Ion Substitution Experiments Research Articles

A calcium-permeable channel in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule.

Calcium is actively reabsorbed in the distal nephron segments and recent studies have demonstrated the presence of Ca2+ channels in these epithelial cells, which could be involved in transepithelial transport. To test this possibility, single-channel currents were recorded by the patch-clamp technique in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule cells (DCTb). In the cell-attached mode with 100 mmol/l BaCl2 in the pipette and 145 mmol/l NaCl in the bath, inward negative currents, consistent with Ba2+ currents, were recorded. In these conditions, the single-channel conductance was 15 pS. In excised inside-out patches, the single-channel conductance was 13 pS and the current reversal potential of +60 mV was close to the Nernst equilibrium potential for Ba2+ (> +58 mV). Similar experiments conducted with Ca2+ as the main charge carrier showed that this ion was less permeant through the channel than Ba2+ (PBa/PCa approximately 1.4). We also showed that the Ca(2+)-channel blocker, lanthanum (1 mumol/l La3+), added on the cytosolic side of the membrane, reversibly blocked the channel activity. On the other hand, verapamil (0.1 mmol/l) and nifedipine (10 mumol/l), perfused on the cytosolic side of the membrane, abolished the channel activity but this effect was not reversible. Another type of channel was also identified in the apical membrane of cultured DCTb cells. Ion-substitution experiments showed that this 21-pS conductance channel did not discriminate between Na+ and K+ and did not conduct Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Right-angle light scattering to assay basal and regulated plasma membrane Cl- conductances.

We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca(2+)-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.

The effects of glutamate agonists on voltage-clamped motoneurons of the lobster cardiac ganglion.

The effects of L-glutamate and its analogues were studied in voltage-clamped motoneurons of the lobster cardiac ganglion. These excitatory amino acids caused a dose-dependent increase in membrane conductance and an inward current at the resting membrane potential. The EC50 for L-glutamate was 150 mumol 1(-1). The rank order of potencies of the various agonists was quisqualate greater than L-glutamate = L-aspartate greater than kainate greater than cysteine. Kainate, unlike the other agonists, showed no desensitization. Of various antagonists studied, only the quinoxalinediones inhibited the response to glutamate. These antagonists also reduced the amplitude and duration of the pacemaker-driven burst potential, suggesting that glutamate may be released by some of the endogenous synapses within the ganglion. The reversal potential of the glutamate-induced current was -15 mV. When Na+ was replaced with K+, the glutamate-induced current still reversed between 0 and -20 mV. When Na+ was replaced with the impermeant ion N-methyl-D-glucamine, the current was inhibited. The amplitude of responses evoked by glutamate and its analogues was reduced in salines containing either high or low concentrations of Ca2+. These results of pharmacological and of reversal potential and ion substitution experiments indicate that glutamate acts on receptors of the non-NMDA (N-methyl-D-aspartate), quisqualate/kainate type to open a channel permeable to both Na+ and K+.

Modulation of inwardly rectifying Na(+)-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech, Haementeria.

The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about -70 mV were associated with the activation of a slow inward current (Ih) which showed no inactivation with time. The time course of activation of Ih was described by a single-exponential function and was strongly voltage dependent. The activation curve of Ih ranged from -72 to -118 mV, with half-activation occurring at -100 mV. Ion-substitution experiments indicated that Ih is carried by both Na+ and K+ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of Ih and its rate of activation. It also produced a positive shift of the activation curve of the conductance underlying Ih (Gh) without altering the slope factor, thus indicating that the voltage dependence of Ih was modulated by 5-HT. Cs+ blocked both Ih and the 5-HT-potentiated current in a voltage-independent manner, whereas Ba2+ had little effect. It is concluded that 5-HT increases Ih by modulating the inwardly rectifying Na(+)-K+ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since Ih was increased by 8-bromo-cyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. In contrast, Ih was reduced by 8-bromo-cyclic GMP and by zaprinast (an inhibitor of cyclic GMP-sensitive phosphodiesterase). Cyclic GMP itself also reduced Ih, and the effect was specific to the 3',5' form; 2',3'-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMP.

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig.

Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (Isc) ranged from -15 to 220 microA.cm-2. Variations in basal Isc resulted from differences in the amount of amiloride (10 microM mucosal addition)-sensitive Na+ absorption. Ion substitution and transepithelial flux experiments showed that 10 microM amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on Isc was independent of Cl- and HCO3- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 microM, little change in Isc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the Isc. The decrease in Isc due to 1 mM amiloride was dependent on both Cl- and HCO3-, and was attributed to reductions in the M-S and net Na+ fluxes as well as the M-S unidirectional Cl- flux. Ion replacement experiments demonstrated that Cl- substitution reduced the M-S and net Na fluxes, while replacement of HCO3- with HEPES abolished net Cl- absorption by reducing the M-S unidirectional Cl- flux. From these data it can be concluded that: (1) Na+ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl- absorption is completely HCO3- dependent (presumably mediated by Cl-/HCO3- exchange) and occurs independently of Na+ absorption.

Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ “leak”.

We studied the factors that determine the intraphagosomal pH (pHp) in elicited murine peritoneal macrophages. pHp was measured in situ by recording the fluorescence of covalently fluoresceinated Staphylococcus aureus ingested by the macrophages. Following spontaneous acidification of the phagosomes, passive (leak) H+ permeability was determined measuring the rate of change of pHp upon complete inhibition of the H+ pump with bafilomycin A1. A significant, but comparatively low passive H+ permeability was detected. The existence of a passive H+ leak implies that continuous energy expenditure is required for the maintenance of an acidic pHp. In combination with ionophores, bafilomycin was also used to estimate the counterion permeability. The counterion conductance was found to be severalfold higher than the H+ leak. Ion substitution experiments in electropermeabilized cells and the inhibitory effects of quinine and 5-nitro-2-(3-phenylpropylamino)benzoic acid suggest that both monovalent anions and cations permeate the phagosomal membrane. The activity of the H+ pump was measured at various pHp levels. In the steady state, the rate of H+ pumping was considerably lower than counterion permeation. These findings suggest that the phagosomal membrane potential is insignificant. Consistent with this notion, increasing phagosomal conductance with ionophores failed to accelerate the rate of H+ pumping. Thus, the transmembrane delta pH is the predominant component of the proton-motive force across the phagosomal membrane in the steady state. The rate of H+ pumping was found to decrease steeply as the phagosomal lumen became acidified. Therefore, the pH sensitivity of the H+ pump, which possibly reflects a kinetic or allosteric effect, is the primary determinant of pHp.

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.

Sodium channel but neither Na(+)-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance.

Normal clearance of alveolar liquid following birth requires active Na transport; however, the contribution of Na channels, Na-H antiports, and Na-glucose symports is unknown. We demonstrated that intraalveolar instillation of amiloride (n = 6) or the more specific Na channel blockers benzamil (n = 13) or phenamil (n = 12) before the first breath impaired lung water clearance relative to control newborns (n = 34). Benzamil and phenamil were more potent than amiloride (P less than 0.05). Neither the Na-H antiport inhibitor dimethyl amiloride (n = 7) nor the Na-glucose symport inhibitor phloridzin (n = 7) impaired lung water clearance. Ion substitution experiments with fetal rat type II alveolar epithelia demonstrated that more than 95% of their resting or terbutaline-stimulated short circuit current (Isc) depended upon Na bathing their apical membrane. Isc was decreased by amiloride (IC50 of amiloride-sensitive Isc = 0.3 x 10(-6) M) and benzamil (IC50 of benzamil-sensitive Isc = 0.3 x 10(-7) M) but was unaffected by dimethyl amiloride (10(-4) M). We conclude that in vivo postnatal clearance of fetal lung liquid can be impaired by Na channel blockers and is unaffected by blockers of Na-H antiports and Na-glucose symports. Na transport in fetal type II cells has high affinity for amiloride, and these cells likely contribute to normal neonatal lung liquid clearance.

