Electrophysiology of the sodium-potassium-ATPase in cardiac cells

Like several other ion transporters, the Na(+)-K(+) pump of animal cells is electrogenic. The pump generates the pump current I(p). Under physiological conditions, I(p) is an outward current. It can be measured by electrophysiological methods. These methods permit the study of characteristics of the Na(+)-K(+) pump in its physiological environment, i.e., in the cell membrane. The cell membrane, across which a potential gradient exists, separates the cytosol and extracellular medium, which have distinctly different ionic compositions. The introduction of the patch-clamp techniques and the enzymatic isolation of cells have facilitated the investigation of I(p) in single cardiac myocytes. This review summarizes and discusses the results obtained from I(p) measurements in isolated cardiac cells. These results offer new exciting insights into the voltage and ionic dependence of the Na(+)-K(+) pump activity, its effect on membrane potential, and its modulation by hormones, transmitters, and drugs. They are fundamental for our current understanding of Na(+)-K(+) pumping in electrically excitable cells.     

Demonstration of an electrogenic Na+-K+ pump in mouse spleen macrophages

The effect of ouabain on the membrane potential of cultured mouse spleen macrophages was examined. Ouabain (10(-3) M) induced a membrane depolarization (6.6 mV) in 18 of 19 cells studied that occurred within several minutes after exposure and was not associated with significant changes in current-voltage relationships. In related studies, cells were placed in K+-free medium in the cold for 2 h to block pump activity. Subsequent exposure to K+ at 37 degrees C resulted in a membrane hyperpolarization. However, addition of K+ also enhanced inward rectification. To differentiate between the effect of K+ on the Na+-K+ pump and its action on inward rectification, two types of experiments were done. Studies performed in the presence of barium, which blocks inward rectification, demonstrated a K+-induced hyperpolarization with no changes in rectification. Additional studies examined the effects of rubidium on Na+-loaded cells. Rubidium, which blocks inward rectification but substitutes for K+ in activating the Na+-K+ pump, induced membrane hyperpolarizations that were reversed by addition of ouabain. These data indicate that macrophages exhibit an electrogenic Na+-K+ pump, which probably contributes to the resting membrane potential under steady-state conditions and can be activated under conditions designed to Na+ load the cells. In addition, they demonstrate that increasing extracellular K+ enhances inward rectification in macrophages.     

The independence of electrogenic sodium transport and membrane potential in a molluscan neurone

1. The current-voltage relations of the Anisodoris giant neurone (G cell) were studied in the presence and absence of Na pump activity.2. Inhibition of the electrogenic Na pump with ouabain had no effect on either the presence at warm temperatures (10-15 degrees C), or absence at cold temperatures (0-5 degrees C), of inward-going rectification.3. Abolition of inward-going rectification in the warm, by replacement of external K with Rb, did not affect the electrogenic Na pump.4. The current generated by the electrogenic pump was essentially constant between the membrane potentials of - 30 and - 100 mV.5. The potential produced by the electrogenic pump can be predicted by a modification of the constant field equation.6. It is estimated that the energy required to extrude Na was between 3160 and 3700 cal/g-atom, and that uncoupled Na efflux during pump activity was typically between 0.2 and 4.0 p-mole/cm(2).sec.     

Effects of ouabain and diphenylhydantoin on transmembrane potentials, intracellular electrolytes and cell pH of rat muscle and liver in vivo

