Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

Does cyclic GMP mediate amylase release from mouse parotid acini?

In mouse parotid acini both cholinergic and beta-adrenergic agonists increased intracellular levels of cyclic-GMP (c-GMP) as well as amylase release. The derivative of c-GMP, 8-bromo-c-GMP, mimicked the effects of cholinergic and beta-adrenergic stimulation on amylase release. Nitroprusside (NP), hydroxylamine (HA) and sodium azide (NaA) increased c-GMP levels and also enhanced amylase release in a dose-dependent manner; cyclic-AMP (c-AMP) levels were not affected. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (MIX) enhanced the effects of carbachol on both c-GMP accumulation and amylase release. These results suggest that c-GMP may mediate the actions of cholinergic agonists and at least partially mediate the actions of beta-adrenergic agonists on mouse parotid enzyme release.     

Observations on the level of cyclic nucleotides in three population of human lymphocytes in culture

The level of cyclic nucleotides in three populations of cultured human lymphocytes were studied. An early conspicuous elevation of c-GMP level and a reciprocal relationship between c-AMP and c-GMP fluctuations were demonstrated in T cells from normal donors. Null cells from patients with ALL showed a constantly low level of c-AMP, while c-GMP fluctuated in apparent relationship with cell doubling time. Persistently low levels of c-AMP and persistently high level of c-GMP were found in B cells from patients with CLL. Possible significance of these findings is discussed.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

The effects of nitroglycerin on cyclic nucleotides in the coronary artery in vivo

We examined the effects of nitroglycerin (NGL) on cyclic AMP (c-AMP) and cyclic GMP (c-GMP) in the coronary artery at 15 sec, 30 sec, 60 sec, and 3 min after the injection of NGL (0.02 mg/kg i.v.) in vivo. The relaxant effect of NGL was significantly correlated to an increase in the c-GMP concentration of the coronary artery. The c-AMP concentrations were not significantly changed at any time during the time response studies. We observed purely in vivo that there was a close correlation between an increase in c-GMP concentration after treatment with NGL and relaxation of the canine coronary artery. This study suggests that intracellular c-GMP may be involved with the biologic events leading to smooth muscle relaxation.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Steroidogenesis in isolated adrenal cells of rat. II. Effect of caffeine on ACTH and cyclic nucleotide-induced steroidogenesis and its relation to cyclic nucleotide phosphodiesterase (PDE)

Corticosterone production in isolated adrenal cells (IAC) of rat has been measured in response to ACTH or ribonucleoside 3′,5′-cyclic phosphate of adenosine (c-AMP), guanosine (c-GMP), inosine (c-IMP) and N⁶-2′-0 dibutyryl adenosine monophosphate (dc-AMP) in the presence and absence of caffeine. Caffeine inhibited ACTH-induced steroidogenesis in a manner independent of its effect on PDE. Study of PDE in whole adrenal homogenate showed hydrolysis of c-AMP, c-GMP and c-IMP but not of dc-AMP and other cyclic nucleotides. No PDE activity was demonstrable in IAC. High sensitivity of IAC to minute quantities of ACTH and various cyclic nucleotides may be due in part to lack of PDE activity in these preparations.     

Regulation by beta-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic GMP levels in rat lung slices

The effects of adrenergic and cholinergic agents, present singly or in combination, on the levels of cyclic AMP and cyclic GMP in slices of rat lung were studied. It was found that isoproterenol increased pulmonary cyclic AMP levels about 3-fold, and this increase was abolished by propranolol, but not by phenoxybenzamine. Acetylcholine increased the cyclic GMP levels also about 3-fold (thus raising its tissue content above that of cyclic AMP), and this increment was largely reduced by atropine, but not by hexamethonium. While without effects on the cyclic GMP levels when present alone, isoproterenol antagonized acetylcholine in increasing cyclic GMP levels. Acetylcholine, while lacking effects on the basal levels of cyclic AMP, on the other hand, depressed the augmented levels caused by isoproterenol.The data presented indicate that cyclic GMP may mediate the cholinergic action in lung and that the pulmonary cyclic GMP levels are also closely regulated by β-adrenergic receptor activation.     

Cyclic AMP and cyclic GMP in hyperresponsiveness

Cyclic-AMP has been shown to cause a hyperresponse in blood pressure change in conjunction with norepinephrine in the anesthetized rat system. Recent experiments show that the antagonist to angiotensin II, Sar1-Thr8 angiotensin II, abolishes the hyperresponse produced by c-AMP. This is interpreted to mean that the added response caused by c-AMP is mediated through angiotensin II. When the antagonist is removed, the hyperresponse is observed to return. The experiments with cyclic-GMP indicate that the hyperresponse seen with c-AMP is not only absent, but a constant decrease in response to norepinephrine is observed as long as c-GMP is present. The decrease in blood pressure change in the presence of c-GMP suggests that the 10-5M c-GMP causes a relaxation of vascular smooth muscle. These two cyclic nucleotides seem to produce their effects by two completely different mechanisms.     

