Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication. (65/1758)

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of alpha1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal ( approximately 75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 microM), prostaglandin F2alpha (PGF2alpha; 1 microM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 microM and 500 microM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 +/- 6% (N = 26 rings) to 57 +/- 7% (N = 26 rings) and 34.9 +/- 6% (N = 13 rings), respectively (P<0.05), and for PGF2alpha from a control response of 75 +/- 10% (N = 16 rings) to 52 +/- 8% (N = 19 rings) and 25.9 +/- 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 microM and 500 microM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 +/- 5. 0% for the control response (N = 38 rings), 59 +/- 6.0% in the presence of 200 microM heptanol (N = 17 rings), and 70 +/- 6.0% in the presence of 500 microM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 +/- 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.  (+info)

Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells. (66/1758)

Connexin43 (Cx43), the predominant gap junction protein of muscle cells in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension on the expression of Cx43 in aorta and heart in three different models of experimental hypertension. Rats were made hypertensive either by clipping one renal artery (two kidney, one-clip renal (2K,1C) model) by administration of deoxycorticosterone and salt (DOCA-salt model) or by inhibiting nitric oxide synthase with N G-nitro-L-arginine methyl ester (L-NAME model). After 4 weeks, rats of the three models showed a similar increase in intra-arterial mean blood pressure and in the thickness of the walls of both aorta and heart. Analysis of heart mRNA demonstrated no change in Cx43 expression in the three models compared to their respective controls. The same 2K,1C and DOCA-salt hypertensive animals expressed twice more Cx43 in aorta, and the 2K, 1C rats showed an increase in arterial distensibility. In contrast, the aortae of L-NAME hypertensive rats were characterized by a 50% decrease in Cx43 and the carotid arteries did not show increased distensibility. Western blot analysis indicated that Cx43 was more phosphorylated in the aortae of 2K,1C rats than in those of L-NAME or control rats, indicating a differential regulation of aortic Cx43 in different models of hypertension. The data suggest that localized mechanical forces induced by hypertension affect Cx43 expression and that the cell-to-cell communication mediated by Cx43 channels may contribute to regulating the elasticity of the vascular wall.  (+info)

Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. (67/1758)

Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, approximately 10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.  (+info)

Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations. (68/1758)

The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil-derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil-derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and alpha-glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co-cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T- and B-lymphocyte co-cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes.  (+info)

PDGF regulates gap junction communication and connexin43 phosphorylation by PI 3-kinase in mesangial cells. (69/1758)

BACKGROUND: Gap junctional intercellular communication (GJIC) plays an important role in the regulation of cell growth, migration, and differentiation. Ultrastructural and histochemical studies indicate the existence of a high density of gap junctions among mesangial cells (MCs), but little is known about their regulation. Because of the close link between growth and GJIC, we examined how platelet-derived growth factor (PDGF) may affect GJIC in cultured MCs. METHODS: MCs were exposed to PDGF in the presence or absence of phosphatidylinositol 3' kinase (PI3K) inhibitors, and GJIC was evaluated by the transfer of Lucifer yellow. The gap junction protein connexin43 (Cx43) was examined by immunohistochemistry, immunoprecipitation, and Western blot. RESULTS: The addition of PDGF into MC culture caused a rapid and transient inhibition of GJIC, with maximal inhibition (80%) occurring 15 minutes after PDGF exposure and returning to control levels after 90 minutes. This action of PDGF could be largely prevented by pretreatment of MCs with the PI3K inhibitor LY294002. Immunochemical staining showed that PDGF did not alter the localization and distribution of Cx43. Immunoprecipitation studies demonstrated that PDGF induced a rapid and transient increase of tyrosine phosphorylation of Cx43 protein, which was dose dependent and in accordance with the time course of the disruption of GJIC. PDGF also elicited activation of extracellular signal-regulated kinase (ERK). Using two structurally unrelated PI3K inhibitors, wortmanin and LY294002, both tyrosine phosphorylation of Cx43 and activation of ERK stimulated by PDGF were largely blocked. CONCLUSION: These results suggest that PDGF abrogates GJIC function in MCs via the PI3K-dependent signaling pathway. Disruption of GJIC by PDGF could be one mechanism by which PDGF modulates MC behavior. Participation of PI3K in the regulation of GJIC demonstrates the complex coordination of molecular events that accompany MC mitogenesis.  (+info)

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. (70/1758)

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.  (+info)

Spontaneous luteinization of antral marmoset follicles in vitro. (71/1758)

Large non-luteinized follicles of the marmoset monkey were cultured for up to 96 h in the presence of substances that are known to induce luteinization, i.e. LH, transforming growth factor (TGF)-beta and cyclic AMP. The state of the basal lamina, and the expression of connexin-43, alpha(2) integrin subunit and TGF-beta receptor type II (TbetaR-II) were chosen as parameters to judge the progress of luteinization. Antral follicles, cultured for 1 h, were not luteinized, as shown by an intact basal lamina, strong immunoreactivity of connexin-43 in granulosa cells, and no expression of TbetaR-II in the theca layer. After 12 h, most follicles showed a dissolution of the basal lamina, a faint reactivity of connexin-43, high expression of TbetaR-II in theca- and outer granulosa cells and high expression of alpha(2) integrin subunit in granulosa cells bordering at the basement membrane; all of which indicate luteinization. After 96 h of culture, luteal structures (e.g. corpora lutea accessoria) had developed. This was true for both non-stimulated and stimulated follicles. Our results strongly suggest that antral follicles luteinize spontaneously. The decisive determinant appears to be the follicular stage.  (+info)

Coexpression of connexins 40 and 43 enhances the pH sensitivity of gap junctions: a model for synergistic interactions among connexins. (72/1758)

Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype. Xenopus oocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from approximately 6.7 when cells expressed only Cx40 or only Cx43 to approximately 7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined.  (+info)