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Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.

Chronic β-adrenoceptor antagonist (β-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K(+) currents. The aim of this study was to investigate the effects of chronic β-blockade on transient outward, sustained and inward rectifier K(+) currents (I(TO), I(KSUS) and I(K1)) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic β-blockade was associated with a 41% reduction in I(TO) density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I(K1) was reduced by 34% at -120 mV (p < 0.05). Neither I(KSUS), nor its increase by acute β-stimulation with isoprenaline, was affected by chronic β-blockade. Mathematical modelling suggested that the combination of I(TO)- and I(K1)-decrease could result in a 28% increase in APD(90). Chronic β-blockade did not alter mRNA or protein expression of the I(TO) pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvβ1, Kvβ2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I(K1). A reduction in atrial I(TO) and I(K1) associated with chronic β-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.

Connexins form hemichannels at undocked plasma membranes and gap-junction channels (GJCs) at intercellular contacting zones. Under physiological conditions, hemichannels have low open probabilities, but their activation under pathological conditions, such as ischemia, induces and/or accelerates cell death. Connexin 46 (Cx46) is a major connexin of the lens, and mutations of this connexin induce cataracts. Here, we report the effects of linoleic acid (LA) on the electrical properties of Cx46 GJCs and hemichannels expressed in Xenopus laevis oocytes. LA has a biphasic effect, increasing hemichannel current at 0.1 μM and decreasing it at concentrations of 100 μM or higher. The effects of extracellular and microinjected LA conjugated to coenzyme A (LA-CoA) suggest that the current activation site is accessible from the intracellular but not extracellular compartment, whereas the current inhibitory site is either located in a region of the hemichannel pore inaccessible to intracellular LA-CoA, or requires crossing of LA through an organelle membrane. Experiments with other fatty acids demonstrated that the block of hemichannels depends on the presence of a hydrogenated double bond at position 9 and is directly proportional to the number of double bonds. Experiments in paired oocytes expressing Cx46 showed that LA does not affect GJCs. The block by unsaturated fatty acids reported here opens the possibility that increases in the concentration of these lipids in the lens induce cataract formation by blocking Cx46 hemichannels.

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca(2+)-activated K(+) channels channels by InsP(3)-induced Ca(2+) release from the endoplasmic reticulum and/or store-operated Ca(2+) entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

The loose-patch clamp technique was used to study voltage-activated currents from the surface of rat neurohypophysial and hypothalamic regions in situ. In the neurohypophysis, depolarizing pulses of 4-8 ms duration yielded tetrodotoxin (TTX)-sensitive sodium currents, a 4-AP-sensitive "A"-type potassium current, and a long-lasting outward TEA- and tetrandrine-sensitive Ca(2+)-activated potassium current. All of these currents were elicited during the application of the pulse. With high external calcium there were long-lasting inward currents blocked by Ni(2+) and Cd(2+), identifying them as voltage-gated calcium currents. Depolarizing pulses of 0.3-0.7 ms duration yielded fast biphasic responses, of 1-3 ms duration, composed of mostly sodium and "A"-type potassium currents. With high external calcium there were fast inward currents blocked by Ni(2+) and Cd(2+), indicating that these were voltage-gated calcium currents. These responses have the characteristics of action potential currents: they were elicited after the cessation of the applied pulse and the "A" component is eliminated together with the sodium component upon application of TTX. Similar responses to long and short pulses were obtained from the surface of the associated magnocellular somata in the supraoptic nucleus, and their projections. The explant currents are similar to those previously characterized using conventional methods from somata and terminals.

Exposure of Xenopus laevis oocytes to NH(4)Cl caused intracellular acidification, cell membrane depolarization and the generation of an inward current. To determine the contribution of uncharged NH(3) and positively charged NH(4)(+), the NH(4)Cl-induced inward current was measured in the presence of increasing [NH(3)] at constant [NH(4)Cl] (10 mM) or increasing [NH(4)Cl] at constant [NH(3)] (0.045 mM) with pH varying in both cases. At -70 mV, the NH(4)Cl-induced current was barely detectable at pH 6.5, 0.01 mM NH(3), but increased successively at pH 7.5, 0.1 mM NH(3) and pH 8.5, 1 mM NH(3). In contrast, NH(4)Cl-associated currents were independent of changes of the [NH(4)Cl] at constant [NH(3)] and variable pH. Similar results with respect to acidification, depolarization and inward current in response to concentration and pH changes were obtained with trimethylamine HCl. Increasing concentrations of the weak acid propionate led to a reduction of the NH(4)Cl-induced current. These data suggest that NH(3) entry may induce local alkalinization that, in turn, may trigger the opening of a conductance for NH(4)(+) or trimethylamine-H(+) entry.

