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PubMed1998

Myosin heavy chain isoform expression in rat smooth muscle development.

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.

Aging↗

PubMed1981

Interaction between macrophages and aortic smooth muscle cells. Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL.

Mouse peritoneal macrophages were cultured for 24 h in Dulbecco-Vogt medium containing 10% calf serum. This medium was replaced with Dulbecco-Vogt medium containing 1% bovine serum albumin to which all subsequent additions were made. Medium changes, accompanied by appropriate additions, were made every 48 or 72 h and the media were used for incubation of aortic smooth muscle cells, prelabeled with [3H]cholesterol. The amount of labeled cholesteryl ester in the smooth muscle cells incubated for 48 h with macrophage media which had been collected 48-144 h after addition of acetylated LDL was increased 3-4 times above that present prior to postincubation. A marked increment in cholesteryl ester mass occurred also after incubation of smooth muscle cells with macrophage media conditioned with acetylated LDL and this effect was shared by maleylated LDL, but not by other negatively charged compounds. The increase in labeled cholesteryl ester in smooth muscle cells was more pronounced with media collected at later time intervals of incubation with macrophages and was evident 8 hr after postincubation. Only the d less than 1.063 fraction of the medium enhanced cholesterol esterification in smooth muscle cells. The acetylated LDL reisolated from macrophage media at d less than 1.063 did not compete with native LDL for degradation by smooth muscle cells. No increase in degradation of 125I-labeled acetylated LDL preincubated with macrophages was observed above that of non-preincubated acetylated LDL. The macrophage medium conditioned with acetylated LDL depressed [14C]acetate incorporation into sterols in smooth muscle cells and this effect was abolished by extraction of the medium with diethyl ether. The ratio of free to total cholesterol in the macrophage media collected after incubation with acetylated LDL increased from 28-70%, and a decrease occurred after incubation with smooth muscle cells. The enhancement of cholesterol esterification could be abolished by addition of high density apolipoprotein/sphingomyelin mixture during incubation with macrophages, even though excretion of free cholesterol into the medium increased 3-fold. It is proposed that when smooth muscle cells are presented with a lipoprotein in which an increase in the free to esterified cholesterol ratio occurred, and which is not recognized by a specific receptor, the enhancement of cellular cholesterol esterification is due mostly to a surface transfer of lipoprotein-free cholesterol. The present results offer another view of the possible interactions between macrophages and smooth muscle cells. A modified lipoprotein, not recognized by smooth muscle cells, is ingested by macrophages, which leads to accumulation of esterified cholesterol. Part of the esterified cholesterol undergoes hydrolysis and is excreted back into the medium, leading to enrichment of the lipoproteins in the medium with free cholesterol. This enrichment with free cholesterol promotes cholesterol esterification in smooth muscle cells.

Acylation↗

PubMed1991

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression.

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.

Actins↗

PubMed1992

O-acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase alpha-smooth muscle actin expression in cultured arterial smooth muscle cells.

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.

Actins↗

PubMed2006

Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells.

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific alpha actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

Actins↗

PubMed1981

Smooth muscle myosin in precursor and mature smooth muscle cells in normal pulmonary arteries and the effect of hypoxia.

Exposure to hypoxia increases pulmonary arterial muscularity-in the intra-acinar arteries "new" muscle appears in the normally nonmuscular regions and in the preacinar arteries, medial thickness increases. In the present study by immunofluorescence techniques, the myosin content of the pulmonary arterial walls at two levels of the circulation (intra-acinar and preacinar) were studied in control rats and those exposed to hypobaric hypoxia of 380 torr for 3,7, 10, or 14 days. In control animals, we show that the precursor smooth muscle cells, pericytes and intermediate cells normally present in the nonmuscular regions of the intra-acinar arteries, contain smooth muscle myosin. With exposure to hypoxia, smooth muscle myosin in the intra-acinar arteries increases to Day 10, both in area of staining and fluorescent intensity. This is in contrast to the preacinar arteries were only the area of myosin increases. Antihuman platelet (non-muscle) myosin shows a little faint staining in both control and hypoxic animals. Adaptations to hypoxia by the intra-acinar precursor and preacinar mature smooth muscle cells is different, and suggests that the functions subserved by the myosin filaments at each of the two levels differs.

Animals↗

PubMed1998

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Actins↗

PubMed1988

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues.

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.

Animals↗

PubMed1996

Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.

Adult↗

PubMed1983

Membrane abnormalities occur in vascular smooth muscle but not in non-vascular smooth muscle from rats with deoxycorticosterone-salt induced hypertension.

