Transmitter Secretion in the Frog Neuromuscular Synapse after Prolonged Exposure to Calcium-Free Solutions

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5–6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings. At initially low extracellular Ca ion concentrations (0.15–0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.     

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.     

Mechanisms of the Non-Neurotransmitter Actions of Acetylcholine in the Neuromuscular Apparatus

Experiments on isolated phrenic-diaphragmatic preparations from rats showed that low acetylcholine concentrations increase the work capacity of exhausted muscle by increasing the level of evoked quantum transmitter release; acetylcholine also induced hyperpolarization of muscle fiber membranes. The effects of acetylcholine persisted for long periods of time. The modulating actions of acetylcholine are mediated by structures with different pharmacological characteristics from those of typical n- and m-cholinoreceptors; these mechanisms involved ouabain-sensitive isoforms of Na⁺,K⁺-ATPase and, perhaps, membrane K⁺channels. The data obtained here support the possible existence of long-term neuronal regulation of the efficiency of neuromuscular transmission involving non-quantal acetylcholine, these mechanisms presumably developing differently in muscle fibers with different functional characteristics and abilities to adapt to physiological loading.     

Membrane cycling after the excess retrieval mode of rapid endocytosis in mouse chromaffin cells

Aim: After exocytosis, neuroendocrine cells and neurones keep constant the plasma membrane and the releasable vesicle pools by performing endocytosis and vesicular cycling. Patch‐clamp capacitance measurements on chromaffin cells showed that strong Ca⁺² entry activates excess retrieval: a rapid endocytosis process that retrieves more membrane than the one fused by preceding exocytosis. The main purpose of the present experiments was to study the recycling pathway that follows excess retrieval, which is unknown. Methods: Membrane recycling after exocytosis–endocytosis can be studied by fluorescence imaging assays with FM1‐43 (Perez Bay et al. Am J Physiol Cell Physiol 2007; 293, C1509). In this work, we used this assay in combination with fluorescent dextrans and specific organelle‐targeted antibodies to study the membrane recycling after excess retrieval in mouse chromaffin cells. Results: Excess retrieval was observed after the application of high‐K⁺ or cholinergic agonists during 15 or 30 s in the presence of FM1‐43. We found that the excess retrieval membrane pool (defined as endocytosis–exocytosis) was associated with the generation of a non‐releasable fraction of membrane (up to 30% of plasma membrane surface) colocalizing with the lysosomal compartment. The excess retrieval membrane pool followed a saturable cytosolic Ca²⁺ dependency, and it was suppressed by inhibitors of L‐type Ca²⁺ channels, endoplasmic reticulum Ca²⁺ release and PKC. Conclusion: Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non‐releasable endosomes. This process is activated by a concerted contribution of Ca²⁺ entry through L‐channels and Ca²⁺ release from endoplasmic reticulum.     

Submandibular salivary secretion in the cat and associated potassium movements: Dependence on temperature and perfusate flow rate

Cat submandibular glands were perfused with Locke solution in a thermostated chamber and intermittently stimulated with 10^–5 M acetylcholine (ACh). In one series of experiments the perfusion pressure was varied within the range 90–60 mm Hg, and secretory flow rate, active K⁺-reuptake, passive K⁺-release, and resting and ACh-induced venous flow rates were measured. The ACh-induced secretory flow rate and the maximal K⁺-fluxes were related to the simultaneous ACh-induced venous flow rates. A proportionality was found between the maximal rate of ACh-induced K⁺-release and ACh-induced venous flow rates below 8 ml/min, while at higher flow rates the K⁺-release leveled off. The maximal rate of the post-stimulatory K⁺-reuptake increased proportionally to the ACh-induced perfusate flow rate throughout the range studied. The secretory flow rate was much less affected by changes in ACh-induced perfusate flow rate. In another series of experiments the gland temperature was varied within the range 12–37°C, and the same parameters were measured. All parameters decreased with cooling being reduced to 50% of their 37°C values at: 24°C for secretion, 19°C for K⁺-reuptake, and 14°C for K⁺-release. It is concluded: that 1) the rate of ACh-induced K⁺-release is limited by the ACh-induced perfusate flow rate (within the physiological range), 2) the capacity of the K⁺-reuptake mechanism is at least one order of magnitude larger than the maximal rate of K⁺-reuptake in vivo, 3) the marked temperature sensitivity of the secretory flow rate reflects the high complexity of the mechanisms involved.     

High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Using the patch clamp technique, one type of K⁺ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K⁺ over Na⁺ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K⁺-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba²⁺ (5 mmol/l) and partially inhibited by Na⁺. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na⁺ competed with K⁺ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca²⁺ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K⁺ secretion to Na⁺ absorption in the fine regulation of cation excretion in renal collecting ducts.     

