Low Chloride Solution Research Articles

GABAergic and glutamatergic inhibition of nonspiking local interneurons in the terminal abdominal ganglion of the crayfish

Nonspiking local interneurons in the terminal abdominal ganglion of the crayfish Procambarus clarkii receive inhibitory inputs from mainly glutamatergic spiking local interneurons and GABAergic nonspiking interneurons. In this study, the inhibitory responses of nonspiking interneurons to local application of glutamate and GABA into the neuropil were compared. Glutamate and GABA injection mediated the hyperpolarization of the nonspiking interneurons with an increase in membrane conductance. The glutamate-mediated membrane hyperpolarization was reversed by injection of 1 or 2 nA hyperpolarizing current. By contrast, more than 3 nA hyperpolarizing current was frequently necessary to reverse the GABA-mediated hyperpolarization. Bath application of a chloride channel blocker, 50 microM picrotoxin (PTX), reduced the glutamate-mediated hyperpolarization, but had no effect on the GABA-mediated hyperpolarization. The GABA-mediated hyperpolarization was not consistently affected by bath application of low chloride solution. These results suggest that the glutamate-mediated inhibition was related to the gating of a Cl(-) conductance, while the GABA-mediated inhibition was not. Electrical stimulation of sensory afferents innervating the exopodite elicited ipsps in uropod opener motor neurons. These sensory-evoked ipsps were also PTX-insensitive, suggesting GABAergic nonspiking interneurons could be the predominant premotor elements in organizing the uropod motor control system.

Airway surface liquid calcium modulates chloride permeability in the cystic fibrosis airway.

Patients with cystic fibrosis (CF) demonstrate a characteristic defect in epithelial chloride movement, which can be demonstrated in vivo by the nasal potential difference technique. After amiloride pretreatment, the CF airway exhibits only a transient response to perfusion with low-chloride solution, contrasting with the sustained hyperpolarization seen in control subjects. This study further investigated the response to low-chloride solution in the CF airway, examining the interaction between surface divalent ions and the low-chloride response. Sequential perfusion with amiloride, low chloride, and isoproterenol was tested in groups of subjects with CF, with the diluent containing different concentrations of calcium and magnesium, on different days. When the low-chloride response was measured with the nominally calcium-free diluents, the subjects with CF had mean (SEM) responses of 8.0 (0.7), 8.6 (2.4), and 9.6 (1.6) mV in the presence of 0, 1, and 3 mM Mg2+, respectively, significantly different from the response in the presence of divalent ions. However, the subsequent response to isoproterenol was not different in the presence or absence of divalent ions. We hypothesize that perfusion of the CF airway with nominally calcium-free solutions reduces tonic inhibition of chloride secretion.

Oxygen consumption and chloride secretion in rat distal colon isolated mucosa.

The aerobic metabolic cost of chloride secretion was studied in rat distal colon isolated mucosa under several conditions by simultaneous measurement of short-circuit current and oxygen consumption under conditions that preserve vectorial ion transport. A low-chloride solution and the presence of bumetanide plus diphenylamine-2-carboxylate reduced short-circuit current by 75% and oxygen consumption by 25%. Ouabain decreased short-circuit current by 93% and oxygen consumption by 32%. Serotonin increased both variables by 59% and 33%, respectively. Bumetanide and diphenylamine-2-carboxylate reduced but did not abolish the effect of serotonin on short-circuit current and oxygen consumption. Changes in short-circuit current and oxygen consumption were linearly correlated under all conditions tested. It is concluded that, in the unstimulated rat distal colon epithelium, chloride secretion accounts for about 75% of ouabain-sensitive short-circuit current and oxygen consumption. Stimulated chloride secretion may demand over 40% of total oxygen consumption.

Use of nasal potential difference and sweat chloride as outcome measures in multicenter clinical trials in subjects with cystic fibrosis.

