Nitric oxide inhibition of cAMP synthesis in parotid acini: regulation of type 5/6 adenylyl cyclase.

The nitric oxide (NO) donor, GEA 3162, inhibited isoproterenol-induced cyclic AMP (cAMP) accumulation in a concentration- and time-dependent manner in mouse parotid acini; SIN-1 mimicked these effects. Inhibition of stimulated cAMP accumulation was independent of phosphodiesterase activity. GEA 3162 also inhibited forskolin-induced cAMP accumulation. Removal of extracellular Ca(2+), addition of La(3+), or the calmodulin (CaM) inhibitor, calmidazolium, did not prevent the NO-mediated response, and addition of the soluble guanylyl inhibitor, ODQ, did not reverse GEA 3162-induced inhibition of cAMP accumulation. GEA 3162 also inhibited adenylyl cyclase in vitro independently of Ca(2+)/CaM. Further studies revealed that the NO synthase (NOS) inhibitor, 7-nitroindazole (7-NI), reduced significantly thapsigargin-induced Ca(2+) release and capacitative Ca(2+) entry and reversed thapsigargin inhibition of the AC Type 5/6 isoform (AC5/6). Data suggest that NO produced endogenously has dual effects on cAMP accumulation in mouse parotid acini, an inhibitory effect on AC activity and a modulatory effect on capacitative Ca(2+) entry resulting in AC5/6 inhibition.

The type 8 adenylyl cyclase is critical for Ca2+ stimulation of cAMP accumulation in mouse parotid acini.

Capacitative Ca(2+) entry stimulates cAMP synthesis in mouse parotid acini, suggesting that one of the Ca(2+)-sensitive adenylyl cyclases (AC1 or AC8) may play an important role in the regulation of parotid function (Watson, E. L., Wu, Z., Jacobson, K. L., Storm, D. R., Singh, J. C., and Ott, S. M. (1998) Am. J. Physiol. 274, C557-C565). To evaluate the role of AC1 and AC8 in Ca(2+) stimulation of cAMP synthesis in parotid cells, acini were isolated from AC1 mutant (AC1-KO) and AC8 mutant (AC8-KO) mice and analyzed for Ca(2+) stimulation of intracellular cAMP levels. Although Ca(2+) stimulation of intracellular cAMP levels in acini from AC1-KO mice was indistinguishable from wild type mice, acini from AC8-KO mice showed no Ca(2+)-stimulated cAMP accumulation. This indicates that AC8, but not AC1, plays a major role in coupling Ca(2+) signals to cAMP synthesis in parotid acini. Interestingly, treatment of acini from AC8-KO mice with agents, i.e. carbachol and thapsigargin that increase intracellular Ca(2+), lowered cAMP levels. This decrease was dependent upon Ca(2+) influx and independent of phosphodiesterase activation. Immunoblot analysis revealed that AC5/6 and AC3 are expressed in parotid glands. Inhibition of calmodulin (CaM) kinase II with KN-62, or inclusion of the CaM inhibitor, calmidazolium, did not prevent agonist-induced inhibition of stimulated cAMP accumulation. In vitro studies revealed that Ca(2+), independently of CaM, inhibited isoproterenol-stimulated AC. Data suggest that agonist augmentation of stimulated cAMP levels is due to activation of AC8 in mouse parotid acini, and strongly support a role for AC5/6 in the inhibition of stimulated cAMP levels.

Nitric oxide acts independently of cGMP to modulate capacitative Ca(2+) entry in mouse parotid acini.

Carbachol- and thapsigargin-induced changes in cGMP accumulation were highly dependent on extracellular Ca(2+) in mouse parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) resulted in complete inhibition of agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca(2+) release and capacitative Ca(2+) entry, whereas the inhibition of sGC had no effect. The effects of NOS inhibition were not reversed by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by the inhibition of sGC. GEA-3162-induced increases in Ca(2+) release from ryanodine-sensitive stores and enhanced capacitative Ca(2+) entry, both of which were unaffected by inhibitors of sGC but reduced by NOS inhibitors. Results support a role for NO, independent of cGMP, in agonist-mediated Ca(2+) release and Ca(2+) entry. Data suggest that agonist-induced Ca(2+) influx activates a Ca(2+)-dependent NOS, leading to the production of NO and the release of Ca(2+) from ryanodine-sensitive stores, providing a feedback loop by which store-depleted Ca(2+) channels are activated.

