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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↗

PubMed1987

pH-dependent electrical properties and buffer permeability of the Necturus renal proximal tubule cell.

Necturus kidneys were perfused with Tris-buffered solutions at three different pH values, i.e. 7.5, 6.0 and 9.0. A significant drop in fluid absorption occurred at pH 6.0, whereas pH 9.0 did not increase volume flow significantly. When acute unilateral, i.e. either in the lumen or the peritubular capillaries, and bilateral pH changes were elicited in both directions from 7.5 to 9.0 at a constant Tris-butyrate buffer concentration, both peritubular membrane potential difference V1 and transepithelial potential difference V3 hyperpolarized, independently of the side where the change in pH was brought about. Acid perfusions at pH 6.0 caused a similar response but of opposite sign. Analysis of the potential changes shows that pH influences not only the electromotive force and resistance of the homolateral membrane, but also the electrical properties of the paracellular path. Interference of pH with Na, Cl or K conductance was assessed. Any appreciable role for sodium or chloride was excluded, whereas the potassium transference number (tK) of the peritubular membrane increased 16% in alkaline pH. However, this increase accounts only for 19 to 36% of the observed hyperpolarization. Since changes in Tris-butyrate buffer concentration at constant pH do not affect V1 or V3 considerably, the hyperpolarization in pH 9 cannot be explained by an elevation in internal pH only, or by a Tris-H+ ion diffusion potential only. The role of the permeability of the buffers: bicarbonate, butyrate and phosphate, in determining electrical membrane parameters was evaluated. Transport numbers of the buffer anions ranked as follows: tHCO3 greater than tbutyrate greater than tphosphate. It is concluded that modulation of membrane potential by extracellular pH is mediated primarily by a change in peritubular cell membrane tK and additionally by membrane currents carried by buffer anions.

Animals↗

PubMed1990

Barium blocks cell membrane and tight junction conductances in Necturus gallbladder epithelium. Experiments with an extended impedance analysis technique.

The site and concentration dependence of the blocking effect of Ba2+ on Necturus gallbladder epithelium has been investigated. A new approach was used which combines time-dependent electrical cell coupling analysis with intermittently performed measurements of transepithelial and apparent intracellular impedance. From the coupling pulse data the sum of apical and basolateral membrane conductances is obtained, which is then held constant during fitting of the impedance data. This combination technique yields more reliable estimates of apical and basolateral membranes resistances (Ra, Rbl) and of tight junction resistance (Rj) than our previous impedance analysis technique. Using the new approach we have found that luminal Ba2+ concentrations between 0.5 and 1.0 mmol/l increase Ra with saturation-type kinetics without affecting Rbl and Rj, while higher luminal Ba2+ concentrations progressively increase Rj. Corresponding effects were observed under serosal Ba2+. The results validate the new impedance analysis approach and demonstrate that millimolar concentrations of Ba2+ block tight junction conductances. Accordingly, Ba2+ can no longer be considered a tool to exclusively alter cell membrane resistances in epithelia.

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↗

PubMed1999

Effects of inhibitory neurotransmitters on the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

Effects of inhibitory neurotransmitters on the locomotor rhythm and pattern generation were investigated using an in vitro preparation isolated from the mudpuppy (Necturus maculatus). The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial nerves. During N-methyl-d-aspartate (NMDA)-induced locomotion, the rhythmic motor output (EMG) was recorded unilaterally from elbow flexor and extensor muscles. While neither glycine nor gamma-aminobutyric acid (GABA)-related substances induced locomotion in the absence of NMDA, they modulated NMDA-induced locomotion. Bath application of glycine and GABA suppressed the rhythmic motor pattern induced by NMDA. Addition of glycine receptor antagonist strychnine or GABA(A) receptor antagonist bicuculline disrupted the phase relationship between antagonistic motor pools during ongoing locomotion, thereby changing the normal alternating pattern into synchronous EMG bursts. Both the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen mimicked the effects of GABA as they either slowed down or stopped locomotion. Nipecotic acid, a GABA uptake blocker, had a similar effect. This suggested that an endogenous release of GABA modulated the locomotor rhythm. The endogenous release was antagonized by the GABA(A) and GABA(B) receptor antagonists bicuculline and CGP-35348, respectively. Immunocytochemistry revealed that glycine and GABA-positive neurons and fibers were present in mudpuppy spinal cord. Although the GABAergic neurons were more numerous than glycinergic neurons, both cell types contributed processes directed towards the white matter and occasionally towards the ependymal lining of the central canal. Our results suggest that inhibitory neurotransmitters exert powerful actions upon the neuronal network governing forelimb locomotion in the mudpuppy. The effects we observed may be mediated by a network of segmentally distributed glycinergic and GABAergic spinal neurons.

