The properties of astroglial gap junction channels and the protein that constitutes the channels were characterized by immunocytochemical, molecular biological, and physiological techniques. Comparative immunocytochemical labeling utilizing different antibodies specific for liver connexin 32 and connexin 26 and antibodies to peptides corresponding to carboxy-terminal sequences of the heart gap junction protein (connexin 43) indicates that the predominant gap junction protein in astrocytes is connexin 43. The expression of this connexin in cultured astrocytes was also established by Western and Northern blot analyses. Cultured astrocytes expressed connexin 43 mRNA and did not contain detectable levels of the mRNAs encoding connexin 32 or connexin 26. Further, the cells contained the same primary connexin 43 translation product and the same phosphorylated forms as heart. Finally, electrophysiological recordings under voltage-clamp conditions revealed that astrocyte cell pairs were moderately well coupled, with an average junctional conductance of about 13 nS. Single-channel recordings indicated a unitary junctional conductance of about 50-60 pS, which is of the same order as that found in cultured rat cardiac myocytes, where the channel properties of connexin 43 were first described. Thus, physiological properties of gap junction channels appear to be determined by the connexin expressed, independent of the tissue type.
Gap junction channels provide a pathway for exchange of ions and small molecules between coupled cells, and this exchange is believed to be critical for normal tissue growth and development. As a test for a role of gap junction-mediated intercellular communication in control of cell growth, we have compared growth rates of communication-deficient human tumor cells (SKHepl) with. clones stably transfected with cDNA encoding the rat liver gap junction protein connexin 32. In culture, growth rates for parental and transfected clones were similar. However, when sizes oftumors were evaluated following hnuection ofthese clones into athymic nude mice, growth rates for two well-coupled clones were significntly lower than for communication-deficient or poorly coupled clones. This study demonstrates that growth rate ofthese tumor cells in situ is negatively correlated with strength of intercellular communication.A role for gap junctions in normal tissue growth and development has long been postulated because of the widespread expression of gap junctions during embryogenesis (1-3) and the progressive delimitation of communication compartments as ontogenesis proceeds (3-6). This hypothesis has been strengthened by the correlation between tumor promotion and partially reduced coupling induced by various agents (7,8), by the low coupling strength and reduced gapjunction abundance between cells of some tumors, especially in highly metastatic cell populations (9)(10)(11)(12)(13), and by demonstration that oncogene expression reduces coupling in cell lines (14,15 MATERIALS AND METHODS Cell Lines. SKHepl cells were cotransfected with plasmid pcEXV3 containing full-length coding sequence for connexin 32 and/or pSV2neo using the calcium phosphate precipitation technique and selected by antibiotic G418 resistance and subsequently (in the case of the connexin 32 transfectants) on the basis of Lucifer yellow transfer as described (19). Cells were grown in a 370C, 5% CO2 incubator in RPMI medium with 10o added fetal bovine serum to which 0.4-0.5 mg of G418 per ml (GIBCO) was added in the case of transfectants (G418 was withdrawn before growth rate studies were begun). Growth rate was determined in 60-mm tissue culture dishes after seeding from confluent cultures at a density of 5 x 104 cells per dish on day 0. On subsequent days, concentration of cells was determined either by counting the number of cells per unit area of the dish (five areas counted per dish; data not shown) or by counting the number of cells per unit volume using a hemocytometer after cells were dissociated using a mild trypsinization protocol [2 min of exposure to 0.5 mg of trypsin per ml/0.2 mg of EDTA per ml (GIBCO) in RPMI medium]. For each time point in each experiment, two or three dishes of cells were counted and mean values from repeated experiments were compared to determine means and variance.Tumor Studies. BALB/c nude mice derived from breeding stocks were obtained from Frederick Cancer Center (Frederick, MD); they were quarantined for 1 week befo...
A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, G~ was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary unitary channel conductance junctional currents was detected corresponding to an " of ~ 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic G~ voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.
The interaction between peptidergic, sensory nerves and sympathetic fibers was examined in the rat iris. The putative peptide neurotransmitter, substance P, was used as an index of the sensory innervation, because the peptide is exclusively localized in the iris to trigeminal sensory fibers. Extirpation of the sympathetic, superior cervical ganglion resulted in an increase in iris content of substance P-like immunoreactivity (henceforth SP), suggesting that sympathetic terminals influence the peptidergic sensory innervation of the iris. The increase in iris peptide after sympathetic ganglionectomy was reversed by implantation of sympathetic ganglia into the anterior chamber of the eye. Pharmacological stimulation or blockade of sympathetic nerve impulse activity and pharmacological blockade of sympathetic axonal transport did not alter iris peptide, suggesting that these procedures did not mediate the sympathetic-sensory interaction. However, injection of nerve growth factor (NGF) systemically or into the anterior chamber increased iris peptide, reproducing the effects of ganglionectomy. Conversely, injection of antiserum to NGF (anti-NGF) into the anterior chamber decreased iris SP suggesting that endogenous trophic protein normally regulates sensory peptide. The effects of anti-NGF were transitory; iris peptide returned to normal after cessation of treatment. Consequently, anti-NGF administration apparently did not lead to sensory neuron destruction, but rather altered either the number of sensory fibers in the iris or the amount of peptide per fiber. Finally, injection of anti-NGF into the anterior chamber reversed the effects of sympathetic ganglionectomy, suggesting that NGF may mediate the sympathetic-sensory interaction. Our observations suggest that competition for target NGF may result in reciprocal regulation of the iris sympathetic and sensory innervation.
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