Effect of vasopressin on intracellular Na+ concentration in cortical collecting duct.

Vasopressin stimulates transepithelial Na+ absorption in the rabbit cortical collection duct (CCD), however, this increase is short-lived and is followed by sustained inhibition of Na+ absorption. Previous studies suggest that increased basolateral Ca2+ influx via a Na+/Ca2+ exchanger may be involved in feedback inhibition of Na+ absorption, accounting for this transient stimulation. The present studies used the Na+ sensitive dye, SBFI, to measure intracellular NA+ ([Na+]i) in the CCD to determine whether either vasopressin or 3'5'cAMP increase cell Na+ in the CCD, thereby increasing Na+/Ca2+ exchange. Resting CCD [Na+]i was 23 +/- 2.5 mM. Both arginine vasopressin (AVP, 23 pM) and 0.1 mM 8-chlorophenylthio-cAMP (8CPTcAMP) transiently increased [Na+]i, which peaked within approximately 250 seconds of their addition and then gradually fell towards baseline. AVP increased [Na+]i from 19.4 +/- 3.6 to a peak of 44.4 +/- 14 mM and 8CPTcAMP increased [Na+]i from 11.8 +/- 3.6 to a peak of 38.7 +/- 6.2 mM. This [Na+]i increase was completely blocked by luminal Na+ removal. Inhibition of the basolateral Na/K ATPase with 10 microM ouabain caused [Na+]i to increase to 71.8 +/- 11.6 mM. These results demonstrate that cAMP and AVP increase CCD [Na+]i via a mechanism involving enhanced luminal Na+ entry. This [Na+]i increase is transient and of sufficient magnitude to enhance basolateral Ca2+ influx, via a Na+/Ca2+ exchanger. This latter event could contribute to feedback inhibition of Na+ absorption in the CCD.

Evidence that separate PGE2 receptors modulate water and sodium transport in rabbit cortical collecting duct.

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.

Muscarinic receptor activation inhibits AVP-induced water flow in rabbit cortical collecting ducts.

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)

Monoclonal antibodies that cross-react with the E1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo.

A panel of four monoclonal antibodies (mAb) were produced that cross-react with representatives of two different togavirus serogroups, namely sindbis (SIN) and Semliki Forest (SF) viruses, by ELISA and ADCMC assays. Three of these mAb, IgG2a and IgG2b isotypes, passively protected C3H/Hej mice against 10 and 100 LD50 of SF challenge and one, IgM, did not protect against either challenge dose, or even at 1 LD50. All these mAb were cross-reactive with the E1 glycoprotein of the viruses by immunoblotting in which three different patterns of reactivity were evident, suggesting that three epitopes were involved. The patterns depended upon whether the mAb recognized E1 extracted from purified virions or infected cells and whether SDS-PAGE and immunoblotting were done in the presence or absence of beta-mercaptoethanol. One mAb (IgM) reacted with nonreduced or reduced E1 from either virions or cells suggesting recognition of a linear epitope. The other three mAb reacted with nonreduced but not reduced E1 from virions suggesting that recognition depends upon conformational epitopes. These three mAb reacted also with nonreduced E1 extracted from SF-infected cells whereas only one reacted with nonreduced E1 extracted from SIN-infected cells.