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.
Light
D B
DB
Department of Biology, Ripon College, Ripon, WI 54971, USA.
Adler
M R
MR
Ter Beest
J K
JK
Botsford
S A
SA
Gronau
R T
RT
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002118:Calcium / Q000502:physiology; D048429:Cell Size; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects* / Q000648:ultrastructure; D006096:Gramicidin / Q000494:pharmacology; D006400:Hematocrit; D007038:Hypotonic Solutions / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D009337:Necturus; D009996:Osmotic Fragility / Q000187:drug effects; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D010617:Phenanthridines / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D013449:Sulfonamides
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D010617:Phenanthridines; D015221:Potassium Channels; D013449:Sulfonamides; C116174:bisindolylmaleimide II; D006096:Gramicidin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; C016299:chelerythrine; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D002118:Calcium