We determined the current-voltage (I-V) relations of the apical and basolateral barriers of frog skins by impaling the cells with an intracellular microelectrode and assuming that the current across the cellular pathway was equal to the amiloride-inhibitable current. We found that : (a) The responses in transepithelial current and intracellular potential to square pulses of transepithelial potential (VT) varied markedly with time . (b) As a consequence of these transient responses, the basolateral I-V relation was markedly dependent on the time of sampling after the beginning of each pulse. The apical 1-V plot was much less sensitive to the time of sampling within the pulse. (c) The I-V data for the apical barrier approximated the I-V relations calculated from the Goldman constant field equation over a relatively wide range of membrane potentials (±100 mV). (d) A sudden reduction in apical bath [Na'] resulted in an increase in apical permeability and a shift in the apical barrier zero-current potential (Ea) toward less positive values . The shift in Ea was equivalent to a change of 45 mV for a 10-fold change in apical [Na'] . (e) The transient responses of the skin to square VT pulses were described by the sum of two exponentials with time constants of 114 and 1,563 ms, which are compatible with the time constants that would be produced by an RC circuit with capacitances of 65 and 1,718 AF . The larger capacitance is too large to identify it comfortably with a true dielectric membrane capacitance .
We measured the effects of insulin on the current-voltage (I-V) relations of frog skins impaled with an intracellular microelectrode. The current across the cell membranes was assumed to be equal to the amiloride-inhibitable current. Insulin increased short-circuit current (Isc) approximately 40% from the control value. The increase in Isc was associated with a depolarization of the cell membrane. In addition there was an increase in the value of the parameters that describe the ease of movement of Na+ across the apical membrane, namely, slope conductance (ga), chord conductance (Ga), and permeability (PNa). The values of these parameters show remarkable linear correlations with membrane current both before and after stimulation. Intracellular Na+ activity (acNa) was determined from the I-V relations of the apical membrane. Insulin did not significantly modify acNa. Insulin also increased the value of the basolateral membrane conductance, however, the relationship between this parameter and current was complex. These experiments show that the stimulatory effect of insulin on Isc is associated with an increase in the conductance of both the apical and basolateral membranes.
Sumimtary. The initiation of perithecia in the homothallic ascomycete Sordaria fimiiicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 uM 5-fluorouracil, 2) 10 to 100 jum 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 uM cyanide or azide, 4) 5 % (w/,v) casein 'hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 um concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequencv of leading hyphae, but not their linear growth rates. Consequently, the branch (lensities wvere diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work.Purine and pyrimidine analogues have been found to intervene at crucial developmental stages in both plants and animals. One of the most direct of these effects on p'aants was the suppression by some of the RNA base analogues of the normal sheet-like (cordate) growth of fern gametophytes while permitting their continued filamentous growth (3,20,21,22,29,30). None of the strictly DNA base analogues had such effect (29). Actinomycin D was effective (30), and so were 2 amino acid analogues, 5-methyltryptophane and ethionine (20-22), but not any of the common respiratory and glycolytic inhibitors (20). The leaf morphology of a water fern could be affected by application of 5-fluorouracil and 2-thiouracil, in the sense that normal lobation was suppressed and the characteristic sunken stomata of the land-form were favored (39). Similarly, 2-thiouracil affected leaf morphogenesis in flowering plants (17), and 5-fluorouracil has been demonstrated as an inhibitor of flower induction (8).
We have studied the effects of the Ca2+ ionophores A23187 and ionomycin on ion transport across amphibian skin and urinary bladder. Both A23187 and ionomycin stimulated transepithelial Na+ transport across the skin. Ionomycin also markedly increased the conductance of an amiloride-insensitive pathway. Both ionophores markedly stimulated the release of prostaglandin E2 (PGE2) into the solution bathing the serosal surface of the skin. Addition of indomethacin to the serosal bathing solution of the skin blocked both the stimulation of short-circuit current (Isc) and the release of prostaglandin caused by the ionophores. Acetylsalicylic acid also blocked the ionomycin-induced stimulation of Isc. These results suggest that the stimulation of Na+ transport caused by Ca2+ ionophores is mediated by the release of a product of the cyclooxygenase pathway, very likely PGE2. Ca2+ ionophores also stimulated the release of PGE2 in urinary bladders; however, they generally depressed Isc. Since the effect on Isc caused by the addition of exogenous PGE2 was different in urinary bladders than in skins, we suggest that at least part of the difference in the action of ionophores is due to the difference in the sensitivity of these epithelia to PGE2. Our results suggest that the heterogeneity of effects that Ca2+ ionophores cause in the physiological parameters of tight epithelia are not always the direct result of increased cytoplasmic Ca2+ but that they may be mediated by other tissue responses triggered by the addition of the ionophores.
Application of transepithelial square voltage pulses to the frog skin leads to responses in the transepithelial current and intracellular potential which include transient components. Determinations at 600 ms allow for meaningful estimates of basolateral membrane responses to transport modifiers. Oxytocin produced a large and sustained increase in the amiloride-inhibitable short circuit current (Im) which was accompanied by a large increase of both apical and basolateral membrane conductance (ga and gb, respectively). While Im and ga increased nearly simultaneously, gb started to increase several minutes after the increase in the two other parameters. Insulin also increased Im, ga and gb. As with oxytocin, the increases in Im and ga often preceded the changes in gb. Ouabain reduced Im and ga. The effects on gb were more complex, since sometimes the inhibition of Im was first accompanied by an increase followed by a decrease while in other instances only minor changes in conductance could be observed. The currently available information regarding the control of cytoplasmic [Ca2+] and the effects of Ca2+ on cell membrane properties are used to construct a model in which changes in cytoplasmic [Ca2+] account for the observed behavior of the basolateral membrane.
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