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At least 235 records · Page 13Linked to original sources
PubMed1988

Dependence of cell membrane conductances on bathing solution HCO3-/CO2 in Necturus gallbladder.

The effects of bathing solution HCO3-/CO2 concentrations on baseline cell membrane voltages and resistances were measured in Necturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO3-/CO2 Ringer's solutions (2.4 mM HCO3-/air or 1 mM HEPES/air) or a high HCO3-/CO2 Ringer's (10 mM HCO3-/1% CO2). The principal finding of these studies was that the apical membrane fractional resistance (fRa) was higher in tissues bathed in the 10 mM HCO3-/CO2 Ringer's, averaging 0.87 +/- 0.06, whereas fRa averaged 0.63 +/- 0.07 and 0.48 +/- 0.08 in 2.4 mM HCO3- and 1 mM HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (Ra) and basolateral membrane (Rb) resistances in tissues bathed in 10 mM HCO3-/1% CO2 Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO3-/CO2 Ringer's, the higher value of fRa was found to be due to both an increase in Ra and a decrease in Rb. The higher values of fRa and lower values of Rb confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K+ and Cl- substitutions. The results of these studies suggest that an electrodiffusive Cl- transport mechanism exists at the basolateral membrane of tissues bathed in a 10 mM HCO3-/1% CO2 Ringer's, which can explain in part the fall in Rb. The above observations are discussed in terms of a stimulatory effect of solution [HCO3-]/PCO2 on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.

Animals↗
PubMed1985

Protamine reversibly decreases paracellular cation permeability in Necturus gallbladder.

Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

Animals↗
PubMed1988

Ca2+-activated K+ channels in the apical membrane of Necturus choroid plexus.

The properties of Ca2+-activated K+ channels in the apical membrane of the Necturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110 mM). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500 nM, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500 nM. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117 mM were calculated as the apparent Ba2+ dissociation constants (KD(0 mV] for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with a KD(0 mV) = 5.6 mM. A second site, with a higher affinity, KD(0 mV) = 0.23 mM, may account for the near total block of channel conductance by 2 mM TEA+ applied to the outside of the membrane. It is concluded that the channels in Necturus choroid plexus exhibit many of the properties of "maxi" Ca2+-activated K+ channels found in other tissues.

Animals↗
PubMed1988

Ca2+-activated K+ currents in Necturus choroid plexus.

The tight-seal whole-cell recording method has been used to study Necturus choroid plexus epithelium. A cell potential of -59 +/- 2 mV and a whole cell resistance of 56 +/- 6 M omega were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110 mM NaCl Ringer solution and the interior of the cell contained a solution of 110 mM KCl and 5 nM Ca2+, stepping the membrane potential from a holding value of -50 to -10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K+ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreased t1/2. For instance, when pipette Ca2+ was increased from 5 to 500 nM, the delay and t1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels. At the resting membrane potential of -60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20-30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.

Animals↗
PubMed1981

Chloride movement across the basolateral membrane of proximal tubule cells.

Electrophysiologic and tracer experiments have shown that Cl- enters Necturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na+, and that Cl- entry is electrically silent. The mechanism of Cl- exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl- ions across the basolateral membrane, the relative conductance of Cl- to K+ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl- and K+ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl- conductance was about 10% of K+ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl- activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl- activity was virtually unaffected by the PD changes. We conclude that Cl- leaves Necturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na+ or K+.

Animals↗
PubMed1988

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel from Necturus enterocytes.

Potassium channels in membranes of isolated Necturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K- selective. Channel activity was modulated by membrane potential and cytosolic Ca2+ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca2+ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca2+ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca2+ concentration, by a three-state model where Ba2+ interacted with the open channel with a dissociation constant of about 10(-4) M at 0 mV.

Animals↗
PubMed1984

Electrical effects of potassium and bicarbonate on proximal tubule cells of Necturus.