1. The effects of ouabain and diphenylhydantoin (DPH) to inhibit and stimulate, respectively, the Na(+)-K(+) pump were used to correlate transmembrane resting potentials (RP), ionic gradients, and cell pH (DMO method) in rat muscle and liver in vivo.2. Ouabain effects included a rise in K(+) and fall in Na(+) concentration in plasma, a rise in intracellular Na(+) and Cl(-) and a fall in K(+) concentration and pH(i) in muscle, and a rise in intracellular K(+) concentration in liver.3. Measured muscle RP was decreased from -90 to -65 mV by ouabain with the RP predictable from the Goldman equation for Na(+) and K(+) with P(Na)/P(K) = 0.01.4. Measured hepatic RP was increased from -44 to -48 mV by ouabain, whereas the Goldman equation predicts the potential should decrease. A change in permeability of some ion or activation of an electrogenic pump component is necessary to explain this result.5. DPH produced no significant effect on muscle electrolytes or RP and failed to reverse the effect of ouabain at the time measured and in the doses used.6. DPH produced a slight rise in hepatic cell K(+) and a rise from -42 to -47 mV in hepatic RP. This hyperpolarization also cannot be explained without invoking a permeability change or activation of an electrogenic pump.In all cases intracellular Cl(-) in both muscle and liver changed in the direction expected from the change in the RP. Muscle Cl(-) appears passively distributed if a constant amount of extra or bound Cl(-) is first subtracted from each group. Hepatic intracellular Cl(-) is always less than expected on the basis of passive distribution, although errors in determination do not allow elimination of the possibility that Cl(-) distribution is determined only by the RP.8. Cell pH and RP data were used to calculate H(+) gradients. DPH had no effect on cell pH and only slightly increased the H(+) gradient in liver. Ouabain produced a slight fall in muscle cell pH but reduced the H(+) gradient by half. In liver only the H(+) gradient was increased slightly. The data support the concept of a loose coupling between active H(+) and Na(+)-K(+) transport.     

Long-term effect of ouabain and sodium pump inhibition on a neuronal membrane

1. The long-term effects of ouabain on the membrane potential of the Anisodoris giant neurone (G cell) were examined in cells maintained for periods of up to 15 hr at 11-13 degrees C.2. In the presence of ouabain (5 x 10(-4)M), the membrane potential depolarized to a constant level for 1-4 hr, then hyperpolarized for 5-7 hr after which it gradually depolarized again.3. During the hyperpolarizing phase, after 6-8 hr in ouabain, [K](1) fell approximately 50%, [Na](1) increased 50-100% and the P(Na)/P(K) ratio decreased to 25% of its initial value.4. After 8 hr in ouabain the membrane conductance increased two- to fourfold. This increase was independent of temperature and membrane rectification.5. The K permeability (P(K)) was calculated from the constant field equation, and showed a fourfold increase after long-term treatment with ouabain. This rise in P(K) probably underlies the membrane hyperpolarization and the decrease in the P(Na)/P(K) ratio.6. It is suggested that inhibition of the Na(+) pump with ouabain causes a gradual rise in [Na](1) which secondarily leads to Ca(2+) uptake, an increase in [Ca](1), and thereby an increase in P(K).     

Activation of the electrogenic sodium pump in guinea-pig auricles by internal sodium ions

1. The effect of various intracellular Na concentrations ([Na](i)) on the membrane potential after hypothermia was studied in guinea-pig auricles.2. For varying [Na](i), the atria were cooled for 4 hr at 4-6 degrees C in a K-poor solution with different concentrations of NaCl. The auricles were rewarmed in normal Tyrode solution at 35 degrees C.3. Extracellular space (ECS), intracellular Na and K concentrations ([Na](i) and [K](i)) and membrane potential of the atria were measured before and after hypothermia.4. The ECS, measured as inulin space, amounted to 350 ml./kg wet wt. at 35 degrees C and to 300 ml./kg wet wt. at 4-6 degrees C.5. [K](i) decreased during cooling and increased during rewarming the auricles. [Na](i) increased during hypothermia in bathing fluids containing NaCl, but decreased in NaCl- and Na-free solutions. At the beginning of rewarming a net Na transport occurred from cells with high [Na](i), while a net Na uptake took place in atria with low [Na](i).6. At the same time, the membrane potential of auricles with increased [Na](i) hyperpolarized beyond the steady-state value recorded at the end of rewarming, or even beyond the calculated K(+) equilibrium potential (E(K)). Afterwards, the hyperpolarization levelled off, while the E(K) values increased further. The membrane potential of atria with decreased [Na](i) showed no transitory hyperpolarization during rewarming.7. The hyperpolarization beyond the steady-state value of membrane potential in rewarmed auricles was significantly correlated to the active Na efflux.8. From these results it is concluded that the membrane potential of guinea-pig atria after hypothermia is affected by an active, electrogenic Na pump activated by intracellular Na ions.     

