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The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

In mammalian astrocytes, calcium waves are transmitted between cells via both a gap junction-mediated pathway and an extracellular, P2 receptor-mediated pathway, which link the cells into a syncytium. Calcium waves in astrocytes have also been shown to evoke calcium transients in neurons, and activity in neurons can elicit calcium waves in astrocytes. In this study, we show that in primary human fetal astrocytes, the P2 receptor-mediated and gap junction-mediated pathways are differentially regulated by the cytokine IL-1beta. Confocal microscopy of astrocytes loaded with Indo-1 demonstrated that intercellular calcium wave transmission in IL-1beta-treated cultures was potentiated compared with controls. However, transmission of calcium waves via the gap junction-mediated pathway was strikingly reduced. The major component of functional gap junctions in human fetal astrocytes was demonstrated to be connexin43 (Cx43), and there was a marked reduction of junctional conductance, loss of dye coupling, loss of Cx43 protein, and down-regulation of Cx43 mRNA expression after IL-1beta treatment of cultures. Conversely, transmission of calcium waves via the P2 receptor-mediated pathway was potentiated in IL-1beta-treated cultures compared with controls. This potentiation was associated with an increase in the number of cells responsive to UTP, and with a transient increase in expression of the P2Y(2) purinoceptor mRNA. Because in inflammatory conditions of the human central nervous system IL-1beta is produced both by resident glia and by invading cells of the immune system, our results suggest that inflammatory events may have a significant impact on coordination of astrocytic function and on information processing in the central nervous system.

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca(2+) waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.

1. The macroscopic and single channel gating characteristics of connexin (Cx) 50 gap junction channels between pairs of N2A neuroblastoma cells transfected with mouse Cx50 DNA were investigated using the dual whole-cell voltage clamp technique. 2. The macroscopic junctional current (Ij) of Cx50-transfected cells decayed exponentially with time in response to transjunctional voltage (Vj) steps (time constant (tau) of approximately 4 s at a Vj of 30-40 mV and 100-200 ms at a Vj of 80-100 mV). The steady-state junctional conductance (gj) was well described by a two-state Boltzmann equation. The half-inactivation voltage (V0), the ratio of minimal to maximal gj (gmin/gmax) and the equivalent gating charge were +/- 37 mV, 0.21 and 4, respectively. 3. The conductance of single Cx50 channels measured using patch pipettes containing 130 mM CsCl was 220 +/- 13.1 pS (12 cell pairs). A prominent residual or subconductance state corresponding to 43 +/- 4. 2 pS (10 cell pairs) was also observed at large Vj s. 4. The relationship between channel open probability (Po) and Vj was well described by a Boltzmann relationship with parameters similar to those obtained for macroscopic gj (V0 = 34 mV, gating charge = 4.25, maximum P= 0.98). The ensemble average of single channel currents at Vj = 50 mV declined in a monoexponential manner (tau = 905 ms), a value similar to the decline of the macroscopic Ij of Cx50 channels at the same voltage. 5. Ion substitution experiments indicated that Cx50 channels have a lower permeability to anions than to cations (transjunctional conductance of KCl vs. potassium glutamate (gammaj, KCl/gammaj,KGlut), 1.2; 6 cell pairs). 6. The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.

Objective: Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression., Methods: Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared., Results: Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone., Conclusion: Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.

When exposed to 20% and 35%, but not to 50% hyposmotic solutions, mouse astrocytes recovered their volume within a few minutes, which coincided with the activation of nonjunctional conductances. Conductance of gap junctions between astrocyte pairs also increased after exposure to a 35% hyposmotic shock; however, this effect began at 3 min after the shock, when cells had partially recovered their initial volumes. During the first minute of exposure to 20% and 35% hyposmotic stimuli, there was a transient monophasic increase in intracellular calcium levels; exposure to 50% hyposmotic solution led to intracellular Ca2+ oscillations. The differences in time courses of nonjunctional conductance changes, Ca2+ alterations, and intercellular coupling suggest that distinct second messenger pathways are involved in each response. The velocity of mechanically evoked calcium waves propagated among the astrocytes increased at 7.5 min after 35% hyposmotic shock. This increase was not seen with 20% or 50% hyposmotic stimuli and is not ascribable to the increase in junctional conductance because it was blocked by suramin, a P2 purinergic receptor antagonist. Given that the transduction pathways activated during cell swelling (e.g., generation of phospholipases, phosphokinases, arachidonic acid) exert inhibitory effects on astrocytic gap junctions (Giaume and McCarthy, 1996), it is proposed that the increased junctional conductance during hyposmotic shock is due to increased number of channels, perhaps triggered by the initial Ca2+ signals (Dolmetsch et al., 1997). As a functional consequence of the increased coupling and enhanced extracellular propagation of Ca2+ waves, spread of signaling molecules throughout the glial network is expected to be significantly enhanced during hyposmotic stress. The increased intercellular communication between mouse astrocytes in response to hyposmotic challenge thus occurs via both gap junction-dependent and -independent mechanisms and presumably provides neuroprotective effects following nervous system injury.

