Some comparative properties and localization of porcine jejunal adenylate cyclase

1978 ◽  
Vol 56 (4) ◽  
pp. 280-286 ◽  
Author(s):  
G. W. Forsyth ◽  
D. L. Hamilton ◽  
K. E. Goertz ◽  
L. W. Oliphant
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Intestinal Secretion ◽  
Hill Coefficient ◽  
Jejunal Mucosa ◽  
Ph Optimum ◽  
Guanosine Diphosphate ◽  
Cyclic Amp Accumulation ◽  
Triton X

Cholera toxin is thought to cause intestinal secretion by activating adenylate cyclase and increasing intracellular 3′,5′-cyclic AMP concentrations in intestinal mucosa. Cholera toxin causes profuse secretion of fluid into ligated intestinal loops of both pigs and rabbits, but cholera toxin-induced increases in 3′,5′-cyclic AMP concentration are much lower in the pig than in the rabbit. Porcine jejunal adenylate cyclase was examined for unusual properties which might account for a lack of 3′-5′-cyclic AMP accumulation after treatment with cholera toxin. The divalent cation requirements, the pH optimum, and the stimulation by fluoride ion were unremarkable. The Km for ATP was 0.11 mM with negative cooperativity indicated by a Hill coefficient of 0.83. Triton X-100 was inhibitory and guanosine diphosphate methylene phosphate stimulated enzyme activity. Adenylate cyclase activity was highest in the basal and lateral membrane fractions of jejunal mucosa and relatively low in brush-border preparations. Pretreatment of pig jejunum with cholera toxin caused a 30–40% activation of the crude and of the partly purified enzyme. A relatively low activation of adenylase cyclase in pigjejunal mucosa, compared with rabbit, may account for the absence of 3′,5′-cyclic AMP accumulation after cholera-toxin treatment in the pig.

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.

Induction And Potentiation Of Platelet Aggregation By Clonidine And 7ρ-Aminoclonidine, Partial Agonists Of The α2-Receptor Which Regulates Platelet Adenylate Cyclase

1981 ◽  
Author(s):  
David C Stump ◽  
Donald E Macfarlane
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Specific Binding ◽  
Phase 1 ◽  
Intrinsic Activity ◽  
Second Phase ◽  
Adrenergic Agents ◽  
Cyclic Amp Accumulation ◽  
Induced Aggregation

Epinephrine induces platelet aggregation, potentiates aggregation by other agents, and blocks the stimulation of the adenylate cyclase by prostaglandins. Synthetic α-adrenergic agents have not been shown to induce aggregation. The effects of clonidine, an α2-agonist, and ρ-aminoclonidine on platelets were examined. Clonidine potentiated aggregation induced by 0.5μM ADP by 1.4-fold (1/2 max 0.5μM). It did not induce significant aggregation itself, and it inhibited aggregation induced by 5μM epinephrine (1/2 max lμM). It inhibited cyclic AMP accumulation induced by PGE1 by a maximum of 25% (1/2 max O.lμM) and it blocked inhibition by epinephrine. No significant specific binding of [3H] clonidine was observed to intact platelets. ρ-Aminoclonidine induced aggregation with delayed second phase (1/2 max 0.2μM), and potentiated ADP aggregation by 2-fold (1/2 max 0.2μM). Aggregation induced by epinephrine was more rapid, and was partially inhibited by ρ-aminoclonidine. It inhibited cyclic AMP accumulation by 50% max (1/2 max O.lμM) and attenuated epinephrine’s effect to the same level. The direct effects of ρ-aminoclonidine were blocked by lμM yohimbine, a selective α2-antagonist. Both clonidine and ρ—aminoclonidine blocked the specific binding of [3H]yohimbine (1/2 max 0.5μM). These results suggest that the platelet bears an α2-receptor with affinity for epinephrine, ρ-aminoclonidine and clonidine as agonists but that these agents display differing intrinsic activity and/or receptor reserve.

Uncoupling of Lipolysis from Cyclic AMP by Procaine: a Tool for Studying the Mechanism of Action of Antilipolytic Agents

1975 ◽  
Vol 53 (4) ◽  
pp. 603-609 ◽  
Author(s):  
Mario D'Costa ◽  
Aubie Angel
Keyword(s):  
Adipose Tissue ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mechanism Of Action ◽  
Initial Rate ◽  
Direct Action ◽  
Inhibitory Effect ◽  
Glycerol Release ◽  
Cyclic Amp Accumulation ◽  
Low K

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40–78%) by procaine–HCl (1–5 mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine–HCl (0.1–1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S®) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine–HCl nor clofibrate had any effect on the low Km 3′:5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.

scholarly journals Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C

1988 ◽  
Vol 253 (1) ◽  
pp. 229-234 ◽  
Author(s):  
P Thams ◽  
K Capito ◽  
C J Hedeskov
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Time Course ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Cyclic Amp Accumulation ◽  
Mouse Pancreatic Islets

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.

scholarly journals Multiple non-specific effects of sphingosine on adenylate cyclase and cyclic AMP accumulation in S49 lymphoma cells preclude its use as a specific inhibitor of protein kinase C

1990 ◽  
Vol 268 (2) ◽  
pp. 507-511 ◽  
Author(s):  
J A Johnson ◽  
R B Clark
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Lymphoma Cells ◽  
Specific Effects ◽  
Kinase C ◽  
Cyclic Amp Accumulation

Recent studies with phorbol esters have suggested that protein kinase C (PKC) may play a role in the regulation of adenylate cyclase in mammalian cells. Since D-sphingosine has been reported to specifically inhibit PKC in many cell types, we evaluated its effects on stimulation of cyclic AMP accumulation by adrenaline in S49 lymphoma cells. We found sphingosine to have multiple non-specific effects which could not be explained by an inhibition of PKC. These effects included: (i) inhibition by sphingosine (50 microM) of adrenaline-stimulated cyclic AMP accumulation and sphingosine permeation of the cells which rendered them leaky to ATP; (iii) sphingosine (20 microMs) augmentation of adrenaline-stimulated cyclic AMP accumulation; (iii) inhibition by sphingosine of adrenaline-stimulated adenylate cyclase in isolated membranes by up to 95%; and (iv) sphingosine (20 microM) inhibition of cellular mechanisms for the elimination of cyclic AMP. These results demonstrate the importance of evaluating the non-specific effects of sphingosine before concluding that its actions are the consequences of a specific inhibition of PKC.

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