Although often used as a Ca(2+) channel blocker, Mn(2+), in fact, permeates through Ca(2+) channels. Under Na(+)-free conditions, depolarizing pulses evoked slowly-decaying Mn(2+) currents ( I(Mn)). Maximal I(Mn) densities in the presence of 5 and 20 mM Mn(2+) were 0.42+/-0.12 pA/pF (mean+/-SEM, n=17) and 1.23+/-0.10 pA/pF ( n=40), respectively. At 5 mM, the ratio of maximal amplitude of I(Mn) to that of the Ca(2+) current ( I(Ca)) was 0.079+/-0.009 ( n=8). I(Mn) elicited from a holding potential of -50 mV was depressed by nitrendipine (1 microM) by approximately 70%. Nitrendipine (0.3 microM) shifted the steady-state inactivation curve to more negative potentials and shifted the potential for half-maximal inactivation ( E(0.5)) from 1.3 to -8.8 mV and also decreased the time constant of decay of I(Mn) at 20 mV from 986.2 to 167.9 ms. BAY K 8644 (1 microM), isoproterenol (10 microM) and forskolin (10 microM) all increased I(Mn) and shifted the current/voltage ( I/ V) relationship to more negative potentials. The small, slowly-inactivating I(Mn) is thus modulated by dihydropyridine Ca(2+) channel modulators and cyclic AMP-mediated phosphorylation in a manner similar to other L-type Ca(2+) channel currents. L-type Ca(2+) channels are involved in the regulation of intracellular [Mn] in ventricular myocytes.

The effect of Cd(2+) on intracellular Ca(2+) homeostasis was examined in renal epithelial A6 cells loaded with Fura-2. Cd(2+) (10 microM to 1 mM) produced a transient spike in cytosolic Ca(2+) in a dose-dependent manner. The phospholipase C inhibitor U73122 and the cation receptor agonist, neomycin, both diminish Cd(2+)-evoked increase in intracellular Ca(2+) ([deltaCa(2+)](Cd)). Further, thapsigargin, an inhibitor of intracellular Ca(2+)-ATPases, significantly reduced [deltaCa(2+)](Cd). Extending these observations, inositol-3-phosphate (IP(3)) binding studies showed that the resting level of intracellular IP(3) underwent a 1.45-fold increase when exposed to Cd(2+). Furthermore, we found that the Cd(2+)-related heavy metals, Zn(2+) and Ni(2+), were even more potent inducers of Ca(2+) mobilization and IP(3) generation than Cd(2+). It can be concluded that Cd(2+), and possibly Zn(2+) and Ni(2+), may act as agonists of a cation-sensing receptor (CSR) belonging to G-protein receptors capable of mediating IP(3) release of Ca(2+) from intracellular stores. The CSR receptor in A6 epithelia could not be stimulated with neomycin or Gd(3+), suggesting that the receptor is different from the calcium-sensing receptor.

Preconditioning ischemia (PI) increases the speed of the initial vasodilatation (vascular preconditioning) of a subsequent coronary reactive hyperemia (CRH) and reduces total hyperemic flow (THF). We investigated whether changes in CRH similar to those induced by PI are obtained with diazoxide, a mitochondrial ATP-sensitive K+ channel opener, and whether diazoxide influences the effects of a subsequent PI on CRH. In anesthetized goats, flow was recorded from the left circumflex coronary artery (LCCA). CRH and PI were obtained with 15-s and 5-min LCCA occlusions, respectively. CRH was studied before and after PI, before and after diazoxide (2.5 mg/kg i.v.) as well as before and after PI was induced after diazoxide pre-treatment. After PI, the time to peak (ttp) of CRH and THF decreased by 51+/-13% and 23+/-8%, respectively. Diazoxide did not change CRH. After diazoxide and PI, when basal flow had returned to the control level, the ttp of CRH was reduced as after PI alone (-45+/-12%), whereas THF was reduced to a greater extent (-41+/-9% versus -23+/-8%; P<0.01). In conclusion, PI alters CRH by decreasing THF and reducing the ttp of CRH. Whilst diazoxide does not reproduce the effects of PI on CRH, pre-treatment with diazoxide potentiates the effects of PI on THF.

In the apical plasma membrane of rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ), besides its inhibiting action on Na+-Cl- symport and some side-effects, opens a SITS-sensitive Cl- conductance (Gcl), resulting in depolarization of the membrane due to a cell-to-lumen Cl- backflux. Some previous indications suggest that GCl is someway related to the Cl-/HCO3- exchanger. Thus the actions on the apical membrane of HCTZ and two inhibitors of the exchanger mainly studied in erythrocytes, namely phlorizin and phenylglyoxal (PG), have been compared. The transapical Cl- influx was measured radiochemically and the anion exchange fraction identified. The apical and transepithelial membrane potentials (Vm, Vm), the apical/basolateral membrane resistance ratio (Rm/Rs) and the transepithelial resistance (Rep) were measured with conventional microelectrodes and the changes in apical electromotive force and conductance were calculated. It has been shown that: (1) 2.5 x 10(-4) mol/l HCTZ abolishes Cl-/HCO3- exchange in a few seconds, (2) exchanger inhibition by HCTZ is direct and not mediated by carbonic anhydrase inhibition, (3) 2 mmol/l phlorizin and 4 mmol/l PG also inhibit the exchanger in a few seconds or at least rapidly and in parallel activate a depolarization of 6-7 mV, like HCTZ, (4) dose/response curves of the three drugs for depolarization activation and anion exchange inhibition overlap, (5) depolarization time courses are similar for the three drugs, (6) a decrease in the Rm/Rs ratio occurs in the presence of the three drugs, with a significant change in apical electromotive force when the luminal Cl- concentration is reduced, all this indicating the appearance of a substantial, quantifiable GCl which is absent in the absence of incubation with the drugs, (7) GCl values are similar, regardless of the drug which generates them, (8) the effects of the three drugs are not additive, and (9) stilbenes and dipyridamole abolish GCl (but not DPC) as well as the basal intrinsic conductance of the exchanger. On this basis it is concluded that some inhibitors of the Cl-/HCO3- exchanger either turn it into an anion channel or, less probably, activate a parallel GCl, as a consequence of the exchanger inhibition.

