Cell and molecular studies of renin secretion.

Renin secretion has been studied in the past at the level of the whole kidney, but the control of the genetic basis of renin synthesis is poorly understood. We have studied the regulation of renin gene expression in the fetus and also in the adult rat in response to angiotensin converting enzyme (ACE) inhibition with enalapril in the presence and absence of angiotensin II (AII). In the fetus, vascular smooth muscle cells of the renal afferent arteriole and feeder vessels contain renin mRNA and immunostain for renin. With maturation, these vessels progressively lose the capacity to synthesize renin, and only the juxtaglomerular cells retain this capacity in the adult. However, in response to ACE inhibition, the adult renal feeder vessels acquire the capacity to synthesize and secrete renin within 7 days. This effect is partially reversed with co-administration of AII. In order to study renin biosynthesis and secretion at the cellular level, we have developed a new method of study of individual renin-secreting cells, the reverse hemolytic plaque assay (RHPA). Utilizing this method, we have demonstrated that ACE inhibition with enalapril increases the number of renin secreting cells by over 15-fold at physiologic calcium concentrations. Enalapril also induced a 3-fold increase in the amount of renin released as estimated by the area of the hemolytic plaques formed. Transmission electron microscopy (EM) of the renin-secreting cell at the center of a hemolytic plaque demonstrates modified vascular smooth muscle cells with cytoplasmic granules. In summary, ACE inhibition stimulates renin mRNA accumulation and redistributes renal renin content toward that observed in early fetal life. AII inhibits renal renin mRNA accumulation. ACE inhibition increases the number of renin secreting cells as well as the amount of renin secreted by each cell. The individual renin secreting cell is a modified vascular smooth muscle cell with cytoplasmic secretory granules. Further studies of the cellular pathways for renin secretion can be provided by EM immunocytochemistry of the individual renin secreting cell.

Comparison of molecular properties of the voltage-sensitive Ca2+ channel from longitudinal smooth muscle of rabbit ileum with the channel purified from transverse tubules of rabbit skeletal muscle.

Dihydropyridine-sensitive calcium channels in membrane preparations from the longitudinal smooth muscle of rabbit ileum bound [3H]PN 200-110 with KD = 0.42 nM and Bmax = 265 fmol/mg. Calcium channels were labelled with [3H]PN 200-110 and solubilized with digitonin. The detergent-solubilized calcium channel had a sedimentation coefficient of 20.9 S in close agreement with the value of 20 S determined for the skeletal muscle calcium channel. Antibodies against the alpha-subunits of the skeletal muscle calcium channel specifically precipitated the calcium channel from smooth muscle. Our results suggest that the calcium channel from smooth muscle has a similar size and homologous alpha-subunits to those of the purified skeletal muscle calcium channel.

Binding of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole to histamine receptors in guinea pig cerebral cortical membranes.

The interaction of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole (AAH), a photolabile histamine receptor antagonist, with the binding of histamine, mepyramine, and tiotidine to guinea pig cerebral cortical membranes was examined to evaluate the specificity of AAH for histamine H1 and H2 receptors. Saturable, specific binding of [3H]histamine, [3H]mepyramine, and [3H]tiotidine to the membranes was observed. Competition assays were used to assess the relative affinity of AAH for H1- and H2-receptors. The rank order of IC50 values obtained was (most to least potent) (i) for competing with [3H]histamine binding: histamine greater than AAH much greater than mepyramine approximately equal to tiotidine; (ii) for competing with [3H]mepyramine binding: mepyramine much greater than AAH greater than histamine greater than tiotidine; and (III) for competing with [3H]tiotidine binding: tiotidine much greater than mepyramine greater than histamine approximately equal to AAH. The affinity of AAH for H1 receptors was ca. 14-fold greater than for H2 receptors. These findings support previous evidence obtained in isolated smooth muscle preparations that AAH shows H1-receptor selectivity as an antagonist.

Sensitivity changes in the smooth muscle of the guinea-pig isolated vas deferens after treatment of animals with 6-hydroxydopamine.

Postjunctional supersensitivity of the smooth muscle of the guinea-pig vas deferens induced by denervation, decentralization and treatment of animals with reserpine has been attributed, in part, to a partial membrane depolarization (8-10 mV) resulting from reduced electrogenic Na+,K+-pumping activity. This study was undertaken to characterize sensitivity changes which occur after treatment of animals with 6-hydroxydopamine (100 mg/kg + 250 mg/kg i.v., 1 day apart). Seven days after the second injection, concentration-response curves for isometric contractile responses to norepinephrine, methoxamine, acetylcholine and histamine were shifted 40.6-, 1.7-, 3.6- and 2.7-fold, respectively, to the left of control; however, the sensitivity to KCl was not increased, which contrasts with the results after denervation, decentralization and reserpine treatment. Ouabain (10(-5) M) produced 1.8- and 1.3-fold leftward shifts of the KCl concentration-response curves in tissues from control and 6-hydroxydopamine-treated animals, respectively. The pronounced effect of ouabain in tissues from treated animals may be an indication that 6-hydroxydopamine treatment does not result in as much inhibition of electrogenic Na+,K+-pumping, and resultant membrane depolarization, as other methods which induce supersensitivity of the guinea-pig vas deferens.