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PubMed1995

The expression of gap junctional proteins during different stages of mouse skin carcinogenesis.

The loss or alteration of gap junctional intercellular communication (GJIC) has long been proposed to play an important role in the process of carcinogenesis. In this study we examined the expression of three gap junction proteins, connexins (Cx)26, 43 and 31.1, in mouse hyperplastic skin, papillomas and squamous cell carcinomas (SCC). Tumors were induced in SENCAR mice by either of two initiation/promotion protocols: 7,12-dimethylbenz-[a] anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA) or DMBA/benzoyl-peroxide (BzPo). Keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but did not express Cx26. Skin hyperplasia induced by one topical application of TPA was accompanied by hyperexpression of both Cx26 and Cx43. In addition, TPA significantly inhibited the expression of Cx31.1. After repetitive application. Connexin 26 and Cx43 were hyperexpressed in most of the papillomas studied (20-40 weeks after initiation). However, in some late papillomas, immunostaining revealed a focal loss of Cx26. Immunostaining of mouse skin SCC revealed decreased Cx43 and Cx26 levels in 65% and 85% of cases respectively. The high levels of Cx26 and Cx43 mRNA in most of the SCC did not correlate with the decreased abundance or disappearance of Cx26 and Cx43 immunoreactive spots from tumor plasma membranes. Thus, the expression of these two connexins in SCC was impaired at the post-translation level. Cx31.1 expression was strongly inhibited during all stages of carcinogenesis. Taken together, our results suggest that three different connexin genes are differentially regulated during mouse skin carcinogenesis and the decrease of connexin expression may be an important marker of skin tumor expression.

Animals↗
PubMed1984

Water flow across the walls of single muscle capillaries in the frog, Rana pipiens.

Individual capillaries of the transilluminated frog muscle cutaneous pectoris were perfused with suspensions of human red cells in frog Ringer solution containing 1 g/dl bovine serum albumin. The modified Landis technique (Michel, Mason, Curry & Tooke, 1974) was used to measure hydraulic conductivities of the capillary wall. Sucrose osmotic reflexion coefficients of the capillary wall were measured in four capillaries when the superfusate contained 100 mM-sucrose. All experiments were made at 22-24 degrees C. The hydraulic conductivity of arterial capillaries varied from 0.3 to 1.26 X 10(-7) cm/(s cmH2O) with a mean of 0.79 X 10(-7) cm/(s cmH2O) (six capillaries). The hydraulic conductivities of mid-capillaries varied from 0.43 to 1.86 X 10(-7) cm/(s cmH2O) with a mean value of 0.72 X 10(-7) cm/(s cmH2O) (six capillaries). The mean reflexion coefficient to sucrose was 0.12 +/- 0.05 (S.D.). The measured reflexion coefficients to sucrose conform to the hypothesis that 90% of the transcapillary water flow crosses the capillary wall via the principal hydrophilic pathway. The remaining 10% crosses via an exclusive water pathway. The distribution of water flow is similar to that previously described in frog mesenteric capillaries. The mean value of the hydraulic conductivity of frog muscle capillaries is about one-seventh the mean value of the hydraulic conductivity of frog mesenteric capillaries measured at the same temperature. The result conforms to the hypothesis that only a small fraction (mean 10%) of the area of junctional contact between adjacent endothelial cells is available for water and solute exchange in frog muscle capillaries. The hydraulic and diffusional conductances per unit length of open junction appear to be very similar when muscle capillaries are compared to mesenteric capillaries in the frog. Our results lead us to speculate that structures within the intercellular junctions determine the extent of open junction and may modulate the hydraulic conductivity of both the principal water pathway and the exclusive water pathway.

Animals↗
PubMed2001

The origin of annular junctions: a mechanism of gap junction internalization.

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3-6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

Animals↗
PubMed2006

Compartmentalized and signal-selective gap junctional coupling in the hearing cochlea.

