Ion channels in vascular smooth muscle: alterations in essential hypertension

The desmocollins are one of two types of putative adhesive proteins present in the desmosome type of cell junctions, the other type being the desmogleins; both are members of the cadherin superfamily. Each type of desmosomal cadherin occurs as a number of isoforms which have differing tissue distribution; within stratifying epithelia some isoforms occur only suprabasally. We have sought to analyse desmocollin function by reducing the amount of protein using antisense gene expression in the widely studied Madin-Darby canine kidney (MDCK) cell line. Although this is a simple epithelial cell line, we show by Northern blot analysis that it expresses multiple isoforms of the desmosomal cadherins. Desmocollins DSC2 and DSC3 and desmogleins DSG2 and DSG3 (the pemphigus vulgaris antigen PVA) were detected, but DSC1 and DSG1, which are present exclusively in the suprabasal layers of the epidermis, were absent. The major desmocollin isoform was the type 2 (DSC2). A DSC2 clone isolated from a MDCK cDNA library had the same cell adhesion recognition sequence (Phe-Ala-Thr) as human, bovine and mouse type 2 isoforms. This sequence appears diagnostic for the three desmocollin isoforms. This cDNA clone was used to isolate a genomic DSC2 clone; antisense expression of this clone in MDCK cells resulted in a drastic reduction of desmocollin protein as judged by Western blots; Dsc3 was not upregulated to compensate for the loss of Dsc2. This antisense expression significantly altered desmosome assembly. There was a loss of punctate staining evident when using a desmosome plaque protein (desmoplakin) antibody. Electron microscopy revealed that there was a reduction in the number of desmosomes and a notable increase in the asymmetry of plaques between adjacent cells. Immunolabelling showed that similar levels of desmogleins and E-cadherin were present. Immunoelectron microscopy also showed that many vesicular structures were labelled, at intervals along the lateral membranes between cells. The distinctive loose organization of the remaining desmosomes may originate in modifications to the targeting and incorporation of proteins into fully assembled plaques. Other junctions were unaffected and the cells maintained their integrity as a confluent monolayer.     

Involvement of L-type calcium channels in hypoxic relaxation of vascular smooth muscle

Immunoelectron microscopy (IEM) was used to analyze the compatible and incompatible host-pathogen interaction between the obligate, biotroph stem rust (Puccinia graminis f.sp. tritici; Pgt) and primary leaves of wheat (Triticum aestivum L.). The investigation was focused on the subcellular localization of a fungal elicitor glycoprotein of stem rust (Pgt-elicitor). Uredospores as well as fungal infection structures of stem rust on wheat leaves were probed with a specific monoclonal antibody, in order to determine the in situ distribution pattern of the antigen. Binding to the anti-elicitor antibody was observed over the cell wall and the germ pore of germinating uredospores. Immunogold staining was found over the infection structures of stem rust within the wheat leaf tissue of both the compatible and incompatible plant-pathogen interaction. Distinct cell wall layers of the intercellular mycelium, of the haustorial mother cells, as well as of the haustoria were clearly labeled. Gold particles were also detected over the intercellular space and the extrahaustorial matrix in between the extrahaustorial membrane and the haustorial cell wall which indicated a release of elicitor molecules from the fungal cell wall. No labeling was observed over the host cell cytoplasm of the compatible and incompatible interaction, respectively. The immunocytochemical detection of elicitor epitopes over the hyphal cell walls of in vitro grown axenic cultures of P. graminis f.sp. tritici confirmed the occurrence of elicitor molecules in young hyphal material. Elicitor molecules were released by the hyphae of axenic cultures of stem rust in vitro.     

Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.     

Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation

Smooth muscle myosin bound phosphatase (MBP) purified from chicken gizzard, which is a holoenzyme of type 1 delta protein phosphatase and dephosphorylated intact myosin, catalyzed the dephosphorylation of calponin phosphorylated by protein kinase C (PK-C). The Km of MBP for calponin was 0.6 microM and the Vmax was 350 nmol/min/mg. All of the multiple sites of phosphorylation by PK-C of calponin were completely dephosphorylated by MBP. Functionally, calponin dephosphorylated by MBP recovered its inhibitory effect on the actin-activated Mg(2+)-ATPase activity of myosin. Therefore, these results suggest that a type 1 delta protein phosphatase causes relaxation of smooth muscle by the dephosphorylation not only of myosin but also of calponin.     

