Smooth Muscle Isoforms Research Articles

Codistribution of NOS and caveolin throughout peripheral vasculature and skeletal muscle of hamsters.

In isolated cell systems, nitric oxide synthase (NOS) activity is regulated by caveolin (CAV), a resident caveolae coat protein. Because little is known of this interaction in vivo, we tested whether NOS and caveolin are distributed together in the intact organism. Using immunohistochemistry, we investigated the localization of constitutive neuronal (nNOS) and endothelial (eNOS) enzyme isoforms along with caveolin-1 (CAV-1) and caveolin-3 (CAV-3) throughout the systemic vasculature and peripheral tissues of the hamster. The carotid artery, abdominal aorta, vena cava, femoral artery and vein, feed artery and collecting vein of the cheek pouch retractor muscle, capillaries and muscle fibers of retractor and cremaster muscles, and arterioles and venules of the cheek pouch were studied. In endothelial cells, eNOS and CAV-1 were present throughout the vasculature, whereas nNOS and CAV-3 were absent except in capillaries, which reacted for nNOS. In smooth muscle cells, nNOS and CAV-1 were also expressed systemically, whereas eNOS was absent; CAV-3 was present in the arterial but not the venous vasculature. Both nNOS and CAV-3 were located at the sarcolemma of skeletal muscle fibers, which were devoid of eNOS and CAV-1. These immunolabeling patterns suggest functional interactions between eNOS and CAV-1 throughout the endothelium, regional differences in the modulation of nNOS by caveolin isoforms in vascular smooth muscle, and modulation of nNOS by CAV-3 in skeletal muscle.

Cell biology of posterior capsular opacification

Posterior capsular opacification (PCO), a major complication of modern cataract surgery, necessitates further surgical intervention in 10-50% of patients. PCO results from the growth and transdifferentiation of lens epithelial cells left on the anterior capsule at the time of cataract surgery. These cells proliferate to form monolayers on the capsular surfaces, and such monolayers continue to line the anterior capsule leaflet many years after surgery. Some cells, however, differentiate or undergo a transition to another cell type, and these processes greatly contribute to PCO. Equatorial differentiation of cells to fibre-like structures leads to Soemmerring's ring formation and peripheral thickening of the capsular bag. Closer to the rhexis, cell swelling can result in globular Elschnig's pearls, which may occlude the visual axis. Cells at the rhexis edge and those in the space around the optic appear to undergo epithelial-mesenchymal transition. The resulting cells are fibroblastic in morphology, express the smooth muscle isoform of actin and secrete extracellular matrix containing proteins not normally present in the lens.

Subcellular distribution of protein kinase C isoforms in gastric antrum smooth muscle of STZ-induced diabetic rats

Contractile responses to carbachol (CCh), protein kinase C (PKC) activity and distribution of PKC isoforms in subcellular fractions isolated from gastric antrum smooth muscle of control and streptozotocin (STZ)-induced diabetic rats were examined. CCh induced concentration-dependent contraction in antrum smooth muscle from controls and diabetics, and this contraction was significantly greater in diabetics than in controls. In diabetics, the PKC activity in the nucleus fraction was significantly decreased by about 63% in the resting condition and that in the cytosol fraction was significantly increased by about 135% after the treatment with 10 μMCCh for 10 min compared to controls. Immunoblot analysis showed that 8 PKC isoforms (-α, -β, -γ, -δ, -ε, -ζ, -ι, -λ) were expressed in rat antrum smooth muscle. The PKC-β isoform was significantly decreased by about 47% in the nucleus fraction in the resting condition in diabetics compared to controls. The nucleus, cytosol and membrane fractions of this isoform were decreased in controls after the treatment with 10 μM CCh for 10 min whereas these fractions were unchanged in diabetics. The PKC-ε significantly increased by about 219% in the cytosol fraction of diabetics in the resting condition, but the distribution of this isoform was unchanged in controls and diabetics after the treatment with 10 μM CCh for 10 min. Results suggest that the diversity of PKC isoform-specific distribution and translocation may be related to abnormal contractility and intracellular signal transduction through the PKC pathway in diabetics.

