Effect of calcium on development of amiloride-blockable Na+ transport in axolotl in vitro.

The axolotl, Ambystoma mexicanum, which has no specific calcium-containing sieve layer in the dermis, provides useful material for the study of the effect of Ca2+ on the development of amiloride-blockable active Na+ transport across the skin of amphibians. We raised axolotls in thyroid hormone or aldosterone or cultured the skin with corticoid plus one of several Ca2+ concentrations and found that 1) although the short-circuit current (SCC) was increased by both aldosterone and 3,3',5-triiodo-L-thyronine in vivo, only corticoid was necessary for such an increase in vitro; 2) the development of the SCC in vitro was both corticoid and Ca2+ dependent, because the SCC was well developed with over 100 microM Ca2+ but not with under 10 microM Ca2+ in the presence of corticoid, nor even with 300 microM Ca2+ without corticoid; and 3) Ca2+, but not corticoid, was necessary for the formation of cell-to-cell junctions, because the resistance of the skin was well developed with 300 microM Ca2+ without corticoid.

In vivo treatment of bullfrog tadpoles with aldosterone potentiates ACh-receptor channels, but not amiloride-blockable Na+ channels in the skin.

Amiloride-blockable Na(+) channels participate in active Na(+) transport across adult, but not larval, bullfrog skin. Their development is induced in vitro by culturing the tadpole skin with aldosterone. When tadpoles were raised in aldosterone (5 x 10(-7) M) for 2 weeks, however, neither development of such channels nor localization of antigen A, a marker of adult-type epidermis, was seen, the skin still being of the larval type. In contrast, aldosterone treatment did potentiate (by a factor of two) the activity of the acetylcholine receptor (ACh-receptor) channel, a functional marker of larval-type skin. The short-circuit current (SCC) across the skin, far from being inhibited by amiloride, was stimulated by both amiloride and ACh. The nystatin-stimulated SCC was about twice its control amplitude, suggesting that the aldosterone treatment also potentiated the activity of the Na(+) pump.

Prolactin antagonizes the corticoid-promoted development of adult-type epidermis in cultured larval bullfrog skin.

EDTA-treated larval bullfrog skin, in which apical and skein cells had been removed and only basal cells remained, was cultured in one of four media. These contained either aldosterone (Aldo) or a mixture of Aldo, hydrocortisone (HC) and corticosterone (C), each either supplemented with prolactin (PRL) or lacking PRL. Skin cultured with Aldo alone or with the corticoid mixture (Aldo + HC + C) developed an adult-type epidermis: (i) both types of skin reacted to human blood group antigen A, a marker for the adult-type epidermis of bullfrog skin; (ii) amiloride decreased the short-circuit current Isc in these skin preparations, but acetylcholine (ACh) had no effect on the Isc. It seemed to make little difference to the results whether the skin was cultured with Aldo or with the corticoid mixture. PRL antagonized the action of Aldo and induced the development of a larval-type epidermis in both skin preparations: (i) the skin preparations did not react to human blood group antigen A; (ii) acetylcholine and amiloride each stimulated Isc in these preparations. Since ACh and amiloride each stimulated the Isc in skin with apical cells, ACh/amiloride-stimulated channels may be located on these cells.

Prolactin enables normal development of ACh-stimulated current in cultured larval bullfrog skin.

The response to acetylcholine (ACh) can be used as a marker for larval-type bullfrog skin because apically applied ACh induces an increase in short-circuit current (SCC) in larval-type but not adult-type skin. EDTA-treated larval skin, which contains only basal cells and does not respond to ACh, was used as the starting material for our culture. ACh, carbamylcholine, and choline stimulated SCC in skin that had been cultured with aldosterone (5 x 10(-7) M) supplemented with prolactin (PRL; 2 micrograms/ml). Atropine and d-tubocurarine each inhibited the ACh-induced stimulation of SCC in skin so cultured. Eserine, an inhibitor of acetylcholinesterase, also inhibited the ACh response. Amiloride stimulated SCC itself, but it reduced the ACh response. All of these results are quite similar to those seen in intact larval skin, suggesting that a larval-skin had differentiated from the basal cells used as the starting point for our culture. This is the first physiological report that PRL induces differentiation in vitro into a true larval-type bullfrog skin.

