Substitution Of Choline For Na Research Articles

Identity, expression and functional role of the sodium-activated potassium current in vestibular ganglion afferent neurons

Vestibular afferent neurons (VANs) transmit information from the vestibular end organs to the central nuclei. This information is encoded within the firing pattern of these cells and is heavily influenced by the K+ conductances expressed by vestibular neurons. In the present study, we describe the presence of a previously unidentified Na+-activated K+ conductance (KNa) in these cells. We observed that the blocking of Na+ channels by tetrodotoxin (TTX) or the substitution of choline for Na+ in the extracellular solution during voltage clamp pulses resulted in the reduction of a sustained outward current that was dependent on the Na+ current. Furthermore, increases in the intracellular concentration of Na+ that were made by blocking the Na+/K+ ATPase with ouabain increased the amplitude of the outward current, and reduction of the intracellular Cl− concentration reduced the TTX-sensitive outward current. The substitution of Li+ for Na+ in the extracellular solution significantly reduced the amplitude of the outward current in voltage clamp pulses and decreased the afterhyperpolarization (AHP) of the action potentials in current clamp experiments. These electrophysiological results are consistent with the presence of mRNA transcripts for the KNa subunits Slick and Slack in the vestibular ganglia and in the sensory epithelium, which were detected using reverse-transcription polymerase chain reaction (RT-PCR). These results are also consistent with the immunolabeling of Slick and Slack protein in isolated vestibular neurons, in the vestibular ganglion and in the vestibular sensory epithelium. These results indicate that KNa channels are expressed in VANs and in their terminals. Furthermore, these data indicate that these channels may contribute to the firing pattern of vestibular neurons.

A Store-operated Calcium Channel in Drosophila S2 Cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

Protection against cellular damage in the perfused rat heart by lowered pH

In this comparative study, rat hearts were perfused at 37°C with three clearly defined protocols: the Ca 2+ paradox, the O 2 paradox and with 20 mM caffeine. Each protocol involved an initial priming (Ca 2+ o depletion or anoxia; stage 1) and subsequent full activation (Ca 2+ o repletion or reoxygenation; stage 2) of the damage system of the sarcolemma. Creatine kinase release in stage 2 was completely inhibited ( P<0.001) in all three protocols when pH was reduced to 6.5 throughout the experiments, or only during stage 1, or only during stage 2. The inhibitor of the Na +/H + antiporter, amiloride (1 mM), completely prevented creatine kinase release in the Ca 2+ paradox ( P<0.001) and markedly reduced damage in the caffeine protocol. Amiloride had no significant effect on creatine kinase release in the O 2 paradox. The possible role of Na + i was studied in the caffeine protocol: ouabain (5×10 −6 M) had little effect whereas substitution of choline for Na + in the perfusion medium reduced creatine kinase release by about 50%. It is suggested that the same damage system is activated in stage 1 in all three protocols and that a key event is the intracellular production of H + which are exported via Na + o/H i exchange. Prevention of H + efflux by lowered pH o, even during stage 2, protected against creatine kinase release. The possible role of Na + movements in the genesis of sarcolemma damage is discussed.

Voltage‐gated Currents in Rabbit Retinal Astrocytes

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was -70 +/- 4.8 mV (mean +/- SEM; n = 54), and the input resistance and cell capacitance were 558 +/- 3.6 M omega and 19.5 +/- 1.8 pF respectively. Depolarization to potentials positive to -50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na+ in the bathing solution, and reduced by 50% in the presence of 1 microM tetrodotoxin. The time-to-peak of the Na+ current was more than twice that for the Na+ current found in retinal neurons. The glial Na+ current was half-inactivated at -55 mV. A transient component of the outward K+ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between -150 and -50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks.

The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10(-4) M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na+ in the bathing solutions abolished the potential difference and the short-circuit current; when Na+ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 micrograms) and T3 (10 microM) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca2+ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na+ dependent and increases with developmental age in the chick embryo.

Sodium-bicarbonate cotransport in retinal Muller (glial) cells of the salamander

An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.

