Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.     

ERK inhibition attenuates 5-HT-induced contractions in fetal and adult ovine carotid arteries

Growth and differentiation-related pathways are much more active in immature than in mature, fully differentiated smooth muscle. Because mitogen-activated protein kinases (MAPK) are intimately involved with growth and differentiation, and the extracellular signal-regulated kinase (ERK) subfamily of MAPKs are involved in some contractile responses, the present studies examined the hypothesis that ERKs play an important and age-dependent role in smooth muscle contraction. The MAPK inhibitors PD098059 and UO126 both inhibited serotonin (5-HT) concentration-response relations more effectively in carotid arteries from term fetal lambs, than in corresponding arteries from mature non-pregnant adult sheep. This inhibition involved significant decreases in both the pD2 (adult: 2-fold; fetus: 4- to 15-fold) and the maximum efficacy (adult: 15-19%; fetus: 34-39%) of 5-HT. Accompanying this age-dependent effect on contraction, quantitative Western blot assays revealed that ERK1 and ERK2 abundances were 39% and 164% greater, respectively, in fetal than in adult carotid arteries. The abundance of the putative ERK target, caldesmon, however, was about 7-fold greater in adult than in fetal arteries. Together, the present results support the view that ERK abundance and activity is upregulated in fetal relative to adult arteries, and that one consequence of this upregulation is that the contribution of ERKs to contraction, at least that initiated by 5-HT2a receptors, is greater in fetal than adult carotid arteries. Whereas the phosphorylation mechanisms through which ERKs augment contraction remain uncertain and controversial, the present results suggest that emphasis should be shifted away from caldesmon and toward other critical contractile proteins, and how these proteins may contribute differently to development of agonist-induced contractile force in immature and mature arteries.     

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.     

Vascular development in early ovine gestation: carotid smooth muscle function, phenotype, and biochemical markers

Vascular smooth muscle (VSM) maturation is developmentally regulated and differs between vascular beds. The maturation and contribution of VSM function to tissue blood flow and blood pressure regulation during early gestation are unknown. The carotid artery (CA) contributes to fetal cerebral blood flow regulation and well being. We studied CA VSM contractility, protein contents, and phenotype beginning in the midthird of ovine development. CAs were collected from early (88-101 day of gestation) and late (138-150 day; term = day 150) fetal (n = 14), newborn (6-8 day old; n = 7), and adult (n = 5) sheep to measure forces in endothelium-denuded rings with KCl, phenylephrine, and ANG II; changes in cellular proteins, including total and soluble protein, actin and myosin, myosin heavy chain isoforms (MHC), filamin, and proliferating cell nuclear antigen; and vascular remodeling. KCl and phenylephrine elicited age- and dose-dependent contraction responses (P < 0.001) at all ages except early fetal, which were unresponsive. In contrast, ANG II elicited dose responses only in adults, with contractility increasing greater than fivefold vs. that shown in fetal or neonatal animals (P < 0.001). Increased contractility paralleled age-dependent increases (P < 0.01) in soluble protein, actin and myosin, filamin, adult smooth muscle MHC-2 (SM2) and medial wall thickness and reciprocal decreases (P < 0.001) in nonmuscle MHC-B, proliferating cell nuclear antigen and medial cellular density. VSM nonreceptor- and receptor-mediated contractions are absent or markedly attenuated in midgestation and increase age dependently, paralleling the transition from synthetic to contractile VSM phenotype and, in the case of ANG II, paralleling the switch to the AT(1) receptor. The mechanisms regulating VSM maturation and thus blood pressure and tissue perfusion in early development remain to be determined.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Sympathetic innervation promotes vascular smooth muscle differentiation

The sympathetic nervous system (SNS) is an important modulator of vascular smooth muscle (VSM) growth and function. Several lines of evidence suggest that the SNS also promotes VSM differentiation. The present study tests this hypothesis. Expression of smooth muscle myosin (SM2) and alpha-actin were assessed by Western analysis as indexes of VSM differentiation. SM2 expression (normalized to alpha-actin) in adult innervated rat femoral and tail arteries was 479 +/- 115% of that in noninnervated carotid arteries. Expression of alpha-actin (normalized to GAPDH or total protein) in 30-day-innervated rat femoral arteries was greater than in corresponding noninnervated femoral arteries from guanethidine-sympathectomized rats. SM2 expression (normalized to alpha-actin) in neonatal femoral arteries grown in vitro for 7 days in the presence of sympathetic ganglia was greater than SM2 expression in corresponding arteries grown in the absence of sympathetic ganglia. In VSM-endothelial cell cultures grown in the presence of dissociated sympathetic neurons, alpha-actin (normalized to GAPDH) was 300 +/- 66% of that in corresponding cultures grown in the absence of neurons. This effect was inhibited by an antibody that neutralized the activity of transforming growth factor-beta2. All of these data indicate that sympathetic innervation increased VSM contractile protein expression and thereby suggest that the SNS promotes and/or maintains VSM differentiation.     