The stilbene- and dipyridamole-sensitive Cl-conductance (GCl), non-additively activated by some inhibitors of the Cl-/HCO3- exchanger (hydrochlorothiazide, phlorizin, phenylglyoxal) after the exchanger inhibition in the apical plasma membrane of rabbit gallbladder epithelium, has been investigated by patch-clamp technique with cell-attached and inside-out configurations. No Cl- channels were observed under basal conditions or after treatment with 2.5 x 10(-4) mol/l 8-Br-cAMP or hydrochlorothiazide (HCTZ) on the cytosolic side. Conversely, with 2.5 x 10(-4) mol/l HCTZ or 2 mmol/l phlorizin in the pipette, a non-rectifying Cl- channel with about 5 pS conductance and 0.3-0.4 voltage-independent open probability was observed; it was inhibited by 10(-4) - 5 x 10(-4) mol/l SITS, 10(-4) mol/l furosemide or 0.6 x 10(-4) mol/l dipyridamole; the effects of HCTZ and phlorizin were not additive. Open probability increased from 0 (after seal formation) to a maximum of 0.3-0.4 reached in 7-9 min. Similar results were obtained with both configurations. With the cell-attached configuration, HCTZ added to the bath did not activate Cl- channels in the patch. The channel was shown to exclude cations, to be selective for Cl-, but also conductive for gluconate (PGluc/PCl = 0.18). On this basis, it is concluded that: (1) GCl, activated either by HCTZ or phlorizin, has the same underlying anion channels, (2) the channels can be activated only on the external side of the membrane, also in the absence of cytoplasm, without cellular mediations or effects at a distance along the membrane, (3) the channels are inhibited by the same drugs which inhibit the very small intrinsic anion conductance of the exchanger, (4) they are either related to a slow conversion of inhibited exchangers into channels or, less probably, they are parallel to the exchanger and slowly activated by intra-membrane (or membrane-bound) mediators in their turn activated by near, inhibited exchangers.

Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.

It has long been known that extracellular Ca2+ and Mg2+ modulate synaptic transmission at the neuromuscular junction, acting both pre- and post-synaptically. Relevant questions concerning the modulation of acetylcholine (ACh) receptors (AChRs) are however still open: are the fetal (gamma-AChR) and adult (epsilon-AChR) receptors modulated differently? Does the ACh concentration influence the effect of divalent cations? Is the effect on channel open duration dependent on type and concentration of divalent cation? These questions were addressed by studying the modulation of the single-channel behaviour of gamma- and epsilon-AChRs by Ca2+ and Mg2+ at the endplate of muscle fibres acutely dissociated from 12- to 14-day-old mice. Ca2+ reduced the conductances of the two receptor channels comparably. Mg2+ had a stronger effect than Ca2+ and reduced the conductance of epsilon-AChR significantly more than that of gamma-AChR. With 0.1 microM ACh, Ca2+ and Mg2+ increased the mean open duration of gamma- and epsilon-AChR channels comparably. At 100 microM ACh, gamma- and epsilon-AChR channels opened in bursts of strikingly similar duration, which was unaffected by divalent cations. These findings indicate that Ca2+, and even more so Mg2+, may regulate synaptic transmission by modulating the function of AChRs in addition to the well-established effects on transmitter release.

The activity of caffeine-activated large conductance channels was recorded in whole-cell, patch-clamped, isolated ventricular myocytes from rabbit heart. The channels were permeable to monovalent and divalent cations and had a unitary monovalent cation conductance of 300-400 pS. Extracellular ruthenium red reduced the unitary conductance of the caffeine-activated channel in a concentration- and voltage-dependent manner. Ryanodine locked the caffeine-activated channels into a subconductance state. Elevating intracellular Ca2+ by photolysis of "caged calcium" increased the number of channel openings. The properties of this caffeine-activated channel were remarkably similar to those of cardiac ryanodine receptors (RyR) and support the novel finding that these channels may also be found on the sarcolemmal membrane.

Biochemical evidence indicates that veratridine (VTD) and batrachotoxin (BTX) share a common binding site in Na+ channels. Under whole-cell voltage-clamp conditions, we examined this single receptor hypothesis by studying the VTD phenotype in BTX-resistant muscle Na+ channels, microl-I433K, N434K, L437K, F1579K, and N1584K. Derived from point mutations at segments D1-S6 and D4-S6, these mutant Na+ channels are resistant to 5 microM BTX when expressed in human embryonic kidney cells. In contrast to the wild-type phenotype, VTD at 200 microM elicits little or no maintained current during a test pulse at +50 mV, and little or no "tail" current after the test pulse in all BTX-resistant mutant channels. Paradoxically, VTD retains its ability to inhibit the peak Na+ current in BTX-resistant mutant Na+ channels. To explain these mutant phenotypes, we propose a two-step binding reaction scheme. An initial VTD-binding interaction with the Na+ channel results in the inhibition of peak current amplitude, and a second binding reaction results in the trapping of VTD within the D1-S6 and D4-S6 domain interface. The failure of BTX-resistant mutant Na+ channels to trap VTD suggests that segments of D1-S6 and D4-S6 form a common receptor for VTD and BTX.