A Single Human Myosin Light Chain Kinase Gene (MLCK; MYLK)Transcribes Multiple Nonmuscle Isoforms

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garciaet al.,1997,Am. J. Respir. Cell Mol. Biol.16, 489–494) except for a deletion of nucleotides 1428–1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081–5233 (MLCK 3a, D3), double deletions of nucleotides 1428–1634 and 5081–5233 (MLCK 3b), and nucleotide deletions 4534–4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5′ and 3′ neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.

Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

Angiotensin II type 1 receptor antagonist downregulates nonmuscle myosin heavy chains in spontaneously hypertensive rat aorta.

The aim of this study was to clarify the differences between the angiotensin II type 1 (AT1) receptor antagonist and the angiotensin-converting enzyme (ACE) inhibitor on smooth muscle and nonmuscle myosin heavy chain isoforms in aortic smooth muscle cells of Wistar-Kyoto rats and spontaneously hypertensive rats. All 4 myosin heavy chain isoforms are heterogeneously expressed in the smooth muscle cells of the aortic tunica media in 20-week-old rats, and the contractile-type myosin heavy chains are highly expressed in smooth muscle cells of the aortic tunica media compared with the synthetic-type myosin heavy chains. Both the AT1 receptor antagonist and the ACE inhibitor had the same effects on hemodynamics, smooth muscle cell hypertrophy and proliferation, fibrosis, and vascular remodeling in spontaneously hypertensive rats. However, the AT1 receptor antagonist had a more potent effect on the downregulation of the synthetic-type myosin heavy chains than the ACE inhibitor in spontaneously hypertensive rat aortic tunica media. In contrast, these effects of the AT1 receptor antagonist and the ACE inhibitor on hemodynamics, morphology, fibrosis, and expression of myosin heavy chain isoforms in smooth muscle cells of the aortic tunica media were not observed in Wistar-Kyoto rats. Thus, within 6 weeks, the AT1 receptor antagonist might modulate the cellular composition of myosin heavy chain isoforms in smooth muscle cells more efficiently than the ACE inhibitor, without morphological changes in the spontaneously hypertensive rat aorta.

Smooth-muscle myosin heavy-chain SM-B isoform expression in developing and adult rat lung.

The smooth-muscle cells composing the vasculature and airways of the lung display a variety of contractile protein phenotypes. To date, however, it has remained unclear how these phenotypes might contribute differentially to contractile activity. To address this issue, we made monospecific rabbit polyclonal antibodies against the difference peptide for the SM-B smooth-muscle myosin heavy chain (SMMHC) and used these to investigate the distribution of the SM-B isoform in lung. SM-B has a seven-amino acid insert in the head region that is known to result in a higher actin-activated adenosine triphosphatase activity and in vitro motility. During development, reactivity is first seen in the trachea and bronchi of saccular lung at the time of birth, when other SMMHC isoforms also are present. Immunoreactivity spreads distally through the airways as development proceeds, reaching the level of alveolar septae in the adult. Although the smaller vessels of the pulmonary vasculature react strongly with the SM-B antibody, reactivity is infrequently observed in large pulmonary vessels. Adult tracheal smooth muscle is highly and more uniformly reactive, commensurate with its relatively high maximal velocity of shortening. The differential expression of the SM-B isoform in vascular and airway smooth muscles demonstrated in this study may provide the molecular basis for functional differences between these smooth-muscle cell types and may provide one mechanism for adapting contractility in response to physiologic stresses in the lung.

Effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit

Background: The disrupted intestinal transit during endotoxemia may be mediated by nitric oxide (NO). We hypothesized that the isoforms of nitric oxide synthase (NOS) are up-regulated in intestinal smooth muscle during endotoxemia and that the scavenging of NO will normalize transit. Methods: Rats were given Escherichia coli lipopolysaccharide (LPS) 10 mg/kg intravenously and were killed 4 hours later. To determine the activity of NOS isoforms in the jejunum and ileum, the conversion of tritiated L -arginine to tritiated L -citrulline was measured. Western immunoblots were performed by incubating the extracted protein with specific polyclonal antibodies. To determine intestinal transit, rats were divided into 4 groups: 0.9% sodium chloride 1 mL/h intravenously for 5 hours, LPS 10 mg/kg intravenous bolus plus 1 mL/h 0.9% sodium chloride intravenously, LPS plus oxyhemoglobin 0.5 g/kg/h intravenously, and oxyhemoglobin 0.5 g/kg/h intravenously. Results: LPS increased the constitutive and inducible NOS enzyme activities in the jejunum and ileum. Western blots demonstrated that LPS up-regulates both the NOS1 and NOS2 isoforms in jejunal and ileal smooth muscle. Oxyhemoglobin alone increased intestinal transit compared with controls, whereas endotoxemia increased intestinal transit, which was ameliorated with infusions of oxyhemoglobin. Conclusions: NO may play a major role in mediating the rapid intestinal transit induced by endotoxemia. (Surgery 1999;125:339-44.)

