Protein kinase C and regulatory volume decrease in mudpuppy red blood cells

This study examined whether protein kinase C (PKC) stimulates K+ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 micrometer), but not with the cyclic nucleotide-dependent kinase antagonists N-(2'-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 micrometer) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 micrometer). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 micrometer). The effect of these three antagonists and H-7 was reversed with gramicidin (5 micrometer in a choline Ringer), indicating PKC was linked to K+ efflux (gramicidin is a cationophore that was used to ensure a high K+ permeability). We also measured cell volume recovery from hypotonic shock (0.5x Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 micrometer), sphingosine (100 nm), chelerythrine (10 micrometer), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 micrometer) nor H-8 (5 micrometer). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 micrometer). Furthermore, using the patch clamp technique, we found H-7 (10 micrometer) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K+ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K+ loss during RVD is regulated, at least in part, by protein kinase C.