pituitrin and indo-1

The change of cytosolic Ca2+ concentration ([Ca2+]i) caused by vasopressin was examined in indo-1-loaded A7r5 smooth muscle cells by use of the high-performance laser cytometer and ratiometric fluorescence method. Vasopressin (100 nM) caused an initial rapid rise and a delayed increase in [Ca2+]i (n = 6). However, in the presence of tetraethylammonium chloride (10 mM), vasopressin consistently triggered sustained Ca2+ oscillations which were preceded by a large peak of [Ca2+]i. The latency for the development of this huge increase in [Ca2+]i prior to the occurrence of sustained Ca2+ oscillations was always the same. The frequency and amplitude of this type of Ca2+ oscillation varied depending upon the extracellular Ca2+ concentration. Ca(2+)-free solution did not completely suppress the sustained Ca2+ oscillations, but caffeine (20 mM) effectively abolished them. The present findings indicate that in A7r5 smooth muscle cells, the sustained Ca2+ oscillations triggered by vasopressin in the presence of tetraethylammonium chloride were mainly due to Ca2+ release from IP3-sensitive Ca2+ stores and Ca2+ influx from extracellular space, and did not require the pacemaker activity derived from the surface membrane. Moreover, the vasopressin-induced change in [Ca2+]i appeared to be linked to pertussis toxin-insensitive GTP-binding protein(s).

Topics: Animals; Aorta, Thoracic; Caffeine; Calcium; Clone Cells; Drug Interactions; Extracellular Space; Fluorescent Dyes; GTP-Binding Proteins; Indoles; Kinetics; Muscle, Smooth, Vascular; Oscillometry; Pertussis Toxin; Rats; Spectrometry, Fluorescence; Tetraethylammonium; Tetraethylammonium Compounds; Time Factors; Vasopressins; Virulence Factors, Bordetella

Many hormones and drugs exert their effects on cells by increasing cytosolic Ca2+ (Cai2+) and activating protein kinase C (PKC). Each of these actions results in cholestasis in the isolated perfused rat liver, but the responsible mechanisms are unclear. We used isolated rat hepatocyte couplets to observe the direct effects of increased Cai2+ and PKC activation on permeability of the hepatocyte tight junction and canalicular volume, two possible determinants of hepatocyte bile secretion. Couplets were stimulated with the Ca2+ agonist vasopressin (10(-8) M) in the absence and presence of the Ca2+ influx antagonist Ni2+ (5 x 10(-3) M) or with the PKC activator phorbol dibutyrate (10(-6) M). Cai2+ was determined by ratio microspectrofluorometry of indo-1, permeability of the couplet tight junctions was assessed by exclusion of horseradish peroxidase from the canalicular space, and changes in canalicular volume over time were measured directly by optical planimetry. Canalicular volume increased by 1.6 +/- 2.5%/min (mean +/- SD) under basal conditions. In response to vasopressin, there was a rapid 15-fold increase in Cai2+, followed first by an increase in paracellular permeability, then by canalicular collapse (15.9 +/- 5.9%/min). Pretreatment with Ni2+ markedly decreased the vasopressin-induced increase in Cai2+ and abolished both the increase in paracellular permeability and the canalicular collapse. Phorbol dibutyrate also increased paracellular permeability but resulted in neither increased Cai2+ nor canalicular collapse. The PKC inhibitor H-7 reversed the effects of both vasopressin and phorbol dibutyrate on tight junction permeability. Bile secretory pressure, measured in isolated perfused rat liver preparations, was acutely increased by vasopressin, but the increase was augmented rather than inhibited by Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Cell Membrane Permeability; Cells, Cultured; Fluorescent Dyes; Indoles; Intercellular Junctions; Isoquinolines; Kinetics; Liver; Male; Models, Biological; Nickel; Phorbol 12,13-Dibutyrate; Piperazines; Protein Kinase C; Rats; Rats, Inbred Strains; Time Factors; Vasopressins

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcium; Cells, Cultured; Cyclic GMP; Cytosol; Fluorescent Dyes; Guanylate Cyclase; Indoles; Inositol Phosphates; Kidney Glomerulus; Rats; Rats, Inbred Strains; Vasopressins