An examination of baseline and drug-induced levels of cyclic nucleotides in the cerebrospinal fluid of control and psychiatric patients

Baseline c-AMP levels, and probenecid-induced accumulations of c-AMP and c-GMP in the lumbar CSF of depressed, manic, and schizophrenic patients failed to show differences when compared to controls screened to exclude affective, schizophrenic, and CNS neurological disorders. Cyclic-GMP baseline levels in all three psychiatric groups did approach a level of significance when compared to controls. The administration of psychotropic drugs to these patients failed to show significant differences in paired comparisons. Levels of c-GMP from lumbar CSF were found to be positively correlated to concentrations of c-AMP. Categorization of data according to sex, and then age did not result in a discernible trend. It seems unlikely on the basis of this investigation that measurement of cyclic nucleotides in lumbar CSF will be of significance in differentiating psychiatric disorders.     

Effect of substance P and eledoisin on K efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland

The undecapeptides, substance P and eledosin, caused a rapid, concentration-dependent increase in K⁺ efflux and amylase release from parotid tissue slices. The effects were not blocked by β-adrenergic, α-adrenergic, or cholinergic anatagonists. Incubation buffer calcium was required for stimulation of K⁺ efflux and amylase release. The action of the undecapeptides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca²⁺ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca²⁺ plays a primary role in agonist regulation of K⁺ efflux from the parotid.     

Effects of a daily temperature cycle on ecdysteroid and cyclic nucleotide titres in adult female crickets, Gryllus bimaculatus

ABSTRACT. Ecdysteroid and cyclic nucleotide titres were determined in ovaries, fat body, muscles, haemolymph and the remaining carcass tissue (cyclic nucleotides only in ovaries and fat body) of females of the Mediterranean field cricket, Gryllus bimaculatus de Geer, during its adult life span. Under a daily temperature cycle 24°: 12°C (16:8h), ecdysteroid levels of the ovaries and fat body reached maximal values 5 times as great and about 10 days earlier than they did under constant 20°C. Under both temperature regimes the highest ecdysone concentrations coincided with the maximum in ovarian fresh weight as well as with the maximum oviposition rate. In the ovaries, titres of c-AMP and c-GMP changed roughly in parallel, the levels of c-GMP, however, were much lower than those of c-AMP. A comparison of the cyclic nucleotide profiles in the ovaries with the ecdysteroid profile shows that the cyclic nucleotide concentrations increase when ecdysteroid titres are still low, and that the highest cyclic nucleotide levels were reached 6–12 days earlier than the highest ecdysteroid titres.     

Selective inhibition of cardiac cyclic nucleotide phosphodiesterases by doxorubicin and daunorubicin

Doxorubicin and daunorubicin, the anthracycline antitumor agents, were evaluated for their in vitro and in vivo effect on phosphodiesterase (PDE) activity in mouse tissues. Doxorubicin at a concentration of 10(-4)M inhibited cardiac c-AMP (adenosine 3',5', monophosphate) PDE activity 50% of the control whereas in lungs and spleen, the activity was inhibited only 20%. On the contrary no effect was seen in kidney and liver. In addition, cardiac c-GMP (guanosine 3',5' monophosphate) PDE appeared less sensitive to doxorubicin than c-AMP PDE though inhibition in heart was more pronounced than in any other tissue. It appears that daunorubicin inhibits c-AMP PDE activity in heart markedly less than doxorubicin. Kinetic studies indicate that both inhibitions of c-AMP and c-GMP PDE by doxorubicin were non-competitive with substrate. Intravenous administration of 20 and 30 mg/kg of free doxorubicin to CDF1 mice resulted in 33 and 39% decreases in cardiac c-AMP PDE activity respectively by 72 hrs. In contrast, similar intravenous injections of same doses of doxorubicin entrapped in cardiolipin liposomes had no effect on c-AMP PDE activity in any tissues. These studies demonstrate the relative selectivity of the cardiac cyclic nucleotide PDE inhibitory effect of doxorubicin suggesting that this inhibition might be one aspect of the mechanism of anthracycline-induced cardiotoxicity.     

Catecholamine inhibition of rat mast cell histamine secretion - a process independent of cyclic AMP levels and counteracted by glucose

The inhibitory effects of the catecholamines norepinephrine, epinephrine and the β-adrenergic agonist isoproterenol were studied and found to be independent of cellular cyclic AMP levels. Glucose counteracted the inhibitory effects observed at high concentrations of the agents employed whereas 2-deoxyglucose abolished the glucose effect.     

