Effects of procaine on the ventricular muscle of bullfrog.

The effects of procaine on the contractility of the bullfrog's ventricular muscle were investigated. The addition of 10(-5) g/ml of procaine potentiated the twitch tension which was accompanied by an elevation as well as a prolongation of the action potential plateau. This positive inotropism of procaine was not induced by endogenous catecholamine because a beta-blocking agent did not influence this twitch potentiation. The twitch potentiation was increased in proportion to the external Ca concentration, suggesting the possibility of augmentation of Ca influx during the action potential. In normal Ringer solution, procaine suppressed potassium contracture which was composed of two components: an initial phasic component and a late tonic one. Potassium contracture after perfusion with Ca-free solution was also suppressed by procaine. However, potassium contracture which had been treated previously with La was composed of only a tonic component and was potentiated by procaine in spite of perfusion with Ca-free solution. The tonic component of potassium contracture may be considered to occur with intracellular Ca. Procaine may increase the Ca inward current, acting on the intracellular Ca storage site and consequently accelerate the excitation-contraction coupling in frog ventricular muscle.     

Different effects of N-ethylmaleimide on the tension components of bullfrog atrium.

The effects of N-ethylmaleimide (NEM) on the tension components of bullfrog atrium were studied under voltage clamp conditions using the double sucrose gap technique. NEM at a dilution of 10(-3) M inhibited the ICa-independent tonic tension elicited by large and long depolarizing pulses (140 mV, 2 sec) time-dependently, whereas the drug produced an initial transient increase and a later decrease of the ICa-dependent phasic tension elicited by small and short depolarizing pulses (60 mV, 100 msec). The effect of NEM was facilitated when the atrial muscles were exposed to a solution having either a reduced Na or an increased Ca concentration. In the presence of Mn ions, 10(-3) M NEM did not evidently inhibit tonic tension, and it led to a slight transient increase in phasic tension. NEM at 10(-3) M, which is known to inhibit sarcolemmal Na, K-ATPase, enhanced both the phasic tension and the tonic tension in the presence of ouabain and when perfused with K-free solution. Even under these conditions (10(-3) M NEM caused an initial transient decrease of ICa-independent tonic tension. This suggests that NEM has a different action on the atrial muscle from those of cardiac glycosides such as ouabain.     

Effects of ouabain on potassium transport in the perfused bullfrog kidney.

We studied the effect of ouabain on transepithelial and cellular potassium transport in the isolated perfused bullfrog kidney. We used recently developed techniques for estimating the unidirectional reabsorptive and secretory K fluxes (Jr and Js) and measuring the kinetics of cellular K transport. Two hours of perfusion with 1 X 10(-6) M ouabain did not affect GFR, reduced fractional Na reabsorption 57%, increased K excretion 41%, and inhibited Jr 34%. Js rose 68% at 60 min and then returned to the control level. Ninety minutes of perfusion with 5 X 10(-6) M ouabain reduced GFR 28% and fractional Na reabsorption 76%. K excretion rapidly increased 71% within 30 min and then fell to 60% of the control level, while Jr fell 64%. Js rose 42% in 30 min and then fell to 23% of the control level. Both doses reduced K uptake into cellular pools from the circulation and increased the rate coefficients for efflux into tubular fluid. The data indicate that ouabain inhibited reabsorption and transiently accelerated the rate of loss of K from the cells into the tubular fluid. This initially masked the ultimate inhibition of K secretion from the circulation into the tubular fluid.     

Effects of ethanol on microsomal drug metabolism in aging female rats. I. Induction

The purpose of this study was to determine how aging affects the induction by ethanol or acetone of the hepatic microsomal monooxygenase system of female Fischer 344 rats. Young-adult, middle-aged and old rats (4, 14 and 25 months) were fed an ethanol-containing or control liquid diet for 15 days. Cytochrome P-450, cytochrome c reductase, aniline hydroxylase, nitrophenol hydroxylase, nitroanisole O-demethylase and benzphetamine N-demethylase activities were measured in hepatic microsomes. All of the drug metabolism activities except benzphetamine N-demethylase were 20-35% lower in old than in young-adult rats fed the control diet. In addition, the increase in drug metabolism produced by feeding the regular ethanol diet (36% of calories as ethanol) was 50-60% lower in the old rats. However, there was no difference in the magnitude of ethanol induction when ethanol intakes were matched. The effects of chronic acetone consumption (1.2g/day per kg body weight for 15 days) paralleled those of ethanol consumption, except that the extent of induction was greater with acetone. Acetone-induced levels of hepatic microsomal cytochrome P-450, nitrophenol hydroxylase, nitroanisole O-demethylase and aniline hydroxylase were similar in all three age groups. The results of this study indicate that induction of hepatic microsomal drug metabolism by ethanol or acetone is unaffected by the aging process.     