Age-dependent modulation of endothelium-dependent vasodilatation by chronic hypoxia in ovine cranial arteries.

Although abundant evidence indicates that chronic hypoxia can induce pulmonary vascular remodeling, very little is known of the effects of chronic hypoxia on cerebrovascular structure and function, particularly in the fetus. Thus the present study explored the hypothesis that chronic hypoxemia also influences the size and shape of cerebrovascular smooth muscle and endothelial cells, with parallel changes in the reactivity of these cells to endothelium-dependent vasodilator stimuli. To test this hypothesis, measurements of endothelial and vascular smooth muscle cell size and density were made in silver-stained common carotid and middle cerebral arteries from term fetal and nonpregnant adult sheep maintained at an altitude of 3,820 m for 110 days. Chronic hypoxia induced an age-dependent remodeling that led to smooth muscle cells that were larger in fetal arteries but smaller in adult arteries. Chronic hypoxia also increased endothelial cell density in fetal arteries but reduced it in adult arteries. These combined effects resulted in an increased (adult carotid), decreased (adult middle cerebral), or unchanged (fetal arteries) per cell serosal volume of distribution for endothelial factors. Despite this heterogeneity, the magnitude of endothelium-dependent vasodilatation to A23187, measured in vitro, was largely preserved, although sensitivity to this relaxant was uniformly depressed. N(G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and endothelium denudation each independently blocked A23187-induced vasodilation without unmasking any residual vasoconstrictor effect. Indomethacin did not significantly attenuate A23187-induced relaxation except in the hypoxic adult middle cerebral, where a small contribution of prostanoids was evident. Vascular sensitivity to exogenous nitric oxide (NO) was uniformly increased by chronic hypoxia. From these results, we conclude that chronic hypoxia reduced endothelial NO release while also upregulating some component of the NO-cGMP-PKG vasodilator pathway. These offsetting effects appear to preserve endothelium-dependent vasodilation after adaptation to chronic hypoxia.     

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).     

Maternal dexamethasone treatment alters myosin isoform expression and contractile dynamics in fetal arteries

We tested the hypothesis that maternal glucocorticoid treatment modulates 17-kDa myosin light chain (myosin LC17) isoform expression and contractile dynamics in fetal ovine carotid arteries. In the single course group, ewes received 6 mg dexamethasone or placebo over 48 h. In the repeated course group, ewes received 6 mg dexamethasone or placebo weekly for 5 wk. In response to 1 microM phenylephrine, arteries from fetuses of dexamethasone-treated ewes exhibited biphasic contractions, characterized by an intermediate relaxation phase. The relaxation rate constant was significantly higher in arteries from the fetuses of dexamethasone than placebo-treated ewes. The observed biphasic contractions suggest the appearance of functional sarcoplasmic reticulum in the arteries from the fetuses of dexamethasone-treated ewes. The myosin LC17(a) isoform expression was lower in the arteries from the fetuses of the placebo-treated ewes than in those from the ewes. Repeated maternal administration of dexamethasone induced an almost twofold increase in myosin LC17(a) isoform expression in the fetal arteries. In contrast, maternal myosin LC17a isoform expression was not affected by dexamethasone treatment. We speculate that dexamethasone-induced increases in fetal myosin LC17(a) isoform expression represent accelerated differentiation of a subpopulation of vascular smooth muscle cells from the fetal to adult phenotype.     

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.     

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.     

Telokin expression and the effect of hypoxia on its phosphorylation status in smooth muscle cells from small and large pulmonary arteries

Small pulmonary arteries (SPA), <500 microm diameter of the cat, constrict when exposed to hypoxia, whereas larger arteries (large pulmonary arteries; LPA), >800 microm diameter, show little or no response. It is unknown why different contractile responses occur within the same vascular bed, but activator or repressor proteins within the smooth muscle cell (SMC) can modify myosin phosphatase and myosin light chain kinase (MLCK), thereby influencing the phosphorylation state of myosin light chain (MLC) and ultimately, contraction. Telokin, a protein with a sequence identical to the COOH-terminal domain of MLCK, is expressed in smooth muscle where in its phosphorylated state it inhibits myosin phosphatase, binds to unphosphorylated myosin, and helps maintain smooth muscle relaxation. We measured telokin mRNA and telokin protein in smooth muscle from different diameter feline pulmonary arteries and sought to determine whether changes in the phosphorylation status of telokin and MLC occurred during hypoxia. In pulmonary arteries, telokin expression varied inversely with artery diameter, but cerebral arteries showed neither telokin protein nor telokin mRNA. Although telokin and MLC were distributed uniformly throughout the SPA muscle cell cytoplasm, they were not colocalized. During hypoxia, telokin dephosphorylated, and MLC became increasingly phosphorylated in SPA SMC, whereas in LPA SMC there was no change in either telokin or MLC phosphorylation. When LPA SMC were exposed to phenylephrine, MLC phosphorylation increased with no change in telokin phosphorylation. These results suggest that in SPA, phosphorylated telokin may help maintain relaxation under unstimulated conditions, whereas in LPA, telokin's function remains undetermined.     