Electric properties of rat liver cell cultures on gas-permeable membranes

In rat hepatocytes grown on gas-permeable membranes (Petzinger et al. In Vitro Cell. Dev. Biol. 24: 491-499, 1988), cellular and canalicular potentials as well as input resistances were measured using two-channel microelectrodes. In HCO3(-)-containing solutions, we found -30.9 +/- 0.4 (SE) (n = 141) and -13.9 +/- 1.4 mV (n = 22) for cell and canalicular membrane potentials, respectively. There was no dependence of these parameters on the age of the primary culture. Canalicular input resistance, however, increased from 13.3 +/- 2.0 M omega (n = 4) at day 1 after seeding to 36.1 +/- 5.0 M omega (n = 9) at day 2 and stabilized thereafter, while cell input resistance continuously decreased from 37.0 +/- 3.3 M omega at 1 h (n = 6) to 5.2 +/- 2.1 M omega (n = 27) at 3 days after preparation. In ion substitution experiments there were no changes in the transference numbers for K+, Na+, or Cl- that could account for this effect. Cable analysis, however, revealed that the decrease in input resistance reflects a time-dependent increase in electrical coupling between cells. We conclude that rat liver cells on gas-permeable membranes are highly suited for the quantitative analysis of cell-to-cell interaction. In addition, cells and canaliculi are readily accessible with two-channel microelectrodes, making this preparation a promising tool for electrophysiological analysis of hepatocellular transport mechanisms.

Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture

The transition of the fetal lung from a fluid-secreting to a fluid-absorbing organ is dependent on ion transport across the pulmonary epithelium. This study examined the ion transport characteristics of distal pulmonary epithelial cells isolated from rat fetuses in late gestation and maintained in differentiation-arrested monolayer cultures. The response to inhibitors of active ion transport suggested the presence of apical to basolateral Na+ transport in monolayers derived from each gestational age. However, amiloride inhibition of short-circuit current (Isc) varied with gestational age, decreasing Isc by 30% in monolayers derived from day 18 fetuses and by 55% in monolayers from day 21 fetuses. A portion (10%) of the residual Isc remaining after amiloride addition to monolayers from day 18 fetuses could be inhibited by bumetanide, suggesting the induction of net Cl- transport. Ion-substitution experiments confirmed the presence of Na+ and inducible Cl- transport mechanisms in monolayers from day 18 fetuses and only Na+ transport mechanisms in monolayers from day 21 fetuses. beta-Adrenergic stimulation increased Isc but maintained the age-dependent characteristics of Na(+)- and Cl(-)-dependent ion transport. In summary, monolayer cultures of fetal pulmonary epithelial cells exhibit age-dependent differences in ion transport properties that are consistent with a transition or maturation of the distal pulmonary epithelium from an epithelium capable of Na+ absorption and Cl- secretion preterm to one capable of only Na+ absorption at term.

Physiological and pharmacological properties of 5-HT 3 receptors — A patch clamp-study

Whole cell and patch clamp techniques were used to investigate the properties of 5-HT 3 receptors of a murine neuroblastoma cell line (N1E-115) and adult rabbit nodose ganglion neurones. In addition, some preliminary results from guinea-pig nodose ganglion neurones are presented. In such cells, voltage-clamped at −60mV, 5-HT (10μM) induced an inward current associated with a conductance increase. The results of ion substitution experiments suggest that the 5-HT activated ion channel is permeable to both Na + and K + ions with a permeability ratio (P Na/P K) of 0.94 and 0.92 for rabbit nodose ganglion cells and N1E-115 cells respectively. On outside out membrane patches excised from rabbit nodose ganglion neurones, 5-HT (1 μM) activated clearly discernible single channel currents with a conductance of 16.6 ± 0.7 pS (n = 4). In contrast, fluctuation analysis of 5-HT induced whole cell currents suggests that the single channel conductance of N1E-115 cells is only 0.3 pS, a value some 50 fold lower. The 5-HT-induced whole cell currents recorded from all three preparations were antagonised by the selective 5-HT 3 receptor antagonist ondansetron (GR38032F) and by the less selective agents metoclopromide, cocaine and (+)-tubocurarine. However, these preparations demonstrate a differential sensitivity to some antagonists. In particular, (+)-tubocurarine was a potent antagonist in N1E-115 cells (IC 50 = 0.85 nM) but was approximately 200 fold (IC 50 = 156 nM) and 1200 fold (IC 50 = 10 μM) less potent in rabbit and guinea-pig nodose ganglion neurones respectively. Additionally, a novel effect of ketamine (10 μM) to potentiate the 5-HT-induced current of rabbit nodose ganglion neurones is described.

Acute electrophysiological responses of bradykinin-stimulated human fibroblasts.

