Cx43 Degradation Research Articles

A Central Role of Connexin 43 in Hypoxic Preconditioning

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

Differential expression and localization of connexins 26 and 43 in the rat gingival epithelium

We investigated the expression and localization of connexins (CX) 26 and 43 in the rat gingival epithelium. RT-PCR analysis revealed CX26 gene expression in both the upper and lower layers of the gingival epithelium and in the total epithelial layer, whereas CX43 gene expression was limited to the lower layer and the total epithelial layer. Immunoreactivity for CX43 was observed in the membranes of adjacent cells from the basal layer to the middle of the prickle cell layer, while immunoreactivity for CX26 was observed in the granular cell layer and lower part of the squamous cell layer. Merged images revealed the co-localization of CX26 and CX43 in the middle of the prickle cell layer. By immuno-electron microscopy, gap junctions appeared curved, hemi-circular, or annular within the cytoplasm, and gold particles indicating the presence of CX43 were localized at the outer edges of these cytoplasmic formations. These results suggest that CX43 is associated with the regulation of cell proliferation and that increased CX26 expression is associated with differentiation of keratinocytes. Thus, degradation of CX43 is considered to play an essential role in differentiation of the rat gingival epithelium.

Ultraviolet A induced modulation of gap junctional intercellular communication by P38 MAPK activation in human keratinocytes

Aberrant gap junctional intercellular communication (GJIC) has been implicated in tumor development and progression. UltravioletA (UVA)-induced oxidative stress has been associated with skin carcinogenesis. We report a potential link between GJIC and the cellular stress response induced by UVA in normal human keratinocytes (NHK). In this study, UVA irradiation (10 J/cm(2)) compromised GJIC integrity in absence of cytotoxic effects as demonstrated by the absence of cell death and by the reversibility of GJIC down-regulation. Inhibition of communication by UVA was associated with hyperphosphorylation and decreased expression of connexin43 (Cx43), the most abundant gap junction protein expressed by keratinocytes. Cx43 hyperphosphorylation induced by UVA is, at least in part, mediated through mitogen-activated protein kinase (MAPK) activation as Ser279 and Ser282 sites, two downstream direct targets of p38 MAPK were found to be phosphorylated after UVA treatment. However, inhibition of p38 MAPK activity did not significantly protect from cell-cell communication inhibition because of a strong cellular cytotoxicity observed with SB202190 and SB203580, two selective inhibitors of p38 MAPK, in combination with UVA that compromises the outcome of dye transfer assay. By contrast, in Hacat cell line, inhibition of p38 activity reduced both phosphorylation and degradation of Cx43, demonstrating that these events are correlated.

The proteasome regulates the interaction between Cx43 and ZO‐1

Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.

Remodeling of connexin 43 in the diabetic rat heart

In the Streptozotocin-induced diabetic rat heart, a decrease in the conductivity and suppression of electrical cell-to-cell coupling were observed. To clarify this mechanism, the present study was performed to investigate alterations of the gap junction connexin 43 (Cx43) using immunoblotting, immunohistochemistry, electron-microscopic analyses. An enhanced activation of PKCepsilon, an augmentation of PKCepsilon-mediated phosphorylation of Cx43, a decrease in the total amount of Cx43, a reduction in the area of immunoreactive particles for Cx43 at the intercalated disk, distribution of Cx43 to cell periphery or cytoplasm and the internalization approximately annular profiles of the gap junction were all characteristically recognized in the diabetic heart. Such abnormalities in the expression of Cx43 were alleviated by treatment with either lysosomal (NH(4)Cl, Leupeptin) or proteasomal inhibitor (ALLN). These results suggest that the PKCepsilon-mediated hyperphosphorylation of Cx43 makes Cx43 vulnerable to proteolytic degradation and that a decrease in the conductivity in the diabetic heart is also caused by a decrease in the number of gap junction channels due to an acceleration of the proteolytic degradation of Cx43. The remodeling of Cx43 induced by the activation of PKCepsilon may therefore contribute to the formation of the arrhythmogenic substrate in the diabetic heart. The cardioprotective effect of the remodeling of Cx43 by PKCepsilon is discussed.

