Secretory transmembrane potentials in acinar cells from the cat submandibular gland during perfusion with a chloride-free sucrose solution

Summary The cat submandibular gland was perfused with a normal NaCl Locke solution and a chloride-free sucrose solution. The numerical increase in acinar membrane potential (secretory potential) was recorded after intra-arterial injection of acetylcholine.There was no significant difference between the size of the secretory potentials recorded during perfusion with the sucrose solution [23.6 mV±1.4 (n=23)] and the size of those recorded during the control periods [20.6 mV±1.2 (n=24)].The maximal value of the membrane potential after injection of acetylcholine was higher [51.8 mV±2.4 (n=23)] during perfusion with the sucrose solution than during the control periods [44.8 mV±1.8 (n=22)].The results show that a pump transporting chloride into the acinar cells cannot be responsible for the generation of the secretory potentials. The results are best accounted for by assuming that an outward passive transport of potassium, being partly short-circuited by an inward passive sodium transport, is responsible for the change in membrane potential after stimulation with acetylcholine.Supported by the Danish State Research Foundation and Johann and Hanne Weimann's legate.     

Membrane potential measurement in parotid acinar cells

1. Intracellular recording of membrane potential was made from acinar cells of the isolated mouse parotid gland superfused with physiological salt solutions.2. The mean acinar resting membrane potential was - 68.5 mV during superfusion with Krebs-Henseleit solution. Shift of the superfusion solution to one containing ACh or adrenaline (10(-5)M) always caused a transient hyperpolarization (about 10-15 mV).3. The membrane potential was mainly dependent on the extracellular K concentration ([K](o)). Increasing [K](o) tenfold decreased the membrane potential by 50 mV. This depolarization was not mediated by ACh release from depolarized nerve endings, since it was seen in the presence of atropine (1.4 x 10(-6)M) and not caused by the accompanying reduction in [Na](o) to 40 mM caused only a small depolarization (less than 10 mV).4. When the superfusion solution was shifted, during intracellular recording, from a normal Krebs-Henseleit solution ([K] = 4.7 mM) to a K-free solution, a hyperpolarization of about 8 mV was measured. Reintroduction of the normal K-containing solution after a longer period of K deprivation (30-70 min) resulted in a short-lasting pronounced hyperpolarization (about 20 mV) which could be blocked by Strophanthin-G (10(-3)M).5. In contrast to previous reports, the present findings indicate that the membrane potential of salivary acinar cells is similar, with respect to magnitude and K-dependence, to that of cells of more thoroughly investigated tissues, such as muscle and nerve, and that the membrane Na-K pump is electrogenic, at least when the cells have been loaded with Na.     

The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres

1. The intracellular Na activity of sheep heart Purkinje fibres has been measured using recessed-tip Na(+)-sensitive glass micro-electrodes.2. The internal Na activity was 7.2 +/- 2.0 mM (mean +/- S.D., n = 32) at the normal external Na concentration, [Na](o), in these experiments of 140 mM (equivalent to an external Na activity of 105 mM). The equilibrium potential for Na across the fibre membrane was therefore approximately + 70 mV.3. When the [K](o) was altered the internal Na activity changed, reaching a new level within about 20 min. Increasing the [K](o) from 4 to 25 mM decreased the internal Na by approximately 30%, while decreasing the [K](o) from 4 to 1 mM increased internal Na by 20%.4. The removal of external K produced an easily reversible increase in the internal Na with an initial rate equivalent to a concentration change of 0.24 +/- 0.07 m-mole/min (mean +/- S.D., n = 8).5. Ouabain produced increases in the internal Na activity that were only very slowly reversible. The threshold concentration for producing an increase was approximately 10(-7)M.6. When [Na](o) was reduced the internal Na activity fell rapidly with a single exponential time course (time constant 3.3 +/- 0.8 min, mean +/- S.D., n = 16) to a new, relatively stable level. The recovery of internal Na on return to the normal [Na](o) did not have a simple time course. It was normally complete within 10-30 min.7. The relationship of the stabilized level of the internal Na activity to the [Na](o) was approximately linear over the range 140-14 mM-[Na](o). When [Na](o) was reduced from 140 to 14 mM the internal Na activity fell by 72 +/- 5% (mean +/- S.D., n = 21).8. When the [Na](o) was reduced, the decrease in the internal Na activity was partially inhibited by Mn or by removal external Ca.9. When the [Ca](o) was altered over the range 0.2-16 mM the internal Na activity was reduced by approximately 50% for a tenfold increase in the [Ca](o).10. The relationship between internal Na and contractility is discussed.     

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.     

Changes of intracellular sodium and potassium ion concentrations in isolated rat superior cervical ganglia induced by depolarizing agents

1. Na and K contents of isolated rat superior cervical ganglia were measured by flame photometry, and intracellular Na and K concentrations ([Na](i) and [K](i)) calculated using Li and (35)SO(4) to determine extracellular space (e.c.s.).2. Resting concentrations after 1-2 hr incubation at 25 degrees C in normal Krebs solution were: [Na](i), 19.8 +/- 0.9 m-mole (kg cell water)(-1); [K](i), 192.7 +/- 2.8 m-mole (kg cell water)(-1) (mean +/- S.E. of mean of thirty-five ganglia). Correction for losses during e.c.s. measurement gave 22 mM [Na](i) and 207 mM [K](i) as probable fresh concentrations.3. Carbachol (180 muM for 4 min) increased [Na](i) by 47.8 +/- 2.9 m-mole (kg cell water)(-1) and decreased [K](i) by 54.6 +/- 4.3 m-mole (kg cell water)(-1). Maximal exchange with carbachol or nicotine (at approximately 1 mM for 4 min) amounted to 80-100 m-mole (kg cell water)(-1). On washing with Krebs solution containing 2.5 mM hexamethonium recovery of ionic concentrations occurred with a rate constant of 0.3-0.4 min(-1).4. Restitution of ganglionic Na and K after carbachol was inhibited by washing with K-free solution, and slowed by ouabain (0.14 mM), cyanide (2 mM) or cooling (Q(10) 2.7 between 17 and 27 degrees C).5. Equilibrium potentials for Na and K (E(Na), E(K)) at rest were calculated to be +49 and -88 mV. At a membrane potential (E(m)) of -70 mV, the permeability ratio P(Na):P(K) was calculated at 0.04:1 (assuming P(Cl):P(K) < 0.1).     

Pancreatic acinar cells: ionic dependence of the membrane potential and acetycholine-induced depolarization

1. Intracellular recordings of membrane potentials have been made in vitro from the exocrine acinar cells of the mouse pancreas using glass micro-electrodes.2. The mean membrane potential of the acinar cells during superfusion with Krebs-Henseleit solution was -39.2 mV. Increasing [K](o) tenfold decreased the membrane potential by 28 mV when [K](o) was above 10 mM. This depolarization was not affected by atropine (1.4 x 10(-6)M). Strophanthin-G (10(-3)M) slowly depolarized the cells at about 10 mV hr(-1).3. Brief exposure to acetylcholine (ACh), 5.5 x 10(-5)M, or pancreozymin resulted in a short lasting depolarization of the acinar cells. Atropine (1.4 x 10(-6)M) blocked the depolarizing action of ACh but not that of pancreozymin. Adrenaline (5.5 x 10(-5)M) or cyclic AMP (10(-3)-10(-4)M) did not influence the membrane potential.4. The amplitude of the ACh-induced depolarization was not dependent on the presence of CO(2)/HCO(3) in the bathing fluid, but it was closely dependent on the extracellular Na concentration. However, ACh was still able to evoke a small depolarization even after prolonged exposure of the tissue to a Na-free solution.5. During exposure of the tissue to a Ca-free solution the resting membrane potential was decreased and the ACh-induced depolarization was significantly reduced. Some substances which are known in other tissues to inhibit membrane Ca(2+) currents, i.e. La(3+), D-600 and tetracaine, were able to reduce, but never abolish, the ACh-induced depolarization.6. These results suggest that the effect of ACh on the pancreatic acinar cell is to increase the permeability of the membrane to commonly occurring ions with a consequent Na-influx and a small Ca-influx.     

Formation of saliva and potassium transport in the perfused cat submandibular gland

1. In the cat submandibular gland perfused with modified Locke solutions, salivary secretion during acetylcholine (ACh) infusion and K uptake from the perfusion fluid after the ACh-induced K loss were measured.2. Strophanthin G (10(-5)M) abolished K uptake, whereas salivary secretion was unaffected.3. Ethacrynic acid (10(-4)-2 x 10(-4)M) hardly affected K uptake whereas salivary secretion was severely inhibited.4. During perfusion with Ca-free solution K uptake was unaffected, whereas salivary secretion was severely reduced.5. The presence or absence of CO(2)/HCO(3) in the perfusion fluid was of no importance for the secretory process and the K uptake. Acetazolamide (2 x 10(-4)-10(-3)M) did not inhibit these transport processes.6. It is suggested that two kinds of Na transport occur in the acinar cells of the salivary glands: a Na extrusion coupled with K uptake, responsible for the maintenance of the concentration gradients across the cell membrane; and a Na transport coupled with Cl transport into the acinar lumen, responsible for the formation of the primary secretion.     

Voltage clamp experiments on ventricular myocarial fibres

1. A voltage clamp method utilizing a sucrose gap and glass microelectrodes was developed and used to study dog ventricular myocardial fibre bundles. The limitations and the reliability of this method are demonstrated by a series of tests.2. A dynamic sodium current, excited at membrane potentials more positive than -65 mV, was measured. The equilibrium potential for this large, rapid inward current depends directly on [Na](o), shifting 29.0 +/- 2.3 mV (+/- S.E. of mean), as opposed to a theoretically expected value of 30.6 mV, when [Na](o) is reduced to 31% of normal.3. Sodium current is inactivated by conditioning depolarizations. Complete inactivation occurs with conditioning potentials more positive than -45 mV, and 50% inactivation occurs at about -55 mV. The location of the inactivation curve shifts along the voltage axis, when [Ca](o) is varied between 0.2 and 7.2 mM.4. A second, much smaller and slower net inward current, with a threshold around -30 mV, and an equilibrium potential above +40 mV was also observed.5. The ;steady-state' current-voltage relationship (after 300-600 msec) exhibits inward-going (anomalous) rectification with negative slope between -50 and -25 mV.6. A small, very slowly developing component of outward current was observed at inside positive potentials. The equilibrium potential for this current, although slightly dependent on [K](o), is neither identical with the potassium equilibrium potential nor with the resting potential in normal Tyrode solution.7. Anatomical limitations, primarily resistance in the extracellular space within the bundle, prevent complete characterization of the rapid, large sodium current, but do not limit the application of the clamp method to the study of other, smaller and slower currents. The evidence for this is discussed extensively in the Appendix.     

Electrical activity in pancreatic islet cells: effect of ions

1. Intracellular micro-electrode recording techniques have been used to study the effects of varying the external ion concentration on the membrane potential and glucose-induced electrical activity in cells from mouse islets of Langerhans.2. Increasing [K](o) to 47 mM depolarized islet cells without inducing electrical activity. Normal action potentials were generated in response to glucose after removal of [K](o) for 60 min.3. Reduction of [Cl](o) to 12 mM did not affect the membrane potential and glucose still induced electrical activity.4. Reduction of [Na](o) to 26 mM increased the amplitude of action potentials; subsequently the cells depolarized.5. Removal of [Ca](o) caused cells, already firing action potentials intermittently in response to glucose, to change their pattern of discharge to one of continuous firing. The time constant of the action potentials was also increased. Depletion of calcium for 60 min before addition of glucose prevented the appearance of electrical activity.6. Increasing [Ca](o) threefold to 7.7 mM in the presence of glucose, 11.1 mM, increased action potential amplitude to 12 mV, but a tenfold increase of [Ca](o) to 25.6 mM completely blocked action potential discharge.7. Exposure of islet cells simultaneously to reduced [Na](o) (26 mM) and increased [Ca](o) (7.7 mM) increased the amplitude of glucose-induced action potentials to about 20 mV.8. Strontium, 2.56 mM, was an effective substitute for [Ca](o), 2.56 mM, and maintained a normal pattern of electrical activity in response to glucose.9. Increasing the magnesium concentration tenfold to 11.3 mM did not block electrical activity whereas manganese, 2 mM, blocked glucose-induced action potentials and depolarized islet cells.10. It was concluded that the action potential induced by glucose in islet beta-cells is due predominantly to calcium entry and that sodium ions tend to repress this calcium influx.     

Membrane potential measurement in cells of the adrenal gland

1. Recordings of transmembrane potentials have been made in vitro from the cells of the adrenal gland using glass micro-electrodes.2. There was only a small species variation in the mean membrane potential of the cortical cells of the rabbit, rat and kitten; 66.2, 70.5, and 71.4 mV respectively.3. The membrane potential of cortical cells was dependent upon the external potassium concentration, [K](o). Raising [K](o) above the normal concentration of 4.7 mM by addition of KCl decreased the membrane potential; lowering [K](o) from normal increased it. The decrease in membrane potential was still evident when chloride was replaced by sulphate. Increasing [K](o) 10-fold decreased the membrane potential of rabbit cortical cells by 44 mV and of kitten cortical cells by 50 mV.4. The mean membrane potential measured in medullary chromaffin cells was for the rabbit 24.2 mV, rat 20 mV, and kitten 31.7 mV. The potentials of medullary cells were much less affected by changes in [K](o) than were cortical cell potentials.5. Age had little influence upon either cortical or medullary membrane potentials of adrenal glands, at least in early life.     

Intracellular concentrations of sodium, potassium and chloride in the salt-gland of the domestic goose and their relation to the secretory mechanism

1. The composition of the nasal salt-glands of geese was found to be Na 57 +/- 3.5 (S.E.), K 52.3 +/- 3.9 and Cl 78.3 +/- 11.0 m-equiv/kg fresh tissue. During secretion, the Na content was significantly raised to 72.4 +/- 3.4 m-equiv/kg.2. Salt-gland slices incubated in Krebs-Henseleit bicarbonate medium plus glucose (6 mM), in the presence of [(14)C]sucrose as an extracellular marker had the following composition, Na 85.3 +/- 3.1, K 37.1 +/- 3.1 and Cl 74.3 +/- 3.6 m-equiv/kg. The calculated intracellular concentrations were for Na 61.5 +/- 2.1, K 105.3 +/- 8.7 and Cl 37.8 +/- 5.0 m-equiv/l. intracellular water.3. Ouabain (10(-4)M) significantly decreased the tissue and cell K concentration and significantly increased the Na concentration.4. Acetylcholine (10(-6)M) and eserine (10(-4)M) in the incubation medium had no effect on intracellular composition.5. Raising the Na concentration of the medium to 172 m-equiv/l. and the Cl to 156 m-equiv/l. in two experiments had no effect on the calculated intracellular composition.6. These results do not support reports that the cells have a very high Na concentration (about 350 m-equiv/l. intracellular water). They are compatible with the hypothesis that the hypertonic secretion is formed across the luminal membrane of the secretory cell by an active Na(+) pump and there are no data to suggest that Na(+) is concentrated across the basal membrane by a ouabain-insensitive process.7. The data are discussed in relation to permeability studies and to electrical potential measurements within the gland by other workers.     

Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane calcium current in ventricular myocardial fibres

1. A slow inward current in ventricular preparations of the dog heart can be measured by the voltage clamp method without interference from the initial rapid sodium current if the sodium system is inactivated by conditioning depolarization.2. The slow inward current is very sensitive to variation in [Ca](o). It occurs above the equilibrium potential of I(Na) immediately after changing the bathing fluid to a sodium-free solution and persists under this condition for a long time without much alteration, while I(Na) is rapidly abolished. Tetrodotoxin and [Mg](o) have no effect on this current component. These results strongly support the view that the slow inward current in cardiac tissue is carried by calcium ions.3. The threshold for initiation of the calcium current is around -35 mV in Tyrode solution and is shifted to more negative potentials by either increasing [Ca](o) or reducing [Na](o).4. Calcium sensitive inward current tails associated with repolarization are assumed to represent a proportional measure of calcium conductance activated during the preceding depolarization. Calcium conductance declines rapidly with time in the inside negative potential range and slowly at positive potentials. The time constants for this ;inactivation' process vary between 40 and 700 msec in the potential range -35 to +50 mV.5. By using instantaneous current-voltage relations the reversal potential of calcium current was estimated to be about +60 mV in normal Tyrode solution. As shown in the Appendix, however, the calcium equilibrium potential cannot be considered to be constant.6. The importance of the calcium current for the plateau of the cardiac action potential is discussed.     

Effects of nerve cross-union on rat intracellular potassium in fast-twitch and slow-twitch rat muscles

1. Electrolytes of normal, self-innervated and cross-innervated extensor digitorum longus and soleus muscles of rats have been determined.2. [K](i) was 173 m-equiv/l. for both normal and self-innervated extensor digitorum longus. In cross-innervated extensor digitorum longus it was reduced to 159 m-equiv/l.3. For normal and self-innervated soleus, [K](i) was 150 m-equiv/l. and 154 m-equiv/l. respectively. In cross-innervated soleus it was increased to 182 m-equiv/l.4. The content and distribution of most other electrolytes of cross-innervated soleus, as well as its weight, were not significantly different from those of controls. On the other hand, cross-innervated extensor digitorum longus weighed about half as much as controls and contained markedly elevated Na(+), Cl(-) and extrafibre water and reduced non-collagenous protein and intrafibre water.5. It is concluded that [K](i) of fast-twitch and slow-twitch muscle fibres are under neural regulation. Possible mechanisms for this regulation are discussed.     