The properties of hyperpolarization-activated current in pregnant rat uterus (17-19 days gestation) were investigated using microelectrode and patch-clamp techniques, and isometric tension recording. The resting membrane potentials were -58.4 mV and -48.5 mV in longitudinal and circular muscle cells, respectively. Application of hyperpolarizing current pulses produced a time-dependent anomalous inward rectification of membrane potential only in circular muscle cells. Under voltage-clamp conditions, inward currents (Ih) were activated by long hyperpolarizing pulses below -60 mV in circular but not in longitudinal muscle cells. Application of extracellular but not intracellular Cs+ reduced the amplitude of I(h) in a concentration-dependent manner (an IC50( of 0.15 mM). The reversal potential for Ih was -26.2 mV and the slope conductance was 5 nS/pF. Changes in [K+]o and [Na+]o shifted the reversal potential, and Ih amplitude increased with excess [K+]o and decreased with low [Na+]o. The steady-state activation of Ih was well fitted by a Boltzmann equation with a half-activation potential of -84.3 mV and a slope factor of 9.6 mV. Time courses of activation and deactivation of the current strongly depended on membrane potential, and were well fitted by a single exponential function. The activation time constant of Ih was dependent on temperature. In isometric tension recording, application of extracellular Cs+ to the circular muscles reduced the frequency, but not the amplitude, of spontaneous contractions in a concentration-dependent manner. It is concluded that in pregnant rat uterus Ih channels are predominantly distributed in smooth muscle cells from the circular layer. Since Ih is activated at the resting membrane potential, it is likely that this current contributes to the maintenance of resting membrane potential and spontaneous activity in circular smooth muscle cells of late pregnant rats.

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.

Ionic currents and the voltage response to injected currents were studied in an acutely dissected preparation of the rat utricle between birth and postnatal day 12 (PN12). Based upon morphological criteria, the sensory cells examined were divided into two classes, "type I" and "type 2 category," the latter of which may include some immature type I cells. The former group comprises a clearly defined electrophysiological population, with one large outwardly rectifying potassium conductance that is sensitive to 4-aminopyridine (4-AP), insensitive to tetraethylammonium (TEA) and displays voltage-dependent activation kinetics. In the absence of enzymatic dissociation procedures, and with the epithelium left largely intact, the mean half activation of this conductance was -30.3 mV at PN3, and -37.5 mV at PN12. At both stages it was almost entirely turned off at -74 mV. Omission of ATP from the intracellular solution appeared to prevent rundown of this conductance. Type II category hair cells formed a more heterogeneous population, exhibiting a distinct TEA-sensitive delayed rectifier potassium conductance; the rapidly activating and inactivating IA; an inward rectifier; and inward sodium currents at around PN3. Both cell types depolarised strongly in response to injected currents, with time courses reflecting the activation kinetics of their major outward conductances.

We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca2+]i. It was demonstrated that ATP acts via a P2Y1 receptor to increase [Ca2+]i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (Vte) and resistance (Rte) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of Vte to lumen-positive values (resting Vte: -2+/-1 mV; peak Vte after 100 micromol/l ATP: +2.4+/-1.1 mV) and a decrease of Rte from 89. 9+/-10.3 to 83.8+/-9.1 Omegacm2 (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC50 values for both nucleotides were approximately 6 micromol/l. The ATP-induced Vte effect was nearly completely sensitive to Ba2+. Addition of the K+ channel blocker Ba2+ (1 mmol/l) to the luminal solution reversibly inhibited 77+/-4% (n=5) of the ATP-induced Vte effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 micromol/l agonist: UTP>/=ATP>>2-methylthio-ATP=ADP>>adenosine> AMP>beta, gamma-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y2 receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y2 receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y2 receptor in the luminal membrane of colonic mucosa to elicit a transient K+ secretion.

In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 microM carbachol (ICCh) at a holding potential of -60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as ICCh was slowly activated by the dialysis of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block ICCh, antibodies (Abs) against Galpha,i (anti-Galpha,i) or Galpha,o (anti-Galpha,o) were tested. Activation of ICCh was blocked by the addition of anti-Galpha,o. However, anti-Galpha,i Abs had no significant effect on ICCh. The expression of Galpha,o in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that Go-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes.

The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (ICCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, ICCh was induced by bath application of 50 microM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]i), ICCh decayed spontaneously (desensitization of ICCh) to around 20% within 10 min. Desensitization of ICCh was significantly attenuated with 2 mM [EGTA]i. At a concentration of 20 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited ICCh at 0.5 mM [EGTA]i but far less at 2 mM [EGTA]i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]) was not inhibited by OAG with 0.5 mM [EGTA]i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 microM chelerythrine or 1 microM peptide inhibitor, attenuated the desensitization of ICCh. [Ca2+]i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]i, [Ca2+]i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]i. These results suggest that the desensitization of ICCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.

Three major ionic currents, Ca2+-dependent K+ current (IK-Ca), delayed rectifier type K+ current (Ikd) and Ca2+ current (ICa), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of IK-Ca and ICa at 0 mV (IC50 values were 4.2 and 5.6 muM, respectively), but did not affect IKd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (Icaf) at -30 mV were reduced by external 10 muM RuR. When 10 muM RuR was added to the pipette solution, IK-Ca during depolarization, STOCs and Icaf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca2+-dependent K+ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca2+ concentration was increased. These results indicate that RuR blocks BK and Ca2+ channels in urinary bladder smooth muscle cells. The decrease in IK-Ca, STOCs and Icaf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca2+ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca2+-binding sites of the channel protein.

The effects of Mg2+ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg2+ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg2+ can be described by a Hill coefficient of 0.9 at +/-100 mV and IC50s of 0.12 mM (-100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg2+ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg2+. The observed voltage dependencies of the IC50 for Mg2+ block and the Km for K+ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K+ self block and anomalous mole fraction effects with K+/Tl+ mixtures indicate a single site pore model.

We studied the effect of brefeldin A, which inhibits the intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on terbutaline (a beta2-specific adrenergic agonist)-induced alterations in ion transport by primary monolayer cultures of fetal rat distal lung epithelium. The amiloride-sensitive short circuit current (Isc) increased 2.5-fold 50 min after application of terbutaline (10 microM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 microg/ml). Brefeldin A did not suppress the Na+/K+ pump capacity. Single channel patch clamp experiments demonstrated that terbutaline increased the density of amiloride-sensitive Na+-permeable nonselective cation channels on the apical cell membrane and this action was blocked by brefeldin A. These observations suggest that beta2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na+-permeable nonselective cation channels to the apical cell surface.

The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (ICCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 micro M PDBu suppressed ICCh by 96.5 +/- 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 microM chelerythrine, a PKC inhibitor, inhibition of ICChby PDBu was not seen. In whole-cell mode, the inhibition of ICCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased ICCh by 98.8 +/- 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on ICCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 +/- 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[gammaS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[gammaS]-induced currents were 6.7 +/- 2.4 pA and 236 +/- 23 pA, respectively. These results suggest that PKC modulates ICCh at postreceptor sites via protein phosphorylation.

We studied the effects of cell swelling on membrane currents of canine ventricular myocytes using the whole-cell patch-clamp method. Cell swelling was induced by lowering the osmolarity of the bath solution to 60% of control. Cell width and currents were measured simultaneously. Cell swelling induced little or no change in the L-type Ca, the inward rectifier, and the transient outward currents, but a marked increase in the slow delayed rectifier current (IKs) was seen. We further examined the role of protein kinase activities in IKs modulation by cell swelling. This modulation was not affected by inhibiting serine/threonine kinases using H-8. On the other hand, the modulation was inhibited by genistein (a protein tyrosine kinase inhibitor) although not by daidzein (an inactive analogue of genistein). Our data suggest that in canine ventricle cell swelling can increase protein tyrosine kinase activity, which can augment IKs and contribute to changes in membrane electrical activity observed under these conditions.

Pharmacological characterization of ion channels and receptors in cultured neurons or transfected cell lines requires microapplication of multiple drug solutions during electrophysiological recording. An ideal device could apply a large number of solutions to a limited area with rapid arrival and removal of drug solutions. We describe a novel "multipuffer" rapid application device, based on a modified T-tube with a nozzle made from a glass micropipette tip. Drug solutions are drawn via suction from open reservoirs mounted above the recording chamber through the device into a waste trap. Closure of a solenoid valve between the device and the waste trap causes flow of drug solution though the T-tube nozzle. Any number of drug solutions can be applied with rapid onset (50-100 ms) after a brief fixed delay (100-200 ms). Recombinant alpha1beta1gamma2S GABAA receptors (GABARs) transfected into L929 fibroblasts were recorded using whole-cell and single-channel configurations. Application of GABA resulted in chloride currents with an EC50 of 12.2 microM and a Hill slope of 1.27, suggesting more than one binding site for GABA. GABAR currents were enhanced by diazepam and pentobarbital and inhibited by bicuculline and picrotoxin. Single-channel recordings revealed a main conductance state of 26-28 pS. This device is particularly suitable for rapid, spatially controlled drug applications onto neurons or other cells recorded in the whole-cell configuration, but is also appropriate for isolated single-channel or multichannel membrane patch recordings.

Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca2+ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (>/= 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of -90 mV. The Ca2+ current recorded at a high holding potential (-50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (-90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (>/= 200 nM) was present in the pipette, the Ca2+ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca2+ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca2+ channel current. To prevent the "run down" of the Ca2+ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca2+ channel activating factor, in the pipette. The unitary Ca2+ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 microM) in the bath completely blocked the open events of the 25-pS Ca2+ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca2+ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca2+ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane.

Changes in electrolytes of pig pancreatic acinar cells following application of gastrin-cholecystokinin (CCK) were investigated using the technique of X-ray microanalysis of hydrated and dehydrated sections of freshly frozen pancreas. After stimulation by CCK (10(-9) M), Na and Cl increased significantly in the cytoplasm [Na, from 10 mmol/kg wet wt. (48 mmol/kg dry wt.) to 19 mmol/kg (95 mmol/kg); Cl, from 22 mmol/kg (105 mmol/kg) to 49 mmol/kg (245 mmol/kg)] as well as in the luminal interspace [Na, from 53 mmol/kg (189 mmol/kg) to 65 mmol/kg (283 mmol/kg); Cl, from 65 mmol/kg (232 mmol/kg) to 102 mmol/kg (443 mmol/kg)]. In the secretory granules Cl increased significantly from 30 mmol/kg (86 mmol/kg) to 67 mmol/kg (203 mmol/kg). K decreased significantly from 120 mmol/kg (571 mmol/kg) to 81 mmol/kg (405 mmol/kg) in the cytoplasm, while both increased from 38 mmol/kg (109 mmol/kg) to 58 mmol/kg (176 mmol/kg) in the granules and from 46 mmol/kg (164 mmol/kg) to 48 mmol/kg (209 mmol/kg) in the luminal interspace. Ca increased significantly in the cytoplasm as well as in the luminal interspace, and decreased significantly in the secretory granules. CCK evoked Ca release from secretory granules in the secretory pole of acinar cells. The values were measured from dehydrated sections, and agreed well with those from hydrated sections. The effect of furosemide, an inhibitor of the Na+-K+-2Cl- co-transporter, on the ion transport of acinar cell was studied. When furosemide (10(-5) M) was added to the external solution, the cytoplasmic Cl and Ca concentrations decreased significantly, while there was a little decrease in Na and K concentrations under the secretory condition. These results indicate that Na+-K+-2Cl- co-transport, and Na+, Cl- and K+ exits into the lumen are involved in the mechanism of ion secretion in pig pancreatic acinar cells.

