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PubMed1989

Alkaline secretion by Necturus proximal duodenal mucosa.

Proximal duodenum from the amphibian Necturus was stripped of muscle layers and the mucosa was mounted as a tube for studies of alkali transport or as a flat sheet for intracellular impalement by voltage-sensitive glass micro-electrodes. The mucosa alkalinized the unbuffered luminal perfusate at a high rate (3.4 muequiv. cm-1 h-1) and developed a transepithelial electric potential difference of 5.7 mV (lumen negative). Transport was inhibited by 2,4-dinitrophenol (10(-4) M) and by furosemide (10(-3) M) and SITS (10(-3) M) on the seros but not on the mucosal side, indicating dependence on tissue metabolism and on serosal membrane Cl-/HCO3- exchange. Prostaglandin E2 (10(-7)-10(-5) M) and dibutyryl cyclic AMP (10(-6)-10(-4) M) had no effects on the secretion or transepithelial electrical potential difference. removal of serosal HCO3- decreased luminal alkalinization by 75%, indicating a contribution by passive migration of HCO3- and/or a dependence of transcellular transport on the nutrient supply of this ion. Administration of HCO3- (17.8 mM) to the luminal perfusate affected neither the transepithelial nor transmembrane electrical potential differences nor the resistance ratio. It is thus unlikely that the luminal membrane possesses any major HCO3- conductance.

2,4-Dinitrophenol↗
PubMed1980

Further studies of the chemical sensitivity of the oscillatory potentials of the electroretinogram (ERG) I. GABA- and glycine antagonists.

The oscillatory potentials (OPs) of the mudpuppy ERG were studied to evaluate the effects of GABA and glycine antagonists. Upon exposing the retina to bicuculline and picrotoxin, blocking agents of the putative inhibitory neurotransmitter GABA, all the OPs were selectively abolished. The earlier OPs (01-03) appeared more sensitive to the drug than the latter ones (04-05). There was no appreciable effect on the range of sensitivity and saturation level of the b-wave. Low concentrations of strychnine, blocking the effect of the putative inhibitory neurotransmitter glycine, produced a selective decrease of the amplitude of the OPs. The first (01) appeared less sensitive to the drug than the later ones. Higher concentrations extinguished all the OPs but also decreased the suprathreshold amplitude of the b-wave. In conclusion, the present results in agreement with previous work, suggest that the OPs appear to have a different origin from the b-wave and seem to be generated by inhibitory feed-back circuits within retina. The differential sensitivity of the individual oscillatory peaks indicate that perhaps chemically different synaptic activities might underlie the individual oscillatory potentials.

Action Potentials↗
PubMed1986

Spatial characteristics of the oscillatory potentials of the electroretinogram.

The spatial properties of the trans- and intra-retinal oscillatory potentials (OPs), a- and b-waves of the mudpuppy electroretinogram (ERG) were analyzed. A comparison of the intra-retinal potentials was made with the proximal negative response (PNR), an extracellular response, predominantly from amacrine cells. The spatial characteristics of the OPs differed from the a- and b-waves and the PNR. The OPs integrated spatially up to full field illumination. They seem to represent summated neuronal activity over broader areas than that of the PNR and the a- and b-waves. The results indicate that the OPs reflect activity of second order neurons to which visual information converge from the whole retina. These neurons may be the bipolar cells. No evidence that the OPs orginate in the laterally extending amacrines was obtained. The b-wave data further suggested a centre-surround organization for the neurons indirectly involved in the b-wave generation.

Animals↗
PubMed1982

Sustained synaptic input to ganglion cells of mudpuppy retina.