Selective effects of bumetanide on chloride transport in bullfrog cornea.

Frog corneas were mounted in a modified Ussing chamber and short-circuit current (SCC) and unidirectional Cl fluxes were measured. Bumetanide, a loop diuretic, at concentrations as low as 10(-7) M, reduced the SCC 29%. At 10(-5) M, bumetanide reduced the SCC 96% and increased transcorneal electrical resistance 20-51%. The forward Cl flux declined from 0.71 +/- 0.04 to 0.20 +/- 0.03 mueq/h.cm2 (n, 7), while, in separate experiments, the backward Cl flux did not change significantly (from 0.22 +/- 0.03 to 0.23 +/- 0.04; n, 7). When corneas were mounted in Cl-free Ringer and the net Na transport was stimulated with amphotericin B, 10(-5) M bumetanide had no effect on the SCC. In separate experiments the effect of 10(-5) M bumetanide on the O2 consumption was measured in a stirrer bath assembly. Bumetanide decreased the O2 consumption from 352 +/- 14 to 297 +/- 19 microliter/h.cm2 (significantly different from sham-treated controls). This decrease was similar to that obtained with furosemide or when Cl was removed from the bathing medium. We infer from these results that bumetanide is a selective inhibitor of active Cl transport in the bullfrog cornea.     

Microdynamics of outer and inner membranes of mitochondria from bullfrog myocardium

Outer and inner mitochondrial membranes were separated from bullfrog myocardium. Membrane viscosity and wobbling angle of phospholipids were measured with a nanosecond time-resolved fluorometer using a fluorophore, DPH, in each membrane. Measurements were also made on liposomes prepared from lipids extracted from each membrane. The anisotropy decay curve for DPH fluorescence was assumed to represent a mean value of decays in several microenvironments in membranes. Phospholipid constituents in membranes were analyzed by HPLC. A high proportion of PE and CL, both of which contain large amounts of unsaturated acyl chain, were found in the inner membrane. The low viscosity and large wobbling angle of phospholipids in the liposome from the inner membrane were consistent with the probable high content of unsaturated acyl chains and the low content of cholesterol in the inner membrane. Measurements of the dynamic microstructure of mitochondrial membranes suggested multifactorial characteristics probably resulting from the lipid-protein interactions. The average viscosity was found to be 0.39 +/- 0.08 P in the outer membrane and 0.58 +/- 0.01 P in the inner membrane. The wobbling angle of phospholipids in the outer and the inner membrane was, respectively, 47 and 49 degrees (non-significant difference). The liposomes prepared from lipid extracts of the membranes showed a lower viscosity and/or higher wobbling angle of phospholipids compared with the membranes themselves. The difference in the viscosity and wobbling angle between the mitochondrial membrane and its respective liposome was large in the inner membrane. These results suggest that the motion of phospholipids is limited by membrane proteins and the limitation of molecular motion of phospholipids results in an increase in the average viscosity. The results also suggest that the microdynamic values obtained in the inner membrane fraction reflect the interaction between phospholipid and protein which is abundant in the inner membrane.     

An exploratory analysis of the competing effects of alcohol use and advanced hepatic fibrosis on serum HDL