Endothelium-derived relaxing factor activates calcium-activated potassium channels of resistance vessel smooth muscle cells

Direct observation was made by using the patch-clamp technique with a specially designed microperfusion system to investigate the effect of acetylcholine (Ach 10(-6) mol/L) elicited endothelium-derived relaxing factor (EDRF) on the calcium-activated potassium channel (IK(Ca)) in the smooth muscle cells of mesenteric resistance vessels in Wistar rats. Activation of IK(Ca) was firstly observed by inducing the elicited EDRF or sodium nitroprusside (SNP 10(-8) mol/L) under various clamping voltages in cell-attached configuration. While the pipette solution contained KCl 126 mmol/L and the bath solution contained KCl 5.9 mmol/L, two types of conductances of calcium-activated potassium current being 76.4 +/- 2.3 pS (mean +/- S.E. n = 7) and 160.3 +/- 7.5 pS (mean +/- S.E. n = 7) were recorded during the EDRF activation, one type of conductance being 100.5 +/- 2.8 pS (mean +/- S.E. n = 6) was activated by nitric oxide (NO) which is an effective component from SNP. Differences in kinetic characteristics of these channels between EDRF and NO activation were found, particularly the probability of the channel being open in EDRF activation was obviously greater than that in NO stimulation. It has been shown that the potassium channel mechanisms involved in the EDRF and NO actions might be different.     

Effects of visnagin on cyclic nucleotide phosphodiesterases and their role in its inhibitory effects on vascular smooth muscle contraction

Several reports have been published which describe the technique of using an Endo GIA to resect submucosal tumors on the anterior wall of the stomach. Lesions on the posterior wall, however, especially near the esophagocardiac junction (ECJ), are difficult to resect using these reported techniques. This is because the surgeon must divide the omentum and enter the omental bursa in order to use a similar extraluminal technique. Furthermore, special care must be taken to ensure that resections do not involve the ECJ and narrow the esophagus. In order to overcome these difficulties, we have proposed a new technique for the laparoscopic excision of a submucosal tumor located on the posterior wall of the gastric fundus. The principle of this procedure involves the intraluminal resection of the submucosal tumor, including the surrounding stomach wall, using the Endo GIA. This technique is safe, simple, and effective. We believe that we are the first to address the excision of a submucosal lesion by resecting the full thickness of the posterior gastric wall lesion intraluminally.     

beta-adrenergic modulation of maxi-K channels in vascular smooth muscle via Gi through a membrane-delimited pathway

Direct modulation of large-conductance Ca2+-activated K+ (maxi-K) channel by receptor-associated G protein in rabbit mesenteric arterial smooth muscle cells was studied using the outside-out patch clamp technique. Applying a beta-adrenoceptor agonist (isoproterenol) increased maxi-K channel activity by 75%, and the effect was almost completely abolished by pretreating the cells with pertussis toxin but not with cholera toxin. When the antibody against Gi protein was present in the pipette solution the stimulatory effect of isoproterenol disappeared. These results suggest that beta-adrenoceptor stimulation increases maxi-K channel activity via a membrane-delimited pathway, probably through pertussis toxin-sensitive G protein (Gi).     

Cyclo-oxygenase-2 in vascular smooth muscle

Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.     

Nitric oxide, a possible mediator of 1,4-dihydropyridine-induced photorelaxation of vascular smooth muscle

