Agonist-induced phosphorylation of the endogenous AT1 angiotensin receptor in bovine adrenal glomerulosa cells.

A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.

Gastrin-producing endocrine cells: a novel source of histamine in the rat stomach.

Gastrin and histamine both potently stimulate secretion of acid into the gastric lumen. How these agents interact and how their release is controlled is poorly understood. Therefore, we decided to look for histamine in the antral portion of the rat stomach where the gastrin-producing G cells are located. We used immunocytochemical methods to visualize histamine, histidine decarboxylase (HDC, the enzyme that converts histidine to histamine), and the type 1 vesicular monoamine transporter (VMAT1, the protein responsible for moving histamine into vesicles for storage and release). We were surprised to find that histamine, HDC, and VMAT1 were all present in G cells. Our results suggest that G cells synthesize and secrete gastrin and histamine. Whether histamine acts in concert with gastrin to stimulate acid secretion, or functions as an autocrine inhibitor of gastrin release remains to be seen.

Susceptibility of dopamine D5 receptor targeted mice to cysteamine.

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.

Localization of two distinct type III phosphatidylinositol 4-kinase enzyme mRNAs in the rat.

Inositol lipid kinases generate polyphosphoinositides, important regulators of several cellular functions. We have recently cloned two distinct phosphatidylinositol (PI) 4-kinase enzymes, the 210-kDa PI4KIIIalpha and the 110-kDa PI4KIIIbeta, from bovine tissues. In the present study, the distribution of mRNAs encoding these two enzymes was analyzed by in situ hybridization histochemistry in the rat. PI4KIIIalpha was found predominantly expressed in the brain, with low expression in peripheral tissues. PI4KIIIbeta was more uniformly expressed being also present in various peripheral tissues. Within the brain, PI4KIIIbeta showed highest expression in the gray matter, especially in neurons of the olfactory bulb and the hippocampus, but also gave a signal in the white matter indicating its presence in glia. PI4KIIIalpha was highly expressed in neurons, but lacked a signal in the white matter and the choroid plexus. Both enzymes showed expression in the pigment layer and nuclear layers as well as in the ganglion cells of the retina. In a 17-day-old rat fetus, PI4KIIIbeta was found to be more widely distributed and PI4KIIIalpha was primarily expressed in neurons. These results indicate that PI4KIIIbeta is more widely expressed than PI4KIIIalpha, and that the two enzymes are probably coexpressed in many neurons. Such expression pattern and the conservation of these two proteins during evolution suggest their nonredundant functions in mammalian cells.

Regulation of angiotensin II-stimulated Ca2+ oscillations by Ca2+ influx mechanisms in adrenal glomerulosa cells.

In adrenal glomerulosa cells, angiotensin II (Ang II) evokes repetitive [Ca2+]i transients and increases Ca2+ influx through voltage-sensitive calcium channels (VSCCs) as well as the capacitative Ca2+ entry pathway. This study analyzed the relationships between these Ca2+ influx pathways and intracellular Ca2+ signaling in bovine glomerulosa cells, in which Ca2+ oscillation frequency was regulated by Ang II concentration over the range of 50-300 p. In the absence of external Ca2+, such oscillations were maintained for prolonged periods of time, but their frequency was significantly reduced (0.23 min-1 versus 0.38 min-1). Restoration of [Ca2+]o to 0.6 mM increased the frequency of Ca2+ oscillations in cells that showed narrow spikes of constant amplitude and caused a plateau response in cells with broad spikes of rapidly decreasing amplitude. In the presence of Ca2+, nifedipine reduced the frequency of the oscillatory Ca2+ response to 100 pM Ang II by 49%, and BAY K 8644 increased oscillation frequency by 86%, or caused plateau-type responses typical of higher Ang II concentrations. In contrast to their prominent actions on Ca2+ spiking frequency, dihydropyridines caused only minor changes in Ang II (100 pM)-induced inositol phosphate production. Dihydropyridines also had minimal effects on the nonoscillatory Ca2+ signals evoked by high Ang II concentrations (10 nM). These findings indicate that Ca2+ influx through VSCCs modulates the frequency of Ca2+ oscillations induced by low agonist concentrations by a mechanism that does not involve major changes in inositol trisphosphate formation. However, VSCCs make relatively little contribution to the nonoscillatory Ca2+ signals generated by high agonist concentrations, when Ca2+ influx occurs predominantly through the capacitative Ca2+ entry pathway.

Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases.

Agonist-sensitive phosphoinositide pools are maintained by recently-identified wortmannin (WT)-sensitive phosphatidylinositol (PI) 4-kinase(s) (Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5317-5321). Two loosely membrane-associated WT-sensitive type III PI 4-kinases were isolated from bovine adrenal cortex as [3H]WT-labeled 110- and 210-kDa proteins. Based on peptide sequences from the smaller enzyme, a 3. 9-kilobase pair (kb) cDNA with an open reading frame encoding a 90-kDa protein was isolated from a bovine brain cDNA library. Expression of this cDNA in COS-7 cells yielded a 110-kDa protein with WT-sensitive PI 4-kinase activity. Northern blot analysis of a human mRNA panel showed a single approximately 3.8-kb transcript. Peptide sequences obtained from the 210-kDa enzyme corresponded to those of a recently described rat 230-kDa PI 4-kinase. A 6.5-kb cDNA containing an open reading frame of 6129 nucleotides that encoded a 230-kDa protein, was isolated from brain cDNA. Northern blot analysis of human mRNA revealed a major 7.5-kb transcript. The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed analysis of their signaling and other regulatory functions in mammalian cells.

Meningococcal cellulitis and sialadenitis.

Neisseria meningitidis is a rare cause of cellulitis. No cases of meningococcal sialadenitis have previously been reported. We recently successfully treated a patient who had meningococcal cellulitis and sialadenitis. We review previously reported cases of cellulitis due to N meningitidis and speculate on the role of underlying disease in the pathogenesis of this infection.

Expanded parietal cell pool in transgenic mice unable to synthesize histamine.

BACKGROUND: The histidine decarboxylase enzyme (HDC) is responsible for the synthesis of histamine in mammals. Histidine decarboxylase-deficient (HDC-/-) mice have recently been developed by targeted mutation of the HDC gene. METHODS: The impact of prolonged histamine deficiency was studied on gastric morphology (by immunohistochemistry and morphometry), gastric acid secretion (by a wash-through method for basal gastric acid secretion and by pylorus ligation for stimulated gastric acid secretion) and gastrin levels (by radioimmunoassay) in homozygous HDC-/- mice kept on a low-histamine diet. RESULTS: A double maximal gastric acid secretory response was found in knockouts after exogenous histamine administration. In contrast, the gastric acid secretion was significantly reduced after gastrinergic and cholinergic stimulation in the absence of histamine. The oxynthic gland area of HDC-/- mice was thickened with an increased parietal cell count compared to wild types. Substantially elevated serum and antral tissue gastrin levels of HDC-/- mice could be possible indications of both an expanded parietal cell mass and/or an increased histamine-induced maximal gastric acid secretory capacity of this genotype. CONCLUSIONS: These data suggest that not enough compensatory mechanisms develop in HDC-/- mice during a prolonged low-histamine diet to maintain/restore normal gastric acid secretion. An expanded parietal cell pool was also demonstrated in HDC-/- mice kept on a low-histamine diet, probably caused by a trophic effect of sustained hypergastrinaemia. The HDC-/- strain is a suitable model for studying the effects of achlorhydria and consequent hypergastrinaemia as an approach to human conditions such as atrophic gastritis or long-term antisecretory therapies.