While alcohol use has been shown to increase serum HDL, advanced liver disease associates with decreased serum HDL. The combined influence of alcohol consumption and liver fibrosis is poorly defined. In this study, we sought to investigate the competing effects of alcohol use and hepatic fibrosis on serum HDL and to determine if the presence of advanced hepatic fibrosis ablates the reported effect of alcohol consumption on serum HDL. We performed a cross-sectional, exploratory analysis examining the interaction between alcohol use and advanced hepatic fibrosis on serum HDL levels in 10,528 patients from the Partners Biobank. Hepatic fibrosis was assessed using the FIB-4 index. We excluded patients with baseline characteristics that affect serum HDL, independent of alcohol use or the presence or advanced hepatic fibrosis. We observed an incremental correlation between increasing HDL levels and amount of alcohol consumed (P?<?0.0001), plateauing in those individuals who drink 1–2 drinks per day, Contrastingly, we found a negative association between the presence of advanced hepatic fibrosis and lower HDL levels, independent of alcohol use (beta coefficient: -0.011075, SEM0.003091, P value: 0.0001). Finally, when comparing subjects with advanced hepatic fibrosis who do not use alcohol to those who do, we observed that alcohol use is associated with increased HDL levels (54.58 mg/dL vs 67.26 mg/dL, p?=?0.0009). This HDL-elevating effect of alcohol was more pronounced than that seen in patients without evidence of advanced hepatic fibrosis (60.88 mg/dL vs 67.93 mg/dL, p?<?0.0001). Our data suggest that the presence of advanced hepatic fibrosis does not blunt the HDL-elevating effect of alcohol use.

     

Kinetic analysis of potassium transport in bullfrog kidney.

Techniques have been developed for studying the distribution and the rates of exchange of K among urine, the tubular cells, and the circulation in the isolated, pump-perfused, bullfrog kidney. Tubular cells were loaded with 42K via the portal circulation, and the subsequent washout of the tracer into the vena cava and into urine was measured. Analysis of the data indicated the existence of at least three cellular pools of K. Pools a and b have half times of exchange of 1.1 and 4.1 min and contain about 25 and 40% of tissue K, respectively. The remainder of cellular K is contained in one or more very slowly exchanging pools. The rate of exchange of K at the basolateral surface of tubular cells is 50-fold greater than at the luminal surface. A pulse-washout method was also devised to permit control and experimental measurements to be made in the same kidney. With this technique, we found that portal perfusion with 10 mM K increased the rate of uptake into pools a and b from the circulation and the rate constants for efflux into the urine from both pools, Acetazolamide increased uptake into pool a and the rate constants for efflux into the urine from both pools.     

The muscarinic effects of acetylcholine on the action potential of bullfrog sympathetic ganglion cells.

The direct effects of acetylcholine (ACh) on Na+- or Ca2+-dependent action potentials of curarized sympathetic ganglion cells in bullfrogs were investigated under a condition where membrane depolarization caused by the muscarinic action of ACh was nullified by means of a hyperpolarizing current. ACh decreased the after-hyperpolarization of Na+-action potentials in Ringer's solution, and increased the after-depolarization of Ca2+-action potentials in the isotonic Ca2+ solution. In both solutions, the maximum rates of rise of the spikes were decreased and the slope membrane resistance at the original resting level was increased. The effects of ACh were abolished by atropine. On the other hand, ACh showed no significant effects on action potentials of bullfrog spinal ganglion cells which possessed no synapses. These results suggest that the ion conductance channels for generation of action potentials of sympathetic ganglion cells are under the direct control of transmitters, such as ACh.     

Stabilizing effects of adenosine on the membrane currents and tension components of the bullfrog atrium.

The effects of exogenously applied adenosine on the membrane potentials, currents and tension components of the bullfrog double sucrose-gap method. Adenosine above 10(-3)M produced a prolongation of action potential accompanied by a slight augmentation of contraction which was followed by a sustained depression. The recovery was slow. Under voltage clamp conditions, adenosine merely produced a negative inotropic effect depressing Ica-dependent and -independent tensions. In the membrane currents, Inaf, Is(Ica), Ix and background current (Ib) were all depressed. In the presence of adrenaline, the increased Is, Ix and contraction were inhibited by adenosine in a lower dose (10(-5) -10 (-3) M), and the inhibition of Is and Ica-dependent tension were more prominent in the presence of than in the absence of adrenaline. Under the effect of ATP, which has a catecholamine-like action, similar selective inhibitions were observed. It was concluded that adenosine has a sustained stabilizing action on the myocardium to survive, especially in the presence of adrenaline, by depressing the augmented Ica and contraction.     

An analysis of the action of ATP and related compounds on membrane current and tension components in bullfrog atrial muscle.