Electrogenic Na-K pump in denervated mouse soleus muscles: prolonged activation by briefly applied acetylcholine

Thin preparations of mouse soleus muscles denervated for 3-11 days were bathed in Cl-free solutions. The membrane potential (microelectrode technique) was an average of -65.6 mV. On application of 10 microM acetylcholine (ACh) the membrane potential fell to -2 to -8 mV. Following the washout of ACh it returned to values 9-24 mV more negative than before ACh. The membrane potential gradually returned toward the initial level during the ensuing 40-60 min. No hyperpolarization occurred when Na ions were absent during the application and washout of ACh or in the presence of 0.1 mM ouabain. The hyperpolarization was enhanced by replacing the Na ions by Li or Tris ions following an application of ACh in the presence of Na+. The hyperpolarization was suppressed by ouabain irrespective of whether the drug was applied in the presence or absence of Na+. The membrane potential was diminished by reducing [K+] from 4 to 1 mM in the absence of Na+ before ACh, but it was increased by the same procedure by up to 20 mV following the application of ACh. This indicates that the hyperpolarization was not entirely due to a K-depleting action of the Na-K pump at the membrane surface.     

Factors related to the low resting membrane potentials of diseased human atrial muscles

We studied the ionic mechanism of low resting potential (RP) of quiescent "diseased" human atrial fibers. The RP was -49.7 +/- 0.8 mV (n = 179) in normal Tyrode's solution (5.4 mM [K]o, 36 degrees C). The changes in RP measured at various levels of [K]o appeared to fit the RP-[K]o relationship predicted by the Goldman-Hodgkin-Katz equation, assuming PNa/PK ratio (alpha) to be 0.102 and [K]i to be 131.9 mM. The alpha far exceeded the normal value (about 0.01) by a factor of 10. Acetylcholine (ACh, 10 microM) led to marked increases in the RP. An application of tetrodotoxin (TTX, 6 microM) and perfusion with low [Na]o (10% of the control) media in the presence and absence of ACh produced considerable hyperpolarizations of the RP. These findings indicate that increased alpha value is due to a combination of decreased PK and increased PNa. Applications of ouabain (5 microM) and a cooling procedure (12.3 degrees C) depolarized the membrane, whereas epinephrine (1 microM) hyperpolarized it. Transient hyperpolarization, which exceeded the steady state levels of RP at 5.4 mM [K]o, was observed with perfusing of 5.4 mM [K]o media following perfusion with K-free media. These findings suggest that electrogenic Na pump current plays a significant role in the maintenance of the RP. In conclusion, partial depolarization of "diseased" human atrial fibers was attributed to both decreases in membrane K+ conductance and increases in Na+ conductance. The electrogenic outward pump current seemed to protect the fibers from severe depolarization produced by the conductance abnormality (increased PNa/PK).     

Effects of nephrectomy and KC1 on transmembrane potentials, intracellular electrolytes, and cell pH of rat muscle and liver in vivo

1. The ability of 24 hr nephrectomy and KCl to raise plasma K(+) concentration was used to correlate transmembrane resting potential (RP), ionic gradients, and cell pH (DMO method) in rat muscle and liver in vitro.2. Effects of 24 hr nephrectomy on electrolytes included a rise in plasma K(+) and fall in Na(+), with a rise in intracellular K(+) and fall in intracellular Na(+) in both liver and muscle. Intracellular Cl(-) concentration rose in muscle and decreased in liver.3. Measured muscle RP was decreased from -91 to -77 mV by 24 hr nephrectomy, with the RP predictable from the Goldman equation for Na(+) and K(+) with P(Na)/P(K) = 0.01 and Cl(-) behaving as if passively distributed.4. Measured hepatic RP was increased from -43 to -48 mV by 24 hr nephrectomy, with a change in ionic permeability or activation of an electrogenic pump necessary to explain the results.5. Plasma acid-base changes consisted of metabolic acidosis with partial respiratory compensation. Cell pH rose slightly in both liver and muscle; the H(+) gradient remained constant in muscle but increased slightly in liver.6. KCl was injected into intact rats while the RPs were continuously measured in muscle or liver. Muscle RP was found to decrease and hepatic RP to increase along a similar time course.7. Infusion of KCl into the portal vein led to an increase in the hepatic RP for values of hepatic venous K(+) of 15-25 mM, whereas infusion sufficient to increase the hepatic venous K(+) concentration to 30-40 mM produced no change or a slight decrease in hepatic RP.8. The rat muscle RP can be adequately described by the Goldman equation for Na(+) and K(+), whereas the hepatic RP may well have both diffusion and electrogenic components which respond differently to an increase in plasma K(+) concentration.     

