Active transport of NA across the cell membrane which depends on the supply of metabolic energy is one of most intriguing and unique living phenomena. Currently it is generally agreed that adenosine triphosphate(ATP) is the prime energizer of the active transport of Na(Caldwell, 1956; Caldwell et al., 1960; Caldwell and keynes, 1957; Hoffman, 1960; Skou, 1965). There are also a number of reports on the substrate specifity of the various organs and tissues as well (Alonso et al., 1967; Ballared et al., 1960; Barac-Nieto and Cohen, 1968; Cohen, 1960;Gold and Spitzer,
1964; Goodale et al., 1959; Harris, 1941; Maizels, 1951; Murphy, 1963; Nieth and Schollmeyer, 1966; Sokoloff, 1960; Willebrands, 1964). These reports thus suggest a possibility that preferred or specific substrates may be utilized with a special relation o the active transport of Na.
Frog skin, perhaps best studied and known as the prototype system for active Na transport, avidly metabolizes pyruvate, citrate, succinate and oxaloacetate (Skjelkvale et al., 1960) while in the toad bladder, one functional analogy to frog skin, both glucose and acetate are principally oxidized. However, there are only scattered reports and rudimentary knowledge of the role of various substrate in the active Na transport. As early in 1935, Huf reported that the reduction of the potential difference across frog skin by monobromoacetate could be prevented or reverved by pyruvate or lactate but not by glucose, whereas active Na transport in the toad bladder can be supported by glucose, lactate, β-hydroxybutyrate and most effectively by pyruvate (Maffly and Edelamn, 1963). No direct simple relationship, however, can be made between the amount of substrate oxidized and the degree of its contribution to the active Na transport. In the frog skin and toad bladder, acetate
is metabolized in greater amount than pyruvate but pyruvate is more effective in supporting active Na transport (Maffly and Edelman, 1963; Van Bruggen and Zerahn, 1960). Hence, the present study deals with the substrate specificity of frog skin as a source of energy for active Na transport.
Though the striking stimulatory effect of antidiuretic hormone on warer and Na transport across frog skin and toad bladder is well documented (Hay and LEaf, 1962; Koefoed-Johnsen and Ussing, 1953; Leaf, 1967; Leaf et al., 1958; Ussing and Zerahn, 1951), the mechanism(s) o action of the hormone, especially on the energy dependency of the action, is controversial. Rasmussen et al.(1960) as well as Sharp and leaf(1965) reported that hormone-induced increase in the permeability to water did not require energy. On the other hand, Hong(1957) and other(Bentley, 1958;
Handler et al., 1966; Hong et al., 1968; Mendoza et al., 1970) provided evidence that the hormone-evoked increase in water and Na transport is dependent upon matebolic energy. Even though ADH action might be an energy dependent one, the role of specific metabolic energy. Even though ADH action might be an energy dependent one, the role of specific metabolic pathways in the hormone-evoked increase in water and Na transport is still poorly defined. Handler et al.(1966) postulated that osmotic water permeability change in response to ADH might be supported by energy derived from glycolysis whereas the increase in Na transport might be from the tricarboxylic acid cycle. Interestingly enough, Hong et al.(1968) observed that the increase in Na transport in response to ADH was marked and sustained in glycogen-rich winter frog skin while it was transient or actually absent in glycogen-poor summer frog skin. In addition, even in glycogen-poor summer frog skin the stimulation action of ADH on Na transport became apparent in the pyruvate fortified frog Ringer's solution. However, this had no bearing on the problem as to whether pyruvate exerts its effect on ADH action as a source of energy for the hormonal action or not. Hence, the present studies were conducted to explore further the relationship between substrate utilization and active Na transport as well as ADH action.
Abdominal skin was removed from frogs, Rana temporalia, freshly captured during summer and winter. In the first series of experiments on the various substrates (10 mM/L), shortcircuit current (SCC), ax an estimate of net Na transport, was measured
in Lucite chambers by the method of Ussing and Zerahb(1951)in the presence or absence of substrate in frog Ringer's solution. In the secone series of experiment the effects of various substrates as well as AD (Parke, Davis and Co., 250mU/ml), either in the presence or absence of substrates, on oxygen consumption of the frog skin were measured by manometric technique (Umbreit et al., 1964). In the third series of experiments the rate of substrateoxidation in the presence or absence of ADH was determined by measuring C**14)^^2 produced from the added C**114 labelled substrates by the method outlined by Maffly and Edelamn(1963). Substrates studied were glucose, pyruvate, acetate, cirate, α-ketoglutarate and succinate for the first series of experiments and were glucose, glucoes-6-C**14, pyruvate, pyruvate-1-C**14 and acetate, acetate-1-C**14 for the remainder of the experiments.
Th result may be summarized as follow:
(1) Among substrates studied, pyruvate, glucose and acetate enhanced, in that order, the active Na transport in the summer but not in winter. TCA cycle intermediates were all without effect in both seasons.
(2)Oxygen consumption was also significantly increased by glucose (7), pyruvate (26) and acetate (17) in the summer while during the winter it was also significantly increased by pyruvate (24) and acetate (38%) but not by glucose.
(3) Oxygen consumption in response to ADH was significantly increased and amounted to 20% above the basal state (without difference either in the presence or absence of substrates) during the winter. During the summer, on the contrary, ADH had no effect in the absence of added substrates, but caused a significant increase averaging 29% in the presence of pyruvate.
(4) The rate of substrate oxidation, on the whole, was greater in winter than summer. By addition of ADH, the rate of oxidation of glucose, pyruvate and acetate was significantly increased in both seasons except pyruvate was no during th winter.
(5) Fractional oxygen consumption for added substrate oxidation was all significantly increased by ADH except for pyruvate during the winter.
On the basis of above results, it may be concluded that energy for active transport of sodium in the basal state is primarily derived from endogenous substrate, possibly skin glycogen, but when the endogenous substrate is depleted or insufficient, exogenous glucose, acetate and most effectively pyruvate may be
utilized for the supply of energy for the transport. On the other hand, for the increase in Na transport induced by ADH or for the action fo ADH itself, energy may be derived from endogenous substrate of from only pyruvate. The possible relation of the action of pyruvate on ADH action, or vice versa, was discussed.