Biomedical subjects
Publications and source records attributed to W R Loewenstein.
Cells of most organized tissues are interconnected at their junctions by aqueous membrane channels, shown in one instance to be at least 14--16 A in diameter. During junction formation, nascent channels are detected as quantal steps in cell-to-cell conductance, growing progressively in number. The channels detected are stable. Elevation of cytoplasmic free Ca2+ concentration can cause graded closure of channels.
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The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of "gap junction"; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.
The permeability of the cell-to-cell membrane channels in salivary gland cell junction (Chironomus thummi) was probed with fluorescent-labeled amino acids and synthetic or natural peptides. Molecules up to 1200 daltons pass through the channels with velocities depending on molecular size. Molecules of 1900 daltons or greater do not pass. This passage failure seems to reflect the normal size limit for junctional channel permeation; the channels continue to be permeated by the molecules up to 1200 daltons when these are mixed with the nonpermeant molecules. From this size limit a channel diameter of 10 to 14 angstroms is estimated.
A cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5 per cent of the contacts (as against 100 per cent normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels. Its possible etiological role in defective growth regulation is discussed.
Divalent cations are microinjected into Chironomus salivary gland cells while the cell-to-cell passage of fluorescein (330 dalton) and electrical coupling are monitored. Injections of Ca and Mg that substantially depolarize the cells produce block or marked slowing fluorescein passage, accompanied by electrical uncoupling. Injections of Ca, Mg or Sr that cause little depolarization, and presumably smaller elevation of divalent cation concentration in the cytoplasm, produce block or marked slowing of fluorescein passage with little or no detectable electrical uncoupling. This partial uncoupling may reflect total closure of a fraction of the channels in junctional membrane or partial closure of all channels.
A 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 hte 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 in 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 X537A or A23187 produces diffuse elevation of [Ca2+]i associated with depression of junctional 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.
The distribution of Ca2+ in the cytoplasm following a local rise in Ca2+ concentration is visualized by means of aequorin luminescence and a television system with an image intensifier. Diffusion of Ca2+ through the cytosol is so constrained that a rise in cytoplasmic Ca2+ concentration produced by local Ca2+ entry through cell membrane or by local Ca2+ injection is confined to the immediate vicinity of these sites. The diffusion constraints are lifted by treatment with cyanide or ruthenium red. Thus, energized calcium sequestering, probably by mitochondria, is the dominant factor in the constraints. In cell regions where the sequestering machinery is sufficiently dense, different Ca2+ message functions inside a cell may be effectively segregated, permitting private-line intracellular communication.
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Human Lesch-Nyhan cells, which are coupling and have gap junctions, were fused with mouse cl-lD cells, which are noncoupling and lack gap junctions. The resulting hybrid cells were coupling and had gap junctions while they contained the nearly complete complement of parent chromosomes. As the hybrid cells lost human chromosomes, clones appeared among the segregants, which had reverted to the noncoupling and junction-deficient trait of the mouse parent cell. The human cell appears to contribute a genetic factor to the hybrids that corrects the junctional deficiency of the mouse cell.
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