Date of Award

1981

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

College

College of Graduate Studies

First Advisor

Perry V. Halushka

Second Advisor

Jerry G. Webb

Third Advisor

George E. Lindenmayer

Abstract

The toad urinary bladder and epithelial cells isolated from it were found to synthesize prostaglandin E (PGE) and thromboxane A2 (TXA2). The syntheses of both of these compounds were found to be stimulated by arginine-vasopressin and by its non-pressor antidiuretic analog I-deamino-8-D-arginine-vasopressin. cAMP, a putative second messenger for vasopressin in the toad bladder, when added to the incubation media, did not affect TXA2 synthesis. However, in the isolated cells both TXA2 and PGE syntheses were found to be stimulated by calcium. In studies using the isolated toad bladder, inhibition of TXA2 synthesis with imidazole (1 mM) and 7-(1-imidazolyl)-heptanoic acid (50-100 µM) was found to inhibit vasopressin-stimulated water flow to a maximum of 30%. Similarly, an antagonist of TXA2 action, trans-13-azaprostanoic acid (50-300 µM) inhibited vasopressin-stimulated water flow in a dose-dependent fashion to a maximum of 35%, while the biologically inactive cis isomer was without effect. Several compounds which exhibit TXA2-like effects in other systems, (15Z)-hydroxy-9α,11α-(epoxymethano)prosta-5Z,13E dienoic acid (U44069), its 9α,11α-(methanoepoxy) isomer (U46619), and TXB2 , mimicked the hydroosmotic effect of vasopressin. This action was inhibited in a dose-dependent fashion by trans-13-azaprostanoic acid while cis-13-azaprostanoic acid was without effect. Vasopressin was found to elicit an enhancement in 45Ca efflux from prelabelled toad bladder epithelial cells. Compartmental analysis of 45Ca efflux from prelabelled cells revealed three components. Studies using EGTA, lanthanum, and mitochondrial inhibitors suggested that the first component of efflux (S1) represented 45Ca bound to heterogeneous sites on the extracellular surface of the plasma membrane, the second component (S2) represented 45Ca bound to some intracellular site(s), and the third component (S3) was composed of two sites, a slowly exchanging site near the plasma membrane, and mitochondria. Vasopressin reduced the sizes of calcium pools S1 and S3. Exogenous cAMP also reduced the size of S3, but increased the size of S2. The TXA2 mimetic, U46619, acted similarly to cAMP; it decreased the size of pool S3 and increased the size of pool S2. The TXA2 synthesis inhibitor 7-(1-imidazolyl)-heptanoic acid blocked the effect of vasopressin to reduce the size of S3. The TXA2 antagonist trans-13- azaprostanoic acid, in concentrations not lethal to the isolated cells, inhibited vasopressin-stimulated water flow only slightly (17%) and did not significantly alter vasopressin's effects on 45Ca kinetics. However, trans-13-azaprostanoic acid significantly blocked the effect of U46619 to decrease the size of S3. Exogenous PGE 1, 1 µM, a concentration which completely inhibits vasopressin-stimulated water flow, enhanced both the efflux and influx of 45Ca. However, net cellular 45Ca was increased. The increased 45Ca was located entirely in pool S3. These results are consistent with a hypothesis wherein vasopressin causes a net release of calcium from an intracellular storage site, perhaps mitochondria. PGE and TXA2 syntheses are stimulated, perhaps by the altered calcium fluxes. TXA2, like vasopressin, reduces the size of a putative mitochondrial calcium compartment, and inhibition of TXA2 synthesis, or antagonism of TXA2 action, inhibits vasopressin-stimulated water flow. PGE enhances 45Ca uptake into the epithelial cells and increases the size of pool S3, resulting in inhibition of vasopressin-stimulated water flow.

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