Fred R. Butcher

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 with 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 alpha-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 effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-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 intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-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 alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,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 alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.

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 eledoisin, caused a rapid, concentration-dependent increase in K+ efflux and amylase release from parotid tissue slices. The effects were not blocked by beta-adrenergic, alpha-adrenergic, or cholinergic antagonists. Incubation buffer calcium was required for stimulation of K efflux and amylase release. The action of the undecapepides 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.

The role of calcium and cyclic nucleotides in α-amylase release from slices of rat parotid: Studies with the divalent cation ionophore A-23187

The divalent cation ionophore A-23187 caused a Ca-2+-dependent increase in alpha-amylase release from slices of rat parotid gland. The effect of A-23187 on alpha-amylase release was not caused by release of endogenous agonists since l-propranolol, phentolamine, and atropine had no effect. The magnitude of alpha-amylase release caused by A-23187 was small compared to the effect of isoproterenol. In this respect it more closely resembles the action of cholinergic and alpha-adrenergic agonists on alpha-amylase release. A-23187 inhibited the increase in the level of parotid adenosine 3,5-monophosphate caused by isoprotrenol. The inhibitory effect required incubation of the slices with the ionophore before the addition of isoproterenol. The ionophore also caused a Ca-2+-dependent increase in the level of guanosine 3,5-monophosphate (cyclic GMP). Theophylline enhanced the effect of A-23187 on the level of cyclic GMP. These results emphasize the role of Ca-2+ in the regulation of parotid cyclic nucleotide levels. Since the effects of the ionophore depended on the presence of Ca-2+, it is possible that some of the effects of agonists on parotid gland physiology are secondary to an action on intracellular Ca-2+ distribution.

Effect of cytochalasin B and colchicine on the stimulation of α-amylase release from rat parotid tissue slices

A role for microfilaments and microtubules in the secretion of α-amylase is indicated since cytochalasin B and colchicine inhibited the stimulation of α-amylase release by epinephrine (30 or 15 μM) but only cytochalasin B inhibited the stimulation by N6, O2′ dibutyryl adenosine 3′,5′monophosphate (1.0 mM). It was necessary to incubate the parotid tissue slices in the presence of cytochalasin B (1 hr.) or colchicine (4 hrs.) before adding the agonist in order to see the inhibitory effects.

Identification and characterization of beta1-adrenergic receptors in rat parotid membranes

Abstract • Beta-adrenergic receptor identification and properties are probed in rat parotid membranes utilizing the high affinity β-adrenergic antagonist(−)-[ 3 H]dihydroalprenolol. • The binding of (−)-[ 3 H]dihydroalprenolol to membrane preparations of parotid is rapid, equilibrium being reached in 5 min. Strict stereospecificity is observed, (−)-propanolol being 100 times more potent than (+)-propranolol in competing with (−)-[ 3 H]dihydroalprenolol for binding sites. Beta-adrenergic agonists compete for the binding sites with (−)-[ 3 H]dihydroalprenolol with the same characteristics, i.e., much higher concentrations of the (+)-stereoisomers than the (−)-stereoisomers are required to produce 50% inhibition, the range varies from 14-fold for epinephrine to 300-fold for isoproterenol. Among the (−)-stereoisomers, the relative potency of inhibitory action is (−)-propranolol > (−)-isoproterenol > (−)-epinephrine ≡ (−)-norepinephrine. (−)-Isoproterenol is about 20 times as potent as norepinephrine, the least potent agonist among all the catecholamine (−)-stereoisomers. • The binding of (−)-[ 3 H]dihydroalprenolol is saturable, with a maximum number of binding sites equalling 450 fmol/mg protein and a dissociation constant of 7.9 nM. The Scatchard plots show no significant curvilinear character. Hill plots consistently give a Hill coefficient close to unity (0.92–1.05). Both pieces of evidence suggest a single-component system with no significant cooperativity. • Dissociation kinetics study after the method of De Metys et al. (1973) Biochem. Biophys. Res. Commun. 155, 154, indicates a lack of site-to-site interactions among the binding sites. The rate of dissociation of bound (−)-[ 3 H]dihydroalprenolol is the same in the presence and absence of 1 · 10 −5 M (±)-alprenolol. • Based on the binding of (−)-[ 3 H]dihydroalprenolol, it is concluded that the beta-adrenergic receptors can be identified in rat parotid and that these binding sites display β 1 character. Results of the study indicate a one-component system with no observable site-to-site interactions.

