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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↗
PubMed1986

Neurotensin actions in the retina: mechanisms and variability.

The effects of neurotensin on mudpuppy retinal cells were studied using extracellular and intracellular electrophysiological recording techniques and bath application of the peptide. Ganglion and amacrine cells (but not bipolar or horizontal cells) were reversibly depolarized by low micromolar concentrations of neurotensin. Depolarizations also occurred with neurotensin application during cobalt block of synaptic transmission and were accompanied by decreased input resistances. This suggests neurotensin may act directly on amacrine and ganglion cells as a conventional excitatory transmitter. However, in many retinas, cells responded to light stimuli and to other drugs but not to neurotensin. These negative results are important in considering the peptide's normal role in retinal function.

Action Potentials↗
PubMed1986

GABA-like immunoreactivity in the vertebrate retina: a species comparison.

Rabbit antisera directed against gamma-amino butyric acid (GABA) conjugated to bovine serum albumin was used to localize neurons containing GABA-like immunoreactivity in the retinas of nine species of animals: human, cat, rabbit, rat, chicken, turtle, frog, mudpuppy and goldfish. The retinas of all species contained GABA-like labeling in several populations of amacrine cells in the inner nuclear layer, cells in the ganglion-cell layer that may include displaced amacrine cells and in fibers in the inner plexiform layer and in the optic nerve fiber layer. Labeled horizontal cells were found in cat and in all non-mammalian retinas. Labeled interplexiform cells were found in rat, rabbit, cat and human retinas. Labeled bipolar cells were restricted to frog and mudpuppy retinas. The distribution of anti-GABA is usually similar to that of anti-glutamic acid decarboxylase and neuronal [3H]GABA uptake, indicating good correspondence between these 'GABAergic' markers. However, several significant differences among these markers are discussed.

Animals↗
PubMed1990

Intracellular pH in isolated Necturus antral mucosa exposed to luminal acid.

Regulation of intracellular pH in gastric epithelial surface cells exposed to luminal acid was investigated in isolated Necturus antral mucosa using microelectrode technique. Exposure of the mucosa to luminal pH 2 acidified intracellular pH from 7.21 +/- 0.01 to 6.95 +/- 0.04 (N = 50). Removal of Na+ from the perfusates or addition of amiloride (1 mM) to serosal perfusate (containing HCO3-) had no influence on intracellular pH during exposure to pH 2 (N = 6), but removal of HCO3-/CO2 from or addition of 4, acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.5 mM) to the serosal perfusate (containing Na+) acidified intracellular pH from 7.02 +/- 0.03 to 6.45 +/- 0.15 (p less than 0.01, N = 10) and from 6.97 +/- 0.06 to 6.58 +/- 0.26 (p less than 0.01, N = 6), respectively, in 15 min. In tissues exposed to mucosal pH 6, epithelial surface pH was about 1.3 pH units higher than pH of the mucosal bulk solution. Removal of Cl-/HCO3- from the serosal perfusate acidified epithelial surface pH by about 0.5 pH units (p less than 0.01, N = 6), suggesting that serosal HCO3- sustains intracellular pH, at least in part, by generating an alkaline buffer layer at the epithelial surface. In the absence of HCO3-/CO2, a stable intracellular pH was obtained when the tissue was exposed to mucosal pH 2.7, but in this situation intracellular pH was sensitive to Na+ removal or amiloride addition, intracellular pH decreasing from 7.00 +/- 0.07 to 6.48 +/- 0.10 (p less than 0.01, N = 6) and from 6.86 +/- 0.06 to 6.32 +/- 0.01 (p less than 0.01, N = 7), respectively, in 15 min. The data suggest that in gastric epithelium exposed to luminal acid, physiological intracellular pH is primarily maintained by the buffer action of serosal HCO3- transported to the epithelial surface to impede the entry of luminal H+ into mucosal tissue. Removal of the sheltering HCO3- unmasks a second line, Na(+)-dependent and amiloride-sensitive intracellular pH regulatory mechanism, presumably a Na+/H+ antiport.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1991

Exogenous surface-active phospholipid protects Necturus gastric mucosa against luminal acid and barrier-breaking agents.

The nature of the protective action of exogenous surface-active phospholipid on gastric mucosa was studied in isolated Necturus antral mucosa by measuring intracellular pH and intraepithelial potentials and resistances with a microelectrode technique. Exposure of the antral mucosa to luminal pH 2 acidified intracellular pH in surface epithelial cells by 0.6-0.3 pH units. A 20-minute pretreatment with exogenous (pulmonary) surfactanlike phospholipid completely abolished this effect. Obviously, phospholipid protected the mucosa against intracellular acidosis by decreasing the apical cell membrane conductance to H+ (and other ions), because it increased apical cell membrane resistance by +108% and total transcellular resistance by +86% but had no significant effects on paracellular or total transepithelial resistances. In mucosas exposed to three barrier-breaking agents, 10 mmol/L acetylsalicylic acid, 20% (vol/vol) ethanol, and 10 mmol/L taurocholate, at acid luminal perfusate (pH 2.0-2.5), a profound intracellular acidification of 0.9-1.3 pH units/15 min occurred. Pretreatment of the tissue with phospholipid significantly opposed intracellular acidification, but the modulatory influences on the changes in intraepithelial potentials or resistances were less conspicuous and mostly insignificant.

Animals↗
PubMed1995

Regulation of intracellular pH in isolated Necturus gastric mucosa during short-term exposure to luminal acid.

BACKGROUND/AIMS: Continuous exposure to gastric acid implies efficient control mechanisms of intracellular pH (pHi) in the gastric epithelium. This study assessed the roles of Na+, H+, and HCO3- transport mechanisms in controlling pHi during short-term exposure of the gastric epithelium to luminal acid. METHODS: pHi and Na+ activity (aiNa) were measured with liquid sensor microelectrodes in isolated Necturus antral mucosa, modulating ion transport mechanisms by ion removal and pharmacological inhibition. RESULTS: Short-term exposure to luminal acid (pH 2.3) acidified pHi by 0.3 pH units, whereafter pHi stabilized. This was associated with transient increase in aiNa. Blocking of Na+/H+ exchange (in the presence of HCO3-/CO2) by removal of Na+ or addition of amiloride eliminated the increase in aiNa and resulted in uncontrolled acidification of pHi. Similarly, blocking of HCO3- transport (in the presence of Na+) by removal of HCO3-/CO2 or addition of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid resulted in uncontrolled acidification of pHi despite increase in aiNa. Blocking of Na+/K+ exchange with ouabain eliminated the recovery of aiNa and also resulted in uncontrolled acidification of pHi. CONCLUSIONS: The data indicate that during short-term exposure of the gastric mucosa to luminal acid, both Na+/H+ antiport and HCO3- transport are needed to control pHi and maintain it within physiological ranges.

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

Aspartate-induced dissociation of proximal from distal retinal activity in the mudpuppy.

The effects of aspartate (Asp) on the ERG and on neuronal, glial, and K+ responses were monitored continuously in the superfused mudpuppy eyecup. Asp induced a time-dependent sequence of events which may be divided into three stages: Stage 1, initially, light-evoked responses throughout the retina are depressed; Stage 2, distal responses (horizontal, bipolar, and K+ responses) return to near pre-drug amplitudes and there is a simultaneous ERG enhancement, but responses in the proximal retina remain suppressed; Stage 3, a second depression of retinal responses leads to a-wave isolation. The dissociation of distal from proximal responses observed during Stage 2 strongly supports the hypothesis that the ERG b-wave results from events arising in the distal retinal network.

Animals↗
PubMed1989

APB increases apparent coupling between horizontal cells in mudpuppy retina.

2-Amino-4-phosphonobutyrate (APB), an agonist at a unique type of glutamate receptor on depolarizing bipolar cells, caused an apparent increase in coupling between horizontal cells as evidenced by a decrease in amplitude of responses to illumination of the receptive field center and an increase in responses to illumination of the peripheral part of the receptive field. APB also caused a hyperpolarization of horizontal cells in darkness and increased the amplitude of responses to full-field illumination, which cannot be explained by an increase in electrical coupling between horizontal cells. Possible mechanisms for these actions are discussed.