Myosin isoforms and cell heterogeneity in vascular smooth muscle. I. Developing and adult bovine aorta

Monoclonal anti-smooth muscle (SM-E7, SM-F11, and BF-48) and anti-nonmuscle (NM-A9 and NM-G2) myosin antibodies, Western blotting, and immunocytochemical procedures were used to study myosin isoform composition and distribution in the smooth muscle (SM) cells of bovine aorta differentiating in vivo and in vitro. Two myosin heavy chain (MHC) isoforms were identified by SM-E7 in adult aorta: SM-MHC-1 (Mr = 205 kDa) and SM-MHC-2 (Mr = 200 kDa), respectively. When tested with the SM-F11 antibody, SM-MHC-2 isoform showed distinct antigenic properties compared to SM-MHC-1. Two bands of 205 and 200 kDa were also present in the aortic SM tissue from 3-month-old fetus and were equally recognized by the BF-48 antibody. The 200-kDa SM myosin isoform was labeled by SM-F11 but not by SM-E7, thus indicating the existence of a fetal-specific SM-MHC-2 isoform. At the cellular level, both developing and adult bovine aortic tissues showed the existence of distinct patterns of myosin isoform expression. Three or even more aortic cell populations are differently distributed in areas which appear as (1) a network of interconnecting sheet-like or compact tissue (early fetus) and (2) enriched of collagenous-elastic or muscular tissue (adult animal). In addition, the SM-MHC-2 isoform of the fetal type appears to be uniquely distributed in cultured SM cells grown in vitro from adult bovine aortic explants. Our data indicate that in bovine aorta (1) MHC isoform expression is developmentally regulated and (2) the distribution of myosin isoforms is heterogenous both among and within aortic cells. These findings may be related to the distinct physiological properties displayed by SM during vascular myogenesis.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Organization of the genetic locus for chicken myosin light chain kinase is complex: Multiple proteins are encoded and exhibit differential expression and localization

We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins. J. Cell. Biochem. 70:402–413, 1998. © 1998 Wiley-Liss, Inc.     

Acute hypoxia modulates 5-HT receptor density and agonist affinity in fetal and adult ovine carotid arteries

In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO(2) = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 +/- 3.5% (fetus) and 20.5 +/- 2.2% (adult), significantly reduced the pD(2) for 5-HT in the fetus (7.01 +/- 0.1 to 6.3 +/- 0.2) but not the adult (6.1 +/- 0.1 to 6.0 +/- 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K(+)) not in the fetus (from 114 +/- 7 to 70 +/- 10%, not significant) but only in the adult (from 83 +/- 15 to 25 +/- 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pK(A), determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 +/- 0.2 to 6.0 +/- 0.2) and adult arteries (from 6.2 +/- 0. 2 to 5.7 +/- 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 +/- 1.1 to 10.9 +/- 1.0) but not in fetal (21.0 +/- 1.0 to 23.2 +/- 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.     

Enzymatic changes in myosin regulatory proteins may explain vasoplegia in terminally ill patients with sepsis

The current study was conducted with the hypothesis that failure of maintenance of the vascular tone may be central to failure of the peripheral circulation and spiralling down of blood pressure in sepsis. Namely, we examined the balance between expression of myosin light chain (MLC) phosphatase and kinase, enzymes that regulate MLCs dephosphorylation and phosphorylation with a direct effect on pharmacomechanical coupling for smooth muscle relaxation and contraction respectively. Mechanical recordings and enzyme immunoassays of vascular smooth muscle lysates were used as the major methods to examine arterial biopsy samples from terminally ill sepsis patients. The results of the present study provide evidence that genomic alteration of expression of key regulatory proteins in vascular smooth muscles may be responsible for the relentless downhill course in sepsis. Down-regulation of myosin light chain kinase (MLCK) and up-regulation of MLCK may explain the loss of tone and failure to mount contractile response in vivo during circulation. The mechanical studies demonstrated the inability of the arteries to develop tone when stimulated by phenylephrine in vitro. The results of our study provide indirect hint that control of inflammation is a major therapeutic approach in sepsis, and may facilitate to ameliorate the progressive cardiovascular collapse.     

Maturational differences between vascular and bladder smooth muscle during ovine development

Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compared by quantifying contractile protein, myosin heavy chain (MHC) isoform contents, and contractile properties of aortas and bladders from female fetal (n = 19) and postnatal (n = 21) sheep. Actin, myosin, and protein contents rose progressively throughout development in both tissues (P 相似文献