Previously, we have shown in porcine inner medullary collecting duct (IMCD) cells that endothelin (ET), probably in an autocrine fashion, suppresses arginine vasopressin (AVP)-induced synthesis of cAMP and thereby, may modify the action of AVP on IMCD fluid transport. In the present study we investigated the effects of various stimuli including extracellular tonicity on ET synthesis in porcine IMCD cells in culture. IMCD cells produced ET in a saturationlike time-dependent manner over a period of 24 h. Neither AVP (10(-7) mol/L), bradykinin (10(-7) mol/L), nor atrial natriuretic peptide (10(-7) mol/L) affected basal ET synthesis of IMCD cells at extracellular isotonicity (323 mOsm/kg H2O). The calcium ionophore A23187 (10(-7) mol/L) increased ET production by 38% within 2 h (P < .05). Preincubation for 48 h with increased osmolality in the incubation media from 323 to 600 mOsm/kg H2O by raising the concentrations of 1) NaCl (n = 6), 2) urea (n = 6), or 3) NaCl+urea (n = 6) increased ET synthesis from a control value of 225 +/- 25 pg/mg cell protein/2 h in isotonic medium to 1) 555 +/- 13 pg/mg cell protein/2 h (P < .01), 2) 354 +/- 18 pg/mg cell protein/2 h (P < .05), and 3) 448 +/- 22 pg/mg cell protein/2 h (P < .05), respectively, in hypertonic media. These data suggest that increases in papillary osmolality are associated with enhanced ET synthesis possibly involving a calcium-dependent process and attenuating AVP-dependent fluid absorption in a short-loop feedback fashion.

In porcine kidneys we investigated the characteristics of endothelin (ET) receptors that are present in papillary tissue but not in glomeruli. Therefore, porcine inner medullary collecting duct (IMCD) cells were separated by Percoll density gradient centrifugation after enzymatic and hypotonic treatment of minced papillary tissue. Studies were performed in fresh cell suspensions and in cells in primary culture. Changes in cytosolic free Ca2+ concentration [Ca2+]i were measured by the use of fura-2. Optimum binding of ET-1 was obtained by incubation for 120 min at 37 degrees C, pH 7.0 when maximal protein content was 40 micrograms. Analysis with the LIGAND program showed an average number of binding sites (Bmax) of 26.0 +/- 30.5 fmol/mg protein and dissociation constant (Kd) of 90.5 +/- 28.6 pmol/L for ET-1 and Bmax of 246.9 fmol/mg protein and Kd of 162.5 pmol/L for ET-3. ET-1, 10(-9) to 10(-6) mol/L, dose dependently raised [Ca2+]i four to tenfold, respectively, from a mean basal level of 41 nmol/L. This rise was significantly attenuated by TMB-8 and by verapamil. Preincubation with Ni2+ almost completely prevented the increment in [Ca2+]i. ET-1 slightly suppressed basal and significantly attenuated arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) synthesis. Thus, porcine IMCD cells possess a single class of super high affinity ETB receptors (ETB1). ET-1 raises [Ca2+]i through release from intracellular stores, activation of L-type calcium channels and, probably to a larger extent, through stimulation of other channels, eg, T-type calcium channels or unselective cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)

1. In the present study we investigated, first, the effects of high Na+ intake and, second, the effects of water deprivation on plasma endothelin-1 concentration and urinary endothelin-1 excretion and on endothelin receptors in membranes of renal glomeruli and papillae and of aortic smooth muscle and lung tissue from 32 female Sprague-Dawley rats. 2. After 5 weeks of high Na+ intake (n = 8) urinary Na+ excretion was 10.5 +/- 1.3 compared with 1.6 +/- 0.2 mmol/24 h in controls. Body weight, plasma osmolarity, plasma endothelin-1 concentration (23 +/- 6 versus 28 +/- 3 fmol/ml) and urinary endothelin-1 excretion (6.1 +/- 1.3 versus 4.7 +/- 0.3 pmol/24 h) remained unchanged. 3. The characteristics of endothelin-1 receptors in glomeruli, papillae, aortic smooth muscle and lung tissue from salt-loaded rats were not different from those of controls. 4. After 48 h water deprivation (n = 8) body weight had decreased, whereas packed cell volume and plasma and urine osmolarities had increased compared with controls (n = 8) (P < 0.05). Plasma endothelin-1 concentration (40 +/- 6 versus 21 +/- 2 fmol/ml) was higher (P < 0.01) and urinary endothelin-1 excretion (1.0 +/- 0.2 versus 2.8 +/- 0.3 pmol/24 h) was lower than in controls (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic fate of endogenous diacylglycerol (DAG) in cultured A10 smooth muscle cells was determined. Preincubation of A10 cells with [3H]myristic acid or [3H]arachidonic acid resulted in preferential labeling of phosphatidylcholine (PC) or phosphatidylinositol (PI), respectively. Addition of PC-specific phospholipase C (PC-PLC) to [3H]myristate-labeled A10 cells resulted in a 10-fold increase in radiolabeled DAG, which was converted to monoacylglycerol (MG) and fatty acid (FA). DAG degradation and MG formation was inhibited by tetrahydrolipstatin, a DAG lipase inhibitor. PC-derived DAG was not converted to phosphatidic acid; in addition, PC resynthesis or triacylglycerol synthesis was not observed. Addition of PI-specific PLC (PI-PLC) to [3H]arachidonate-labeled A10 cells resulted in a modest increase in radiolabeled DAG that was also hydrolyzed to MG and FA. Therefore, the principal metabolic fate of endogenous DAG generated from membrane phospholipids by treatment of A10 cells with PC-PLC and PI-PLC was hydrolysis by a DAG lipase pathway.

The metabolism of endogenous and exogenous diacylglycerol (DG) was studied in SM-3 cells derived from rabbit aortic smooth muscle to determine the biochemical step(s) responsible for degrading DG second messengers. Incubation of growth-arrested SM-3 cells with [3H]myristate for 2 h resulted in selective labelling of cellular phosphatidylcholine (PC). Addition of bacterial PC-specific phospholipase C (PC-PLC, 20 U) to [3H]myristate-labelled SM-3 cells resulted in a 7.4-fold increase in endogenous radiolabelled DG and activation of the epsilon isoform of protein kinase C. Subsequent incubation of PC-PLC-treated SM-3 cells resulted in the incorporation of radioactivity primarily into products of a lipase pathway, monoacylglycerol and fatty acid. The hydrolysis of endogenous PC-derived DG was inhibited by 0.25 microM tetrahydrolipstatin, a DG lipase inhibitor. Similar results were obtained when SM-3 cells were incubated with 1,2-dioctanoyl-[2-3H]glycerol, a cell-permeable DG analog; radioactivity was mainly recovered in the glycerol product of the lipase pathway. Therefore, hydrolysis by a lipase pathway represents the principal metabolic fate of both endogenous and exogenous DG in SM-3 smooth muscle cells.