Gap junctions are regarded as the primary pathway underlying propagation of Ca2+ waves between astrocytes, although signaling through extracellular space may also contribute. Results obtained from astrocytes cultured from sibling Cx43 knockout (KO) and wild-type (WT) mice in six litters showed that Ca2+ waves propagated more slowly in Cx43 KO than in WT astrocytes; however, because this difference in velocity was only seen in conditions where cell confluence was higher in WT than KO astrocytes, it is attributable to differences in plating density. By contrast, density-independent differences were observed in the amplitudes of the Ca2+ responses (15% smaller in KO astrocytes) and efficacy of spread (to 14% fewer cells in KO astrocytes). Blockade of purinergic receptors with suramin reduced the velocities of the waves by 40% in WT and KO astrocytes and reduced the amplitudes by 20% and 6%, respectively. In the presence of heptanol, Ca2+ waves spread to only 30% of the cells, with a 70% reduced velocity and 30% reduced amplitude. It is concluded that the propagation of Ca2+ waves between astrocytes from Cx43 KO mice is not so greatly affected as expected by deletion of the major gap junction protein between these cells. The residual 5% coupling contributed by the additional connexins (Cx40, Cx45, and Cx46) expressed in KO astrocytes still suffices to provide a more substantial portion of Ca2+ wave propagation than does signaling through extracellular purinergic pathways. These studies demonstrate that, even with severely reduced junctional conductance, Cx43 KO astrocytes are capable of performing long-range Ca2+ wave signaling, perhaps preserving one mechanism critical to neural function.

Purpose: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization., Methods: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections., Results: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions., Conclusions: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.

Trypanosoma cruzi and Toxoplasma gondii are protozoan parasites capable of causing infections of the nervous system. In order to determine effects of infection by these organisms on intercellular communication in the brain, dye coupling and connexin abundance and distribution were examined in leptomeningeal cells and astrocytes infected with T. cruzi or T. gondii. For both cell types infected with either type of protozoan parasite, intercellular diffusion of intracellularly injected Lucifer Yellow was dramatically reduced. Immunocytochemistry with antibodies specific for connexin43 (in astrocytes) or both connexin43 and connexin26 (for leptomeningeal cells) demonstrated that punctate gap junctional staining was much reduced in infected cells, although uninfected neighbors could display normal connexin abundance and distribution. Western blot analyses revealed that connexin43 abundance in both cell types infected with either parasite was similar to that in uninfected cells. Phosphorylation state of connexin43 (inferred from electrophoretic mobility of connexin43 isoforms) was not significantly affected by the infection process. Immunocytochemistry of whole brains from animals acutely infected with either parasite also showed a marked reduction in connexin43 expression. We conclude that infection of both types of brain cells with either protozoan parasite results in a loss of intercellular communication and organized gap junction plaques without affecting expression levels or posttranslational processing of gap junction proteins. Presumably, these changes in gap junction distribution result from altered targeting of the junctional protein to the plasma membrane, and/or from changes in assembly of subunits into functional channels., (Copyright 1998 Elsevier Science B.V.)

RT4 is a family of cell lines derived from a rat peripheral neurotumor. The RT4 family consists of a multipotential stem-cell line that spontaneously gives rise to three derivative cell types, one glial and two neuronal. The three derivative cell types are capable of further lineage-specific maturation under appropriate culture conditions. Gap-junctional communication is postulated to be important during nervous-system development by allowing and/or controlling the transmission of both electrical current and signaling molecules, which may affect growth and differentiation. Our characterization of gap-junctional communication in the RT4 cell line family revealed that: (1) the glial-derivative and the stem-cell line were extensively coupled, while the two neuronal derivatives were significantly less coupled, and (2) all of the RT4 cell lines, including the stem-cell line, expressed Cx43 mRNA and protein, and the levels were generally consistent with the observed degree of functional coupling. These observations are consistent with data from in vivo studies and establish the RT4 cell line family as a potentially useful in vitro model system for understanding the role(s) of gap-junctional communication during differentiation in the peripheral nervous system.

The cellular mechanisms that regulate progenitor cell lineage elaboration and maturation during embryonic development of the mammalian brain are poorly understood. Conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) were found to be strongly coupled by gap junctions comprising connexin 43 (Cx43) during early neuronal ontogeny; the presence of this Cx type was confirmed by electrophysiological, molecular biological, and immunocytochemical assays. However, as progenitor cells underwent intermediate stages of neuronal differentiation under the influence of interleukin 7 (IL-7) alone or terminal differentiation after composite exposure to basic fibroblast growth factor, IL-7, and transforming growth factor alpha, coupling strength and the level of Cx43 expression declined. An additional population of junctional channels with distinct properties was detected at an intermediate stage of neuronal differentiation. Reverse transcription-PCR assays detected mRNA encoding Cx40 in IL-7-treated cells and Cx33 after both treatment conditions. Because functional channels in exogenous expression systems are not formed by pairing Cx40 with Cx43 or by pairing Cx33 with itself or additional connexins, these experimental observations raise the possibility that the progressive loss of coupling during differentiation of neural progenitor cells may involve downregulation of Cx43 coupled with potentiation of expression of Cx33 and Cx40. Furthermore, continued expression of Cx43 in differentiating neuroblasts could mediate intercellular communication between neuronal precursor cells and astrocytes by direct signaling via homotypic gap junction channels.

