Modulation of cyclic AMP in purified rat mast cells. II. Studies on the relationship between intracellular cyclic AMP concentrations and histamine release.

Changes in intracellular and extracellular rat mast cell adenosine 3':5' monophosphate (cAMP) concentrations during stimulation of histamine release by 48/80 were studied. There was a rapid and progressive fall in intracellular cAMP beginning within 10 sec after the addition of 48/80. The lowest cAMP values were obtained at 10 min, with return to control levels by 30 min. The fall in cAMP was dose-related with progressive decreases in 10-min cAMP measurements as the 48/80 concentration was increased from 0.25 to 1.00 mug/ml. There was a graded increase in histamine release over the same concentration range. Attempts to demonstrate significant amounts of cAMP in the medium during 48/80 stimulation were unsuccessful, indicating that the changes in cAMP intracellularly are not due to altered cellular permeability. There was a general correlation between the ability of pharmacologic agents to sustain high intracellular levels of cAMP in the presence of 48/80, and inhibition of histamine release. Theophylline (20 mM) which increased cAMP levels 2- 3-fold prevented a detectable decrease in cAMP after 1 mug/ml 48/80 (measured at 10 min) and almost completely inhibited histamine release. Prostaglandin E1 (27 muM) also raised cAMP levels, decreased the 48/80-induced fall in cAMP (by 42%). Epinephrine increased mast cell cAMP levels, but did not prevent the subsequent 48/80-induced decrease in cAMP and did not inhibit histamine release. Carbamylcholine (1 nM), adenine (1 muM), and diazoxide (10 muM) lowered mast cell cAMP and potentiated 48/80 induced release. In view of previous studies from this laboratory indicating that 48/80 stimulates mast cell phosphodiesterase, it seems likely that the 48/80-induced fall in cAMP is due, at least in part, to increased cAMP destruction. Since agents which prevent the fall in cAMP inhibit histamine release, it is apparent that cAMP is an important part of the control mechanism of histamine secretion. On the other hand, it cannot be concluded that a decrease in cAMP alone is sufficient to produce a response since carbamylcholine, diazoxide, and adenine which lower cAMP do not alter histamine release unless 48/80 is also present.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.     

beta-adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of beta-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked beta-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of beta-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1724 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro greater than IBMX greater than theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low Km cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

Modulation of cyclic AMP in purified rat mast cells. III. Studies on the effects of concanavalin A and anti-IgE on cyclic AMP concentrations during histamine release.

Changes in rat mast cell cyclic adenosine 3',5' monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM alpha-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.     

β-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes

Effects of β-adrenoceptor antagonists propranolol and alprenolol in the oocyte maturation of the catfish (Clarias batrachus) were investigated under in vitro. Cyclic AMP (cAMP) levels were also measured in the control, propranolol and phosphodiesterase (PDE) inhibitor treated oocytes. When full-grown folliculated oocytes were cultured in vitro in the presence of different concentrations of propranolol or alprenolol, both the substances induced germinal vesicle breakdown (GVBD) in a dose-dependent manner. The maturational effect of alprenolol at the concentration of 1.0 mM was similar to that of the 1.5 mM dose of propranolol inducing more than 88% GVBD. In the time course study, when the oocytes were treated with 1.5 mM propranolol or with 1.0 mM alprenolol for various times, both the antagonists induced more than 80% GVBD after 4 h of incubations and this induction gradually increased with the increased duration of treatments. On the other hand, 1.5 mM propranolol treatment caused a significant decrease in oocyte cAMP which was maintained upto the duration of the study (36 h). When the oocytes were first stimulated by 1.5 mM propranolol or 1.0 mM alprenolol for 4 h and then treated with various doses of cAMP or PDE inhibitors (IBMX and theophylline), all these substances effectively blocked β-adrenoceptor antagonist-induced GVBD. Both these PDE inhibitors promoted the accumulation of cAMP in the oocytes. These results provide the first example of an existence of a cAMP-mediated mechanism of action of β-adrenoceptor antagonists in the induction of oocyte maturation in fish.     

Reduction of adenine nucleotide content of clone 4 MDCK cells: effects on multiplication, protein synthesis, and morphology

The antitumor agent hadacidin (N-formyl-hydroxyamino-acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug-treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16-fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H-leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.     