Animals↗

PubMed1997

Fluid recirculation in Necturus intestine and the effect of alanine.

Fluid absorption by Necturus small intestine has been studied using radiolabeled dextrans as molecular probes of the paracellular pathway under voltage-clamped conditions. Fluxes of H3-dextrans of MW up to 20K were followed in both directions between mucosal (M) and serosal (S) baths by fractionating those that passed the epithelium as a function of molecular radius. Consideration of the unstirred layers in the baths and the surface geometry rules out any contribution made by solute polarization. The geometry of the paracellular system was measured by light microscopy, TEM and SEM, and values were used in conjunction with a program that calculates convective-diffusive coupling in the tight junctions, intercellular spaces and subepithelium in series. The results indicate that the net fluxes are due to the convection of fluid through two opposing paracellular fluid circuits with different size selectivity, resulting in net absorption at small radii. Alanine at 20 mM stimulates fluid and salt uptake by a factor of 2. Its effect on the two convective components is to increase the M to S flux and decrease the S to M. The selectivities are not significantly different from those before alanine treatment. The volume absorption predicted from the net probe fluxes is very close to that measured gravimetrically across the epithelium.

Alanine↗

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↗

PubMed1989

Effects of aspirin and acetic acid on intracellular pH in necturus gastric mucosa.

Intracellular microelectrode techniques were employed to examine the effects of luminal aspirin and acetic acid on intracellular pH and cell membrane potential in the surface epithelial cells of Necturus antrum. Antral mucosa was mounted in a modified Ussing chamber, and intracellular pH was determined from the difference between the potentials recorded by intracellular conventional and pH-sensitive microelectrodes. Under neutral conditions (pH7), aspirin (5 mM) hyperpolarized (-7.5 +/- 1 mV, p less than 0.0001) and acetic acid (5 mM) depolarized (+4 +/- 0.08 mV, p less than 0.001) cell membrane potential. Neither agent had any significant effect on intracellular pH. Under acidic conditions (pH 4.5), aspirin (5 mM) reduced the intracellular pH from 6.99 +/- 0.03 to 6.87 +/- 0.04 (p less than 0.001) and depolarized cell membrane potential from -36.7 +/- 1.5 to -30.3 +/- 1.6 mV, p less than 0.001). Similarly, acetic acid (5 mM) acidified the cells (-0.20 +/- 0.02, p less than 0.001) and depolarized cell membrane potential (+9.6 +/- 1.9 mV, p less than 0.01). These changes suggest that, in the absence of luminal acid, small organic acids, such as aspirin and acetic acid, may have complex effects on the ionic conductances of the surface cell membranes without altering intracellular pH. In contrast, under acidic conditions, these agents increase the permeability of the apical cell membrane-to-acid back-diffusion from the gastric lumen.

Acetates↗

PubMed1984

Permeability properties of the subepithelial tissues of Necturus gallbladder.

The permeability properties of the subepithelial connective tissue of Necturus gallbladder were evaluated by measurement of electrical resistance, dilution potentials and hydraulic water permeability. The gallbladder epithelial cells were removed by scraping and the underlying connective tissue placed in an Ussing chamber. The electrical resistance was 2.2 +/- 0.8 omega X cm2; the tissue was slightly cation selective relative to free solution. The subepithelial tissues restricted the rate of diffusion of small solutes to 50% of the free solution value. The hydraulic water permeability averaged 2.1 X 10(-2) cm/s per atm. We conclude that limitations of the area of subepithelium available for fluid movement are the most important factors in determining the restrictions to solute and water flow offered by the subepithelial tissues.

Animals↗

PubMed1986

Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport.

Exposure of Necturus small intestine to a galactose-containing perfusate that is 20% hypertonic compared to the galactose-free (control) perfusate results in a rapid depolarization of the electrical potential difference across the apical membrane, psi mc, and a decrease in the ratio of the resistance of the apical membrane to that of the basolateral membrane, (rm/rs); however, the slow repolarization of psi mc and increase in (rm/rs), observed under isotonic conditions, is blocked. These findings are consistent with the notion that the increase in the conductance of the basolateral membrane in response to Na+-coupled sugar (or amino acid) transport across the apical membrane may be a 'volume regulatory response' to cell swelling.

Animals↗

PubMed1987

Changes in the apparent chloride permeability of Necturus enterocytes during the sodium-coupled transport of alanine.