1. Intracellular responses were recorded from on-centre and off-centre ganglion cells in isolated eyecups of the mudpuppy, Necturus maculosus.2. Current-voltage relations were measured in darkness, during illumination of the receptive field centre, and after chemically mediated synaptic inputs were blocked by 4 mM-cobalt chloride.3. In on-centre cells the membrane potential in darkness was -56+/-6 mV (mean+/-S.D.). Addition of Co(2+) resulted in an average depolarization of 10 mV and an average decrease in conductance of 2.1 nS. These results suggest that in darkness on-centre cells are tonically inhibited by synaptic input which increases conductance and has a reversal potential more negative than the dark membrane potential. In off-centre cells the membrane potential in darkness was -46+/-5 mV. Addition of Co(2+) caused an average hyperpolarization of 6 mV and an average decrease in conductance of 1.5 nS. These results suggest that in darkness off-centre cells receive a tonic excitatory input which increases conductance and has a reversal potential more positive than the dark membrane potential.4. In on-centre cells light causes a sustained depolarization. This response involves an increase in a tonic excitatory input which increases conductance and has a reversal potential more positive than the dark membrane potential.5. In off-centre cells, light causes a sustained hyperpolarization. This response involves an increase in a sustained inhibitory input which increases conductance and has a reversal potential more negative than the dark membrane potential.6. The depolarizing off-response of off-centre cells is associated with an increase in an excitatory input which increases conductance and has a reversal potential more positive than the dark membrane potential. This response may be due to a temporary increase in the excitatory input which is tonically active in darkness or may reflect an additional excitatory input.7. It is suggested that in both on- and off-centre ganglion cells the balance of sustained excitatory and inhibitory synaptic inputs determines the resting potential in darkness. Centre illumination alters the balance of these inputs, by increasing one and decreasing the other, to produce the characteristic sustained light responses.8. The possible presynaptic sources of the sustained excitatory and inhibitory inputs are discussed.

Animals↗
PubMed1987

Steady-state analysis of ion fluxes in Necturus gall-bladder epithelial cells.

1. The steady-state fluxes of Na, K and Cl ions have been measured in Necturus gall-bladder epithelium by a technique that involves labelling the cells with tracer ions from the mucosal bath only, whilst the serosa is kept at low specific activity. After removing tracer, the efflux is followed into the serosal bath, revealing two exponential components. 2. The time constant of the fast component lies between 0.03 and 0.04 s-1 and corresponds to that of the extracellular space. The slow component closely matches the cellular efflux, with constants which lie between 0.14 and 0.46 X 10(-2) s-1. 3. Full unstirred-layer calculations have been performed to determine the specific activities in the mucosal solution, the cell and the corium (subepithelium). These involved measuring the diffusion coefficients of Na and Cl in the isolated corium: they are restricted by factors of 0.17 and 0.11. 4. The partial flux equations for this double-membrane system have been solved to obtain the cellular fluxes for all three ions. The results indicate that: (i) the net transcellular Na flux is 190 pmol cm-2 s-1, equivalent to the transepithelial salt flux during fluid secretion; (ii) the net transcellular K flux is effectively zero because this ion recirculates across the serosal membrane; (iii) the net transcellular Cl flux is 27 pmol cm-2 s-1, or 15% of the net transepithelial salt flux. 5. The permeability of the paracellular pathway to Cl is 1.65 X 10(-5) cm s-1 and the available driving forces will allow a maximum net electrodiffusive Cl transport of 10% through the shunt pathway. 6. 1:1 coupling of Na and Cl net fluxes at the mucosal membrane of this epithelium cannot be present, and processes other than simple electrodiffussion are required to effect net Cl transport by another route. 7. The serosal fluxes of K and Cl do not obey the flux-ratio equation. A component of these fluxes must be present which is neither active (pumped) nor passive (electrodiffusive and independent). If they are symmetrical in the steady state then the ratio of these exchange fluxes lies between 2:1 and 3:1 depending upon the size of the pump flux. They support the view that a mode of coupled K and Cl transport may be operating at the basolateral membrane of these cells.

Animals↗
PubMed1987

Transcellular sodium fluxes and pump activity in Necturus gall-bladder epithelial cells.

1. Transepithelial Na transport in Necturus was determined by measuring the rate of isotonic fluid flow. The rate at 20 degrees C was equivalent to 175 pmol cm-2 s-1. 2. Ouabain was effective in Necturus, binding to the Na pump in gall-bladder cells with a mean rate constant of 5.4 X 10(3) M-1 s-1. Measurement of the diffusive time constant of the free space for [3H]ouabain shows that the pump must be fully inhibited within 20 s when ouabain is applied to the serosa at 10(-3) M. 3. The serosal Na efflux from loaded cells was inhibited 36% by ouabain equal to a flux of 73 pmol cm-2 s-1. The remaining flux could not be attributed to either exchange diffusion or electrodiffusion induced by ouabain. 4. The transepithelial potential was 0.3 mV serosa positive. The short-circuit current measured was 6.33 +/- 1.9 microA cm-2, equal to a positive univalent ion flux of 65.6 pmol cm-2 s-1 or 38% of the net Na transfer. The current was inhibited within 1-5 min by 5 X 10(-5) M-amiloride. 5. Fluid secretion was immediately inhibited 34% by ouabain, equivalent to an isotonic transport of Na of 59.7 pmol cm-2 s-1. Thereafter it continued for at least an hour, sometimes declining slowly. Amiloride had little effect (13%). 6. The Na pump rate was measured by titrating the cell content with tracer Na at different times after ouabain treatment. The initial slope was equal to a rate of 61.6 pmol cm-2 s-1 or 35% of the net flux at time zero. 7. The Na pump rate has also been measured by recording the rise in cell Na activity with ion-specific micro-electrodes, and correcting for swelling effects. The Na pump rate was very similar to that estimated from the rise in tracer Na content, equal to 59.3 pmol cm-2 s-1 or 31.4% of the transepithelial rate. Examination of the same experiment in the literature shows a closely similar value, about one-third of that expected from fluid secretion or net flux measurements. 8. A scheme is proposed to explain the results, which requires a flow of NaCl through a parallel pathway of small Na content involving exchange en route with the cytoplasmic Na.

