Search

The transport pathways mediating regulatory volume increase (RVI) and beta-adrenergic responses in red cells of the European flounder Platichthys flesus have been investigated. Hypertonic treatment under a low-PO2 atmosphere led to a complete RVI and to a three- to fourfold increase in Na+ influx. The RVI and the activated Na+ influx were blocked by the transport inhibitors amiloride and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), both at a concentration of 10(-4 )mol l-1, and the RVI was abolished in a Na+-free saline, indicating the involvement of a hypertonically induced Na+/H+ exchanger and an accompanying Cl-/HCO3- exchanger. Both the hypertonically induced Na+ influx and the RVI were blocked by oxygenation of shrunk cells. The beta-adrenergic agonist isoproterenol also strongly activated a Na+ influx and caused cell swelling. This response was also inhibited by amiloride and DIDS but was unaffected by oxygenation. Simultaneous application of isoproterenol and hypertonic shrinkage did not lead to additive Na+ influxes, suggesting that both responses were mediated by the same pool of exchangers. Mild cell acidification activated a Na+ influx under iso-osmotic conditions; amiloride caused partial inhibition of this influx, but oxygenation had no effect. Acid-induced and isoproterenol-induced Na+ fluxes were again non-additive. Thus, the Na+/H+ exchanger of flounder red cells is strongly activated by three physiological stimuli: hypertonic shrinkage, beta-adrenergic hormones and cell acidification. Of these responses, only the first is affected by oxygenation, indicating some differentiation of their respective transduction mechanisms. These characteristics contrast with those of the corresponding exchangers from rainbow trout and eel red cells.

The volume sensitivity of different K flux pathways has been determined in trout red blood cells subjected to volume perturbation. Gentle hyposmotic swelling induced a K influx in a Cl-containing saline but not in NO3- or methanesulfonate (MeSF)-containing salines, consistent with the activation of a Cl-dependent flux. Extreme hyposmotic swelling led to larger K fluxes in all salines but with reduced anion discrimination of the Cl-dependent flux. In contrast to these graded responses, isosmotic swelling using ammonium chloride or beta-adrenergic stimulation activated only Cl-dependent fluxes in an all-or-none fashion. The relationship between the hyposmotically and isosmotically induced pathways was studied by coactivation using either ammonium chloride or isoproterenol with anisosmotic treatment. Cells in ammonium chloride-containing hyposmotic salines showed no additive K flux over that induced by hyposmotic treatment alone, indicating that the isosmotically induced Cl-dependent flux was identical to the hyposmotically induced Cl-dependent flux. However, cells coactivated by hyposmotic and beta-adrenergic treatment showed a small Cl-dependent flux in addition to that induced by hyposmotic treatment alone. This small third component was unaffected by anisosmotic treatment. We conclude that the major Cl-dependent and Cl-independent K flux pathways are distinct and separate and that the former has an anion dependence that varies with cell volume and a volume sensitivity that varies with ionic strength.

Electroneutral salt transporters are activated and deactivated by changes to the phosphorylation status either of the transporter itself or of other, as yet unidentified, regulatory proteins. We have studied the effects of an inhibitor of protein tyrosine kinase (PTK), genistein, upon KCl cotransport in trout erythrocytes. We show that Cl-dependent K fluxes activated by physiological stimuli, i.e. oxygenation and beta-adrenergic agonists, are rapidly and completely blocked by genistein, whilst the inactive analogue of genistein, daidzein, had no effect. By contrast, the protein tyrosine phosphatase (PTP) inhibitor, vanadate (V), caused a slow but strong activation of an inactive cotransporter. This vanadate (V) activated flux was inhibited by genistein as well as by the serine/threonine phosphatase (PSP) inhibitor, calyculin A. However, genistein had no effect upon the activation of the cotransporter by the protein (serine/threonine) kinase (PSK) inhibitor, staurosporine, or by N-ethylmaleimide, which also appears to act by inhibiting a PSK. These results are consistent with a sequential scheme of at least two tyrosine phosphorylation events which lie upstream to the serine/threonine phosphorylation sites in the signal transduction pathway leading from stimulus to transporter activation. The regulation of the activity of KCl cotransporter appears to involve a complex series of phosphorylation reactions.

The role of protein phosphatases in the regulation of K flux pathways of the trout red blood cell has been investigated using the phosphatase inhibitors calyculin A and okadaic acid. Both inhibitors completely blocked an oxygenation-activated Cl-dependent K flux with a 50% inhibitory concentration of 17 and 675 nmol/l, respectively, but not the hypotonically activated Cl-independent K uptake. N-ethylmaleimide (NEM) and staurosporine caused an increase in the Cl-dependent flux. In both cases preincubation with calyculin A blocked activation but, when added during activation, it prevented any further increase with NEM but abolished the staurosporine-induced uptake. K uptake that was activated by NEM and "clamped" by calyculin A was volume sensitive, indicating a dual influence on this pathway. Chelerythrine, a protein kinase inhibitor, activated a Cl-independent K uptake that was unaffected by calyculin A. It is concluded that activation and deactivation of both Cl-dependent and Cl-independent pathways require changes in the phosphorylation of an as yet unidentified target protein(s), although with different sets of protein kinase and/or phosphatases. These observations also suggest a complex model of kinase-phosphatase regulation and provide drugs for the pharmacological definition and manipulation of Cl-dependent and Cl-independent K flux pathways.