Synergistic effects of PDGF-BB and cAMP-elevating agents on expression of connexin43 in mesangial cells

The gap junction plays an important role in the regulation of cell growth, migration, and differentiation. Platelet-derived growth factor (PDGF) is reported to be a potent inhibitor of gap junctional intercellular communication (GJIC). Short-term exposure of cells to PDGF causes rapid and transient disruption of GJIC without altering connexin43 (Cx43) protein level. In this study, we investigated long-term effects of PDGF-BB on Cx43 expression in mesangial cells (MCs). Exposure of MCs to PDGF-BB affected neither the Cx43 protein level nor GJIC. However, in the presence of cAMP-elevating agents, PDGF-BB dramatically increased the expression of Cx43, which was accompanied by obviously augmented membrane distribution of Cx43 and functional GJIC. The increased expression of Cx43 was closely correlated with reduction in alpha-actin, a dedifferentiation marker of MCs. The effect of PDGF on Cx43 was largely prevented by inhibitors of phosphatidylinositol 3'-kinase or mitogen-activated protein kinase, but not by inhibition of protein kinase C. Exposure of MCs to PDGF-BB caused elevation in intracellular cAMP, and it was abolished by indomethacin, a cyclooxygenase inhibitor. However, indomethacin did not affect the synergistic effect. In addition, PDGF-BB also did not affect the degradation of Cx43. With the use of MCs transfected with a Cx43 promoter-luciferase vector, cooperative activation of Cx43 promoter by PDGF and cAMP was found. Together, our data reveal, for the first time, unexpected synergy between PDGF-BB and cAMP-elevating agents in the induction of Cx43 and MC differentiation. Regulation of GJIC could be an important mechanism via which PDGF modulates MC phenotypes.

Inhibition of Gap Junction–Mediated Intercellular Communication by TNF-α in Cultured Human Corneal Fibroblasts

Keratocytes are connected to each other by gap junctions, which mediate intercellular communication and contribute to maintenance of corneal homeostasis. The possible effect of tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts was examined. GJIC activity was measured by observing the intercellular diffusion of the fluorescent dye Lucifer yellow. The expression of the gap junction protein connexin43 (Cx43) was evaluated by immunofluorescence and immunoblot analyses with a specific monoclonal antibody. The abundance of Cx43 mRNA was determined by quantitative reverse transcription and polymerase chain reaction analysis. TNF-alpha induced a time- and concentration-dependent decrease in GJIC activity in human corneal fibroblasts. Immunofluorescence analysis revealed that TNF-alpha reduced the level of specific staining for Cx43 at sites of contact between adjacent cells. Immunoblot analysis detected four specific Cx43 bands, one corresponding to the nonphosphorylated form of the protein and three corresponding to phosphorylated forms. Exposure of cells to TNF-alpha reduced the relative abundance of the three phosphorylated forms of Cx43. The amount of Cx43 mRNA was not affected by TNF-alpha. TNF-alpha inhibited GJIC in cultured human corneal fibroblasts, an effect that was possibly mediated by dephosphorylation and consequent degradation of Cx43. The downregulation of GJIC among keratocytes in response to TNF-alpha may contribute to the breakdown of corneal homeostasis during corneal inflammation.

Ubiquitination and Down-regulation of Gap Junction Protein Connexin-43 in Response to 12-O-Tetradecanoylphorbol 13-Acetate Treatment

Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells

The tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell–cell communication via gap junctions.

High Glucose Down-regulates Intercellular Communication in Retinal Endothelial Cells by Enhancing Degradation of Connexin 43 by a Proteasome-dependent Mechanism

Intercellular communication through gap junctions (GJIC) is most likely relevant to maintaining the integrity of the blood-retinal barrier. In this study, we investigated the mechanism whereby high glucose enhances degradation of connexin 43 (Cx43), thus contributing to a decrease in GJIC. The levels of Cx43 in bovine retinal endothelial cells exposed to high glucose (25 mm) decreased about 50% as compared with controls (5.5 mm glucose). Consistently, the half-life of the protein decreased from 2.3 to 1.9 h. The proteasome inhibitors MG132 and lactacystin prevented the loss of Cx43 induced by high glucose and extended Cx43 half-life. The amount of phosphorylated Cx43 increased in high glucose and after proteasome inhibition. Scrape-loading dye transfer experiments show that high glucose is associated to a decrease of 40% in GJIC. Significantly, this reduction can be reversed by proteasome inhibitors. The decrease in GJIC in cells exposed to high glucose is associated with a loss of Cx43 from the plasma membrane, as demonstrated by immunofluorescence and biotinylation of cell-surface proteins. Results indicate that increased phosphorylation of Cx43 under high glucose is the mechanism targeting Cx43 for degradation by a proteasome-dependent mechanism. Increased degradation of Cx43 and reduction of GJIC in high glucose may be of physiological importance by contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier in diabetic retinopathy.

