Evidence for the involvement of K+ channels and K+-Cl- cotransport in the regulatory volume decrease of newborn rat cardiomyocytes

Tn order to delineate ion transport mechanisms involved in volume homeostas is of freshly isolated newborn rat ventricular myocytes, we investigated th e effects of ion substitutions and pharmacological maneuvers upon (1) isoto nic volume, (2) hypotonically induced initial swelling, and (3) the subsequ ent regulatory volume decrease (RVD), as determined by electronic cell sizi ng. Cardiomyocytes exposed to hypotonic medium (176 mosmol/l) swelled by 51 +/-1% of isotonic volume, and they underwent a partial regulatory volume de crease (RVD), reaching a maximum regulation after 30 min (51+/-1% of initia l swelling), with a half-time (T-1/2) Of 6+/-1 min (n=60). RVD was associat ed with significant cardiomyocyte K+ loss (12+/-4% at 5 min and 15+/-2% of isotonic control after 30 min: n=6, P<0.001), 71% of which was Cl- dependen t (P<0.05). Within the 30-min experimental time frame, ouabain, a Na+/K+ pu mp inhibitor, had no significant effect on RVD (despite an inhibitory trend ), cell swelling or on isotonic volume (n=6). Bumetanide (50 mu M), a Na+-K +-Cl- cotransport blocker, induced a significant reduction of isotonic cell volume (3+/-2%, n=6, P<0.05), potentiated initial swelling by 16+/-1% (n=8 , P<0.02), and it partially inhibited RVD (24+/-11% at 30 min, n=6), wherea s Na+ omission had no significant effect on isotonic cell volume, cell swel ling or RVD. The effects of bumetanide on initial swelling and RVD were pre vented by gadolinium ion (10 mu M), a stretch-activated cation channel bloc ker (n=5). Quinidine (500 mu M), a non-selective Ca2+-activated potassium c hannel blocker with no side-effects on K+-Cl- cotransport, did not modify i nitial cell swelling, but inhibited RVD (50+/-3% at 5 min, n=9, P<0.01; 22/-3% at 30 min), an effect which was can celled by external Ca2+ chelation with EGTA (n=5), and reproduced by tetraethylammonium (TEA, 20 mM), another K+ channel blocker. 4,4'-Diisothiocyanatostilbene 2,2'-disulfonic acid (DI DS, 100 mu M), a non-selective swelling-activated Cl- channel blocker with marginal side-effects on K+-Cl- cotransport, did not modify initial swellin g, but inhibited RVD to the same extent as quinidine (42+/-3% at 5 min, and 23+/-3% at 30 min, n=15, P<0.05), whereas hypotonic Cl--free solution had no effect on isotonic volume, but potentiated initial swelling by 16+2% (P< 0.05) and fully inhibited RVD (n=5, P<0.001). R(+)-[(2-n-Butyl-6,7-dichloro -2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5yl)-oxy] acetic acid) (DIOA, 80 mu M), a K+-Cl- cotransport blocker (with inhibitory potency toward Ca2+-ac tivated K+ channels), inhibited 87+/-5% of the RVD process at 5 min (P<0.00 1) and 56+/-16% at 30 min (P<0.001), whereas it had a small effect on isoto nic volume (+4%, P<0.01) and initial cell swelling (+2%, N.S.; n=9). In con trast to quinidine, DIOA was able to inhibit Ca2+-omission-resistant RVD (f ull inhibition at 5 min, and 56+/-9% at 30 min; P<0.01, n=5). In conclusion , our results suggest that at least three distinct ion transport mechanisms are involved in the RVD in newborn rat cardiomyocytes: (1) K+ and Cl- chan nels, (2) K+-Cl- cotransport, and (3) Na+-K+-Cl- cotransport.