1. In rat aortic tissues pre-contracted with phenylephrine, certain 1,4-dihydropyridines (DHPs) such as Bay K 8644 (0.1 microM), PN 202791 (1 microM), RK 30 (1 microM), NI 104 (1 microM) and NI 105 (1 microM) enhanced photoactivated relaxations (photorelaxation or PR) whereas NI 72, NI 85, NI 99, NI 102, amlodipine, felodipine, nifedipine and nimodipine were inactive. 2. The PR inducing effects of Bay K 8644 were mimicked by the diabetogenic agent, streptozotocin (STZ). 3. Solutions of Bay K 8644 which had been irradiated for various periods of time initiated light independent transient relaxations followed by contractile responses in aortic tissue partially contracted with phenylephrine. With exposure times to light of 30 to 120 min, the intensity of the relaxation response to irradiated Bay K 8644 increased from 26 +/- 3.3 to 71 +/- 3.7% of the maximum contractile response to phenylephrine (n = 5). Conversely the contractile responses decreased, from 84.2 +/- 4.1 to 19.8 +/- 10.4% of the maximum contractile response to phenylephrine (n = 5). 4. Superoxide ions, generated by incubation of xanthine (2mM) plus xanthine oxidase (10 mu ml-1) in physiological saline solution (PSS) NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 12.5 and glucose 11.1 (mM) for 1 h. reduced the PR induced by DHPs, STZ, and also NO-induced relaxations of rat aortic preparations. 5. Direct measurements of NO indicate that, following exposure to a polychromatic light source, equimolar concentrations (0.1 mM) of the DHP compounds that enhance PR, as well as STZ, photodegrade to release NO (25 +/- 2-40.3 +/- 5.9 nmol min-1, n = 6). 6. Structure-activity studies indicate that a nitro group at the -3 position of the dihydropyridine ring is essential for DHPs to support PR. 7. These data suggest that the photodegradation of DHPs and STZ leading to the release of NO provides the primary cellular process underlying the PR response.     

MAP kinases and vascular smooth muscle function

Integrins are a subclass of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. Integrins influence transendothelial migration of lymphocytes and monocytes and are suitable targets for experimental immunotherapy. They are critically involved in the pathogenesis of autoimmune neuritis and abnormally expressed in human neuropathies. Also, the role of integrins in myelination, neurite outgrowth, and nerve regeneration suggests that they could be involved in the recovery phase of immune-mediated neuropathies. We investigated by immunohistochemistry the expression of a number of integrin subunits during the course of experimental autoimmune neuritis (EAN). Results were compared with the human immune neuropathy Guillain-Barre syndrome (GBS) and extended in vitro. Inflammation and demyelination in both EAN and GBS induced the down-regulation of beta4 integrin in Schwann cells (SCs), whereas loss of alpha2 was noted only in EAN. When axonal loss was present, SCs displayed alpha5 integrin, in both EAN and GBS. In vitro, basal lamina and inflammatory cytokines modulated the expression of beta4 in SCs, but they did not influence alpha2 and alpha5 expression. Finally, integrins were differentially expressed in blood vessels during EAN. In conclusion, the spatiotemporal changes in integrin expression may be used to characterize, stage, and better understand the pathogenesis and evolution of inflammation during GBS and EAN. This may help to establish useful, novel therapy for immune-mediated neuropathies.     

Sarcoplasmic reticulum-sarcolemma interactions and vascular smooth muscle tone

A characteristic of vascular smooth muscle cell morphology is a close apposition of its peripheral sarcoplasmic reticulum (SR) with the sarcolomma; this arrangement gives rise to important functional interactions whereby the peripheral SR regulates Ca2+ influx and vascular tone. We review here the key evidence supporting the following aspects of SR-sarcolemma interactions while establishing a conceptual framework encompassing (i) the SR ultrastructure and functions, (ii) the integration of the sarcolemmal Na+-Ca2+ exchanger and the peripheral SR in the mediation of a bidirectional Ca2+ exchange between the peripheral SR and the extracellular space, (iii) the existence of a higher myoplasmic free Ca2+ concentration [Ca2+]myo in the subsarcolemmal space formed between the sarcolemma and the peripheral SR relative to the [Ca2+]myo of the inner myoplasm in the resting smooth muscle cell, (iv) the division of the subsarcolemmal space into functional microdomains, (v) the existence of spontaneous localized bursts of Ca2+ release from the peripheral SR (Ca2+ sparks) towards the sarcolemma, (vi) the physiological triggering of nonlocalized Ca2+ release from the peripheral SR by Ca2+ influx (Ca2+-induced Ca2+ release), and (vii) capacitative Ca2+ entry in vascular smooth muscle. We present an overview of the physiological and pathological implications of these interactions.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca2+-activated K+ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 microM) had no effect in the absence of intracellular adenosine 5'-triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 microM) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5'-[beta gamma-methylene]-triphosphate (AMP-PCP). NPY inhibited Ca2+-activated K+ channel activity when ATP was replaced by adenosine 5'-O-(3-thiotriphosphate) (ATP [gamma-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 microM), a specific inhibitor of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 microM) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca2+-activated K+ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