One of the goals of current research in cystic fibrosis (CF) is to develop treatments that correct or compensate for defects in function of the cystic fibrosis transmembrane regulator (CFTR) gene. The use of outcome measures that assess CFTR function such as nasal potential difference (NPD) measurements and sweat chloride determinations will be required to evaluate the efficacy of such treatments in multicenter clinical trials. The purpose of this work was to identify the sources and magnitude of variability in NPD and sweat chloride measurements when performed at multiple centers. For the variance component analysis presented here, we used NPD and sweat chloride measurements from 37 subjects with CF participating in a phase I, four-center clinical trial of CPX (8-cyclopentyl-1,3-dipropylxanthine), a drug intended to enhance trafficking of Delta F508 CFTR to the cell membrane. The specific techniques used to measure these outcomes were not standardized, and varied between the four sites. Variability of both NPD measurements (baseline potential difference during infusion with Ringer's solution; change in response to addition of 0.1 mM amiloride; and subsequent change in response to perfusion with low chloride solution containing 0.1 mM amiloride and 0.01 mM isoproterenol) and sweat chloride measurements differed significantly between study sites. For change in NPD, one study site had significantly greater variability (lower reproducibility) of measurement than the other three sites. For sweat chloride measurements, reproducibility was lower at two of the sites relative to the other two sites. Sample size calculations showed that lower reproducibility at one or more sites can substantially reduce the power of studies using NPD or sweat chloride determinations as outcome measures. Standardization of measurement protocols, careful operator training and certification, and ongoing monitoring of individual operator performance may help to improve reliability in multicenter trials.

Mechanosensitive chloride channels on the growth cones of cultured rat dorsal root ganglion neurons

We developed an experimental system to investigate mechanosensitivity of a single neuron, using cultured rat dorsal root ganglion cells. Highly precise mechanical stimulation was applied to various sites of the cells, using a piezo-driven glass microcapillary whose movement was computer-controlled, while whole-cell patch-clamp recordings were made from the cell bodies. When the growth cones and lamillipodia from the cell soma were mechanically stimulated, inward currents were recorded at the holding potential of −60 mV. Filopodia were most sensitive to mechanical stimulation. However, when neurites or soma of dorsal root ganglion cells were stimulated in the same way, electrical responses were hardly recorded. Two types of currents varying in time-course were observed: fast type of 100–200 ms and slow type of several seconds in duration. When the membrane potential was held at around 0 mV, both types of currents were almost abolished or even reversed, and the reversal potential was estimated to be about −2.2 mV. Replacement of extracellular sodium by tetraethylammonium did not significantly change the reversal potential. In the low-chloride solution ([Cl −] o=11.7 mM), the reversal potential was about +60 mV, as expected from the Nernst equation for chloride. These inward currents were almost completely inhibited by extracellular application of chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 μM). These results indicate that the inward currents are due to activation of mechanosensitive chloride channels, preferentially located on the growth cones of cultured dorsal root ganglion neurons.

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects.

Previous studies have indicated that milrinone, a specific type III phosphodiesterase inhibitor, may be able to induce chloride secretion in cystic fibrosis (CF) tissues. We have now assessed the effect of this agent in vivo on the nasal epithelium of CF mutant mice and also in the nose and lungs of human subjects with CF. Wild-type mice showed a small hyperpolarization of the nasal potential difference (PD) in response to milrinone (100 microM, 1.6 +/- 0.6 mV, n = 8, P < 0.05). In contrast, CF mice carrying either the most common human mutation of the gene for the CF transmembrane regulator (CFTR), DeltaF508 (protein mislocalized), or the G551D mutation (protein normally localized) failed to demonstrate this response. Milrinone perfused alone had no significant effect on the baseline nasal PD of human subjects without CF (14.7 +/- 4.0 mV preperfusion; 15.3 +/- 4.6 mV postperfusion), but significantly (P < 0.05) augmented the hyperpolarization induced by a subsequently perfused low-chloride solution (with milrinone, 36.8 +/- 3.0 mV, n = 6; without milrinone, 18.1 +/- 2.2 mV, n = 19). In contrast, in human subjects with CF (n = 6), milrinone alone significantly (P < 0. 05) altered the nasal baseline PD (52.2 +/- 3.3 mV preperfusion; 57. 4 +/- 4.2 mV, postperfusion) but not the subsequent responses to the low-chloride solution (with milrinone, 1.1 +/- 2.2 mV, n = 4; without milrinone, 0.6 +/- 0.5 mV, n = 28) or to isoproterenol (100 microM). In a separate study in subjects (n = 6) with the DeltaF508 mutation, nasal coadministration of milrinone with isoproterenol produced no effect in the presence of amiloride and a low-chloride solution (-0.8 +/- 0.5 mV). This was also the case in the nasal epithelium of CF subjects (n = 4) carrying at least one G551D allele (-0.3 +/- 0.8 mV). Similarly, milrinone did not hyperpolarize the PD of either the tracheal (n = 6) or segmental (n = 6) airways of CF subjects (DeltaF508) when applied topically in vivo in the presence of amiloride, isoproterenol, or adenosine triphosphate (all 100 microM) in a low-chloride solution. These data do not support the use of milrinone to induce chloride secretion in CF airways in vivo.

Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.

1. Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea-pig urethra using conventional microelectrode techniques. 2. Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3. STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4. When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5. Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either alpha,beta-methylene-ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co-application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6. In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+-activated chloride channels. STDs sum to activate L-type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.

Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra.

1. Intracellular microelectrode recordings were made from circular smooth muscle of rabbit urethra. 2. The smooth muscle of urethra was spontaneously active exhibiting large, regularly occurring depolarizations, termed slow waves (SWs), 1-3 s in duration, up to 40 mV in amplitude and generated every 3-15 s and small irregularly occurring events (or summations there of) termed spontaneous transient depolarizations (STDs) of < 1 s in duration. 3. The SWs and STDs were not sensitive to 10(-6) M atropine, 10(-6) M phentolamine, 10(-5) M guanethidine or 10(-6) M tetrodotoxin, indicating that they were myogenic in origin. 4. Application of 3 x 10(-6) M nifedipine or 5 x 10(-5) M GdCl3 did not inhibit the generation of SWs or STDs, indicating that activation of L-type Ca2+ channels and non-selective cation channels are not essential for their generation. However, the duration of SWs but not STDs was reduced by nifedipine, indicating L-type Ca2+ channels contribute to the plateau-like potential of SWs. 5. Application of low chloride solution (6.4 mM), niflumic acid (10(-5) - 10(-4) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 10(-4) -5 x 10(-4) M) inhibited the generation of SWs and STDs, suggesting an involvement of chloride channels. 6. Application of nominally Ca2+ free solution, 5 x 10(-5) M BAPTA-AM, 10(-5) M cyclopiazonic acid, 10(-2) M caffeine or 10(-3) M procaine inhibited the generation of SWs and STDs, indicating that Ca2+ released from intracellular stores was required for the generation of SWs and STDs. 7. Exogenously applied noradrenaline (10(-7) - 10(-5) M) increased the frequency of SWs through stimulation of alpha-adrenoceptors which was inhibited by sodium nitroprusside (SNP, 10(-4) M). SNP also reduced the frequency of SWs without altering the membrane potential, an effect mimicked by 8-bromocyclic GMP (10(-3) M) indicating that SNP acted by elevating the production of cyclic GMP. 8. It is concluded that smooth muscle cells of the rabbit urethra exhibit SWs and STDs which are likely to be induced by stimulation of Ca(2+)-activated chloride channels evoked by release of Ca2+ from intracellular stores.

Purinergic cation channels in neurons of rabbit vesical parasympathetic ganglia

Membrane current was recorded from neurons in rabbit vesical parasympathetic ganglia, utilizing single electrode voltage clamp techniques. ATP (0.1–1 mM) caused an inward current ( I ATp) associated with an increased conductance at a holding potential of −50 mV. ADP (0.1–1 mM) and 5′- O-3-thiotriphosphate (0.1–0.6 mM) but not AMP (0.3–2 mM) and adenosine (0.1–2 mM) mimicked the actions of ATP. The I ATp reversed its polarity at −12.1 ± 1.4 mV. The amplitude of the I ATp was depressed in low sodium solutions and in nominally calcium-free solutions but not in low chloride solutions. Suramin (10–100 μM) and reactive blue 2 (10–100 μM), P 2-antagonists, reversibly depressed the I ATp. In contrast, hexamethonium (100 μM) did not affect the I ATp. These data suggest that ATP activates cation channels through P 2X receptor subtypes in rabbit parasympathetic neurons.

