Treatment of intact hepatocytes with the calcium ionophore A23187 perturbs both the synthesis and the degradation of the second messenger cyclic AMP. Actions on adenylate cyclase and cyclic AMP phosphodiesterase activities

The presence of the calcium ionophore A23187 augmented glucagon's ability to elevate intracellular cyclic AMP concentrations in intact hepatocytes. However, when the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine (IBMX) was added to prevent the degradation of cyclic AMP then the presence of A23187 attenuated the ability of glucagon to increase intracellular cyclic AMP concentrations. Treatment of intact hepatocytes with A23187 led to a dose-dependent persistent inhibition of the glucagon-stimulated adenylate cyclase activity expressed by a membrane fraction isolated from such ionophore-treated hepatocytes. In hepatocytes where glucagon-stimulated adenylate cyclase activity was desensitized then A23187-treatment of hepatocytes failed to exert any inhibitory action on adenylate cyclase. Treatment of isolated membranes directly with A23187 did not elicit any changes in glucagon-stimulated adenylate cyclase activity. Such actions of A23187 were blunted when Ca2+ (2.5 mM) was not added to the extracellular medium. It is suggested that treatment of hepatocytes with A23187 leads to the functional uncoupling of glucagon-stimulated adenylate cyclase activity in a manner which appears to mimic the desensitization process. A23187-treatment also exerted an overall inhibitory effect on the cyclic AMP phosphodiesterase activity displayed by intact hepatocytes. Thus treatment of hepatocytes with A23187 exerted a profound effect on cyclic AMP metabolism in these cells.     

Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes

Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.     

Studies on the mechanism of the inhibitory effect of ethanol on the gastric acid output in the rat

Summary The mechanism of the ethanol-induced decrease of the output of gastric acid was studied in the perfused stomach preparation of urethaneanaesthetised rats. Hypotonic (0.8%) ethanol caused a transient decrease in the spontaneous output of acid. Isotonic (1.7%) or higher concentrations of ethanol induced a constant inhibition of the gastric acid output. At the concentration of 10% ethanol strongly inhibited the gastric acid secretion, stimulated by carbachol, histamine or pentagastrin. In the presence of 20% ethanol in the gastric lumen a significant disappearance of added hydrogen ions was observed, whereas 10% ethanol did not cause a loss of acid. This indicates that the decrease of gastric acid output by 10% or lower concentrations of ethanol is due to the inhibition of the secretory process. Higher concentrations may decrease the gastric acid output also by inducing a backdiffusion of hydrogen ions into the gastric mucosa. Pretreatment of the rats with inhibitors of prostaglandin synthesis, viz. indomethacin, mefenamic acid, meclofenamic acid or phenylbutazone, antagonised the ethanol-induced inhibition of the spontaneous gastric acid secretion. Pretreatment with the phosphodiesterase inhibitors, papaverine (20 mg/kg i.v.) and theophylline (60 mg/kg i.v.) increased the secretion of gastric acid. Papaverine antagonised the inhibition of gastric acid output induced by 10% ethanol, but theophylline failed to do so. At the concentration of 10% ethanol strongly depressed the secretory response to dibutyryl cyclic AMP. The inhibitory effect of ethanol was abolished by a pretreatment with meclofenamic acid. The results suggest that in the gastric mucosa ethanol increases the formation of prostaglandins which exert an inhibitory effect on the gastric acid secretion.     

Effects of adenosine analogues on force and cAMP in the heart. Influence of adenosine deaminase

The effects of the adenosine receptor agonists (-)-N6-phenylisopropyladenosine (PIA) and 5'-N-ethylcarboxamideadenosine (NECA) on the force of contraction, adenylate cyclase activity and cAMP content in the presence of isoprenaline (Iso) were studied in ventricular preparations of the guinea-pig heart. Only in the presence of adenosine deaminase (ADA) and 50 mM sodium chloride, i.e. under 'optimal' conditions, did PIA and NECA reduce the Iso-stimulated adenylate cyclase activity in broken cell preparations, with a maximal effect of about 25%. In electrically driven (1 Hz) papillary muscles from guinea-pigs, both compounds concentration dependently reduced the Iso-stimulated force of contraction maximally by about 50% in the presence of ADA (1 microgram/ml). cAMP was measured in the same preparations. Low concentrations (0.1-1 microM) of PIA reduced the cyclic AMP content while higher concentrations increased the cyclic AMP content. The negative inotropic effect of NECA was accompanied by a concentration-dependent increase in the cyclic AMP content. We conclude that the negative inotropic effect of PIA in the presence of Iso is only in part due to a decrease in the cyclic AMP content resulting from inhibition of adenylate cyclase activity. Such an effect was only detected in the presence of ADA so that endogenous adenosine can obviously mask small effects of PIA on adenylate cyclase activity or the cyclic AMP content. In addition, the negative inotropic effect of NECA in the presence of isoprenaline was not accompanied by a reduction but an increase in the cyclic AMP content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP and Ca-binding in microsomal fractions isolated from rabbit colon smooth muscle.