Gap junctional intercellular communication (GJIC) plays a major role in cochlear function. Recent evidence suggests that connexin 26 (Cx26) and Cx30 are the major constituent proteins of cochlear gap junction channels, possibly in a unique heteromeric configuration. We investigated the functional and structural properties of native cochlear gap junctions in rats, from birth to the onset of hearing [postnatal day 12 (P12)]. Confocal immunofluorescence revealed increasing Cx26 and Cx30 expression from P0 to P12. Functional GJIC was assessed by coinjection of Lucifer yellow (LY) and Neurobiotin (NBN) during whole-cell recordings in cochlear slices. At P0, there was restricted dye transfer between supporting cells around outer hair cells. Transfer was more extensive between supporting cells around inner hair cells. At P8, there was extensive transfer of both dyes between all supporting cell types. By P12, LY no longer transferred between the supporting cells immediately adjacent to hair cells but still transferred between more peripheral cells. NBN transferred freely, but it did not transfer between inner and outer pillar cells. Freeze fracture further demonstrated decreasing GJIC between inner and outer pillar cells around the onset of hearing. These data are supportive of the appearance of signal-selective gap junctions around the onset of hearing, with specific properties required to support auditory function. Furthermore, they suggest that separate medial and lateral buffering compartments exist in the hearing cochlea, which are individually dedicated to the homeostasis of inner hair cells and outer hair cells.

Animals↗
PubMed2006

Current understanding of the mechanisms of gastro-oesophageal reflux disease.

Gastro-oesophageal reflux disease (GERD) covers a broad range of signs and symptoms arising from the orad movement of gastric contents into the oesophagus, oropharynx, larynx or airway. Most commonly, contact with and damage to the oesophageal epithelium by acidic refluxate causes micro or macroscopic defects leading to the symptom of heartburn. However, GERD can also give rise to extra-oesophageal manifestations such as pharyngitis, laryngitis, asthma and other disorders, identifiable as acid-mediated events by a favorable response to acid suppression. Only one-third of individuals with heartburn have endoscopic evidence of erosive oesophagitis; the remainder have endoscopy-negative or non-erosive reflux disease (NERD). Improved investigative technologies are increasing our understanding of the pathophysiology of NERD. For example, although a number of microscopic abnormalities have been identified, oesophageal damage in NERD has been shown to be characterized by the presence of 'dilated intercellular spaces' within oesophageal stratified squamous epithelium. Dilated intercellular spaces that reflect damage to the intercellular junctions enable levels of acidity that would be considered innocuous when present in the oesophageal lumen to initiate pathological responses within oesophageal nociceptors when present within the intercellular spaces. This effectively gives rise to the symptom of heartburn. Excessive acidity within the intercellular spaces in NERD also presages its evolution to erosive disease, the latter through inflammation-mediated disruption of the antireflux and luminal clearance mechanisms. Support for this scenario is evident by the ability of effective acid control with proton pump inhibitors both to control symptoms, and lead to resolution of dilated intercellular spaces in patients with both erosive and non-erosive disease. This article examines these concepts and how they shape our current understanding of GERD.

Esophagus↗
PubMed1996

Synovial sarcoma of the vulva: a report of two cases.

We report two cases of synovial sarcoma arising in the vulva. The patients were 30 and 37 years old and presented with a painless mass that was interpreted clinically as a cyst. The tumors were 2.0 and 1.2 cm in greatest diameter. Histologically, they were composed of epithelial cells forming solid nests and gland-like and papillary structures surrounded by spindle-shaped cells. Immunohistochemically, the epithelial cells stained for cytokeratin and the spindle-shaped cells for vimentin. Ultrastructurally, the epithelial cells had prominent intercellular junctions and narrow microvilli and were separated from the spindle-shaped cells by a basal lamina. The spindle-shaped cells were closely apposed with focal intercellular contacts. One tumor recurred locally 3.5 years after excision, but the patient was alive and well 1 year after a re-excision and radiation therapy. The other patient was alive and well 4 years after an excision. These tumors are the first reported examples of synovial sarcoma arising in the vulva.