Hypothesis: Cisplatin causes the generation of reactive oxygen species (ROS), which interferes with the antioxidant defense system of Corti's organ and results in damage to the hair cells., Background: Cisplatin is a widely used chemotherapeutic agent with the dose-limiting side effect of ototoxicity. Evidence is accumulating that cisplatin interferes with the antioxidant defense system of Corti's organ., Methods: Organotypic explants of P-3 rat organ of Corti were the in vitro model system. Presence of intact auditory hair cells and stereocilia bundle integrity was assayed by phalloidin-FITC staining. Fluorescent dye probes detected H2O2 and intracellular thiol [e.g., glutathione (GSH)]. Spectrophotometric analysis determined antioxidant enzyme levels., Results: There was a rapid dose-dependent cisplatin cytotoxicity in the explants after 48 h of exposure. An accumulation of H2O2 and a reduction of GSH levels were observed within cisplatin-exposed hair cells. L-buthionine sulfoximine, an inhibitor of GSH formation, enhanced cisplatin ototoxicity, whereas N6-(2-phenylisopropyl) adenosine, an adenosine agonist, elevated antioxidant enzyme levels and ameliorated cisplatin toxicity. The following molecules protected hair cells from cisplatin-induced damage: GSH; glutathione diethyl ester (GSHe); ebselen (EBS); 4-methylthiobenzoic acid (MTBA); and D-methionine (D-MET). EBS, MTBA, and D-MET in vitro protection correlates with in vivo protection in rats., Conclusions: Organotypic culture of Corti's organ has been validated as a model for studying cisplatin toxicity and for screening otoprotective molecules. Some of the events that contribute to cisplatin's ability to damage auditory hair cells are generation of ROS (e.g., H2O2), depletion of intracellular GSH, and interference with antioxidant enzymes within the cochlea. Agents that bolster the cochlea's antioxidant system can prevent cisplatin destruction of auditory hair cells. Identified protective agents may prove to be clinically useful in limiting or completely protecting from cisplatin ototoxicity.

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.

Oligodendrocytes, the myelin-forming glia of brain, are connected by gap junctions in situ and in culture. Cultured oligodendrocytes from adult bovine and porcine brains were studied using immunocytochemical, molecular, and electrophysiological techniques in order to characterize the gap junction types. The expression of connexin32 was substantiated by the detection of low, but significant, signals using connexin-specific probes in Northern and Western blot analyses. Connexin43, which comprises gap junctions in astrocytes, was not detectable in pure oligodendrocytic cultures; mRNAs of connexin40 and connexin37 and connexin26 were also not detected. By means of two specific antibodies directed to the recently cloned connexin45 and by RT-PCR we were able to identify this connexin as a second oligodendrocytic gap junction protein. Whole cell voltage clamp recording provided evidence for electrical coupling between pairs of cultured oligodendrocytes (mean junctional conductance 3.9 nS, n = 38 pairs) and intracellular Lucifer Yellow injection indicated that oligodendrocytes were usually only weakly dye coupled, with spread generally being restricted to nearest neighbors. Unitary conductances ranged from > 20 to < 150 pS with modes of distribution at about 100 to 120pS and 40 to 20 pS, respectively. These unitary conductances are consistent with the channel events expected for connexin32 and connexin45. The low degree of functional coupling between oligodendrocytes in vitro corresponds with the low levels of connexin32 and connexin45 messenger RNAs and protein expression.

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk+) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk+ cells) exerted bystander cytotoxicity on gap junction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiments also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk+ cells. In conclusion, these results indicate that in several cellular environments the bystander effect is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.

1. Trophoblast invasion during embryo implantation in some aspects resembles tumour cell invasion but, unlike tumour cells, trophoblast cells are able to differentiate and establish a placenta. Because direct cell-cell communication is believed to be involved in growth control and differentiation, we have investigated connexin (cx) gene expression during trophoblast development. 2. Pre-implantation embryos expressed cx43 as well as cx31 proteins from the 8-cell stage onwards. Following implantation, compartmentalization of both connexins occurred: cx31 expression was restricted to the invasive trophoblast cell population, whereas the embryo proper was characterized by cx43. Trophoblast differentiation was indicated by induction of cx26 in the labyrinth and cx43 in the spongiotrophoblast accompanied by a disappearance of cx31. Comparison with trophoblast cell lines revealed that rat trophoblast HRP-1 cells express connexin43, while malignant choriocarcinoma cells express cx31. Treatment with retinoic acid led to a disappearance of cx31 in the choriocarcinoma. Both cell lines reduced their invasion properties after retinoic acid treatment, but growth retardation was only observed in the malignant trophoblast. 3. It seems that the cx31 channel is needed for trophoblast cell populations to maintain the highly proliferative properties but does not alter their invasion properties.