Synchronized oscillatory activity in leech neurons induced by calcium channel blockers.

1. Leech ganglia were superfused with salines in which Ca2+ was replaced with equimolar concentrations of Co2+, Ni2+, or Mn2+. These salines elicited rhythmic membrane potential oscillations with cycle periods ranging from 8 to 25 s in all neurons examined within the ventral nerve cord. 2. Rhythmic activity consisted of a rapid depolarization to a prolonged (3-6 s) plateau level, followed by a rapid repolarization. Each depolarization elicited a burst of action potentials. Peak-to-trough amplitudes of the plateau depolarizations were up to 40 mV in some cells. The plateau depolarizations were separated by slowly depolarizing ramp potentials. 3. Oscillations in all neurons were synchronized (in phase) both within individual ganglia and between ganglia linked by connective nerves. Rhythmic activity in isolated ganglia persisted after the interposed connective nerves were cut. 4. The occurrence of oscillatory activity was strongly correlated with the block of chemical synaptic transmission. 5. Electrotonic interactions persisted during oscillatory activity and may be one mechanism by which oscillations are synchronized. 6. The phase of rhythmic impulse bursts monitored with extracellular electrodes could be reset by electrical stimulation of connective nerves but not by injection of current pulses into individual neurons. Phase reset appeared to occur within one cycle and to a fixed phase point (plateau termination). 7. Oscillatory activity was eliminated by 75-100% reductions of [Na+]o (Na+ replaced with N-methyl-D-glucamine). Smaller reductions of Na+ (by 25-50%) increased the cycle period of oscillations. 8. The Na(+)-K+ pump inhibitors ouabain and strophanthidin disrupted oscillations. Cells were depolarized by approximately 20 mV and fired tonically. After the initial washout of the inhibitors, cells repolarized and became quiescent. After several minutes of continued washing, oscillatory activity resumed. 9. A conceptual model is proposed to explain the mechanisms underlying oscillatory activity induced by Ca2+ channel blockers. According to this model, depolarizing plateaus are generated by a noninactivating Na+ conductance. Na+ influx during the plateau leads to an increase in [Na+]i, which activates an electrogenic Na(+)-K+ pump that contributes to plateau termination. 10. A quantitative computer simulation incorporating six types of currents (capacity, outward rectifying potassium, inward rectifying potassium, sodium, leakage, and an electrogenic sodium pump) demonstrates the plausibility of the conceptual model. 11. These data suggest that a novel Na(+)-based mechanism for membrane potential oscillation is revealed by blockade of Ca2+ channels in leech ganglia.     

Mechanisms of neuronal hyperexcitability caused by partial inhibition of Na+-K+-ATPases in the rat CA1 hippocampal region