Two splice variants of the ligand-gated 5-hydroxytryptamine or serotonin 5-HT3 receptor that differ in a six-amino-acid deletion were cloned by polymerase chain reaction from the hippocampus x neuroblastoma cell line HN9.10e. When expressed in Xenopus oocytes, both variants individually formed 5-HT3 receptors that revealed no significant differences in current responses to the agonists 5-HT and 1-phenylbiguanide and block by the specific antagonist LY-278, 584-maleate. For both receptors, the monovalent cations Na+, K+, Rb+ and Li+ showed the same relative permeability; NH4(+)permeated approximately 2.7 times better than Na+, and Tris+ was only poorly permeable. In contrast to other reports, the receptors were completely and reversibly blocked by extracellular Cs+ in both oocytes and native HN9.10 cells. Moreover, Ca2+ was not permeant and exhibited a concentration-dependent decrease (0.9-18 mM) of the 5-HT-induced currents without affecting the inward rectification of the current/voltage relation. The two receptors were reversibly inhibited by nanomolar concentrations of the specific inhibitor of protein kinase C (PKC) bisindolylmaleimide, but not by the equipotent and less specific inhibitor staurosporine. A regulatory effect on both 5-HT3 receptor subunits by PKC-mediated protein phosphorylation might be possible, however, a functional role of the two splice variants present in one cell remains to be determined.

Capsaicin activates a non-specific cation conductance in mammalian sensory neurones. If capsaicin is applied continuously or repeatedly then there is a progressive decline in responsiveness. We have studied the mechanism of this desensitization using electrophysiological methods in cultured dorsal root ganglion neurones from adult rats. The rate of desensitization of capsaicin-induced responses is partly dependent on the extracellular calcium concentration and is slower when extracellular calcium is reduced. Desensitization is strongly inhibited by intracellular application of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid (BAPTA). These data suggest that desensitization is due to a rapid rise in intracellular calcium levels which occurs when capsaicin-sensitive ion channels are activated. Desensitization is not reduced by the non-specific protein kinase inhibitors H7 or staurosporine or by okadaic acid, a selective inhibitor of protein phosphatases 1 and 2A. Desensitization is greatly reduced by cyclosporin A complexed to cyclophilin, which is a specific inhibitor of protein phoshatase 2B (calcineurin). A mechanism for desensitization of capsaicin responsiveness is proposed whereby the evoked rise in calcium activates calcineurin leading to dephosphorylation and desensitization of the capsaicin-sensitive ion channels.

Synaptically released dopamine is known to evoke fast as well as slow synaptic potentials in neurones of gastropod molluscs. Here evidence is presented that the fast excitatory response to dopamine is mediated by the direct activation of a ligand-gated channel: unitary currents were observed in outside-out patches of neurones exposed to dopamine, and the response persisted in the presence of intracellular guanosine 5'-o-(2-thiobiphosphate), GDP[beta-S], a condition known to block G-protein-coupled responses to dopamine and other agents. In whole-cell recordings, the fast response desensitized very rapidly; it was less desensitized in outside-out patches, suggesting dependence of desensitization on an intracellular factor. The response to dopamine was blocked by D-tubocurarine and strychnine (both probably by channel blockade), by apomorphine, chlorpromazine and relatively high doses of (+/-)-sulpiride and spiperone. The channel conducts predominantly monovalent cations. Unexpectedly, the fast response to dopamine was also observed in an identified dopaminergic neurone when maintained in isolation in culture. The receptors on the dopaminergic neurone were unevenly distributed, being more abundant on the axon-hillock, axon and neurite terminals.

Chicken enterocytes present a H+-conducting pathway involved in the recovery of intracellular pH (pHi) from an acid load. In the current study we have tested the effect of protein kinase C (PKC) activators on the rate of proton efflux through the H+-conducting pathway. The rate of proton efflux was increased by the addition of 1,2-dioctanoyl-rac-glycerol (DOG) or phorbol 12-myristate 13-acetate (PMA), but it was not affected by the addition of the inactive phorbol ester analogue, 4alpha-phorbol 12, 13-didecanoate. DOG stimulated the process in a dose-dependent manner with a half-maximal effect at 45 microM. Staurosporine and Zn2+ prevented the DOG-dependent increase in the rate of proton efflux. The rate of proton efflux was affected by the pH, and DOG shifted this relationship upward and to the right. These results suggest that the proton-conducting pathway is regulated by PKC.

The presence and properties of thrombin receptors have been investigated in Xenopus Laevis follicles. In follicles, voltage-clamped at -60 mV or -80 mV, sub-micromolar concentrations of thrombin (Thr) induce smooth inward currents carried by chloride ions, which desensitize over tens of minutes. This desensitization is prevented by PKC inhibitors (staurosporine and H7). Responses are inhibited by hirudin. Inconsistent responses can be observed in defolliculated oocytes. Thr responses, readily recorded in hypo-osmotic solution are abolished by perfusion of normal frog "Ringer" or by the addition of 50 mM sucrose. Thus Thr acts on receptors present in the follicular cells triggering the opening of chloride channels by acting on receptors, likely to be located on follicular cells, via a PKC-regulated pathway.

