Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Introduction of cyclic AMP phosphodiesterase into rat submandibular acini prevents isoproterenol-stimulated cyclic AMP rise without affecting mucin secretion

Cyclic AMP phosphodiesterase has been incorporated into isolated rat submandibular acini by hypotonic swelling. This resulted in complete inhibition of the cyclic AMP rise stimulated by isoproterenol (10 microM), but had no effect on the stimulation of mucin secretion. Acini swollen in the absence of cyclic AMP phosphodiesterase showed similar cyclic AMP and mucin secretion responses to those of unswollen acini. The dissociation between cyclic AMP rise and mucin secretion was not due to stimulation of different beta-receptor subtypes since both responses to isoproterenol were inhibited by the beta 1 antagonist atenolol, but not by the beta 2 antagonist, butoxamine. The results are the first to directly demonstrate that a maximally effective concentration of isoproterenol can increase mucin secretion in the absence of a detectable increase in cyclic AMP.     

Adenosine 3',5'-cyclic monophosphate stimulates secretion of alpha-melanocyte-stimulating hormone from permeabilized cells of the intermediate lobe of the rat pituitary gland

The effect of Mg-ATP and cyclic AMP on the secretion of alpha-melanocyto-stimulating hormone (alpha-MSH) from electrically permeabilized cells of rat intermediate lobe (IL) were investigated. Addition of exogenous Ca2+ stimulated alpha-MSH secretion in a concentration- (EC50 = 4.8 microM) and temperature-dependent manner. This Ca2+-evoked secretion was further enhanced by Mg-ATP and cyclic AMP. Mg-ATP was required for the fully secretory response in the electrically permeabilized IL cells and the maximal secretion was reached at 1 mM. Cyclic AMP in the presence of GTP gamma S also potentiated Ca2+-evoked alpha-MSH secretion to the same magnitude as Mg-ATP. In the absence of Ca2+ both the cyclic AMP and Mg-ATP did not stimulate alpha-MSH secretion from IL cells. The data suggest that Mg-ATP and cyclic AMP may modulate directly the secretory components rather than change intracellular concentration of free Ca2+.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.     

The effect of 5-hydroxytryptamine and other indole derivatives on the formation of adenosine 3',5'-cyclic monophosphate in pigeon erythrocytes.

1. The effect of insulin, acetylcholine, histamine, 5-hydroxytryptamine and prostaglandins E1, E2 and F2alpha on basal and adrenalin-stimulated cyclic AMP content in intact pigeon erythrocytes was investigated. 2. None of these compounds influenced basal cyclic AMP contest, and only 5-hydroxytryptamine antagonized the effect of adrenalin. The increase in cyclic AMP with 0.55 micronM adrenalin was inhibited by approx. 60% in the presence of 10 muM 5-hydroxytryptamine. The interaction between adrenalin and 5-hydroxytryptamine was competitive. 3. 5-Hydroxytryptamine did not affect the rate of degradation of cyclic AMP in intact cells, but did inhibit adrenalin-stimulated cyclic AMP formation in permeable or resealed cell "ghosts". 4. The effect of 5-hydroxytryptamine to inhibit cyclic AMP accumulation was not dependent on the presence of Ca2+, in either intact cells or "ghosts". 5. Various indole derivatives and other compounds were tested for their ability to inhibit the effect of adrenalin on cyclic AMP accumulation. Only those derivatives with a free amino group and net positive charge in the side chain were effective. 6. It was concluded that 5-hydroxytryptamine inhibits adrenalin-stimulated adenylate cyclase activity in pigeon erythrocytes, possibly by competing with adrenalin for binding to the beta-adrenergic receptor.     

Alpha-melanocyte-stimulating hormone secretion from permeabilized intermediate lobe cells of rat pituitary gland. The role of guanine nucleotides

The non-hydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cyclic AMP potentiated the Ca2+-evoked secretion of alpha-melanocyte-stimulating hormone (alpha-MSH) from permeabilized neurointermediate lobe (IL) cells of rat pituitary gland. The enhancement by Mg-GTP gamma S (100 microM) and cyclic AMP (1 microM) depended on the intracellular Ca2+ concentration (EC50 = 4.8 +/- 1.8 and 4.6 +/- 1.7 microM; mean +/- SE, with and without Mg-GTP gamma S and cyclic AMP, respectively). A similar effect was observed with guanine nucleotide triphosphate (GTP and GppNHp). Mg was absolutely required for this event. Neither Mg-GTP gamma S nor cyclic AMP alone was effective in potentiating alpha-MSH secretion. GDP beta S blocked the Mg-GTP gamma S (100 microM) and cyclic AMP augmented secretion of alpha-MSH. Neither neomycin (which affects the process of inositol 1,4,5-triphosphate-mediated Ca2+ mobilization) or colchicine (which influences microtubule assembly) had an effect on the cyclic AMP and Mg-GTP gamma S potentiation of alpha-MSH secretion. These data suggest that the GTP-binding protein may be involved in the regulation of alpha-MSH secretion after Ca2+ entry into the cells, since the intracellular environment is controlled in the permeabilized cells.     

Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion.

Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli.     

Calcium metabolism and amylase release in rat parotid acinar cells.

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).     

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.     