Arachidonic-acid-activated membrane conductances in dissociated cardiac parasympathetic neurons from Necturus.

1. Characteristics of the membrane currents activated by arachidonic acid (AA) in dissociated mudpuppy parasympathetic neurons have been determined using the perforated-patch whole cell recording technique. 2. In a sodium-containing physiological solution with 12.5 mM potassium, AA (10-50 microM) increased total membrane current produced by either depolarizing or hyperpolarizing voltage steps delivered from a holding potential of -40 mV. Decreasing the external potassium concentration from 12.5 to 2.5 mM shifted the reversal potential of the AA-induced current by 10 mV rather than the approximately 42 mV predicted for a highly potassium-selective channel. 3. In cells kept in sodium solution plus 12.5 mM potassium and treated with 20 microM nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, AA activated only inward currents following hyperpolarizing voltage steps. In this condition, the shift in reversal potential of the AA-induced current was 40 mV when extracellular potassium concentration was changed fivefold. Consequently, in cells treated with NDGA, AA appeared to activate only an inwardly rectifying potassium current. 4. Decreasing the extracellular chloride concentration by approximately 90% did not alter the reversal potential of the AA-activated current when the extracellular sodium concentration was kept constant and the external potassium concentration was 2.5 mM. In the low-chloride solution, AA potentiated both inward and outward current amplitudes. These results suggested that AA did not activate a chloride current in these cells. 5. In a sodium-deficient, N-methyl-D-glucamine (NMG)-containing solution, AA only activated currents for voltage steps to potentials more negative than the holding potential. In the NMG-substituted solution, changing the extracellular potassium concentration fivefold shifted the reversal potential of the AA-induced current by 40 mV. Therefore, in the NMG solution, AA primarily activated an inwardly rectifying potassium current. 6. Exchanging the control solution containing AA to an external solution containing AA and barium (barium blocks the inwardly rectifying potassium current) shifted the current-voltage relationship to more positive voltages such that the extrapolated reversal potential was approximately 0 mV. In other experiments, using the barium-containing solution, the reversal potential for the AA-induced current was -3.3 +/- 2.4 (SE) mV. 7. In conclusion, the results of the present study indicate that at least two membrane currents are activated in the presence of AA: an inwardly rectifying potassium current and an NDGA-sensitive, sodium-dependent current that has a reversal potential more positive than the potassium equilibrium potential. We suggest the second current component is due to the activation of a nonselective cationic conductance.

Activation of calcium-dependent chloride channels causes post-tetanic depolarization in rabbit parasympathetic neurons