Prolactin inhibits corticoid-induced differentiation of active Na+ transport across cultured frog tadpole skin.

Active Na+ transport differentiates in larval bullfrog skin cultured with corticoids. After 2 wk in culture, the epidermis became positive against human blood group antigen A, the marker for the adult-type cells of the epidermis, but was negative to the antibody against the acetylcholine receptor, the marker for the larval-type epidermis. Amiloride (10(-5) M) did not inhibit the differentiation of active Na+ transport. On the other hand, in skin cultured with prolactin (2 micrograms/ml), the epidermis remained negative against antigen A and positive against acetylcholine receptor, and the differentiation of active Na+ transport was inhibited. Thyroid hormone did not antagonize the inhibitory action of prolactin on this transport differentiation. Prolactin affected the basal cells of the larval epidermis and inhibited development of corticoid-induced adult features in the epidermis.

Corticoid-induced differentiation of amiloride-blockable active Na+ transport across larval bullfrog skin in vitro.

The hormone-induced differentiation of an active Na+ transport across larval bullfrog skin during metamorphosis was investigated in vitro and in vivo. In in vitro experiments, EDTA-treated larval dorsal skin from which apical cells were removed was used. Even in the absence of thyroid hormone, corticoids induced the differentiation. Although aldosterone was the most potent hormone, hydrocortisone or corticosterone was also effective. Prolactin inhibited the corticoid-induced differentiation. The differentiation of the transport system coincided almost exactly with the appearance of adult features of the epidermis, namely, the epidermis at 7 days carried the human blood group antigen A, a specific molecular marker of adult-type bullfrog epidermis. The transport system appeared to develop in cells that had been newly generated from basal cells. On the contrary, in in vivo experiments, the effect of amiloride on the short-circuit current of the skin of tadpoles raised in the presence of aldosterone was very small, suggesting that a mechanism exists to inhibit the ability of aldosterone to induce the differentiation of the transport system in vivo.

Increase in chloride permeability of snail neurons during high potassium-induced hyperpolarization.

High K+-induced hyperpolarization was recorded intracellularly from the snail neurons, Euhadra subnimbosa, and the ionic mechanism underlying this hyperpolarization was analyzed in comparison with the ACh-induced hyperpolarization of the same cell, the latter known to be Cl(-)-dependent. The membrane resistance always decreased during both hyperpolarizing responses to high K+ (24mM) and ACh (0.1 mM). Both hyperpolarizing responses to high K+ and ACh were reversed in Cl(-)-free Ringer to the depolarizing responses. Both hyperpolarizing responses to high K+ and ACh were markedly augmented immediately after returning to normal Cl- from Cl(-)-free Ringer perfusion. Increase in intracellular Cl(-)-concentration by a leak from KCl-electrode reversed both hyperpolarizing responses to high K+ and ACh. Reversal potential of high K+-response was always 10-20 mV more positive than that of ACh-response, when measured in normal Ringer perfusion. Intracellular Cl(-)-concentration of the cells which were hyperpolarized by high K+ was estimated to be one half of that of the cells which were depolarized by high K+. Above results indicated that the high K+-induced hyperpolarization is due to the permeability increase of the postsynaptic membrane toward Cl-, masking the depolarizing effect of high K+ on the same membrane.

Effects of various enzymes and chemical modification reagents on the Na+- and Cl- -dependent responses of the ganglion cells to acetylcholine.