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2α (PGF2α) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 µM [Ca2+]e, both PGF2α and U46619 (10-5 M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 µM [Ca2+]e, PGF2α and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx - PKC activity - contraction relationship showed a 45Ca2+-influx threshold of ~7 µmol·kg-1·min-1 for maximal PKC activation by PGF2α and U46619. 45Ca2+ influx > 10 µmol·kg-1·min-1 was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed α-, δ-, ε-, and ζ-PKC in porcine coronary artery. In unstimulated tissues, α- and ε-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of ε-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of α-PKC was observed at 600 µM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent ε-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent α-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.Key words: protein kinase C, isoform, calcium influx, eicosanoids, vascular smooth muscle, contraction.

Vascular smooth muscle nitric oxide synthase anomalies in Dahl/Rapp salt-sensitive rats.

Salt-sensitive hypertension in the Dahl/Rapp rat (S strain) is prevented by L-arginine. Based on the observations that dexamethasone prevented the antihypertensive effect of L-arginine in these animals and the suggestion that a locus in or near an inducible nitric oxide synthase (NOS) gene on chromosome 10 cosegregated with hypertension in some F2 crosses that utilized the S rat, the present study explored the hypothesis that the vascular smooth muscle isoform of inducible NOS (NOS2) was abnormal in S rats. Primary cultures of aortic smooth muscle cells from S rats demonstrated impaired inducible NO production, which improved with increased L-arginine in the medium. Sequence analysis identified a single T-->C transversion that produced an amino acid substitution (S714P) between the FAD and FMN binding sites and a restriction fragment length polymorphism. This restriction fragment length polymorphism was present only in S rats. The mutation of NOS2 and the role of this enzyme in the pathogenesis of salt-sensitive hypertension in the Dahl/Rapp rat require further investigation.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.

Skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase gene expression in congestive heart failure.

Congestive heart failure leads to skeletal muscle abnormalities, one of which is a prolongation of sarcoplasmic reticulum Ca2+ flux. The purpose of this study was to determine whether skeletal muscle of spontaneous hypertensive and heart failure rats have alterations in the expression of the sarcoplasmic (or endoplasmic) reticulum Ca(2+)-ATPase (SERCA) gene. Northern analysis revealed that SERCA1, the predominant skeletal muscle isoform, was decreased by 45%, 43%, and 58% in the tibialis anterior, plantaris, and diaphragm muscles, respectively. Ribonuclease protection assay showed that the decrease was due to the adult isoform, SERCA1a, with minor changes in the alternatively spliced neonatal isoform, SERCA1b. There was no change in SERCA1 mRNA levels in gastrocnemius muscles. No change was found in SERCA2a (cardiac/slow skeletal isoform) mRNA or protein levels or in SERCA2b (smooth muscle isoform), dihydropyridine receptor, or alpha-actin mRNA levels in diaphragm muscle. Northern blot and ribonuclease protection assays showed that SERCA2a decreased 61% in the heart while the alternatively spliced isoform, SERCA2b, decreased 27%. Western analysis of the tibialis anterior, diaphragm, and gastrocnemius muscles showed a decrease in SERCA1 protein levels by 46%, 64%, and 42%, respectively, whereas sarcoplasmic reticulum Ca(2+)-ATPase activity, a functional correlate of SERCA expression, was decreased by 38%, 38%, and 40% in the same muscles, SERCA2 protein expression decreased by 36% in the failing heart. Decreases in both mRNA and protein suggest pretranslational control of SERCA1 expression, whereas the lack of decreased SERCA1 mRNA in gastrocnemius muscle suggests translational regulation. The decreased SERCA1 protein expression in all muscles studied probably contributes to contractile abnormalities related to excitation-contraction coupling function in heart failure.

