Total creatine in muscle: imaging and quantification with proton MR spectroscopy

Liver cell volume and intracellular ion concentrations are maintained within a narrow physiologic range by regulated changes in membrane ion permeability. These studies of homozygous HTC hepatoma cells, a model liver cell line, evaluate the relationship between cell volume and membrane ion permeability, and assess the possibility that cell swelling allows the efflux of the intracellular osmolite taurine through the opening of a conductive pathway. Cell swelling induced by exposure to hypotonic solutions (203 mOsm) caused a rapid increase in cell volume, followed by recovery toward basal values. Volume recovery was inhibited by Cl- depletion or by exposure to the putative Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (25 micromol/L). Swelling increased the efflux rates of 36Cl (181% +/- 15%, P < .01) and 125I (310% +/- 21%, P < .01). In whole cell patch clamp recordings, cell swelling induced by 1) exposure to hypotonic solution or 2) intracellular perfusion with hypertonic sucrose-containing solutions activated an anion-selective current which was outwardly rectified and showed time-dependent inactivation at depolarizing potentials. The current density at -80 mV increased proportionally with increases in the transmembrane osmotic gradient from basal values of -1 pA/pF to maximal values of 70 pA/pF with 100 mmol/L sucrose in the pipette. Basal taurine permeability was low, but cell swelling increased the efflux of [1,2-3H]taurine to 1,587% +/- 172% of basal levels (P < .05). Intracellular perfusion with hypertonic solutions activated currents carried by anionic taurine, with an estimated taurine/Cl- permeability ratio of .88 +/- .17 for whole cell currents. These studies demonstrate that the HTC membrane anion permeability is closely coupled to changes in cell volume, and that the recovery from swelling depends upon activation of anion-selective conductance pathways permeable to both Cl- and taurine.     

Ammonia-induced depolarization of cultured rat cortical astrocytes

Exposure of cultured rat cortical astrocytes to increased concentrations of ammonia has been shown to induce morphological and biochemical changes similar to those found in hyperammonemic (e.g., hepatic) encephalopathy in vivo. Alterations of electrophysiological properties are not well investigated. In this study, we examined the effect of ammonia on the astrocyte membrane potential by means of perforated patch recordings. Exposure to millimolar concentrations of NH4Cl induced a slow dose-dependent and reversible depolarization. At steady state, i.e., after several tens of minutes, the cells were significantly depolarized from a resting membrane potential of -96.2 +/- 0.6 mV (n = 83, S.E.M.) to -89.1 +/- 1.6 mV (n = 7, S.E.M.) at 5 mM NH4Cl, -66.3 +/- 3.6 mV (n = 9, S.E.M.) at 10 mM NH4Cl and -50.4 +/- 2.5 mV (n = 12, S.E.M.) at 20 mM NH4Cl, respectively. In order to examine the underlying depolarizing mechanisms we determined changes in the fractional ion conductances for potassium, chloride and sodium induced by 20 mM NH4Cl. No significant changes were found in the fractional sodium or chloride conductances, but the dominating fractional potassium conductance decreased slightly from a calculated 0.86 +/- 0.04 to 0.77 +/- 0.04 (n = 9, S.E.M.). Correspondingly, we found a significant fractional ammonium ion (NH4+) conductance of 0.23 +/- 0.02 (n = 10, S.E.M.) which was blocked by the potassium channel blocker barium and, hence, most likely mediated by barium-sensitive potassium channels. Our data suggest that the sustained depolarization induced by NH4Cl depended on changes in intracellular ion concentrations rather than changes in ion conductances. Driven by the high membrane potential NH4+ accumulated intracellularly via a barium-sensitive potassium conductance. The concomitant decrease in the intracellular potassium concentration was primarily responsible for the observed slow depolarization.     

Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle

The effects of niflumic acid on whole-cell membrane currents and mechanical activity were examined in the rat portal vein. In freshly dispersed portal vein cells clamped at -60 mV in caesium (Cs+)-containing solutions, niflumic acid (1-100 microM) inhibited calcium (Ca2+)-activated chloride currents (IC1(Ca)) induced by caffeine (10 mM) and by noradrenaline (10 microM). In a potassium (K+)-containing solution and at a holding potential of - 10 mV, niflumic acid (10-100 microM) induced an outward K+ current (IK(ATP)) which was sensitive to glibenclamide (10-30 microM). At concentrations < 30 microM and at a holding potential of -2 mV, niflumic acid had no effect on the magnitude of the caffeine- or noradrenaline-stimulated current (IBK(Ca)) carried by the large conductance, Ca(2+)-sensitive K+ channel (BKCa). However, at a concentration of 100 microM, niflumic acid significantly inhibited IBK(Ca)) evoked by caffeine (10 mM) but not by NS1619 (1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3 H) benzimidazolone; 20 microM). In Cs(+)-containing solutions, niflumic acid (10-100 microM) did not inhibit voltage-sensitive Ca2+ currents. In intact portal veins, niflumic acid (1-300 microM) inhibited spontaneous mechanical activity, an action which was partially antagonised by glibenclamide (1-10 microM), and contractions produced by noradrenaline (10 microM), an effect which was glibenclamide-insensitive. It is concluded that inhibition of ICl(Ca) and stimulation of IK(ATP) both contribute to the mechano-inhibitory actions of niflumic acid in the rat portal vein.     

Response to Pneumocystis infection in an immunocompetent host

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Endothelium-derived relaxing factor activates calcium-activated potassium channels of resistance vessel smooth muscle cells

Direct observation was made by using the patch-clamp technique with a specially designed microperfusion system to investigate the effect of acetylcholine (Ach 10(-6) mol/L) elicited endothelium-derived relaxing factor (EDRF) on the calcium-activated potassium channel (IK(Ca)) in the smooth muscle cells of mesenteric resistance vessels in Wistar rats. Activation of IK(Ca) was firstly observed by inducing the elicited EDRF or sodium nitroprusside (SNP 10(-8) mol/L) under various clamping voltages in cell-attached configuration. While the pipette solution contained KCl 126 mmol/L and the bath solution contained KCl 5.9 mmol/L, two types of conductances of calcium-activated potassium current being 76.4 +/- 2.3 pS (mean +/- S.E. n = 7) and 160.3 +/- 7.5 pS (mean +/- S.E. n = 7) were recorded during the EDRF activation, one type of conductance being 100.5 +/- 2.8 pS (mean +/- S.E. n = 6) was activated by nitric oxide (NO) which is an effective component from SNP. Differences in kinetic characteristics of these channels between EDRF and NO activation were found, particularly the probability of the channel being open in EDRF activation was obviously greater than that in NO stimulation. It has been shown that the potassium channel mechanisms involved in the EDRF and NO actions might be different.     

Effect of niflumic acid on noradrenaline-induced contractions of the rat aorta

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline-evoked contractions in isolated preparations of the rat aorta. 2. The cumulative concentration-effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration-dependent manner. The degree of inhibition of the maximal contractile response to NA (1 microM) produced by 10 microM niflumic acid (38%) was similar to the effect of 1 microM nifedipine (39%). 3. Contractions to brief applications (30 s) of 1 microM NA were inhibited by 55% and 62% respectively by 10 microM niflumic acid and 1 microM nifedipine. 4. In the presence of 0.1 microM nifedipine, niflumic acid (10 microM) produced no further inhibition of the NA-evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5. In Ca-free conditions the transient contraction induced by 1 microM NA was not inhibited by niflumic acid (10 microM) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6. Niflumic acid 10 microM did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 microM levcromakalim but were unaffected by niflumic acid. 7. It was concluded that niflumic acid produces selective inhibition of a component of NA-evoked contraction which is probably mediated by voltage-gated calcium channels. These data are consistent with a model in which NA stimulates a calcium-activated chloride conductance which leads to the opening of voltage-gated calcium channels to produce contraction.     

Black widow spider venom: effect of purified toxin on lipid bilayer membranes

A purified toxin (the B5 fraction) from black widow spider venom added to the solution on one side of a lipid bilayer membrane interacts irreversibly with the membrane to produce a continuous, linear rise of membrane conductance with time. Conductances greater than 10(-4) reciprocal ohm per square centimeter can eventually be attained without any loss of membrane stability. Membranes treated with toxin are ideally selective for alkali cations over anions and are substantially permeable to calcium ion. These effects of the toxin result from the formation of permanent channels in the membrane of uniform conductance, 3.6 X 10(-10) reciprocal ohm (in 0.1 molar potassium chloride), that remain open almost all the time. Both the divalent cation permeability and the smaller conductances at low pH of toxin-treated membranes suggest that there is negative charge (possibly from carboxyl groups) associated with the channels. We discuss the possible relation of the action of this toxin on lipid bilayer membranes to its ability to stimulate massive transmitter release at the neuromuscular junction and to produce profound morphological changes on tissue cultured neurons.     

Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.     

The pharmacology of mesocortical dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area and prefrontal cortex of the rat brain

The development of receptor function at corticothalamic synapses during the first 20 days of postnatal development is described. Whole cell excitatory postsynaptic currents (EPSCs) were evoked in relay neurons of the ventral posterior nucleus (VP) by stimulation of corticothalamic fibers in in vitro slices of mouse brain from postnatal day 1 (P1). During P1-P12, excitatory postsynaptic conductances showed strong voltage dependence at peak current and at 100 ms after the stimulus and were almost completely antagonized by -2-amino-5-phosphonopentoic acid (APV), indicating that N-methyl--aspartate (NMDA) receptor-mediated currents dominate corticothalamic EPSCs at this time. After P12, in 42% of cells, excitatory postsynaptic conductances showed no voltage-dependence at peak current but still showed voltage-dependence 100-ms poststimulus. This voltage-dependent conductance was antagonized by APV. The nonvoltage-dependent component was APV resistant, showed fast decay, and was antagonized by the nonNMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In the remaining 58% of cells after P12, excitatory postsynaptic conductances showed moderate voltage dependence at peak conductance and strong voltage dependence 100 ms after the stimulus. Analysis of EPSCs before and after APV showed a significant increase in the relative contribution of the non-NMDA conductance after the second postnatal week. From P1 to P16, there was a significant decrease in the time constant of decay of the NMDA EPSC but no change in the voltage dependence of the NMDA response. After P8, slow EPSPs, 1.5-30 s in duration and mediated by metabotropic glutamate receptors (mGluRs), could be evoked by high-frequency stimulation of corticothalamic fibers in the presence of APV and CNQX. Similar slow depolarizations could be evoked by local application of the mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) but from P0. Both conductances were blocked by the mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. Hence functional mGluR receptors are present on VP cells from birth, but their synaptic activation at corticothalamic synapses can only be detected after P8. In voltage clamp, the extrapolated reversal potential of the t-ACPD current, with potassium gluconate-based internal solution, was +12 +/- 10 (SE) mV, and the measured reversal potential with cesium gluconate-based internal solution was 1.5 +/- 9.9 mV, suggesting that the mGluR-mediated depolarization was mediated by a nonselective cation current. Replacement of NaCl in the external solution caused the reversal potential of the current to shift to -18 +/- 2 mV, indicating that Na+ is a charge carrier in the current. The current amplitude was not reduced by application of Cs+, Ba2+, and Cd2+, indicating that the t-ACPD current was distinct from the hyperpolarization-activated cation current (IH) and distinct from certain other previously characterized mGluR-activated, nonselective cation conductances.     

Differential regional hemodynamic effects of corticotropin in conscious sheep

The regional hemodynamic effects of 5 days of intravenous infusion of corticotropin (ACTH) (5 micrograms/kg per day) were examined in conscious sheep (n = 8). Mean arterial pressure increased from 81 +/- 2 to 93 +/- 3 mm Hg (P < .001) on day 2 of ACTH and remained at this level during the infusion. Cardiac output increased from 5.13 +/- 0.19 to 6.06 +/- 0.33 L/min (P < .01) because of an increase in stroke volume from 65 +/- 4 to 79 +/- 8 mL per beat (P < .01); heart rate remained unchanged. ACTH did not alter total peripheral conductance but had differential effects on regional conductances. Mesenteric conductance fell from 5.8 +/- 0.2 to a minimum of 4.9 +/- 0.3 (mL/min)/mm Hg (P < .05), and renal conductance increased from 3.5 +/- 0.3 to 4.6 +/- 0.3 (mL/min)/mm Hg (P < .001). There was a small increase in iliac conductance (P < .05) and no change in coronary conductance. Mesenteric and iliac conductances fell progressively over 24 to 48 hours, whereas renal conductance increased rapidly after 3 hours of ACTH, reaching a maximum after 6 hours. Renal blood flow was increased during ACTH infusion from 278 +/- 18 to 403 +/- 23 mL/min (P < .001); mesenteric blood flow was unchanged; there was a small increase in iliac blood flow (P < .01); and coronary blood flow increased (P < .05), paralleling the change in cardiac output.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological changes that accompany reactive gliosis in vitro

An in vitro injury model was used to examine the electrophysiological changes that accompany reactive gliosis. Mechanical scarring of confluent spinal cord astrocytes led to a threefold increase in the proliferation of scar-associated astrocytes, as judged by bromodeoxyuridine (BrdU) labeling. Whole-cell patch-clamp recordings demonstrated that current profiles differed absolutely between nonproliferating (BrdU-) and proliferating (BrdU+) astrocytes. The predominant current type expressed in BrdU- cells was an inwardly rectifying K+ current (KIR; 1.3 pS/pF). BrdU- cells also expressed transient outward K+ currents, accounting for less than one-third of total K+ conductance (G). In contrast, proliferating BrdU+ astrocytes exhibited a dramatic, approximately threefold reduction in KIR (0.45 pS/pF) but showed a twofold increase in the conductance of both transient (KA) (0.67-1. 32 pS/pF) and sustained (KD) (0.42-1.10 pS/pF) outwardly rectifying K+ currents, with a GKIR:GKD ratio of 0.4. Relative expression of GKIR:GKD led to more negative resting potentials in nonproliferating (-60 mV) versus proliferating astrocytes (-53 mV; p = 0.015). Although 45% of the nonproliferating astrocytes expressed Na+ currents (0.47 pS/pF), the majority of proliferating cells expressed prominent Na+ currents (0.94 pS/pF). Injury-induced electrophysiological changes are rapid and transient, appearing within 4 hr postinjury and, with the exception of KIR, returning to control conductances within 24 hr. These differences between proliferating and nonproliferating astrocytes are reminiscent of electrophysiological changes observed during gliogenesis, suggesting that astrocytes undergoing secondary, injury-induced proliferation recapitulate the properties of immature glial cells. The switch in predominance from KIR to KD appears to be essential for proliferation and scar repair, because both processes were inhibited by blockade of KD.     

Characterization of maxi-K-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate

PURPOSE: Electrophysiological characterization of trabecular meshwork cells and investigation of their response to elevation of cytosolic cyclic guanosine monophosphate (cGMP). METHODS: Bovine trabecular meshwork cells were cultured according to established methods and were studied, using the whole-cell and single-channel configurations of the patch-clamp technique. RESULTS: In single-channel experiments, cells expressed a channel with characteristics typical of maxi-K-channels. The channel was densely distributed in the membrane and had a high conductance of 326 +/- 4 pS (Pico Siemens) (symmetrical 150 mmol/l KCl; 37 degrees C) for potassium and negligible conductance for sodium (0.9 +/- 1 pS). The open probability could be elevated by depolarization, increasing cytosolic calcium, or adding adenosine triphosphate (1 mmol/ l). The channel could be blocked by external charybdotoxin (10(-8) mol/1), external TEA+ tetraethyl ammonium chloride (1 mmol/l) and by internal Ba2+ (10 mmol/l), whereas external Ba2+ and internal TEA+ (10 mmol/l) had no effect. In whole-cell experiments, trabecular meshwork cells displayed a strong outward conductance. Part of this conductance (35 +/- 5%) could be blocked by charybdotoxin and stimulated by ionomycin (10(-5) mol/1). Addition of 8-bromo-cGMP (10(-3) mol/1) stimulated the current to 290 +/- 57% (n = 4) of the original level, charybdotoxin led to a reduction of this current to 156 +/- 28% of the initial value. CONCLUSIONS: Trabecular meshwork cells express maxi-K-channels. These channels can be stimulated by raising internal cGMP levels and are known for their importance in smooth muscle relaxation. The results in this study supply further evidence that trabecular meshwork displays smooth muscle-like properties and contributes to the clarification of the mechanism leading to the relaxation of trabecular meshwork by nitrate and nonnitrate vasodilatators.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses

In an in-vitro preparation of gastric mucosae of Rana pipiens, the effect of adding melittin to a concentration of 5x10-6 M in the secretory solution on the transepithelial potential difference (PD), resistance (R) and short-circuit current (Isc) was studied. In 20 min, melittin decreased the PD by 9.3 mV and R by 148 ohm cm2. These changes can be explained by a decrease in the resistance, RP, of the paracellular pathway. To determine whether specific-ion pathways were responsible for the decrease in R, the effect of melittin on the partial conductances of Cl-, K+ and Na+ was also studied using the ion substitution method. Melittin decreased the PD response to changes in nutrient Na+, K+ and Cl- and the PD response to changes in secretory Cl-, but did not affect PD responses to changes in secretory Na+ or K+. Therefore, melittin decreased the nutrient membrane partial conductances of Cl-, K+ and Na+ and secretory membrane partial conductance of Cl-, without affecting the secretory partial conductances of Na+ or K+. Initially, melittin increased Isc in regular and Cl--free but not in Na+-free solutions. There was a delayed decrease in Isc. The results indicate that melittin decreases RP, increases the Na+ conductance of the secretory membrane and inhibits, eventually, the Na+/K+-ATPase pump.     

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas

Nonselective cation channels have been identified and linked to important cell functions in rat hepatocytes. In this study, we characterized inward rectifying nonselective cation channels in detail by the patch clamp technique in human HepG2 cells. Channel properties were studied with high resistance borosilicate pipettes in cell-attached and inside-out configurations. With Ringer's solution and KCl as pipette solutions, the conductances were 19.7 +/- 2.1 and 22.2 +/- 0.0 picosiemens (pS), and reversal potentials were 30.9 +/- 3.5 and 31.3 +/- 4.6 mV, respectively. The channel was permeable to Ba2+, and the sequence of permeability ratios was Na+ > K+ > Cs+ > Ba2+. In the cell-attached configuration, the channel had a higher opening probability at depolarizing potential than at hyperpolarizing. In the inside-out patches with symmetric Ringer's solution, the current voltage curve was linear with conductance of 19.8 +/- 0.9 pS. Reversal potential shifted from -0.2 +/- 1.0 mV to 23.2 +/- 1.0 mV when the bath solution was replaced by dilute Ringer's solution. In the inside-out configuration, the gating was Ca(2+)-dependent, and the opening probability increased with increasing intracellular calcium concentration ([Ca2+]i). An outward rectifying channel appeared when [Ca2+]i was less than 1 mumol/L. The nonselective channel was reversibly blocked by 10 mumol/L internal flufenamic acid. We conclude that Ca(2+)- and voltage-dependent nonselective cation channels are present in human HepG2 cells. The channels might be involved in the regulation of Ca2+ influx and are associated with activation of other ion channels.     

Intracellular potassium activities in Amphiuma small intestine

Intracellular potassium activity (aKi) has been determined in absorptive cells lining the villi of isolated, stripped proximal segments of Amphiuma small intestine. With single-barreled liquid ion-exchanger microelectrodes aKi = 41.6 +/- 1.5 mM in normal chloride buffer; with double-barreled microelectrodes constructed by a new method aKi = 38.5 +/- 2.4 mM. Also, by the latter approach aKi = 41.1 +/- 2.1 mM in buffer in which potassium was elevated to 5 meq/liter and aKi = 44.2 +/- 1.3 mM in sulfate buffer with the same bath potassium concentration. Since the calculated potassium equilibrium potential exceeds the membrane potential this ion is accumulated by the intestinal absorptive cell. A major portion of cellular potassium is bound or compartmentalized since the intracellular potassium activity coefficient is very low. A layer exists near the villi in which the potassium activity exceeds that in the bath buffer solution.     

Glutamate currents in morphologically identified human dentate granule cells in temporal lobe epilepsy