Ionic permeability changes as the basis of the thermal dependence of the resting potential in barnacle muscle fibres

1. The thermal dependence of the resting potential of isolated barnacle muscle fibres was larger (1-2 mV/ degrees C) than predicted by Nernst's equation (about 0.2 mV/ degrees C). A comparative study was made of the influence on thermal dependence of parameters related to (a) passive permeability and to (b) Na extrusion.2. High [K](o) decreased the thermal dependence reversibly. [K(i)], [Na](i) and [Cl](i) were determined by chemical analysis, and Goldman's equation was fitted to data relating V to [K](o) at different temperatures, in the presence and absence of ouabain 5 x 10(-5)M. In both cases the behaviour of V when T was lowered from 20 to 4 degrees C was accounted for by increases in the calculated P(Na/PK) and P(Cl/PK) (from 0.006 to 0.043 and from 0.17 to 0.34 on the average, respectively.)3. Other parameters related to passive permeability (and which caused reversible depolarization): decreased [Cl](o) (methanesulphonate or gluconate substituted), and decreased pH(o) (below 5.0), also decreased the thermal dependence reversibly.4. Inhibitors (ouabain 5 x 10(-5)M, cyanide 2-10 x 10(-3)M, 2,4-dinitrophenol 2 x 10(-4)M) externally applied did not affect either resting potential or its thermal dependence for several hours.5. Increasing [Na](i) three- to fourfold by intracellular injection decreased both resting potential and its thermal dependence.6. Although a small effect by a Na electrogenic pump cannot be excluded, the largest part of the thermal effect on the resting potential is concluded to depend on temperature-induced variations in relative ionic permeabilities to cations and anions. A model is proposed which can account for the data assuming that (a) each permeant ion associates to a separate site in the membrane, and (b) the ion-site equilibrium is temperature-dependent.     

Some factors influencing stimulation-induced release of potassium from the cat submandibular gland to fluid perfused through the gland

1. The release of K from the cat submandibular gland to the extracellular fluid (ECF) after stimulation with acetylcholine (ACh) and the subsequent uptake of K from the ECF was studied in glands perfused artificially with Locke solutions.2. The first injection of ACh after shift of the perfusion fluid from control to K-free Locke solution evoked a normal loss of K and a normal secretion of saliva. The second injection only evoked a small release of K and a reduced secretion.3. Perfusion with dinitrophenol (DNP) (10(-4)M) containing solutions, Na-free Li Locke solutions and chloride-free nitrate Locke solutions inhibited salivary secretion and the uptake of K. The first injection of ACh after shift of the perfusion fluid from control to test solution gave a normal K loss, but thereafter the ACh-induced K-loss declined.4. Perfusion with g-strophanthin (10(-5)-10(-4)M) always inhibited K uptake whereas K release was not affected primarily. The sensitivity of the secretory mechanism of different glands to strophanthin varied considerably.5. Perfusion with tetraethylammonium Locke solution inhibited secretion, K uptake and release of K.6. It is suggested that the release of K from salivary glands to the ECF after stimulation with ACh can be explained by diffusion as a consequence of an enhanced permeability of the cell membranes to K. Concomitantly with the release of K, Na is taken up. It is suggested that the subsequent uptake of K and extrusion of Na is due to active transport processes probably involving a Na-K activated ATP-ase.     

Ionic dependence of adrenal steroidogenesis and ACTH-induced changes in the membrane potential of adrenocortical cells

