Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 +/- 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 +/- 6.1%) or a control peptide (32.1 +/- 6.5%). Administration of 18alpha-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 +/- 3.2% vs. 23.8 +/- 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (-48.6 +/- 1.4 mV) at the higher pressure compared with vehicle (-41.4 +/- 1.5 mV) and Gap20-treated (-38.4 +/- 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.     

Intercellular calcium signaling via gap junctions in glioma cells

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.     

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Upregulation of connexin43 gap junctions between neointimal smooth muscle cells

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.     

Regulation of RGS2 and second messenger signaling in vascular smooth muscle cells by cGMP-dependent protein kinase

RGS2, a GTPase-activating protein (GAP) for G(q)alpha, regulates vascular relaxation and blood pressure. RGS2 can be phosphorylated by type Ialpha cGMP-dependent protein kinase (cGKIalpha), increasing its GAP activity. To understand how RGS2 and cGKIalpha regulate vascular smooth muscle signaling and function, we identified signaling pathways that are controlled by cGMP in an RGS2-dependent manner and discovered new mechanisms whereby cGK activity regulates RGS2. We show that RGS2 regulates vasoconstrictor-stimulated Ca(2+) store release, capacitative Ca(2+) entry, and noncapacitative Ca(2+) entry and that RGS2 is required for cGMP-mediated inhibition of vasoconstrictor-elicited phospholipase Cbeta activation, Ca(2+) store release, and capacitative Ca(2+) entry. RGS2 is degraded in vascular smooth muscle cells via the proteasome. Inhibition of cGK activity blunts RGS2 degradation. However, inactivation of the cGKIalpha phosphorylation sites in RGS2 does not stabilize the protein, suggesting that cGK activity regulates RGS2 degradation by other mechanisms. cGK activation promotes association of RGS2 with the plasma membrane by a mechanism requiring its cGKIalpha phosphorylation sites. By regulating GAP activity, plasma membrane association, and degradation, cGKIalpha therefore may control a cycle of RGS2 activation and inactivation. By diminishing cGK activity, endothelial dysfunction may impair RGS2 activation, thereby blunting vascular relaxation and contributing to hypertension.     

缝隙连接在同型半胱氨酸介导的自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。     

Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture

Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.     

Innervation and gap junctions of intestinal striated and smooth muscle cells in the loach

Summary The tunica muscularis of the proximal intestine of the loach consisted of intermingling striated and smooth muscle cells without forming any distinct sublayers. Close contacts devoid of intervention by a basal lamina sometimes occurred between these different types of muscle cells. Gap junctions were occasionally found between heterologous as well as homologous muscle cells. In freeze-fracture replicas, striated muscle cells were distinguished from smooth muscle cells by numerous, evenly distributed subsurface caveolae. These were relatively rare and linearly arranged in smooth muscle cells. Variously-sized and -formed aggregations of connexon particles were found in the protoplasmic fracture-face of both muscle cells. Striated muscle cells had aggregates of connexon particles taking the form of either a small solid polygon or an annulus with a particle-free central region. In smooth muscle cells, the particles were arranged either in variously-sized patches or in straight lines. Topologically, heterologous gap junctions observed in ultrathin section were thought to correspond to the small patchy aggregations. Striated muscle cells in the gut had neuromuscular junctions, which differed morphologically from cholinergic nerve terminals at neuromuscular junctions of typical skeletal muscle cells. The smooth muscle cells had close apposition with axonal terminals containing many granular vesicles and a variable number of small, clear vesicles. Occasionally, a cholinergic-type axonal terminal with a presynaptic active site was found close to a smooth muscle cell.     

A stepwise method for the isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries

Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.     