Immunohistochemical evaluation of chemically induced rhabdomyosarcomas in rats: diagnostic utility of MyoD1.

Monoclonal antibodies (mAbs) to selected muscle proteins were assessed as potential immunohistochemical markers to assist in the definitive diagnosis of poorly differentiated soft tissue sarcomas in rats. A series of 7 rat rhabdomyosarcomas (RMS) induced with nickel subsulfide were studied by light microscopy and were evaluated for immunoreactivity to desmin, vimentin, fast (type II isoform) skeletal myosin, alpha-actin (smooth muscle isoform), or MyoD1 (myogenic regulatory protein) mAbs using an avidin-biotin-chromogen technique. Consecutive RMS slices were fixed in 10% neutral buffered formalin (the fixative routinely used in carcinogenicity bioassays) for periods of 3 days or 2 mo prior to paraffin embedding to determine the effect of fixation time on immunoreactivity. Desmin and vimentin mAbs bound to many cells of all tumors, but fixation for 2 mo resulted in irretrievable loss of desmin and vimentin binding. Fast myosin and alpha-actin mAbs bound to many cells in 1 RMS but to < 1% of the cells in the remainder. MyoD1 mAb bound to tumor cell nuclei in 5/7 RMS with no loss of staining in tissue fixed for 2 mo. Results indicate that MyoD1 immunostaining, in contrast to desmin, maintains its sensitivity following prolonged formalin fixation and may be of value to distinguish RMS from other soft tissue sarcomas in the rat.

Caldesmon

Caldesmon is a protein that is found in smooth muscle and in non-muscle cells. Two isoform classes produced by alternative splicing of one gene have been characterized. The smooth muscle, high molecular weight (89–93 kDa), caldesmon isoforms are found in non-muscle and in de-differentiated smooth muscle cells. The conserved regions of all isoforms contain caldesmon's properties such as binding to actin, tropomyosin, Ca 2+-calmodulin, myosin and phospholipids. All isoforms are also very potent inhibitors of the actin-tropomyosin activated myosin MgATPase. Non-muscle and smooth muscle isoforms of caldesmon perform different roles in vivo. This may be reflected by the distinct cellular distribution of these isoform classes. Non-muscle caldesmon is a regulatory factor in the microfilament network and is thus involved in the assembly and stabilization of microfilaments. Smooth muscle caldesmon together with tropomyosin is a mediating factor for Ca 2+-dependent inhibition of smooth muscle contraction.

Distribution of Myosin Heavy Chain (MHC) Isoforms in Tracheal Smooth Muscle From Preterm And Newborn Lambs. • 1822

Distribution of Myosin Heavy Chain (MHC) Isoforms in Tracheal Smooth Muscle From Preterm And Newborn Lambs. • 1822

Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type.

1. Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H2 (PGH2) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined. 2. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE2, thromboxane B2 or 6-ketoPGF1 alpha by radioimmunoassay. 3. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) each at 10 ng ml-1) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 microM; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 microM) and cyclohexamide (10 micrograms ml-1; added 30 min before the cytokines). 4. Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1 beta, TNF alpha, IFN gamma each at 10 ng ml-1 for 24 h) caused the accumulation of PGE2 and 6-keto-PGF1 alpha. 5. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1 beta in combination with TNF alpha caused a similar release of PGE2 to that when the three cytokines were given in combination. 6. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 microM for 15 min) after which PGE2 was measured. IL-1 beta and TNF alpha increased COX-2 activity and an additional small increase was produced by the three cytokines in combination. 7. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

Immunohistological localization and expression of alpha-actin in the baboon (Papio anubis) corpus luteum.