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V0 = 18.5 mV, n = 4.5 and gmin/gmax = 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels.

Examination of rat hepatic cell lines has revealed a correlation between the differentiated state of the cells and the gap junctional proteins, or connexins, they express. The cell lines RLC (Gershenson et al, 1970) and FTO.2B (Killary et al, 1984) were examined and compared to primary adult hepatocytes for expression of fetal and adult hepatic antigens under various tissue culture conditions. Maximal expression of fetal antigens was observed in cells grown in serum-supplemented medium, on either tissue culture plastic or type IV collagen. Maximal expression of adult specific antigens was seen in cells grown in a hormonally defined medium containing heparin, on type I or type IV collagen. The cell line RLC strongly expressed fetal antigens, while FTO.2B expressed both fetal and adult antigens. These cell lines and another poorly differentiated hepatic cell line, WB-F344 (Tsao et al., 1984) were used to assess the developmental profile of mRNAs encoding isoforms of gap junctions: connexins 26, 32, and 43. The cell lines each transcribed mRNAs of all three connexins, as determined by transcriptional elongation analysis. By contrast, only certain of the connexin mRNAs could be detected in specific cell lines by Northern analysis: RLC expressed only connexin 43 mRNA; WB-F344 expressed connexin 32 and 43 mRNAs. Selection among the connexin mRNAs appears to occur post-transcriptionally. Culture of the cell lines in hormonally defined medium vs. serum supplemented medium did not affect the patterns of connexin mRNA abundance. When the cell lines were cultured in hormonally defined medium containing heparin, however, the level of connexin mRNAs did vary: Connexin 26 mRNA increased in WB-F344 cells, and connexins 32 and 43 mRNAs increased in FTO.2B, but connexin 43 mRNA decreased in WB-F344 and RLC. The abundance of connexin mRNAs also varied when the cell lines were analyzed at different cell densities: connexin 43 mRNA increased with cell density in RLC and WB-F344, and connexin 26 mRNA peaked at an intermediate density and fell at higher cell densities in WB-F344. The differences in connexin mRNA expression among cell lines characteristic of different stages of hepatic differentiation, and the differences in regulation of connexin mRNAs in the hepatic cell lines, suggest distinct biological roles of the highly homologous proteins. Moreover, connexin gene expression may be a marker of hepatic development: as hepatocytes differentiate the proportions of connexin 43 then 26 mRNAs decrease while that of connexin 32 mRNA increases.

Integration and coordination of responses among vascular wall cells are critical to the local modulation of vasomotor tone and to the maintenance of circulatory homeostasis. This article reviews the vast literature concerning the principles that govern the initiation and propagation of vasoactive stimuli among vascular smooth muscle cells, which are nominally the final effectors of vasomotor tone. In light of the abundance of new information concerning the distribution and function of gap junctions between vascular wall cells throughout the vascular tree, particular attention is paid to this integral aspect of vascular physiology. Evidence is provided for the important contribution of intercellular communication to vascular function at all levels of the circulation, from the largest elastic artery to the terminal arterioles. The thesis of this review is that the presence of gap junctions, in concert with the autonomic nervous system, pacemaker cells, myogenic mechanisms, and/or electrotonic current spread (both hyperpolarizing and depolarizing waves through gap junctions), confers a plasticity, adaptability, and flexibility to vasculature that may well account for the observed diversity in regulation and function of vascular tissues throughout the vascular tree. It is hoped that the summary information provided here will serve as a launching pad for a new discourse on the mechanistic basis of the integrative regulation and function of vasculature, which painstakingly accounts for the undoubtedly complex and manifold role of gap junctions in vascular physiology/dysfunction.

Astrocytes form functional networks that participate in active signaling in which external stimuli are generated and amplified in many of the same ways as in neurons. Gap junctions between astrocytes offer the structural avenue by which the electrical and metabolic signals are propagated from one cell to another. Little is known about the trafficking, assembly, and degradation mechanisms of the major astrocytic gap junction protein connexin43. We have studied a glial cell line transfected with the C-erbB2/neu oncogene (neu+), finding severe interruption of gap junctional communication after stable transfection. Evidence from Western blotting and phosphorylation studies showed that the processing of connexin43 to its higher phosphorylated isoforms is disturbed. Confocal laser imaging indicates that the major deficit in the neu+ cells is attributable to a lack in plaque assembly of connexin43. Because the neu+ cells also lack N-CAM proteins and because work from others has indicated a close relationship between communication competence and constitutive CAM expression, our data suggest that expression of C-erbB2/neu oncogene alters cell-cell association via CAM proteins, which thereby affects gap junction plaque assembly and appropriate phosphorylation of connexin43.

The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin-43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch-clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.