Prostaglandin-stimulated second messenger signaling in bone-derived endothelial cells is dependent on confluency in culture

New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E₂ and F_2α (PGE₂, PGF_2α), it was investigated if these PGs were agonists to bone-derived endothelial cells (BBE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca²⁺]i) second messenger generation. We found that confluent cultures of BBE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 μM PGE₂ by an increase in cAMP. PGF_2α at the same concentration was less potent in stimulating an increase in cAMP production in confluent BBE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE₂-stimulated cAMP generation. PGF_2α failed to elicit any cAMP production in subconfluent cultures. PGE₂ and PGF_2α both stimulated an increase in [Ca²⁺]i concentration in a dose-dependent manner. The potency of PGE₂ was similar to that of PGF_2α in stimulating an increase in [Ca²⁺]i. The Ca²⁺ response was mostly independent of extracellular Ca⁺, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PG-induced increase in [Ca²⁺]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE₂ or PGF_2α were either negligible, or only small increases in [Ca²⁺]i were noted with high concentrations of these two PGs. Consistent, dose-dependent increases in [Ca²⁺]i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BBE cells respond to parathyroid hormone (PTH) by the production of cAMP, we tested if bovine PTH(1-34) amide bPTH(1—34) also increased [Ca²⁺]i in these cells. No change in [Ca²⁺]i was found in response to bPTH (1—34), although bPTH (1—34) stimulated a nine to tenfold increase in cAMP. We conclude that BBE cells respond to PGE₂ and PGF_2α but not to bPTH(1—34) by an increase in [Ca²⁺]i probably secondary to stimulation of phospholipase C and that the cAMP and [Ca²⁺]i second messenger responses in BBE cells are dependent on the state of confluency of the cells. © 1994 Wiley-Liss, Inc.     

Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells

Homocysteine found in the plasma of patients with coronary heart disease, induces vascular smooth muscle cell (VSMC) proliferation and increases deposition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is largely unknown. We hypothesized that homocysteine induces ECM production via intracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeling for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with fura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca(2+)](i) transients with an EC(50) of 60 +/- 5 nM. The EC(50) for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular calcium; however, thapsigargin pretreatment, which depletes intracellular Ca(2+) stores, abolished the homocysteine effect, demonstrating its dependence on intracellular Ca(2+) stores. Extracellular sodium depletion significantly (P < 0.05) increased [Ca(2+)](i) also suggesting a possible role of sodium-calcium exchange in the process. To begin to elucidate the intracellular pathways by which homocysteine might act, VSMC were pretreated with specific inhibitors and stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented the release of Ca(2+) by homocysteine. Interestingly, pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) greatly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretreatment with either the phospholipase A(2) activator neomycin, the antioxidant and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium channel blocker, felodipine completely inhibited the homocysteine-induced Ca(2+) signal in VSMC. This suggests the role of multiple signaling pathways in the homocysteine effect on VSMC Ca(2+). Effects of homocysteine on collagen production, as ascertained by immunoblot analysis, correlated with its effect in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM deposition, has the potential to affect vascular reactivity. To determine the effect of homocysteine on the ability of VSMC to react to potent agonist such as angiotensin II, VSMC were pretreated with homocysteine and exposed to a range of angiotensin II concentrations which normally have no effect on intracellular Ca(2+). After homocysteine pretreatment, VSMC were extremely responsive to angiotensin II at concentrations well below the physiologic range. These data taken together suggested that an initial effect of homocysteine is to induce release of intracellular Ca(2+) in VSMC and may induce vascular reactivity. The transient in Ca(2+) correlates with the effect on ECM associated with homocysteine.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells

Myoendothelial gap junctions are involved in regulating systemic arterial smooth muscle cell phenotype and function, but their role in the regulation of pulmonary arterial smooth muscle cell (PASMC) phenotype is unknown. We therefore investigated in cocultured pulmonary arterial endothelial cells (PAECs) and PASMCs whether myoendothelial gap junctional signaling played a role in PAEC-dependent regulation of PASMC phenotype. Rat PAECs and PASMCs were cocultured on opposite sides of a porous Transwell membrane that permitted formation of heterotypic cell-cell contacts. Immunostaining showed expression of the gap junctional protein connexin 43 (Cx43) on projections extending into the membrane from both cell types. Dye transfer exhibited functional gap junctional communication from PAECs to PASMCs. PASMCs cocultured with PAECs had a more contractile-like phenotype (spindle shape and increased expression of the contractile proteins myosin heavy chain, H1-calponin, and α-smooth muscle cell-actin) than PASMCs cocultured with PASMCs or cocultured without direct contact with PAECs. Transforming growth factor (TGF)-β1 signaling was activated in PASMCs cocultured with PAECs, and the PASMC differentiation was inhibited by TGF-β type I receptor blockade. Inhibition of gap junctional communication pharmacologically or by knock down of Cx43 in PAECs blocked TGF-β signaling and PASMC differentiation. These results implicate myoendothelial gap junctions as a gateway for PAEC-derived signals required for maintaining TGF-β-dependent PASMC differentiation. This study identifies an alternative pathway to paracrine signaling to convey regulatory signals from PAECs to PASMCs and raises the possibility that dysregulation of this direct interaction is involved in the pathogenesis of hypertensive pulmonary vascular remodeling.     

Evidence for high molecular weight proteins in arthropod gap and smooth septate junctions

The proteins that make up arthropod gap and septate junctions have not been identified with any certainty. Several candidate proteins for both types of junctions have been proposed in the literature, but there has been no agreement on any of these. Arthropod gap junctions do not label with antibodies to vertebrate gap junction connexins; it thus appears that unrelated proteins form these rather similar structures. Gap junctions inManduca sextamidgut epithelium are unusual since they function only during the molt and are non-functioning during the larval instars. We have developed a preparation from this tissue that is highly enriched in both gap and smooth septate junctions when examined by electron microscopy. SDS-PAGE gels of this preparation have two major protein bands, at 75 and 90 kDa. The presence of gap junctions correlates best with the 75 kDa protein and smooth septate junctions with the 90 kDa protein. Further, the 75 kDa band is stained by an antibody to a putative gap junction protein fromC. elegans. We propose that the 75 kDa protein is a major structural component of gap junctions inManduca sextamidgut epithelium and that the 90 kDa protein forms the smooth septate junctions.     

Peroxynitrite induces apoptosis in rat aortic smooth muscle cells: possible relation to vascular diseases

An emerging body of evidence is accumulating to suggest that in vivo formation of free radicals in the vasculature, such as peroxynitrite (ONOO-), and programmed cell death (i.e., apoptosis) play important roles in vascular diseases such as atherosclerosis, hypertension, and restenosis. The present study was designed to determine whether primary rat aortic smooth muscle cells (SMCs) undergo apoptosis following treatment with ONOO-. Direct exposure of primary rat aortic SMCs to ONOO--induced apoptosis in a concentration-dependent manner, as confirmed by means of quantitative fluorescence staining and TUNEL assays. ONOO--induced apoptosis in rat aortic SMCs appears to involve activation of Ca2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis in rat aortic SMCs need to be further investigated, the present, preliminary findings could be used to suggest that ONOO- formation in the vasculature may play roles in the processes of vascular diseases, such as atherosclerosis, hypertension, and restenosis, via adverse actions on blood vessels.     

The nanostructure of myoendothelial junctions contributes to signal rectification between endothelial and vascular smooth muscle cells

Micro-anatomical structures in tissues have potential physiological effects. In arteries and arterioles smooth muscle cells and endothelial cells are separated by the internal elastic lamina, but the two cell layers often make contact through micro protrusions called myoendothelial junctions. Cross talk between the two cell layers is important in regulating blood pressure and flow. We have used a spatiotemporal mathematical model to investigate how the myoendothelial junctions affect the information flow between the two cell layers. The geometry of the model mimics the structure of the two cell types and the myoendothelial junction. The model is implemented as a 2D axi-symmetrical model and solved using the finite element method. We have simulated diffusion of Ca(2+) and IP(3) between the two cell types and we show that the micro-anatomical structure of the myoendothelial junction in itself may rectify a signal between the two cell layers. The rectification is caused by the asymmetrical structure of the myoendothelial junction. Because the head of the myoendothelial junction is separated from the cell it is attached to by a narrow neck region, a signal generated in the neighboring cell can easily drive a concentration change in the head of the myoendothelial protrusion. Subsequently the signal can be amplified in the head, and activate the entire cell. In contrast, a signal in the cell from which the myoendothelial junction originates will be attenuated and delayed in the neck region as it travels into the head of the myoendothelial junction and the neighboring cell.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.