The effects of extracellular adenosine 5'-triphosphate (ATP) on voltage-dependent Ca2+ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 microM) reversibly suppressed Ca2+ currents in the presence of 5mM Ca2+ in the extracellular solution. The inhibitory effect of ATP on Ca2+ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10mM EGTA, on the other hand, the inhibitory effect did not change regardless of the amplitude of current responses to ATP. In the presence of 10 mM Ba2+, ATP (100 microM) reduced Ba2+ currents in a manner similar to Ca2+ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]; 1 mM) or guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]; 100 microM). A depolarizing prepulse to + 100 mV partly relieved ATP-induced reduction of Ba2+ currents. ADP, AMP and adenosine also reduced Ba2+ currents and the effect of adenosine was the most potent. Adenosine (0.5 and 1 mM) significantly inhibited adrenaline secretion induced by nicotine (50 microM). These results suggest that Ca2+ entry through ATP-activated non-selective cation channels results in the inactivation of voltage-dependent Ca2+ channels. In addition, ATP seems to modulate Ca2+ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.

We have investigated the effect of genistein on the hormone-stimulated Ca2+ influx and on a 28pS nonselective cation channel in mouse pancreatic acinar cells using the Ca2+ indicator fluo3 and the patch-clamp technique. The identity of the Ca2+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca2+-dependent nonselective cation channel as a potential pathway for Ca2+ influx. Capacitative Ca2+ entry was induced by depletion of intracellular Ca2+ stores with 500nM acetylcholine or with the Ca2+ ATPase inhibitor 2,5di-tert- butylhydroquinone. In the presence of 100microM genistein, Ca2+ release was unimpaired, whereas Ca2+ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca2+-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28pS nonselective cation channel does not contribute to Ca2+ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca2+ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca2+ entry mechanism in mouse pancreatic acinar cells is unclear.

To investigate the effect of basic fibroblast growth factor (bFGF) on ion channel expressions in developing cardiac cells, a short-term primary culture of ventricular myocytes isolated from day-old newborn rats was established. Functional expressions of the transient outward currents (Ito) were measured by whole-cell voltage-clamp method. Addition of 40 ng/ml bFGF to the serum-free culture medium for 72 hours increased the Ito density without influences on membrane capacitance of the myocytes, an estimate of cell size. bFGF also promoted expressions of the steady-state outward currents. Methyl 2,5-dihydroxycinnamate (3 microM), a tyrosine kinase inhibitor, showed preferential inhibition of the bFGF-stimulated increase in Ito density. These results suggest that bFGF may play a role in regulating ion channel expressions of developing cardiac cells via activation of bFGF receptor tyrosine kinase activity.

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

5-Hydroxytryptamine (5-HT) is present in nerve fibres descending from the brainstem Raphe nuclei to motoneurones and its release is thought to exert excitatory actions. 5-HT, applied from the outside, directly depolarizes spinal and cranial motoneurones in slices. This action of 5-HT is mediated, in part, by an inwardly rectifying cationic current, Ih. In cardiac cells, an equivalent current, if, has been shown to be directly activated by adenosine 3':5'-cyclic monophosphate (cAMP) applied to the inside of the patch membrane. By applying the whole-cell method to thin slices of brainstem and spinal cord, we have shown that intracellularly applied camp and extracellularly applied dibutyryl camp or forskolin mimics the inward current induced by 5-ht. The selective cAMP phosphodiesterase inhibitor, Ro 20-1724, clearly prolonged the 5-HT-induced current. Maximal doses of dibutyryl cAMP or forskolin occluded the 5-HT-induced current. The broad spectrum protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methlypiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) had no effect on the currents induced by 5-HT and forskolin. From these results, we suggest that activation of 5-HT receptors on the motoneuronal membrane stimulates formation of intracellular cAMP, thereby inducing the inward current, possibly by a direct action on Ih.

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to guinea-pig gastric myocytes, which were held at -20 mV using the whole-cell patch-clamp method. ICCh was suppressed by nicardipine or removal of Ca2+ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7[N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of ICCh. However, @-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5'-O-(3-thiotriphosphate) (GTP¿gammaS] 0.2 mM) in the pipette solution. This GTP[gammaS]-induced cationic current was still markedly suppressed by the Ca2+-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca2+ channels as well as the effects on ICCh. These data suggest that multiple Ca2+-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca2+/calmodulin-dependent pathway would be one of them.

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K+ currents, as described for microglia from untreated cultures. These currents activated negative to the K+ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-D-glucamine (NMG), thereby indicating a partial block of this K+ conductance by Na+. Inward rectifying currents were also blocked by extracellularly applied Cs+ or Ba2+. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H+ current which could be reduced by application of inorganic polyvalent cations (Ba2+, Cd2+, Co2+, La3+, Ni2+, Zn2+) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H+ current of macrophages.

Some epithelial nonselective cation channels although in an open conformation are impermeable because they are blocked by extracellular Ca2+ ions. Such a channel is present in the apical membrane of the ectodermal cells of the chick gastrula. This single-channel study shows that protonation of a site located inside the pore of this embryonic channel reduces the Ca2+ block thus allowing Na+ to pass. The apparent pK for Ca2+ at -50 mV shifts from 5.7 at pH 7.4 to 3.5 at pH 4.5. Unexpectedly, the change in Ca2+ affinity does not originate from a destabilization of the binding but comes from a large reduction of the association rate.

