Werner R. Loewenstein

Permeability of membrane junctions.

The general and familiar idea of the cell membrane is that of a barrier to diffusion encompassing the entire cell surface. This idea derives largely from work with a few cell types: nerve, striated muscle, blood, and gamete cells. These are, however, rather peculiar cells. They are generally not in direct contact with each other but are separated by wide fluid-filled spaces. I shall deal here with the question of what happens to the diffusion barrier when two cell membranes are brought close together. This states the question for the general case of cell association; most tissues, by far, are built of cells in close surface contact. I chose epithelial cells for a study of this question. These cells have several advantages. Their areas of membrane contact are large, and their size permits direct conductance measurements. The giant salivary gland cells of Drosophila and Chironornus were particularly suitable in this respect (FIGURE 1 ) .

Growth inhibition of transformed cells correlates with their junctional communication with normal cells.

The growth of various chemically and virally transformed cell types in culture is inhibited when they are in contact with normal cell types. We show that this growth inhibition is contingent on the presence of junctional communication between the normal and transformed cells (heterologous communication), as probed with a 443 dalton microinjected fluorescent tracer. In cell combinations where heterologous communication is weak or absent there is no detectable growth inhibition; the inhibition appears when communication is induced by cyclic AMP-dependent phosphorylation, and only then. In cell combinations where heterologous communication is spontaneously strong, the growth inhibition is present, but it is abolished when the communication is blocked by retinol or retinoic acid. The cell-to-cell membrane channels of gap junctions are the likely conduits of the signals for this growth control.

The cell-cell channel in the control of growth

Several lines of evidence indicate that the cell-cell channels in gap junction are conduits for growth-regulating signals. Experimental upregulation of the channels by retinoids causes inhibition of cellular growth and, conversely, their downregulation by oncogenes, e.g. activated src, stimulates growth. In either direction, the extent of growth correlates tightly with the degree of communication. Cogent evidence of the channels function in growth regulation is now on hand: incorporation of a channel-protein gene into the genome of a transformed communication-deficient cell line normalizes communication and growth. The current data conform to a model of growth control with discrete regulatory centers.

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.

Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cyclic nucleotide or phosphodiesterase inhibitor

SummaryMammalian cells in culture were exposed to cyclic AMP, dibutyrul cyclic AMP, the phosphodiesterase inhibitor caffeine, or a combination of the last two, while junctional molecular transfer was probed with the series of microinjected, fluorescentlabelled linear molecules Glu, Glu-Glu, Glu-Glu-Glu, and Leu-Leu-Leu-Glu-Glu. The junctional permeability for these molecules increased with each of the agents, most markedly with the dibutyryl cyclic AMP-caffeine combination, as the intracellular cyclic nucleotide concentration rose. The junctional permeability effect developed over several hours. When probed with molecules close to the limit of cell-to-cell channel permeation (the most sensitive setting), the effect was detectable both, as an increase in the (relative) junctional transit rate and as an increase in the number of transferring cell interfaces in the test populations. The number of transferring cell interfaces reached a maximum by 4 hr, when the junctional transit rate, hence the junctional permeability, was still rising. Nonjunctional membrane permeability for the probe molecules, as determined by intracellular fluorescence loss, was not significantly changed (nor was there significant nonjunctional cell-to-cell transfer of molecules before or after the treatments). The rise in junctional permeability was associated with an increase in the number of gap junctional membrane particles, as determined by freeze-fracture electron microscopy: the average size of the particle clusters increased, and the frequency of the clusters increased, particularly that of the smaller (and presumably newer) clusters. This effect was blocked by treatments with the protein synthesis inhibitors cycloheximide or puromycin. These agents caused particle diminution (diminution of cluster frequency but not of average cluster size), with or without cyclic nucleotide. The junctional effects may represent a cyclic AMP-promoted proliferation of cell-to-cell channels. Some physiological implications, in particular, implications for hormone-regulated tissues, are discussed.

Intercellular Communication: Renal, Urinary Bladder, Sensory, and Salivary Gland Cells.