We have recently shown the presence of E-cadherin and of alpha- and gamma-catenins in human and baboon corpora lutea. These are components of adherens junctions between cells. The cytoplasmic catenins link the cell membrane-associated cadherins to the actin-based cytoskeleton. This interaction is necessary for the functional activity of the E-cadherins. Our aim therefore was to determine the presence of alpha-actin in the baboon corpus luteum, to further establish whether the necessary components for E-cadherin activity are present in this tissue. An antibody specific for the smooth muscle isoform of actin, alpha-actin, was used for these studies. The results using immunohistochemistry show that (a) alpha-actin is present in steroidogenic cells of the active corpus luteum, theca externa of the corpus luteum, cells of the vasculature, and the tunica albuginea surrounding the ovary. The intensity of immunoreactivity for alpha-actin varied, with the cells of the vasculature reacting more intensely than the luteal cells. A difference in intensity of immunoreactivity was also observed among the luteal cells, with the inner granulosa cells showing stronger immunoreactivity than the peripheral theca lutein cells. There was no detectable immunoreactivity in the steroidogenic cells of the atretic corpus luteum. However, in both the active and atretic corpora lutea, alpha-actin-positive vascular cells were dispersed within the tissue. (b) Total alpha-actin (luteal and non-luteal), as determined by Western blot analyses, does not change during the luteal phase and subsequent corpus luteum demise (atretic corpora lutea). (c) hCG stimulated the expression of alpha-actin and progesterone secretion by the early luteal phase (LH surge + 1-5 days) and mid-luteal phase (LH surge + 6-10 days) cells in culture, but only progesterone in the late luteal phase (LH surge + 11-15 days). The data show that alpha-actin is present in luteal cells and that its expression is regulated by hCG, thus suggesting that E-cadherin may form functional adherens junctions in the corpus luteum.

Strain increases airway smooth muscle contractile and cytoskeletal proteins in vitro.

Mechanical stress contributes to lung development and the progression of some lung diseases, although its effects on individual lung cells are unknown. Because increased airway smooth muscle (ASM) is found in lung diseases where abnormal stress is present, we determined if strain (change in resting length) causes ASM hypertrophy independently of other in vivo influences. Cultured canine ASM cells were subjected to two levels of cyclic deformational strain for 14 days and compared with nonstrained cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Cells subjected to 16-30% strain demonstrated increases in total cellular protein, myosin, myosin light chain kinase (MLCK), and desmin, whereas the cellular contents of actin, vimentin, and tubulin were similar. Changes in myosin appeared mostly due to the smooth muscle isoform, whereas nonmuscle myosin was unchanged. The increases in myosin and MLCK were disproportionate to increases in total protein, suggesting selective changes in contractile proteins. These relative increases in content of proteins were not as pronounced with 0-16% strain, suggesting a graded response. These data suggest that strain per se can increase the contractile proteins of ASM cells independently of other in vivo factors and modulates cultured cell phenotype to a more differentiated state, since it increases smooth muscle-specific proteins, such as smooth muscle myosin isoforms and desmin.

Fibroblast transgelin and smooth muscle SM22alpha are the same protein, the expression of which is down-regulated in many cell lines.

In this report we investigate the expression and relationship of transgelin (Tg), a transformation and shape-change sensitive actin gelling protein found in fibroblasts and smooth muscle [Shapland et al., 1988: J. Cell. Biol. 107:153-161; Shapland et al., 1993: J. Cell. Biol. 121:1065-1073], to SM22alpha, a smooth muscle protein of unknown function [Lees-Millar et al., 1987: J. Biol. Chem. 262:2988-2993; Solway et al., 1995: J. Biol. Chem. 270:13460-13469]. To clarify their relationship we have cloned and sequenced the cDNA encoding Tg from cultures of rat embryo fibroblasts. The sequences of fibroblast Tg and the smooth muscle isoform SM22 are identical [Prinjha et al., 1994: Cell Motil. Cytoskeleton 28:243-255; Shanahan et al., 1993: Circ. Res. 73:193-204; Solway et al., 1995]. These data, coupled with our immunoblot analysis and previous observations [Shapland et al., 1988; Shapland et al., 1993], demonstrate that Tg expression is not restricted to smooth muscle since this protein is also present in normal mesenchymal cells. However, we also show that Tg, although present in secondary cultures of mouse and rat embryo fibroblasts, is absent in many apparently normal fibroblast cell lines. Tg down-regulation may therefore be an early and sensitive marker for the onset of transformation. A functional role for Tg is unlikely to directly involve Ca2+ since it neither contains a functional EF hand nor binds 45Ca2+.