GTP-binding proteins and regulated exocytosis.

Regulated exocytosis, which occurs in response to stimuli, is a two-step process involving the docking of secretory granules (SGs) at specific sites on the plasma membrane (PM), with subsequent fusion and release of granule contents. This process plays a crucial role in a number of tissues, including exocrine glands, chromaffin cells, platelets, and mast cells. Over the years, our understanding of the proteins involved in vesicular trafficking has increased dramatically. Evidence from genetic, biochemical, immunological, and functional assays supports a role for ras-like monomeric GTP-binding proteins (smgs) as well as heterotrimeric GTP-binding protein (G-protein) subunits in various steps of the vesicular trafficking pathway, including the transport of secretory vesicles to the PM. Data suggest that the function of GTP-binding proteins is likely related to their localization to specific cellular compartments. The presence of both G-proteins and smgs on secretory vesicles/granules implicates a role for these proteins in the final stages of exocytosis. Molecular mechanisms of exocytosis have been postulated, with the identification of a number of proteins that modify, regulate, and interact with GTP-binding proteins, and with the advent of approaches that assess the functional importance of GTP-binding proteins in downstream, exocytotic events. Further, insight into vesicle targeting and fusion has come from the characterization of a SNAP receptor (SNARE) complex composed of vesicle, PM, and soluble membrane trafficking components, and identification of a functional linkage between GTP-binding and SNARES.

Beta-adrenergic-induced cytosolic redistribution of Rap1 in rat parotid acini: role in secretion.

Rap1 has recently been identified on the secretory granule membrane and plasma membrane of rat parotid acinar cells (N. J. D'Silva, D. DiJulio, C. B. Belton, K. L. Jacobson, and E. L. Watson. J. Histochem. Cytochem. 45: 965-973, 1997). In the present study, we examined the cellular redistribution of Rap1 following treatment of acini with isoproterenol (ISO), the beta-adrenergic agonist, and determined the relationship between translocation and amylase release. In the presence of ISO, Rap1 translocated to the cytosol in a concentration- and time-dependent manner; this effect was not mimicked by the muscarinic agonist, carbachol. Translocation was maximal at 1 microM ISO and paralleled amylase release immediately after ISO stimulation. Rap1 translocation and amylase release were blocked by the beta-adrenergic antagonist, propranolol, whereas okadaic acid, a downstream secretory inhibitor, significantly blocked amylase release but did not inhibit Rap1 redistribution. Results suggest that the translocation of Rap1 is causally related to secretion and that the role of Rap1 in secretion is at a site proximal to the exocytotic event.

Capacitative Ca2+ entry is involved in cAMP synthesis in mouse parotid acini.

Muscarinic receptor interaction leading to augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The effectiveness of capacitative Ca2+ entry and intracellular Ca2+ release on this response was determined in time course studies by using three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic agonist carbachol, thapsigargin, and ionomycin. Time course studies revealed that Ca2+ release from intracellular stores by carbachol produced an early rapid increase (0.25-0.5 min) in stimulated cAMP levels, whereas capacitative Ca2+ entry resulted in a sustained increase in stimulated cAMP levels that was blocked by La3+. Capacitative Ca2+ entry, alone, was involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. The inability of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and milrinone, to prevent agonist augmentation of cAMP levels, as well as the finding that the type VIII adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this adenylyl cyclase isoenzyme.

The heterotrimeric GTP-binding protein, GS, modulates the Cl- conductance of rat parotid acinar secretory granules.

Gsalpha has been reported to be present in rat parotid acinar secretory granule membrane (SGM) fractions. In the present study, we evaluated epitope orientation of Gsalpha on the secretory granule (SG) and the ability of Gs to modulate the Cl- conductance of isolated granules by measuring granule lysis. Gsalpha was found to be associated with the cytoplasmic face of the SGM. Aluminum fluroide (AlF4-, 20 microM Al3+ and 10 mM F-) significantly increased granule lysis and this effect was blocked by GDPbetaS. Cholera toxin (5 microg/ml) mimicked the effects of AlF4- on granule lysis, whereas pertussis toxin (0.5 microg/ml) was without effect. GTPgammaS, however, reduced granule lysis in a concentration-dependent manner. The orientation of Gsalpha on the SGM as well as the effects of AlF4- and cholera toxin on granule lysis lends support for a role of Gs in the exocytotic process.