The effects of stepwise concentration changes of K+ and HCO3- in the basolateral solution on the basolateral membrane potential (Vbl) of proximal tubule cells of the doubly-perfused Necturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes in Vb1 after alterations in external K+ concentration from 1.0 to 2.5 mM (tK, 1.0-2.5), 2.5 to 10, and in external HCO3- concentration (at constant pH) from 5 to 10 mM (tHCO3, 5-10), 10 to 20, or 10 to 50. tK, 2.5-10 was 0.38 +/- 0.02 under control conditions but was sharply reduced to 0.08 +/- 0.03 (P greater than 0.001) by 4 mM Ba++. This concentration of Ba++ reduced Vb1 by 9 +/- 1 mV (at 2.5 external K+). Perfusion with SITS (5 X 10(-4)M) for 1 hr hyperpolarized Vb1 by 10 +/- 3 mV and increased tK, 2.5-10 significantly to 0.52 +/- 0.01 (P less than 0.001). Ba++ application in the presence of SITS depolarized Vb1 by 22 +/- 3 mV. In control conditions tHCO3, 10-50 was 0.63 +/- 0.05 and was increased to 0.89 +/- 0.07 (P less than 0.01) by Ba++ but was decreased to 0.14 +/- 0.02 (P less than 0.001) by SITS. In the absence of apical and basolateral chloride, the response of Vb1 to bicarbonate was diminished but still present (tHO3, 10-20 was 0.35 +/- 0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42 +/- 0.19 to 7.57 +/- 0.17 (P less than 0.02) when basolateral bicarbonate was increased from 10 to 20 mM at constant pH. These data show that the effects of bicarbonate on Vb1 are largely independent of effects on the K+ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Regulation of the basolateral potassium conductance of the Necturus proximal tubule.

Two methods, the measurement of the response of the basolateral membrane potential (Vbl) of proximal tubule cells of Necturus to step changes in basolateral K+ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (GK) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K+ activity (aiK) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0 mM K+ basolateral solution for 15 min followed by 45 min of 1 mM K+ solution resulted in a fall in basolateral potassium (apparent) transference number (tK), Vbl and aiK. Results of cable analysis showed that total basolateral resistance, Rb, rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium of Necturus, were also investigated. Ouabain caused a fall in tK accompanied by large decreases in aiK and Vbl. Lowering luminal sodium caused a fall in tK and a small reduction in Vbl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease in tK. These results suggest that GK varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.

Animals↗
PubMed1984

Intracellular activities during volume regulation by Necturus gallbladder.

Necturus gallbladder epithelial cells regulate their volume after a change in solution osmolality. We determined the intracellular activities of Na, K and Cl when the mucosal bathing solution osmolality was increased 18% by the addition of mannitol. The gallbladder was mounted in a rapid flow chamber and punctured simultaneously with two single-barrelled microelectrodes. One electrode sensed membrane potential and the other was sensitive to the activity of Na, K or Cl. Cell volume measurements, made in previous studies utilizing quantitative light microscopy, indicated that hypertonicity of the mucosal bath first caused a cell shrinkage of 15% followed by volume readjustment. Some loss of Na, K and Cl was observed during shrinkage; subsequently during volume regulation, the intracellular quantities of all three ions increased. The loss of Na during the initial cell shrinkage could be blocked by ouabain and was therefore due to increased transport. K and Cl losses were probably related to the increase in their concentrations during shrinkage. The gain of Na, K and Cl during volume regulation was similar in magnitude to the loss of these solutes during cell shrinkage. The increase of Na, K and Cl during volume regulation accounted for about 60% of the increase of cell solutes during this period indicating that other solutes also contributed to the volume regulation response.

Animals↗
PubMed1980

Cryoprotection of human bone marrow committed stem cells (CFU-c) by dextran, glycerol and dimethyl sulfoxide.

Dextran, glycerol and dimethyl sulfoxide (DMSO), alone or in combination, were used for cryoprotection of human bone marrow cells. The viability of cryopreserved cells was assessed by culture of myelopoiesis-committed stem cells (CFU-c) in vitro. A significantly better protection against freezing injury was obtained by 9% dextran in combination with 3 or 5% DMSO, and also with 5 or 10% DMSO alone, than with either 15% glycerol or 9% dextran with 1% DMSO.

Animals↗
PubMed1983

KCl cotransport: a mechanism for basolateral chloride exit in Necturus gallbladder.