1. The effects of changes of ionic environment upon corticosteroid production by rabbit adrenal glands have been investigated in vitro using a superfusion technique and on-line steroid analysis by an automated fluorescence method. In some experiments micro-electrode recordings of adrenocortical transmembrane potentials were made concomitantly with measurement of steroid output.2. Adrenocorticotrophic hormone (ACTH), 10 m-u./ml., induced a sevenfold increase in corticosteroid production rate in normal Krebs solution.3. The steroidogenic response to ACTH was not impaired after omission of [K](o) for 1 hr but was inhibited following exposure to K(+)-free medium for 3 hr. Increase of [K](o) tenfold to 47 mM increased the basal but not the ACTH-stimulated output of corticosteroid whereas raising [K](o) twentyfold to 94 mM enhanced both the basal and ACTH-stimulated steroid production rate. In K(+)-free solution the adrenocortical cells hyperpolarized from - 67 to - 86 mV; subsequently on addition of ACTH they depolarized. Reintroduction of K(+) restored the membrane potential.4. Omission of Ca(2+) partially depolarized the cells but only affected the steroidogenic response to ACTH in the presence of EDTA. A threefold increase of [Ca](o), to 7.68 mM, had no effect on either membrane potentials or steroid formation, but increasing [Ca](o) tenfold to 25.6 mM partially blocked ACTH action. Increasing [Mg](o) twentyfold to 22.6 mM had little effect on ACTH-stimulated corticosteroid output and Sr 2.56 mM, in substitution for Ca(2+), supported ACTH action, but La, 0.25 mM, completely blocked the steroidogenic effect of ACTH.5. Replacement of NaCl, 118 mM by choline chloride, 118 mM, was without effect on ACTH-induced steroidogenesis, whereas LiCl, 118 mM, reduced it by 50%. NaF, 1 and 10 mM, inhibited ACTH-induced steroidogenesis by approximately 60%.6. Nupercaine, 10(-4)M, inhibited the steroid response to ACTH with no effect upon membrane potentials: increasing the nupercaine concentration to 10(-3)M inhibited the steroid response and depolarized the cells. Ouabain, 10(-5)M, induced complete depolarization and suppression of the steroidogenic response to ACTH.7. Action-potential-like changes in membrane potential appeared in cells exposed to ACTH in a K(+)-free medium. The amplitude of the action potentials ranged from 10 to 60 mV according to cell, with a frequency up to 36/min; the frequency tended to increase with time. Tetrodotoxin, 10(-6) g/ml., did not inhibit ACTH-induced action potentials in K(+)-free medium.8. These observations are discussed in relation to the ionic requirements for the steroidogenic action of ACTH. The results further emphasize the dissociation of membrane polarization and the secretion of steroid. The mechanism of output of steroid hormone from the adrenocortical cell may thus differ fundamentally from the secretory mechanisms in other, particle-storing cells.     

Whole-cell K+ currents in isolated rabbit corneal epithelial cells.

Membrane currents were recorded from enzymatically isolated cells from basal layers of rabbit corneal epithelium by the whole-cell clamp technique. Pipettes contained 140.4 mM KCl and extracellular K+ concentration was varied. The membrane currents on step voltage changes were rectangular currents with some fluctuations. The fluctuations disappeared near the zero-current potential. The reversal potential in normal Tyrode's solution with 5.4 mM K+ was -57.8 +/- 6.2 mV (mean +/- S.D., n = 10). Increasing [K+]o from 5.4 to 140.4 mM shifted the reversal potentials in the positive direction with a slope of 41.0 mV/decade. Concomitant depolarization of the resting potential was observed on increasing [K+]o. The whole-cell currents were blocked by Cs+ or Ba2+. These suggest that the major current component in the corneal epithelial cells in K+.     

Dependence of endocochlear potential on basolateral Na+ and Cl- concentration: a study using vascular and perilymph perfusion.

Inside-positive endocochlear potential (EP) and high potassium concentration in the endocochlear duct are generated by transepithelial K+ transport in marginal cells of the stria vascularis. In order to estimate the degree of involvement of Na+ and Cl- in K+ transport in marginal cells, EP in guinea pigs was measured under artificial vascular and perilymphatic perfusion in situ. Na+ depletion due to both vascular and perilymphatic perfusion decreased EP by -10.0 +/- 4.1 mV (delta EP = -86 +/- 5.2 mV, n = 5) from the control value of 78 +/- 4.3 mV (p < 0.01). Cl- depletion due to vascular and perilymphatic perfusion also decreased EP by -10.0 +/- 4.9 mV (delta EP = 8.5 +/- 4.8 mV, n = 6) from the control value of 77 +/- 5.1 mV (p < 0.01). However, under either vascular or perilymphatic perfusion, even lowering of Na+ or Cl- concentration in the perfusate decreased EP only slightly compared to the results under both vascular and perilymphatic perfusion. Furosemide, a blocker of Na+/K+/2Cl- symport, decreased EP under vascular perfusion. This dependency of EP on basolateral Na+ and Cl- concentration strongly suggests that K+ transport by the marginal cell is dependent on the basolateral Na+ and Cl- concentration, and that Na+/K+/2Cl- symport is raised as a possible mechanism for Na+ and Cl- dependency of EP.     

Sodium-activated potassium current in guinea pig gastric myocytes

This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.