Depolarization Of Em Research Articles

Acid-induced two-step depolarization in the peritubular membrane of bullfrog kidney proximal tubules.

Using ion-selective microelectrode technique, we investigated acid-induced changes of peritubular membrane potential (EM) and intracellular activity of Na+ and H+ ((Na)i and pHi) in the proximal tubule of perfused bullfrog kidney in vivo. When peritubular pH was reduced at constant PCO2, EM was depolarized in two steps: i.e., an initial sharp fall followed by an additional deeper fall. This was termed as the acid-induced two-step depolarization. During this change, pHi was decreased gradually from 7.4 to 6.9 in response to 1/10 HCO3- reduction (pH from 7.7 to 6.7), whereas (Na)i was increased after a transient decrease. This result supports the peritubular rheogenic HCO3- exit coupled to Na+ movement during the initial depolarization period. Complete removal of peritubular HCO3- at constant pH produced a less marked initial depolarization with a transient rise of pHi, followed by a partial repolarization without appreciable change in (Na)i. Peritubular SITS administration inhibited these depolarization responses to the acid perfusion. The above findings suggest that 1) the initial part of the two-step EM depolarization produced by low HCO3- or HCO(3-)-free perfusion resulted mainly from the peritubular rheogenic exist of HCO3-, 2) the magnitude of initial depolarization would also be affected by pHi, and 3) the subsequent delayed changes of EM were mainly determined by the pHi-sensitive K+ conductance of the peritubular membrane.

The roles of ion fluxes in skeletal muscle fatigue

Intense muscle contractions result in large changes in the intracellular concentrations of electrolytes. The purpose of this study was to examine the contributions of changes in intracellular strong ions to calculated changes in steady-state membrane potential (Em) and muscle intracellular H+ concentration ([H+]i). A physicochemical model is used to examine the origin of the changes in [H+]i during intense muscle contraction. The study used the isolated perfused rat hindlimb intermittently stimulated to contract at high intensity for 5 min. This resulted in significant K+ depletion of both slow (soleus) and fast (white gastrocnemius, WG) muscle fibers and a release of K+ and lactate (Lac-) into venous perfusate. The major contributor to a 12- to 14-mV depolarization of Em in soleus and WG was the decrease in intracellular K+ concentration ([K+]i). The major independent contributors to [H+]i are changes in the concentrations of strong and weak ions and in CO2. Significant decreases in the strong ion difference [( SID]i) in both soleus and WG contributed substantially to the increase in [H+]i during stimulation. In WG the model showed that the decrease in [SID]i accounted for 35% of the increase in [H+]i (133-312 nequiv/L; pHi = 6.88-6.51) at the end of stimulation. Of the main contributors to decreased [SID]i, increased [Lac-]i and decreased [K+]i contributed 40 and 60%, respectively, to increased [H+]i, whereas a decrease in [PCr2-]i contributed to reduced [H+]i. It is concluded that decreased muscle [K+]i during intense contractions is the single most important contributor to reduced Em and increased [H+]i. Depletion of PCr2- simultaneous to the changes in [Lac-]i and [K+]i prevents larger increases in [H+]i and helps maintain the intracellular acid-base state.

Veratridine-induced oscillations in membrane potential of cultured rat skeletal muscle: role of the Na-K pump.

1. The acute effects of veratridine on membrane potential (Em) and Na-K pump activity in cultured skeletal muscle were examined. 2. At a concentration of 10(-4) M, veratridine caused depolarization of Em and a decrease in Na-K pump activity. At concentrations of 10(-5) and 10(-6) M, veratridine caused oscillations of Em and an increase in Na-K pump activity compared to untreated, control cells. The oscillations consisted of depolarization to about -40 mV followed by hyperpolarization to about -90 mV; the level of hyperpolarization was higher at 37 than at 23 degrees C. 3. Veratridine-induced oscillations could be prevented by pretreatment with tetrodotoxin (10(-6) M) and blocked or prevented by ouabain, which depolarizes Em of cultured myotubes. In contrast, depolarization of Em to -60 mV by excess K+ did not alter the amplitude or frequency of the oscillations. 4. The results demonstrate that veratridine-induced increase in Na influx both depolarizes cultured myotubes and increases the activity of the Na-K pump, which repolarizes Em to levels higher than control. This sequence accounts for veratridine-induced oscillations in Em. High concentrations of veratridine cause only depolarization of Em and inhibition of Na-K pump activity.

