大鼠心脏缺血对间隙连接蛋白Cx43分布的影响

目的：探讨大鼠心脏缺血区心肌间隙连接蛋白43（Connexin 43．Cx43）的分布特征。方法：结扎大鼠冠状动脉前降支造成心肌缺血模型。用免疫组织化学法显示心肌缺血区、边缘带及非缺血区Cx43的分布。结果：缺血中心区和边缘区Cx43表达明显紊乱。缺血中心区心肌细胞端-端相接处的Cx43严重消失．重接排列到细胞侧-侧相接处。边缘区Cx43阳性颗粒开始从细胞端-端处向四周扩散,非缺血区Cx43的表达与止常心肌相同。结论：急性短时间心肌缺血时Cx43已开始大量扩散和重新分布,可能是导致心功能异常的结构基础。     

左旋多巴诱发异动症大鼠纹状体缝隙连接蛋白CX36表达增强

目的:观察左旋多巴诱发异动症(LID)大鼠模型缝隙连接蛋白36(CX36)表达,初步探讨缝隙连接在LID形成机制中的作用。方法:制备帕金森病(PD)和LID大鼠模型,将实验动物分3组:LID模型组、PD未治疗组、正常对照组。各组大鼠分2亚组(缝隙连接阻断剂处理组和生理盐水对照组),观察系统途径给予缝隙连接阻断剂甘珀酸(carbenoxolone)对各组大鼠不自主运动行为的影响。利用免疫组化法检测各组大鼠脑皮层运动区和纹状体区CX36表达并进行分析比较。结果:腹腔注射缝隙连接阻断剂甘珀酸对阿扑吗啡诱导的LID不自主运动和PD旋转行为均无明显影响(P>0.05)。免疫组化结果显示LID组皮层运动区和纹状体区CX36表达较PD组和正常组均有显著增多(P<0.05),PD组与正常对照组亦有明显差别(P<0.05)。结论:LID大鼠基底节及大脑皮层CX36表达增加,缝隙连接可能参与了LID的形成机制。     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

黄芪甲苷减轻镉致TM3细胞Cx43表达下降及缝隙连接细胞间通讯功能减退

目的探讨黄芪甲苷(As)对镉(Cd)致TM3细胞连接蛋白43(Cx43)表达改变及由Cx43介导的缝隙连接细胞间通讯(GJIC)功能改变的保护作用。方法用TM3细胞系作为研究对象,设对照组、As(20 mg/L)组、Cd(10μmol/L)组、Cd加As组,免疫组织化学方法分析Cx43阳性产物表达,荧光定量PCR检测Cx43 mRNA表达,Western blot检测Cx43蛋白表达,划痕标记染料示踪技术(SLDT)检测GJIC功能。结果与对照组相比,Cd组TM3细胞Cx43阳性产物表达的平均吸光度值(A_(OD))显著降低(P0.01),Cx43 mRNA和蛋白表达下降(P0.01),TM3细胞GJIC功能降低(P0.01);Cd+As组TM3细胞Cx43阳性产物表达虽较对照组减弱但明显高于相应Cd组(P0.01),Cx43 mRNA、蛋白表达及GJIC功能虽比对照组降低,但较Cd组高(P0.01)。结论黄芪甲苷对镉致TM3细胞Cx43表达下降及GJIC功能减退有明显的拮抗作用。     

Wound healing: the role of gap junctional communication in rat granulation tissue maturation.

Granulation tissue maturation is dependent upon the orientation of collagen fibers and cell differentiation. Gap junctions are intercellular membrane gated channels that facilitate direct communication between cells known as gap junctional intercellular communication (GJIC). The hypothesis is that GJIC modulates the maturation of granulation tissue during wound repair. In vitro, GJIC optimizes fibroblast-populated collagen lattice contraction and influences cell morphology. It is reported that LiCl increases GJIC in cultured cardiac myocytes. Polyvinyl alcohol (PVA) sponge implants with central reservoirs were placed within separate subcutaneous pockets on the backs of adult male Sprague-Dawley rats. Each PVA implant received either 20 mM LiCl or saline injections on days 5, 7, and 10 after implantation. On day 11 implants were harvested and processed for light microscopy. By H&E staining LiCl-treated implants showed increased vascularization and decreased cell density compared to saline controls. Polarized light microscopy of Sirius red-stained specimens revealed more intense collagen fiber birefringence secondary to dense, parallel-organized collagen fiber bundles after LiCl treatment. This suggests that LiCl enhancement of GJIC between fibroblasts advances the maturation of granulation tissue. It is proposed that the degree of GJIC between granulation tissue fibroblasts influences both the quantity and the quality of granulation tissue deposited during the wound healing process.     