From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density grandient at 35% and 35-45% sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10(-5)M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 X 10(-6)M) stimulated the Ca-accumulation in the 35-45% fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35% sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic beta-receptor blocking agent sotalol. In the 35-45% fraction papaverine (1 X 10(-3)M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

The absence of stimulation of lipolysis by papaverine, a strong inhibitor of phosphodiesterase

Quantitative studies of the action of theophylline and papaverine were performed in rat epididymal fat pads, both on the lipolytic effect and on the activity of phosphodiesterase, adenylate cyclase and protein kinase. Papaverine, a stronger inhibitor of phosphodiesterase than theophylline, did not produce lipolysis. The maximum lipolytic effect (glycerol release) of theophylline was much higher than that of epinephrine and nearly approached the effect exerted by dibutyryl cyclic AMP. While theophylline potentiated or was without any effect on lipolysis produced by epinephrine and dibutyryl cyclic AMP, papaverine at concentration 10⁻³M reduced the effect of both drugs as well as of theophylline by 90%. These concentrations of papaverine also strongly inhibited the activity of adenylate cyclase. Neither papaverine nor theophylline prevented the activation of protein kinase by cyclic AMP.

The data suggest that the lack of a lipolytic effect of papaverine might be caused by a combination of its inhibitory effect on adenylate cyclase and direct inhibition of activation of triglyceride lipase     

Role of cyclic AMP in cardiac beta-adrenoceptor desensitization: studies using prenalterol and inhibitors of phosphodiesterase

We examined the effects of prolonged exposure of cardiac cells in primary culture to the partial beta-adrenoceptor agonist prenalterol and inhibitors of phosphodiesterase on their subsequent ability to increase intracellular cyclic AMP during a 5-min exposure to 50 microM isoprenaline (receptor responsiveness). Although prenalterol possesses only 7% of the agonist activity of isoprenaline on adenylate cyclase, it induces extensive beta-adrenoceptor desensitization. Three hours after exposing the cells to 1 microM prenalterol, beta-adrenoceptor responsiveness was reduced by 40% (p less than 0.05), whereas after 12 h the reduction averaged 55%. Prolonging the incubation time to 48 h had no further effect on the magnitude of receptor desensitization. The magnitude of the desensitization was concentration dependent. On exposure of cells to 10(-8) M prenalterol for 16 h, receptor responsiveness was reduced by 19%, and at concentrations of 1 microM and higher responsiveness was reduced by 60% (p less than 0.01). Receptor desensitization appeared to be due to an inability of receptors to activate adenylate cyclase as well as to receptor loss. To investigate if beta-adrenoceptor desensitization as well as receptor loss could be mediated by cyclic AMP, the cells were exposed for 16 h to inhibitors of phosphodiesterase. Exposure of cells to the phosphodiesterase inhibitor isobutylmethylxanthine (0.1 mM) (which increased intracellular cyclic AMP by between 50 and 150%) also induced receptor desensitization. The reduction in receptor responsiveness averaged 62% (p less than 0.01). The loss in responsiveness could be accounted for by an inability of receptors to activate adenylate cyclase as well as by receptor loss.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ethanol on the cyclic AMP system in rat brain.

The effects of ethanol on adenylate cyclase and phosphodiesterase activity in vitro, and on cyclic AMP, ATP and adenosine levels in vivo were studied. Ethanol appeared to affect the cyclic AMP system indirectly, probably through its effect on neurotransmitter release or on adenosine formation.     

