Prostaglandin E2 (PGE2) modulates both water and sodium transport in the rabbit cortical collecting duct (CCD). To determine whether these effects are mediated by separate PGE2 receptors, we compared the effects of PGE2 and its analogue sulprostone in the isolated perfused rabbit CCD. PGE2 increased basal water permeability (hydraulic conductivity), whereas sulprostone did not. PGE2 and sulprostone were equipotent inhibitors of water absorption when it was prestimulated by vasopressin. Pertussis toxin completely reversed the inhibitory effect of sulprostone but only partially reversed the inhibitory effect of PGE2. In contrast, a protein kinase C (PKC) inhibitor, staurosporine, partially reversed the inhibitory effect of PGE2 but had no effect on sulprostone. PGE2 also raised intracellular calcium ([Ca2+]i). This effect is coupled to its capacity to inhibit Na+ absorption. Sulprostone was 10-fold less potent than PGE2 both in raising [Ca2+]i or inhibiting sodium transport. The results suggest sulprostone selectively interacts with a PGE2 receptor coupled to pertussis toxin-sensitive inhibition of water permeability. Sulprostone less potently activates a PGE2 receptor coupled to [Ca2+]i, PKC activation, and sodium transport and completely fails to interact with the PGE2 receptor that stimulates water permeability in the collecting duct. These results suggest distinct PGE2 receptors modulate sodium and water transport in the CCD.
Endothelin-1 (Et) has profound effects on glomerular microcirculation and mesangial cell contraction. A parameter of mesangial cell contraction was examined by measuring myosin light chain phosphorylation (MLCP) in glomerular mesangial cells in the presence and absence of a newly developed endothelin-1 receptor antagonist (EtA). Addition of Et alone (10 nM) caused a marked increase in MLCP, which, on average, rose by 53 +/- 6% above the level in cells exposed to vehicle (P less than 0.0005). This effect was shown to continue for at least one hour; MLCP at 60 minutes was 64 +/- 12% higher than controls, (P less than 0.025), constituting a unique observation of an in vitro parameter which parallels the characteristic in vivo effect of Et. Treatment of cells with EtA virtually abolished this Et-induced increase in MLCP, which rose by only 2 +/- 3% and -1 +/- 4% for doses of EtA of 44 nM and 66 nM, respectively. Examination of the intracellular calcium concentration, [Ca2+]i, revealed that EtA almost completely abolished the transient increase in [Ca2+]i evoked by Et and also suppressed the early portions of the sustained increase in [Ca2+]i. EtA was ineffective in abolishing [Ca2+]i increase in response to arginine vasopressin. Finally, to evaluate EtA's efficacy in a pathophysiologic setting, we also studied mesangial cells exposed to cyclosporine (Cs). Exposure of mesangial cells to Cs (10(-5) M) for 60 minutes caused a significant increase in MLCP, on average, by 38 +/- 6% above control (P less than 0.0005), while cells exposed to Cs in the presence of EtA increased MLCP significantly less, by only 15 +/- 9%. These data provide further evidence for Et's long-lasting cellular actions, and demonstrate inhibitory effects of an Et receptor antagonist after direct cellular exposure to Et and also after Cs exposure, a pathophysiologic setting which likely involves Et.
We examined the effect of carbachol, an acetylcholine analogue, on hydraulic conductivity (Lp) response to 10 microU/ml arginine vasopressin (AVP) in rabbit cortical collecting duct (CCD). In CCDs in which water flow had been established with AVP, subsequent addition of carbachol caused Lp (X10(-7) cm.atm-1.s-1) to fall from 251 +/- 32 to 146 +/- 19. Carbachol washout resulted in recovery of Lp to 217 +/- 38. In CCDs in which water flow had been established using 10(-4) M 8-chlorophenylthioadenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), addition of carbachol had no effect. These posttreatment studies suggest that carbachol's effects on modulating established water flow occur at a "pre-cAMP" step. With carbachol added first, AVP-induced Lp was reduced from 233 +/- 24 (controls) to 105 +/- 19 (carbachol-pretreated). Pretreatment with 10(-6) M atropine, a muscarinic receptor antagonist, totally reversed the inhibitory effect of carbachol, consistent with a receptor-mediated effect of carbachol. Carbachol pretreatment also inhibited 8-CPT-cAMP-induced Lp, indicating that carbachol's effects also occur at a "post-cAMP" step. Pretreatment with 10(-7) M staurosporine, a protein kinase C (PKC) inhibitor, reversed inhibitory effect of carbachol on AVP-induced Lp (193 +/- 26), suggesting that carbachol's effects are mediated by PKC. Intracellular calcium concentration [( Ca2+]i) was measured in fura-2-loaded CCDs. Carbachol also increased [Ca2+]i from 229 +/- 120 to 389 +/- 160 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
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