Thromboxanes (TX), prostaglandins (PG), and leukotrienes (LT) (eicosa­ noids) are metabolites of the the 20-carbon fatty acid arachidonic acid. Current evidence supports potential pathophysiologic roles for these autocoids in many diseases (1-3). While the synthesis and metabolism ofthese auto­ coids have been studied extensively in normal and pathologic states, the study of their receptors has lagged far behind. With the development of drugs that will be targeted to these receptors, there is a pressing need to improve our understanding of the fundamental characteristics of these important receptors.

Thromboxane, prostaglandin and leukotriene receptors.

Selective COX-2 Inhibitors

Effect of prostaglandins on renal salt and water excretion.

Direct inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol.81:643–666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive22Na+ uptake could not be blocked by Ca2+ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 μm at pH 7.8 to >10 μm at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of22Na+ uptake in vesicles that contained 0.5 μm Ca2+. This Ca2+ activity produced a maximal inhibition of22Na+ uptake at pH≥7.4 but had no effect at pH≤7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions.

Fusion of cytoplasmic vesicles with the plasma membrane represents an important mechanism whereby additional transport proteins can be introduced into the cell membrane. Although it is apparent that some epithelial hormone responses, such as the vasopressin-induced change in water permeability, utilize this mechanism, the mechanism of some other responses remains uncertain. In addition, many details concerning the intracellular systems responsible for controlling vesicle translocation and membrane fusion in epithelial hormone responses remain to be elucidated.

Role of Membrane Fusion in Hormonal Regulation of Epithelial Transport

Vasopressin enhances osmotic water flow and sodium transport across the toad urinary bladder by mechanisms involving cyclic AMP and calcium. It is believed that changes in intracellular calcium concentration or in its binding to membranes may in part mediate the effects of vasopressin. In addition, several agents which alter the response of the toad bladder to vasopressin may also act by altering cellular calcium metabolism. The effects of vasopressin and several agents which modify its effects in the toad bladder were studied on 45Ca fluxes in isolated epithelial cells from the toad bladder. Compartmental analysis of 45Ca exchange revealed three components. Vasopressin reduced the amount of calcium in the most rapidly exchanging pool from 1.67 +/- 0.20 to 0.86 +/- 0.12 nmol/mg of protein (P less than .025) and the most slowly exchanging pool from 2.72 +/- 0.26 to 1.90 +/- 0.34 nmol/mg of protein (P less than .001), while not affecting the intermediate pool. Theophylline, which mimics the natriferic and hydroosmotic effects of vasopressin, also mimicked the effects on 45Ca exchange by vasopressin. Exogenous cyclic AMP and the prostaglandin endoperoxide analog U46619, which mimic the hydroosmotic effect of vasopressin, also reduced the amount of calcium in the most slowly exchanging pool. Prostaglandin E1, which inhibits the hydroosmotic effect of vasopressin at the concentrations used increased the size of the most slowly exchanging pool. These studies suggest that prostaglandins and other agents which alter the effect of vasopressin in the isolated toad bladder may elicit their effects in part by influencing the calcium concentration at some critical site.

45Ca fluxes in isolated toad bladder epithelial cells: effects of agents which alter water or sodium transport.

Ca2+ is thought to play a role in the enhancement of water permeability of toad urinary bladder epithelial cells by antidiuretic hormone (ADH) or theophylline. This study examined the effects of ADH and theophylline on intracellular free Ca2+ ([Ca2+]i) and total cellular exchangeable Ca2+ in isolated toad bladder epithelial cells. ADH or theophylline enhanced water permeability maximally by 15-25 min after a 4-min lag. 45Ca2+ efflux, a probe for total cellular exchangeable (plasma membrane plus intracellular) Ca2+, was enhanced by ADH within 2 min and returned to control by 8 min. Chlortetracycline fluorescence, a probe for intracellular Ca2+ only, was not affected, suggesting that ADH released only plasma membrane-bound Ca2+. Theophylline enhanced 45Ca2+ efflux and decreased chlortetracycline fluorescence, suggesting release of Ca2+ from intracellular sources. Both agents decreased [Ca2+]i as assessed by quin-2 fluorescence with a time course similar to the enhancement in water permeability. The results suggest that the changes in membrane-bound Ca2+ and [Ca2+]i induced by ADH and theophylline may play a role in the enhanced permeability to water in response to these agents.

P. V. Halushka

Papers

45Ca fluxes in isolated toad bladder epithelial cells: effects of agents which alter water or sodium transport.

ADH or theophylline-induced changes in intracellular free and membrane-bound calcium.