; n = 5; P less than 0.05); in the presence of GTP plus isoproterenol (10(-4) M), the acetylcholine-induced decreases were from 163 +/- 29 to 101 +/- 15 pmol cyclic AMP X min-1 X mg protein-1 (P less than 0.05). These decreases were dose dependent and they were altered by a series of cholinergic agents in a pattern consistent with a muscarinic effect. Our results suggest that one biochemical effect of vagal stimulation in the central airways of dogs may be attenuated adenylate cyclase activity in the smooth muscle.

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