1. Acute responses to bradykinin in human dermal fibroblasts were studied at 20-24 degrees C using both the patch-clamp technique to monitor ion currents and Fura-2 fluorescence to monitor [Ca2+]i. 2. During subconfluent culture, human dermal fibroblasts can express a diversity of ion channels as described in the preceding paper. 3. When GTP (1 mM) was included in the pipette solution, two additional ion channel populations were transiently augmented in response to bradykinin stimulation. 4. The first is a component of outwardly rectifying current which reached maximal induction within 10-15 s after bradykinin addition (1 microM) and then decayed back to near baseline over 60 s. 5. Ion substitution experiments combined with tail current analysis indicate that the outward current is carried predominantly by K+. 6. Video imaging of single-cell Fura-2 fluorescence from both intact cells and patch-clamped cells showed temporal correlation of the K+ current modulation and the Ca2+ transients in response to bradykinin stimulation. 7. The calcium ionophore, ionomycin, caused both an increase in intracellular calcium and the augmentation of the outward K+ current. The amount of additional K+ current was correlated with [Ca2+]i levels and could be elicited even without the presence of GTP in the pipette. 8. Apamin, a blocker of Ca(2+)-activated K+ channels, inhibited (at 1 microM) the ionomycin-induced modulation of K+ current. 9. In addition, an inward current was transiently induced in response to bradykinin. This current was strictly dependent on the presence of GTP in the pipette solution. This current showed little voltage dependence, as evidenced by a linear current vs. voltage relation, and a reversal potential near but measurably more positive than 0 mV. 10. This current could be decoupled from the Ca2+ transient and be irreversibly induced by including GTP gamma S (100 microM) in the pipette solution. 11. Ion substitution experiments show that this is a non-specific cation channel. This current prefers monovalents but exhibits a small permeability to divalents. 12. GTP gamma S-induced single channels from isolated outside-out patches showed similar ion selectivity and voltage dependence. These channels are 32 pS in size with an estimated reversal potential of 17 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Plateau potentials drive axonal impulse bursts in insect motoneurons

Intracellular recordings were made from the soma of the `fast' coxal depressor motoneuron of the hind leg of the cockroach Periplaneta americana under current- and voltage-clamp. In response to depolarizing current injection, this neuron was able to generate plateau potentials that were able to far outlive the duration of the applied depolarization. These events constitute an inherent membrane property of the neuron because they could be evoked in somata that had been surgically isolated from other parts of the neuron (the soma is devoid of synaptic contacts); these experiments also showed that the soma of this neuron can participate in the generation of plateau potentials. The amplitude of these events at the spike-initiating zone was sufficient to evoke action potentials that correlated 1:1 with axonal impulses recorded extracellularly from the axon of the neuron. The ability to generate plateau potentials is associated with a region of negative slope resistance in the current--voltage relation of the neuron. Ion substitution experiments showed that the plateau potentials recorded from this neuron are calcium dependent. Our observations show for the first time that an insect motoneuron can produce plateau potentials. Accordingly, bistable membrane properties may play a role in shaping motor output in insects.

Analysis of a decreased Na+ conductance by tumor necrosis factor in identified neurons of Aplysia kurodai.

The ionic mechanisms of the effect of extracellularly ejected recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on the membrane of identified neurons R9 and R10 of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion substitution techniques. Micropressure-ejected rhTNF caused a marked hyperpolarization in the unclamped neuron. Clamping the same neuron at it resting potential level (-60 mV) and reejecting rhTNF-alpha with the same dose produced a slow outward current [Io (TNF)] associated with a decrease in input membrane conductance. Io (TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io (TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that Io (TNF) in identified neurons R9 and R10 of A. kurodai is due to a decreased Na+ conductance but not due to an activation of the Na(+)-K+ pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the immune system.

Serotonin is released from isolated leech ganglia by potassium-induced depolarization

1. The quantities of serotonin that are released from isolated leech ganglia in vitro were measured with the sensitive neurochemical technique of HPLC-EC. 2. Segmental ganglia were exposed to elevated concentrations of potassium that depolarize leech serotonin-containing neurons by approximately 35 mV per decade. 3. Each segmental ganglion released on average 0.20 pmol of serotonin during 10 min of incubation in a solution containing 64 mM K +. 4. The rate of serotonin release increased nearly four-fold to 0.74 pmol 10 min when ganglia were incubated in 120 mM K +. 5. The rates of ganglionic serotonin release in 120 mM K + were quantitatively similar in these three, experimentally important species of leeches: Hirudo medicinalis, Macrobdella decora and Haementeria ghilianii. 6. Ionic substitution experiments with the divalent cations Mg 2+ and Co 2+ indicated that the release of serotonin from leech ganglia is mediated by a Ca 2+ dependent process. 7. The serotonin-uptake blockers, imipramine and chlorimipramine, did not increase the amount of serotonin released in elevated potassium. 8. Vitally staining the identified serotonin-containing neurons with Neutral Red dye did not reduce the quantity of serotonin that was released from the ganglia in elevated potassium. 9. This study demonstrates the capacity of leech ganglia to release the neurochemical serotonin, and the rates of transmitter release increase with the degree of depolarization of serotonin-containing neurons.

