Birgit Rose

Permeability of a cell junction and the local cytoplasmic free ionized calcium concentration: A study with aequorin

SummaryA technique is devised to determine the spatial distribution of the free ionized cytoplasmic calcium concentration ([Ca2+]i) inside a cell:Chironomus salivary gland cells are loaded with aequorin, and the Ca2+-dependent light emission of the aequorin is scanned with an image-intensifier/television system. With this technique, the [Ca2+]i is determined simultaneously with junctional electrical coupling when Ca2+ is microinjected into the cells, or when the cells are exposed to metabolic inhibitors, Ca-transporting ionophores, or Ca-free medium. Ca microinjections elevating the [Ca2+]i the junctional locale produce depression of junctional membrane conductance. When the [Ca2+]i elevation is confined to the vicinity of one cell junction, the conductance of that junction alone is depressed; other junctions of the same cell are not affected. The depression sets in as the [Ca2+]i rises in the junctional locale, and reverses after the [Ca2+]i falls to baseline. When the [Ca2+]i elevation is diffuse throughout the cell, the conductances of all junctions of the cell are depressed. The Ca injections produce no detectable [Ca2+]i elevations in cells adjacent to the injected one; the Ca-induced change in junctional membrane permeability seems fast enough to block appreciable transjunctional flow of Ca2+. Control injections of Cl− or K+ do not affect junctional conductance. The Ca injections that elevate [Ca2+]i sufficiently to depress junctional conductance also produce under the usual conditions an increase in nonjunctional membrane conductance and, hence, depolarization. But injections that elevate [Ca2+]i at the junction while largely avoiding nonjunctional membrane cause depression of junctional conductance with little or no depolarization. Moreover, elevations of [Ca2+]i in cells clamped near resting potential produce the depression, too. On the other hand, complete depolarization in K medium does not produce the depression, unless accompanied by [Ca2+]i elevation. Thus, the depolarization is neither necessary nor sufficient for depression of junctional conductance. Treatment with cyanide, dinitrophenol and ionophores X537 A or A23187 produces diffuse elevation of [Ca2+]i associated with depression of nunctional conductance. Prolonged exposure to Ca-free medium leads to fluctuation in [Ca2+]i where rise and fall of [Ca2+]i correlate respectively with fall and rise in junctional conductance.

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.

SummaryIncorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A.

Abstract The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca 2+ , which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.

Growth factors modulate junctional cell-to-cell communication

SummaryThe epidermal growth factor (EGF) and the platelet-derived growth factor (PDGF) inhibit gap junctional communication in the mammalian cell lines NRK and BalbC 3T3: cell-to-cell transfer of a 400-dalton tracer molecule is reduced and junctional conductance is reduced. The inhibition of cell-to-cell transfer is reversible and dose dependent; half-maximal effects are obtained at 10−9 and 10−11m concentrations of EGF and PDGF, respectively. The response of junctional conductance is detectable within 2 min of EGF application and reaches a maximum within 10 min. It is among the earliest cellular responses to this growth factor and may be significant in the regulation of growth. The response is lacking in EGF receptor-deficient NIH 3T3 cells. The transforming factor β (TGFβ) enhances junctional communication in BalbC 3T3: cell-to-cell transfer is increased over a period of 8 hr. But in NRK cells, where it upregulates EGF receptors, TGFβ reduces junctional communication synergistically with EGF.

Cell-to-cell channels with two independently regulated gates in series: Analysis of junctional conductance modulation by membrane potential, calcium, and pH

SummaryWe study cell-to-cell channels, in cell pairs isolated fromChironomus salivary gland, by investigating the dependence of junctional conductance (gj) on membrane potentials (E1,E2), on Ca2+, and on H+, and we explore the interrelations among these dependencies; we use two separate voltage clamps to set the membrane potentials and to measuregj. We findgj to depend on membrane potentials whether or not a transjunctional potential is present. The pattern ofgj dependence on membrane potentials suggests that each channel has two closure mechanisms (gates) in series. These gates pertain, respectively, to the two cell faces of the junction. By treating the steady-stategj as the resultant of two simultaneous but independent voltage-sensitive open/closed equilibria, one within each population of gates (i. e., one on either face of the junction), we develop a model to account for the steady-stategj vs.E relationship. Elevation of cytosolic Ca2+ or H+ at fixedE lowersgj, but at moderate concentrations of these ions this effect can be completely reversed by clamping to more negativeE. Overall, the effect of a change inpCai or pHi takes the form of a parallel shift of thegj vs. E curve along theE axis, without change in slope. We conclude (1) that the patency of a cell-to-cell channel is determined by the states of patency of its two gates; (2) that the patency of the gates depends on membrane potentials (not on transjunctional potential), onpCai, and on pHi; (3) thatpCai and pHi determine the position of thegj vs. E curve on theE axis; and (4) that neither Ca2+ nor H+ at moderate concentrations alters the volrage sensitivity ofgj.

Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect.