Using the abdominal ganglion cells of Aplysia, we analyzed the effects of various enzymes and chemical modification reagents on the acetylcholine (ACh)-induced responses of the excitatory (Na+ -dependent) and inhibitory (Cl- -dependent) types. (1) Phospholipase A (2 mg/ml) caused no appreciable effects on either type of response. (2) Phospholipase C (2 mg/ml) markedly depressed both types of response. These suggested that the phosphoryl group of the phospholipid is an important site related to the binding of ACh, common to both types of ACh-receptors. (3) Carboxypeptidase A (10 mg/ml) caused no observable effects on either type of response. (4) Carboxypeptidase B (10 mg/ml) depressed the inhibitory type of response without affecting the excitatory one. (5) Pyridoxal-5'-phosphate (1 mM) also depressed the inhibitory response without affecting the excitatory one. These findings (4, 5) suggested that the Cl- -channel in the inhibitory ACh-receptor complex includes a C-terminal lysine which may play an active role in the movement of Cl- across the receptor membrane. (6) L-Leucine aminopeptidase (1 mg/ml) depressed the excitatory response without altering the inhibitory one. (7) p-Nitrothiophenol (1 mM)-also depressed the excitatory response without affecting the inhibitory one. These findings (6, 7) suggested the presence of a certain N-terminal amino acid near a glutamate or aspartate residue within a molecular moiety of Na+ -channel included in the excitatory ACh-receptor complex.

Suppressive effect of cadmium on high potassium-induced hyperpolarization in snail neurons.

Some molluscan ganglion cells were hyperpolarized by an excess of external K, contrary to expectations by the Nernst equation. The membrane resistance of the cells was markedly decreased with the hyperpolarization. This phenomenon was considered to represent a result of the summated IPSP's elicited by the presynaptic inhibitory fibers which were primarily depolarized by high K. We examined the effect of Cd in low pH saline on the membrane potential of snail ganglion cells in order to analyze this phenomenon further. Since Cd has been reported to block Ca channels, it might prevent neurotransmitter release from the presynaptic ending. In pH 8 control saline, the excess of K (22 mM) caused significant hyperpolarization and an evident decrease in membrane resistance, and these changes were not modified much after 1mM Cd addition. In pH 6.5 and 5 saline, the high K-induced hyperpolarization was markedly suppressed or even changed to depolarization by 1 mM Cd. The resistance change was also decreased in low pH Cd saline. Cadmium exerted a greater effect at rather higher concentrations. The results obtained suggest that the high K-induced hyperpolarization is due to the release of inhibitory neurotransmitter.

Hyperpolarization caused by external high potassium in snail neurons.

Two-thirds of the tested subesophageal ganglion cells of Japanese snail, Euhadra peliomphala, was hyperpolarized when they were perfused by high K-Ringer. This hyperpolarization was accompanied with a marked decrease in membrane resistance and was independent of cell types classified by ACh-induced response. Pentobarbital was found to convert high K-induced hyperpolarization into depolarization, or to augment K-depolarization. The high K-induced hyperpolarization was considered as the result of summated IPSP's elicited by the presynaptic inhibitory fibers which were primarily depolarized by high K. Pentobarbital may remove this synaptic inhibition and disclose the original K-depolarization underlying the cell. In Cl-free media, high K caused a marked depolarization instead of hyperpolarization, or augmented the depolarization observed in normal Ringer. This suggested that the subsynaptic membrane became permeable to Cl ions during the inhibitory presynaptic activity when high K was applied. ACh was found not to be responsible for this synaptic inhibition, because d-tubocurarine, which is known to block the Cl-dependent ACh response of the snail neurons, did not affect the high K-induced hyperpolarization. The possibility of indirect action of high K on the membrane potential through synaptically mediated inhibition was discussed as a cause of its hyperpolarizing effect.

The ionic permeability changes during acetylcholine-induced responses of Aplysia ganglion cells.

ACh-induced depolarization (D response) in D cells markedly decreases as the external Na(+) is reduced. However, when Na(+) is completely replaced with Mg(++), the D response remains unchanged. When Na(+) is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl(-) is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl(-)-free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na(+)-free or Cl(-)-free Ringer's solution. High [K(+)](0) often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K(+)](0) on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K(+) depolarization. In perfusates containing normal [Na(+)](0), the effects of Ca(++) and Mg(++) on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K(+)](0) and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl(-) permeability of the H membrane.