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Acupuncture treatment has been used as physiotherapy to treat gastrointestinal disorders in traditional Oriental medicine. In the present study, we selected anesthetized animals as the subjects of this experiment in order to eliminate emotional factors, and investigated the responses to acupuncture stimulation at only a single point by changing animal conditions. 1) There was marked enhancement of gastric acid secretion by electroacupuncture stimulation of the hindlimb based on pH values of the perfusate. However, the enduring response after the momentary initial decrease suggested the existence of humoral factors. 2) The electroacupuncture-specific response was not observed in rats after sciatic nerve denervation. However, the pH values of the perfusate decreased in response to electroacupuncture stimulation of the hindlimb on the sciatic nerve intact side of the same rat. This suggests that the input pathway of acupuncture stimulation was a somatic nerve efferent pathway from the hindlimb. 3) No response to electroacupuncture stimulation was observed in the rats after vagotomy, but a clear enhancement response was seen after sympathectomy. These findings show that the major efferent path of the responses to acupuncture stimulation is the vagus nerve. The results of this experiment suggest that electroacupuncture stimulation of the hindlimb of anesthetized rats increases gastric acid secretion with somatic nerves as the afferent pathway and branches of the vagus nerve to the stomach as the efferent pathway.

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.

An impedance locus consisting of two superimposed semicircular arcs was revealed on the abdominal skin of the bullfrog. The decrease of skin resistance upon epidermal application of Cd (2 mM), which was related to Na active transport, proved to be due solely to the reduction in the size of the high frequency arc.

Effects of cadmium on the electrical properties of frog skin were investigated by using the microelectrode technique. The epidermal application of 2 mM Cd for 5 min increased both the short circuit current and the skin potential, and decreased the skin resistance (RM). Cadmium also decreased intracellular potential with reference to the epidermal solution (PDi) and the electrical resistance between the epidermal solution and the tip of a microelectrode (RM1) located in the stratum germinativum. deltaRM that is RM,Cd-RM, control seemed to approximately coincide with deltaRM1 which is RM1,Cd-RM1,control. Comparing with our previous results, i.e., Cd decreased RNa (the resistance to the active Na current) and increased Rsigma (the resistance to the passive ions) but did not altered ENa (the electromotive force of active Na current) in the later stage (15 min or later). Cd seemed to decrease the RNa component at the epidermal side of the skin in the early stage (less than 5 min).

Effects of Cd on the active transport of Na in SO4-Ringer's solution on the abdominal skin of a bullfrog (Rana catesbeiana) were investigated. The short circuit current (SCC), potential difference (PD), and Na flux were determined. The skin resistance (Rm), the electromotive force of active Na transport (Ena), and the resistance to the active Na current (Rna) were calculated from the measured data. The application of Cd to the epidermal side at 2 mM was more effective in increasing SCC than that to the dermal side at the same concentration. The time course of changes in SCC induced by Cd were classified into three types: (I) a simple increase in SCC during Cd application, (II) an early decrease followed by an increase in SCC, and (III) a decrease in SCC except for a small increase in the initial short period. Type I response was observed in the majority of cases (about two-thirds). Types II and III were observed in 7 and 32 percent of 28 cases, respectively. The Cd-induced changes in SCC were analyzed on the basis of changes in ENa and RNa. In Type I, the increase in SCC was due to a remarkable decrease in RNa without an appreciable change in ENa (since SCC = ENa/RNa). In Types II and III, the changes in SCC in the early and the later stages could be explained by the differences in the extents to which ENa and RNa decreased. The experimental and calculated ratios of PDcd to PDcontrol in these stages were in good agreement with each other.

Epidermal application of 2 mm-cadmium (Cd2+) to the SO2-4-Ringer's solution bathing the whole frog skin increased its short circuit current (SCC) significantly, whereas the dermal application did not, in most cases. When Cd2+ was applied to the epidermal side, the uptake of cadmium by the skin was approximately 150 μM/kg wet weight, but the uptake by the skin from the dermal side was about eight times as large. In the isolated epidermis, the epidermal application of Cd2+ increased the SCC, whereas the dermal application decreased it. The amount of cadmium taken up by the isolated epidermis was approximately the same when the cation was applied to either epidermal or dermal side. These seemingly contradictory results can probably be explained by the barrier-hypothesis which is supported by the results of an electron probe X-ray microanalysis. In the case of the epidermal application, the junction between s. corneum and s. granulosum may act as an outside barrier for cadmium translocation. For the dermal application of Cd2+, the sieve layer in the dermis, where a large amount of calcium is deposited, may probably exert its effects as an inside barrier by the mechanism of Cd-Ca exchange process. Tela subcutanea, the basement membrane and its adjacent tissue may be also inside barriers, though their role as a diffusion barrier may be considered to be weaker than that of the sieve layer.

