Protein kinase C inhibitor H‐7 alters the actin cytoskeleton of cultured cells

Abstract

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 microM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 microM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 microM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.

Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes

“…However, we cannot rule out the possibility that the differences observed are due to the fact that staurosporine is a more potent and less specific inhibitor than H-7. When the effects of staurosporine and H-7 on actin microfilaments in PTK2 cells were compared, 20 nM staurosporine (Hedberg et al 1990) gave results similar to those obtained using > 1000 fold more H-7 (30 M, Birrell et al 1989). Similarly, staurosporine inhibits protein kinase C activity at concentrations 1000-fold lower than H-7 (Watson et al 1988).…”

“…Morphological changes in cells treated with staurosporine (Sako et al 1988;Hedberg et al 1990), as well as with H-7 (Birrell et al 1989), have been reported. This phenomenon is thought to result from the inhibition of protein kinase C. However, no evidence for a protein kinase C-like activity in Leishmania has been found to date (Hermoso 1989), though treatment of L. amazonensis with phorbol myristate acetate, an activator of protein kinase C, resulted in membrane and cytoplasmic alterations (Vannier-Santos et al 1988).…”

Combined Effects of H-7 and Cytochalasin B on Outflow Facility in Monkeys

“…Addition of H-7 to cultured cells inhibits acto-myosin contractility, leading to deterioration of the actin microfilament system and perturbation of its membrane anchorage. This, in turn, weakens cell-extracellular matrix focal contacts (more so than cell-cell adherens junctions) in Swiss 3T3 cells, PTK2 cells, chick lens epithelial cells and fibroblasts, porcine or bovine aortic endothelial cells, and HTM cells (Birrell et al, 1989 ;Yu and Gotlieb, 1992 ;Volberg et al, 1994 ;Epstein, Skinner and Roberts, 1997 ;Liu et al, 1998 ;Tian et al, 1998). In living monkeys, H-7 increased outflow facility to a similar degree as CB (Tian et al, 1998).…”

ML-7, Chelerythrine and Phorbol Ester Increase Outflow Facility in the Monkey Eye

“…The serine-threonine kinase inhibitor H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] inhibits actomyosin-driven contractility, leading to deterioration of the actin micro®lament system and perturbation of its membrane anchorage, in several types of cultured cells including human trabecular cells (Birrell et al, 1989;Volberg et al, 1994;Gills, Roberts and Epstein, 1998;Tian et al, 1998a). In living monkeys, H-7 dramatically increases out¯ow facility and decreases intraocular pressure (Tian et al, 1998a), probably by inducing generalized relaxation and apparent expansion of the trabecular meshwork (TM) and Schlemm's canal (Sabanay et al, 2000).…”