Effect of ethanol on cyclic AMP levels in intact PC12 cells

Two subclones of the rat pheochromocytoma cell line, PC12, were used to compare the effects of ethanol on adenylate cyclase activity in isolated membranes with its effects on cyclic AMP accumulation in intact cells. Consistent with previous reports, ethanol increased basal and 2-chloroadenosine-stimulated adenylate cyclase activity in isolated membrane preparations from both subclones. However, ethanol had opposite effects on agonist-stimulated cyclic AMP accumulation in intact cells of the two subclones, enhancing accumulation in one subclone, and inhibiting it in the other. The inhibition of cyclic AMP accumulation did not result from stimulation of phosphodiesterase activity, activation of the inhibitory guanyl nucleotide regulatory protein, Gi, or stimulation of protein kinase C. The results indicate that extrapolation of the effects of ethanol from one cell type to another, or from in vitro to in vivo systems, may be complicated by the interaction of ethanol with regulatory processes that influence second messenger systems, and can differ in various types of intact cells.     

Cyclic AMP and Ca-binding in Microsomal Fractions Isolated from Rabbit Colon Smooth Muscle

Abstract From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density gradient at 35 % and 35-45 % sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10-⁵ M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 × 10-⁶ M) stimulated the Ca-accumulation in the 35-45 % fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35 % sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic β-receptor blocking agent sotalol. In the 35-45 % fraction papaverine (1 × 10-³ M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

Studies on responsiveness of hepatoma cells to catecholamines. I. Lack of beta-adrenergic responsiveness in rat ascites hepatoma AH13 cells

The beta-adrenoceptor density and the activities of adenylate cyclase and cyclic AMP phosphodiesterase were examined to compare AH13 cells having lower beta-adrenergic responsiveness with other rat ascites hepatoma cells and normal rat liver cells. Normal rat liver cells used were cultured for 24 hr after the collagenase digestion of liver. The density of binding sites of 3H-dihydroalprenolol in AH13 cell plasma membrane was very similar to the density in AH44 and normal liver cell membrane, but that in AH130 cell plasma membrane was about 10-fold greater than those in the other three cell lines. The activity of cyclic AMP phosphodiesterase was about 2.5- to 7-fold higher in hepatoma cells than in rat liver cells, but this enzyme activity of AH13 cells was not especially high among the hepatoma cells examined. The basal adenylate cyclase activity was lower in AH44 cells, but was higher in AH13 and AH130 cells than in rat liver cells. However, adenylate cyclase of AH13 cells was hardly activated by isoproterenol, while the enzyme of the other cells was activated 3- to 5-fold. On the other hand, adenylate cyclase of each cell line including AH13 was activated 4- to 14-fold by NaF. From these results, it is suggested that AH13 cells can hardly produce cyclic AMP by the beta-adrenergic stimulation because of the disordered interaction of beta-adrenoceptors with adenylate cyclase.     

Influence of vasoactive intestinal polypeptide on net water flux and cyclic adenosine 3′,5′-monophosphate formation in the rat jejunum

1. The effects were studied of vasoactive intestinal polypeptide (VIP), theophylline, and morphine on net water flux and cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo and of VIP and morphine on adenylate cyclase activity in rat epithelial cell membranes in vitro. 2. Infusion of VIP (0.1-2 x 10(-9) mol/min/kg) dose dependently caused a reversal from net water absorption to net secretion; 2 x 10(-9) mol/min/kg enhanced the mucosal cyclic AMP content by 67%. 3. Theophylline (5 mg/ml, intraluminally) enhanced the effect of intra-arterial infusion of VIP (2 x 10(-9) mol/min/kg) as to net water secretion and increase in mucosal cyclic AMP content. 4. Pretreatment with morphine (5 mg/kg, s.c.) did not influence the effects of VIP on net water flux and on mucosal cyclic AMP content. 5. Atropine (2 mg/kg, s.c.) also failed to reduced the effect of VIP (0.4 x 10(-9) mol/min/kg) on net water flux. 6. Stimulation of adenylate cyclase activity was a function of VIP concentration over a range of 1 x 10(-10)-1 x 10(-7) M. Morphine (up to 1 x 10(-3) M) failed to influence stimulation of adenylate cyclase by VIP. 7. The finding that low doses of VIP, which already have an effect on net water flux, fail to increase cyclic AMP levels makes it likely that other mediators besides cyclic AMP are involved in the effect of VIP on net water flux. Some of the present results, however, support the assumption that VIP stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels.     