The epidermal effects of activator (cadmium) or inhibitors (calcium and amiloride) as well as their interactions in active Na transport through bullfrog abdominal skin were analyzed kinetically. Cadmium at 1 mM increased the short circuit (SCC) to 126%. The Hill coefficient (n) for the relation between [Cd2+] and SCC was 0.737, which indicated negative cooperation. Calcium at over 8 mM decreased the SCC to 80% at its maximum effect. Calcium inhibited the Cd2+-induced SCC at lower [Cd2+], and facilitated it at higher [Cd2+]. Calcium acted uncompetitively on the binding reaction of Na+ with a Na entry channel. Cd2+ alone or Cd2+ under the influence of Calcium also acted uncompetitively on the reaction. Amiloride at 5 X 10-5 M inhibited the SCC almost completely. The Hill coefficient in the relation between amiloride and SCC was 0.757, indicating the presence of negative cooperation. In contrast to the effect of calcium, the Cd2+-induced SCC was much reduced at higher [amiloride]. Amiloride inhibited the channel-Na binding reaction in a mixed manner (competitive + noncompetitive), while Cd2+ activated the reaction under the influence of amiloride noncompetitively. From the SCC model based on the hypothesis that the kinetic constants are invariable even under interaction of the activator and inhibitors, the percentage of the various forms of channel.ion complex were calculated. The SCC was then calculated, assuming that is was directly proportional to the sum of the rate of decomposition of channel.Na complex and the higher rate of decomposition of channel.Na.Cdn complex. The above assumption is supported by the fact that there is good agreement between the calculated SCC and experimentally obtained SCC in the mixture of the activator and the inhibitor.

We have developed a new simple procedure for separating the epidermis from the dermis of the bullfrog (Rana catesbeiana) (freezing and scraping off the skin), and extracting microsomal fraction from the epidermis. The effects of Cd on the ATPase activity of the microsomal fraction was investigated by varying Na and/or K concentrations in the reaction medium. The higher the concentration of Cd, the larger the decreases were found in both activities of ouabain-sensitive ATPase and ouabain-insensitive ATPase in the range of Cd concentration from 10(-7) M to 10(-3) M. The activity of ouabain-sensitive ATPase (Na, K-ATPase) was completely inhibited by 1 mM Cd. The amount of Cd uptake into the whole skin was about 150 micrometer/kg wet weight. This concentration of Cd was sufficient to inhibit the Na, K-ATPase activity of the microsomes. The mechanisms underlying the three types of Cd effects on the frog skin were discussed in relation to the Na, K-ATPase activity.

In order to examine the pH dependency of Cd effects on cardiac muscle, the electrical and mechanical activities in the bullfrog heart were investigated in relation to various external pHs and concentrations of Cd. The amplitude and duration of the action potential or the spontaneously beating frequency in the atrium were not significantly affected during the 3-min administration of various concentrations of Cd in a range from pH 6 to 10, but the isotonic contraction at the end of the same period was greatly altered: the lower the external pH the more Cd decreased the contractility of the atrium. This pH dependency of Cd effects was also observed in SO4-Ringer's solution whose anions, SO4, were more impermeable than Cl. Decrease in contractility in Cd-Ringer's solution was counteracted by excess Ca. The lower the pH of the Cd-Ringer's solution, the more Ca was necessary to counteract an equal amount of Cd. The amount of Cd-uptake into the atrium was analyzed after soaking the atrium in various concentrations of Cd-Ringer's solution. The higher the concentration of external Cd, the larger the Cd-uptake into the atrium. No pH dependency, however, was observed in this relationship. This suggests that probably only a small fraction of total Cd-uptake interacts with Ca-binding sites which is specific to contraction, and causes the Cd-induced decrease in contractility. This process is considered to be pH-dependent. However, most Cd-uptake into cardiac muscle is pH-independent and may be nonspecific to contraction.