The effect of adenosine compounds (ATP, ADP, AMP and adenosine) on membrane potential, current and contractile tension on the bullfrog atrium were studied under voltage clamped and unclamped conditions. The compounds produced immediate positive and late negative inotropic effects in unclamped conditions. The positive inotropic effect and the potency of drugs appeared less marked in the order of ATP, ADP, AMP, and adenosine. Under voltage clamped conditions, only the energy rich compounds, ATP and ADP, produced an enhancement of calcium inward current (Ica) and Ica-dependent phasic tension, while AMP and adenosine elicited a negative inotropic effect. The delayed outward current was initially depressed but later augmented epecially in case of ATP and ADP where Ica was enhanced. All adenosine compounds, however, inhibited the Ica-independent tonic tension. This effect, appearing nonspecific, was ascribable to the action of common structure of these compounds, purine-riboside moiety.     

5-Hydroxytryptamine effects on the somata of bullfrog primary afferent neurons

Intracellular recordings were made from neurons in the isolated dorsal root ganglia of bullfrogs. 5-Hydroxytryptamine was applied by superfusion and by ionophoresis. The most common response to 5-hydroxytryptamine in C neurons was a membrane hyperpolarization and this was observed in 80% of cells. This was due to an increase in membrane potassium conductance because it reversed its polarity at about -90 mV. It was blocked by removal of calcium or addition of calcium blockers. (+)-Tubocurarine, methysergide, ketanserin, quipazine, picrotoxin, caffeine and ouabain blocked this response. The next most common response in C neurons was a fast depolarization, particularly readily observed when 5-hydroxytryptamine was applied by ionophoresis. Since this response reversed its polarity at about -10 mV and was blocked by removal of sodium, this was due to an increase in membrane conductance to both sodium and potassium ions. This response was reduced by superfusion of acetylcholine and gamma-aminobutyric acid. (+)-Tubocurarine, quipazine, picrotoxin and caffeine blocked the response. A small proportion of C neurons (16%) responded to superfusion of 5-hydroxytryptamine with a slow depolarization accompanied by an increase in input resistance. This response reversed its polarity at about -90 mV and, therefore, is presumed to result from potassium inactivation. It was blocked by methysergide and ketanserin but not by (+)-tubocurarine or quipazine. A few type A neurons (8%) caused a fast and transient depolarization like the fast depolarization of C neurons. About half of the A neurons showed a slow depolarization associated with a fall in input resistance. This slow response was assumed to be due to an increase in membrane conductance to both potassium and calcium ions because the response reversed its polarity at about -65 mV and was sensitive to change in external concentrations of those ions. This slow response was blocked by (+)-tubocurarine, methysergide, ketanserin, picrotoxin, caffeine and ouabain but not by quipazine. The effects of 5-hydroxytryptamine are discussed in relation to the similar actions described on a variety of other vertebrate and invertebrate nerve cells. The findings imply that dorsal root ganglion cells of bullfrogs are sensitive to 5-hydroxytryptamine and causes multiple types of 5-hydroxytryptamine responses.     

Alcohol and immune regulation. I. In vivo effects of ethanol on concanavalin A sensitive thymic lymphocyte function

Alcohol is known to suppress the immune response, but the underlying mechanism to account for this immune suppression is still not clearly elucidated. In an attempt to clarify such mechanisms, experimental rats were fed for 50 days on a 36% ethanol, Lieber diet (LED) while control (LCD) rats were fed a similar diet supplying the same amount of calories but lacking ethanol. It was found that both LCD and LED animals grew at a linear rate (LCD: r = 0.981, LED: r = 0.961) but that LCD animals grew more rapidly. While thymic weights in the LED group were significantly smaller (P less than 0.05) than in the LCD group, the ratios of thymic weight/body weight between these groups were not significantly different. To identify the effects of ethanol on immune response, thymic (Th) or splenic (S) cells were prepared and incubated in culture with the mitogen, Con A and rat serum prepared from LCD or LED groups. It was found that lymphocytes prepared from thymus of LED animals appeared to be depressed in mitogen-driven blastogenic transformation when incubated in LCD serum but not LED serum. Furthermore, lymphocytes prepared from the spleen of LED animals appeared to be depressed in mitogen driven blastogenic transformation when incubated in LED serum but not LCD serum. Since lymphocytes of the thymus and spleen are undergoing maturation and replication this implies that ethanol may alter these processes.