Cholecystokinin-induced contraction of dispersed smooth muscle cells

Isolated smooth muscle cells were prepared from the fundus of guinea pig stomach by incubation with collagenase. Incubating the cells with the C-terminal octapeptide of cholecystokinin induced contraction, which was measured by micrometry and expressed as percent decrease in mean cell length. Cholecystokinin-induced contraction was maximal within 30 s and reduced cell length by approximately 37%. The threshold concentration of cholecystokinin was 0.1 pM, and the maximally effective concentration was 0.3 nM. Contraction caused by cholecystokinin could be inhibited by proglumide and by glucagon. Inhibition by proglumide was competitive and resulted in a parallel rightward shift of the cholecystokinin dose-response curve. In contrast, inhibition by glucagon was noncompetitive and resulted in a reduction in the efficacy of cholecystokinin without a change in its potency. Furthermore, proglumide-induced inhibition was specific for cholecystokinin, whereas glucagon-induced inhibition of contraction was nonspecific and reduced the contraction caused by carbamylcholine and the calcium ionophore A23187.     

Tuning smooth muscle contraction by molecular motors

As in striated muscle, smooth muscle cells (SMC) contract by Ca²⁺ activated cyclic interaction between actin and type II myosin. However, smooth muscle maintains tone at basal activating Ca²⁺ and low energetic cost during sustained activation. This review analyzes the regulation of phasic and tonic contraction of SMC on the molecular level. Type II myosin is the molecular motor also of smooth muscle contraction. Six myosin heavy chain (MHC) isoenzymes (four smooth muscle, two nonmuscle) and five myosin light chain (MLC) isoforms (two 17 kDa, two 20 kDa, one 23 kDa) are expressed in SMC. These myosin subunits could be generated by alternative splicing or by differential gene expression. Thus different myosin isoenzymes are generated which may be modified posttranslationally by phosphorylation, affecting the contractile state of the SMC. Furthermore, they may be part of distinct contractile systems which are targeted by different second messenger cascades and are recruited differentially during activation, electromechanical, and pharmacomechanical coupling. Low energy consumption, shortening velocity, and MLC20 phosphorylation at low Ca²⁺ activation levels during tone maintenance ("latch") could be explained by a switch from smooth muscle myosin to nonmuscle myosin activation upon prolonged activation.Abbreviations MHC Myosin heavy chains - MLC Myosin light chains - MLCK Myosin light chain kinase - MLCP MLC20 phosphatase - NM Nonmuscle - nt Nucleotide - SM Smooth muscle - SMC Smooth muscle cells     

Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural study of contraction of pulmonary vascular smooth muscle cells.

The pulmonary vessels of rats treated with fulvine were studied electron microscopically for morphologic signs of contraction of smooth muscle cells. Except for a number of indirect indications of vasoconstriction such as medial smooth muscle cell hypertrophy and excessive crenation of elastic laminae, conspicuous smooth muscle cell excrescences were observed and were interpreted to be a direct result of contraction. The close relationship between contraction and smooth muscle cell excrescences was confirmed by their simultaneous occurrence within 1 minute after administration of histamine to isolated perfused guinea pig lungs. The images of pulmonary vessels of rats with prolonged survival times after fulvine administration suggested a gradual increase in the size of the excrescences with a simultaneous degeneration of their cytoplasmic content, and in some cases their eventual detachment from the main cell body. The latter changes were possibly associated with the widespread vasculitis that often occurred at longer intervals after fulvine application. The smooth muscle cell excrescences in pulmonary veins were generally much more prominent than those in pulmonary arteries. This difference was probably caused by the more rigid structure of the arterial wall whcih prevented the formation of large excrescences. The mechanism of the formation of smooth muscle cell excrescences, their possible general validity as markers of vascular smooth muscle cell contraction, and the implications for the mechanism of action of fulvine are briefly discussed.     

Influence of microtubules on vascular smooth muscle contraction

Microtubules are ubiquitous in eukaryotic cells and play key roles in many cellular activities. The purpose of this study was to investigate the influence of microtubules on vascular smooth muscle contraction. Quantitative immunocytochemical analysis of rat aortic tissue revealed that, relative to the control group, colchicine (15 M, 90 min) and nocodazole (15 M, 90 min) decreased the microtubule density by 40–50% while taxol (10 M, 90 min) increased the microtubule density by 33%. Isometric contraction studies demonstrated that both colchicine and nocodazole caused an upward shift in the phenylephrine (10^–8 to 10^–5 M) dose–response curve while taxol caused no significant change when compared to the control group. Potassium chloride (30 mM) induced 55 ± 5% P ₀ contraction in DMSO treated vessel rings. The active tension increased to 73 ± 5% P ₀ and 71 ± 6% P ₀ after pretreatment of the aortic rings with colchicine or nocodazole, respectively. Taxol did not cause a significant change in the active tension (56 ± 7% P ₀). These results indicate that microtubule depolymerization enhances isometric contraction of vascular smooth muscle and this enhanced contraction is not receptor dependent. Pretreatment of the aortic rings with an inhibitor of nitric oxide synthase (NOS) (N -nitro-L-arginine) did not change the increased contractile response to phenylephrine due to microtubule depolymerization suggesting that this phenomenon is not mediated by endothelium dependent relaxation.     

Detection of contraction of isolated smooth muscle cells in suspension.

A Coulter counter has been utilized to assess the contractile state of suspensions of isolated smooth muscle cells prepared by enzymatic digestion of stomach muscularis of Bufo marinus. Pulse-duration and pulse-hieght histograms were constructed from the pulses that occur as cells pass through the Coulter counter orifice. Contraction of cells in suspension causes shifts in these pulse histograms to a greater percent of shorter duration pulses and of larger pulse heights. These shifts are consistent with teh assumption made in out studies that the duration and amplitude of the pulses generated are related to the length and cross-sectional area (or square of the diameter), respectively, of the cell. Shifts in the pulse-height histogram were found to be more reliable. The results of a calibration experiment in which shifts in the histograms of pulse heights were linerrly related to shifts in the histograms of all lengths measured from photomicrographs suggested that the shifts in the pulse-height histogram could be used as a continuous index of changes in the contractile state of a population of cells in suspension.     

Pentobarbital and contraction of vascular smooth muscle.

This study, with isolated rat aortic strips and portal veins, was undertaken to : 1) study the effects, if any, of pentobarbital Na (PTB) (5 x 10(-5) to 2 X 10(-3) M) on reactivity to epinephrine, serotonin, and KCl; 2) determine whether certain concentrations of PTB induce direct actions on aortic strips and portal veins; and 3) gain some insight into how these effects are brought about. The results indicate that PTB can: a) inhibit development of spontaneous mechanical activity in these vessels in anesthetic concentrations; b) dose-dependently attenuate contractions induced by epinephrine, serotonin, and KC1; c) cause a noncompetitive type displacement of the dose response curves of these vasoactive agents; d) attenuate Ca2+- induced contractions of potassium-depolarized aortic strips and portal veins concomitant with a dose-dependent displacement of these dose-response curves to the right; and e) rapidly relax drug as well as Ca2+ -induced contractions of aortas and portal veins. In addition, the data indicate that rat portal venous smooth muscle is more sensitive to the inhibitory actions of PTB than rat aortic smooth muscle. Overall, these data suggest that concentrations of PTB used to induce surgical anesthesia can exert profound depressant effects on at least two different types of vascular smooth muscle that may be related to actions on movement and/or translocation of Ca2+.     

Rho-kinase-mediated regulation of receptor-agonist-stimulated smooth muscle contraction

Rho kinase was shown to regulate smooth muscle contraction through modulating myosin phosphatase (MLCP) activity, but the in vivo mechanism remains to be clarified. This study examined the effects of Rho kinase inhibition on the phosphorylation time course of MLCP subunit MYPT1 at Thr697 and Thr855 and MLCP inhibitory protein CPI-17 at Thr38 and on actin polymerization during the contraction of rat tail artery (RTA) smooth muscle. Rho kinase inhibitor Y27632 suppressed force activated by α₁-adrenergic agonist phenylephrine or thromboxane A₂ analog U46619 with concomitant decreases in MLC₂₀ phosphorylation. Phenylephrine and U46619 significantly increased MYPT1^Thr855 phosphorylation that was eliminated by Y27632 pretreatment, whereas MYPT1^Thr697 phosphorylation was not stimulated. Phenylephrine increased CPI-17^Thr38 phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor Ro 31-8220; in contrast, U46619 did not stimulate CPI-17 phosphorylation. Both agonists increased actin polymerization that was diminished by Y27632 under phenylephrine but not U46619 activation. These results demonstrated a temporal correlation between MYPT1^Thr855 phosphorylation, MLC₂₀ phosphorylation, and contraction in a Rho-kinase-dependent manner for both phenylephrine and U46619 stimulation, suggesting that Rho kinase regulates MLCP activity through MYPT1^Thr855 phosphorylation during RTA smooth muscle contraction. Furthermore, Rho kinase regulates actin polymerization activated by α₁-adrenoceptors but is less significant in thromboxane receptor stimulation.     

Effect of genistein on agonist-induced airway smooth muscle contraction

Inflammation Research - .     

Magnesium dependent contraction of glycerinated smooth muscle

The effect of Mg²⁺ on the contraction of glycerinated smooth and skeletal muscles was examined. In the absence of Ca²⁺, glycerinated smooth muscle contracted slowly when exposed to 5 mM adenosine triphosphate (ATP) and Mg²⁺. The magnitude of this contraction depended on the concentration of Mg²⁺ in a range of 1–20 mM. When 20 mM Mg²⁺ was used with ATP, glycerinated smooth muscle contracted almost maximally in a Ca²⁺ free environment, and it did not contract further on subsequent addition of 0.1 mM of free Ca²⁺ (EGTA-CaEGTA buffer). In contrast, in skeletal muscle a Ca²⁺ dependent contraction could always be elicited in the presence of 1–20 mM Mg²⁺ and 5 mM ATP. Tension development in smooth muscle induced by Mg²⁺ and ATP was not due to a contamination by Ca²⁺ or the removal of a Ca²⁺-sensitive protein. In the present experimental conditions, glycerinated smooth muscle developed a Mg²⁺-dependent contraction in a Ca²⁺ free medium, but glycerinated skeletal muscle did not.     

Cytokeratins in smooth muscle cells and smooth muscle tumours

A keratin positive metastatic leiomyosarcoma in the lung, which resulted in diagnostic error, is reported. The results of additional studies of 17 benign and malignant leiomyogenic tumours with various keratin antibodies are presented and discussed in the light of recent bibliographical data.