Isoprenaline-induced changes in rat parotid and submandibular glands are age- and dosage-dependent.

Neonatal rats treated with chronic injections of isoprenaline (isoproterenol) for 10 days revealed differential induction of proline-rich proteins and glycoprotein synthesis between the parotid and submandibular glands. Biosynthesis of proline-rich proteins (Mr 17000-35000) and a Mr-220000 glycoprotein were detectable by solubilization in 10%-trichloroacetic acid extracts from parotid glands 14 days after birth. The enzyme lactose synthase (UDP-galactose: 2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase) (EC 2.4.1.22) is also induced 4-7-fold in specific activity compared with control neonatal rats, but again only after 14 days post partum, with isoprenaline treatment. This is in accord with the ability of the parotid gland to respond to beta-receptor stimulation and subsequent increases in intracellular cyclic AMP necessary for induction of protein synthesis [Grand, Chong & Ryan (1975) Am. J. Physiol. 228, 608-612]. Induction of the proline-rich proteins and a Mr-190000 glycoprotein in the soluble fraction from the submandibular gland were not detected until 49 days after birth under identical conditions in the same animal. Cyclic AMP in the submandibular gland undergoes increases on beta-receptor stimulation similar to those achieved in the adult animal, 1 day after birth (Grand et al., 1975). This same differential induction between parotid and submandibular gland was obtained with a range of isoprenaline dosages in adult animals. Trichloroacetic acid-soluble proline-rich proteins were isolated from parotid glands at a dosage of 4.0 mg of isoprenaline/kg body wt., but 7.0 mg/kg was required to induce also biosynthesis of these proteins in the submandibular gland. Gland hypertrophy showed the same differential dosage kinetics, based on gland weight, between the two glands; however, hypertrophy could be accomplished at a lower dosage of isoprenaline than that used to induce proline-rich-protein biosynthesis.     

Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla

Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation-evoked secretion by 80-90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 micrograms), nicotine (5 microM), muscarine (50 micrograms), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine- and K(+)-evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine-evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetylcholine-evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.     

Involvement of cyclic AMP and calcium in exocrine protein secretion induced by vasoactive intestinal polypeptide in rat parotid cells

The effects of vasoactive intestinal polypeptide (VIP) on exocrine protein secretion were studied in enzymatically dispersed cell aggregates from rat parotid glands. VIP (10(-9) - 10(-7) M) stimulated secretion of alpha-amylase in a dose-dependent manner. The VIP-induced release of alpha-amylase was potentiated in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP of the dispersed cells were increased 6.7-fold after stimulation for 10 min by VIP (10(-7) M). The VIP-induced release of alpha-amylase was reduced by 40% when cells were incubated in a Ca2+-free medium in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Efflux of 45Ca2+ was significantly increased over basal levels by stimulation with VIP (10(-8) and 10(-7) M), but this increased efflux was approximately only half the increased efflux induced by carbachol (10(-5) M). VIP had no effect on the incorporation of [14C]leucine into protein by parotid cells, whereas incorporation was reduced to 30% of the control value by carbachol (10(-5) M). Thus, the VIP-ergic secretory response in the rat parotid gland is associated with a raised intracellular cyclic AMP level and the mobilisation of a different intracellular Ca2+ pool than that mobilised by carbachol. It is, therefore, closely analogous to the beta-adrenergic response.     

Secretion of epidermal growth factor. The role of calcium in stimulcule during secretion.

Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca2+ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of alpha-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth facto; occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.     

Regulation of pyruvate dehydrogenase by Ca2+, 3'5' cyclic AMP and adrenalin in isolated rat kidney tubules.

Renal pyruvate dehydrogenasea activity was measured in suspensions of kidney cortex tubules from fed rats after incubation with various concentrations of Ca2+, 3'5' cyclic AMP or adrenalin. At certain concentrations these agents all decreased pyruvate dehydrogenasea activity and increased glucose formation from pyruvate. There appeared to be some interdependence between the effects of Ca2+ and 3,5, cyclic AMP on pyruvate dehydrogenasea activity. Effects of adrenalin on pyruvate dehydrogenasea activity were no longer observed when palmitate was included in incubations. The possibility that regulation of pyruvate dehydrogenasea may indirectly contribute to the control of gluconeogenesis by influencing pyruvate metabolism is briefly discussed.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).     

An in vitro evaluation of adrenergic action on rat pancreatic amylase secretion: lack of stimulatory effect.

To assess direct evidence of adrenergic stimulation in pancreatic amylase secretion, effects of catecholamines on amylase release and intracellular cyclic AMP accumulation were examined with rat dispersed pancreatic acini. We first carried out control studies with CCK-8 and carbamylcholine to evaluate the usefulness of the material for the examination of amylase secretion, and examined VIP-induced cyclic AMP accumulation to assess the agonist evoked intracellular response. As a result, significant effects of CCK-8, carbamylcholine and VIP were observed, which confirmed that dispersed pancreatic acini used in this study were useful in examining exocrine pancreatic secretion. However, catecholamines failed to stimulate amylase release from pancreatic acini, although a significant increase in intracellular cyclic AMP accumulation was observed. Thus the present study strongly suggests that direct involvement of catecholamine is unlikely in pancreatic amylase secretion, in contrast to results reported previously.     

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.     

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.