Effects of isoproterenol, theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle.

Abstract The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10 −6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol. The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.

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.

Abstract 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.

Cholinergic regulation of cyclic nucleotide levels, amylase release, and K+ efflux from rat parotid glands.

Carbachol increased amylase release and K+ efflux from rat parotid tissue slices. The amount of amylase released was small compared to that released by isoproterenol. The effect of carbachol on amylase release and K+ efflux was a direct effect. This conclusion was based on the finding that the stimulatory effects of carbachol were blocked only by atropine and not by propanolol or phentolamine. In addition to the above effects, carbachol also caused a rapid increase in the parotid guanosine-3, 5 cyclic monophosphate (cGMP) levels without a discernable effect on adenosine-3,5 cyclic monophosphate (cAMP) levels. The increase in cGMP level caused by carbachol was blocked by atropine and not by phentolamine. The stimulatory effect of carbachol on amylase release was not additive with that of isoproterenol or dibutyryl cAMP. Although carbachol had no effect on basal cAMP levels it did inhibit increases in cAMP caused by isoproterenol. Similarly isoproterenol inhibited increased in parotid cGMP levels caused by carbachol. Unlike the apparent nonadditivity between the effects of isoproterenol and carbachol on amylase release and cAMP and cGMP accumulation, the effects on K+ efflux were additive. The possibility of a role for cGMP in mediating the effects of cholinergic agonists on K+ efflux was lessened by our observations that 1-methyl-3-isobutylxanthine enhanced the effect of limiting concentrations of carbachol on cGMP accumulation while not enhancing the effects of carbachol on K+ efflux.

Cyclic nucleotide levels in regenerating liver

Abstract Cyclic AMP and cyclic GMP levels were measured in rat liver at various times following partial hepatectomy. Cyclic AMP levels show an initial rapid dropfollowed by a biphasic increase while cyclic GMP remains at an almost constant level throughout the time period examined.

Cyclic nucleotides and gluconeogenesis by rat liver cells.

Gluconeogenesis from lactate, pyruvate, fructose, alanine, and other substrates was accelerated by glucagon or epinephrine in hepatocytes isolated from rat liver. Glucagon and epinephrine also increased cyclic AMP accumulation by rat hepatocytes. Isoproterenol increased cyclic AMP but not gluconeogenesis, while phenylephrine accelerated gluconeogenesis. The activation of gluconeogenesis by epinephrine was unaffected by propranolol but blocked by dihydroergotamine. Dibutyryl cyclic AMP added to hepatocytes stimulated gluconeogenesis at concentrations as low as 1 muM. Exogenous cyclic GMP (0.1- muM) inhibited gluconeogenesis due to either glucagon or epinephrine without affecting basal gluconeogenesis. However, carbamylcholine did not affect gluconeogenesis by hepatocytes. Basal gluconeogenesis and the increases due to all agents were inhibited by removal of extracellular calcium or the presence of A-23187, D-600, or tetracaine. In contrast, added 0.1 muM cyclic GMP, 2 mM NH-4-Cl, and 10 muM phenethylbiguanide inhibited glucagon- or epinephrine-stimulated gluconeogenesis without affecting basal values. Studies with hepatocytes indicate that the hormonal activation of gluconeogenesis is not limited to substrates entering prior to triose phosphate formation. Glucagon may act by increasing cyclic AMP which acts via unknown mechanisms to increase gluconeogenesis. In contrast, epinephrine acts via a cyclic AMP-independent mechamism which does not appear to involve cyclic GMP, Ca-2+ flux, of K+ flux.