Effects of dopamine on the transport of Na, H, and Ca in the bullfrog proximal tubule.

To study effects of dopamine (DA, 10(-6) M) on ion transport processes in proximal tubular cells, we used Na(+)-, H(+)-, and Ca2(+)-selective microelectrodes in doubly-perfused bullfrog kidneys. The peritubular membrane potential difference (EM) and cytosolic Na+, Ca2+, and H+ activities ((Na)i, (Ca)i, and pHi) were measured continuously after peritubular administration of DA or dibutyryl-cyclic AMP (db-cAMP, 10(-4) M). Results obtained are as follows. 1) DA induced a decrease of (Na)i by 4.2 mM, but during the luminal perfusion of 0.5 mM Na+ solution, DA decreased (Na)i only by 1.4 mM. 2) DA produced a delayed fall of (Ca)i by 8 nM. 3) DA produced hyperpolarization of EM by 5-7 mV. 4) Peritubular perfusion with high K+ (35 mM) solution produced about 30 mV depolarization of EM. DA added to high K+ perfusate induced 38 mV depolarization. 5) DA increased pHi by 0.07. 6) DA produced almost the same changes in (Na)i, (Ca)i, EM, and pHi as those induced by db-cAMP. 7) Effects of DA on EM and (Na)i were inhibited by dopamine1 (DA1) antagonist. Effects of DA on ion transport in the proximal tubular cells are mediated by the adenylate-cyclase-cAMP system. The activation of this system induces 1) suppression of Na+ entry step across the luminal membrane, 2) an increase of K+ permeability in the peritubular membrane, 3) cytosolic alkalinization with vesicular accumulation of H+ by enhancing H+ pump, and 4) a decrease of (Ca)i possibly by restricting Ca2+ entry across the luminal membrane and activating Ca2+ pump in the peritubular membrane or subcellular organelles.

Assessment of the early cellular membrane response to live Escherichia coli bacteremia

Alterations in skeletal muscle cellular function during septic shock have been previously demonstrated. However, whether these alterations represent a specific response to the septic state or are simply a consequence of low flow is uncertain. The present study was designed to evaluate the cellular membrane response to the early bacteremic state, prior to the onset of hemodynamic compromise. A clinically relevant model of sepsis was achieved in six mongrel dogs by intraarterial infusion of live Escherichia coli organisms and concurrent volume loading with lactated Ringer's solution. Four sham-treated dogs served as controls. Forty-eight hours after induction of sepsis, resting transmembrane potential (Em) was measured in a hindlimb adductor muscle. Contemporaneous muscle biopsy was performed for determination of transmembrane water and electrolyte distribution. The bacteremic state was associated with depolarization of Em to −79.7 ± 1.2 mV from a basal value of −89.3 ± 0.2 mV ( P < 0.01), while Em in the sham-treated group remained unchanged over the same time course. In addition, there was a significant increase in the calculated intracellular Na + and Cl − concentrations in the septic group ( P < 0.02), while intracellular K + was unchanged. These data are consistent with a selective increase in cell membrane permeability to Na + and indicate that cellular alterations in skeletal muscle occur early in the septic course, in the absence of hemodynamic compromise. This alteration in membrane permeability appears to be common to cells of disparate organ systems in response to sepsis, and may represent a protean manifestation of cellular injury.

A chloride conductance activated by adenosine 3',5'‐cyclic monophosphate in the apical membrane of Necturus enterocytes.

1. Intracellular potentials, Cl- activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl- replacement experiments using conventional or Cl- -selective double-barrelled microelectrodes. A Cl- conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion-substitution experiments. 2. The mean mucosal membrane potential (Em) was -35.5 mV. Removal of Cl- from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl- to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low-Cl- solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl- and the preponderance of a K+ membrane permeability in the absence of Cl-. This emphasizes the role of Cl- in establishing Em. 4. Removal of mucosal Cl- produced a fast decrease in intracellular Cl-, as measured with Cl- -selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl- activity, indicating the presence of a large Cl- conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl- which on average corresponds to an approximately 15-fold increase in the relative Cl- permeability. These results are consistent with an activation of apical Cl- conductance. 6. The selectivity of Cl- channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN- greater than I- greater than or equal to Br- greater than NO3- greater than Cl- much greater than HCO3- greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9-anthracene which are potent inhibitors of Cl- channels in other systems failed to block the apical Cl- conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane potential change induced by infection by Phytophthora infestans of potato cells.