Adult↗
PubMed1997

Comparative effects of clofibrate and methyl clofenapate on morphological transformation and intercellular communication of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell system was used to evaluate the ability of two hepatocarcinogenic structurally related peroxisome proliferators (PPs) to induce morphological transformation (MT) of SHE colonies and to inhibit gap junctional intercellular communication (GJIC). Clofibrate and methyl clofenapate (MCP), which was shown to be a more active PP and a more potent carcinogen in vivo than clofibrate, were compared. MCP appeared slightly more active in vitro than clofibrate in affecting MT and GJIC of SHE cells. The morphological transformation of SHE colonies was induced by 50 microM MCP, against 100 microM clofibrate. Moreover, 50 microM MCP potentiated the transforming effects of both benzo[a]pyrene and 12-O-tetradecanoylphorbol-13-acetate. The inhibition of GJIC, measured by transfer of lucifer yellow, was transient and occurred at concentrations inducing morphological transformation. MCP inhibited dye transfer at 50 microM and the inhibition lasted up to 24 h at 100 microM. Inhibition of communication lasted only 4 h with clofibrate and occurred at a higher concentration (175 microM). This study showed that both the SHE cell transformation and dye transfer assays were able to display the different activities of the two PPs, even though the difference in potency observed was smaller than in vivo. It also revealed interactions between non-genotoxic carcinogens and the ability of the SHE cell transformation assay to detect these combined effects.

Animals↗
PubMed1998

The polarized hepatic human/rat hybrid WIF 12-1 and WIF-B cells communicate efficiently in vitro via connexin 32-constituted gap junctions.

Gap junction intercellular communication (GJIC) plays an essential role in the control of growth, differentiation, and functions of different tissues. The expression of connexins (Cxs), the structural proteins of gap junctions, is developmentally regulated and tissue-specific. In vivo hepatocytes express Cx32 and Cx26. Most currently available in vitro hepatic cell systems express Cx43 instead of the expected Cxs. This work analyzes the GJIC competence and Cx expression of the highly differentiated and polarized hepatoma-derived hybrid cell lines, WIF 12-1 and WIF-B. It shows (using two dye transfer assays) that both lines communicate efficiently and that the acquisition of GJIC competence precedes the formation of bile canaliculi. Interestingly, these cells communicate via Cx32 expression, whereas Cx26 and Cx43 are not expressed, as demonstrated by Western and Northern blotting, immunocytochemistry, and confocal microscopy. The human fibroblast W138 parent communicates via Cx43, whereas the rat hepatoma parent Fao and the subclone WIF 12-1 TGdelta, that has lost the human X chromosome, do not communicate, the expression of Cx32 being restricted to the mRNA in these two lines. The GJIC competence of WIF cells could thus result from the activation of the human X chromosome-linked Cx32 gene.

Animals↗
PubMed2003

Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters.

Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

Animals↗
PubMed1990

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

Actin Cytoskeleton↗
PubMed2003

Differential regulation of junctional complex assembly in renal epithelial cell lines.

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and beta-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

3T3 Cells↗
PubMed1994

[Studies on the reduction of malignant phenotypes in a highly metastatic human lung carcinoma--correlated changes of intercellular communication, cytoskeletons, oncogenes and antioncogene].