The vesicle cycle in motor nerve endings of the mouse diaphragm

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1–2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5–6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 2, pp. 129–141, February, 2008.     

Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

Neuronal death leading to gross brain atrophy is commonly seen in Alzheimer's disease (AD) patients. Yet, it is becoming increasingly apparent that the pathogenesis of AD involves early and more discrete synaptic changes in affected brain areas. However, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently, we have identified dynamin 1, a protein that plays a critical role in synaptic vesicle endocytosis, and hence, in the signaling properties of the synapse, as a potential molecular determinant of such dysfunction in AD. In the present study, we analyzed beta-amyloid (Abeta)-induced changes in synaptic vesicle recycling in rat cultured hippocampal neurons. Our results showed that Abeta, the main component of senile plaques, caused ultrastructural changes indicative of impaired synaptic vesicle endocytosis in cultured hippocampal neurons that have been stimulated by depolarization with high potassium. In addition, Abeta led to the accumulation of amphiphysin in membrane fractions from stimulated hippocampal neurons. Moreover, experiments using FM1-43 showed reduced dye uptake in stimulated hippocampal neurons treated with Abeta when compared with untreated stimulated controls. Similar results were obtained using a dynamin 1 inhibitory peptide suggesting that dynamin 1 depletion caused deficiency in synaptic vesicle recycling not only in Drosophila but also in mammalian neurons. Collectively, these results showed that Abeta caused a disruption of synaptic vesicle endocytosis in cultured hippocampal neurons. Furthermore, we provided evidence suggesting that Abeta-induced dynamin 1 depletion might play an important role in this process.     

CGRP-induced activation of KATP channels in follicular Xenopus oocytes

The two-microelectrode voltage-clamp technique was used to monitor K⁺ channel activity in Xenopus oocyte follicular cells, which are electrically coupled to the oocyte itself by gap junctions. Endogenous vasodilators such as calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), prostaglandin E₂ (PGE₂) and adenosine activate glibenclamide-ATP-sensitive K⁺ (K_ATP) channels in Xenopus oocyte follicular cells. The mechanism of action of CGRP was studied in detail. CGRP effects undergo a rapid desensitization. CGRP acts via CGRP_I receptors. Its effects are antagonized by the amino-truncated CGRP analog hCGRP(8–37). The second messenger for CGRP activation of K_ATP channels is cAMP. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine enhances the CGRP response while adenyl cyclase inhibition by either 2,5-dideoxyadenosine or progesterone nearly completely depresses the CGRP response. Vasoconstrictors such as ACh and angiotensin II also have receptors in follicular cells. ACh strongly inhibits the CGRP activation of K⁺ channels as it inhibits the activation of K_ATP channels by P1060, but angiotensin II does not. It is concluded that as in vascular smooth muscle cells, CGRP and probably other hyperpolarizing vasodilators open K_ATP channels in follicular cells by protein kinase A activation.Thanks are due to C. Roulinat and F. Aguila for expert technical assistance. This work was supported by the Centre National de la Recherche Scientifique (CNRS).     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel. The most common mutation, ΔF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of ΔF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca²⁺-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca²⁺-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca²⁺-activated K⁺ channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K⁺ channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca²⁺-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca²⁺-activated secretion.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion

The involvement of large-conductance, voltage- and Ca²⁺-activated K⁺ channels (maxi-K⁺ channels) in basolateral Ca²⁺-dependent K⁺-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K⁺ efflux was monitored by measuring the change in K⁺ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na⁺/K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K⁺ efflux, followed by a smaller K⁺ efflux, which gradually returned to the basal level within 10 min. These two components of the K⁺ efflux appear to be dependent on an increase in cytosolic Ca²⁺ concentration. The initial transient K⁺ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K⁺ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba²⁺ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca²⁺-dependent transient large K⁺ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K⁺ current attributable to maxi-K⁺ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K⁺ channels are not responsible for the major part of the Ca²⁺-dependent basolateral K⁺ efflux and fluid secretion by the rat submandibular gland.     