Slicing of potato shoot tissue depolarized very soon (possibly almost instataneously) the membrane electropotential (Em) of their cells. Once depolarized Em (about -70--80mV at first) was, however, restored and repolarized up to about -120mV in average in the first several or 1-1.5hr, when measured by inserting microelectrode into the cut surface cell or into inner cell of slice tissue, respectively. This transient depolarization and restoration of Em was mostly due to change of the respiration dependent component (Ep) of Em, which decreased at first and then increased in absolute value. The passive component (Ed) of Em was almost constant (measured with surface cells) or slightly decreased (measured with inner tissue cells) in its absolute value. Several hr after slicing, Ed began to increase in its absolute value up to nearly about -100mV within a day, while Ep, instead, decreased again, resulting in maintenance of nearly constant level of Em with small fluctuations (about -120--130mV for at least one day). The time when Ed began to increase coincided with the time of initiation of wound-responding physiological processes which need precedent activation of genetic system and protein svnthesis. The time when the hypersensitivity potential of potato slice against the infection by an incompatible race of Phytophthora infestans began to rise after slicing, also coincided with this time. The electrophysiological response of potato cell to infection by the incompatible race was roughly classified into rapid and slow types response. In both types, absolute value of Ed began to decrease almost simultaneously with the penetration by the parasite and continued to decrease to -56--57mV in average. The rapid and slow types differed in the response of Ep to the infection. In the rapid response, the infection had little effect on Ep at first, but later gradually decreased its absolute value. In the slow type, Ep increased at first (later decreased) and appeared to compensate the decrease in Ed, resulting in slowing down of depolarization of Em. Electric coupling between a cell infected by the incompatible race and adjacent uninfected cells appeared to be shut off in early stage of infection. The results suggested that plasmodesmata between the infected cell and its adjacent uninfected tissue might be shut off in early stage of infection.

Intracellular Free Calcium Measured with Calcium-Selective Microelectrodes From Salivary Gland Cells of the Snail Planorbis Corneus

ABSTRACT Double-barrelled Ca2+-selective microelectrodes have been used to make simultaneous measurements of the intracellular concentration of free Ca2+ ([Ca2+]i) and membrane potential (EM) from single salivary gland acinar cells of the pond snail Planorbis corneus. The mean [Ca2+]i recorded from nine cells in unstimulated glands was l·24×10−7moll−1 (S.D. ± 0·84×10−7moll−1), while the mean EM was −67·1 ±4·1 mV. The equilibrium potential for Ca2+ in these cells was calculated to be +132mV. Stimulating the glands with 10−4moll−1 acetylcholine (ACh) produced a simultaneous depolarization of EM and an increase in [Ca2+]i by 2·9×10−7moll−1 (S.D. ± 10× 10−7moll−1, N= 12), followed by a transient hyperpolarization of EM and the restoration of [Ca2+]i to its original level. ACh-induced increases in [Ca2+]i were abolished, or much reduced in size, after the removal of extracellular Ca2+, though the EM response to ACh was unaffected in Ca2+-free saline. This finding indicates that the application of ACh leads to an influx of Ca2+ from the medium bathing the glands. Li+ caused an increase in the size of the ACh-induced elevation in the Ca2+ signal and inhibited the recovery of [Ca2+]i following stimulation. The implications of this observation are discussed.

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential.

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.

Responses of Regularly-Firing Aplysia R3-R13 Neurones to Normoxia and Hypoxia

ABSTRACT Exposure of 10 R3-R13 neurones to a 115-min period of hypoxia resulted in depolarization of their membrane potentials (EM) from a mean of −46·9 ± 3·1 to −20·8 ± 4·4mV (S.E.). Intracellular potassium ion activities decreased significantly from 118·9 ± 5·1 to 67·7 ± 8·5 mm-K+. This is equivalent to a change in EK from − 70·9 mV to −54·5 mV, which is insufficient to account for depolarization of approximately 26 mV. During reoxygenation of the saline surrounding the ganglion, there was a continued depolarization of EM to − 11·5 ± 3·2 mV and progressive fall in to 49·2 ± 4·9 mm. Decreases in the membrane slope resistance were also observed in these depolarizing neurones. The depression in resistance remained irreversible for as long as experiments were conducted. Computations of PNa /PK ratios were made using a steady-state calculation. Increases in the PNa/PK ratio from 0·030 to 0·045 were observed during hypoxic depolarization using a modification of the Goldman equation which neglects the contribution of chloride ions. Subsequent depolarization and loss of a’K during reoxygenation elevated this value to 0·183. Whether or not the observed depression of the membrane resistance is linked to a change in either the sodium or potassium ion permeability is unknown. Release of neurotransmitter and related permeability changes cannot be ruled out as an effect of hypoxia.

Effects of acid base disturbances on basolateral membrane potential and intracellular potassium activity in the proximal tubule of Necturus.

The effects of extracellular acid-base disturbances on intracellular potential (Em) and potassium activity (aiK) in the early proximal tubule of Necturus were examined. Using conventional and double barreled potassium ion selective microelectrodes it was possible to measure both the transient and steady-state responses to various states of extracellular acidosis and alkalosis. The results show that (i) when extracellular [HCO-3] is varied at constant pCO2, Em and aiK decrease in acidosis and increase in alkalosis. The greatest sensitivity in Em is between pH 7.6 and 6.8 with apparent saturation above and below these extremes; (ii) decreased [HCO-3] at constant pH = 7.6 also causes a depolarization of Em and reduces aiK, suggesting a major effect of extracellular [HCO-3] on intracellular potential and aiK; (iii) rapid perfusions and transient delta Em analysis suggest a high basolateral conductance for K+ and HCO-3 and a low Cl- conductance; (iv) increasing extracellular [K+] decreases the response of both Em and aiK to reduced [HCO-3] at constant pCO2. The results of this study demonstrate the important role of extracellular pH and/or [HCO-3] on the maintenance of cellular K+ homeostasis.

Stimulation of pancreatic acinar secretion: increases in cytosolic calcium and sodium.

Na+-selective and Ca2+-selective microelectrodes were used to examine the ionic mechanisms regulating acetylcholine (ACh) stimulation of pancreatic secretion. The cytosolic concentrations of free ionized Na+ and Ca2+ ([Na]i, [Ca]i) were determined in unstimulated acinar cells to be 10.5 +/- 0.4 mM and 0.43 +/- 0.03 microM, respectively. By measuring the induced changes in intracellular Ca2+, Na+, and membrane potentials (ECa, ENa, Em), we were able to demonstrate that 5 X 10(-8) M ACh depolarized Em by 4.3 +/- 0.2 mV and increased [Na]i and [ca]i to 12.2 +/- 0.3 mM and 0.58 +/- 0.02 microM, respectively. Stimulation with ACh at concentrations ranging from 10(-8) to 10(-5) M increased [Ca]i from 0.4 microM to between 0.5 and 1.0 microM. Amylase release reached a maximum at 10(-7) M ACh stimulation and progressively decreased at higher concentrations of stimulus. Increasing the stimulus above an optimal concentration appears to reduce or inhibit enzyme release. These experiments provide direct evidence supporting the concept that acinar cell secretion is triggered by increases in [Ca]i and of calcium's ability to act as primary intracellular mediator. Stimulation after removal of extracellular Ca2+ eliminated the increase in [ca]i that is usually observed in secreting cells, while producing the normal depolarization of Em and increase in [Na]i. These studies demonstrate the increases in [Ca]i are derived from an increase in membrane permeability to Ca2+ and the ability of ACh to depolarize the Em by a transmembrane movement of Na+ that is independent of the change in intracellular Ca2+.

Transmembrane and transepithelial movement of calcium during stimulus-secretion coupling.