Ethanol-induced changes in gastric mucosal content of cyclic AMP and ATP in the rat.

The stomach of urethane-anaesthetised rats was perfused with 10% (v/v) ethanol. At 40 min, the secretion of acid was strongly inhibited and the contents of cyclic AMP and ATP were lowered in the superficial mucosa, but not in whole gastric mucosa. After discontinuation of the ethanol perfusion, the rate of gastric acid output as well as the cyclic AMP and ATP levels recovered. Most of the acid-secreting parietal cells were found in the superficial mucosa where the changes of cyclic AMP and ATP took place. The results suggest that lowering of the mucosal contents of cyclic AMP and ATP may be involved in the ethanol-induced inhibition of the gastric acid output.     

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.     

Stimulation of adenylate cyclase of guinea pig gastric mucosa by histamine,sodium fluoride and 5′-guanylylimidodiphosphate and inhibition by histamine H1- and H2-receptor antagonists in vitro

Summary There are experimental data indicating that cyclic AMP is involved in the regulation of gastric acid secretion in various mammalian species. In a broken cell preparation of guinea pig gastric mucosa the effects of some stimulants of gastric acid secretion on the activity of adenylate cyclase were studied. The basal adenylate cyclase activity was 483±43 pmoles cyclic AMP/mg proteinx10 min. The activity could be stimulated by histamine maximally 5-fold, by sodium fluoride (NaF) maximally 20-fold and by 5-guanylylimidodiphosphate (GMP-PNP) maximally 10-fold. Neither pentagastrin nor carbachol were able to stimulate the adenylate cyclase. Stimulants of adrenergic - or -receptors (phenylephrine, isoproterenol) were also ineffective.The activation of the adenylate cyclase by histamine was inhibited by the histamine H₁-receptor antagonists diphenhydramine and mepyramine as well as by the histamine H₂-receptor antagonist metiamide. On the other hand, the stimulatory action of NaF or GMP-PNP could be antagonized only by high concentrations of dipenhydramine or mepyramine while metiamide showed no antagonizing effect in this respect. Thus this preparation can be used as a tool to determine the activity and specificity of histamine H₂-receptor antagonists.     

Cardiac adenylate cyclase, cyclic nucleotide phosphodiesterase and lactate dehydrogenase in normotensive and spontaneously hypertensive rats.

To understand altered physiological responses of hypertrophied spontaneously hypertensive rat (SHR) myocardium to beta-adrenergic receptor stimulation in vivo, myocardial tissues from SHR, normotensive Wistar (NR) and normotensive Wistar-Kyoto (WKY) rats were analyzed for adenylate cyclase, phosphodiesterase and lactic dehydrogenase. While WKY left ventricular adenylate cyclase activity exceeded that of SHR at low (1 and 5 μM) norepinephrine concentrations, there were no further differences. Norepinephrine stimulation of NR and SHR left ventricular adenylate cyclase was the same. In all rat strains similar responses of adenylate cyclase to glucagon were observed. Cyclic AMP phosphodiesterase activity of whole left ventricular homogenates were not significantly different in NR, WKY or SHR when assayed at either 1 mM or μM cyclic AMP. While it was not possible to entirely explain previous hemodynamic findings in vivo on the basis of abnormal cAMP-related enzyme activities, certain other interstrain enzymatic differences were observed. The hypertrophied SHR left ventricle contained higher levels of lactate dehydrogenase (LDH) and an altered isozymic composition as compared to both normotensive strains. These changes may indicate a shift in glycolytic enzyme needs as also seen with artificially produced cardiac hypertrophy. In all rat strains the right ventricular wall has less norepinephrine-stimulatable adenylate cyclase activity and more cyclic AMP phosphodiesterase activity than the left ventricle. These results demonstrate differences in cyclic AMP-related activities between the ventricles and increased LDH activity in the hypertrophied SHR left ventricle.     

Effect of carbenoxolone on lipolysis in rat adipose tissue.