Glutamate-receptor-mediated synaptic transmission was studied in morphologically identified hippocampal dentate granule cells (DGCs; n = 31) with the use of whole cell patch-clamp recording and intracellular injection of biocytin or Lucifer yellow in slices prepared from surgically removed medial temporal lobe specimens of epileptic patients (14 specimens from 14 patients). In the current-clamp recording, low-frequency stimulation of the perforant path generated depolarizing postsynaptic potentials that consisted of excitatory postsynaptic potentials and phase-inverted inhibitory postsynaptic potentials mediated by the gamma-aminobutyric acid-A (GABA(A)) receptor at a resting membrane potential of -62.7 +/- 2.0 (SE) mV. In the voltage-clamp recording, two glutamate conductances, a fast alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitatory postsynaptic current (EPSC; AMPA EPSC) and a slowly developing N-methyl-D-aspartate (NMDA)-receptor-mediated EPSC (NMDA EPSC), were isolated in the presence of a GABA(A) receptor antagonist. NMDA EPSCs showed a voltage-dependent increase in conductance with depolarization by exhibiting an N-shaped current-voltage relationship. The slope conductance of the NMDA EPSC ranged from 1.1 to 9.4 nS in 31 DGCs, reaching up to twice the size of the AMPA conductance. This widely varying size of the NMDA conductance resulted in the generation of double-peaked EPSCs and a nonlinear increase of the slope conductance of up to 37.5 nS with positive membrane potentials, which resembled "paroxysmal currents," in a subpopulation of the neurons. In contrast, AMPA EPSCs, which were isolated in the presence of an NMDA receptor antagonist (2-amino-5-phosphonovaleric acid), showed voltage-independent linear changes in the current-voltage relationship and were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The AMPA conductance showed little variance, regardless of the size of the NMDA conductance of a given neuron. The average AMPA slope conductance was 5.28 +/- 0.65 (SE) nS in 31 human DGCs. This value was similar to AMPA EPSC conductances in normal rat DGCs (5.35 +/- 0.52 nS, mean +/- SE; n = 55). Dendritic morphology and spine density were quantified in the individual DGCs to assess epileptic pathology. Dendritic spine density showed an inverse correlation (r2 = 0.705) with a slower rise time and a longer half-width of the excitatory postsynaptic potentials mediated by the NMDA receptor. It is concluded that both AMPA and NMDA EPSCs contribute to human DGC synaptic transmission in epileptic hippocampus. However, a wide range of changes in the slope conductance of the NMDA EPSCs suggests that the NMDA-receptor-mediated conductance could be altered in human epileptic DGCs. These changes may influence the generation of chronic subthreshold epileptogenic synaptic activity and give rise to pathological excitation leading to epileptic seizures and dendritic pathology.     

Comparative analysis of latrotoxin channels of different conductance in planar lipid bilayers. Evidence for cluster organization

It has been established that channels induced by Latrodectus tredicimguttatus alpha-toxin (LT) in lipid bilayers have a cluster organisation. So far as: (i) the LT-channels had practically identical sizes of its water pores (r = 9.4 +/- 0.6 A) independently on the lipid composition of planar bilayer lipid membrane (BLM) although their conductances might differ from each other more than 10 times (100 mM KCl (pH 7.5)). (ii) affinity of permeable ions to channels had a small variation with distinct group of BLM, although LT-channels conductances varied from 112 +/- 8 pS till 1110 +/- 40 pS for phosphatidylcholine-BLM and from 75 +/- 6 pS till 170 +/- 14 pS for phosphatidylserine-BLM. (iii) Ca/K selectivity was greater in negatively charged membranes but did not also depend on the channel amplitude for the same BLM. Cation-anionic selectivity was identical for all studied channels.     

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.     

Inhibition by P1075 and pinacidil of a calcium-independent chloride conductance in conditionally-immortal renal glomerular mesangial cells

1. Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H-2Kb-tsA58 transgenic mouse possess a significant whole-cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors. 2. The effects on the whole-cell chloride conductance of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the potassium channel openers, (KCOs) P1075 and pinacidil were investigated in mesangial cells from the H-2Kb-tsA58 transgenic mouse cultured in permissive conditions (at 33 degrees C in the presence of 50 u ml-1 murine gamma-interferon). 3. In symmetrical solutions of 140 mM tetramethylammonium chloride (TMAC1) the whole-cell chloride conductance was 1.08 +/- 0.05 nS (n = 63) and this could be reversibly inhibited by 5 x 10(-5) M NPPB. 4. Both P1075 and pinacidil inhibited the whole-cell chloride conductance. This inhibition was not reversible after drug washout and was demonstrated only when drugs were applied to the extracellular surface of the cells. Very low concentrations of the drugs were found to reduce the chloride conductance after 16 h incubation but under no circumstances studied was the conductance totally inhibited, leaving a mean residual current of 0.33 +/- 0.03 nS (n = 12). 5. The effects of different peptide calcium concentrations on the magnitude of the residual current in the presence of the drugs were investigated. The residual current was reduced with 10(-8) M calcium in the pipette and increased with 10(-3) M pipette calcium. Therefore, these data suggest that P1075 and pinacidil selectively inhibit a calcium-independent chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse.