Contractile rabbit aortic smooth muscle cells in culture. Preparation and characterization.

Investigations were conducted to determine whether rabbit aortic smooth muscle cells (SMC) reproduce their essential in situ features in culture. Enzymatically isolated cells in culture were compared with their in situ state in terms of myosin and caldesmon isoform expression, sensitivity to Ca(2+)-mobilizing agonists, and contractility. Protein marker expression was assessed by electrophoresis and quantitative immunoblotting, and intracellular free Ca2+ ([Ca2+]i) measurements were accomplished using indo-1, a Ca(2+)-sensitive fluorescent dye. Contraction of SMC grown on deformable silicone films was monitored optically. Before the onset of cell division (3 to 6 days in culture), SMC still contained significant although decreasing amounts of smooth muscle myosin (SM1 and SM2 isoforms) and they started to express nonmuscle-type myosin. The relative content of 150-kDa caldesmon decreased, whereas the expression of 77-kDa caldesmon increased during this period. In the confluent primary culture (11 days), SM1 was expressed, but 150-kDa caldesmon was hardly detectable. Histamine (10(-5) mol/L), serotonin (10(-6) mol/L), and thrombin (1.5 units/mL) contracted deendothelialized rings of rabbit aorta, but only histamine was able to elevate [Ca2+]i 2.5- to 3-fold and induce reversible contraction of primary nondividing cells. [Ca2+]i elevation in response to histamine was due both to Ca2+ mobilization from intracellular stores and Ca2+ flux across the plasma membrane. After the onset of proliferation, SMC regained the ability to elevate [Ca2+]i in response to serotonin and thrombin but lost the ability to contract. Thus, primary cultured quiescent rabbit aortic SMC (3 to 6 days in culture) retain the essential features of vascular SMC in situ (eg, smooth muscle specific contractile and regulatory proteins, vasoactive hormone sensitivity, and contractility).

Mechano-chemical control of human endothelium orientation and size.

Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.

Agonist-induced polyphosphoinositide breakdown in cultured human endothelial and vascular smooth muscle cells.

Human aortic endothelial cells and smooth cells (SMC) from human aorta and coronary arteries were grown in culture. Subcultured vascular SMC retained several important features of human vascular SMC in situ, for example, vimentin-type intermediate filaments, smooth muscle myosin, a well-developed microfilament system, and expression of caldesmon protein involved in the regulation of contraction in smooth muscle. Aortic endothelial cells were shown to possess functional receptors to histamine, thrombin, serotonin, acetylcholine, bradykinin, platelet activating factor (PAF), angiotensin II, vasopressin, prostaglandin E2 (PGE2), and U46619, a stable analog of thromboxane A2. All these substances stimulated polyphosphoinositide (PPI) breakdown in endothelium. Thrombin, histamine, and PAF were the most potent activators. The response of aortic SMC to the same panel of agonists were different. Serotonin, histamine, and angiotensin II produced higher levels of inositol phosphates (IP, IP2, IP3) in SMC than in endothelium. Responses to acetylcholine, bradykinin, and PGE2 were weak and inferior to those of endothelial cells. Other agents evoked approximately equivalent responses in both cell types. Coronary artery SMC resembled aortic SMC in the high extent of PPI hydrolysis after stimulation with serotonin and histamine. The complete inability of angiotensin II and vasopressin to cause accumulation of inositol phosphates in coronary SMC contrasted with the presence of functional receptors to these hormones on aortic SMC. We conclude that the effect of vasoactive agents on human vascular cells may be realized via activation of PPI hydrolysis. Agonists with reported strong vasoconstrictor action seem to stimulate preferential PPI hydrolysis in SMC, whereas endothelium-dependent relaxers cause more pronounced PPI breakdown in endothelial cells. Peculiarities of angiotensin II and vasopressin receptor expression and/or coupling in human aorta and coronary artery SMC may be relevant for understanding the selective action of agonists on human vessels.