Action Potentials↗
PubMed1987

Push-pull effect of surround illumination on excitatory and inhibitory inputs to mudpuppy retinal ganglion cells.

1. Changes in membrane potential and conductance were measured in on-centre and off-centre ganglion cells during the responses to illumination of different portions of the receptive field. 2. In on-centre ganglion cells the sustained depolarizing response to steady illumination of the receptive field centre was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus of light caused a hyperpolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a negative direction. In the absence of centre illumination the same annular stimulus caused a hyperpolarization and a net increase in conductance. 3. In off-centre ganglion cells the sustained hyperpolarizing response to centre illumination was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus caused a depolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a positive direction. In the absence of centre illumination the same annulus caused a depolarization and a net increase in conductance. 4. The results indicate that illumination of the receptive field surround can affect both the excitatory and inhibitory sustained inputs to a given ganglion cell in a 'push-pull' manner, by decreasing the synaptic input that was increased by centre illumination and increasing the synaptic input of opposite sign. The relative effect of a given surround illumination on these two inputs, and hence the sign and magnitude of the net conductance change, varied with the amount of centre illumination.

Action Potentials↗
PubMed1988

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)

Animals↗
PubMed1991

Cytosolic pH regulates maxi K+ channels in Necturus gall-bladder epithelial cells.

1. The patch clamp technique was used to study the effects of internal and external pH on the Ca(2+)- and voltage-activated maxi K+ channel present in the apical membrane of Necturus gall-bladder epithelial cells. 2. When the pH of the solution bathing the cytosolic side of inside-out patches (pHi) was lowered from 7.9 to 6.9, with internal free Ca2+ concentration ([Ca2+]i) buffered below saturation levels for the channel gating sites, channel open probability (Po) decreased. At saturating Ca2+ concentrations, Po was near 1.0, and unaffected by pHi. The results are consistent with a competitive interaction between Ca2+ and H+ at regulatory binding sites. Kinetic analysis assuming competitive binding yields a Hill coefficient for H+ of 1.3. 3. At sub-maximal [Ca2+]i, changing the pH of the solution bathing the extracellular surface of the patch (pHo) between 8 and 7, had no effect on maxi K+ channel Po, but lowering pHo to 6 or 5 significantly reduced Po. At saturating [Ca2+]i, Po was independent of pHo. 4. There were no effects of either pHi or pHo on single-channel conductance. 5. Inasmuch as reductions in either pHo or pHi decrease maxi K+ channel Po, changes in maxi K+ channel activity account in part for the reduction of apical membrane K+ conductance elicited by acidification of the bathing medium. The dominant effect of pH on maxi K+ channels is on the cytosolic surface of the membrane. 6. The change in Po elicited by small changes in [H+]i (delta Po/delta [H+]i) is -7.6 microM-1, compared to delta Po/delta [Ca2+]i = 2.6 microM-1, both at Vm = -30 mV and at physiological intracellular [H+] and [Ca2+]. This implies that [H+]i and [Ca2+]i have opposite effects on channel Po at physiological levels and underlines the importance of pHi in channel gating.

Action Potentials↗
PubMed1993

Whole-cell currents in isolated resting Necturus gastric oxynticopeptic cells.

