Dihydropyridine Ca2+ channel antagonists inhibit the salvage pathway for DNA synthesis in human vascular smooth muscle cells

doi:10.1016/0922-4106(93)90152-Y

European Journal of Pharmacology: Molecular Pharmacology

Volume 244, Issue 3, 15 February 1993, Pages 269-275

https://doi.org/10.1016/0922-4106(93)90152-Y Get rights and content

Abstract

We examined the mechanisms by which Ca²⁺ channel antagonists inhibit the growth of smooth muscle cells by determining their effect on epidermal growth factor (EGF)-stimulated (i) induction of the early signalling gene, c-fos, (ii) incorporation of [³H]thymidine into cells as a measure of DNA synthesis, and (iii) increase in cell number. Verapamil, diltiazem, and the dihydropyridines felodipine, MDL 72892 A-15 (MDL) and nisoldipine had no effect on EGF-stimulated c-fos mRNA induction. Furthermore, only small inhibitory effects were observed on EGF-stimulated increases in cell number; felodipine, MDL, and nisoldipine at 0.3 μM inhibited EGF-stimulated cell proliferation by 9, 11, and 15%, respectively. In contrast, the dihydropyridine Ca²⁺ channel antagonists were found to be potent inhibitors of [³H]thymidine incorporation suggesting that they inhibit DNA synthesis. However, further examination revealed that the potent effects of dihydropyridine Ca²⁺ channel antagonists on [³H]thymidine incorporation were due not to an effect on incorporation of [³H]thymidine into DNA, but to a marked inhibitory effect on the cellular uptake of [³H]thymidine. Thus, we conclude that the small antiproliferative effects of the dihydropyridine antagonists are predominantly due to their ability to inhibit the activity of the salvage pathway for thymidylate synthesis in human vascular smooth muscle cells.

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Citation Excerpt :
In earlier studies, cell proliferation was measured by the uptake of [3H]-thymidine into DNA. The problem with this approach was revealed in later studies, which showed that calcium channel antagonists inhibit the uptake of [3H]-thymidine at the cellular level rather than inhibition of uptake into DNA [18,19]. New approaches using cell cycle specific antibodies have shown that calcium channel antagonists do not exert an inhibitory effect simply by decreasing [Ca2+]i, but that other more complex factors are probably involved [20].
Background: The potential of the calcium channel antagonist verapamil to cause apoptosis (programmed cell death) is of considerable importance in arterial injury where the loss of smooth muscle cells may contribute to a reduction in intimal hyperplasia development. The aim of this study was to determine whether verapamil induces vascular cell apoptosis after carotid artery synthetic grafting.
Methods: Thirty-two adult-female Merino sheep received gelatin sealed fusiform shape-Dacron grafts into the left common carotid artery at day 0. After operation animals were randomly allocated to either a control group or one of three treatment groups (groups 2, 3, and 4). Group 1 animals (n = 9) received no treatment. For the treatment groups, intravenous verapamil was given at a rate of 0.5 mg/kg per day in two divided doses. Group 2, 3, and 4 sheep were treated for 1, 2, and 4 weeks, respectively. Animals were sacrificed at 4 weeks. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-fluorescent labelling. Proliferating cells and their phenotype were determined by doublestaining with antiproliferation cellular nuclei antigen and anti-α-actin or anti-HAM-56.
Results: There were significantly more apoptotic cells in the perigraft adventitia in the 4-week treatment group than in the control group (P <0.05). The average number of proliferating cells at 2 and 4 weeks in the intima were significantly less than in the control (P <0.05). The average numbers of macrophages inside graft matrix in the 2 and 4 weeks treatment groups were significantly less than for the control (P <0.05). The number of proliferating cells inside the graft was significantly lower at 4 weeks compared with control (P <0.05). There was negative correlation between intimal PCNA expression and perigraft apoptotic expression level (P <0.05).
Conclusion: The antihypertensive agent verapamil inhibits intimal hyperplasia through enhancing adventitial cell apoptosis and inhibiting intimal cell proliferation after vascular grafting.
Differential effects of Ca2+ channel blockers on Ca2+ transients and cell cycle progression in vascular smooth muscle cells
1998, European Journal of Pharmacology
We examined the differential effects of Ca²⁺ channel blockers on the elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and G₀/G₁ transition induced by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells in primary culture. The phase of the cell cycle was determined by an immunocytochemical analysis of cell cycle-specific nuclear antigens. [Ca²⁺]_i was monitored by fura-2 microfluorometry. The efficacy of Ca²⁺ channel blockers for the inhibition of [Ca²⁺]_i elevation induced by PDGF (NiCl₂>isradipine>verapamil=diltiazem) did not parallel that for the inhibition of cell cycle progression induced by PDGF (verapamil=diltiazem>NiCl₂>isradipine). In addition, no significant correlation was observed between the extent of [Ca²⁺]_i elevation and the extent of G₀/G₁ transition. We thus conclude that the inhibitory effects of Ca²⁺ channel blockers on the G₀/G₁ transition induced by PDGF are not simply due to their inhibitory action on the [Ca²⁺]_i elevations but instead are due to more complex unknown factors.
Inhibitory effect of calcium channel blockers on human mesangial cell growth: Evidence for actions independent of L-type Ca2+ channels
1996, Kidney International
Inhibitory effect of calcium channel blockers on human mesangial cell growth: Evidence for actions independent of L-type Ca²⁺ channels. 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 10⁻⁵M) 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 10⁻⁵M) (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 10⁻⁸M) 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 benzodiazepine diltiazem was conspicously less effective. Even at the lowest effective concentration (10⁻⁸M) 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.
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Regular paperDihydropyridine Ca2+ channel antagonists inhibit the salvage pathway for DNA synthesis in human vascular smooth muscle cells

Abstract

J. Clin. Invest.

Anal. Biochem.

J. Pharmacol. Exp. Ther.

Hypertension

Atherosclerosis

Atherosclerosis

Biochim. Biophys. Acta

J. Cell. Physiol.

New Engl. J. Med.

Eur. J. Biochem.

J. Clin. Invest.

J. Clin. Invest.

In situ detection of TGF-α and EGF receptor mRNA in human atheromatous plaque tissue

Arteriosclerosis Thromb.

Growth factor activity of endothelin on vascular smooth muscle

Am. J. Physiol.

Growth factor activity of endothelin on vascular smooth muscle

Cell Physiol.

Ethylisopropylamiloride-sensitive pH control mechanisms modulate vascular smooth muscle cell growth

Am. J. Physiol.

Ethylisopropylamiloride-sensitive pH control mechanisms modulate vascular smooth muscle cell growth

Cell Physiol.

Is epidermal growth factor present in human blood? Interference with the radioreceptor assay for epidermal growth factor

Biochem. Biophys. Res. Commun.

Regular paper
Dihydropyridine Ca²⁺ channel antagonists inhibit the salvage pathway for DNA synthesis in human vascular smooth muscle cells