Immunolocalization of rap1 in the rat parotid gland: detection on secretory granule membranes.

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [alpha-32P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.

Ryanodine receptor type III (Ry3R) identification in mouse parotid acini. Properties and modulation of [3H]ryanodine-binding sites.

Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 degrees C, and in the presence of 0.5 M KCl and 100 microM Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (Kd = 6 nM); maximum binding capacity (Bmax) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mM and 0.5 microM, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein.RyR complex, increased [3H]ryanodine binding 3-4-fold by enhancing Kd. The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 microM and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.

Identification of muscarinic receptor subtypes in mouse parotid gland.

Immunoprecipitation of muscarinic receptors from mouse parotid membranes by specific subtype antisera showed that M3 and M1 receptors represented 75 and 15% of the total number of precipitable receptors, respectively. [N-methyl-3H]methylscopolamine (NMS) labeled a single class of high-affinity binding sites in membranes from parotid glands with a dissociation constant of 0.67 +/- 0.02 nM and a maximum binding capacity of 176 +/- 15 fmol/mg protein. Competition curves for NMS, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydro-sila-difenidol fit best to a one-site binding model, whereas pirenzepine and methoctramine fit best to a two-site binding model, indicating 76-90% M3 receptors. Results from the use of pirenzepine indicated that the second mouse parotid receptor subtype, unlike that of the submandibular gland, has atypical characteristics for an M1 receptor. The rank order of potency of muscarinic antagonists in inhibiting phosphoinositide turnover and biphasic effects of carbachol on isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was atropine > or = 4-DAMP >> pirenzepine > AF-DX 116. A specific M1 antagonist, m1-toxin, had no effect on carbachol augmentation or inhibition of isoproterenol responses. Results suggest that M3 receptors couple to both augmentation and inhibition of stimulated cAMP levels.

Case record analysis: biopsied lesions in a high-risk white and African American Community.

OBJECTIVES: The purpose of this secondary data analysis was to characterize biopsied intraoral lesions documented from neighborhood health center records in a lower socioeconomic biracial community over an eight year period, (1982-1989.) METHODS: A thorough intraoral examination, which included radiographs, was completed by either a general dentist or an oral surgeon. Questionable lesions were referred to and biopsied in the Department of Pathology at Johns Hopkins Medical Center. Biopsied lesions were classified into three groups for analysis: benign, malignant/premalignant, and dental-related. RESULTS: Of 13,317 recorded dental visits, 10,819 (81.2%) visits were made by African Americans. There were 341 lesions biopsied, representing 69 different histologic diagnoses. Of identified lesions, 76.5% were in African Americans; 41.6% were in males, ranging in age from 41 to 94 years old (a mean age of 73 +/- 7 years). Approximately 50% of identified lesions in African American and Whites were benign. Among African Americans, 11.9% of the lesions were malignant premalignant, while 16.3% were among Whites. Within the malignant/premalignant category, squamous cell carcinoma was the most frequently identified malignant lesion. Hyperkeratosis was the most common premalignant lesion type. CONCLUSIONS: Neighborhood health centers can provide a valuable screening service identifying intraoral lesions in vulnerable communities. Future research should explore how socioeconomic factors affect differences in the prevalence of intraoral pathologies among White and African American subgroups as well as other subpopulations.

The measurement of mandibular cortical bone height in osteoporotic vs. non-osteoporotic postmenopausal women.