Aminobutyrates↗
PubMed1986

Glutamate depolarization of glial cells in Necturus optic nerve.

L-Glutamate, 10(-5) to 10(-2) M, depolarized glial cells in the normal and axon-free optic nerve of Necturus by up to 80 mV. The depolarization was maintained if lithium, but not tetramethylammonium, replaced sodium in the bathing solution. It was not blocked by either strophanthidin or tetrodotoxin. The response was accompanied by a membrane conductance increase and was followed by a strophanthidin-sensitive hyperpolarization. These results suggest that the depolarization resulted from sodium entry. D-Glutamate and L-aspartate were less potent but produced a comparable depolarization. gamma-Aminobutyric acid, glycine, taurine or glutamine (up to 10(-3) M) were ineffective.

Amino Acids↗
PubMed1997

Luminal acid increases apical cell membrane resistance in isolated Necturus antral mucosa.

BACKGROUND & AIMS: The gastric mucosa must have efficient protective mechanisms to maintain physiological intracellular pH. The aim of this study was to investigate the effect of low luminal pH on apical membrane permeability. METHODS: Chambered Necturus antral mucosa was perfused with Ringer's/95% O2-5% CO2 at pH 7.25. The mucosal side was exposed to pH 4.0-2.0 with four microelectrodes placed in surface cells. Two-dimensional cable analysis was used to measure apical, basolateral, and shunt resistances. In some experiments, liquid sensor pH or Na(+)-selective microelectrodes were used. RESULTS: Luminal acidification hyperpolarized apical cell membrane potential and increased apical cell membrane resistance from 21.3 +/- 2.6 (pH 7.25) to 38.0 +/- 2.3 k omega.cm2 (pH 3.0; n = 8). The increase in apical cell membrane resistance was preceded by transient intracellular acidosis from 7.32 +/- 0.07 (pH 4.0) to 7.23 +/- 0.06 (pH 3.0; n = 6). Similar intracellular acidosis (provoked by NH4+ prepulse) failed to cause the effects observed with luminal acid. The increase in apical cell membrane resistance caused by luminal acid was eliminated when N-methyl-D-glucamine+, but not Na+, was substituted for all cations in the luminal solution. CONCLUSIONS: Luminal acidification (pH 3.0-2.0) closes apical amiloride-blockable Na+ channels. Protons are probably able to pass and even block these channels, but their effect in closing the channels does not occur intracellularly.

Acids↗
PubMed1997

Two phenylglycine derivatives antagonize responses to L-AP4 in ON bipolar cells of the amphibian retina.

Light responses of retinal ON bipolar cells are mediated by metabotropic glutamate receptors selectively activated by L-2-amino-4-phosphonobutyric acid (L-AP4). Antagonists to L-AP4 receptors in ON bipolar cells have not previously been identified. This study examines the electrophysiological effects of (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), (RS)-4-4-chloro-3,5-dihydroxyphenylglycine (CDHPG) and (RS)-3,4,5-trihydroxyphenylglycine (THPG), at L-AP4 receptors in ON bipolar cells of the amphibian retina. Unlike its actions in spinal cord, in retinal ON bipolar cells MAP4 is a weak agonist which exhibits no detectable antagonism to L-AP4. On the other hand, CDHPG exhibits a mixture of agonist and antagonist properties. Addition of Co2+ and oxygenation of CDHPG turns the solution brown and enhances antagonist effects, suggesting that the antagonism reflects actions of a breakdown product of CDHPG. Although THPG did not prove to be this breakdown product, it also has electrophysiological effects consistent with an L-AP4 receptor antagonist. The results suggest that THPG and breakdown products of CDHPG may be antagonists to L-AP4 receptors in retinal ON bipolar cells, although the possibility that these compounds antagonize effects of L-AP4 by acting at some site in the transduction pathway of L-AP4 receptors cannot yet be excluded.

Aminobutyrates↗
PubMed1996

Cholinergic modulation of dopamine release and horizontal cell coupling in mudpuppy retina.

The effects of cholinergic agonists and antagonists on electrical coupling between horizontal cells were studied in dark-adapted mudpuppy retinas. Carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) uncoupled horizontal cells, but the muscarinic agonist oxotremorine did not. The uncoupling effects of carbachol and DMPP were blocked by the nicotinic antagonist D-tubocurarine and by the dopamine antagonist fluphenazine, indicating that carbachol uncoupled horizontal cells by stimulating dopamine release via nicotinic receptors. Carbachol also caused an increase in release of [3H]dopamine from retinas. D-Tubocurarine increased horizontal cell coupling, indicating that tonic cholinergic input was present in dark-adapted retinas. D-Tubocurarine did not reduce light-evoked uncoupling of horizontal cells, suggesting that cholinergic neurons are not an essential part of the direct pathway by which light causes an immediate increase in dopamine release.

Acetylcholine↗
PubMedDate not supplied

Serial inhibitory synapses in retina.

Whole-cell voltage clamp in the retinal slice and intracellular current clamp in the intact retina were used to study inhibitory interactions in the inner plexiform layer. Picrotoxin or strychnine reduced inhibitory, light-evoked currents in a majority of ganglion cells. However, in nearly a third of the ganglion cells, each of these antagonists enhanced the inhibitory synaptic current. All inhibitory current was blocked by the addition of the other antagonist. This indicates a cross-inhibition between GABAergic and glycinergic feedforward pathways. Blocking of GABAARs with SR95531 shortened the time course of both excitatory and inhibitory synaptic currents in ganglion cells. Application of picrotoxin, which blocked both GABAARs and GABACRs, produced the opposite effect. Recordings in the intact retina indicated that the light responses of ON bipolar cells, sustained ON, and transient ON-OFF third-order neurons were all made more transient by SR95531 and made more sustained by picrotoxin. The data suggest that a GABAC feedback pathway to bipolar cells makes light responses more phasic and that this feedback is inhibited through a GABAAR pathway. Consequently, the balance between GABAAR and GABACR inhibition regulates the time course of inputs to ganglion cells.

Ambystoma↗
PubMed2000

Topical prostaglandin E2 protects isolated gastric mucosa against acidified taurocholate-, but not ethanol- or aspirin-induced injury.

This study investigates whether topical prostaglandins protect isolated gastric mucosa against injury provoked by acidified "barrier-breaking" agents. Intracellular pH (pHi), apical cell membrane potential (Vcm) and intraepithelial resistances in isolated Necturus antral mucosa were measured using double-barreled liquid sensor microelectrodes. Topical PGE, treatment protected the antral mucosa against acidified taurocholate-induced injury, reducing significantly (P<0.05) intracellular acidification (pHi from 7.39+/-0.05 to 7.08+/-0.08 vs. from 7.30+/-0.02 to 6.62+/-0.15), and opposing significantly the changes in Vcm (hyperpolarization followed by depolarization), and completely abolishing the decrease in transmembrane resistance (Rt from 702+/-37 to 723+/-39 Ohms x cm2 vs. from 721+/-34 to 270+/-105 Ohms x cm2). Also the ratio of apical and basolateral membrane resistances (Ra/Rb) remained at a significantly higher level in PGE2-treated tissues. In contrast, PGE2 treatment had no protective influence on the changes of the respective parameters in acidified ethanol or acetylsalicylic acid injured mucosas. Topical prostaglandin E2 protects isolated gastric mucosa against acidified taurocholate, but not against ethanol- or acetylsalicylic acid-induced injury.

16,16-Dimethylprostaglandin E2↗
PubMed2003

Effect of luminal ethanol on epithelial resistances and cell volume in isolated Necturus gastric mucosa.