Human lung giant cell carcinoma cell line PG is characterized by its highly metastatic (100%) behavior in nude mice. When compared with cultured normal human fetal lung cells, PG cells were deficient in gap junctional intercellular communication (GJIC) function as detected by Scrape Loading and Dye Transfer method. Tubulin immunofluorescent and rhodamine-phalloidin staining revealed disorganization of microtubules and disruption of stress fibers with appearance of reorganized F-actin-bodies in PG cells. Northern or dot blot hybridization results showed that PG expressed high levels of c-myc and c-Ha-ras oncogenes and low level of antioncogene P53. Southern hybridization demonstrated that PG also exhibited c-myc gene amplification. When PG was treated with calmodulin antagonist calmidazolium (CDZ, 100-200nmol/L) or a Chinese medicinal mixture L2 (3-13mg/ml), cell proliferation was inhibited, GJIC function restored, and microtubule network recovered. But only L2 was efficient in (1) improving the stress fiber organization, (2) inhibiting the colony formation in soft agar, (3) reduction of c-myc amplification and expression, and (4) up-regulation of P53 mRNA level. The correlation between markers of malignant phenotypes and the reversion of PG cells is discussed.

Animals↗
PubMed1991

Carotenoids enhance gap junctional communication and inhibit lipid peroxidation in C3H/10T1/2 cells: relationship to their cancer chemopreventive action.

We have previously demonstrated that diverse carotenoids inhibit chemically induced neoplastic transformation in 10T1/2 cells. To address their mechanism of action, the effects of six diverse carotenoids, with or without provitamin A activity, on gap junctional communication and lipid peroxidation have been investigated. beta-Carotene, canthaxanthin, lutein, lycopene and alpha-carotene increased gap junctional intercellular communication in a dose-dependent manner in the above order of potency, whereas m-bixin was inactive at concentrations up to 10(-5) M. alpha-Tocopherol, a potent chain-breaking antioxidant, caused a marginal enhancement of junctional communication. The enhancement of junctional communication by diverse carotenoids showed a strong statistical correlation with their previously determined ability to inhibit methylcholanthrene-induced neoplastic transformation (r = -0.75). All carotenoids tested inhibited lipid peroxidation, but with differing potencies. alpha-Tocopherol was the most active inhibitor followed by m-bixin. The capacity of carotenoids or alpha-tocopherol to inhibit lipid peroxidation was neither consistent with their ability to inhibit neoplastic transformation (r = 0.30) nor to increase junctional communication (r = 0.12). Since junctional communication appears to play an important role in cell growth control and carcinogenesis, we propose that in this system carotenoid-enhanced intercellular communication provides a mechanistic basis for the cancer chemopreventive action of carotenoids. These data also imply that carotenoids function in a manner analogous to retinoids in the 10T1/2 assay system. Interestingly this activity appears independent of their provitamin A status.

Animals↗
PubMed1978

Reconstruction of mammalian heart tissue from embryonic heart cell suspension with reference to the aggregation of adult heart cells.

Aggregations of isolated embryonic and adult heart cells were studied so as to examine in detail the formation of cell contacts, the assembly of cells into multicellular systems, and cell co-operation in forming organized and differentiating tissues. At selected intervals after initiating rotation cultures, aggregates were examined microscopically for evidence of contractility, and subsequently processed for scanning and transmission electron microscopy (TEM, SEM). By continuous accretion of single cells, and the joining of small clusters, the aggregates increased in size. Cells within the aggregates exhibited rhythmic and synchronous contractility by 3--12 h of culture, suggesting the formation of low-resistance inter-cellular junctions between apposed cells. Two populations of cells could be recognized by 9--12 h with SEM. One was spherical in surface view and the other was flattened. Spherical cells possessed myofibrils, and were classified as cardiac myocytes which occupied the core of the aggregate. The flattened cells were devoid of myofibrils and non-muscle in nature. They covered the surface in a multilayered epithelium. At 12h the aggregates were round to oval, covered by flattened cells, but individual round cells could still be recognized. Intercalated discs were frequently observed in 12 h aggregates. The junctional complexes observed in 12--72 h aggregates include desmosomes, fascia adherens and gap junctions. Most of the aggregation was completed by 24 h, and at later time periods, i.e. 48--72 h, the external surface of the aggregates was smoothed out with epithelial investment. In these aggregates myofibrils and intercellular junctions became reconstructed in less than 24 h. Unlike embryonic myocardial cells, adult cells did not form aggregates of numerous cells. Instead, they formed irregular clusters of 2--5 cells during 3--48 h of culture. Intercellular contacts and suggestive desmosomal materials were observed between adherent cardiac muscle cells. When culture continued for 48--72 h, the cells underwent supercontraction and became non-viable, suggesting that the terminally differentiated adult myocardial cells are incapable of regenerating constituents obligatory for histogenetic reconstruction.