Extra- and intracellular records were made from rat acute hippocampal slices to examine the effects of partial inhibition of Na(+)-K(+)-ATPases (Na(+)-K(+) pumps) on neuronal hyperexcitability. Bath application of the low-affinity cardiac glycoside, dihydroouabain (DHO), reversibly induced interictal-like epileptiform bursting activity in the CA1 region. Burst-firing was correlated with inhibition of the pumps, which was assayed by changes in [K(+)](o) uptake rates measured with K(+)-ion-sensitive microelectrodes. Large increases in resting [K(+)](o) did not occur. DHO induced a transient depolarization (5-6 mV) followed by a long-lasting hyperpolarization (approximately 6 mV) in CA1 pyramidal neurons, which was accompanied by a 30% decrease in resting input resistance. Block of an electrogenic pump current could explain the depolarization but not the hyperpolarization of the membrane. Increasing [K(+)](o) from 3 to 5.5 mM minimized these transient shifts in passive membrane properties without preventing DHO-induced hyperexcitability. DHO decreased synaptic transmission, but increased the coupling between excitatory postsynaptic potentials and spike firing (E-S coupling). Monosynaptic inhibitory postsynaptic potential (IPSP) amplitudes declined to approximately 25% of control at the peak of bursting activity; however, miniature TTX-resistant inhibitory postsynaptic current amplitudes were unaffected. DHO also reduced the initial slope of the intracellular excitatory postsynaptic potential (EPSP) to approximately 40% of control. The conductances of pharmacologically isolated IPSPs and EPSPs in high-Ca/high-Mg-containing saline were also reduced by DHO by approximately 50%. The extracellular fiber volley amplitude was reduced by 15-20%, suggesting that the decrease in neurotransmission was partly due to a reduction in presynaptic fiber excitability. DHO enhanced a late depolarizing potential that was superimposed on the EPSP and could obscure it. This potential was not blocked by antagonists of NMDA receptors, and blockade of NMDA, mGlu, or GABA(A) receptors did not affect burst firing. The late depolarizing component enabled the pyramidal cells to reach spike threshold without changing the actual voltage threshold for firing. We conclude that reduced GABAergic potentials and enhanced E-S coupling are the primary mechanisms underlying the hyperexcitability associated with impaired Na(+)-K(+) pump activity.     

Role of Na,K pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle

BACKGROUND AND AIM: In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na(+),K(+) pumps in force recovery after EP. METHODS: Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100-800 V cm(-1), and changes in tetanic force, Na(+),K(+) contents, membrane potential, (14)C-sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na(+),K(+) pump stimulation or inhibition were followed. RESULTS: Electroporation caused voltage-dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K(+) and gain of Na(+), which was not suppressed by tetrodotoxin, but associated with increased (14)C-sucrose space and release of LDH. In soleus, EP at 500 V cm(-1) induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na(+),K(+) transport by adrenaline, the beta(2)-agonist salbutamol, calcitonin gene-related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183-433% and steady-state force level by 104-143%. These effects were blocked by ouabain (10(-3) m), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol. CONCLUSION: Electroporation induces reversible depolarization, partial rundown of Na(+),K(+) gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na(+),K(+) pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na(+),K(+) pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.     

Contributions of the sodium pump and ionic gradients to the membrane potential of a molluscan neurone

1. The membrane potential of the gastro-oesophageal giant neurone of the marine mollusc, Anisodoris nobilis, was examined during changes of temperature and of the ionic medium.2. The response of the membrane potential to rapid changes in the external K concentration was prompt, stable, and reversible up to 200 mM-K, and was independent of the external Cl concentration.3. Warming the cell produced a prompt hyperpolarization that was approximately 10 times greater than predicted by the Nernst or constant field equations. Electrogenic activity of the Na-K exchange pump was shown to be responsible for this effect.4. At temperatures below 5 degrees C, the relationship between the membrane potential and the external K concentration could be predicted by a constant field equation.5. At temperatures above 5 degrees C, the membrane potential could not be predicted by the constant field equation except after inhibition of the electrogenic Na pump with ouabain or the reduction of internal Na.6. Inhibition of the electrogenic Na pump by low external K concentrations was dependent upon the external Na concentration.7. It is concluded that the membrane potential is the sum of ionic and metabolic components, and that the behaviour of the ionic component can be predicted by a constant field type equation.     

Metabolism and the electrical activity of anoxic ventricular muscle

1. The action potential duration of anoxic guinea-pig ventricular muscle was related to ATP generated by glycolysis. In 50 mM glucose medium the action potential duration was maintained; in 5 mM glucose medium the action potential duration shortened, the glycolytic rate declined and the ATP content was reduced.2. The action potential amplitude was related to the metabolic state of the muscle but not to the intracellular sodium concentration.3. It is suggested that changes in the action potential duration and overshoot in anoxic muscle may be due to an influence of metabolism on the slow inward current.4. Anoxic muscle incubated for 8 hr in 5 mM glucose medium had an E(m) of -77.1 mV compared to -81.1 mV in fresh muscle. The calculated E(k) of anoxic muscle was -47.4 mV.5. The resting potential of anoxic muscle was separated into two components, one dependent on potassium distribution and the other on the activity of an electrogenic sodium pump.6. The electrogenic pump component was stimulated upon raising the glucose concentration of the medium or upon raising the external potassium concentration.7. The electrogenic pump component was inhibited by ouabain or by reduction of the temperature from 35 to 8 degrees C.     