In four epithelial cell systems (salivary gland, renal, urinary bladder, and sensory cells) cells are interconnected as far as much of their ion content is concerned. In the salivary gland and renal epithelia, all cells of the epithelium are interconnected; and communication between a given cell and any of its nearest neighbors is equally good. In the bladder and sensory epithelia, communication appears to be more restricted, manifesting itself in chains of connected cells in the former, and in small groups of connected cells in the latter. The permeability of the cell membrane at the junction between connected cells is several orders of magnitude greater than it is at the cell surface bordering the exterior of the cells. Each connected cell ensemble functions as a system with a fairly continuous cytoplasmic core bounded by a diffusion barrier which is continuous along the entire outer surface of the system. As a result, ions move rather freely from cell to cell, but not from cell interior to exterior. Intercellular communication in at least three epithelia is associated with the presence of certain close-junctional membrane complexes.

Junctional membrane permeability : Effects of divalent cations.

SummaryJunctional membranes ofChironomus salivary gland cells were exposed to test media of varying divalent cation concentration through a hole (estimated diameter ∼10 μ) in a cells nonjunctional surface membrane. Junctional conductance is markedly depressed by Ca++, Mg++, Sr++, Ba++ and Mn++. The order of potency is Ca++>Mg++>Sr++>Ba++; the minimal effective concentration for Ca is 4 to 8×10−5m. Tests with Ca++ show that, at least, this ion also depresses junctional permeability to fluorescein (mol. wt. 330). The permeability depression is confined to the junctional membranes to which (exogenous) Ca++ has direct access via the hole. The permeability change produced by Ca++ is apparently fast enough to limit transjunctional flux of this ion. The depression is reversed by repolarization of the nonjunctional membrane with inward current when the junctional membrane is exposed to divalent cation-free medium, but not when it is exposed to medium containing 10−3m Ca.Perforation of the nonjunctional membrane in divalent cation-free medium leads to transient depression of junctional permeability when the membrane hole is large enough to cause nearly complete cell depolarization. This depression can be prevented by clamping the membrane potential with inward current. Smaller holes (estimated diameter ∼2 μ) seal in the presence of divalent cations; the ion diffusion barrier is restored within 14 to 30 min of divalent cation application.

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.

SEPARATION OF TRANSDUCER AND IMPULSE-GENERATING PROCESSES IN SENSORY RECEPTORS.

New evidence is presented that spike and transducer processes in sensory receptors are independent events; impulse activity in tile crustacean stretch receptor neuron and the mammalian pacinian corpuscle was selectively blocked by a compound (tetrodotoxin) without affecting any of the parameters of the generator potential.

Intercellular communication and the control of growth: X. Alteration of junctional permeability by thesrc gene. A study with temperature-sensitive mutant Rous sarcoma virus

SummaryTo study changes of junctional membrane permeability associated with transformation, the junctions and the nonjunctional membranes of quail embryo-, chick embryo- and mouse-3T3 cell cultures, infected with temperature-sensitive mutant Rous sarcoma virus, were probed with fluorescent-labelled glutamate. Junctional permeability fell in the transformed state. In the quail cells, the fall was detectable within 25 min of shifting the temperature down to the level (permissive) at which tyrosine-phosphorylation by the viralsrc gene product is expressed. This reduction of junctional permeability is one of the earliest manifestations of viral transformation. Normal permeability was restored within 30 min of raising the temperature to the nonpermissive level, a reversibility that could be displayed several times during the span of a cell generation. The reversal seems to reflect a reopening of cell-to-cell channels rather than a synthesis of new ones; it is not blocked by protein-synthesis inhibition. Treatments with cyclic AMP and phosphodiesterase inhibitor or with forskolin, which stimulate serine and threonine phosphorylation—the type of phosphorylation on which normal junctional permeability depends (Wiener & Loewenstein, 1983,Nature 305∶433)—did not abolish, in general, the junctional effect of the virus;src tyrosine-phosphorylation apparently overrides the junctional upregulation mediated by cyclic AMP. Nonjunctional membrane permeability was not sensibly affected by the virus. It was affected, however, by temperature: lowering the temperature from the nonpermissive to the permissive level caused the nonjunctional permeability to fall, andvice versa. This change was unrelated to transformation. Its secondary effect on junctional transfer is in the opposite direction to that produced by the temperature-activated viral transformation.