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Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

Second messengers have been implicated in the control of communication between cells of various tissues and of a number of cell lines. To assess whether protein kinase C (PKC) is involved in the regulation of gap junctions between primary differentiated cells, we studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on PKC translocation and junctional conductance of rat pancreatic exocrine cells. Our results show that although TPA induced the translocation of PKC from a "cytosolic" to a "microsomal" fraction within minutes, it failed to block the junctional conductance of acinar cell pairs up to 30 min after application. By contrast, analogous experiments on a liver-derived cell line (WB cells) showed that TPA-induced PKC translocation was paralleled by a marked and irreversible inhibition of intercellular coupling. These results indicate that, in contrast to the effects on transformed or dedifferentiated permanent cell lines, PKC is not involved in gating gap junctional channels between primary differentiated secretory cells of the pancreas.

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to “oval” cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 +/- 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca2+ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca2+ in response to Ins(1,4,5)P3 was more variable than for Ca2+ injections. These studies demonstrate that Ca2+, and perhaps Ins(1,4,5)P3 as well, can diffuse between smooth muscle cells through gap junction channels.

Properties of gap junction channels present between specific cell types constituting the hepatopancreas of the crayfish (Procambarus clarkii) were investigated using the dual whole cell voltage clamp technique. Four different cell types (E, Fe, R and B) were identified on the basis of their morphology using light and electron microscopy. Although junctional conductance (Gj) could not be measured in B-B cell pairs, junctional currents were resolved in both homologous and heterologous combinations of the other cell types. E-E, Fe-Fe, and E-Fe cell pairs exhibited strong dependence on inside-out voltage (Vi-o), such that Gj increased with hyperpolarization to a maximal plateau reached at approximately -40 mV and was abolished with depolarization > 10 mV. The Gj-Vi-o relationship can be described by a squared Boltzmann relation with A = 0.101 and V0 = 0.135 mV. In this system, sensitivity of the junctions to transjunctional voltage was slight, if present at all. Gating mechanisms were complex, as evidence by the presence of multiple unitary channel conductance states. Single channel recordings showed that large unitary conductances (> 200 pS) were generally found between E-E, Fe-Fe, and E-Fe cell pairs, whereas smaller channel sizes (< 90 pS) were detected between R-R cell pairs.

Ventricular myocytes were isolated from adult rat hearts using the technique of Wittenberg and Robinson (Cell Tissue Res. 216: 231-251, 1981). These cells exhibited morphology, input resistance, time constant, and excitability expected for cells in intact cardiac tissue. Pairs of these cells were electronically coupled, and junctional conductance was unaffected by transjunctional potential or hyperpolarization of both cells. Brief exposure of cell pairs to medium equilibrated with 100% CO2 or containing 0.1 mM octanol quickly and reversibly decreased junctional conductance. We conclude that gap junctions between pairs of ventricular myocytes possess physiological properties like those of junctions in many other tissues. This preparation will be useful in evaluating drug action on junctional communication in heart.

The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We have investigated the involvement of protein residues in regulating the resistance of crayfish junctional channels by determining effects of group-specific protein reagents. When applied to well-coupled axons, the sulfhydryl group reagents N-ethylmaleimide (NEM) and diamide uncoupled the segments; junctional resistance (Rj) was increased without changing membrane resistance or axoplasmic pH (pHi). The uncoupling produced by NEM could be reversed by alkalinization of the cytoplasm (addition of ammonium chloride to the external medium). Another sulfhydryl reagent (p-chloromercuribenzoic acid) increased Rj to a lesser extent. A disulfide reagent and three amino and three carboxyl group reagents had no effect on the Rj of these axons. The effect of group-specific reagents on partially uncoupled axons was tested by applying the drugs to axons previously exposed to weak acids. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline recoupled partially uncoupled axons by decreasing Rj and prevented subsequent uncoupling of the junction by low pHi. Another carboxyl group reagent, as well as sulfhydryl and amino group reagents, either had no effect or uncoupled the axons further by increasing Rj. These experimental results suggest that amino acid residues, possibly containing carboxyl and sulfhydryl groups, control the opening and closing of junctional channels and may thus be associated with the channels' active sites.

