Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy

Joshua K. Salabei, Arun Balakumaran, Justin C. Frey, Paul J. Boor, Mary Treinen-Moslen, Daniel J. Conklin

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation ( 3H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80μM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140μM) markedly decreased MTT absorbance (40%) at 120h. VR or VS treatment inhibited 3H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP + puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.

Original languageEnglish (US)
Pages (from-to)265-272
Number of pages8
JournalToxicology and Applied Pharmacology
Volume262
Issue number3
DOIs
StatePublished - Aug 1 2012

Fingerprint

Stereoisomerism
Autophagy
Calcium Channel Blockers
Verapamil
Vascular Smooth Muscle
Smooth Muscle Myocytes
Muscle
Cells
Diltiazem
Vacuoles
Transmission Electron Microscopy
Thymidine
Cell Nucleus Division
Transmission electron microscopy
Blood pressure
Cell proliferation
Lysosomes
Rats
Cell Survival
Western Blotting

Keywords

  • Autophagosome
  • Calcium channel blockers
  • Diltiazem
  • LC3 conversion
  • Mitophagy
  • MTT assay
  • Proliferation
  • Restenosis
  • Smooth muscle cell

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Salabei, J. K., Balakumaran, A., Frey, J. C., Boor, P. J., Treinen-Moslen, M., & Conklin, D. J. (2012). Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy. Toxicology and Applied Pharmacology, 262(3), 265-272. https://doi.org/10.1016/j.taap.2012.04.036

Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy. / Salabei, Joshua K.; Balakumaran, Arun; Frey, Justin C.; Boor, Paul J.; Treinen-Moslen, Mary; Conklin, Daniel J.

In: Toxicology and Applied Pharmacology, Vol. 262, No. 3, 01.08.2012, p. 265-272.

Research output: Contribution to journalArticle

Salabei, JK, Balakumaran, A, Frey, JC, Boor, PJ, Treinen-Moslen, M & Conklin, DJ 2012, 'Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy', Toxicology and Applied Pharmacology, vol. 262, no. 3, pp. 265-272. https://doi.org/10.1016/j.taap.2012.04.036
Salabei, Joshua K. ; Balakumaran, Arun ; Frey, Justin C. ; Boor, Paul J. ; Treinen-Moslen, Mary ; Conklin, Daniel J. / Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy. In: Toxicology and Applied Pharmacology. 2012 ; Vol. 262, No. 3. pp. 265-272.
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