K+ and Cl--selective double-barreled microelectrodes were used to study the effect of changes in external K+ concentration on intracellular Cl- activity (aiCl) in epithelial cells of Necturus gallbladder. Decreasing the K+ concentration simultaneously in both bathing solutions produced a decrease in aiCl. Steady-state values of aiCl were related to the values of the chemical potential gradient for K+ (delta microK) across either the apical or the basolateral cell membrane. A similar dependence between aiCl and delta microK appeared when the K+ concentration was changed in the serosal solution only. This indicates that aiCl depends on delta microK across the basolateral membrane. aiCl was virtually independent of the membrane potential. This supports the idea that both the mucosal and the basolateral membranes of Necturus gallbladder cells have very low passive permeabilities to Cl-. These results indicate that the exit of Cl- from Necturus gallbladder cells is driven by delta microK across the basolateral membrane, and suggest that KCl electroneutral coupled mechanism in this membrane plays an important role in transcellular Cl- transport.

Animals↗
PubMed2002

Effects of cholinergic and noradrenergic agents on locomotion in the mudpuppy (Necturus maculatus).

Some neurotransmitters act consistently on the central pattern generator (CPG) for locomotion in a wide range of vertebrates. In contrast, acetylcholine (ACh) and noradrenaline (NA) have various effects on locomotion in different preparations. The roles of ACh and NA have not been studied in amphibian walking, so we examined their effects in an isolated spinal cord preparation of the mudpuppy ( Necturus maculatus). This preparation contains a CPG that produces locomotor activity when N-methyl- D-aspartic acid (NMDA), an excitatory amino acid agonist, is added to the bath. The addition of carbachol, a long acting ACh agonist, to the bath disrupted the walking rhythm induced by NMDA, while not changing the level of activity in flexor and extensor motoneurons. Adding clonidine, an alpha(2)-noradrenergic agonist, had no effect on the NMDA-induced walking rhythm. Physostigmine, an ACh-esterase inhibitor, disrupted the walking rhythm, presumably by potentiating the effects of endogenously released ACh. Atropine, an ACh antagonist that binds to muscarinic ACh receptors, blocked the effects of carbachol, indicating that the action is mediated, at least in part, by muscarinic receptors. In the absence of carbachol, atropine had no effect. Locomotion was not induced by carbachol, atropine or clonidine in a resting spinal cord preparation. Cholinergic actions do not seem to be essential to the CPG for walking in the mudpuppy, but ACh may convert a rhythmic walking state to a more tonic state with occasional bursts of EMG activity for postural adjustments.

Acetylcholine↗
PubMed2001

Extracellular ATP activates a P2 receptor in necturus erythrocytes during hypotonic swelling.

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPgammaS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, alpha,beta-methylene-ATP, and 2'-& 3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K+ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K+ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2'4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K+ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype.

Adenosine↗
PubMed1998

Improved intraepithelial two-dimensional cable analysis with application to necturus gastric antral mucosa.

Previously 2D cable analysis has been performed with two microelectrodes, one for passing intraepithelial current and the other for measuring the voltage response in multiple cells along the distance scale. This requires that the epithelium must be in a stable state for a considerably long period because of the multiple impalements. To follow changes of intraepithelial resistances in chambered Necturus antral mucosa with good temporal resolution, four/five electrodes were used to impale cells in the same preparation and the intraepithelial current (6.5-20 nA) was conducted sequentially to three/four of the electrodes, one at a time, to obtain six/ten independent voltage response measurements along the distance scale. The solution to the 2D cable equations was fitted to results and apical, basolateral and shunt resistances were calculated. It was found that an incorrect distance configuration can ruin the precision of the measurement. The distance configuration can, however, be optimized. The resistance values calculated with the 2D cable analysis were very close to those obtained by the amiloride exposure technique in the same tissues. The improvement gained with this work is better temporal resolution (even <10 s) when measuring epithelial resistances.

Amiloride↗
PubMed1999

The activation of an apical Cl- conductance by extracellular ATP is potentiated by genistein in Necturus gallbladder epithelium.