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

Cardiac gap junction channels play the important roles of synchronizing pacemaker cells and allowing impulse propagation along the conduction system and throughout the ventricular myocardium. These channels, which support current flow in both longitudinal and tranverse directions, are permeable to anions and cations with radii less than approximately 0.5 nm and in rat heart have unitary conductances on the order of 50 pS. This unitary conductance is consistent with channel geometry described by a right cylindrical pore with diameter large enough for the brilliantly fluorescent dye molecule lucifer yellow to pass between cells. These channels, like others in biological systems, are opened and closed by various treatments, a process termed gating. Cytoplasmic acidification reduces junctional conductance (gj), an effect that is apparently potentiated by elevated myoplasmic Ca ions. Reduced gj also occurs in response to a variety of lipophilic molecules, including halothane, heptanol, and unsaturated fatty acids; the mechanism of action may involve disruption of the protein-lipid microenvironment of the gap junction channel. Arachidonic acid uncouples, and this effect is partially, but incompletely, blocked by an inhibitor of the lipoxygenase metabolic pathways. Cyclooxygenase inhibitors have no protective effects. Certain cyclic nucleotides can rapidly increase gj [adenosine 3',5'-cyclic monophosphate (cAMP)] or slightly decrease it [guanosine 3',5'-cyclic monophosphate (cGMP)], and agents that use these cyclic nucleotides as second messengers (isoproterenol and perhaps carbachol, respectively) produce consistent effects. Agents expected to cause protein kinase C activation (tumor-promoting phorbol esters and diacylglycerol) increase gj rapidly. The gap junction protein from rat heart has been cloned and sequenced. From the primary sequence for the protein, plausible sites of action within the putative cytoplasmic domains are proposed for each of these treatments. In response to gating stimuli that close the channel (halothane, CO2, heptanol), unitary channel conductance is unchanged, suggesting that these agents act by reducing open time probability. Together, these properties constitute the beginnings of our endeavor to define pharmacological agents that are potentially useful in therapeutic manipulation of synchronous discharge, conduction velocity, and isochronous wavefront propagation in cardiac tissue.

The sensitivity of gap junctions connecting crayfish lateral axons to uncoupling by axoplasmic acidification was studied after altering the hormonal balance of animals by 1) ablation of eyestalks or sinus glands or 2) inducing long-lasting defensive posturing behavior (stress). Internal pH (pHi) was measured with microelectrodes, and junctional resistance (Rj) was calculated from input and transfer resistances. In isolated nerve cords from intact animals, the maximal Rj (Rjmax) reached after acidification varied diurnally (Rjmax approximately 10 and 0.6 M omega at 0900 and 1800 h, respectively). Basal Rj (20-30 k omega) did not change during the 24-h period. Organ ablation (eyestalks or sinus glands) or stress rendered gap junctions less sensitive to uncoupling by low pHi within 1 h or 2 days; recovery toward control values had different time courses. The reduced pH sensitivity of crayfish junctions seen after eyestalk ablation is attributable to stress in its early phase (lasting 1-2 days) and to ablation of the endocrine organs in its late phase (2-7 days). No striking structural differences accompanied these changes, indicating that the altered properties are not due to major changes in gap junction expression.

We previously showed that corpus cavernosum smooth muscle cells are connected via gap junctions in situ and in culture and that a major protein component of these gap junctions is connexin43. To characterize the physiological properties of the gap junctions between corpus cavernosum smooth muscle cells, we now demonstrate that the cells are dye and electrically coupled and describe some of the gating properties of these gap junctional channels at macroscopic and single-channel levels. Junctional conductance (gj) between corporal smooth muscle cells was moderately voltage sensitive; was reduced rapidly, reversibly, and completely by halothane; and was increased by treatment with a tumor-promoting phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] and decreased by isoproterenol. Histograms of unitary junctional currents revealed multiple conductance peaks with events of approximately 90 pS being the most abundant. TPA and phenylephrine produced large increases in relative frequencies of the smaller events, whereas isoproterenol and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) slightly increased the relative frequencies of the larger events. None of the tested drugs substantially affected the steady-state voltage dependence of gj. These second messenger systems also affected expression of connexin43 by corpus cavernosum smooth muscle cells, as judged by immunoblots. At 6 h of treatment, both TPA- and 8-BrcAMP-treated cultures showed markedly elevated levels of connexin43, whereas at 24 h, the level of connexin43 in TPA-treated cultures had returned to control levels. Together, these data indicate that second messenger molecules involved in penile erection produce changes in gap junction expression and function; it is plausible that these changes could be physiologically relevant in altering and propagating changes in vasomotor tone.

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.

Pages C1–C11: J. A. Sáez, W. A. Gregory, T. Watanabe, R. Dermietzel, E. L. Hertzberg, L. Reid, M. V. L. Bennett, and D. C. Spray. “cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.” Page C8: Fig. 8B should appear as follows:

Pages C1–C11: J. A. Sáez, W. A. Gregory, T. Watanabe, R. Dermietzel, E. L. Hertzberg, L. Reid, M. V. L. Bennett, and D. C. Spray. “cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.” Page C8: Fig. 8B should appear as follows: