Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia.

Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.

Connexin and gap junction degradation.

Many of the subunit proteins (connexins) that form gap junctions are rather dynamic, with half-lives of only a few hours. Thus, alterations in connexin turnover and degradation may represent significant mechanisms for the regulation of intercellular communication. We describe a pharmacological approach to determining pathways of connexin degradation. Cell cultures are left untreated or treated with inhibitors of lysosomal or proteasomal proteolysis. Changes in connexin levels, localization, or decay curves (derived from pulse-chase experiments) are assessed by immunoblotting, immunofluorescence, and immunoprecipitation, respectively. Such experiments have provided evidence that connexin43 degradation involves both the lysosome and the proteasome.

Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes.

OBJECTIVE: The present studies were performed to examine the degradation of connexin43-containing gap junctions by the lysosome or the proteasome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. METHODS: Primary cultures were prepared from neonatal rat ventricular myocytes. Connexin43 was detected by immunoblotting, immunofluorescence, or immunoprecipitation. Gap junction profiles were detected by transmission electron microscopy. RESULTS: Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures treated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or ammonium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse-chase experiments showed that the half-life of connexin43 was 1.4 h in control cultures, but was prolonged to 2.0 or 2.8 h in cultures treated with chloroquine or lactacystin, respectively. Immunofluorescence and electron microscopy showed a significant increase in the number of gap junction profiles in myocytes treated with either chloroquine or lactacystin. Heat treatment of cultures (43.5 degrees C for 30 min) produced a rapid loss of connexin43 as detected by immunoblotting or immunofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexin43 levels and distribution returned to normal by 3 h following a heat shock and were resistant to a subsequent repeat heat stress. The heat shock also led to production of HSP70 as detected by immunoblotting. CONCLUSIONS: These data suggest that Cx43 gap junctions in myocytes are degraded by the proteasome and the lysosome, that this proteolysis can be augmented by heat stress, and that inducible factors such as HSP70 may protect against Cx43 degradation.

Mouse connexin40: gene structure and promoter analysis.

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3'-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5' of the transcriptional start site and 400 bp 3' of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297.

Degradation of connexin43 gap junctions involves both the proteasome and the lysosome.

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1-3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of approximately 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.

Rat uterine myometrium contains the gap junction protein connexin45, which has a differing temporal expression pattern from connexin43.

OBJECTIVE: Our purpose was to determine whether myometrial cell lines and rat myometrial tissue contained additional gap junction proteins besides connexin43. STUDY DESIGNS: Syrian hamster myocytes (SHM-ER) and human SK-UT-1 myometrial cell lines were analyzed for intercellular coupling by microinjection of Lucifer yellow. These cell lines and myometrial tissue isolated from pregnant rats were analyzed for connexin expression by ribonucleic acid blotting and immunofluorescence. RESULTS: SHM-ER and SK-UT-1 cells showed functional gap junctional coupling by intercellular passage of microinjected dye. Both cell lines contained connexin43 and connexin45 messenger ribonucleic acids but did not contain any other detectable connexin messenger ribonucleic acids. Immunofluorescence confirmed the presence of connexin43 and connexin45 proteins in these cells. Connexin 43 and connexin45 messenger ribonucleic acids and immunoreactive proteins were detected in pregnant rat myometrium. Connexin 43 messenger ribonucleic acid levels increased dramatically at term. In contrast, connexin45 messenger ribonucleic acid was present in nonpregnant myometrium, remained relatively constant early in gestation, fell just before term, and more than doubled post partum. CONCLUSIONS: Rat uterine myometrium contains connexin45 and connexin43. Coexpression of connexin45 with connexin43 in uterine myometrium may regulate gap junctional coupling between these cells. The different temporal expression patterns suggest that connexin45 and connexin43 may have different roles or that the ratio of these connexins may be important in the increased cellular coupling coincident with the onset of labor.

Lack of contribution of nitric oxide to basal vasomotor tone in heart failure.

Patients with heart failure have reduced forearm vasodilator responses when endothelial cell nitric oxide production is stimulated by muscarinic agonists. The aim of this study was to determine if activity of the nitric oxide pathway was also abnormal under basal conditions. Forearm blood flow (FBF) was measured with strain-gauge plethysmography in response to the intraarterial infusion of a subsystemic dose range of L-N-monomethylarginine (L-NMMA), a competitive inhibitor of nitric oxide synthase. In 18 normal subjects, the baseline FBF of 3.6 +/- 1.4 was decreased by 0.3 +/- 0.5 (p < 0.01), 1.0 +/- 0.7 (p < 0.01), 1.4 +/- 0.9 (p < 0.01), and 1.3 +/- 1.3 (p < 0.01) ml/min/100 ml forearm volume during infusions of 1, 4, 8, and 16 mumol/min of L-NMMA, respectively. In 10 patients with heart failure, the baseline FBF of 2.6 +/- 0.9 was decreased by 0.4 +/- 0.5 (p < 0.05), 0.4 +/- 0.5 (p < 0.05), 0.9 +/- 0.8 (p < 0.01), and 0.9 +/- 0.7 (p < 0.01) ml/min/100 ml forearm volume with the 4 doses of L-NMMA, respectively. There was no difference in the L-NMMA response between the 2 groups in terms of absolute flow, percent change, or with analysis of covariance to adjust for different baselines. The stable end products of nitric oxide (nitrite and nitrate) were measured in the forearm venous effluent. Nitrite and nitrate levels at baseline were not reduced in patients with heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)