Effects of electrogenic sodium pumping on the membrane potential of longitudinal smooth muscle from terminal ileum of guinea-pig

1. The membrane potential of the separated longitudinal muscle of the guinea-pig terminal ileum was recorded intracellularly with glass micro-electrodes.2. In tissues kept at room temperature and then brought to 35 degrees C for 15-30 min or about 1 hr, the fall in membrane potential upon changing to potassium-free solution was 21.4 +/- 3.5 mV and 13.4 +/- 1.8 mV respectively. Ouabain (1.7 x 10(-6)M) produced a fall in membrane potential of 8.1 +/- 1.1 mV. Returning potassium to potassium-free solution, or changing from ouabain-containing to ouabain-free solution, resulted in an increase in membrane potential which was greater than the initial fall.3. Readmitting potassium to potassium-free solution produced an increase in membrane potential which began within 10 sec and reached a maximum within 15-30 sec. This response was reduced, abolished, or converted to a depolarization by ouabain. In chloride-deficient (13 mM) solution in which membrane resistance was increased, the response to readmitting potassium was increased 2(1/2)-fold so that the membrane potential sometimes exceeded -100 mV, which was probably more negative than E(K). On the basis of these results it was assumed that the response to readmitting potassium was due to the electrogenic activity of the sodium pump.4. The response to briefly readmitting a fixed concentration of potassium increased during the first 30 min in potassium-free solution. This increase was not due to an increase in membrane resistance as this fell with time in potassium-free solution. It was suggested that the increase in the response resulted from the progressive rise in internal sodium concentration which is known to occur in smooth muscle in potassium-free solution.5. Increasing the concentration of potassium over the range approximately 0.1-20 mM, increased the size of the electrogenic potential observed upon readmitting potassium to potassium-free solution. There was a fall in membrane resistance upon readmitting potassium (0.6, 5.9, or 20 mM) which was greater the larger the concentration of potassium. When allowance was made for the fall in membrane resistance, the dependency of the electrogenic response upon the concentration of potassium over the range 0.6-20 mM was much increased.6. The results indicate that the rate of electrogenic sodium pumping in this tissue is increased by increasing the external potassium concentration, and probably by increasing the internal sodium concentration. It was suggested that a rise in the latter could sensitize the pump to an increase in the former.     

Parotid acinar cells: ionic dependence of acetylcholine-evoked membrane potential changes

Segments of mouse parotid were placed in a superfusion chamber. Surface acini were impaled by one or two micro-electrodes for measurement of membrane potential and resistance. The acinus under investigation was stimulated by micro-iontophoretic application of acetylcholine (ACh) or adrenaline.Neighbouring acinar cells were electrically coupled. Electrical coupling between acinar cells only occurred within restricted domains probably corresponding to an acinus or a group of acini.Passing direct current through one intracellular electrode, the resting potential of an acinus could be set at desired levels and the dependency of the ACh-evoked potential change on the resting potential investigated. The ACh null potential (initial effect) was about –60mV. A delayed hyperpolarizing effect of ACh could not be reversed.The initial ACh-evoked potential change was sensitive to alterations in extracellular Na, K and Cl concentration. The delayed ACh-evoked hyperpolarization was blocked by ouabain, exposure to Na-free or K-free solutions.It is concluded that ACh increases mainly K and Na membrane conductance causing K efflux and Na influx with a subsequent Na activation of an electrogenic Na pump.     