We have investigated the properties of gap junction channels of three human malignant trophoblast (choriocarcinoma) cell lines: BeWo, Jeg-3 and JAr, as well as in Jeg-3 cells stably transfected with rat connexin40 (Cx40). Reverse-transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis and immunostaining demonstrated expression of Cx40 in BeWo and JAr cell lines. JAr cells also expressed minor amounts of Cx43. Very low levels of Cx40 transcripts were revealed by RT-PCR in parental Jeg-3 cells, but Cx40 protein was not detected. To compare properties of endogenously and exogenously expressed Cx40 channels we have transfected Jeg-3 cells with rat Cx40. Recordings with dual whole-cell methods were used to determine the junctional conductance (gj) in the various cell lines and transfectants. Cx40 channels exogenously expressed in Jeg-3 cells demonstrated steep voltage sensitivity in the transjunctional voltage range of +/-30 to +/-40 mV and a unitary mainstate conductance of 175 pS, values which are similar to the data obtained from endogenously expressed Cx40 in BeWo cell pairs. In addition, greater driving forces resulted in a lower unitary conductance of about 30 pS, exclusively in BeWo cells. Between JAr cell pairs we determined a gj of 10 nS and unitary conductances were predominantly 100 and 152 pS. Voltage dependence was less sensitive in JAr cells compared to Cx40 transfectants and BeWo cells. Thus, coexpression of Cx43 and Cx40 leads to a macroscopic conductance with a mixture of properties expected for each connexin, whereas single-channel properties of each connexin type are maintained.

Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-alpha or the mitogen neu differentiation factor (NDF)-beta. TNF alpha inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNF alpha also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both Po and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDF beta initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDF beta stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU+) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNF alpha may be a signal which terminates proliferation as well as junctional communication.

Cytoplasmic calcium ([Ca(2+)](i)) changes evoked by adenosine 5(1)-triphosphate (ATP) were recorded in cultured individual Leydig cells within 10-18 h after cell dispersion. [Ca(2+)](i) was monitored using Fura-2AM loaded cells with a digital ratio imaging system. Five micromolars ATP induced biphasic [Ca(2+)](i) responses in most cells (94%,n=100), characterized by a fast increase from a basal level (126±5 nMSE,n=60 cells) to a peak (5-7 times above basal levels) within seconds, followed by a slow decrease toward a plateau level (2-3 times above basal) within 5 min. The peak phase of the [Ca(2+)](i) response increased with ATP concentrations (1-100 μM ATP) in a dose-dependent manner with an IC(50) of 5.9±1.2 μM, and it desensitized in a reversible manner with repeated application of 5 μM ATP at <5-min intervals. The [Ca(2+)](i) peak response was dependent on Ca(2+) release from an intracellular pool, whereas the plateau phase was dependent on extracellular [Ca(2+)]. ATP did not appear to induce formation of nonspecific membrane pores, since stimulation for 10 min with ATP (10-100 μM) in the presence of extracellular Lucifer yellow (LY) (5 mg/mL) did not result in dye loading of the cells. [Ca(2+)](i) transients were elicited by other adenosine nucleotides with an order of potencies (ATP>Adenosine diphosphate [ADP]>Adenosine> Adenosine monophosphate [AMP]) that was compatible with the expression of P(2) receptors. [Ca(2+)](i) responses were suppressed by the purinergic P(2) receptor antagonist, suramin. These results provide functional evidence for the expression of purinergic P(2) receptors in Leydig cells.

The identification of connexin32 (Cx32) in myelinating Schwann cells and the association of Cx32 mutations with peripheral neuropathies suggest a functional role for gap junction proteins in the nerve. However, after nerve crush injury, Cx32 expression dramatically decreases in Schwann cells in the degenerating region, returning to control levels at newly formed nodes of Ranvier and Schmidt-Lantermann incisures by 30 days. The present study examined increases in expression of other connexins that occur after peripheral nerve injury. A 56/58-kDa connexin46 (Cx46) protein species was detected in adult rat sciatic nerve, along with very low levels of Cx46 mRNA. However, by 3 days after crush injury, coincident with changes in Schwann cell phenotype, Cx46 mRNA rapidly increased in the degenerating regions. Additionally, the 56/58-kDa Cx46 protein species present in adult nerve decreased and a 53-kDa Cx46 species, which was also present in cultured Schwann cells, became apparent. Connexin43 (Cx43) mRNA and protein, which was localized to perineurial cells in adult nerve, dramatically increased in endoneurial fibroblasts in the crush and distal regions by 3 days, coincident with macrophage infiltration. By 12 days after injury, Cx43 decreased and was comparable to normal nerve. These results suggest that enhanced expression of Cx46 and Cx43, by nonneuronal cells, may be important for the injury and regenerative responses of peripheral nerves.

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca2+]i), and was additive to that of high K+. Survival was optimized when Ctx B was present for several days during the early culture period. 45Ca2+ and cell survival studies indicated the mechanism to involve enhanced influx of Ca2+ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K+, caused [Ca2+]i to rise to 0.2-0.7 microM in a higher proportion of cells than 25 mM K+ alone. A significant change in the nature of GM1 modulation of Ca2+ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca2+]i below the level attained with 25 mM K+. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca2+ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca2+ homeostasis in cultured neurons of the CNS.

Tumor cells transduced with the herpes simplex virus thymidine kinase (HSVtk) gene are sensitive to the anti-viral drug ganciclovir (GCV). However, nearby untransduced tumor cells are also efficiently killed. The mechanism of this 'bystander effect' was studied by comparing pairs of tumor cell lines transfected with connexin genes that differed only in their degree of gap junctional communication. More efficient cell killing was uniformly seen in connexin transfectants compared with the less coupled cell lines. These results provide direct evidence that gap junctional communication plays an important role in mediating the bystander effect of the HSVtk/GCV system in vitro and have important prognostic and therapeutic implications for future gene therapy trials.

Direct intercellular coupling through gap junction channels has been implicated in diverse processes including cellular differentiation, growth control, metabolic cooperativity and electronic coupling and natural and induced mutations in connexin genes have been described in human and experimental disease states. Genetic systems in which the extent of coupling could be reversibly regulated would provide an important approach for examining these potential functional roles, both in vitro and in vivo. Here we describe the generation and characterization of cell lines in which the extent of coupling is reversibly controlled at the transcriptional level. Plasmids encoding a tetracycline-controlled transactivator and a tetracycline-responsive connexin32 target gene were introduced in the communication-deficient SKHep1 cell line. Quantitative immunoblotting and confocal immunofluorescence microscopy with connexin32-specific antibodies demonstrated that expression of connexin32 in stable transfectants was tightly regulated by tetracycline treatment. Moreover, transfectants exhibited a highly coupled phenotype which was rapidly and reversibly converted to the communication deficient parental state after tetracycline treatment. Time constants for decay of the messenger RNA, protein and functional coupling were similar (approximately 4 hrs), implying that transcription was rate-limiting and that separate long-lived pools of connexin32 protein were absent. In contrast to other approaches in which the extent of coupling is pharmacologically regulated by altering channel gating characteristics or by generalized blockade of transcription or translation, in this system intercellular communication is regulated by directly controlling connexin gene expression.

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 (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj 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 (gammaj) 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 gammaj 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 gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

We developed and characterized a coculture system composed of a fat-storing cell clone (CFSC-2G) and freshly isolated hepatocytes that can reproduce in vitro some of the physical and functional relationships observed in vivo. Hepatocytes in the coculture are polarized, are smaller in size than hepatocytes plated on plastic, maintain a cuboidal shape, and have a tendency to form cords. Fat-storing cells, which are initially extended, retract and leave spaces that resemble liver sinusoids. Both cell types in the coculture system are functional for at least two weeks as determined by the expression of high levels of liver-specific protein mRNAs as well as by the production and secretion of liver-specific proteins into the culture medium. The hepatocytes maintain relatively high levels of asialoglycoprotein receptor on their cell surface and form functional gap junctional complexes with fat-storing cells. Hence, this coculture system retains a number of differentiated functions of hepatocytes, making it a useful model to study cell-cell interactions in culture and to analyze regulation of hepatocyte functions.

Gap junctions play important roles in the exchange of information and metabolites in the nervous system. These roles are highlighted by peripheral neuropathy (X-linked dominant Charcot-Marie-Tooth disease) that is associated with mutations in a gap-junction protein (connexin32), resulting in loss of function, and by somatic dysfunctions where changes in expression, organization or function of gap junctions are associated with neuronal hyper- or hypoexcitability. In this review, the causes and consequences of this gap-junction-related peripheral neuropathy and other pathological conditions of the nervous system, where dysfunctions of junctional communication are considered to play a casual role, are considered.

We have observed sympathetic hyperinnervation and hyperplasia of an unknown cell population within the base of the hearts from transgenic mice in which nerve growth factor (NGF) expression was targeted to cardiac tissue (Hassankhani et al., 1993, 1995). To characterize this ectopic cell population, cells from the base of hearts of 3- to 4-week-old transgenic mice were dissociated and established in tissue culture, and their properties were analyzed using morphological, immunocytochemical, and physiological techniques. Morphological studies of the ectopic cells revealed the temporal expression of two different cellular phenotypes: (i) a spindle-shaped cell type, present by 1 to 2 days in vitro (DIV) and (ii) cells with a fiber-like morphology, detected by 7 DIV. In contrast to transgenic cardiac myocytes that express immunoreactivity to antibodies against sarcomeric myosin, beat spontaneously (approximately 60/min), and are electrically excitable, the ectopic cell types did not stain with an antibody against sarcomeric myosin, never showed contractile activity, and did not manifest membrane excitability. Moreover, these two types of ectopic cells demonstrated other distinctive characteristics. The spindle-shaped subpopulation typically formed small clusters of cells that were immunostained by antibodies to GFAP, vimentin, and low-affinity NGF receptor (LNGFR) and demonstrated a low incidence of dye coupling (30%), whereas the fiber-like cells aligned themselves along their long axes, immunostained for gp140trkA, LNGFR, and anti alpha-smooth muscle actin and showed extensive (100%) and diffuse dye spread to numerous other contiguous fiber-like cells. Dye spread was not observed between the adjacent fiber-like and the spindle-shaped cells and was reversibly blocked between homologous cells after treatment with halothane, a gap junction channel blocking agent. In comparison to cardiac myocytes, macroscopic junctional conductance of the spindle-shaped cells was more voltage-sensitive. At the single channel level, unitary junctional conductances of approximately 60 pS were predominant. Overall, these results indicate that these ectopic cells are likely derived from neural crest. The spindle-shaped cells appear to be immature Schwann cells, whereas the fiber-like cells may be related to the ectomesenchymal cells contributing to the morphogenesis of the cardiac outflow tract.

We recently established primary cultures from dissociated second trimester human fetal brains using a novel spin seeding method and characterized cellular populations with distinct phenotypes in these cultures. Here, we report that these neural cultures can be dissociated to single-cell suspensions, sorted by size using flow cytometry and re-seeded to yield cultures selectively enriched for the neuronal and glial cell populations. Sorted neurons were highly homogeneous, viable and extended processes, by one day after re-seeding. These neurons expressed immunoreactivity for neurofilament protein, retained their GABAergic phenotype and were electrically excitable. Re-seeded astrocytes proliferated in culture and expressed glial fibrillary acidic protein. We describe the conditions required for the flow cytometric sorting and tissue culture assays as well as the morphological, immunocytochemical and electrophysiological characteristics of the sorted neuronal population.

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.

A multiparametric study was carried out to investigate the presence and possible role of communicating junctions in the thymus, particularly in the thymic epithelium, the major component of the thymic microenvironment. The presence of direct cell-cell communication mediated by gap junctions was demonstrated in human and murine thymic epithelial cells (TEC) by means of in situ and in vitro immunohistochemical labeling as well as in vitro fluorochrome injection and double whole-cell patch clamp experiments. Moreover, both immuno- and Northern blot studies revealed that the gap junction protein connexin 43 and its mRNA were present in TEC. Importantly, we showed that thymic endocrine activity, as ascertained by thymulin production, could be specifically down-modulated in vitro by a gap junction inhibitor, octanol. We also investigated the existence of gap junctions between TEC and thymocytes. In thymic nurse cells we were able to detect cell-cell communication, although only a minor percentage of epithelial/thymocyte pairs were coupled in a given moment. In contrast, intercellular communication was not detected between cultured phagocytic cells of the thymic reticulum and the respective rosetting thymocytes. We suggest that gap junctions formed by connexin 43 may represent a novel (and rather cell type-specific) pathway for intrathymic cellular communication, including TEC/TEC as well as possible TEC/thymocyte interactions.

Mechanisms regulating the expression of intercellular coupling, development of membrane excitability, and cellular responsiveness to neurotransmitters during neuronal ontogeny are largely unknown. To define the temporal relationship among these properties during neurogenesis, murine embryonic hippocampal progenitor cells immortalized with a temperature-sensitive allele of the SV40 large T-antigen were examined during successive stages of neuronal differentiation in vitro using patch clamp, dye coupling, and Ca2+ imaging techniques. Electrotonic and dye coupling between untreated neuroblasts were frequent in cells maintained at the temperature (39 degrees C) nonpermissive for T-antigen expression. However, as neuroblasts differentiated into neurons under the influence of interleukin-7 added alone or concurrently with transforming growth factor-alpha after basic fibroblast growth factor, both junctional conductance and the extent of dye coupling progressively decreased. Voltage-dependent inward currents were present within 2 to 6 days after differentiating treatments began. During intermediate developmental stages (3 to 5 days in culture), cells became responsive to GABA (> or = 100 microM) but not to glutamate, glycine, or to acetylcholine (< or = 1 mM), as indicated by [Ca2+]i measurements and patch clamp recordings. In contrast, voltage- and ligand-gated responses but not electronic coupling were frequently observed in mature neuronal primary cultures. Together, these results indicate that certain cytokines may orchestrate the progressive expression of functional neuronal phenotypes in vitro, in which the gradual disappearance of intercellular coupling parallels the onset of voltage-dependent responses and both of which precede the expression of neurotransmitter chemosensitivity.