Intracellular recordings were made from neurons in rabbit and feline vesical parasympathetic ganglia in vitro. In response to cathodal current injection (0.1–1 nA for 2–20 ms) the majority of rabbit neurons (229 out of 250) exhibited a single action potential that was followed by a fast and slow after-hyperpolarization (sAHP neuron). The remainder of the cells exhibited an action potential followed by only a fast after-hyperpolarization (fAHP neuron). fAHP neurons did not exhibit anomalous rectification and a spontaneous rhythmic hyperpolarization, which were common membrane properties in sAHP neurons. In response to a train of cathodal current pulses (5–20 Hz for 0.1–10 s), fAHP neurons exhibited action potentials followed by a post-tetanic depolarization (PTD). The PTD was associated with a decrease in membrane input resistance. The amplitude and duration of the PTD were a function of the number of action potentials in the train. The amplitude of the PTD was increased by membrane hyperpolarization and its estimated reversal potential was approximately −30 mV. Low-chloride solution and intracellular injection of chloride ions augmented the amplitude and duration of the PTD, whereas low-sodium, high-potassium and low-potassium solutions did not affect them. Tetraethylammonium (5–10 mM) and barium (0.5–1 mM) increased the amplitude and duration of the PTD. Nominal calcium-free solutions and ω-conotoxin (500 nM) abolished the PTD. The data suggest that activation of chloride channels by calcium influx through ω-conotoxin-sensitive calcium channels mediates the PTD. Repetitive stimulation of the pelvic nerve evoked a train of orthodromic action potentials followed by the PTD of fAHP neurons. (+)-Tubocurarine (10 μM) and hexamethonium (200 μM), but not atropine (1 μM), abolished orthodromic action potentials and the PTD, whereas these cholinergic antagonists did not depress the PTD evoked by direct action potentials. In summary, the data suggest that the PTD may function as a slow synaptic potential in fAHP neurons. This appears likely because neither slow excitatory nor inhibitory postsynaptic potentials are present in neurons of rabbit vesical parasympathetic ganglia. In contrast, slow inhibitory and excitatory postsynaptic potentials were recorded from neurons in feline vesical parasympathetic ganglia.

Stimulation of guinea-pig tracheal afferent fibres by non-isosmotic and low-chloride stimuli and the effect of frusemide.

1. Inhalation of low-chloride or non-isosmotic solutions evokes cough or reflex bronchoconstriction in humans that is inhibited by frusemide (furosemide), whilst capsaicin-evoked cough is unaffected. Here we have examined the responses of single vagal afferent fibres innervating the isolated guinea-pig trachea to these stimuli, and tested the effect of frusemide on fibre responses. 2. Both distilled water and hypertonic saline applied for 30 s onto identified receptive fields produced marked excitation of all A delta and C fibres tested. Isotonic glucose, a low-chloride solution, was a less potent stimulant and caused excitation in 37% of A delta fibres and 69% of C fibres. There was no difference in the distribution of low-chloride sensitive and insensitive receptive fields. 3. In the presence of frusemide, responses of A delta fibres to isotonic glucose were significantly inhibited to 34.2 +/- 6.2% of the pre-drug control level. However, frusemide was without effect either on responses of A delta fibres to distilled water or hypertonic saline, or on responses of C fibres to capsaicin. 4. These data support a role for tracheo-bronchial A delta and C fibres in airway reflexes evoked by hypotonic, hypertonic and low-chloride stimuli. The protective effect of frusemide against airway responses to low-chloride but not to non-isosmotic solutions may reflect an action on sensory nerve endings.

Protocols for in vivo measurement of the ion transport defects in cystic fibrosis nasal epithelium

New treatments for cystic fibrosis (CF), including gene therapy, are currently being assessed. These aim to correct the basic defects of increased sodium absorption and decreased chloride secretion in airway epithelia. Assessment of these bioelectric parameters, particularly in the nasal epithelium, is likely to be used as a measure of treatment efficacy. However, the optimal in vivo protocol to discriminate cystic fibrosis from non-cystic fibrosis subjects is unclear. We have, therefore, compared three protocols for measurement of the cystic fibrosis ion transport defects in vivo in the nasal epithelium. Sodium absorption was measured using both the baseline potential difference and the response to the sodium channel blocker, amiloride. Chloride secretion was assessed in the presence of amiloride, using perfusion with isoprenaline, or terbutaline, or a low chloride solution followed by isoprenaline. Baseline potential difference (PD) and the absolute response to amiloride clearly differentiated the increased sodium absorption in the cystic fibrosis subjects. The responses both to terbutaline (delta PD: non-CF: -0.8 (SEM 0.7) mV; CF: -3.6 (0.5) mV) and isoprenaline (non-CF: 1.5 (0.6) mV; CF: -2.9 (0.6) mV) differentiated the two groups of subjects, but there was considerable overlap of values. Perfusion with a low chloride solution (non-CF: 12.6 (1.2) mV; CF: 0.6 (0.4) mV), as well as subsequent perfusion with isoprenaline (non-CF: 10.0 (1.1) mV; CF: -1.4 (0.4) mV) allowed clear separation of the two groups, with no overlap of values. Some CF subjects showed a transient hyperpolarization to these stimuli, which could clearly be differentiated from the sustained responses seen in non-cystic fibrosis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of furosemide on the response of laryngeal receptors to low-chloride solutions

