Peptidoglycan in the cell wall of the primary intracellular symbiote of the pea aphid.

Primary intracellular symbiotes of the pea aphid, Acyrthosiphon pisum (Harris), when fixed with potassium permanganate, revealed a distinctly staining area between the cytoplasmic membrane and the outer cell-wall envelope. This area is thought to be analogous to the peptidoglycan complex of the Eubacteriales. In addition, the diagnostic bacterial peptidoglycan amino compounds, muramic acid and diaminopimelic acid, were detected in a hydrochloric acid hydrolyzate of isolated symbiotes.

Effects of ouabain and potassium on protein synthesis and angiotensin-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells.

Ouabain (1 microM and below) inhibited both basal and angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Ouabain had no effect on binding of 125I-labeled angiotensin, on angiotensin's effects on 45Ca2+ fluxes, or on 32PO4 incorporation into phosphatidylinositol. This spectrum of activities resembles that of the protein synthesis inhibitor cycloheximide, which also blocks aldosterone synthesis. Ouabain was, therefore, tested for its effect on protein synthesis, as measured by uptake of [3H]leucine into acid-precipitable material. Ouabain inhibited protein synthesis at concentrations similar to those that depressed aldosterone synthesis, but did not block uptake of the nonmetabolized amino acid [carboxyl-14C]aminocyclopentane-1-carboxylic acid, nor the entrance of [3H]leucine into cells. When cells previously loaded with 86Rb+ were treated with 1 microM ouabain, they lost approximately half of the accumulated radioactivity in 30 min. When cells were incubated in potassium-free buffer, both protein and aldosterone synthesis were severely inhibited. Increased extracellular potassium reversed ouabain's inhibition of protein and aldosterone synthesis in parallel. Pregnenolone synthesis was inhibited by ouabain, and elevated potassium overcame that blockade. Ouabain did not block aldosterone synthesis from exogenous progesterone. These data fit a model in which ouabain causes loss of cell potassium, which, in turn, depresses protein synthesis. Since protein synthesis is necessary for angiotensin II stimulation of cholesterol side-chain cleavage, ouabain depresses that step, pregnenolone synthesis, and thus, aldosterone synthesis.

Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells.

We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of 45Ca+2 into cells and increased efflux of 45Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of 45Ca+2 in the presence of EGTA and retarded uptake of 45Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools.

Beta-adrenoceptor agonists block corticosteroid inhibition in eosinophils.

Although beta-adrenoceptor agonists are primary agents in therapy of asthma, epidemiological studies have suggested that frequent or prolonged used of these drugs could be associated with exacerbation of disease. Mechanisms of any adverse effects remain unclear although in vitro studies have suggested that beta-adrenoceptor agonists can block glucocorticoid actions. Because asthma is an inflammatory disease characterized by eosinophil infiltration of the airways, actions of beta-agonists and corticosteroids that alter eosinophil survival and mediator generation may be of importance. Eosinophil generation of superoxide anion, a potent mediator that can damage respiratory epithelium, was markedly increased after 2-24 h of in vitro beta-adrenoceptor agonist exposure. These proinflammatory effects are in contrast to inhibition of superoxide generation, which is observed with acute beta-agonist exposure. Corticosteroid treatment to reduce inflammation is combined with beta-agonist therapy in current asthma guidelines. Although dexamethasone independently decreased eosinophil superoxide anion generation, in the presence of beta-adrenoceptor agonist dexamethasone inhibition was minimal and not statistically significant. Eosinophil survival is a relevant factor to pulmonary inflammation. Although beta-adrenoceptor agonists did not independently increase eosinophil survival, glucocorticoid actions that increase apoptosis were blocked. Thus, in vitro beta-agonists can independently increase inflammatory mediator generation and block anti-inflammatory actions of corticosteroid.

Ionophores for monovalent cations inhibit angiotensin-stimulated aldosteronogenesis.

We examined the effects of monensin (a sodium ionophore), valinomycin (a potassium ionophore), and nigericin (a nonspecific ionophore) on steroid production and its stimulation in bovine adrenal glomerulosa and fasciculata cells. All three ionophores at nanomolar concentrations inhibited angiotensin (AII)-stimulated aldosterone production; potassium-stimulated aldosteronogenesis was more sensitive, and cortisol synthesis by fasciculata cells was much less sensitive. Ionophores completely inhibited the early pathway of aldosteronogenesis and partially inhibited conversion of progesterone to aldosterone. Ionophores had no effect on pregnenolone production by isolated glomerulosa mitochondria. Monensin had no effect on AII binding, calcium flux, calcium transient, protein phosphorylation, or protein synthesis; valinomycin slightly inhibited these processes. Valinomycin lowered cell potassium and raised cell sodium, but its inhibition of aldosteronogenesis was not overcome by increasing extracellular potassium. Monensin and nigericin had no effect on cell potassium or sodium. Cellular ATP was decreased by valinomycin, but not by monensin or nigericin. Our results show that stimulation of aldosteronogenesis by AII and potassium is highly sensitive to ionophores of monovalent cations. Monensin and nigericin inhibit steroidogenesis at concentrations that have no other observed deleterious effects on glomeulosa cells. These results identify a distinguishing characteristic of adrenal glomerulosa cells and suggest a new pharmacologic approach to inhibition of aldosteronogenesis.

Beta-adrenergic regulation of the eosinophil respiratory burst as detected by lucigenin-dependent luminescence.

Because beta-adrenoceptor agonists are commonly used in the treatment of disease states associated with eosinophil activation, beta-adrenergic regulation of the eosinophil respiratory burst (as monitored with lucigenin-dependent luminescence) was evaluated. Normodense, nonprimed eosinophils from healthy volunteer subjects were potently inhibited by very low concentrations of isoproterenol. The inhibitory concentration of 50% for isoproterenol was approximately 2 nmol/L. The beta-agonist was able to inhibit the eosinophil respiratory burst induced by receptor-mediated (chemotactic peptide) and nonreceptor-mediated (calcium ionophore and phorbol ester) stimuli. Thus beta-agonist inhibition was unlikely to be isolated to an effect at the receptor or G protein linkage. To determine whether cyclic adenosine 3',5' monophosphate (cAMP) may mediate beta-agonist effects, studies were performed with the type IV cAMP phosphodiesterase inhibitor Ro-201724. beta-Agonist inhibition of the respiratory burst was clearly synergistic with effects of Ro-201724. We conclude that beta-adrenoceptor agonists can regulate the eosinophil respiratory burst at least partially through an effect mediated by cAMP. Because regulation of the eosinophil by isoproterenol was observed at very low concentrations, these results may be relevant to pharmacologic effects of beta-agonists in disease states complicated by eosinophil activation during asthma.