Hepatic transport of a fluorescent stearate derivative: electrochemical driving forces in intact rat liver

We determined the effect of varying the transmembrane Na+ electrochemical gradient on extraction of a fluorescent derivative of stearate, 12-N-methyl-7-nitrobenzo-2-oxa-1,3,-diazol-amino stearate (NBD-stearate), by the isolated perfused rat liver. Membrane potential difference (PD) of individual hepatocytes and extraction of NBD-stearate were measured simultaneously under basal conditions and during changes in PD induced by perfusate ion substitutions. Under basal conditions, PD average -30 +/- 1 mV, and extraction of 10 microM NBD-stearate from 1% albumin solutions averaged 0.54 +/- 0.03. Fluorescence microscopy indicated that uptake exhibited a declining portal-to-central gradient in the presence but not absence of Na+. Substitution of nitrate for Cl- hyperpolarized PD to -59 mV and increased extraction to 131% of control values. Withdrawal of nitrate and substitution of gluconate for Cl- depolarized PD to -3 and -15 mV, respectively, and decreased extraction to 63 and 73% of control values. Substitution of choline for Na+ eliminated the out-to-in Na+ gradient, depolarized PD to -16 mV, and decreased extraction to 27% of control values, an effect greater than expected for membrane depolarization alone. Uptake of NBD-stearate was saturable and caused Na(+)-dependent membrane depolarization at higher concentrations (300 microM). These studies indicate that uptake of NBD-stearate occurs in large part by an efficient Na(+)-dependent mechanism compatible with electrogenic Na(+)-fatty acid cotransport.

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander.

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

The effects of acute omission of extracellular Na + on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na + hyperpolarized the B-cell membrane, inhibited 86Rb + and 45Ca 2+ efflux, but did not affect insulin release. In contrast, Li + substitution for Na + depolarized the B-cell membrane and caused a Ca 2+-independent, transient acceleration of 45Ca 2+ efflux and insulin release. Na + replacement by choline in the presence of 10 mM glucose and 2.5 mM Ca 2+ again rapidly hyperpolarized the B-cell membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb + efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca 2+ efflux and insulin release. In the absence of Ca 2+, 45Ca 2+ and 86Rb + efflux were inhibited and insulin release was unaffected by choline substitution for Na +. Na + replacement by Li + in the presence of 10 mM glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 45Ca 2+ efflux, 86Rb + efflux and insulin release. In the absence of extracellular Ca 2+, Li + still caused a rapid but transient increase in 45Ca 2+ and 86Rb + efflux and in insulin release. Although not indispensable for insulin release, Na + plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.

The Na +/H + antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion

We used the pH-sensitive fluorescent probe 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na +/H + exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pH i) of freshly isolated alveolar type II cells was 7.36 ± 0.05 ( n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pH i dropped to 6.59 ± 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCl, pH i increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pH i. When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H + in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pH i of 7.48 ± 0.05 ( n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaCl increased the pH i of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O -tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pH i. Inhibition of the Na +/H + antiporter by the addition of amiloride to a Na + containing medium or the substitution of choline for Na + did not inhibit stimulated phosphatidylcholine secretion. We conclude that pH i regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na +/H + antiporter, but this system appears not to be involved in TPA- or terbutaline-induced pulmonary surfactant secretion in primary culture.

The action of N-methyl-D-aspartic acid on mouse spinal neurones in culture.

Neurones from the ventral half of mouse embryo spinal cord were grown in dissociated culture and voltage clamped. The current-voltage relation of responses evoked by N-methyl-D-aspartic acid (NMDA), L-glutamic acid and kainic acid was recorded in media of different ionic composition. On removal of Mg2+ from the extracellular solution, responses to NMDA and L-glutamate became less voltage sensitive, such that NMDA responses were no longer associated with a region of negative slope conductance. The antagonism of NMDA responses produced by application of Mg2+ to neurones bathed in nominally Mg2+-free solutions shows voltage dependence and uncompetitive kinetics. Voltage-jump experiments showed that the voltage-dependent action of Mg2+ occurred rapidly, and with complex kinetics. Ni2+ and Cd2+, two potent blockers of calcium currents in spinal cord neurones, had significantly different potencies as NMDA antagonists, Ni2+ being of greater potency than Mg2+, and Cd2+ considerably weaker. The voltage-dependent block of NMDA responses produced by physiological concentrations of Mg2+ is sufficient to explain the apparent increase in membrane resistance produced by NMDA in current-clamp experiments, and the ability of NMDA to support repetitive firing. Substitution of choline for Na+ produced a hyperpolarizing shift in the reversal potential for responses evoked by kainic acid consistent with an increase in permeability to Na+ and K+. In choline-substituted solutions, the reversal potential of NMDA responses was more positive than that recorded for kainic acid, and in addition NMDA responses showed enhanced desensitization.

Action and binding of palytoxin, as studied with brain membranes.

Palytoxin in concentrations as low as 10(-11) to 10(-12) M promotes the outflow of the lipophilic [3H]tetraphenylphosphonium ion from particulate brain cortex of guinea-pigs and rats, and from preloaded crude synaptosomes of rats, which indicates depolarization. The outflow is not influenced by tetrodotoxin or the calcium channel blocker nimodipin, or by substitution of choline for Na+ ions. It is increased by Ca2+ and by borate, the latter interacting with the toxin itself. To assess the fixation of palytoxin to biological membranes, a binding step was installed before the depolarization step. Palytoxin binds to membranes from rat brain, liver, kidney, human and dog erythrocytes, and to a lesser degree to liposomes made from rat brain or erythrocyte lipids. Binding is reversible. It is decreased by mild physical pretreatments of crude synaptosomes. Palytoxin binding is increased in the presence of micromolar concentrations of Ca2+ or borate. It is concluded that the potentiation of palytoxin actions by Ca2+ or borate is at least partially due to the promotion of its binding.

Effect of kainic acid, glutamate, and aspartate on CO2 production by goldfish tectal slices.

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.

Influences of tetrodotoxin, amiloride, phenytoin, aconitine, tetraethylammonium on ACTH-and isoproterenol-mediated lipolysis in isolated rat adipocytes

The effects of tetrodotoxin, amiloride, phenytoin, aconitine, and tetraethylammonium on isoproterenol- and ACTH-mediated Free Fatty Acids (FFA) and glycerol release from rat adipocytes are investigated in order to understand more about the role of Na + or K + in the lipolytic process. Lipolysis induced by 2×10 −6 M isoproterenol was mildly affected by the drugs tested. Only tetraethylammonium and aconitine inhibited the lipolytic action of 10 −7 M isoproterenol but only slightly. Amiloride significantly enhanced both FFA and glycerol release induced by ACTH. Ionophore X537A inhibited this effect, but the substitution of choline for Na + or the omission of Ca 2+ did not. In these latter experimental conditions, ACTH-mediated lipolysis was strongly inhibited. The sum of these results suggests that the widely observed Na + and K + involvement in lipolytic process is independent from operating ion channels in adipocytes.

Discrete states of responsiveness of a locust muscle γ-aminobutyric acid receptor: The influence of extracellular ion concentrations

The membrane conductance of locust muscle fibers was measured with intracellular microelectrodes. The relationship between γ-aminobutyric acid concentration and increase in membrane conductance was studied in solutions of different ionic composition. The limiting slope of the log-log and Hill plots for small responses was interpreted as a minimum estimate of the number of γ-aminobutyrate molecules required to activate a γ-aminobutyrate receptor. Doubling the Mg 2+ concentration from 2 to 4 mM was associated with an increase in limiting slope from a mean of approximately 3 to 4. Neither partial substitution of propionate for Cl − nor increasing the Ca 2+ concentration from 2 to 10 mM influenced the limiting slope. Complete substitution of choline for Na +, in the presence of 4 mM Mg 2+, decreased the mean limiting slope back to 3. In a Mg 2+-free solution, to which it was necessary to add Tris buffer, the mean limiting slope was approximately 2. The rates of equilibration of responses to γ-aminobutyrate approximated a single exponential with half-time independent of γ-aminobutyrate concentration within a limited low concentration range. The equilibration half-time, like the limiting slope tended to adopt discrete values. We conclude that the receptor for γ-aminobutyrate can exist in multiple states differing in responsiveness to γ-aminobutyrate. A general function of state is derived to describe the equilibrium properties of the interaction based on the assumption that different numbers of γ-aminobutyrate molecules are able to activate the receptor.

Chloride, sodium, potassium and hydrogen ion transport in isolated canine gastric mucosa.

1. The fluxes of isotopically labelled Na+, Cl- and K+ in each direction and H+ secretion across isolated dog gastric mucosa were measured under short-circuit conditions. 2. In the non-stimulated state, the net flux of Na+ was 6.61 micronequiv/cm2.hr from mucosal (luminal, secretory) to serosal (nutrient, blood) side, whereas the net flux of Cl- was only 0.79 micronequiv/cm2.hr, and the direction was from serosal to mucosal side. 3. There was a positive correlation between the net flux of Cl- and acid secretion, however, net flux of Na+ was not correlated with acid secretion initiated by secretagogue treatment. 4. With ion substitution studies, only replacement of mucosal Na+ with choline produced a highly significant decrease in potential difference (p.d.). This indicates that active transport of Na+ from the mucosal to the serosal side is the most important source for the generation of the gastric p.d. in dog gastric mucosa. 5. From ion substitution studies, it was also observed that Cl- in either mucosal or serosal solution is necessary for maintaining acid secretion; whereas only serosal Na+ and K+ are essential for acid secretion. Removal of either Na+ or K+ from the mucosal solution had no effect on acid secretion. 6. Substitution of SO2-(4) for Cl- had no effect on active transport of Na+, but choline substitution for Na+ diminished active transport of Cl-.

Membrane electrical properties of vascular smooth muscle from the guinea pig superior mesenteric artery.

Some electrical membrane properties of an isolated small artery, namely, the superior mesenteric artery of the guinea pig, were studied by intracellular microelectrodes. The mean resting membrane potential (E m) was −54 mV. The average slope of theE m vs. log [K]o curve (between 10 and 100 mM [K]o) was 32 mV/decade, and the curve extrapolated to a [K]i of 160 mM. The ratio of Na+ permeability to K+ permeability (P Na/P K) at 4.0 mM [K]o calculated from the Goldman constant-field equation (assuming Cl− to be passively distributed) was 0.18 (E m=−46 mV after a 5 min exposure to ouabain to suppress any electrogenic pump potential). The normal input resistance (R in) averaged 8.5 mΩ. Choline substitution for Na+ or amiloride, an agent known to depressP Na, hyperpolarized the muscle to about −63 mV without a significant change inR in. Ba2+ (0.5 mM) depolarized the muscle to −37 mV, increasedR in to 15 mΩ, and produced spontaneous action potentials in this normally quiescent artery; tetraethylammonium (TEA, 5 mM) enabled large overshooting action potentials to be produced upon stimulation. Glutamate of NO 3 − substitution for Cl− produced an initial depolarization followed by a return to the original resting potential within 10 min; readdition of 25 mM Cl− transiently hyperpolarized the muscle markedly, followed by a return to the originalE m. These data indicate that Cl− is passively distributed and does not contribute to the steady-state resting potential in this vascular muscle. The data also suggest that the relatively lowE m in this arterial muscle is not due to a low [K]i, but is due to a highP Na/P K ratio, presumably related to a low K+ conductance (g K). Since Ba2+ and TEA+ are known to decrease restingg K and K+ activation, the data also suggest that K+ activation could inhibit action potential generation.

Effects of aldosterone on Na+ transport in the toad bladder. II. The anaerobic response.

The action of aldosterone on active Na+ transport was assessed under aerobic and anaerobic conditions in the isolated urinary bladder of the toad, Bufo marinus. Aldosterone augmented the short-circuit current (Isc) under rigorous anaerobiosis. Four lines of evidence indicate that the increase in anaerobic Isc does not represent an equivalent increase in active Na+ transport: 1. Net Na transport, determined by isotopic fluxes, was the same in the aldosterone-treated and control quarter-bladders, and significantly greater than the simultaneously measured Isc-2. Amiloride, an inhibitor of the apical entry of Na+, did not reduce the steroid-dependent increase in the anaerobic Isc-3. Substitution of choline for Na+ in the mucosal medium reduced the magnitude of the anaerobic Isc values but did not eliminate the effect of aldosterone. 4. Addition of ouabain, a potent inhibitor of the Na+ pump, partially inhibited the effect of aldosterone on the anerobic Isc but a significant hormonal increment remained. The source of the anaerobic Isc was not identified; an effort was made, however, to determine the dependence of this current on glycolysis. During anaerobiosis, aldosterone increased the integral Isc by 42% but did not alter lactate production. These results suggest that the steroid-dependent increase in the anaerobic Isc may involve effects on permeability properties of the epithelium rather than on active transport systems.

Glycogen in isolated mucosal and serosal fractions of turtle bladder

1. 1. The amount and behavior of glycogen in isolated fractions of turtle bladder were determined. The mucosal (epithelial) fraction contained approximately 30 % of whole bladder glycogen in both summer and winter. The remainder was contained in the serosal (muscle) fraction. 2. 2. The mucosal fraction made up a significantly greater proportion of total bladder wet weight in August than in January–March. 3. 3. Anoxia and 2,4-dinitrophenol caused rapid depletion of glycogen in both fractions. The substitution of choline for Na + in bathing fluids reduced glycogen losses both in the presence and absence of dinitrophenol. 4. 4. In each set of conditions tested, glycogenolysis was more extensive in the mucosal fraction than in the serosal fraction. 5. 5. Although the magnitudes of glycogen losses varied under different conditions, mucosal and serosal losses maintained an approximately constant relationship. It was suggested that glycogenolytic systems with similar characteristics (response patterns) but different intrinsic activities are operative in the two fractions.