Effects of guanine nucleotides on vasopressin-induced water flow and sodium transport of the frog bladder

Abstract

In the present study, we examined the effects of guanine nucleotides on vasopressin-induced osmotic water flow and sodium transport in the 14-h preincubated frog bladder. We also examined the effects of the adenylate cyclase-cyclic AMP and cyclic AMP-dependent protein kinase system in the bladder's epithelial cells. Gpp(NH)p significantly enhanced vasopressin-induced water flow while it did not affect cyclic AMP-induced water flow. However, Gpp(NH)p did not enhance the vasopressin-induced increment of sodium transport across the frog bladder. The adenylate cyclase activity of the crude homogenate was enhanced by vasopressin, Gpp(NH)p and NaF. The effects of Gpp(NH)p and vasopressin, at their maximum doses, on the enzyme activities were additive, while other combinations were not. Specific Gpp(NH)p binding sites were found in the pellet fraction after 2,400 X g centrifugation. No direct effect on the protein kinase activity was observed in the presence of 10(-6) M nucleotides, such as GTP, GDP, GMP, CTP, UTP, ITP and Gpp(NH)p. Cyclic AMP stimulated the phosphorylation of discrete protein bands, however, Gpp(NH)p did not influence cyclic AMP-dependent protein phosphorylation of crude homogenate of the bladder's epithelial cells. These results suggest the guanine nucleotides stimulate the vasopressin-induced osmotic water flow in frog bladder by enhancing the vasopressin-mediated adenylate cyclase activity, so that accumulated cyclic AMP might activate cyclic AMP-dependent protein kinase.

The effects of PGE2 on vasopressin- and guanine nucleotide-mediated adenylate cyclase activity in toad bladder membrane.

“…By 12 to 14 hr incubation in Ringer's solution containing no glucose, substrate (Maffly and Edelman 1963) and hormone (Marumo 1968) in the toad bladder cells are depleted. In these condition, guanine nucleotides such as Gpp(NH)p enhance vasopressin-induced water flow in the toad bladder (Marumo 1978) and stimulate the adenylate cyclase activity in the frog bladder (Shimada et al 1983), as previously reported. POE stimulates the adenylate cyclase activity in the kidney (Herman et al 1980) and the platelet membrane (Stein and Martin 1983).…”

“…Each hemibladder was mounted in a glass chamber. To prevent mechanical distortion of the membrane, both orifices of the chamber were covered with a nylon mesh as previously described (Shimada et al 1983). To measure the osmotic water flow volumetrically, Ringer's solution, diluted by one-fifth, was introduced into the mucosal chamber.…”

“…Endogenous protein phosphorylation of a crude homogenate of toad bladder epithelial cells was determined by the method of Rudolph and Krueger (1979), with slight modification (Shimada et al 1983). The total volume of the reaction mixture was 200 containing 50 mM Hepes, pH 7.0, 10 mM MgC12 crude homogenate of the toad bladder epithelial cells (100 pg protein), with and without cyclic AMP.…”