Laryngeal irritant receptors are stimulated by water and solutions lacking chloride ions, such as isotonic dextrose. It has been reported that furosemide (frusemide) reduces cough evoked by inhalation of low-chloride solutions. We studied the effect of furosemide on the response of laryngeal receptors to isotonic dextrose. Experiments were performed on nine dogs anaesthetized, spontaneously breathing through a tracheostomy, and with the upper airway functionally isolated. We recorded the activity of 13 laryngeal irritant receptors. Isotonic dextrose (4 ml) was instilled into the laryngeal lumen, before and after administration of a furosemide solution (3.75 mg.ml-1) into the upper airway. Before furosemide, dextrose increased the activity of the 13 receptors from 1.0 +/- 0.5 to 25.0 +/- 3.5 impulses (imp).s-1 (average discharge in the first 10 s of activation) and, 1-2 min after furosemide, from 0.3 +/- 0.2 to 13.4 +/- 3.2 imp.s-1; the difference between the stimulation by dextrose before and after furosemide was statistically significant. In contrast, the response to distilled water of four respiratory-modulated mechano-receptors (known to be activated by low-osmolality solutions) was not modified by furosemide. These results suggest that the furosemide-mediated inhibition of cough induced by inhalation of low-chloride solutions is, at least in part, due to the inhibitory effect of this substance on irritant receptor stimulation.

Effect of growing conditions on the corrosivity and ascorbic acid retention in canned tomato juice

AbstractTomatoes (Lycopersicon esculentum) (M82‐1‐8) grown hydroponically in a greenhouse experiment at 16 combinations of nitrate/chloride nutrient solutions were processed as juice and the canned product was stored at 22°C and 36°C for 27 months to study corrosion of the packs from the point of view of detinning or delamination of the internal lacquer and comparative retention of ascorbic acid content.Detinning in plain tinplate cans was greater with juices from tomatoes grown in high nitrate, low chloride solution as a result of the high nitrate content of tomatoes. Addition of chloride decreased the nitrate uptake of the tomatoes, thereby reducing detinning in plain cans significantly. A relevant positive correlation between detinning and high nitrate, high chloride nutrient regime after 27 months storage was attributed to an increase in total acid (as anhydrous citric acid) content of the respective juices and hence increased corrosivity.Delamination, with underfilm corrosion, in lacquered cans was correlated with tomatoes grown under a higher nitrate, medium chloride regime, giving medium levels of nitrate (0.30‐0.65 mmol litre−1) and chloride (20‐25 mmol litre−1) but higher levels of acidity (24‐37 mmol litre−1 citric acid) in the juice. Tomatoes grown under a higher nitrate, low chloride regime exhibited nitrate concentrations as high as 1.0 mmol litre−1 and high corrosivity. Tomatoes grown under low nitrate and increasing levels of chloride regimes gave juices containing as low as 0.18 mmol litre−1 of nitrate but higher chloride concentrations up to 42 mmol‐litre−1; no delamination was observed in lacquered cans of these juices but some pitting corrosion and perforations occurred at the end of 27 months at 36°C.Ascorbic acid retention was mainly correlated with the type of can as well as with the initial ascorbic acid level of the raw tomatoes, itself the result of the interaction of Cl− and NO3− in the nutrient solution. During 27 months storage, the ascorbic acid retention was consistently higher in tomato juice canned in plain tinplate cans (88 % at 22°C or 70% at 36°C) than in lacquered cans (24% at 22°C or 18% at 36°C).

