Abstract
Cerebral blood vessels have unique properties when compared to most peripheral vascular beds. One such property is that large cerebral vessels are involved in the regulation of cerebral vascular resistance. Studying smooth muscle cells isolated from these vessels will determine how phenotypic properties of these cells contribute to unique cerebrovascular function. Therefore we developed a method of culturing smooth muscle cells from explants of cerebral arteries of porcine brains obtained, gratis, from a local slaughter house. Cells isolated and cultured by the methods described herein were of smooth muscle origin as indicated by histochemical staining for smooth muscle α-actin. Further, we examined the response of the cultured cells to agonists which activate the cGMP dependent vasodilator system by stimulating soluble guanylyl cyclase (nitroglycerin and sodium nitroprusside) or particulate guanylyl cyclase (C-type natriuretic peptide). Also, forskolin activation of adenylyl cyclase was examined. These agents stimulated an increase in intracellular cGMP and cAMP in a manner that was reproducible in every cell isolation (20 brains) and which remained unchanged through nine passages. Additionally, the cells could be frozen, thawed and replated without loss of responsiveness to these agents. The protocol reported here provides a method for culturing cerebral artery smooth muscle cells that is inexpensive, relatively simple, and which yields cells that can be utilized through multiple passages.
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Abbreviations
- DMEM:
-
Dulbecco's modified Eagle's medium
- FBS:
-
Fetal bovine serum
- HEPES:
-
N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid
- PBS:
-
Phosphate buffered saline
References
Abboud FM (1981). Special characteristics of the cerebral circulation. Fed Proc 40: 2296–2300.
Albuquerque MLC, Lowery-Smith L, Hsu P, Parfenova H, Leffler CW (1995). Low CO2 stimulates inositol phosphate turnover and increased inositol 1,4,5,-trisphosphate levels in piglet cerebral microvascular smooth muscle cells. Proc Soc Exp Biol Med 209: 14–19.
Altura BM, Zhang A, Cheng TP-O, Altura BT (1993). Ethanol promotes rapid depletion of intracellular free Mg in cerebral vascular smooth muscle cells: Possible relation to alcohol-induced behavioral and stroke-like effects. Alcohol 10: 563–566.
Altura BM, Zhang A, Cheng TP-O, Altura BT (1993). Cocaine induces rapid loss of intracellular free Mg2+ in cerebral vascular smooth muscle cells. Eur J Pharmacol 246: 299–301.
Baron DA, Lofton CE, Newman WH, Currie MG (1989). Atriopeptin inhibition of thrombin-mediated changes in morphology and permeability of endothelial monolayers. Proc Nat Acad Sci USA 86: 3394–3398.
Bennett BM, Leitman DC, Schroder H, Kawamoto JH, Nakatsu K, Murad F (1989). Relationship between biotransformation of glyceryl trinitrate and cyclic GMP accumulation in various cultured cell lines. J Pharmacol Exp Ther 250: 316–323.
Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
Black RS, Thompson MM, Owens GK (1988). Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells. J Cell Biol 107: 299–306.
Buljubasic N, Flynn NM, Marijic J, Rusch NJ, Kampine JP, Bosnjak ZJ (1992). Effects of isoflurane on K+ and Ca2+ conductance in isolated smooth muscle cells of canine cerebral arteries. Anesth Analog 75: 590–596.
Catravas JD, Rubin JW, Stone CD, Cziraki A, Papapetropoulos A (1995). Enzymatic activity and gene expression of soluble guanylate cyclase (sGC) in human vascular tissue. FASEB J 9: A40.
Heistad DD, Marcus ML, Abboud FM (1978). Role of large arteries in regulation of cerebral blood flow in dogs. J Clin Invest 62: 761–768.
Heistad DD, Marcus ML, Sandburg G, Abboud FM (1977). Effect of sympathetic nerve stimulation on cerebral blood flow and on large cerebral arteries of dogs. Circ Res 41: 342–350.
Heistad DD, Busija DW, Marcus ML (1981). Neural effects on cerebral vessels: alteration of pressure-flow relationship. Federation Proc 40: 2317–2321.
Hong Y, Puil E, Mathers DA (1994). Effect of halothane on large-conductance calcium-dependent potassium channels in cerebrovascular smooth muscle cells of the rat. Anesthesiology 81: 649–656.
Lofton CE, Newman WH, Currie MG (1990). Atrial natriuretic peptide regulation of endothelial permeability is mediated by cGMP. Biochem Biophys Res Commun 172: 793–799.
Madden JA, Vadula MS, Kurup V (1992). Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol (Lung Cell Mol Physiol 7) 263: L384-L393.
Nagatani K, Masciopinto JE, Letarte PB, Haworth RA, Duff TA (1995). The effect of hemoglobin and its metabolits on energy metabolism in cultured cerebrovascular smooth muscle cells. J Neurosurg 82: 244–249.
Owens GK, Loeb A, Gordon D, Thompson MM (1986). Expression of smooth muscle-specific α-isoactin in cultured vascular smooth muscle cells: Relationship between growth and cytodifferentiation. J Cell Biol 102: 343–352.
Parfenova H, Hsu P, Leffler CW (1995). Dilator prostanoid-induced cyclic AMP formation and release by cerebral microvascular smooth muscle cells: Inhibition by indomethacin. J Pharmacol Exp Ther 272: 44–52.
Quayle JM, McCarron JG, Asbury JR, Nelson MT (1993). Single calcium channels in resistance-sized cerebral arteries from rats. Am J Physiol 264 (Heart Circ Physiol 33): H470-H478.
Robertson BE, Schubert R, Hescheler J, Nelson MT (1993) cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 265 (Cell Physiol 34): C299-C303.
Schulz R, Krueger C, Manickavel V, Steele JA, Cook DA (1993). Production of 15-HETE by cultured smooth muscle cells from cerebral artery. Pharmacology 46: 211–223.
Tao H, Zhang LM, Castresana MR, Shillcutt SD, Newman WH (1995). C-natriuretic peptide but not atrial natriuretic peptide increases cyclic GMP in cerebral arterial smooth muscle cells. Life Sci 56: 2357–2365.
Wang Y, Baimbridge KG, Mathers DA (1991). Effect of serotonin on intracellular free calcium of rat cerebrovascular smooth muscle cells in culture. Can J Physiol Pharmacol 69: 393–399.
Wang Y, Mathers DA (1993). Ca2+-Dependent K+ channels of high conductance in smooth muscle cells isolated from rat cerebral arteries. J Physiol 462: 529–545.
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Tao, H., Zhang, LM., Castresana, M.R. et al. A method for isolating and culturing vascular smooth muscle cells from porcine cerebral arteries. Methods Cell Sci 18, 41–46 (1996). https://doi.org/10.1007/BF00123522
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DOI: https://doi.org/10.1007/BF00123522