Following peripheral nerve injury, Schwann cells undergo a series of cellular alterations that are thought to assist the regenerative process. Some of these changes are stimulated by the local release of cytokines and mitogenic factors. To test the hypothesis that cytokine regulation of gap junctional communication between cells helps to coordinate Schwann cell responses, cultured rat Schwann cells, from sciatic nerve, were utilized to study phasic changes induced by transforming growth factor-beta 1 (TGF beta 1), a cytokine released after nerve injury, or forskolin in combination with bovine pituitary extract (F-BPE), known for its mitogenic effects in vitro. In mitotically quiescent cultures, TGF beta 1 significantly decreased both electrical and dye coupling mediated by gap junctions. Single-channel analysis revealed that cultured Schwann cells expressed gap junctions with two distinct channel sizes of about 26 pS and 44 pS. TGF beta 1 treatment reduced coupling due to both populations of channels. Exposure to TGF beta 1 had a minimal effect on proliferation but significantly altered cellular morphology; cell bodies became flattened with multipolar processes within 72 hr. Additionally, immunolabeling for both low-affinity nerve growth factor receptor (L-NGFR) and glial fibrillary acidic protein (GFAP) were reduced, suggesting increased differentiation. In contrast, treatment with F-BPE significantly enhanced both electrical and dye coupling and stimulated Schwann cell proliferation. Additionally, cell bodies became more rounded with polarized, cytoplasmic processes contiguously aligned with adjacent cells. F-BPE reduced immunolabeling for L-NGFR but increased expression of both GFAP and the major peripheral myelin protein, P0. These data indicate that TGF beta 1 and/or F-BPE induce phenotypic changes in Schwann cells, including the coordinated regulation of proliferation and modulation of intercellular communication via gap junctions. Such mechanisms may underlie phasic responses that orchestrate recovery from nerve injury, indicating that Schwann cell gap junctions may be critical for peripheral nerve function.

We established high density primary cultures of neural cells from dissociated second trimester human fetal brains using a novel spin seeding method. Under our culture conditions, the majority of the cells exhibited neuronal phenotypes as evidenced by morphological criteria, immunoreactivity to the 66 kDa neurofilament protein and expression of TTX-sensitive Na+ channels and cell excitability. These cultures were enriched in glutamic acid decarboxylase (GAD), the synthetic enzyme of the neurotransmitter GABA, and responded with Ca2+ influx to acute application of glutamate. Interestingly, the human fetal neurons in culture did not express either dopaminergic or cholinergic phenotypes. In addition, the population of neurons obtained express a high incidence of gap junction-mediated intercellular communication. These studies provide evidence that functional neuronal properties arise early during prenatal development in humans and offer the potential to evaluate pharmacological agents on primary human neurons.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neuronal choice of neurotransmitter. In this present study, these factors were shown to specifically and differentially regulate levels of both muscarinic (subtypes m1, m2, m3, m4, and m5) and substance P receptor (SPR) mRNAs in sympathetic neurons of the rat superior cervical ganglion (SCG) using solution hybridization/RNase protection analysis. In vivo, neonatal rat SCG expressed predominantly m2 (10.31 +/- 0.43 pg mRNA/micrograms total RNA) and some m1 (1.54 +/- 0.84 pg/microgram) muscarinic receptor mRNA, which increased developmentally to adult levels (m2 mRNA levels being 60% higher than those in neonates). By contrast, m3, m4, and m5 subtype mRNAs were much less abundant at all time points measured. A similar developmental regulation was found in dissociated SCG neurons in vitro. After 16 days in culture, m2 mRNA increased 334% to 15.76 +/- 0.68 pg/microgram, while m1 mRNA changed little (2.03 +/- 1.00 pg/microgram). However, LIF or CNTF treatment (5 ng/ml, 14 days) in sister cultures completely blocked this developmental increase. Further, LIF treatment blocked the normal muscarinic receptor-mediated increase in intracellular calcium (fura-2 imaging), indicating a functional change in receptor phenotype. By contrast, levels of SPR mRNA, which were low in untreated cultures (0.037 +/- 0.025 pg SPR mRNA/microgram total RNA), were elevated by LIF or CNTF treatment, to 0.866 +/- 0.034 pg/microgram and 0.662 +/- 0.148 pg/microgram, respectively. These observations indicate that muscarinic and SPR receptor expression are differentially regulated by the same factors in SCG neurons and that neuronal choice of receptor phenotype may be, at least in part, specifically regulated by cytokines/growth factors in the cellular milieu.

The protozoan parasite Trypanosoma cruzi causes Chagas' disease, a major cause of cardiac dysfunction in Latin Americans. Chagas' disease exhibits both acute and chronic phases, and each may be characterized by cardiac conduction disturbances. In acutely infected cultures of rodent heart cells, synchronized spontaneous beating becomes less regular, and coupling between cells is reduced. The basis of this decreased conduction is apparently in localization of the gap junction protein (Cx43) inside infected cells. Although total Cx43 is normal in infected cells, little is recognizable at appositional membranes. Electrophysiological properties are also altered by this infection. Action potentials are shortened, resting Ca2+ levels are elevated, and response to alpha-adrenergic agonists was altered, compared to controls. Humoral factors may contribute to the conduction defects in chronic Chagas' disease. Sera from chronically infected rabbits produced ECG abnormalities in Langendorff-perfused rabbit hearts. These findings indicate that chagasic infection may modify ion channel function in the heart, and we suggest that these changes may be manifested in the conduction disturbances that characterize this disease.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.