Nitric oxide depresses connexin 43 after myocardial infarction in mice

Aims: Heart failure (HF) is a major cause of death and morbidity. Connexin 43 (Cx43) content is reduced in the failing myocardium, but regulating factors have not been identified. In HF, inducible nitric oxide synthase (iNOS)‐induced high levels of nitric oxide (NO) cause apoptosis and cardiac dysfunction. However, a direct iNOS–Cx43 link has not been demonstrated. We investigated this relationship in mice after myocardial infarction. Methods: Effects of myocardial infarction were evaluated 2 weeks after coronary artery ligation in wild‐type C57BL/6 (WT) and iNOS^?/? knockout mice. Myocardial Cx43 and Cx45 content were assessed by immunofluorescence confocal imaging and western blotting. Cardiac function was evaluated in anaesthetized mice using a micro pressure‐tipped catheter inserted into the left ventricle. Results: Despite similar infarct size, deficiency in iNOS resulted in significantly lower plasma nitrate/nitrite levels, better haemodynamic performance and lower mortality 2 weeks after coronary ligation. Myocardial Cx43, but not Cx45, content was lower in WT mice following ligation. The reduction in Cx43 was less in iNOS^?/? compared with WT mice. To assess the direct effect of NO on Cx43 expression, cultured neonatal mouse cardiomyocytes were employed. Incubation with the NO donor, S‐nitroso‐N‐acetylpenicillamine, elicited a dose‐dependent decrease in Cx43 content in cultured neonatal cardiomyocytes. Conclusions: Increased NO production from iNOS depressed cardiac performance and contributed to the decreased myocardial Cx43 content 2 weeks after myocardial infarction.     

Regional differences in cell shape and gap junction expression in rat Achilles tendon: relation to fibrocartilage differentiation

Tendon cells have complex shapes, with many cell processes and an intimate association with collagen fibre bundles in their extracellular matrix. Where cells and their processes contact one another, they form gap junctions. In the present study, we have examined the distribution of gap junction components in phenotypically different regions of rat Achilles tendon. This tendon contains a prominent enthesial fibrocartilage at its calcaneal attachment and a sesamoid fibrocartilage where it is pressed against the calcaneus just proximal to the attachment. Studies using DiI staining demonstrated typical stellate cell shape in transverse sections of pure tendon, with cells withdrawing their cell processes and rounding up in the fibrocartilaginous zones. Coincident with change in shape, cells stopped expressing the gap junction proteins connexins 32 and 43, with connexin 43 disappearing earlier in the transition than connexin 32. Thus, there are major differences in the ability of cells to communicate with one another in the phenotypically distinct regions of tendon. Individual fibrocartilage cells must sense alterations in the extracellular matrix by cell/matrix interactions, but can only coordinate their behaviour via indirect cytokine and growth factor signalling. The tendon cells have additional possibilities — in addition to the above, they have the potential to communicate direct cytoplasmic signals via gap junctions. The formation of fibrocartilage in tendons occurs because of the presence of compressive as well as tensile forces. It may be that different systems are used to sense and respond to such forces in fibrous and cartilaginous tissues.     

Deletion of neuronal gap junction protein connexin 36 impairs hippocampal LTP

In the mammalian CNS, deletion of neuronal gap junction protein, connexin 36 (Cx36), causes deficiencies in learning and memory. Here we tested whether Cx36 deletion affects the hippocampal long-term potentiation (LTP), which is considered as a cellular model of learning and memory mechanisms. We report that in acute slices of the hippocampal CA1 area, LTP is reduced in Cx36 knockout mice as compared to wild-type mice. Western blot analysis of NMDA receptor subunits indicates a higher NR2A/NR2B ratio in Cx36 knockout mice, indicating that there is shift in the threshold for LTP induction in knockout animals. Data suggest a possibility that learning and memory deficiencies in Cx36 knockout mice are due to deficiencies in LTP mechanisms.     

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down-regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special-type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up-regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up-regulation of Cx26 and/or Cx43 in the carcinoma cells of over two-thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems. © 1998 John Wiley & Sons, Ltd.     

Ischaemia-induced temporal expression of connexin43 in rat heart

 To investigate the regulation of cell-to-cell coupling in myocardial ischaemia, the three-dimensional expression of connexin43 (Cx43) during experimental ischaemia was examined using a confocal laser scanning microscope. After induction of myocardial infarction in rats, serial optical sections were obtained from the left ventricular myocardium at various times (3 h to 60 days after ligation). The expression of Cx43 was detected immunohistochemically with FITC-labelled anti-rat Cx43 antibody. Fluorescent dots of Cx43 remained along the intercalated disc and decreased in number around the infarct up to 12 h after ligation. Cx43-expression disappeared completely within 48 h after ligation. After day 4, and especially on days 8 and 15 after ligation, the edges of the cardiomyocytes bordering the infarcted area manifested numerous sarcoplasmic tentacles that reacted positively to anti-desmin antibody. Distinct expression of Cx43 was observed extensively on the tentacles, although no cardiomyocytes remained viable around them. By day 60 after ligation, atypical expression of Cx43 had disappeared. These findings suggest that ischaemia induces temporally abnormal expression of Cx43, which might be responsible for abnormal conduction around the infarct. Received: 4 April 1997 / Accepted: 19 June 1997     

肝癌细胞中connexin32、connexin43的表达及其与间隙连接通讯功能的相关性

目的：研究肝细胞肝癌和正常肝细胞间隙连接蛋白connexin32（Cx32)、connexin43(Cx43）的表达,及其对间隙连接通讯功能（GJIC）的影响。方法：应用应用培养及流式细胞分析技术（FCM）,研究肝癌细胞系HHCC、SMMC－7721和正常肝系QZG细胞中Cx32和Cx43的表达。结合Ｌucifer Yellow划痕标记荧光传输技术（SLDT）,检测上述细胞的间隙连接通讯功能。结果：流式细胞仪分析证实,Cx32蛋白在肝癌细胞系HHCC、SMMC－7721和正常肝细胞系QZG细胞中表达的阳性率分别为1．9％、0．7％和99．0％;Cx43蛋白在HHCC、SMMC－7721和QZG细胞中表达的阳性率分别为7．3％、26．5％和99．1％。SLDT检测发现肝癌细胞HHCC,SMMC－7721的间隙连接通讯功能较正常肝细胞QZG明显减弱。结论;Cx3、Cx43蛋白在正常肝细胞中具有较高水平的表达,在肝癌细胞中表达水平显著降低,肝癌细胞的间隙连接通讯功能较正常肝细胞亦明显减弱。Cx32、Cx43表达调控异常引起的间隙连接通讯障碍可能与肝癌的发生密切相关。     

Hydroxy apatite microspheres enhance gap junctional intercellular communication of human osteoblasts composed of connexin 43 and 45

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.     

Neoplastic reversal of human ovarian carcinoma cells transfected with connexin43

Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.     

