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PubMedDate not supplied

[Features of the preparation and estimation of homogeneity and native state of smooth muscle actin].

It is shown that smooth muscle actin preparations, produced with the help of techniques described in literature, have contaminations of tropomyosin and proteins with the molecular weight of 250 000, 80 000 and 18 000. The composition of contaminations depends on the method of preparation and conditions of dissociation of native actomyosin material. A method is proposed for actin separation from acetone-treated actomyosin followed by salting out with 50 mM MgCl2. Gel-electrophoresis in the presence of Na-dodecylsulphate has shown that the actin preparations are homogeneous and have the same cofactor activity for the myosin ATPase as actin from the rabbit skeletal muscle. It is supposed that in the cytoplasm of smooth muscle cells as well as in the non-muscle ones there is a considerable part of actin is the monomer form. It gets easily lost in the process of fibrillar actin separation. This may account for low actin output in spite of its relatively high contents in smooth muscles.

Actins↗
PubMed1990

Myosin light chain kinase phosphorylation in tracheal smooth muscle.

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.

Animals↗
PubMed2006

LIM-only protein, CRP2, switched on smooth muscle gene activity in adult cardiac myocytes.

Smooth muscle alpha-actin gene activity appears in promyocardial cells well before cardiac myocyte differentiation and is down-regulated during the onset of rhythmic contractility and cardiac morphogenesis. The levels of LIM-only CRP2 correlated well with smooth muscle gene activity. Cardiomyocyte-specific expression of CRP2 in transgenic mice showed robust expression of smooth muscle cell-specific transcripts and protein filaments in the adult heart. Protein transduction of a recombinant CRP2 protein, fused to the protein transduction domain of HIV, into neonatal heart cells induced de novo synthesis of smooth muscle cell-specific transcripts and proteins. The LIM zinc fingers in CRP2 were found to collaborate with Brg1 of the SNF/SWI complexes, recruited serum response factor, and remodeled smooth muscle target gene chromatin through histone acetylation. CRP2 may have a cytoskeletal role, but as a nuclear protein, CRP2 acted as a potent transcription coadaptor that remodeled silent cardiac myocyte chromatin and directed serum response factor-dependent smooth muscle gene activity.

Actins↗
PubMed1994

Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture.

Cultured smooth muscle cells obtained from rat lung periphery (RPC) and proximal pulmonary artery (RSMC) expressed mRNA for serotonin (5-HT) type 2 receptor (5-HT2) and 5-HT transporter (by Northern blot analysis). Functional expression of these genes was evident since both cell types 1) bound 125I-labeled lysergic acid diethylamide (LSD; 5-HT2 receptor antagonist) that was equally effectively displaced by either ketanserin or mianserin; and 2) transported 5-[3H]HT in an imipramine-sensitive manner. Serotonin (10(-9)-10(-5) M) stimulated DNA synthesis (as measured by [3H]thymidine uptake) in RPC and RSMC. The 5-HT-induced increase in DNA synthesis was significantly inhibited in both cell types by the 5-HT2 receptor antagonist, ketanserin (10(-7)-10(-6) M), and by fluoxetine (10(-6) M), a putative 5-HT transport inhibitor. Acute exposure to 5-HT (1-100 microM) caused an abrupt rise in intracellular calcium ([Ca2+]i) in single pulmonary vascular smooth muscle cells as microspectrofluorometrically determined using the calcium-sensitive dye, fura 2. The 5-HT-induced change in [Ca2+]i was completely abolished in the presence of 10(-6) M ketanserin as well as imipramine or fluoxetine (10(-6) M). The calcium transients due to 5-HT persisted in a Na(+)-free condition (in which the transporter activity was completely abolished) and imipramine and fluoxetine (and ketanserin) were effective inhibitors of 5-HT under these conditions. Therefore, the 5-HT2 receptor, but not the transporter, is responsible for initiating the acute effects (e.g., calcium transients) of 5-HT in cultured rat pulmonary vascular smooth muscle cells and fluoxetine (1 microM) may have 5-HT2-receptor antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Direct contractile effect of endothelin-1 on isolated circular smooth muscle cells of guinea pig caecum.

Smooth muscle cells isolated from the circular muscle layers of the guinea pig caecum were used to determine whether endothelin-1 (ET-1) can cause contraction by exerting a direct action on smooth muscle cells. In addition, the inhibitory effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of intracellular Ca2+ release, verapamil, a Ca2+ channel blocker, and removal of extracellular Ca2+ on the ET-1-induced muscle contraction were examined. ET-1 elicited a contractile response of isolated smooth muscle cells in a dose-dependent manner (ED50: 2 nM). TMB-8, verapamil, and removal of extracellular Ca2+ significantly inhibited the contraction produced by ET-1. These results strongly suggest that ET-1 has a direct contractile effect on circular smooth muscle cells of the guinea pig caecum, and that this contraction depends on both intracellular and extracellular Ca2+.

Animals↗
PubMed1975

Changes in myosin distribution in dedifferentiating and redifferentiating smooth muscle cells in tissue culture.

