Cx43-containing Gap Junctions Research Articles

Morphology of the myocardium of the interventricular septum in children with hypertrophic cardiomyopathy

To carry out a comparative analysis of the morphology of the interventricular septum (IVS) myocardium in children with hypertrophic cardiomyopathy (HCM) and without cardiovascular pathology. A study of myocardial biopsies of the IVS in children with HCM (n=18, 1.2-17 years) and children without cardiovascular pathology (n=11, 1-16 years) was carried out. The volume of interstitial tissue in the IVS myocardium was determined, a morphometric study of the size of cardiomyocytes (CMCs), the myofibrillogenesis level and the ploidy of CMCs was carried out, the ultrastructure of the CMCs was studied, and the localization of the gap junction protein, connexin43 (Cx43), was revealed by immunohistochemistry. The proportion of interstitial tissue in the myocardium of children with HCM was 9-10% and did not differ from its proportion in the myocardium of children in the control group. The diameter of the CMCs of the IVS in children with HCM reached the limit of ontogenetic growth and exceeded the parameters of the control group (average 18.9±5.7 µm vs 9.3±4.4 µm). CMCs ploidy in children with HCM was 2 times higher than CMCs ploidy in control patients (5.3c vs 2.7c). In the myocardium of children with HCM, the assembly of myofibrils most actively occurred in small CMCs. At the ultrastructural level, signs of immaturity of the contractile apparatus and intercalated discs of the CMC in HCM were demonstrated. In the myocardium of children with HCM, Cx43-containing gap junctions were more often located on the lateral surfaces of the CMC than in the myocardium of the control group. In children with HCM, a morphological picture of an increase in the size of the CMCs and their ploidy during accelerated ontogenetic development was demonstrated in combination with ultrastructural signs of immaturity of the contractile apparatus and intercalated discs and the lack of growth of interstitial tissue of the IVS myocardium compared with patients in the control group.

Connexin 43 expression in satellite glial cells contributes to ectopic tooth-pulp pain.

Pulpitis often causes referred pain in opposing teeth. However, the precise mechanism underlying ectopic pain associated with tooth-pulp inflammation remains unclear. We performed the present study to test the hypothesis that functional interactions between satellite glial cells (SGCs) and trigeminal ganglion (TG) neurons are involved in ectopic orofacial pain associated with tooth-pulp inflammation. Digastric muscle electromyograph (D-EMG) activity elicited by administration of capsaicin into the upper second molar pulp (U2) was analyzed to evaluate noxious reflex responses. D-EMG activity was significantly increased in rats with lower first molar (L1) inflammation relative to saline-treated rats. Significantly increased expression of glial fibrillary acid protein (GFAP), a marker of activated glial cells, and connexin 43 (Cx43), a gap-junction protein, was observed in activated SGCs surrounding U2-innervating TG-neurons after L1-pulp inflammation. Daily administration of Gap26, a Cx43-inhibiting mimetic peptide, into the TG significantly suppressed capsaicin-induced D-EMG activity enhancement and reduced the percentage of fluorogold-labeled (U2-innervated) cells that were surrounded by GFAP-immunoreactive (IR) and Cx43-IR cells after L1-pulp inflammation. These findings indicate that tooth-pulp inflammation induces SGC activation and subsequent spread of SGC activation in the TG via Cx43-containing gap junctions. Thus, remote neuron excitability becomes enhanced in the TG following tooth-pulp inflammation, resulting in ectopic tooth-pulp pain in the contralateral tooth.

Potentiation of Amitriptyline Anti-Hyperalgesic-Like Action By Astroglial Connexin 43 Inhibition in Neuropathic Rats.

Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants.

Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.

Human endometrial stromal decidualization is required for embryo receptivity, angiogenesis, and placentation. Previous studies from our laboratories established that connexin (Cx)-43 critically regulates endometrial stromal cell (ESC) differentiation, whereas gap junction blockade prevents it. The current study evaluated the plasticity of ESC morphology and Cx43 expression, as well as other biochemical markers of cell differentiation, in response to decidualizing hormones. Primary human ESC cultures were exposed to 10 nM estradiol, 100 nM progesterone, and 0.5 mM cAMP for up to 14 days, followed by hormone withdrawal for 14 days, mimicking a biphasic ovulatory cycle. Reversible differentiation was documented by characteristic changes in cell shape. Cx43 was reversibly up- and down-regulated after the estradiol, progesterone, and cAMP treatment and withdrawal, respectively, paralleled by fluctuations in prolactin, vascular endothelial growth factor, IL-11, and glycodelin secretion. Markers of mesenchymal-epithelial transition (MET), and its counterpart epithelial-mesenchymal transition, followed reciprocal patterns corresponding to the morphological changes. Incubation in the presence of 18α-glycyrrhetinic acid, an inhibitor of gap junctions, partially reversed the expression of decidualization and MET markers. In the absence of hormones, Cx43 overexpression promoted increases in vascular endothelial growth factor and IL-11 secretion, up-regulated MET markers, and reduced N-cadherin, an epithelial-mesenchymal transition marker. The combined results support the hypothesis that Cx43-containing gap junctions and endocrine factors cooperate to regulate selected biomarkers of stromal decidualization and MET and suggest roles for both phenomena in endometrial preparation for embryonic receptivity.

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.

The "connexin" between bone cells and skeletal functions.

The processes of bone modeling and remodeling are crucial in the skeleton's functions as a supportive and protective structure, a mineral reservoir, and an endocrine organ. The coordination between bone cell activities (bone formation and bone resorption), necessary to maintain the integrity of the skeleton during these processes, is mediated at least in part by cell-cell and cell-environment interactions across gap junctions and hemichannels. The increasing number of genetically engineered Connexin 43 (Cx43) knockout and missense mouse models have provided insight into the complex and critical roles of Cx43-containing gap junctions and hemichannels in the development and turnover of the skeleton, in differentiation, activity and survival of the bone cell lineages, and in the cellular and molecular mechanisms by which Cx43 functions and assists in mediating cellular responses to stimuli in bone. Cx43 may be an important potential therapeutic target, making it crucial that we continue to gain understanding of the multiple and complex roles of Cx43 in bone.

Monoclonal Antibodies Against the Connexin43-interacting Protein CIP85

The connexin43 (Cx43)-interacting protein of 85 kDa CIP85 has been identified as an interacting partner for the cytoplasmically located, carboxyl-terminal tail of Cx43. Further characterization has shown that the interaction between Cx43 and CIP85 is associated with increased turnover of Cx43 that may be lysosome-mediated. This suggests that CIP85 may regulate the endocytic trafficking of Cx43 from the plasma membrane and its degradation, and thus, indirectly influence gap junction function. This study reports the first successful production of monoclonal antibodies (MAbs) against CIP85. These antibodies are useful in detecting CIP85 expressed in several species in immunoblotting, immunoprecipitation, and immunofluorescence microscopy experiments. These MAbs will assist in defining the functional roles of CIP85, including its influence on Cx43 trafficking and intercellular communication through Cx43-containing gap junctions.

Identification of the Calmodulin Binding Domain of Connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.

Connexin 43 mediates spread of Ca2+ -dependent proinflammatory responses in lung capillaries

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 microm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

Ca 2+ communication through connexin 43 in lung capillaries

The extent to which endothelial (EC) cells support gap junctional intercellular communication (GJIC) remains unknown. To determine GJIC, by fluorescence microscopy we viewed septal capillaries of isolated, blood-perfused lungs from wild type (WT) mice, and from mice with targeted deletion of the gap junctional protein, connexin 43 (Cx43) in EC (Cx43-/-) (Liao, et al., PNAS, 2001). Lung artery, left atrial and airway pressures were held at 10, 3 and 5 cm H2O, respectively. We co-loaded vessels with the Ca2+ cage, NP-EGTA and the cytosolic Ca2+ (Ca2+cyt) indicator, fluo 4. At baseline, EC Ca 2+cyt was 88±17 gray levels. To increase Ca2+cyt focally, we targeted high-intensity UV light to a single EC of a septal capillary, thereby uncaging Ca2+ from NP-EGTA. In WT mice, uncaging increased EC Ca2+cyt locally as well as at a site 100 μm distant by 33±2 and 29±2 gray levels, respectively (mean±SE, n=3, P<0.05). By contrast, in Cx43-/- mice uncaging increased Ca2+cyt locally (n=3, P<0.05), but not at a distant site. Thus, the uncaging-induced Ca2+ signal was conducted 100 μm from the uncaging site in WT, but not in Cx43-/- mice. We conclude, EC of lung capillaries rapidly communicate Ca2+ signals through Cx43-containing gap junctions. Such spatial communication may amplify Ca2+-dependent proinflammatory responses in the lung capillary bed (supported by HL75503, HL57556).

Effects of human pituitary FSH isoforms on mouse follicles in vitro

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10–5.99 (pool I), pI 5.62–4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Manipulation of gap junction proteins: effect on cellular coupling and impulse propagation

www.elsevier.com/locate/jelectrocard Manipulation of gap junction proteins: effect on cellular coupling and impulse propagation S.M. Taffet, S. Shah, R.L. Lewandowski, W. Coombs J. Shibayama, P. Sorgen, M. Delmar (Upstate Medical University, Syracuse, NY, USA; University of Nebraska, Omaha, NE, USA) The cardiac gap junction protein, connexin43 (Cx43), is not a passive pore but an actively regulated channel. Under conditions of cellular stress, such as acidification, the pore closes resulting in alterations in impulse propagation. We have previously demonstrated that the carboxyl terminal (CT) domain of Cx43 (Cx43CT) is involved in various intraand intermolecular interactions that regulate gap junctions. Here, we used a nonbiased phage display library to identify novel peptidic sequences that bind Cx43CT and hopefully will alter the properties of the channel. A library of phage containing a randomized series of 12–amino acid peptides (approximately 10 different sequences) was presented to immobilized Cx43CT (pH, 6.5). Analysis of the selected phage revealed a total of 48 Cx43CTinteracting peptides. One third of the peptides had a preserved arginine-X-proline (RXP) sequence. Surface plasmon resonance confirmed binding of 6 of these peptides to Cx43CT. Two of these peptides (RXP-1 and RXP-4) were further analyzed. The interaction of RXP-4 with the CX43-CT was further analyzed by NMR revealing 0022-0736/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.jelectrocard.2005.06.091 that this peptide modifies the structure of region 375-379 of Cx43CT. Dual-patch clamp analysis revealed that RXP-4 (but not RXP-1) reduced the frequency with which Cx43 channels transit into the residual state. Unitary conductances were not modified by this peptide. To further analyze the optimal RXP sequences for binding and modifying the function of Cx43, we generated a biased phage display library where the RXP sequence was flanked on each side by 4 random amino acids. Approximately 40000 distinct sequences were presented to Cx43CT. A minor fraction of the inserts resulted from concatenation of 2 peptides in tandem (a bdoublet Q). No doublets were found in a random sampling of 63 inserts obtained before binding. Yet, of the 485 clones sequenced after selection by binding, 85% were doublets, suggesting a high selectivity of Cx43CT for doublets during the binding step. This increased affinity of doublets for CT interaction was confirmed by surface plasmon resonance. Further analysis of the sequences obtained by phage display indicated that there was an increased occurrence of acidic amino acids at position 3 (relative to RXP) but an absence of acidic residues at positions 2 and +1. Basic amino acids were absent from position 1. Overall, these data show that RXP peptides can lead to structural modifications in Cx43CT and changes in the function of Cx43 channels. These results open the door for further development of peptide-based strategies to modify the regulation of Cx43-containing gap junctions in native tissues such as heart. ardiology 38 (2005) 51

Drebrin Is a Novel Connexin-43 Binding Partner that Links Gap Junctions to the Submembrane Cytoskeleton

Background: Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Many aspects of connexin function, for example cellular transport, plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to connexins' cytoplasmic domains. However, little is known about such regulatory proteins. To identify novel proteins that interact with the COOH-terminal domain of Connexin-43 (Cx43), the most widely expressed connexin family member, we applied a proteomics approach to screen fractions of mouse tissue homogenates for binding partners.Results: Drebrin was recovered as a binding partner of the Cx43 COOH-terminal domain from mouse brain homogenate. Drebrin had previously been described as an actin binding protein that diminishes in brains during Alzheimer's disease. The novel Drebrin-Cx43 interaction identified by proteomics was confirmed by colocalization of endogenous proteins in astrocytes and Vero cells, coimmunoprecipitation, electron microscopy, electrophysiology, coexpression of both proteins with fluorescent tags, and live-cell FRET analysis. Depletion of Drebrin in cells with siRNA results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway.Conclusions: We conclude that Drebrin is required for maintaining Cx43-containing gap junctions in their functional state at the plasma membrane. It is thus possible that Drebrin may interact with gap junctions in zones of cell-cell contacts in a regulated fashion in response to extracellular signals. The rearrangement or disruption of interactions between connexins and the Drebrin-containing submembrane cytoskeleton directs connexins to degradative cellular pathways.

Presence and Importance of Connexin43 During Myogenesis

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C(2)C(12) cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C(2)C(12) myoblasts in the presence or absence of the gap junction blocker 18beta-glycyrrhetinic acid (beta-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with beta-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in beta-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43(Cre-ER(T)/fl) transgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.

Gap Junction Remodeling in Hypertrophied Left Ventricles of Aortic-banded Rats: Prevention by Angiotensin II Type 1 Receptor Blockade

Remodeling of gap-junctional organization in hypertrophied left ventricle (LV) in response to pressure overload in rats induced by abdominal aorta banding was investigated by immunoconfocal and electron microscopy. Eight to 12 weeks after banding, rats developed significant LV hypertrophy. In contrast to control LV myocytes, which showed connexin43 (Cx43) labeling largely confined to the intercalated disks, LV myocytes from aortic-banded rats showed dispersion of punctate Cx43 labeling over the entire cell surface. In LV tissues sectioned longitudinally, the proportion of Cx43 label at the intercalated disk decreased significantly (control, 0.87 v aortic-banded, 0.62). En-face views of intercalated disks of hypertrophied myocardium revealed a reduction of Cx43 gap junctions in the disk center, giving rise to a significant decrease in the proportion of the disk occupied by gap-junctional membrane (control, 0.32 v aortic-banded, 0.24). Electron microscopy of hypertrophied LV tissue revealed that Cx43-containing gap junctions were frequently displaced from their usual locations to form side-to-side contacts distant from the disk, and also appeared as annular profiles. In aortic-banded rats treated with the angiotensin II (AII) type 1 receptor (AT1) antagonist, losartan (10 mg/kg/day, 11 weeks) not only LV hypertrophy, but also the gap junction disorganization was markedly reduced. These results suggest that LV hypertrophy induced by pressure overload is associated with Cx43 gap junction disorganization and that AII may play an important role either directly or indirectly in gap-junctional remodeling.

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C.

In this study, we investigated whether the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB-F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin-specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.