Stimulation of Phosphoinositides by Agents that Stimulate Proton Secretion in Toad Urinary Bladder 1

Abstract

Abstract

The urinary bladder of Bufo marinus excretes H+ and this excretion is increased by metabolic acidosis (MA), insulin (IN), prostaglandin E₂ (PGE₂), ↑CO₂, and aldosterone. The purpose of this experiment was to determine whether MA, IN, PGE₂, CO₂, and aldosterone stimulate inositol phosphate's (IP) formation in isolated cells of toad urinary bladder. Cells were prepared by treating bladder sacs with collagenase. Cells were obtained from 10 toads in MA and 10 normal toads, suspended in 2 ml of Ringer's solution containing LiCI (10 mM), myo-inositol (5 mM), and [³H]myo-inositoi (10 μCi), and then incubated for 2 hr at 25°C. Cells were homogenized and the IP fractions quantitated by column chromatography and liquid scintillation counting. The results were expressed as dpm (μMPO₄)^-1 (hr)^-1. The IP in MA cells was 44,202 ± 4,646 and in normal toad cells it was 31,637 ± 3,613 (P < 0.05). In a separate experiment, cells from 10 paired hemibladders were isolated from normal toads. The cells were treated exactly as above except there was no LiCI in the bath. LiCI was added to all baths after 2 hr and the experimental cells were challenged with IN, PGE₂, ↑CO₂, and aldosterone for 20 min. The IP were quantitated as above. IN treatment stimulated inositol bisphosphate and inositol triphosphate (P < 0.01). PGE₂ and ↑CO₂ also stimulated inositol triphosphate (P < 0.05). Aldosterone did not alter formation of any of the IP fractions. We conclude that MA, IN, PGE₂, and ↑CO₂ stimulate IP formation in cells of toad urinary bladder and inositol triphosphate may be an important second messenger in mediating the response of MA, IN, PGE₂, and ↑CO₂.