The asymptomatic progression of osteoporosis, in conjunction with the possibility of catastrophic disability, makes this disorder a major public health priority. Various body sites, including the mandible, have shown susceptibility to decreasing bone density. In 1986, Benson et al. proposed a radiomorphometric technique called the Panoramic Mandibular Index (PMI) as an inexpensive noninvasive dental technique for osteoporosis screening, although no osteoporotic subjects were included in their study. The purpose of our study was to determine whether osteoporotic postmenopausal women would show a decrease in mandibular cortical bone height, as measured by the PMI index, when compared with nonosteoporotic postmenopausal women. Seventy-two Caucasian females (33 cases/39 controls), aged 54-71 years old, were selected through records and screening via a dual-energy x-ray absorptiometry scan (LUNAR-DEXA). ANOVA analysis indicated no differences in the mean PMI between case and control groups (0.37 +/- 0.15 and 0.38 +/- 0.13, respectively; p = 0.69). Other techniques, such as computer digitized radiography should be explored to test the validity of the PMI.

Localization of the G-protein G(o) in exocrine glands.

The GTP-binding protein G(o) was localized immunohistochemically in the rat parotid gland and in other exocrine glands with specific G(o) antibodies. Immunohistochemical studies revealed that affinity-purified G(o alpha) polyclonal antibody (GO/85) immunoreacted primarily with duct cells of the rat parotid gland; immunoreactivity was also noted in duct cells of the rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. G(o alpha) antiserum (9072) differing in specificity for epitopes within G(o alpha) produced similar results. This antiserum also immunoreacted with rat submandibular duct cell secretory granule membranes. In contrast, in rat and mouse pancreas G(o alpha) antibodies immunoreacted primarily with islet cells. Duct cells were negative but there was light labeling of rat pancreatic acinar cells. The apparent duct specificity of G(o alpha) staining was further verified by demonstrating that G(o alpha) antibodies immunoreacted with HSG-PA cells, a human transformed salivary duct cell line. Specificity in immunohistochemical labeling of HSG-PA cells was confirmed by Western blot analysis. The results demonstrate that G(o) appears to be selectively expressed in the duct cells of rat parotid gland and other salivary glands. The selective enrichment of G(o) in duct cells suggests that this G-protein plays an important role in duct cell physiology.

Biphasic effects of carbachol on stimulated cAMP accumulation in mouse parotid acini.

Carbachol (0.1-10 microM) augmented the isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by approximately 50% in mouse parotid acini; at carbachol concentrations > 10 microM the stimulatory trend was reduced. These effects were time dependent. In the presence of 3-isobutyl-1-methylxanthine (IBMX), the overall response to carbachol was an inhibition of the isoproterenol response. Pretreatment of acini with pertussis toxin failed to reverse this inhibition, suggesting that the effects of carbachol were not related to effects on the GTP binding protein, Gi. A-23187 mimicked the effects of carbachol on isoproterenol-stimulated cAMP accumulation in the presence and absence of IBMX. In the presence of IBMX, carbachol failed to inhibit isoproterenol-stimulated cAMP accumulation when calcium was absent from the extracellular media and depleted from intracellular stores by thapsigargin. By contrast, in the absence of IBMX, removal of calcium abolished augmentation of isoproterenol responses by low concentrations of carbachol, whereas at higher carbachol concentrations isoproterenol responses were significantly inhibited; the time to maximal cAMP accumulation was decreased approximately eightfold. The results show that the mechanisms underlying the effects of carbachol on cAMP metabolism involve both the enzymes that synthesize and degrade cAMP.

Glucose stimulates cAMP accumulation in the oral bacterium Actinomyces viscosus.

Actinomyces viscosus T14V, a Gram-positive bacterium found in the oral cavity, was found to be insensitive to glucose-mediated catabolite repression. Basal levels of beta-galactosidase (18-26 U) were observed at all phases of growth regardless of the culture conditions. Further, beta-galactosidase could not be induced with lactose, or with a known inducer of the enzyme, isopropyl-beta-D-thiogalactoside, or with dibutyryl cAMP. Glucose, on the other hand, stimulated cAMP accumulation in a concentration-dependent manner. Fructose and sucrose mimicked the effects of glucose on cAMP accumulation, whereas galactose, mannose and maltose had lesser stimulatory effects. Other carbon sources, i.e., lactose, alpha-methylglucoside, ribose, xylose and succinate were without effect. Glucose and alpha-methylglucoside were found to stimulate cAMP accumulation in toluene-permeabilized cells, in the presence of the phosphodiesterase inhibitor, theophylline. Glucose did not stimulate cAMP levels in other Gram-positive bacteria including Streptococcus mutans, S. sanguis and S. salivarius but did cause cAMP accumulation in other strains of A. viscosus. The results suggest that glucose effects on cAMP metabolism are independent of the induction of beta-galactosidase as presently defined for Escherichia coli, and that the effects appear to be selective to the A. viscosus bacteria. The results also suggest that glucose stimulates cAMP accumulation via activation of adenylate cyclase.