A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells

An intraperitoneal injection of the β-adrenergic agonist dl-isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400-fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two-fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA-synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl-propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65-75%) reduced the incorporation of [³H]-thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP-dependent pre-DNA-synthetic step because it did not reduce [³H]-thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β-adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.     

The effect of isoproterenol and its analogs upon adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate levels in mouse parotid gland in vivo: Relationship to the stimulation of DNA synthesis

The ability of a large number of catecholamine analogs to stimulate DNA synthesis in the mouse parotid gland in vivo was compared to their effect on the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) in this tissue. In the normal parotid gland the level of cyclic GMP is very low (10^?9 moles/kg wet wt), being only 1/800th of the cyclic AMP concentration. Isoproterenol increases the levels of cyclic AMP and cyclic GMP 30- and 3-fold, respectively. The increase in cyclic AMP is biphasic with an apparent early maximum at 2.5 min and a main peak at 15 min while the increase in cyclic GMP is monophasic with maximum levels at 15 min. Other analogs showed a similar effect on cyclic AMP levels but the time course of increases in cyclic GMP was very variable with peak stimulation as early as 1 min in some cases. The ability of analogs to cause the accumulation of cyclic AMP was correlated with their capacity to activate adenylate cyclase in parotid extracts and to act as β-adrenergic agonists in other systems. All compounds which raised cyclic AMP levels stimulated DNA synthesis but a number of other analogs also stimulated DNA synthesis. The effects of these analogs have been correlated with their ability to raise the intracellular concentration of cyclic GMP. Cholinergic agents also cause the accumulation of cyclic GMP but the effect of the analogs does not appear to be mediated through the cholinergic system as atropine does not block their effects and cholinergic agonists do not stimulate DNA synthesis. It is suggested that cholinergic agonists and the catecholamine analogs act primarily on the duct and acinar cells, respectively.Significant with inhibitors of the rises in cyclic nucleotide levels suggest that in isoproterenol stimulation it is the rise in cyclic GMP which is the more significant event in relation to stimulation of DNA synthesis.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Alterations in activities of cyclic nucleotide systems and in β-adrenergic receptor-mediated activation of cyclic amp-dependent protein kinase during progression and regression of isoproterenol-induced cardiac hypertrophy

Initial and transient increases in the basal levels of cyclic GMP in the heart were noted prior to cardiac hypertrophy in rats administered isoprotenol. Increased levels of cyclic AMP-phosphodiesterase (in both the soluble and particulate fractions) and stimulatory modulator of cyclic GMP-dependent protein kinase, however, were associated with the progression, or the state, of cardiomegaly, with their levels returning to the control values upon regression of the hypertrophy. The levels of cyclic GMP phosphodiesterase in the soluble fraction were lower, whereas those in the particulate fraction were higher, in the hypertrophied heart than the control. In cardiac hypertrophy, the maximal activity ratio (?cyclic AMP/+cyclic AMP) of cyclic AMP-dependent protein kinase in the incubated minced heart caused by isoproterenol was lower, whereas the concentration of isoproterenol required to increase the activit ratio half-maximally was higher than controls; The reduced responsiveness to the drug, however, was reversed when the hypertrophy regressed. These observations, taken collectively, appear to suggest that the desensitization of the β-adrenergic mechanism seen in the cardiac hypertrophy produced by repeated administration of isoproterenol is associated with adaptive modifications in certain parameters of the cyclic nucleotide systems.     

ADRENERGIC α- AND β-RECEPTORS EXPRESSED BY THE SAME CELL TYPE IN PRIMARY CULTURE OF PERINATAL MOUSE BRAIN

Abstract— The β-adrenergic agonist, isoproterenol and the α- and β-adrenergic agonist. NA. raise the intracellular concentration of cyclic AMP in cultures of dissociated perinatal mouse brain. This rise is prevented by a β- but not by an α-adrenergic antagonist. The maximal level of cyclic AMP reached in the presence of isoproterenol is markedly higher than that found after exposure to NA. However, if NA is used along with an α-adrenergic antagonist, cyclic AMP levels as high as those after isoproterenol are measured. Agonists with α-adrenergic activity including NA decrease the response to isoproterenol. The decrease is blocked by α-adrenergic antagonists. From this and additional evidence it is concluded: (1) The increase in the level of cyclic AMP caused by β-adrenergic agonists is due to β-receptor-mediated stimulation of adenylate cyclase; (2) the inhibition of this effect by α-adrenergic agonists is mediated by adrenergic α-receptors; (3) the α- and β-adrenergic receptors are likely to be located on the same cells, probably the most abundant putative glial precursor cells. The simultaneous stimulation of α- and β-adrenergic receptors on the same cell may be of significance in the regulation of the response to NA.