Electrical impedance of a bullfrog ventricle was recorded epicardially by the V-I method and the bridge method together with an electrocardiogram (ECG) and a contraction curve, in terms of shortening. Impedance change during one cardiac cycle of a contracting ventricle corresponded completely to the contraction curve in its time course: impedance decreased during systole and attained minimum at the peak of contraction, and the durations of both curves were almost identical. The ratio of impedance just before T-wave to that just after R-wave (|ZT|/|ZR|) of an in situ heart was increased when the ventricle contracted isovolumetrically by means of mineral oil: the ratio was far less than 1.0 in control ventricle, while it was more than 1.0 in oilfilled one (2/3 cases). Upon isometric condition or treatment of cadmium the motion restricted ventricle caused an increase in the impedance ratio (|ZT|/|ZR|) to higher than 1.0, as in the case of oil-filled ventricle. The epicardial recording of ventricular impedance seems to consist of two components: contraction (shortening)-dependent impedance change and contraction (shortening)-independent impedance change, which reflects the membrane activity. Vigorous contraction will probably mask the latter and reveal only the former.

Hyperpolarizing responses of the somata of median giant fibers of crayfish were investigated under current clamp and voltage clamp conditions in normal saline. The responses consisted of a peak [mean delta Vm (peak)=-217 mV] followed by a plateau (-107 mV), and could be classified into N-type and G-type. In the N-type cells, the current clamp I-V curve showed a transient phase to the peak. The phase corresponded to the negative slope region of the voltage clamp I-V curve. There was no negative slope region in the G-type cells. In the transient and the peak phases, the instantaneous I-V curves showed nonlinearity, and the shifts of the electromotive forces (delta EMF) from the resting level were slightly negative or zero. In the plateau phase, however, the instantaneous I-V curve was linear and delta EMF was large and positive (33.2 mV). The membrane resistance, which was larger (X2.3) at the peak than at the resting state, decreased (X0.23) gradually during the plateau phase. delta EMF during the hyperpolarizing response consisted of two components: a fast component (1.7 msec) and a slow one (113 msec). The amplitudes of delta EMF (27.4 mV) of these components in the depolarizing direction increased gradually, once the response had reached the peak. The fast and slow delta EMF's attained 17.0 and 10.4 mV respectively at the end of 100 msec current pulse. It is shown that constant charge triggers the plateau process and that this threshold voltage-current curve is hyperbolic. It is a possibility that the plateau is caused by membrane breakdown.

Dermally applied ouabain decreased the short circuit current (SCC) in frog skin, whereas epidermal cadmium increased it. The percent increase in SCC induced by Cd2+ was never modified by the presence of ouabain. This suggests that Cd2+ and ouabain may exert their effects on Na transport independently.

The effect of stimulation of the vagaosympathetic trunk or the vagal nerve on the cardiac interval of the spontaneously beating heart of the bullfrog was studied in the presence and absence of Cd. The prolongation of the cardiac interval following the nerve stimulation was abolished by Cd (5 micrometer). Such an effect of Cd was completely antagonized by increasing the external Ca to 10 times the normal concentration. Cd (10 micrometer) did not alter the compound action potential of the nerve trunk, nor did it affect the pacemaker activity of the heart. Bioassay of acetylcholine in the effluent from the heart after cardiac nerve stimulation showed that Cd reduced the acetylcholine release from the cardiac nerve. It is concluded that Cd may act on the cardiac nerve terminals where Cd suppresses the release of acetycholine.

The effects of the sympathomimetic amines (adrenaline and noradrenaline) upon the transmembrane potential of both atrial and ventricular fibers of the toad were studied. Transmembrane potentials were recorded by glass-pipette microelectrode.1. In the atrial fibers, these amines increased the resting potential at an early stage. An increase was also observed on the overshoot and duration of the action potential. At the same time a slowing of the terminal phase of depolarization and a slowing of the initial phase of repolarization was observed in some cases.At a later stage, these changes were replaced by a marked shortening of the action potential duration especially after a longer exposure to higher concentrations of the amines. This shortening of the action potential duration at the later stage was occasionally followed by fibrillation or flutter. All these later effects were abolished by atropine. This suggests that a liberation of acetylcholine or some allied substance from the atrium in an amount sufficient to mask the effects of adrenaline occurs.2. Similar changes were observed in the ventricular fiber as a result of the administration of sympathomimetic amines.3. Premedication of the animal with reserpine sensitized both atrium and ventricle to the sympathominetic amines.4. Acetylcholine and sympathominetic amines are antagonistic and counteract each other in their effect on the duration of the action potential.5. The effect of adrenaline on the atrial membrane potential was not essentially altered by changing the cationic concentrations of the external fluid.6. The mode of action of adrenaline on the membrane potential is discussed on the basis of the results obtained.