Intracellular chloride in submucosal gland cells

The chloride ion concentration within isolated tracheal submucosal gland cells was studied micro-spectrofluorometrically using a fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), that is quenched by Cl −. Cells from normal weanling swine and from a cystic fibrosis (CF) patient were used. Ion substitution experiments showed that cell fluorescence increased in both cell types when bath Cl − was replaced with the impermeant anion glucuronate. Following a Donnan-type ion substitution that kept the product of the bath K + and Cl − concentrations constant, reducing bath chloride had little effect on fluorescence for normal cells, but caused a marked increase for CF cells. Thus, K + and Cl − ions have approximately the same Nernst potential in control submucosal gland cells; in contrast, cells from a CF patient concentrated Cl i, resulting in a Cl − Nernst potential that was more positive than the K + Nernst potential. This finding is consistent with the hypothesis that CF submucosal gland cells have a decreased Cl − permeability.

Potassium currents in canine airway smooth muscle cells

The electrical properties of dissociated canine tracheal smooth muscle cells were examined using the whole cell patch-clamp technique. In current clamp mode, current clamp steps did not initiate action potentials but showed clear outward rectification, which was abolished when cells were loaded with Cs+ ions and when tetraethylammonium (TEA+) ions replaced Na+ in the bath solution. In voltage-clamp experiments, depolarizations positive to -45 mV evoked brief voltage-dependent inward Ca2+ currents [Am. J. Physiol. 254 (Cell Physiol. 23): C793-C801, 1988], followed by sustained outward currents, which did not completely inactivate. Outward currents were identified as K+ currents on the basis of the reversal potential of the current and by ion-substitution experiments. The currents were further defined as Ca2(+)-insensitive delayed rectifier currents, since they were unaltered under conditions in which 1) the Ca2+ current was completely blocked by Mn2+ or nifedipine (10 microM); 2) Ba2+ ions were substituted for Ca2+ as the inward current charge carrier; or 3) charybdotoxin (40 nM) or TEA+ (up to 10 mM) were added to the bath. A Ca2(+)-activated potassium [K(Ca)] current was activated by application of methacholine (100 microM), or A23187 (1 microM), under conditions of low Ca2+ buffering capacity in the internal solution [0.3 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)]. The K(Ca) current was blocked by 10 mM TEA+ and was not observed under conditions of high intracellular Ca2+ buffering (11 mM EGTA). These data indicate that canine airway smooth muscle cells contain voltage-dependent delayed rectifier channels that underlie membrane rectification and K(Ca) channels that are activated by agents which release intracellular Ca2+ stores.

Stretch‐activated channels in single early distal tubule cells of the frog.

1. Single stretch-activated channels have been studied in cell-attached and excised patches from single early distal tubule (diluting segment) cells of Rana temporaria. 2. The channels can be reversibly activated, in both cell-attached and excised patches, by the application of negative pressure to the pipette causing mechanical stretching of the cell membrane. In cell-attached patches, application of 14.8 cmH2O negative pressure to the patch pipette increased reversibly the open probably from 0.11 to 0.87. 3. The channel conductance in the cell-attached configuration with standard Ringer solution in the pipette is 21.3 pS. 4. The channel is non-specific. In excised inside-out patches ion substitution experiments show that the channel does not discriminate between sodium and potassium ions, nor does it appear to select for cations over anions. 5. The channel is voltage sensitive such that depolarizing the cell opens the channel. The open probability at the resting membrane potential, 0.89, was reduced to 0.26 at a hyperpolarizing potential of 100 mV (holding pressure of -20.1 cmH2O or -206 Pa). 6. The sensitivity of the channel to mechanical stretching suggests that the channel may be involved in cell volume regulation.