Inhibitory effect of calcium channel blockers on human mesangial cell growth: evidence for actions independent of L-type Ca2+ channels

Abstract

Calcium channel blockers (CCB) are known to affect the outcome of glomerulosclerosis in vivo and to suppress mesangial cell proliferation and cytokine production in vitro. It is uncertain, however, whether (i) human adult mesangial cells (HMC) express L-type Ca2+ channels and (ii) whether the effect of CCB on HMC is mediated by inhibition of L-type Ca2+ channels. In single cell preparations of HMC, the L-type Ca2+ channel agonist Bay K 8644 and K+-depolarization of the cell membrane caused a transient increase of cytosolic free Ca2+ ([Ca2+]i) in 60 to 80% of the cells. The CCB verapamil and nifedipine partially inhibited the effect of Bay K 8644 and K+-depolarization on [Ca2+]i. Binding experiments confirmed these functional studies by showing specific binding at the phenylalkylamine binding site of L-Type Ca2+ channels. Quiescent HMC were stimulated with fetal calf serum (FCS) or growth factors (platelet derived growth factor A/B, epidermal growth factor, angiotensin II, endothelin 1) in the presence of various concentrations (10(-10) to 10(-5) M) of different CCB: either (R)-verapamil, (S)-verapamil or the raceme of verapamil, and nifedipine or diltiazem, respectively. In addition, the enantiomers of devapamil were studied, because their action on the L-type Ca2+ channel is more stereoselective than that of the enantiomers of verapamil. At high concentrations (10(-6) to 10(-5) M) (R,S)-verapamil decreased cell numbers in cultures of quiescent HMC, increased LDH in the supernatant, and caused loss of trypan blue exclusion (cytotoxicity). At lower concentrations (R,S)-verapamil showed no cytotoxicity, but had two effects: (1.) concentration dependent (down to 10(-8) M) inhibition of indices of cell proliferation, that is, (i) stimulated (FCS or growth factor) 3H-thymidine incorporation and (ii) increment in cell number; and (2.) inhibition of indices of cell or matrix protein synthesis, that is, (i) stimulated 3H-methionine incorporation and (ii) 3H-proline incorporation. At equimolar concentrations the dihydropyridine nifedipine was equipotent with verapamil, whereas the benzothiazepine diltiazem was conspicuously less effective. Even at the lowest effective concentration (10(-8) M) comparison of (R)- and (S)-verapamil showed no significant difference between the enantiomer with weak or with strong effect on L-type Ca2+ channels, and this was true even when the more stereoselective enantiomers of devapamil were tested. These observations argue against the notion that effects of CCB result from specific interaction with L-type Ca2+ channels. The data are more consistent with the idea that interactions with targets other than L-type Ca2+ channels are involved.