Microsomal fractions were isolated from the smooth muscle of gastric fundus, vasa deferentia and mesenteric arteries of rats made hypertensive by deoxycorticosterone-salt treatment. Several enzymatic activities, Ca2+ binding and ATP-dependent Ca2+ accumulation of the microsomal fractions from these hypertensive rats were compared with those from the control of rats which remained normotensive under similar treatment. Altered membrane properties were observed in microsomal fractions isolated from vascular smooth muscle but not in those isolated from non-vascular smooth muscles in this form of experimental hypertension. These alterations included decreased Mg2+ ATPase activity, enhanced alkaline phosphatase activity, decreased Ca2+ binding in the absence of ATP and decreased ATP-dependent Ca2+ accumulation. This result is in contrast to our previous findings that decreased ATP-dependent Ca2+ accumulation was observed in microsomal fraction isolated from non-vascular smooth muscles of rats with genetic hypertension. The present study, together with our previous findings, support the contention that altered Ca2+ handling by vascular smooth muscle is associated with the pathogenesis of hypertension, whereas altered Ca2+ handling by non-vascular smooth muscles previously observed in spontaneous hypertension may be associated with genetic factors not related to hypertension.

Animals↗

PubMed1999

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic pulmonary hypertension.

The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in an in vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 micrometers diameter) and in larger vessels (100-700 micrometers) quantitatualy by the labeled [strept]avidin-biotin technique (day 1-28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time, we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH.

Animals↗

PubMed2001

CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo.

Expression of smooth muscle myosin heavy chain (SM-MHC) is tightly controlled depending on the differentiated state of smooth muscle cells (SMCs). To better understand the mechanisms that regulate transcription of the SM-MHC gene in vivo, we tested the function of several conserved CArG elements contained within the -4200 to +11,600 region of this gene that we had previously shown to drive SMC-specific expression in transgenic mice. CArG1 in the 5'-flanking sequence was required for all SMCs, while CArG2 and a novel intronic CArG element were differentially required in SMC subtypes. Of particular note, mutation of the intronic CArG selectively abolished expression in large arteries. A promoter construct containing three repeats of a conserved 227-bp intronic CArG-containing region was sufficient to direct transcription in vascular SMCs in transgenic mice, although this construct was also expressed in skeletal and cardiac muscle. These results support a model in which transcriptional regulation of SM-MHC is controlled by multiple positive and negative modular control regions that differ between SMCs and non-SMCs and among SMC subtypes. We also demonstrated that the CArG elements of the endogenous SM-MHC gene were bound by SRF in chromatin.

Animals↗

PubMed2003

Evidence for the primary role for 4-aminopyridine-sensitive K(v) channels in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles.

Gastrointestinal smooth muscles exhibit relaxation in response to the stimulation of beta-adrenoceptors with catecholamines. Subtypes of beta-adrenoceptors which mediate catecholamine-elicited relaxations in gastrointestinal smooth muscles are predominantly atypical beta-adrenoceptors including beta(3)-adrenoceptors. Gastrointestinal smooth muscle relaxations mediated via beta(3)-adrenoceptors can occur independently of intracellular cyclic adenosine monophosphate (AMP) elevation. One of the mechanisms responsible for cyclic AMP-independent smooth muscle relaxation following activation of G(s) protein-coupled receptors could be activation of voltage-gated K(+) channels. In the present study, possible contribution of two types of K(+) (large-conductance, Ca(2+)-sensitive and voltage-gated K(+), BK(Ca); voltage-gated, K(v)) channels to beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations was compared in gastric fundus and duodenum smooth muscles isolated from the guinea-pig. In these gastrointestinal smooth muscles, three catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and two beta(3)-adrenoceptor agonists ((R(*), R(*))-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) elicited a concentration-dependent relaxation in the presence of beta(1)- and beta(2)-adrenoceptor antagonists. The relaxations were unaffected by an adenylyl cyclase inhibitor, SQ-22536 (100 microM), which indicates their characteristic of cyclic AMP-independency. On the other hand, the SQ-22536-resistant, beta(3)-adrenoceptor-mediated relaxant components were potently attenuated when the tone was raised using high-KCl (80 mM) or in the presence of a K(v) channel blocker, 4-aminopyridine (4-AP, 1-3 mM). Iberiotoxin (100 nM), a selective blocker of BK(Ca) channels which significantly contribute to cyclic AMP-independent vascular smooth muscle relaxations induced through activation of G(s) protein-coupled receptors, did not apparently show any inhibitory effects on SQ-22536-resistant, beta(3)-adrenoceptor-mediated relaxations in these gastrointestinal smooth muscles. The present results indicate that 4-AP-sensitive K(v) channels play a primary role in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles. In these smooth muscles, BK(Ca) channels seem to apparently contribute insignificantly to cyclic AMP-independent relaxations following stimulation of beta(3)-type of adrenoceptors.

4-Aminopyridine↗

PubMed2003

Interstitial cells: involvement in rhythmicity and neural control of gut smooth muscle.

Many smooth muscles display spontaneous electrical and mechanical activity, which persists in the absence of any stimulation. In the past this has been attributed largely to the properties of the smooth muscle cells. Now it appears that in several organs, particularly in the gastrointestinal tract, activity in smooth muscles arises from a separate group of cells, known as interstitial cells of Cajal (ICC), which are distributed amongst the smooth muscle cells. Thus in the gastrointestinal tract, a network of interstitial cells, usually located near the myenteric plexus, generates pacemaker potentials that are conducted passively into the adjacent muscle layers where they produce rhythmical membrane potential changes. The mechanical activity of most smooth muscle cells, can be altered by autonomic, or enteric, nerves innervating them. Previously it was thought that neuroeffector transmission occurred simply because neurally released transmitters acted on smooth muscle cells. However, in several, but not all, regions of the gastrointestinal tract, it appears that nerve terminals, rather than communicating directly with smooth muscle cells, preferentially form synapses with ICC and these relay information to neighbouring smooth muscle cells. Thus a set of ICC, which are distributed amongst the smooth muscle cells of the gut, are the targets of transmitters released by intrinsic enteric excitatory and inhibitory nerve terminals: in some regions of the gastrointestinal tract, the same set of ICC also augment the waves of depolarisation generated by pacemaker ICC. Similarly in the urethra, ICC, distributed amongst the smooth muscle cells, generate rhythmic activity and also appear to be the targets of autonomic nerve terminals.

Animals↗

PubMed1997

Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth muscle cell-specific markers.

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 microM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle alpha-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs.

Actins↗

PubMed1999

Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function.

Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

Amino Acid Sequence↗

PubMed1997

Galpha16 mimics vasoconstrictor action to induce smooth muscle alpha-actin in vascular smooth muscle cells through a Jun-NH2-terminal kinase-dependent pathway.

Prolonged exposure of vascular smooth muscle cells (VSMC) to vasoconstrictors such as vasopressin or angiotensin II induces hypertrophy and increases expression of muscle-specific genes including smooth muscle alpha-actin (SM-alpha-actin). These vasoconstrictors signal through G-proteins, including members of the Gq family. To further investigate the role of Gq family members, VSMC were transfected with a constitutively active mutant of a Gq family member, Galpha16 (Galpha16Q212L). Stable expression of Galpha16Q212L persistently stimulated phospholipase C, resulting in increased basal levels of inositol phosphates. These cells were hypertrophied and expressed elevated levels of SM-alpha-actin compared with wild-type VSMC or cells transfected with a control plasmid (Neo). SM-alpha-actin promoter activity was markedly increased in cells stably or transiently expressing Galpha16Q212L. Basal c-Jun-NH2-terminal kinase (JNK) activity was increased 3-9-fold in cells stably expressing Galpha16Q212L, while basal activity of the p42/44 mitogen-activated protein kinases (ERKs) was unaffected. Transient expression of a kinase inactive JNK kinase partially inhibited induction of SM-alpha-actin promoter activity in response to vasoconstrictors or expression of Galpha16Q212L. These results indicate that expression of constitutively active Galpha16 in VSMC mimics the effects of vasoconstrictors on hypertrophy and muscle-specific gene expression, and activation of JNK may play a role in these responses.

Actins↗

PubMed2006

Small lungs and suspect smooth muscle: congenital diaphragmatic hernia and the smooth muscle hypothesis.

Lung hypoplasia and congenital diaphragmatic hernia (CDH) represent an unsolved clinical and scientific problem. Early lung morphogenesis is coupled to development and function of pulmonary smooth muscle. Activity of the latter is abnormal from the earliest stages of hypoplastic lung development and before supervening CDH. A "smooth muscle hypothesis" is advanced to help explain embryonic lung malformations, fetal failure of lung growth, and postnatal susceptibility to barotrauma, airway hyperreactivity, and pulmonary hypertension in CDH. Exploring the interaction of smooth muscle function and airway pressures may help optimise tracheal occlusion and provide support for both an adequately powered trial of glucocorticoids and also for experimental "preventilation" strategies in fetal CDH.

Fetal Diseases↗