The membrane potential and intracellular Cl- activity of Necturus enterocytes were measured with double-barrelled ion-selective microelectrodes and apparent permeability coefficients (PCl) for the apical membrane calculated from Cl(-)-replacement experiments. In the presence of L-alanine in the mucosal solution an increase in PCl took place. It is proposed that this might reflect the activation of a Cl- conductance during active substrate transport.

Alanine↗

PubMed1987

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine.

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.

Amino Acids↗

PubMed1988

K+ channels activated by L-alanine transport in isolated Necturus enterocytes.

Using the patch-clamp technique, we demonstrate here the opening of K+ channels evoked by the actively transported amino acid L-alanine in isolated Necturus enterocytes. These channels had a conductance of about 30 pS and their activation was dependent on transmembrane electrical potential and cytosolic Ca2+.

Alanine↗

PubMed1987

Effects of luminal hyperosmolality on cellular and paracellular ion transport pathways in necturus antrum.

Using microelectrode techniques, the electrical properties of the cell membranes and paracellular pathway of the surface epithelium in Necturus antrum were studied under control conditions and during exposure to mucosal solutions made hyperosmotic by addition of sucrose or urea. Sucrose (500 mmol/kg) significantly decreased apical membrane resistances (Ra, from 5501 +/- 841 to 3789 +/- 597 omega X cm2, p less than 0.01), and basolateral membrane resistances (Rb, from 3805 +/- 646 to 2594 +/- 429 omega X cm2, p less than 0.05). The paracellular pathway resistance (Rs) increased significantly from 720 +/- 57 to 822 +/- 71 omega X cm2 (p less than 0.001). Urea (500 mmol/kg) more markedly diminished the apical and basolateral resistances (Ra, from 4303 +/- 663 to 1914 +/- 286 omega X cm2, p less than 0.001; and Rb, from 2600 +/- 474 to 1034 +/- 213, p less than 0.001). In contrast to sucrose, urea-containing solutions significantly decreased Rs (from 682 +/- 78 to 398 +/- 57 omega X cm2, p less than 0.001). Electron micrographs revealed dilatation of lateral intercellular spaces and disruption of desmosomes in tissues exposed to urea, but no visible alterations in tissues exposed to solutions containing sucrose. The resistance of the cellular pathway to ion permeation was thus diminished during exposure to both solutes. In contrast, changes in resistance of the paracellular pathway appeared to depend on the effects of each solute on dimensions and structures of the intercellular pathways.

Animals↗

PubMed1988

Intracellular pH in isolated Necturus antral mucosa in simulated ulcerogenic conditions.

Intracellular pH (pHi) was measured with proton-sensitive liquid sensor microelectrodes in isolated Necturus antral mucosa, paying special attention to arranging experimental conditions to simulate conditions frequently associated with in vivo "stress ulceration." Intracellular pH in mucosas perfused under standard conditions (Ringer's solution containing HCO3-/CO2) was 7.22 + 0.02 (n = 27). Removal of Na+ and HCO3- or addition of amiloride or 4-acetamido-4-isothiocyanostillbene-2,2-disulfonic acid (blockers of Na+/H+ and Cl-/HCO3-exchangers) had no influence on steady-state pHi, suggesting that these ion exchangers do not significantly contribute to the maintenance of pHi in the presence of normal external pH. Acidification of mucosal (luminal) perfusate to pH 3 (mimicking the presence of gastric acid) had no influence on pHi, but mucosal pH 2 (10 mM HCl) acidified pHi to 6.93 +/- 0.07. Acidification of serosal (nutrient) perfusate to pH 6 (mimicking intramucosal acidosis caused by back-diffusion of luminal H+) acidified pHi to 6.72 +/- 0.10. Removal of Na+ from and addition of amiloride to the serosal perfusate during exposure to serosal pH 6.0 induced further acidification of pHi, suggesting that in this acidotic situation (with very low ambient HCO3- concentration) a Na+/H+ exchanger does contribute to the maintenance of steady-state pHi. Increased PCO2 (10% vol/vol in the gas) in a slightly acidic milieu (mimicking mucosal ischemia) likewise acidified pHi to 6.73 +/- 0.05. A combination of mucosal acid (pH 3), high PCO2 (10% CO2), and low serosal pH (pH 6) (mimicking conditions that prevail, for example, during hemorrhagic shock) acidified pHi and ultimately resulted in cell death. These derangements of intracellular acid-base balance may have pathogenetic importance also in in vivo stress ulceration.

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