Altered expression of connexin43 and phosphorylation connexin43 in glioma tumors

In this study, we aim to evaluate the connexin (Cx43) and phosphorylation Cx43 (p-Cx43) expression of human glioma tumors and correlate their expression with degrees of malignancy and proliferation, apoptosis, and migration activity of tumors. Cx43 and p-Cx43 expression were examined by Western blot analysis and immunohistochemical staining. The U251 cell viability was measured by MTT analysis. The apoptosis and migration were also evaluated by flow cytometric analysis and fluoroblok transwell chambers, respectively. We found that the Cx43 expression were significantly downregulated in in malignant glioma (WHO grade III and IV), compared to the malignant glioma (WHO grade I and II) and the p-Cx43 expression levels of malignant glioma (WHO grade III and IV) were significantly increased (P<0.05), compared to the malignant glioma (WHO grade I and II) at immunohistochemical analysis. After treatment of cells with a specific inhibitor of PKC, MAPK, and PTK inhibitors, the cell viability and migration were significantly decreased, while the apoptosis was slightly induced. In conclusion, the Cx43 expression level is inversely correlated with the tumor grade and proliferation and migration activity of tumor. Higher p-Cx43 expression level in high tumor grade suggests that a complex mechanism is involved in the suppression of tumor growth by connexins.     

Changes in connexin 43 protein expression in human endometrial carcinoma

The expression of connexin 43 was studied using immunohistochemical and Western blot analyses on cell lines of endometrial epithelial origin. Connexin proteins were examined because decreases in their expression and function have been correlated with carcinogenesis. The cell lines were chosen to represent increasing grades of endometrial cancer progression starting from FEEC (fetal endometrial epithelial cells; transformed with SV40 large T antigen) to HEC-1A (stage 1A endometrial carcinoma) to RL-95-2 (grade 2 endometrial carcinoma). Parallel studies using connexin 43 polyclonal antibodies for both Western blots and immunofluorescence showed that the levels of connexin 43 expression were normal endometrial stromal cells = FEEC > HEC-1A > RL-95-2. Consequently, we applied the immunofluorescence assay to analyze paraffin-embedded uterine sections from hysterectomy specimens of patients with normal endometrium and from patients diagnosed with grade 1, 2, and 3 endometrial cancer. Using five different cases from each category, we found an inverse correlation between connexin 43 expression and tumor grade. Our data indicate that connexin 43 expression may be useful as an adjunctive marker of progression for endometrial carcinoma.     

Drebrin (developmentally regulated brain protein) is associated with axo-somatic synapses and neuronal gap junctions in rat mesencephalic trigeminal nucleus

Drebrin (developmentally regulated brain protein)-like immunoreactivity was investigated in the adult rat mesencephalic trigeminal nucleus (MTN) using light and electron microscope. Intense immunoreactive puncta were observed on the cytoplasmic membrane and within the cytoplasm. The cytoplasm was also faintly immunopositive for drebrin, and thus MTN somata other than multipolar cells were distinguishable from non-MTN somata. These immunoreactive cell bodies were localized from the level of the superior colliculus to the pons. Electron microscopic observation showed that the post-synaptic cytoplasmic membrane at axo-somatic synapses was immunoreactive for drebrin. Drebrin-like immunoreactivity was also observed on spine-like processes emanating from MTN somata. In addition, the post-synaptic cytoplasmic membrane at axo-somatic synapses was also immunopositive for drebrin. Within the cytoplasm of MTN cell bodies, a part of the rough endoplasmic reticulum and neighboring structures were also immunopositive. Further, both ends of the somato-somatic close appositions that contained neuronal gap junctions harbored immunoreactive structures. We can infer from the results that drebrin is an ideal marker protein for MTN cell bodies. The abundance of drebrin-like immunoreactivity in the MTN neurons suggests that the MTN has highly flexible synaptogenesis.     

Endometrial connexin expression in the mare and pig: Evidence for the suppression of cell-cell communication in uterine luminal epithelium

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction–associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction–mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness. Anat. Rec. 251:277–285, 1998. © 1998 Wiley-Liss, Inc.     

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile

Background: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected smallmolecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. Methods: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. Results: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. Conclusions: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall. © 1995 Wiley-Liss, Inc.     

Induction of gap junctional intercellular communication,connexin43 expression,and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell–cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whether the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24–48 h treatment of SVG cells with 5 μM forskolin or 5 μM forskolin+200 μM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5–65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin+3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin+3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin+3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to ≈41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin+3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin+3-isobutyl-1-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes.We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the appropriate signals.