Carbenoxolone slightly but significantly decreased the release of FFA from rat epididymal fat pads. The antilipolytic action of carbenoxolone was not blocked by 10(-3)M 3-isobutyl-1-methylxanthine, a potent inhibitor of phosphodiesterase. The findings suggest that carbenoxolone exerts its antilipolytic activity by acting on adenylate cyclase, thereby decreasing cyclic AMP concentrations and the activity of the hormone-sensitive lipase in adipose tissue.     

The effects of alloxanate, nicotinic acid and imidazole on secretory processes and the activities of adenylate cyclase and 3′,5′-AMP phosphodiesterase in cat pancreas

1 Nicotinic acid and alloxanate inhibited water and electrolyte secretion in a dose-dependent fashion when added to the perfusate of the isolated saline-perfused pancreas of the cat stimulated by a supramaximal dose of secretin.

2 There were no changes in the concentration of sodium or potassium secreted into the juice, but the anions exhibited changes which were related to flow rate. As the flow rate declined the chloride concentration increased with a reciprocal decrease in bicarbonate concentration.

3 Nicotinic acid and alloxanate inhibited enzyme secretion stimulated by carbachol.

4 Imidazole inhibited pancreatic electrolyte secretion, but stimulated amylase secretion. Atropine (0.14 μM) reduced the secretion of amylase but did not abolish the effect.

5 Adenylate cyclase prepared from cat pancreas, was stimulated by the octapeptide of cholecystokinin-pancreozymin, secretin and sodium fluoride.

6 Alloxanate strongly inhibited both basal and hormone-stimulated adenylate cyclase activity. Nicotinic acid and imidazole stimulated basal adenylate cyclase activity but had little effect on secretin-stimulated activity.

7 Alloxanate, nicotinic acid and imidazole were all without effect on phosphodiesterase when tested in the presence of micromolar concentrations of adenosine 3′,5′-monophosphate (cyclic AMP). At higher cyclic AMP concentrations (2 mM) alloxanate and nicotinic acid were without effect, whereas imidazole had a slight stimulatory effect at 10 mM which was more marked at 50 mM.

8 Alloxanate (10 mM) strongly inhibited both basal and secretin-stimulated adenylate cyclase activity.

9 It is concluded that the effects of nicotinic acid, alloxanate and imidazole on pancreatic secretion are not mediated entirely through their effects on the adenylate cyclase or phosphodiesterase enzyme systems.

     

Effect of carbenoxolone on lipolysis in rat adipose tissue

Carbenoxolone slightly but significantly decreased the release of FFA from rat epididymal fat pads. The antilipolytic action of carbenoxolone was not blocked by 10^?3m 3-isobutyl-1-methylxanthine, a potent inhibitor of phosphodiesterase. The findings suggest that carbenoxolone exerts its antilipolytic activity by acting on adenylate cyclase, thereby decreasing cyclic AMP concentrations and the activity of the hormone-sensitive lipase in adipose tissue.     

Mechanism of isoprenaline-induced refractoriness of the beta-adrenoceptor--adenylate cyclase system in chick embryo cardiac cells

We examined the role of beta-adrenoceptors in regulating cardiac cell cyclic adenosine monophosphate (cyclic AMP) in concentrations during beta-receptor activation. Chick embryo cardiac cells in primary tissue culture respond to 50 microM isoprenaline with a rapid, but transient, increase in intracellular cyclic AMP. Continued exposure of these cells to this concentration of isoprenaline results in a rapid time-dependent decrease in maximum beta-receptor responsiveness. In vitro determination of adenylate cyclase and 125I-iodohydroxybenzyl pindolol specific beta-receptor binding sites in 35,000 x g particulate cell fractions and phosphodiesterase activity in cell homogenates suggest that the decrease in the cells' ability to raise intracellular cyclic AMP concentration is mainly due to an initial "uncoupling" of the beta-receptor--adenylate cyclase system. During 16 hr exposure of cells to varying isoprenaline concentrations, the magnitude of the cells' ability to increase intracellular cyclic AMP and the concentration of beta-receptor binding sites were inversely related to the isoprenaline concentration to which they had been previously exposed. On removal of isoprenaline, both beta-receptor response and receptor concentration returned slowly towards control levels over the next 24 hr. These results suggest that loss of beta-receptor responsiveness of cardiac cells during prolonged exposure to isoprenaline involves both alterations in receptor number and alterations of the adenylate cyclase system distal to the receptor.