Electrophysiological studies were undertaken to determine the transmembrane and transepithelial changes in free Ca2+ concentration that occur during serotonin-induced secretion in the salivary glands of the blowfly, Phormia regina. Ca-selective and conventional microelectrodes were used to measure intracellular and luminal Ca2+ concentrations ([Ca]L), serosal membrane and transepithelial potentials (Em, Etr), and their changes during serotonin (5-hydroxytryptamine, 5HT)-induced salivary secretion. The effect of stimulus concentration on these parameters and enzyme release was also determined. Previous studies provided evidence that serosal stimulation with 10(-8) M 5HT caused a hyperpolarization of Em and short phasic two- to threefold increases in [Ca]i. In these studies, higher concentrations of 5HT (10(-7) M) resulted in depolarization of Em by 13 +/- 1.2 mV and of ECa by 64 +/- 2.1 mV, a dramatic increase in [Ca]i, and a decrease in enzyme release. In addition, serotonin (10(-8) M) reduced the normal spontaneous Etr (+19.6 +/- 1 mV) to near zero while causing an increase in [Ca]L from 1.3 +/- 0.3 X 10(-5) mM to 2.0 +/- 0.1 X 10(-3) mM, a concentration isomolar with that of the bathing medium. These results provide direct electrochemical evidence that, during stimulus-secretion coupling of the salivary epithelial cells, the neurohormone serotonin controls the secretory response by the regulation of intracellular Ca2+ and induces transepithelial transport of Ca2+, thereby suggesting that, during secretion, the neurohormone causes the salivary gland to behave as a "leaky epithelium" by activating the paracellular shunt pathways.

Different actions of calcium channel blocking agents on resting membrane conductance in developing skeletal muscle.

The effects of Co2+, Mn2+, and La3+ (2 mM) and verapamil (5 x 10(-6) M) on membrane conductance (Gm) and resting potential (Em) were studied in chick skeletal muscle fibres developing in culture. Cobalt and manganese had no effect on Gm at any time during myogenesis but verapamil caused a decrease in Gm in immature myotubes. This effect diminished with time and was absent by 3 days after myoblast fusion. Lanthanum caused an increase in Gm at all stages of development. All the agents studied caused a significant depolarization of Em. It is concluded that there is no resting calcium conductance in developing skeletal muscle but that there may be a resting sodium conductance which declines with maturation. Lanthanum may increase Gm by displacing membrane-bound calcium and destabilizing membrane structure. All the agents studied were thought to induce depolarization by an inhibitory action on (Na+ + K+)-ATPase.

Regulation of chloride in quiescent sheep-heart Purkinje fibres studied using intracellular chloride and pH-sensitive micro-electrodes.

1. The intracellular Cl activity, alpha iCl was measured inside quiescent sheep cardiac Purkinje fibres, bathed in normal Tyrode at pH 7.40, buffered with approximately 22 mM-bicarbonate/approximately 5% CO2 + 95% O2. The measurements were made using liquid ion-exchanger Cl-sensitive micro-electrodes. 2. After internal Cl levels had been depleted by prolonged exposure to Cl-free media (glururonate-substituted) when external Cl was restored, there was a rapid re-accumulation of Cl inside the fibres to levels that were much higher than those expected for a passive Cl distribution. Such a process can be conveniently defined as an active inward Cl pump. 3. The inward-pumping was noticeably temperature-sensitive (Q10 approximately 2.6), its rate was reduced about eighteenfold in the nominal absence of external bicarbonate/CO2 and it was substantially inhibited by the drug SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid). 4. The fall of alpha iCl in Cl-free solution was slow and was also equally temperature-sensitive and substantially inhibited by SITS, but was only slightly impaired in the nominal absence of external bicarbonate/CO2. 5. pHi was measured using recessed-tip pH-sensitive micro-electrodes, and in some experiments both pHi and alpha iCl were monitored simultaneously. When alpha iCl slowly declined in Cl-free solution then pHi slowly became alkaline. Upon restoring external Cl, then there was, as usual, a rapid recovery of a high alpha iCl and this was accompanied by a rapid re-acidification of pHi. Both the recovery of alpha iCl and pHi were exponential with virtually the same time constant. 6. Both the slow alkalinization of pHi in Cl-free solution and the rapid re-acidification upon restoring external Cl were substantially inhibited by the drug SITS. 7. When [k]O was raised to 45 mM or more (by removing equivalent amounts of [Na]O), there was a large depolarization of Em and a slow rise of alpha iCl, which was not accompanied by a large change of pHi. The rise of alpha iCl appeared to be unaffected by SITS. 8. It is suggested that a Cl/CHO-3 exchange mechanism can operate reversibly across the membrane of quiescent Purkinje fibres, and that it can account, at least in part, for the high levels of alpha iCl measured in the resting state. It is also concluded that Cl can cross the membrane in other ways, especially in high-K solution possibly by moving passively through conductance channels that are open under these conditions.