EGF-Induced Gap Junction Degradation

Gap junctions allow cytoplasmic molecules to move from one cell to another, and such communication appears to influence a broad range of processes, including development, cell proliferation, and behavior of cancer cells. Epidermal growth factor (EGF) is known to regulate conductance of gap junctions through mitogen-activated protein kinase-dependent phosphorylation of the connexin proteins that form gap junction channels. Leithe and Rivedal now present evidence that such phosphorylation may also promote internalization and proteasome-dependent degradation of connexin43 (Cx43). In a rat liver cell line, exposure of cells to EGF caused internalization and degradation of Cx43. Ubiquitination of Cx43 was also stimulated by EGF, and studies with proteasomal inhibitors implicated the proteasome in degradation of Cx43. Because loss of gap junction communication has been associated with carcinogenesis and enhanced EGF signaling also contributes to some forms of cancers, the authors note that the newly described regulatory pathway could have a role in tumorigenesis. E. Leithe, E. Rivedal, Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43. J. Cell Sci. 117 , 1211-1220 (2004). [Abstract] [Full Text]

Luteinizing hormone-induced connexin 43 down-regulation: inhibition of translation.

The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, [(35)S]methionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK.

Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Lysosomal and Proteasomal Degradation Play Distinct Roles in the Life Cycle of Cx43 in Gap Junctional Intercellular Communication-deficient and -competent Breast Tumor Cells

The present study was designed to determine the specific roles played by lysosomes and proteasomes in the degradation of Cx43 in both gap junctional intercellular communication-deficient MDA-MB-231 and -competent BICR-M1Rk cells. In MDA-MB-231 cells, immunolocalization and brefeldin A protein transport blocking studies revealed that there was a propensity for newly synthesized Cx43 to be transported to lysosomes. On the other hand, light and electron microscopic analysis of BICR-M1Rk cells showed that Cx43 gap junctions were prevalent with a subpopulation of intracellular Cx43 localized to lysosomes. In both cell types, Western blots revealed a notable increase in total cellular Cx43 in response to lysosome inhibitors. Interestingly, lactacystin inhibition of proteosomal degradation in MDA-MB-231 cells resulted in a marked increase in phosphorylated Cx43 at the expense of non-phosphorylated Cx43, and this change corresponded with an increase in "oversized" gap junction plaques. In BICR-M1Rk cells, lactacystin treatment partially prevented the BFA-induced loss of gap junctions. Together, our data suggests that lysosomes play a key role in not only degrading internalized gap junction in BICR-M1Rk cells but also in degrading Cx43 delivered from early secretory compartments to lysosomes in MDA-MB-231 cells. Overall proteasomal degradation regulates the stability of phosphorylated Cx43 and appears to promote the internalization of Cx43 from the cell surface.

Rapid turnover of connexin43 in the adult rat heart.

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.

Attenuation of Gossypol Cytotoxicity by Cyclic AMP in a Rat Liver Cell Line

The effect of pretreatment of a rat liver cell line (Clone 9) with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), an agent previously shown to increase gap junctional intercellular communication (GJIC), was used to determine if increased GJIC would attenuate the cytotoxic effects of gossypol acetic acid (GAA). Pretreatment with 8-Br-cAMP increased the inhibitory concentration 50% (IC50) for gossypol from 4.1 to 6.1 μM in Clone 9 cells. GJIC was suppressed by about 57% within 5 min when Clone 9 cells were treated with 1.0 μM GAA alone, whereas cells treated with 3.0 or 10 μM GAA were completely uncoupled within the same time frame. In contrast, GJIC was maintained near control levels in Clone 9 cells preincubated with 1.0 mM 8-Br-cAMP for 10 min prior to 1.0 μM GAA exposure. 8-Br-cAMP partially restored GJIC in cells treated with 3.0 μM GAA but was unable to protect cells exposed to 10 μM GAA. The effects of GAA and 8-Br-cAMP pretreatment on connexin43 (Cx43) protein expression was analyzed with Western blots. GAA treatment at concentrations of 1 and 3 μM caused a time- and dose-dependent increase in phosphorylation of Cx43 over a 20-min period, whereas 10 μM GAA caused a time-dependent degradation of Cx43 over the same interval. Pretreatment of cells for 10 min with 8-Br-cAMP completely reversed the effect of 1 μM GAA and partially blocked the effect of 3 μM GAA on Cx43 phosphorylation and suppressed Cx43 degradation at 10 μM. Additional fluorescence endpoints associated with cellular homeostasis mechanisms were also monitored to evaluate cytotoxicity and potential protective effects of 8-Br-cAMP pretreatment in cells exposed to GAA, including generation of reactive oxygen species (ROS), cytoplasmic acidification, glutathione (GSH) content, intracellular calcium levels ([Ca2+]i), and mitochondrial and plasma membrane potential. The adverse effects of GAA on the production of ROS, cytoplasmic acidification, GSH content, and [Ca2+]iwere also attenuated. The primary protective effect of 8-Br-cAMP was observed at or below the IC50of GAA, with greatest protective effects detected on early endpoints affected by GAA exposure. These studies suggest that the protective effect of 8-Br-cAMP in GAA-treated cells results from enhanced GJIC, which facilitates cellular homeostasis by providing cell–cell diffusion of essential metabolites, ions, and regulatory and informational molecules.

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

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. 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. 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. 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.