Chloride-mediated inhibitory junction potentials in opossum esophageal circular smooth muscle

Intracellular recordings were made from circular smooth muscle cells of the opossum esophagus. Inhibitory junction potentials (IJPs) 8.3 +/- 0.7 mV in amplitude were observed in response to a single pulse (1 ms, 15 mA) of transmural nerve stimulation. Potassium channel blockers apamin (1 microM), tetraethylammonium (10 mM), 4-aminopyridine (250 microM), and cesium chloride (10 mM) did not reduce IJP amplitude. Conditioning hyperpolarizations to the equilibrium potential for potassium were associated with a significant increase in IJP amplitude. A small increase in membrane resistance was observed during the IJP. Changes in external potassium concentration had no significant effect on IJP amplitude acutely. However, prolonged perfusion with potassium-free Krebs solution resulted in a marked decrease in IJP amplitude as did prolonged perfusion with ouabain (0.1 mM). Low-chloride solution (12.4 mM) resulted acutely in an increase in IJP amplitude. Prolonged low-chloride perfusion resulted in a significant decrease in IJP amplitude. The anion exchange chloride channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (600 microM) significantly reduced IJP amplitude. These findings suggest that the IJP observed in opossum esophageal circular smooth muscle in response to a single pulse of stimulation is due to a decrease in membrane chloride conductance. The ability of prolonged application of Na-K pump inhibitors to abolish the IJP appears to be due to known secondary effects of these agents in depleting intracellular chloride.

Chloride-mediated junction potentials in circular muscle of the guinea pig ileum

Junction potentials were recorded from circular muscle cells of the guinea pig ileum at various sites oral and anal to a transmural stimulus in the presence of atropine, apamin, and substance P antagonism (desensitization) at 30 degrees C. A short train of pulses produced an inhibitory junction potential (slow IJP), which preceded an excitatory junction potential (slow EJP). The slow IJP was observed up to 56.4 +/- 2.9 mm oral and 65.4 +/- 2.2 mm anal to the stimulus. The slow EJP was observed up to 50.4 +/- 1.9 mm oral and 58.3 +/- 2.1 mm anal to the stimulus. Hexamethonium (400 microM) decreased the amplitudes of the slow IJP and slow EJP at all sites. After hexamethonium, the slow IJP was observed up to 37.3 +/- 2.3 mm oral and 39.8 +/- 2.1 mm anal to the stimulus and the slow EJP was 44.2 +/- 2.5 mm oral and 43.3 +/- 2.6 mm anal to the stimulus. The slow IJP and slow EJP were associated with an increase and decrease in membrane resistance, respectively. Conditioning depolarizations of the circular muscle cells reduced the amplitudes of the slow IJP and slow EJP. Both were abolished at a membrane potential of approximately -25 mV. Conditioning hyperpolarizations increased the amplitude of both the slow IJP and slow EJP. Ionic substitution experiments with low external chloride solution (12.4 mM) resulted in an immediate increase in slow IJP and slow EJP amplitudes, whereas more prolonged perfusion resulted in a significant decrease in slow IJP and slow EJP amplitudes. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) resulted in decreases in slow IJP and slow EJP amplitudes. These results suggest that the slow IJP and slow EJP are due to a decrease and increase in membrane chloride conductance, respectively. The noncholinergic neural pathways responsible for the slow IJP and slow EJP extend approximately 40 mm orally and anally along the longitudinal axis of the guinea pig ileum.

Spontaneous and noradrenaline‐induced transient depolarizations in the smooth muscle of guinea‐pig mesenteric vein.

1. Recordings of membrane current were made in the smooth muscle of short segments of mesenteric vein before or during stimulation with noradrenaline (NA). 2. Small veins (diameter less than 150 microns) when cut into short segments (of length less than 250 microns) had the passive electrical characteristics of short cables both before and during activation with NA. 3. Spontaneous transient depolarizations (STDs) or the underlying inward currents (STICs) were recorded in these preparations. STDs were of myogenic origin as they were not blocked by tetrodotoxin or antagonists to the alpha-adrenoreceptor and persisted after either denervation or disruption of the endothelium. 4. STDs had time courses similar to the underlying currents and were generally slow compared to the membrane time constant of the short segments. 5. STDs and the underlying currents showed large variability in frequency and amplitude both within and between short segments. Currents were typically less than 0.3 nA, were characteristic in shape, had half-durations normally in the range 0.1-0.7 s and reversed at about -25 mV. 6. STDs persisted, but at markedly reduced frequencies, after exposure (3-10 min) to a solution in which cobalt ions had been used to substitute for Ca2+. STDs were also substantially suppressed by exposure to low-chloride solution. 7. Caffeine induced excitatory and inhibitory conductances. An initial component of the caffeine-induced responses showed similar voltage dependence to STDs and was also suppressed by exposure to low-chloride solution. 8. NA, through activation of alpha-adrenoreceptors, caused a sustained depolarization or inward current (under voltage clamp) with considerable membrane potential or current noise often in the form of agonist-induced spontaneous transient depolarizations (ASTDs) or currents (ASTICs). There were marked increases in amplitude and frequency of ASTDs with increase in NA concentrations. 9. ASTDs appeared to be generated within the smooth muscle as they were activated in preparations which had been denervated or in which the endothelium had been disrupted. 10. Except for the pathway of activation, ASTDs were indistinguishable from STDs having half-durations in the same range (0.1-2 s with the majority less than 0.7 s). The underlying currents again showed large variation in amplitude (typically less than 0.3 nA; maximum recorded 0.9 nA). They reversed at about -25 mV, could still be elicited in cobalt solution (but at reduced intensity for long exposures to this low-Ca2+ solution) and were reduced by long term exposure to low-chloride solution.(ABSTRACT TRUNCATED AT 400 WORDS)

The nature of noncholinergic membrane potential responses to transmural stimulation in guinea pig ileum

The effect of substance P antagonism on membrane potential responses to transmural nerve stimulation in the presence of atropine was examined in circular smooth muscle of the guinea pig ileum. Intracellular recordings of membrane potential responses recorded 3–5 mm oral to the transmural stimulus consisted of an inhibitory junction potential followed by two distinct depolarizations referred to as early and late excitatory junction potentials. Substance P antagonism was achieved by desensitization with high doses of substance P or use of the antagonist Spantide (Sigma Chemical Co., St. Louis, MO). Substance P antagonism had no effect on the amplitude of the inhibitory junction potential, caused an increase in the latter portion of the early excitatory junction potential, and abolished the late excitatory junction potential. The excitatory junction potential potentiated by substance P receptor antagonism was associated with a decrease in membrane resistance, increased in amplitude with conditioning hyperpolarizations to the estimated equilibrium potential for K+, and was blocked by the Cl−/HCO3− exchange inhibitor DIDS or prolonged perfusion with low-chloride solution. These studies suggest that a noncholinergic, non-substance P neurotransmitter is released from enteric motoneurons that produces excitation through an increase in smooth muscle chloride conductance.

Cyclic AMP effects on chloride transport and carbonic anhydrase activity in frog skin

Unidirectional (36Cl) chloride fluxes across isolated and short-circuited frog skin were measured, with both sides bathed in low chloride solution. Transepithelial chloride influx was inhibited by exogenous cAMP as well as by substances enhancing its cellular concentration, such as epinephrine, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX). Epinephrine and isoproterenol addition resulted in an increase of transepithelial chloride outflux, but exogenous cAMP or IBMX had no significant effect on this unidirectional flux. Phenylephrine had no significant effect on influx or outflux. Carbonic anhydrase (CA) activity in extracts obtained from frog skin epithelium was inhibited by pretreatment with IBMX at 4-5 degrees C and prolonged exposure to cAMP at freezing point. cAMP or IBMX alone had no significant effects on CA activity. This catalytic activity was chloride insensitive and was abolished by 0.1 microM acetazolamide. Results suggest a Cl(-)-HCO3- exchange inhibition by cAMP via carbonic anhydrase inactivation. Chloride outflux stimulation by beta-adrenergic agonists does not seem to depend solely on an increase in cAMP concentration.