Membrane potential and resistance measurement in acinar cells from salivary glands in vitro: effect of acetylcholine

1. Cell membrane potential and input resistance measurements were made on segments of submaxillary glands from mice, rabbits or cats placed in a tissue bath, which was perfused with physiological salt solutions.2. During exposure to a standard Krebs-Henseleit solution, ACh stimulation always evoked a marked decrease in input resistance and time constant. The change in potential evoked by ACh stimulation was either a monophasic hyperpolarization (low resting potential) or a depolarization followed by hyperpolarization (high resting potential).3. Increasing [Ca](o) from 2.56 to 10 mM resulted in an enhanced input resistance. Under this condition it was sometimes possible to obtain current-voltage relations. The relationship was linear in the range -50 to -10 mV. In the absence of extracellular Ca the resting potential was reduced and ACh mostly evoked hyperpolarizations. In those cases when the resting potential remained high biphasic potentials were still observed.4. During exposure to Na-free solutions the resting potential was either unchanged or slightly enhanced. ACh never evoked biphasic potentials, but always large hyperpolarizations.5. In the first period (1 hr) after exposure to a K-free solution ACh normally evoked very large hyperpolarizations, often to more than -100 mV. After several hours of exposure to K-free solution the input resistance gradually increased and ACh evoked a tremendous fall in input resistance and time constant with only a small potential change. Re-introducing control solution, ([K](o) = 4.7) for a short period at this stage, caused a very marked hyperpolarization (about 30 mV) unaccompanied by a change in input resistance and time constant.6. Replacing extracellular Cl by SO(4) hyperpolarized the cell membrane. ACh mostly evoked hyperpolarization under this condition, but occasionally biphasic potentials were observed. Increasing [K] of the sulphate solution depolarized the cell membrane by about 49 mV per tenfold increase in [K]. In the presence of ACh the membrane behaved as a K-selective membrane with a slope of the linear curve relating membrane potential to [K](o) of 59 mV per tenfold increase in [K](o).7. It is concluded that ACh evokes a marked increase in surface cell membrane permeability of salivary acinar cells. The ACh evoked hyperpolarization is due to an increase in P(K): the depolarization frequently preceding the hyperpolarization is probably mainly related to an increase in P(Na). The membrane Na-K pump can act electrogenically at least under conditions of Na loading.     

Development of neuromuscular transmission in a larval tunicate

1. The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo.2. The fertilized tunicate egg was incubated in natural sea water at 9 degrees C. Sixty-eight hr after fertilization the free-swimming larva was hatched, which had six striated muscle fibres in the tail. The developmental stage of the embryo was indicated by the developmental hours after fertilization.3. The transmitter at the neuromuscular junction in the hatched larva is ACh. (i) Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 x 10(-5)M). (ii) Eserine (5-10 x 10(-7)M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (e.j.c.s). (iii) The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of e.j.c.s. (iv) AChE was present on the muscle membrane surface.4. AChE activity became visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 hr after fertilization, 12 hr before the ACh response appeared).5. The response to iontophoretically applied ACh was present at 39 hr after fertilization but could not be evoked at 38 hr.6. Between 39 and 41 hr after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching.7. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane.8. The first sign of neuromuscular transmission was appearance of a giant excitatory junctional potential (e.j.p.) with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant e.j.c. was 23.4 +/- 6.9 msec, mean and S.D., at -60 mV and 11 degrees C).9. Within a few hours, these giant e.j.p.s disappeared and were successively replaced by medium-sized e.j.p.s and then e.j.p.s similar to those seen in hatched larvae (time constant of the falling phase of a miniature e.j.c. was 8.5 +/- 1.8 msec at -60 mV and 11 degrees C).     

Depolarizing effects of anoxia on pyramidal cells of rat neocortex.

The response of rat neocortical pyramidal neurons (layers II III) in vitro to brief periods of anoxia is a reversible depolarization of 3.8 +/- 1.01 mV (mean +/- S.E.M.; n = 114), which is accompanied by a moderate decrease in input resistance and significant depression of evoked synaptic activity. This effect is mimicked by ouabain, and is partially attenuated by the excitatory amino acid (EAA) antagonist, kynurenic acid. The estimated reversal potential (Vrev) for the anoxic depolarization (AD) is between -35 and -40 mV; in the presence of TTX a Vrev of -65 mV is obtained. Although a partial failure of Na(+)-K(+) pump activity and release of EAAs may contribute the generation of the AD, other processes are likely to be involved.     

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system.

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.