Phorbol ester has different effects on forskolin and beta-adrenergic-stimulated cAMP accumulation in mouse parotid acini.

Phorbol 12-myristate 13-acetate (TPA) augmented the effects of forskolin, and inhibited the effects of isoproterenol on cAMP accumulation in mouse parotid acini. Treatment of intact cells with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), blocked TPA inhibition of isoproterenol but not forskolin-stimulated cAMP accumulation. TPA also caused the translocation of protein kinase C (PKC) from cytosol to membrane. Pre-treatment of parotid acini with TPA for 30 min enhanced the forskolin and isoproterenol-stimulated adenylate cyclase activity in isolated parotid membranes. Addition of purified PKC (pPKC) to parotid membranes mimicked the effects of TPA in increasing cAMP synthesis; the effects were blocked in the absence of calcium and phospholipid, and in the presence of the synthetic peptide (PKC 19-36). Purified PKC also mimicked the effects of TPA in augmenting forskolin and isoproterenol-stimulated adenylate cyclase activities in the cell-free system. Data suggest that the differential regulation of forskolin and isoproterenol-stimulated cAMP accumulation by TPA results from modification of enzymes that synthesize and degrade cAMP.

Identification and localization of G proteins in exocrine glands.

GTP-binding proteins were identified in rat parotid acinar plasma-enriched membranes (PM) by immunoblot analysis and localized immunohistochemically in the parotid gland as well as in other exocrine glands by using affinity-purified antisera specific for alpha subunits of the G proteins. Isolated rat parotid acinar PM immunoreacted strongly to antisera directed against Gs alpha, Gi alpha 1/alpha 2, Gi alpha 3, and Go alpha; the signal for Go alpha, however, was weak with crude Go antisera. Immunohistochemical studies to identify and localize Go in rat parotid tissue revealed that antisera to Go alpha immunoreacted with ductal cells. In addition, strong immunoreactivity to Go alpha antisera was noted in ductal cells of other salivary glands including rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. In contrast, in the rat and mouse pancreas, Go antisera immunoreacted primarily with islet cells. Ductal cells were negative, but there was light labeling of rat pancreatic acinar cells. The apparent ductal specificity of Go alpha staining was further verified by demonstrating that Go alpha antisera immunoreacted strongly with HSG-PA cells, a human transformed salivary ductal cell line. The results demonstrate that rat parotid acinar plasma membranes express a number of G proteins including Go and that Go appears to be selectively expressed in the ductal cells of rat parotid gland and other salivary glands. The selective enrichment of Go in ductal cells suggests that this G protein may play an important role in ductal cell physiology.

Evidence for G proteins in rat parotid plasma membranes and secretory granule membranes.

G proteins were identified in rat parotid plasma membrane-enriched fractions and in two populations of isolated secretory granule membrane fractions. Both [32P]ADP-ribosylation analysis with bacterial toxins and immunoblot analysis with crude and affinity-purified antisera specific for alpha subunits of G proteins were utilized. Pertussis toxin catalysed the ADP-ribosylation of a 41 kDa substrate in the plasma membrane fraction and both secretory granule membrane fractions. Cholera toxin catalysed the ADP-ribosylation of two substrates with molecular masses of 44 kDa and 48 kDa in the plasma membrane fraction but not in the secretory granule fractions. However, these substrates were detected in the secretory granule fractions when recombinant ADP-ribosylating factor was present in the assay medium. Immunoblot analysis of rat parotid membrane fractions using both affinity-purified and crude antisera revealed strong immunoreactivity of these membranes with anti-Gs alpha, -Gi alpha 1/alpha 2 and -Gi alpha 3 sera. In contrast Gs alpha was the major substrate found in both of the secretory granule fractions. Granule membrane fractions also reacted moderately with anti-Gi alpha 3 antiserum, and weakly with anti-Gi alpha 1/alpha 2 and -G(o) alpha sera. The results demonstrate that the parotid gland membranes express a number of G proteins. The presence of G proteins in secretory granule membranes suggests that they may play a direct role in regulating exocytosis in exocrine glands.