SummaryWe tested the question whether junctional cell-to-cell communication is regulated by the diacylglycerol branch of the phosphoinositide transmembrane signal pathway. Cultured epithelial rat liver cells were treated with the synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol, while their junctional permeability was probed with the microinjected 443-dalton fluorescent tracer Lucifer Yellow. The treatment reduced junctional permeability (without affecting Lucifer permeability of nonjunctional cell membrane). The effect was dose dependent, with a threshold of about 25 μg diacylglycerol/ml in sparse cultures and about 50 μg/ml in confluent cultures. The reduction of junctional permeability began within 3 min of diacylglycerol application, peaked within 20 min, and reversed spontaneously within 90 min. The phorbol ester TPA mimicked the diacylglycerol effect, but the (spontaneous) reversal was slower. We propose that cell-to-cell communication is under dual physiological control: an upregulatory one, as exerted by the cyclic AMP signal route (Loewenstein, W.R., 1985,Biochem. Soc. Symp. London,50: 43–58), and a downregulatory one, by the diacylglycerol signal route.TMB-8 (54–70 μm)—a blocker of intracellular Ca2+ mobilization-impeded the diacylglycerol action on junctional permeability. It prevented the effect of low diacylglycerol doses completely and it markedly reduced the effect of high doses. (It also counteracted the effect of TPA.) Ca2+ thus emerges as a possible candidate for a role in the junctional downregulation by the diacylglycerol signal route. We tentatively advance two models. In one, leaning closely on the Calcium Hypothesis of cell-to-cell channel regulation (Loewenstein, W.R., 1966,Ann. N.Y. Acad. Sci.137:441–472), Ca2+ mediates the action of the route on the channel. In the other, Ca2+ acts farther removed from the channel, on protein kinase C.Calmidazolium (5–10 μm)—an inhibitor of calmodulin-activated proteins—did not prevent the diacylglycerol-induced reduction of junctional permeability. Nor did sodium orthovanadate (25 or 50 μm)—an inhibitor of tyrosyl phosphatase-prevent the reversal of diacylglycerol-induced (or TPA-induced) reduction of junctional permeability.

Permeability properties of cell-to-cell channels: Kinetics of fluorescent tracer diffusion through a cell junction

SummaryWe have analyzed the intracellular and cell-to-cell diffusion kinetics of fluorescent tracers in theChironomus salivary gland. We use this analysis to investigate whether membrane potential-induced changes in junctional permeability are accompanied by changes in cell-to-cell channel selectivity. Tracers of different size and fluorescence wavelength were coinjected into a cell, and the fluorescence was monitored in this cell and an adjacent one. Rate constants,kj, for cell-to-cell diffusion were derived by compartment model analysis, taking into account (i) cell-to-cell diffusion of the tracers; (ii) their loss from the cells; (iii) their binding (sequestration) to cytoplasmic components; and (iv) their relative mobility to cytoplasm, as determined separately on isolated cells. In cell pairs, we compared a tracerskj with the electrical cell-to-cell conductance,gj.At cell membrane resting potential, thekjs ranged 3.8–9.2×10−3 sec−1 for the small carboxyfluorescein (mol wt 376) to about 0.4×10−3 sec−1 for a large fluorescein-labeled sugar (mol wt 2327). Cell membrane depolarization reversibly reducedgj andkj for a large and a small tracer, all in the same proportion. This suggests that membrane potential controls the number of open channels, rather than their effective pore diameter or selectivity. From the inverse relation between tracer mean diameter and relativekj we calculate an effective, permeation-limiting diameter of approximately 29 Å for the insect cell-to-cell channel. Intracellular diffusion was faster than cell-to-cell diffusion, and it was not solely dependent on tracer size. Rate constants for intracellular sequestration and loss through nonjunctional membrane were large enough to become rate-limiting for cell-to-cell tracer diffusion at low junctional permeabilities.

INHIBITION OF GLYCOSYLATION INDUCES FORMATION OF OPEN CONNEXIN-43 CELL-TO-CELL CHANNELS AND PHOSPHORYLATION AND TRITON X-100 INSOLUBILITY OF CONNEXIN- 43

We transfected the cDNA for the cell-to-cell channel protein connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express little Cx43 and lack gap junctional communication (open cell-to-cell channels). We found that cells overexpressing Cx43 nonetheless lacked open cell-to-cell channels, but that inhibition of glycosylation by tunicamycin induced open channels in these cells. Tunicamycin also induced biochemical changes in Cx43 protein; the level increased, and a considerable fraction became phosphorylated and Triton X-100 insoluble, in contrast to untreated cells where Cx43 was non-phosphorylated and Triton X-100 soluble. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.

Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

SummaryThe viralsrc gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36–72 μm) raises junctional permeability when applied during activity ofsrc protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity ofsrc protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of thesrc protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 μm) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca2+ and protein kinase C.

Permeability of the cell-to-cell membrane channel and its regulation in an insect cell junction

SummaryCells of organs and tissues commonly communicate directly with one another via permeable membrane junctions. Cell-to-cell channels, spanning the width of both membranes of a junction, are thought to provide the pathways between the cytoplasms of adjacent cells for the immediate exchange of ions and small molecules. We study these cell-to-cell channels in a cell model system, the salivary gland ofChironomus. Using intracellularly injected fluorescent labelled peptides and oligosaccharides of various molecular dimensions as channel permeability probes we find the channels to have a bore of about 2 nm. The channel permeability can be modulated and, in the extreme, the channels can be closed under various experimental conditions. With the aid of the Ca2+-sensitive photoprotein aequorin as monitor of cytoplasmic free Ca2+ concentration, we show that a determining factor in this modulation of channel permeability is the cytoplasmic free Ca2+ concentration. Moreover, results obtained by injection of different-sized and different-labelled channel permeability probes together with Ca2+ indicate that closure of the individual channels may occur in more than one step, i.e., by a graded reduction of channel bore.