Mechanisms underlying spontaneous rhythmical contractions in irideal arterioles of the rat.
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1. Mechanisms underlying spontaneous rhythmical contractions have been studied in irideal arterioles of the rat using video microscopy and electrophysiology. 2. Rhythmical contractions (4 min-1) were more common during the second and third postnatal weeks and were always preceded by large, slow depolarizations (5-40 mV). 3. Spontaneous contractions were unaffected by tetrodotoxin (1 microM), neurotransmitter receptor antagonists, the sympathetic neurone blocker, guanethidine (5 microM) or sensory neurotoxin, capsaicin (1 microM). 4. Stimulation of sensory nerves inhibited spontaneous activity and this was not prevented by L-NAME (10 microm). 5. L-NAME (10 microm) caused an increase in frequency of spontaneous contractions, while forskolin (30 nM), in the presence of L-NAME, abolished spontaneous, but not nerve-mediated, contractions. 6. Spontaneous activity was not affected by felodipine (1 nM) or nifedipine (1 microM), but was abolished by cadmium chloride (1 microM) or superfusion with calcium-free solution. 7. Caffeine (1 mM), thapsigargin (2 microM) and cyclopiazonic acid (3 microM), but not ryanodine (3 microM), abolished spontaneous and nerve-mediated contractions. After preincubation in L-NAME (10 microM), cyclopiazonic acid abolished spontaneous contractions only. 8. Spontaneous depolarizations and contractions were abolished by 18alpha-glycyrrhetinic acid (20 microM). 9. Results suggest that spontaneous rhythmical contractions are myogenic and result from the cyclical release of calcium from intracellular stores, without a contribution from voltage-dependent calcium channels. Intercellular coupling through gap junctions appears to be essential for co-ordination of these events which could be modulated by nitric oxide and increases in cAMP. The possibility that different intracellular stores underly spontaneous and nerve-mediated contractions is discussed. (+info)
Inhibition of endothelial cell migration, intercellular communication, and vascular tube formation by thromboxane A(2).
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The eicosanoid thromboxane A(2) (TXA(2)) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA(2) might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA(2) mimetic [1S-(1alpha, 2beta(5Z),3alpha(1E,3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-o xab icyclo- [2.2.1]heptan-2-yl]-5'-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC(50) of 25 nM. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18beta-glycyrrhetinic acid (1 microM) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA(2) mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA(2) signaling might enhance vascularization of ischemic tissue. (+info)
Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.
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The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification. (+info)
Role of gap junctions in the responses to EDHF in rat and guinea-pig small arteries.
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1. In guinea-pig internal carotid arteries with an intact endothelium, acetylcholine (10 microM) and levcromakalim (10 microM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K(+) was without effect. 2. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 microM) or gap 27 (500 microM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine-induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K(+) channel opener, 1-ethyl-2-benzimidazolinone (600 microM). 3. In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 microM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine-induced hyperpolarization was inhibited by carbenoxolone. 4. Phenylephrine (1 microM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine-induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. 5. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium-derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea-pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF. (+info)
A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.
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Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor. (+info)
Disruption of cadherin-related junctions triggers autocrine expression of vascular endothelial growth factor in bovine aortic endothelial cells : effects on cell proliferation and death resistance.
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The mechanisms involved in the blockade of proliferation in confluent endothelial cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of endothelial monolayer cell growth. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both endothelial cell proliferation and autocrine production of growth factors. With this purpose, we assessed the changes in growth, death resistance, and expression of vascular endothelial growth factor (VEGF) under conditions of disruption of the intercellular junctions between endothelial cells. Disruption of cell junctions was produced by means of a specific anti-vascular endothelial cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the 121-, 165-, and 189-amino acid isoforms of VEGF. Further evidence of the relationship between endothelial cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical endothelial injury and in endothelial cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with growth-promoting and cytoprotective effects, as assessed by [(3)H]thymidine uptake, (51)Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic interendothelial junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of endothelial cells. (+info)
Gap junctional coupling and patterns of connexin expression among neonatal rat lumbar spinal motor neurons.
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Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)-P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state. (+info)
Ultrastructural characterization of the interstitial cells of Cajal.
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Recent studies on the interstitial cells of Cajal (ICC) have determined ultrastructural criteria for the identification of these previously enigmatic cells. This review deals with the electron microscopic findings obtained by the author's research group in different tissue regions of the gut in mice, rats and guinea-pigs, comparing these with reports from other groups in different species and in humans. ICC are characterized by the following morphological criteria: numerous mitochondria, abundant intermediate filaments and large gap junctions which connect the cells with each other and with smooth muscle cells. Due to their location in the gut and the specific species, the ICC are markedly heterogeneous in appearance, ranging from cells closely resembling smooth muscle cells to those similar to fibroblasts (Table 1). Nevertheless, the above-mentioned morphological features are shared by all types of ICC and serve in identifying them. Recent discoveries on a significant role of c- kit in the maturation of the ICC and their specific immunoreactivity to anti-c-Kit antibody have confirmed the view that the ICC comprise an independent and specific entity of cells. This view is reinforced by the findings of the author's group that the ICC characteristically possess vimentin filaments and are stained with the zinc iodide-osmium tetroxide method which provides a staining affinity similar to methylene blue, the dye used in the original work by Cajal, (1911). Developmental studies indicate that the ICC are derived from a non-neuronal, mesenchymal origin. This paper further reviews advances in the physiological studies on the ICC, in support of the hypothesis by THUNEBERG (1982) that they function as a pacemaker in the digestive tract and a mediator transmitting impulses from the nerve terminals to the smooth muscle cells. (+info)