1. Necturus gastric mucosa secretes Cl- actively across the gastric glands which are composed almost entirely of acid- and enzyme-secreting oxynticopeptic cells. Single channel studies on Necturus oxynticopeptic cells have shown that the basolateral membrane possesses multiple K(+)-selective channels but no observable Cl- channels while the apical membrane has Cl- channels but no observable K+ channels. To relate these channel properties to the conductance of the whole cell we have investigated the macroscopic membrane currents with conventional whole-cell patch-clamp techniques. 2. When bathed in amphibian Ringer solution, gastric oxynticopeptic cells had a membrane resistance of 47.8 +/- 2.8 M omega and a membrane capacitance of 75.5 +/- 2.7 pF (n = 82). This gave a specific membrane resistance of 3260 +/- 160 omega cm2 (n = 82). Reversal potentials of the oxynticopeptic cells were -13.8 +/- 1.2 mV (n = 45) for an intracellular Cl- concentration ([Cl-]i) of 42 mM and were significantly more negative -24.4 +/- 3.1 mV (n = 31, P < 0.001) for [Cl-]i = 22 mM. 3. In the absence of ATP in the pipette solution, there was an 80% reduction of the whole-cell current with a typical half-time (t1/2) of 5 min. The run-down was not observed when the pipette solution contained 4 mM ATP. 4. A slow and voltage-independent inhibition of 80% of the whole-cell currents occurred after addition of NPPB (35 microM). Ba2+ (10 mM) produced a reversible inhibition of 20% of the total current. Together, 35 microM NPPB and 10 mM Ba2+ eliminated 95% of the whole-cell currents. These data suggest that in the resting oxynticopeptic cells Cl- carried the major fraction of the current while K+ ions carried only a small fraction. 5. Total replacement of Cl- in the pipette and bath solution by gluconate- increased the membrane resistance to 751 +/- 104 M omega (n = 53) and shifted the reversal potential to -38.1 +/- 2.8 mV (n = 53). 6. Increasing the bath K+ concentration from 6 to 91 mM activated a current which had a high selectivity for K+ over choline+, Li+, Na+, Rb+ and Cs+ and was independent of Cl-. The activation of this K+ current (IK*) by high external K+ was not seen with ATP-free pipette solution. 7. Ba2+ or Cs+ had a voltage-dependent blocking effect of this inward K+ current. Ouabain (1 mM) or SCH 28080 (200 microM), specific inhibitors of the Na+,K(+)-ATPase and H+,K(+)-ATPase, had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1989

Spatial buffering of light-evoked potassium increases by retinal Müller (glial) cells.

Activity-dependent variations in extracellular potassium concentration in the central nervous system may be regulated, in part, by potassium spatial buffering currents in glial cells. The role of spatial buffering in the retina was assessed by measuring light-evoked potassium changes in amphibian eyecups. The amplitude of potassium increases in the vitreous humor was reduced to approximately 10 percent by 50 micromolar barium, while potassium increases in the inner plexiform layer were largely unchanged. The decrease in the vitreal potassium response was accurately simulated with a numerical model of potassium current flow through Müller cells, the principal glial cells of the retina. Barium also substantially increased the input resistance of Müller cells and blocked the Müller cell-generated M-wave, indicating that barium blocks the potassium channels of Müller cells. Thus, after a light-evoked potassium increase within the retina, there is a substantial transfer of potassium from the retina to the vitreous humor by potassium current flow through Müller cells.

Ambystoma↗
PubMed1984

Sodium-coupled sugar transport: effects on intracellular sodium activities and sodium-pump activity.

Intracellular sodium activities, (Na)c, were determined in Necturus small intestine before and after addition of galactose to the mucosal bathing solution. In the absence of galactose, (Na)c averaged 12 millimoles per liter. Within 2 minutes after the addition of galactose to the mucosal solution, (Na)c increased to a mean value of 20 millimoles per liter and then declined, in parallel with an increase in transcellular sodium transport, to a value that did not differ significantly from that observed in the absence of the sugar. The final steady state in the presence of galactose was characterized by a three- to fourfold increase in the rate of transcellular Na+ transport in the absence of a significant increase in (Na)c. Thus, the increase in steady-state basolateral pump activity cannot be attributed to an increase in the intracellular sodium transport pool.

Animals↗
PubMedDate not supplied

Activation of NMDA receptors is required for the initiation and maintenance of walking-like activity in the mudpuppy (Necturus Maculatus).

