gramicidin-a and chelerythrine

The effects of angiotensin II (Ang II) on L-type Ca2+ current (I(Ca,L)) remains controversial. We studied the effects of Ang II on I(Ca,L) in single adult rabbit ventricular myocytes using a perforated patch-clamp technique with gramicidin D. Ang II increased I(Ca,L) in a concentration-dependent manner (EC(50)=0.75 nM). In contrast, in conventional whole-cell patch-calmp, I(Ca,L)ran down gradually and the I(Ca,L) response to Ang II was variable, suggesting the potential loss of diffusible components crucial for the Ang II-induced signaling process. An AT(1) antagonist, CV11974 (0.1 microM), completely inhibited the increase in I(Ca,L) induced by Ang II (0.1 microM), whereas an AT(2) antagonist, PD123319 (10 microM), did not influence the I(Ca,L) increase. Neither pre- nor after-treatment with a Na+/H+ exchange (NHE) inhibitor HOE642 (1 microM) affected the Ang II-induced increase in I(Ca,L). The protein kinase C (PKC) inhibitor chelerythrine (1 microM) did not affect the Ang II-induced I(Ca,L) increase. The present findings indicate that Ang II increases I(Ca,L) via AT(1) receptors in adult rabbit ventricular myocytes. Neither the activation of NHE nor PKC may contribute to the Ang II-induced activation of I(Ca,L).

Topics: Action Potentials; Alkaloids; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Anti-Bacterial Agents; Benzimidazoles; Benzophenanthridines; Biphenyl Compounds; Calcium Channels, L-Type; Cell Separation; Electrophysiology; Enzyme Inhibitors; Gramicidin; Guanidines; Heart; Heart Ventricles; In Vitro Techniques; Male; Myocardium; Patch-Clamp Techniques; Phenanthridines; Protein Kinase C; Rabbits; Receptor, Angiotensin, Type 1; Sodium-Hydrogen Exchangers; Sulfones; Tetrazoles

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.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cell Size; Enzyme Inhibitors; Erythrocytes; Gramicidin; Hematocrit; Hypotonic Solutions; Indoles; Ion Channel Gating; Ion Transport; Isoquinolines; Maleimides; Models, Biological; Necturus; Osmotic Fragility; Osmotic Pressure; Patch-Clamp Techniques; Phenanthridines; Potassium Channels; Protein Kinase C; Sulfonamides