Antiviral activity of lambda-, beta-, and gamma-interferons in the presence of theophylline: involvement of 2',5'-oligo(A) synthetase

Theophylline, an inhibitor of cAMP phosphodiesterase, induces in human ovary carcinoma cells (CaOv) a 2-2.5-fold elevation of intracellular cAMP. This rise in the cAMP level is followed by an increase of the activity of 2',5'-oligo(A) synthetase in CaOv cells -insignificant (1.5-fold) after 16 hr incubation, and substantial (3.7-fold) after 30 hr incubation, as well as the development of antiviral resistance. Once CaOv cells have been incubated with the mixtures containing theophylline (2 mM) and lambda-, beta-, and gamma-interferon preparations (0.5-13 IU/ml), the total antiviral effect of the mixtures exceeds that generated by interferon or theophylline separately; the action of the above agents being additive. These data agree with the previously obtained results and support the suggestion that cAMP phosphodiesterase inhibitors partially mimic the antiviral action of interferon.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1274 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro > IBMX > theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low K_m cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness.

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.     

Cyclic AMP, cyclic GMP and glucose utilization by diabetic rat fat cells

Fat cells from epididymal adipose tissue from normal and streptozotocin-diabetic rats were studied to determine glucose utilization and cyclic nucleotide levels. Diabetic rat fat cells present a higher cAMP content (P less than 0.05) compared with controls. Addition of insulin decreases within 10-min incubation the cAMP content in both normal and diabetic cells (P less than 0.05). However, the value obtained in the latter remains by 25% higher than that of normal cells not exposed to insulin. No changes in cGMP were detected. Pretreatment of the diabetic animals during two days with propranolol (1 mg kg body wt-1 day-1) induces the decrease to normal levels of the fat cell cAMP content. However, it persists the impairment on glucose utilization observed in fat cells from diabetic animals. It seems that the increase in the intracellular amount of cAMP found in fat cells from diabetic rats is not involved, at least directly, to the impaired glucose utilization found in the diabetic state. Furthermore, through an unknown mechanism, pretreatment with propranolol can induce a drop in fat tissue cAMP toward normal values without normalizing glucose utilization.     

Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

Cyclic AMP-mediated control of oocyte maturation in the catfish, Clarias batrachus (Bloch): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phosphodiesterase inhibitors

An increase in the percentage of germinal vesicle breakdown (GVBD) with a corresponding decrease in cAMP was found in the oocytes which were incubated for 36 hr with different concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP). At its highest concentration (1 microgram/ml), 17 alpha,20 beta-DP induced 91.9 +/- 2.3% GVBD and decreased cAMP level to 0.8 +/- 0.1 pmol/oocyte from 2.9 +/- 0.2 pmol/oocyte (control). The two different known inhibitors of phosphodiesterase viz. 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline inhibited GVBD in vitro and promoted the accumulation of cAMP in a dose-dependent manner irrespective of whether the oocytes were treated for a short duration (2 hr) or for a long duration (36 hr). Evaluation of time course response to 1 mM IBMX or 1 mM theophylline revealed that cAMP levels increased at all the time points when compared with their respective controls and blocked maturation. In contrast, 1 microgram/ml 17 alpha,20 beta-DP not only induced oocyte maturation but also caused an immediate decrease in cAMP within the first 2 hr (from 3.2 +/- 1.3 to 1.3 +/- 0.1 pmol/oocyte) of incubation which was maintained till the end of experiment (36 hr). Likewise, a significant inhibition of GVBD and accumulation of cAMP was recorded even in oocytes pre-stimulated with 1 microgram/ml 17 alpha,20 beta-DP for 6 hr and then treated with different concentrations of IBMX or theophylline. Taken together, these data strongly suggest that in C. batrachus a decrease of oocyte cAMP concentration is a prerequisite for the induction of oocyte maturation, and its increase is associated with the maintenance of meiotic arrest.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.     

Target-effector interaction in the natural killer cell system. IV. Modulation by cyclic nucleotides.

Dibutyryl cAMP (dB-cAMP) and the cAMP elevating agents, prostaglandin E1, theophylline, and histamine markedly suppressed NK cytolytic function in a dose- and rate-dependent manner. The inhibition was rapidly induced and persisted in the presence of the drugs. Separate pretreatment of targets and highly purified NK cells, isolated by a target binding and velocity sedimentation technique, revealed that PGE1 and dB-cAMP acted at the level of the effector cell in a short-term cytolytic assay. In contrast to the inhibitory effects of cAMP elevating agents, dB-cGMP and carbamylcholine caused a small but significant acceleration in the rate of lysis and could compete with inhibitory doses of dB-cAMP to reduce the level of suppression thereby suggesting that the cAMP-cGMP ratio might be important in NK-mediated lysis. Insulin had no effect on NK activity, whereas T cell-mediated cytolysis was augmented by insulin and cGMP if the effector cells were taken early after alloimmunization but not later. Neither cAMP- nor cGMP-elevating agents affected the frequency of NK-target cell conjugates. These results are compatible with the hypothesis that cyclic nucleotides may be involved in triggering the lytic event within NK cells.