Regulation of cAMP metabolism in mouse parotid gland by cGMP and calcium.

The interaction of hormones acting via the mobilization of calcium and stimulation of cAMP levels in cells was examined by determining the effects of carbachol and forskolin on cAMP and cGMP accumulation in mouse parotid gland. Treatment of isolated acini with either carbachol (0.01 to 20 microM) or forskolin (1 microM) alone produced little or no increase in cAMP levels; carbachol, however, augmented the effect of forskolin on cAMP accumulation approximately 3- to 4-fold. The effects of carbachol on forskolin-stimulated cAMP levels were further augmented approximately 10-fold in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (MIX) but not in the presence of "low Km" cGMP-inhibited phosphodiesterase inhibitor milrinone. Augmentation of cAMP levels also occurred in the presence of carbachol plus the beta-adrenergic agonist isoproterenol (0.01 microM). In either the presence or absence of forskolin, carbachol increased cGMP levels independently of the inclusion of MIX and in a fashion parallel to that observed for cAMP accumulation. In the presence of forskolin (1 microM), the concentration of carbachol that produced half-maximal effects on cAMP and cGMP levels was 0.62 and 0.72 microM, respectively. Similar values were obtained in the presence of MIX. Cyclic GMP levels were also enhanced by carbachol plus isoproterenol. Hydroxylamine, as well as dibutyryl-cGMP and 8-bromo-cGMP in combination with forskolin, mimicked the effects of carbachol plus forskolin on cAMP levels. LY83583 (6-anillino-5,8-quinolinedione), an agent that lowers cGMP by inhibiting guanylate cyclase, reduced basal levels of cGMP and also completely prevented the increase in cGMP caused by carbachol plus forskolin. In these experiments, however, the augmentation of forskolin-stimulated cAMP levels by carbachol was reduced by approximately 50%. Additional studies suggest that calcium is also required for carbachol augmentation of forskolin-stimulated cAMP accumulation by effects on the adenylate cyclase complex. Augmentation of cAMP levels by carbachol did not involve effects on cAMP degradation. The results suggest that, when cAMP synthesis is stimulated by forskolin or isoproterenol, the muscarinic agonist carbachol augments cAMP accumulation by mechanisms involving cGMP and calcium in mouse parotid gland.

Monovalent ion enhancement of beta-adrenergic-stimulated adenylate cyclase activity in mouse parotid gland.

Sodium activated basal adenylate cyclase at all concentrations of sodium examined (5-100 mM) and independently of GTP. Stimulation of adenhylate cyclase by the beta-adrenergic agonist, isoproterenol, was enhanced at all concentrations (5-100 mM) of sodium ions tested in the presence of GTP. Maximal enzyme activation under all conditions occurred between 25 and 50 mM NaCl. Enhancement of forskolin-activated adenylate cyclase by sodium did not require GTP nor was it affected by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), a competitive inhibitor of GTP. The selectivity of adenylate cyclase for monovalent cations was Na+ congruent to K+. Lithium chloride produced an inhibition of hormone-activated adenylate cyclase. Sodium ions also enhanced isoproterenol- and forskolin-activated adenylate cyclase of submandibular gland membranes. In contrast to mouse parotid and submandibular glands, activation of mouse liver and brain adenylate cyclase activities by forskolin and isoproterenol was not enhanced by sodium ions. The tissue differences were not related to differences in potency of the agonists. These results suggest (1) that sodium ions may have a selective and positive regulatory role in hormonal activation of adenylate cyclase in mouse exocrine tissue, and (2) that sodium ions enhance hormonal activation of enzyme by interacting at a site on the adenylate cyclase complex which is independent of the hormone receptor (Rs) and the stimulatory guanine nucleotide binding protein (Ns).