Effect of serum withdrawal on the contribution of L-type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells.
Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.
Patel
Mahendra K
MK
Clinical Pharmacology, NHLI Division, Faculty of Medicine, Imperial College London, St Mary's Campus, South Wharf Road, London W2 1NY.
Clunn
Gerard F
GF
Lymn
Joanne S
JS
Austin
Oneka
O
Hughes
Alun D
AD
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D017311:Amlodipine / Q000494:pharmacology; D000077214:Becaplermin; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects* / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D012501:Saphenous Vein / Q000166:cytology; D014700:Verapamil / Q000494:pharmacology
D000199:Actins; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D016895:Culture Media, Serum-Free; C419385:L-type calcium channel alpha(1C); D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D017311:Amlodipine; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D014700:Verapamil; D002118:Calcium