K+ recirculation in A6 cells at increased Na+ transport rates

Homocellular regulation of K⁺ at increased transcellular Na⁺ transport implies an increase in K⁺ exit to match the intracellular K⁺ load. Increased K⁺ conductance, g_K, was suggested to account for this gain. We tested whether such a mechanism is operational in A6 monolayers. Na⁺ transport was increased from 5.1±1.0 A/cm² to 20.7±1.3 A/cm² by preincubation with 0.1 mol/l dexamethasone for 24 h. Basolateral K⁺ conductances were derived from transference numbers of K⁺, t _K, and basolateral membrane conductances, g_b, using conventional microelectrodes and circuit analysis with application of amiloride. Activation of Na⁺ transport induced an increase in g_b from 0.333±0.067 mS/ cm² to 1.160±0.196 mS/cm² and t _K was reduced to 0.22±0.01 from a value of 0.70±0.05 in untreated control tissues. As a result, g_K remained virtually unchanged at increased Na⁺ transport rates. The increase in g_b after dexamethasone was due to activation of a conductive leak pathway presumably for Cl^–. Increased K⁺ efflux, I _K, was a consequence of the larger driving force for K⁺ exit due to depolarization at an elevated Na⁺ transport rate. The relationship between calculated K⁺ fluxes and Na⁺ transport rate, measured as the I _sc, is described by the linear function I _K=0.624×I _Na–0.079, which conforms with a stoichiometry 23 for the fluxes of K⁺ and Na⁺ in the Na⁺/K⁺-ATPase pathway. Our data show that homocellular regulation of K⁺ in A6 cells is not due to up-regulation of g _K .     

Intracellular potassium activity in epithelial cells of frog fundic gastric mucosa

Microelectrodes were used to measure membrane potential and intracellular potassium activity in surface epithelial cells (SEC) of frog (Rana esculenta) fundic gastric mucosa in vitro. Separate measurements were carried out by applying fine-tipped, single barrelled, KCl filled non-selective electrodes and liquid K⁺-selective electrodes. Membrane potentials with respect to the mucosal and serosal surfaces, measured with non-selective electrodes, were –54.5±1.0 S.E. mV (n=59) and –73.0±1.1 S.E. mV (n=59) respectively. The electrical potential difference referred to the mucosal surface, when measured with K⁺-sensitive electrodes, was +21.2±0.8 S.E. mV (n=35), and intracellular K⁺ activity was 98.5 mmol/l. Assuming that intracellular and extracellular K⁺ activity coefficients are equal (_K=_K), the K⁺ concentration is 135.0 mmol/l. The K⁺ equilibrium potential,E _K, was calculated as –90.0 mV i.e. more negative than both membrane potentials. This result indicates active potassium accumulation in the SEC and provides direct evidence of the presence of an active K⁺ pump in either both or in only one of the cell membranes.     

Trans- and paracellular K+ transport in diluting segment of frog kidney

In frog diluting segment transepithelial K⁺ net flux (J _te ^K ) occurs via trans- and paracellular transport routes. Inhibition of transcellular K⁺ transport disclosesJ _te ^K across the shunt-pathway. By means of K⁺-sensitive microelectrodes we have measured secretoryJ _te ^K induced by an acute K⁺ load, in the diluting segment of the isolated and doublyperfused frog kidney. Transcellular K⁺ transport was inhibited by blocking the luminal K⁺ permeability either directly by barium or indirectly by the diuretic drug amiloride (via intracellular acidification induced by inhibition of Na⁺/H⁺ exchange), by the the Na⁺/K⁺ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretoryJ _te ^K to about 50% of the controlJ _te ^K . The apparent permeability coefficient for K⁺ of this nephron portion after inhibition of transcellular secretoryJ _te ^K was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellularJ _te ^K is close to unity. This ratio represents a minimum estimate because inhibition of the transcellular K⁺ pathway by barium, amiloride or an acute acid load may have been incomplete. Acidosis and/or amiloride exert large antikaliuretic effects due to the inhibition of the luminal K⁺ permeability.     

Characterization of potassium channels in respiratory cells

Potassium channels present in the basolateral membrane of respiratory epithelial cells play an important role in the process of chloride secretion. Utilizing the patch clamp technique, we examined human cultured respiratory epithelial cells derived from patients with cystic fibrosis (CF) and normals individual (N) for the existence of and for the properties of K+ channels. We obtained qualitatively and quantitatively identical results for both preparations (CF and N). K+ channels were spontaneously present in cell attached patches. The channels showed burst appearance with rapid flickering within the bursts. When the pipette was filled with 145 mmol/l KCl, a mean conductance of 131 +/- 25 pS (n = 15) was read from the I/V-curve at a clamp voltage (Vc) of 0 mV. After excision, the conductance read from the I/V-curve at Vc = 0 mV was 212 +/- 11 pS (Pipette: 145 mmol/l KCl, bath: 145 mmol/l NaCl) (n = 61). With NaCl in the pipette and KCl in the bath, a similar conductance was obtained (g = 210 pS; n = 2). When both, pipette and bath contained KCl, the conductance was increased to 302 +/- 19 (n = 7). The channel was highly selective for potassium over sodium: PK + /PNa + greater than 40. The channel open probability was only slightly voltage dependent i.e. the open probability increased slightly with depolarisation. For most of the channels one open time constant (to = 6.3 +/- 1.6 ms; n = 22) and one closed time constant (tc = 1.8 +/- 0.3 ms; n = 21) was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)