Necturus gallbladder epithelium (NGE) expresses a CFTR-homologous apical Cl- conductance (Ga,Cl) which can be activated either by elevation of intracellular cAMP or by extracellular ATP. Here we show by microelectrode experiments and impedance analysis that genistein (50 microM), which is known to potentiate the stimulation of Ga,Cl in several cell culture models, also potentiates the stimulation of Ga,Cl by low doses of forskolin in NGE. Moreover, we show that genistein also potentiates the stimulation of Ga,Cl by ATP. In addition genistein renders gallbladders that initially do not respond to ATP sensitive to this stimulant, and it delays the conductance inactivation after ATP removal. Under control conditions Ga,Cl inactivates within < 5 min, but in the presence of genistein a significant Ga,Cl persists even after 60 min. These effects of genistein are not related to inhibition of protein tyrosine kinases, since structurally different inhibitors of the tyrphostin family do not mimic the genistein effects. The data support our conclusion that stimulation of Ga,Cl by ATP is mediated by activation of the cAMP pathway and involves a CFTR-homologous protein. They also favour the view that genistein acts via inhibition of protein phosphatases which dephosphorylate CFTR, but cannot exclude the possibility of a direct interaction with CFTR.

Adenosine Triphosphate↗
PubMed1999

Activation of an apical Cl- conductance by extracellular ATP in Necturus gallbladder is mediated by cAMP and not by [Ca2+]i.

Necturus gallbladder epithelium (NGE) expresses a CFTR-like apical Cl- conductance that can be activated by cAMP. Here, we show that extracellular ATP (100 microM), which is known to elevate intracellular Ca2+ and to hyperpolarize cells by stimulating apical and basolateral K+ conductances, also stimulates an apical Cl- conductance (Ga,Cl), however with a much slower time course. The selectivity sequence of Ga,Cl was SCN- > I- > NO3- > Br- > Cl- >> isethionate (ISE-), but SCN- and I- partially blocked it, which is analogous to observations of CFTR Cl- channels. To disclose a possible role for intracellular Ca2+, gallbladders were incubated with the Ca2+ chelator BAPTA/AM or bathed in solutions containing only submicromolar Ca2+ concentrations. BAPTA partially inhibited the Ca(2+)-mediated hyperpolarization, but did not reduce the ATP-dependent activation of Ga,Cl and the latter was also seen in low extracellular Ca2+. On the other hand, the cAMP-antagonist Rp-8-Br-cAMPS strongly inhibited the stimulation of Ga,Cl by ATP (as well as by forskolin), but left the ATP-induced hyperpolarization unchanged. Preincubation with a low concentration of forskolin markedly enhanced the stimulatory effect of ATP, and this effect was not modified by the selective inhibition of protein kinase C. These data suggest the involvement of different signal transduction pathways in the ATP-dependent activation of K+ and Cl- conductances in NGE. The stimulation of the Ga,Cl appears to be mediated by cAMP but not by elevation of intracellular Ca2+.

Adenosine Triphosphate↗
PubMed2005

Cell volume regulation during hyperosmotic shrinkage is mediated by Na+/K+-ATPase and Na+-K+-2Cl- cotransporter in Necturus gastrics surface epithelial cells.

Cell volume regulation was investigated in gastric surface epithelial cells during hypertonic conditions. Isolated Necturus antral mucosa was perfused on the serosal side with Ringer's solution (pH 7.25, 95%O2/5%CO2) and on the mucosal side successively with 150-500 mM NaCl. Amiloride, ouabain, and bumetanide were used to experimentally inhibit Na+/H+, Na+/K+ ATPase or Na+-K+-2Cl- ion transporters. Intracellular sodium activity and cell volume changes were measured with liquid sensor microelectrodes. The increase in intracellular sodium activity caused by luminal hyperosmolar exposure was mainly due to cell shrinkage. Inhibition of Na+/K+ ATPase or Na+-K+-2Cl- cotransporter increased hyperosmotic cell shrinkage (-52 +/- 5%, -85 +/- 19%, and -77 +/- 9% for control, ouabain, and bumetanide, respectively). Inhibition of Na+/K+ ATPase increased intracellular sodium activity (from 18 +/- 4 to 52 +/- 12 mM). Cell volume regulation in gastric epithelial surface cells during mucosal hyperosmolar exposure is maintained by the basolateral Na+-K+-2Cl- cotransporter, while Na+/K+ ATPase maintains sodium balance, but Na+/H+ antiport seems to have a less important role.

Amiloride↗