Calphostin C

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Calphostin C is a potent inhibitor of protein kinase C (IC50 = 0.05 mM). Calphostin C also inhibits protein kinase A at much higher concentrations (IC50>50mM).

Calphostin-c inhibits protein kinase C (PKC) isoenzymes by covalent modification of the lipid binding regulatory domain. Exposure of cells to calphostin-c elicits PKC independent effects including disruption of intracellular transport, growth inhibition, and stimulation of apoptosis suggesting actions at additional targets. Phospholipase D (PLD) enzymes are targets for activation by PKC /so/ ... the PKC isoenzyme selectivity for activation of two mammalian PLD enzymes, PLD1 and PLD2, by PKC /were investigated/. ..../Examining/ the sensitivity of this process to widely used PKC inhibitors /resulted in/ the surprising finding that calphostin-c is a potent direct inhibitor of PLD1 and PLD2. In vitro, calphostin-c inhibits activity of both PLD1 and PLD2 with an IC(50) of approximately 100 nM. Inhibition is not overcome by protein and lipid activators of these enzymes and does not involve blockade of phosphatidylinositol 4,5-bisphosphate-dependent PLD binding to substrate containing liposomes. Studies using a series of deletion and point mutants of the enzymes suggest that calphostin-c targets the PLD catalytic domain. Inhibition of PLD by calphostin-c in vitro involves stable and apparently irreversible modification of the enzyme. Activity of both PLD1 and PLD2 can be inhibited by calphostin-c treatment of intact cells in a manner that is independent of upstream actions of PKC. Our results suggest that inhibition of PLD1 and PLD2 may explain some of the PKC-independent effects of calphostin-c observed when the compound is applied to intact cells.

Vascular smooth muscle cells (VSMCs) play a major role in the development of atherosclerotic and restenotic lesions. The apoptotic process has been implicated in the development of this pathology. ... This study ... characterized the induction of apoptosis by calphostin C (CC), a protein kinase C (PKC) inhibitor, in primary human coronary artery smooth muscle cells in the presence and absence of insulin-like growth factor-I (IGF-I). Additionally, ... signal transduction pathways important for IGF-I mediated protection /were investigated/. Calphostin C induced apoptosis, as measured by terminal deoxy-UTP nick-end labeling (TUNEL), in a time- and dose-dependent manner, approaching 20% within 6 hr of 50 nM calphostin C treatment. The amount of apoptosis increased to 44.58+/-8.08%, 47.54+/-1.66% and 78.1+/-11.9% after 8, 10 and 12 hr of treatment, respectively (p<0.01 vs. control). IGF-I offered significant protection (p<0.05) at 8 and 10 hr of treatment (60.6% and 52.5% protection, respectively). DNA ELISA confirmed the apoptotic effect of calphostin C and the protective effect of IGF-I. After 6 hr of calphostin C treatment, DNA ELISA revealed 11.20+/-1.53 fold greater apoptosis as compared to baseline values. IGF-I treatment offered a level of protection of 46.6% as measured by DNA ELISA (p=0.06). Apoptosis was further qualitatively confirmed by time-lapse video microscopy and scanning electron microscopy. Interestingly, inhibitors of phosphatidylinositol-3-kinase (PI-3-K), p38 and extracellular regulated kinase (ERK) activation significantly (p<0.05 vs. calphostin C only treatment) increased apoptosis when used in conjunction with calphostin C. Inhibitors of phospatidylinositol-3-kinase and ERK activation reversed IGF-I protection. However, the p38 inhibitor SB203580 failed to reverse IGF-I protection. ...