血管平滑肌细胞的增殖因素及机制

血管平滑肌细胞(VSMC)增殖已成为目前心血管疾病研究领域里的热点,VSMC增殖、迁移及凋亡在冠状动脉粥样硬化(AS)、冠状动脉搭桥术、经皮冠状动脉介入治疗(PCI)术后血管再狭窄的发生和发展过程中起重要作用.本文作者对VSMC增殖及促增殖因素和机制进行综述.     

Control of vascular smooth muscle cell growth in fowl

In adult domestic fowl, angiotensin (ANG) receptors are present in the vascular smooth muscles (VSM) and in the endothelium, mediating vasorelaxation via endothelium-derived relaxing factor/cGMP. ANG II-induced relaxation is minor in chicks and becomes more marked as they mature but diminishes in adult birds, whereas ANG II neither relaxes nor contracts endothelium-denuded aortae from mature chickens. The present study examines in cultured fowl aortic SM cells whether (1) ANG II stimulates or inhibits VSM cell growth and, if so, whether this growth-stimulatory or -inhibitory effect changes with maturation/aging, and (2) S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, and cGMP attenuate the basal or stimulated VSM cell growth. [Asp1, Val5]ANG II (native fowl ANG II, 10(-6) M) markedly increased (increase from vehicle control, 226.5%; P < 0.01) [3H]thymidine (Thd) incorporation into DNA of quiescent VSM cells (first subculture) from 6-week-old chicks. This growth-stimulating effect was reduced with age (41.4, 29.6, and 3.2% at 9, 19, and 43 weeks of age, respectively). In contrast, platelet-derived growth factor (PDGF, 20 ng/ml) increased [3H]Thd incorporation similarly in chicks, pullets, and hens. Furthermore, ANG II significantly (45.9%, P < 0.01) attenuated the growth-promoting effect of fetal calf serum in cultured VSM cells from 6-week-old chicks. This inhibitory effect also decreased in older birds. ANG II showed neither a growth-stimulatory nor -inhibitory effect in cultured neointimal cells. SNAP attenuated dose dependently (20-60 microM) the basal and PDGF-induced VSM cell growth, whereas cGMP inhibited basal growth only at a high dose (100 microM). These results indicate that in fowl VSM cells, ANG II is mitogenic and antimitogenic in chicks but not in mature birds, suggesting that phenotypic modulation occurs in the ANG receptors/signaling mechanism with maturation/age or in neointimal cells, whereas the mitogenic mechanism via PDGF remains in both young and mature birds.     

Lipid second messengers derived from glycerolipids and sphingolipids, and their role in smooth muscle function

The processes that link activation of an external receptor to the internal mechanisms that elicit a physiological response have been the subject of extensive investigation. It has been established that rather than just being an inert barrier to protect the cell from environmental damage, there are populations of phospholipids located within the plasma membrane that act as a reservoir for signalling molecules and when a receptor binds its appropriate activating ligand a chain of events is initiated which leads to the breakdown of these lipids and the release of second messengers. Such processes are rapid enough for physiological responses to be effected. The purpose of this review is to examine the profile of lipid second messengers derived from glycerophospholipids and sphingolipids. In the former class are included phosphoinositide and phosphatidylcholine and the latter includes sphingomyelin. Hydrolysis of such parent compounds is mediated by phospholipases and the profile of metabolites appears to be agonist specific and modulated by a number of mechanisms including heterotrimeric G-protein subunits, small G-proteins, alterations in intracellular calcium concentration, protein kinase C and tyrosine kinases. The recent interest in sphingolipids, particularly in vascular smooth muscle cells, has been provoked by the observation that ceramide and sphingoid base formation is observed in response to vasoconstrictor hormones.