We hypothesized that blocking the activation of N-methyl-D-aspartate (NMDA) receptors prevents the initiation of walking-like activity and abolishes the ongoing rhythmic activity in the spinal cord-forelimb preparation from the mudpuppy. Robust walking-like movements of the limb and rhythmic alternating elbow flexor-extensor EMG pattern characteristic of walking were elicited when continuous perfusion of the spinal cord with solution containing D-glutamate. The frequency of the walking-like activity was dose-dependent on the concentration of D-glutamate in the bath over a range of 0.2 to 0.9 mmol/L. Elevation of potassium concentrations failed to induce walking-like activity. Application of the selective antagonist 2-amino-5-phosphonovalerate (AP-5) produced dose-dependent block of the initiation and maintenance of walking-like activity induced by D-glutamate. Complete block of the activity was achieved when the concentration of AP-5 reached 20 micromol/L. Furthermore, application of L-701,324 (a selective antagonist of the strychnine-insensitive glycine site of NMDA receptor) (1-10 micromol/L) also resulted in complete block of the walking-like activity. In contrast, application of the non-NMDA receptor antagonist 6-cyno-7-nitroquinoxaline-2,3-dione (CNQX) (1-50 micromol/L) induced a dose-dependent inhibition of the burst frequency but failed to result in a complete block. Only at concentration as high as 100 micromol/L, did CNQX cause complete block of the rhythmic activity, presumably through nonspecific action on the strychnine-insensitive glycine site of NMDA receptors. These results suggest that activation of NMDA receptors is required for the initiation and maintenance of walking-like activity. Operation of non-NMDA receptors plays a powerful role in the modulation of the walking-like activity in the mudpuppy.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1982

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange.

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1985

Polarity of volume-regulatory increase by Necturus gallbladder epithelium.

Necturus gallbladder epithelial cells respond to the presence of a hypertonic perfusate in either bathing solution by first shrinking due to osmotic water loss and then swelling back to their original volume (volume-regulatory increase). Previous investigations involving increases in the osmolality of the mucosal bath had suggested that volume-regulatory increase was due to the activation of ion exchangers in the apical cell membrane. In the present study the sidedness of the transport processes involved in volume-regulatory increase was investigated. The osmolality of the serosal bath was increased by 18% either in the absence of HCO3- or when an inhibitor of volume-regulatory increase, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), was added to the mucosal or serosal bath. Volume regulation was HCO3- dependent. DIDS was only effective in inhibiting volume regulation when it was added to mucosal bathing solution, suggesting that volume-regulatory increase depended on transport across the apical membrane. Volume-regulatory increase could also be activated by first swelling the cells in hypotonic solution and then returning the tissue to control Ringer solution. The volume-regulatory increase that occurred upon return to control Ringer was also shown to be sensitive to DIDS in the mucosal bath.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Protamine alters apical membrane K+ and Cl- permeability in gallbladder epithelium.

Protamine addition to the solution bathing the mucosal side of Necturus gallbladder epithelium (25-100 mg/l) caused depolarization of both cell membranes, a mucosa-negative change in transepithelial voltage, an increase in the apical membrane resistance (Ra) followed by a decrease, and a monotonic increase in transepithelial resistance (Rt). In protamine (25 mg/l), the change in apical membrane voltage elicited by elevating mucosal solution [K+] from 2.5 to 92.5 mM was reduced from 66 +/-2 to 38 +/- 5 mV (P less than 0.001). The K+-induced fall in Ra was also reduced in protamine. These effects could also be elicited by elevating mucosal solution [K+] simultaneously with the addition of protamine and by transient addition of protamine during exposure to the high K+ medium. The effect of protamine on the electrodiffusive Cl- permeability of the apical membrane (PCl) was studied both in control and forskolin-treated tissues. In the absence of forskolin, the hyperpolarization of Vmc produced by lowering mucosal [Cl-] to 10 mM was reversed to a small depolarization; in forskolin, the initial depolarization produced by lowering [Cl-] was significantly increased. Finally, exposure to protamine in the absence of forskolin produced an initial fall in intracellular Cl- activity. Our results indicate that protamine decreases apical membrane K+ permeability and increases apical membrane PCl. The time course of the effects of protamine suggests the possibility of an initial effect on surface potential, followed by secondary actions mediated by intracellular events.

Animals↗
PubMed1992

Regulation of apical membrane ion transport in Necturus gallbladder.

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Animals↗
PubMed1993

Effects of a Shaker K+ channel peptide and trypsin on a K+ channel in Necturus enterocytes.

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Animals↗