Animals↗
PubMed2002

In vitro generation of differentiated cardiac myofibers on micropatterned laminin surfaces.

Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.

Animals↗
PubMed1995

Changes in connexin expression and distribution during chick lens development.

Gap junctions are composed of the connexins, a family of proteins which have been shown to be the structural and functional building blocks of gap junctional intercellular channels. In the chick lens, three members of the connexin (Cx) family have been characterized: Cx 43 has been shown to be a component of interepithelial cell gap junctions, and Cx 45.6 and Cx 56 have been shown to be part of fiber-to-fiber junctions. Early in chick development, gap junctional communication between differentiating lens fibers in the chick loses its sensitivity to blockade by elevated carbon dioxide (CO2). The lens epithelial cells, however, remain CO2-sensitive throughout development. A possible explanation for the change in CO2 sensitivity is that the lens fibers express differentiation-specific connexins with different physiological properties. Using specific antibodies for each of the three lens connexins, we show here that the lens fibers began to express Cx 45.6, and Cx 56 at the developmental stage when lens fibers acquired CO2-insensitivity. However, electron microscopic immunocytochemistry revealed that both Cx 45.6 and Cx 56 were found in interepithelial cell gap junctions, in addition to Cx 43. Conductances between paired Xenopus oocytes injected with Cx 43, Cx 45.6 and Cx 56 mRNAs revealed that all three connexins were CO2-sensitive in this expression system. Taken together, these data ruled out the possibility that the change in CO2 sensitivity observed in vivo could be explained on the basis of the distribution of connexins alone; other parameters of cellular context, such as post-translational processing, must be involved in the observed developmental changes in physiology.

Animals↗
PubMed2000

PDGF regulates gap junction communication and connexin43 phosphorylation by PI 3-kinase in mesangial cells.

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.

Animals↗
PubMed1994

Expression of multiple gap junction proteins in human fetal and infant hearts.

Mammalian cardiac myocytes express multiple gap junction channel proteins or connexins. Expression patterns of the avian homologues of the mammalian cardiac connexins change during cardiac morphogenesis in association with changes in the electrophysiologic properties of intercellular junctions in chick cardiac myocytes. To determine whether expression of cardiac connexins is developmentally regulated in humans, we characterized connexin mRNA and protein content and distribution in hearts of 11 human fetuses (74 to 122 d gestational age), seven children (0.5 mo to 3 y of age), and two adults. Northern blot analysis identified transcripts of connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45) genes in all hearts analyzed. Cx40 mRNA was approximately 5-fold more abundant in samples from fetal hearts than in hearts of children or adults. However, fetal samples used for RNA extraction included atrial as well as ventricular myocardium, whereas samples from children and adults were exclusively ventricular. Northern analysis of adult human right atrial appendages revealed abundant Cx40 mRNA, thus suggesting that the greater amount of Cx40 signal seen on Northern blots from fetal hearts could have been attributable to atrial contributions. Neither Cx43 nor Cx45 mRNA varied significantly in amount in samples from the different developmental stages analyzed. Immunofluorescence identified abundant Cx43 in the known distribution of gap junctions in myocytes in sections of all hearts. Cx45 staining was inconspicuous in fetal hearts but was readily apparent in cardiac myocytes in hearts of older subjects. In contrast, Cx40 staining in the ventricle was confined to mural coronary arteries, apparently in endothelial cells, whereas in the atrium Cx40 staining at myocyte junctions was abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