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.

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.

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).

Pulmonary granuloma caused by Pseudomonas andersonii sp nov.

Pulmonary granuloma is a common lesion for which gram-negative bacteria are rarely implicated as a cause. Hence, most physicians are unaware of this etiology. We isolated a gram-negative bacterium from a surgically resected pulmonary granuloma in a 42-year-old, nonimmunocompromised woman. Within the necrotizing granuloma, numerous organisms also were demonstrated by Gram stain, suggesting a cause-disease relationship. Characterization of the bacterium by sequence analysis of the 16S ribosomal gene, cellular fatty acid profiling, and microbiologic studies revealed a novel bacterium with a close relationship to Pseudomonas. We propose a new species for the bacterium, Pseudomonas andersonii. These results suggest that the differential diagnosis of a lung granuloma also should include this gram-negative bacterium as a potential causative agent, in addition to the more common infections caused by acid-fast bacilli and fungi. This bacterium was shown to be susceptible to most antibiotics that are active against gram-negative bacteria.

Mycobacterium tuberculosis at a comprehensive cancer centre: active disease in patients with underlying malignancy during 1990-2000.

Thirty HIV-seronegative cancer patients with active tuberculosis were evaluated. Eighteen (60%) were immigrants, 19 (63%) had haematological malignancy, and fever was the most common presentation (97%). Of 19 (63%) patients with pulmonary tuberculosis, 11 (58%) were misdiagnosed initially as suffering from cancer following radiography. Death was attributed to tuberculosis for six (21%) of 29 patients who received anti-mycobacterial therapy. All four patients who had received high-dose systemic corticosteroids within 4 weeks of diagnosis of infection died, whereas two (8%) deaths occurred in 25 individuals without corticosteroid exposure (p < 0.001; OR 8.67). At this institution, active tuberculosis was rare, and was seen mostly in immigrants. Recent high-dose corticosteroid therapy is a significant predictor of mortality in cancer patients with tuberculosis.

Calcium-dependent protein kinase reactions associated with parotid gland secretory granule membranes.

Rat parotid secretory granule membranes were examined for the presence of calcium-dependent protein kinase activities and kinase substrates. Protein kinase C (C-kinase), which is stimulated by certain phospholipids, was present in the membranes, as indicated by its ability to catalyze the phosphorylation of histone. Two substrates for protein kinase C were seen in the granule membranes. The cytosolic fraction from the cell contained kinase activity, which was stimulated by phosphatidylserine and which caused the phosphorylation of two granule membrane polypeptides. In addition, when both granule membranes and cytosol were incubated together, phosphorylation of the cytosolic substrates was inhibited, indicating that the granule membrane substrates were phosphorylated preferentially. The results indicate that the granule membranes may react with cytosolic protein kinase C activity in a way which would direct an intracellular calcium and diacylglycerol signal toward the granule membrane. Since these signals occur during stimulation by various agonists, the mechanism may contribute to secretion.

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.

Does cyclic GMP mediate amylase release from mouse parotid acini?

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

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

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

Protein-energy malnutrition in infant non-human primates (Macaca nemestrina). I. Correlation of biochemical changes with fine structural alterations in the liver.

Male infant pigtail monkeys (Macaca nemestrina) aged 6-9 months and born in captivity were fed restricted amount of either an adequate protein diet (20% casein) or a protein-deficient diet (2% casein). After 3-5 months of receiving the low protein containing diet, ultrastructural evaluation of hepatic parenchymal cells revealed extensive fatty metamorphosis, marked fragmentation and vesiculation of the rough endoplasmic reticulum, some evidence of distension of the nuclear membrane, variable degree of mitochondrial polymorphism and gigantism, increased intracytoplasmic accumulation of glycogen particles and enhanced prominence of collagen fibres in the intercellular and perisinusoidal spaces. Biochemical studies indicated severe distortion in the plasma profile of free amino acids by Week 10 of malnutrition, with a prominent decrease in valine/glycine ratio (9% in the malnourished animals compared to a ratio of 55-63% in the control group). While plasma concentration of glycine was elevated above baseline value throughout the period of malnutrition, the level of alanine showed an initial rise followed by a significant drop (-58% at Week 20). Although showing wide individual variation, plasma level of corticosteroid in general tended to increase with duration of protein malnutrition, an observation consistent with the accumulation of glycogen and enlargement of mitochondria in the hepatic cells.

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.

Calmodulin activation and calcium regulation of parotid gland adenylate cyclase.

The effect of Ca2+ on the adenylate cyclase activity associated with membranes prepared from mouse parotid gland has been examined. Ca2+ stimulated then inhibited adenylate cyclase activity, with values for half-maximal stimulation and inhibition of 0.6 and 10 microM, respectively. Maximal activation (1.4-fold) was observed at 2 microM free Ca2+. These membranes contained 1.2 microgram calmodulin/mg protein. Exogenous calmodulin (0.2-1.2 microgram) activated, in a concentration-dependent manner, adenylate cyclase activity, with maximal activation being 2.5-fold at 12 micrograms calmodulin. Preparation of membranes in 2 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacet ic acid (EGTA) resulted not only in a significant decrease in calmodulin levels (0.5 microgram calmodulin/mg protein) but also in a loss of the ability of Ca2+ to stimulate the enzyme. Exogenous calmodulin restored the ability of Ca2+ to stimulate the adenylate cyclase activity associated with EGTA-treated membranes. Trifluoperazine (50 microM) blocked the ability of Ca2+ to activate adenylate cyclase activity in control membranes. The effect of trifluoperazine could be reversed by exogenous calmodulin (0.5 or 5.0 micrograms). These data indicate that calmodulin mediates the activation of parotid gland adenylate cyclase by Ca2+ and that Ca2+, at concentrations which stimulate and inhibit amylase secretion, can activate and inhibit adenylate cyclase activity.

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.

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.

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.

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.

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.

Central venous catheter-associated Nocardia bacteremia: an unusual manifestation of nocardiosis.

Nocardia bacteremia is very rare. We report 2 cases of central venous catheter-associated Nocardia bacteremia and review the literature. The limited clinical experience suggests that discontinuing the catheter and embarking on a relatively short course of appropriate antibiotics results in a good outcome.

Mycobacterium kansasii infections in patients with cancer.

Mycobacterium kansasii was isolated from 25 patients with cancer who were cared for at the University of Texas M. D. Anderson Cancer Center (Houston) from January 1987 through December 1996. Two patients (8%) had disseminated disease, and 23 (92%) had pleuropulmonary isolates only. Signs and symptoms of mycobacterial infection at the time of diagnosis were often minimal or absent despite substantial radiographically evident involvement. The infections responded well to rifampin-based antimycobacterial regimens. M. kansasii is an infrequent but serious cause of pulmonary and, occasionally, disseminated disease in patients with cancer.