Ethanol is a well-established "barrier breaker" in gastric mucosa, but its effects at the cellular level remain to be elucidated. Isolated Necturus antral mucosa was exposed luminally to 5-15% (v/v) ethanol at pH 3.0. Apical, basolateral, shunt, and internal resistances in surface epithelium were measured using 2-D cable analysis. Cell volume changes were determined from tetramethylammonium-loaded surface cells. Low luminal ethanol (5%) decreased basolateral resistance, presumably by opening of K+ channels, since this decrease was partially inhibited by the K+ channel blocker, quinine. Low ethanol decreased also epithelial cell volume, which was opposed by quinine, suggesting that efflux of intracellular K+ underlies this shrinkage. High luminal ethanol (15%) markedly decreased shunt and apical cell membrane resistances, and partially closed gap junctions as judged from increased epithelial internal resistance. Opening of basolateral K+ channels with resultant epithelial cell shrinkage might be among the initial steps in ethanol induced gastric injury. The changes in intraepithelial resistances provoked by stronger ethanol probably reflect emerging structural epithelial damage.

Animals↗
PubMed1980

Hydrostatic pressure changes related to paracellular shunt ultrastructure in proximal tubule.

We examined the effets of changes in hydrostatic pressures on the ultrastructural geometry of the lateral intercellular space and tight junctions in proximal tubules of contrtol (C) and volume-expanded (VE) Necturus kidney. The following groups of tubules were studied: (1) C, free-flow pressure, (2) C, stopped-flow, high-luminal pressure, (3) C, stopped-flow, low-luminal pressure, (4) VE, free-flow pressure, and (5) VE, stopped-flow, high-luminal pressure. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy, and complete cross-sections of all sampled tubules were subjected to morphometric analysis. Average lateral intercellular space widths decreased significantly in C and VE stopped-flow tubules with high-luminal pressures but widened greatly in C stopped-flow tubules with low-luminal pressures. The length or width of the tight junctions did not change between the five experimental conditions. The ultrastructural changes correlate with the applied transepithelial pressure gradients rather than with transepithelial volume fluxes. The narrowing of lateral intercellular spaces in high pressure tubules correlate with the previously described increase in electrical resistance expressed per unit length tubule indicating that in these conditions part of the paracellular resistance is located in the free interspaces. The geometry of the lateral intercellular space in the proximal tubule of Necturus favors models of near-isotonic transport that do not depend on long and narrow interspaces.

Animals↗
PubMed1993

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Actions of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in retinal ON bipolar cells indicate that it is an agonist at L-AP4 receptors.

Metabotropic glutamate receptors (mGluRs) include receptors sensitive to L-2-amino-4-phosphonobutyrate (L-AP4) and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). To determine whether 1S,3R-ACPD is an agonist at retinal L-AP4 receptors, whole cell voltage clamp recordings were obtained from mudpuppy ON bipolar cells in a superfused retinal slice and L-AP4 and 1S,3R-ACPD were bath applied. Both compounds evoked similar outward currents which reversed near 0 mV and were accompanied by an increased input resistance. Responses to both agonists washed out in parallel suggesting they act through the same second messenger pathway(s). Inhibitors of cGMP-PDE activity suppressed responses to both L-AP4 and 1SR,3RS-ACPD, suggesting that both compounds activate cGMP-PDE. Responses to 1S,3R-ACPD were occluded by prior activation of L-AP4 receptors, but not blocked by the non-AP4, mGluR antagonists, L-aminophosphonopropionic acid (L-AP3) or 4-carboxy-3-hydroxyphenylglycine (4C3H-PG). These results indicate that 1S,3R-ACPD is an agonist at L-AP4 receptors. 1S,3S-ACPD and 4C3H-PG evoked outward currents similar to L-AP4 suggesting they may also be L-AP4 receptor agonists. Using the b-wave of the ERG as an assay for ON bipolar cell responses, concentration/response curves were obtained for ACPD enantiomers. The rank-order potency of ACPD enantiomers at L-AP4 receptors in ON bipolar cells is similar to their rank-order potency at non-AP4, mGluRs in brain which suggests that the receptors possess similar binding sites and may be members of a common receptor family.

Aminobutyrates↗