The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.     

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Cyclic AMP metabolism in mouse parotid glands properties of adenylate cyclase,protein kinase and phosphodiesterase

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands.Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity with a pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a K_a of 1.2·10^?7 M. Parotid cyclic AMP and cyclic GMP phosphoriesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least six peaks of enzyme activity in the pI range of 4–6.Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Independent regulation of beta-adrenergic receptor and nucleotide binding proteins of adenylate cyclase. Developmental and denervation-dependent responses in rat parotid

Rat parotid gland secretion cannot be activated through beta-adrenergic stimulation of adenylate cyclase until after 2 weeks postnatal. We have studied the relationship of the levels of putative guanine nucleotide-binding regulatory components (G/F) of the cyclase system to the onset of hormone responsiveness. The effect of sympathetic denervation on the components of this system during development of secretory function also has been examined. Nucleotide-dependent, hormone-stimulated, and fluoride-stimulated adenylate cyclase activities in parotid membranes are present at low levels at birth and increase 2-fold between 14 and 18 days postnatal while beta-adrenergic receptor levels remain constant. G/F proteins, regulatory for adenylate cyclase activation, were quantitated by ADP-ribosylation in the presence of cholera toxin. Labeling of two cholera toxin-specific substrates occurs at low levels in neonatal rats and increases sharply at the critical 14-18-day period. This provides a plausible explanation for the increase in adenylate cyclase sensitivity at this time, although increases in cyclase catalytic units and/or coupling efficiency of receptor and cyclase may also be involved. In previous studies we found that animals chemically sympathectomized with 6-hydroxydopamine at birth developed elevated levels of membrane-bound beta-adrenergic receptors. The functional consequence is that treated animals show a shift (1.7-fold) toward increased sensitivity in the dose-response curve for adenylate cyclase activation by isoproterenol. However, the levels of maximal hormone- and fluoride-stimulated adenylate cyclase activities do not change, suggesting that some component distal to the receptor is limiting under both control and treated conditions, or that there are deficiencies in coupling of the receptor pool.     

Forskolin activation of adenylate cyclase in mouse parotid membranes

In mouse parotid membranes forskolin activated adenylate cyclase four-fold; maximal activation of the enzyme occurred with 10 microM forskolin. Activation was not dependent on the guanyl nucleotide GTP nor on the inhibitory guanine nucleotide 5'-0-(2-Thiodiphosphate), GDP beta S. In contrast, stimulation of adenylate cyclase by isoproterenol required GTP and was antagonized by GDP beta S in a dose-dependent manner. These results indicate that the guanyl-binding protein of mouse parotid adenylate cyclase is not a requisite for forskolin activation and lends support for direct interaction of forskolin at the catalytic subunit.     

Chymotrypsin selectively decreases forskolin stimulation of adenylate cyclase

We studied the effects of chymotrypsin on turkey erythrocyte membrane adenylate cyclase activity. Proteolysis with chymotrypsin led to a concentration- and time-dependent increase in activation of adenylate cyclase by isoproterenol + guanine nucleotides, and fluoride, and to a decrease in activation by forskolin. Maximal effects (up to 10-fold increases in fluoride- and isoproterenol + guanine nucleotide-stimulated activity, and up to 100% inhibition of forskolin-stimulated activity) occurred under similar conditions (10-20 micrograms/ml chymotrypsin for 10-15 min at 30 degrees C). Augmentation of isoproterenol + guanosine-3'-O-thiotriphosphate (GTP-gamma-S)-stimulated activity by chymotrypsin occurred only if proteolysis preceded stimulation with isoproterenol + GTP-gamma-S. Addition of isoproterenol + GTP-gamma-S to membranes before proteolysis, however, did not prevent chymotrypsin from augmenting subsequent stimulation by these agents. In contrast, addition of forskolin during proteolysis with chymotrypsin prevented the time- and concentration-dependent decline in forskolin stimulation observed with chymotrypsin. Proteolysis decreased the magnitude of stimulation at any concentration of forskolin, but did not alter the concentration dependence of forskolin stimulation (apparent half-maximum = 3 microM). The data are consistent with the existence of a chymotrypsin-sensitive site essential for forskolin stimulation of adenylate cyclase. In view of the simultaneous effect of chymotrypsin to augment fluoride- and isoproterenol + guanine nucleotide-stimulated activities, it is highly unlikely that the site is on the stimulatory guanine nucleotide binding protein. Since forskolin is thought to act directly on the catalytic unit of adenylate cyclase, and since forskolin can protect against the effect of proteolysis with chymotrypsin, the site involved may be on the catalytic unit itself.     

Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and beta-adrenergic receptors in spontaneously hypertensive rats

[3H]Dihydroalprenolol binding and adenylate cyclase activity in the myocardial membranes of Kyoto Wistar normotensive rats and spontaneously hypertensive rats were compared at various stages of postnatal development ranging from 2 to 36 weeks. Basal as well as agonist-stimulated myocardial adenylate cyclase activity was consistently decreased in spontaneously hypertensive rats as compared to normotensive rats as early as 2 weeks of age with significant differences (P < 0.05) observed after 6 weeks of age. When results were expressed as percent stimulation over the basal activity, only isoproterenol plus GTP-stimulated enzyme activity was reduced by 25--30% in spontaneously hypertensive rats, suggesting a specific loss of stimulation by isoproterenol in hypertensive animals. The number of [3H]dihydroalprenolol binding sites of KD for dihydroalprenolol binding were comparable between spontaneously hypertensive and normotensive rats at 3, 6 and 12 weeks of age. The competition of isoproterenol with [3H]dihydroalprenolol for the specific binding sites showed that the affinity of isoproterenol binding was decreased 3--4-fold in spontaneously hypertensive compared with normotensive rats. With postnatal development in age, basal as well as agonist-stimulated activities decreased progressively in both spontaneously hypertensive and normotensive rats. Similarly, the number of [3H]dihydroalprenolol binding sites decreased with the development in age, whereas affinity of dihydroalprenolol binding increased up to 12 weeks of age. These results therefore suggest that adenylate cyclase activity and the number of beta-adrenergic receptors in rat heart, decrease with age and that in hypertension, specific decrease in isoproterenol stimulation of cyclase appears at all stages of development.     

Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.

beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.     

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.     

Heterologous desensitization of pigeon erythrocyte adenylate cyclase by the catalytic subunit of cAMP-dependent protein kinase

Preincubation of pigeon erythrocyte plasma membranes with the catalytic subunit of cAMP-dependent protein kinase results in the desensitization of erythrocyte adenylate cyclase. The adenylate cyclase activity measured in the presence of 10 microM isoproterenol and 50 microM GTP-gamma-S decreases by 40% after 10 min incubation; that in the presence of 50 microM GTP-gamma-S by 35% (20 min). The decrease of the adenylate cyclase activity is due to the prolongation of the lag phase of the enzyme activation in the presence of a hydrolysis-resistant GTP analog and to the drop in activity in the steady state of the activation. The heterologous desensitization of adenylate cyclase induced by cAMP-dependent protein kinase is also coupled with the decrease of the number of beta-adrenoreceptors capable of acquiring a high affinity for the agonists in the absence of guanyl nucleotides. The effect of the catalytic subunit on adenylate cyclase is fully compatible with the process of the enzyme desensitization in erythrocytes treated with isoproterenol or cAMP.     

Biological activity of agarose-immobilized catecholamines.

Catecholamines substituted to agarose were synthesized in various ways. Norepinephrine and isoproterenol were linked to p-aminobenzamidohexyl agarose by an azo linkage to the catechol ring. Norepinephrine was also couple to hexyl agaros via the amino group, forming an amino, guanidino or amido bond. Biological activity of the immobilized catecholamines was determined by assessing their abilities to interact with adenylate cyclase in several membrane preparations and intact preparations of erythrocytes. In dog heart membranes, stimulation of adenylate cyclase by the catecholamine-gels could be accounted for by leached hormone which had been released from the gels. In frog erythrocyte membranes, leaching was minimal and no significant stimulation of adenylate cyclase was observed. Agarose-immobilized catecholamines, however, competitively inhibited isoproterenol stimulation of adenylate cyclase in these erythrocyte membranes indicating that catecholamines which are bound to agarose interact with the beta-adrenergic receptors as antagonists rather than agonists. When tested on intact frog erythrocytes, agarose immobilzed catecholamines did not increase the intracellular levels of cyclic AMP, although isoproterenol caused as 8-10 fold rise in these levels. Similarly, when tested for antagonist activity in the intact cells the agarose-catecholamines failed to inhibit the stimulation of cyclic AMP caused by isoproterenol. The difference observed in the beta-adrenergic antagonist activity of the agarose-bound catecholamines in membrane preparations and intact cells can be attributed to steric factors which could have prevented the access of the bead-bound ligands with the surface of the cell or to the possibility that receptors might be buried in the membrane matrix.     

Adenylate cyclase activity in rat submandibular gland during postnatal development

Adenylate cyclase activity was measured in homogenates of submandibular glands of 1 to 42 day old rats. During this period of time the gland reached its final stage of differentiation. Adenylate cyclase activity was higher in the glands of one day old rats than in those of 7 and 14 day old animals. Between 14 and 28 days of age the enzyme activity more than doubled and approached the level that characterized the glands of adult animals. Fluoride (10mM) stimulated the enzyme activity in all age groups but the stimulation was less in the case of one day old rats as compared to older animals. Isoproterenol (10^?4 M) stimulated adenylate cyclase by 50–60% in the gland of adult rats but had no effect on the enzyme activity in 7 to 28 day old animals. Administration of isoproterenol for 5 days to 9 day old rats increased the weight of the submandibular gland by 70 per cent. Total adenylate cyclase activity increased parallel with the weight of the gland but the specific activity of the enzyme remained unchanged. It is concluded that during the postnatal development of the submandibular gland the rapid increase in adenylate cyclase activity occurs after weaning and it coincides with an accelerated rate of functional differentiation of the acinar cells.     

Regulation of postnatal beta-adrenergic receptor/adenylate cyclase development by prenatal agonist stimulation and steroids: alterations in rat kidney and lung after exposure to terbutaline or dexamethasone

Glucorticoids and adrenergic stimulation are both thought to control the development of beta-adrenergic receptors/responses. In the current study, rats were exposed to dexamethasone or terbutaline during late gestation and the development of beta-receptor binding capabilities and adenylate cyclase activity evaluated in membrane preparations from kidney and lung. Prenatal dexamethasone exposure produced postnatal adrenergic hyperreactivity of kidney adenylate cyclase; the effect resulted from increases in the enzyme itself, as both basal adenylate cyclase and forskolin-stimulation of the enzyme were also increased by dexamethasone. Similarly, prenatal terbutaline exposure evoked increases in basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase in the kidney. In the lung, dexamethasone produced an initial postnatal deficit in basal adenylate cyclase and deficient responsiveness to isoproterenol, but the deficit resolved shortly after birth. Terbutaline selectively promoted the ability of isoproterenol to stimulate lung adenylate cyclase in the first few days after birth, without alterations in basal adenylate cyclase; this was followed by a period of prolonged subsensitivity of both basal and isoproterenol-stimulated activity. Although dexamethasone and terbutaline also caused significant changes in development of beta-receptor binding capabilities, in neither tissue could these effects account for the direction or magnitude of the changes in adenylate cyclase reactivity. Thus, glucocorticoids and beta-agonists can participate in the programming of development of postsynaptic reactivity by exerting actions upon post-receptor coupling mechanisms.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.