Isolated smooth muscle cells and fibroblasts from the newborn guinea-pig vas deferens were grown in culture. In the first 2 days, all cells characterized as smooth muscle by phase-contrast microscopy reacted intensely with fluoresceinated antibodies against smooth muscle myosin. The fluorescence was in the form of particles (termed here "myosin aggregates"), which were often aligned to give the cell a striated appearance. After 3-5 days, coarse fluorescent fibrils were also visible. These were termed "attachment fibrils" ("A-fibrils") since they were thought to represent myosin in microfilament bundles. Between 6 and days in culture, the smooth muscle cells began to dedifferentiate morphologically. At this time, the "myosin aggregates" became clumped and less intensely fluorescent. "A-fibrils" also decreased in fluorescence intensity. By 8 days in culture, the dedifferentiated cells had undergone intense proliferation and gave only a minimal reaction with myosin antibodies. However, when a confluent monolayer of cells formed on day 9 or 10, they immediately began to redifferentiate ultrastructurally and to regain immunofluorescence in both "myosin aggregates" and "A-fibrils". Throughout the entire culture period, cells characterized as fibroblasts by phase contrast microscopy gave only a weak reaction with fluoresceinated antibodies to myosin showing "A-fibrils" but no "myosin aggregates".

Animals↗
PubMed1996

G-proteins in guinea pig airway smooth muscle: identification and functional involvement.

In airway smooth muscle, G-proteins have not been identified directly as yet. This study was an attempt to detect various types of conventional Gi- and Gs-proteins in purified membranes of guinea pig airway smooth muscle and to assess the involvement of the G-proteins in agonist-induced contractile response of the smooth muscle. Immunoblotting using AS/7 antibody which recognizes Gi-1/2 demonstrated the presence of polypeptides of M(r) 34, 41 and 75 kDa. Polypeptides of M(r) 43, 46 and 48 kDa were identified with RM/1 antibody that detects Gs-type G-proteins. The AG/1 antibody that recognizes alpha-subunits common to all Gi- and Gs-proteins detected two polypeptides of M(r) 41 and 75 kDa. Heterotrimeric structure of the G-proteins was confirmed by the identification of a single dense beta-subunit band at M(r) 39 kDa with SW/1 antibody. Pertussis toxin (PT) ADP-ribosylated three Gi alpha polypeptides of M(r) 41, 43, and 62 kDa. On the other hand, cholera toxin (CT) catalysed the ADP-ribosylation of two Gs alpha polypeptides of M(r) 46 and 62 kDa. Both PT and CT attenuated the maximum contractile responses of the airway smooth muscle to the muscarinic agonist, methacholine. Pretreatment of the tissues with the sulphydryl alkylating G-protein inhibitor, N-ethylmaleimide, also inhibited the maximum contractions to methacholine. These data suggest that plasma membranes of guinea pig airway smooth muscle contain a variety of conventional, including Gs and Gi, and other types of G-proteins, and at least a portion of the proteins present may be involved in mediating the contractile responses of the smooth muscle to an agonist such as methacholine.

Animals↗
PubMedDate not supplied

Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells.

Using freshly isolated single smooth muscle cells prepared by collagenase treatment, membrane currents were recorded by whole-cell voltage clamp. Intracellular constituents were modified by using an intracellular perfusion technique, i.e., pipette solutions were continuously exchanged from control to test solutions during current recording. In smooth muscle cells, intracellular application of ATP, but not cyclic AMP, enhanced the amplitude of Ca2+ currents and prevented current run-down. In addition, with this stabilization of Ca2+ current recording by ATP, introduction of various chemicals into the cell using the intracellular perfusion technique is useful for investigations of regulation of ion channels in smooth muscle cells.

Adenosine Triphosphate↗
PubMed1986

Na-H exchange is a major pathway for Na influx in rat vascular smooth muscle.

Rat aortic segments and aortic smooth muscle cells in primary culture were used to examine the importance of the Na-H exchange system in transporting Na into smooth muscle. Ethylisopropylamiloride was approximately 100 times more potent than amiloride at inhibiting Na influx into smooth muscle. In a 135 mM Na-containing medium approximately 80% of the Na influx rate could be inhibited by 100 microM ethylisopropylamiloride. The rate of Na entry into cells was markedly influenced by extracellular and intracellular pH. Elevating extracellular pH from 6.0 to 8.0 increased the Na influx rate. The dependence of the Na influx rate on intracellular pH was demonstrated by acidification of cells with nigericin or preincubation with ammonium chloride. These two procedures increased 22Na+ influx rate by about 3.5-fold. In both instances the increases in 22Na+ influx rate could be completely attenuated by ethylisopropylamiloride. Increases in Na influx rate via the Na-H exchange also increased the activity of the Na-K pump, thereby maintaining intracellular Na content approximately constant. These results indicate that Na-H exchange is a major influx pathway for Na in rat vascular smooth muscle. Activation of this system activates the Na-K pump, which maintains intracellular Na constant.

Amiloride↗