A patch-clamp study was done to characterize the recently found GABAergic (i.e. gamma-aminobutyric acid) inhibitory synaptic channels of crayfish deep extensor abdominal muscle. Outside-out patches were rapidly activated by GABA to measure the dose/response curves for the open probability of the channels, Po, and the rise time, tr, (time from Po = 0.1 to Po = 0.9). In some of the patches the GABA-activated currents decayed due to desensitization and such patches were not studied further. Rare channel openings were elicited with 0.1 mM GABA. The Po at this low concentration of GABA was 0.0005 to 0.01. Application of 10 mM GABA was necessary to reach the maximal Po of 0.9. The slope of the dose/response relationship in the double logarithmic plot was 5.4 +/- 1.1 (mean +/- SD; n = 9) between 0.1 mM and 0.2 mM GABA. The plot of tr versus GABA concentration had a peculiar shape, recently found to be characteristic for positive cooperativity of the binding sites. tr increased from a minimum at 10 mM GABA with declining concentrations of GABA and reached a peak at 0.4 mM GABA. Below 0.4 mM GABA, tr decreased again. With 0.2 mM GABA tr was 0.40 +/- 0.1 (mean +/- SD; n = 4) of the peak value measured at 0.4 mM GABA. Simulations were compared with the experimental results and a linear reaction scheme with five binding sites for GABA was established to describe the dose/response curves for Po and tr.

Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of Ip.

A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 microliters) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 microliters) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of Silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 microliters) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the chamber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M omega). Additionally, a radioactive calcium channel ligand [3H]isradipine was added to the MPC and the appearance of radioactivity in the Petri dish (outside the MPC) subsequently measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K(+)-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5 +/- 3.1 pS and 17.3 +/- 1.8 pS, respectively (n = 4). In contrast, cell-attached patches were usually inactive when a Na(+)-rich solution filled the patch pipette. However, another type of channel with a conductance of 20-30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1 +/- 1.2 pS (n = 17), excluded anions (PCl/PNa = 0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa = 1.5; PK/PNa = 0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa = 0.09; PBa/PNa approximately 0). The cation channel was moderately voltage-dependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 mumol/l-0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition of Po at 1.2 mumol/l AMP. As in other cell models, 3',5'-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (IH) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately -60 mV in nominally zero-Ca2+ Krebs solution containing tetrodotoxin (1 microM), tetraethylammonium (40 mM), Cd2+ (500 microM) and 4-aminopyridine (1 mM). IH developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time. IH was selectively blocked by extracellular Cs+ (1 mM). The activation of the H-channel conductance (GH) ranged between -55 and -120 mV. The GH was 80-150 pS (n = 4) at the half-activation voltage of -84 +/- 7 mV (n = 4). The reversal potential of IH obtained by instantaneous current voltage (I/V) relations was -41 +/- 6 mV (n = 4); it shifted to -51 +/- 8 mV (n = 3) in low-Na+ (20 mM) solution and to -24 +/- 4 mV (n = 4) in high-K+ (20 mM) solution. Forskolin (1-10 microM) produced an inward current and increased the amplitude of IH. Forskolin did not change the half-activation voltage of GH. 8-Bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 0.1-1 mM) and dibutyryl-cAMP (0.1-1 mM) enhanced IH. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhanced IH. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.

Under conditions of low Ca buffering of the pipette intracellular dialyzing solution, the opening of Ca dependent large conductance (approximately 310 pS) channels (LCCs) was observed in isolated ventricular myocytes using the whole-cell patch clamp technique. With Na-Ca exchange current (INaCa) suppressed by elimination of intracellular and extracellular Na, sustained LCC activity, which is markedly enhanced by caffeine-stimulated Ca release from the sarcoplasmic reticulum (SR), was increased by application of the inhalational anesthetic halothane, but not isoflurane. Halothane (0.90 mM in solution) reversibly increased the frequency of LCC openings, fo, by a factor of approximately 30 with a concurrent rise in the observed probability of opening, NPo, by a factor of approximately 50. The effect of halothane on LCC activation was suppressed by either strong Ca buffering in the whole-cell pipette solution or pretreatment of the myocytes with ryanodine (10 microM) to decrease SR Ca release. In the presence of intracellular and extracellular Na, a transient inward current was evoked by application of caffeine (5 mM) or halothane (1.80 mM) suggesting that Ca release from the SR by either agent can activate INaCa. Our findings are consistent with the notion that halothane, in contrast to isoflurane, causes SR release of Ca. The eventual depletion of SR activator Ca may account, at least in part, for the differential effects of these anesthetics on myocardial tension development.

This paper describes the first voltage-clamp recordings from an arthropod cuticular sensory neuron. In the femoral tactile spine neuron of the cockroach Periplaneta americana, a rapidly activating and inactivating outward current, IA, appeared when the neuron was hyperpolarized for a short period before a depolarizing test pulse. IA could be separated from the other outward currents using 5 mM 4-aminopyridine (4-AP), which specifically blocked it. Tetraethylammonium (TEA), (50 mM) did not remove IA, but decreased the steady-state outward current by about 50%. The threshold for IA activation was about -75 mV. The minimum activation and inactivation time constants were approximately 0.2 ms and 15 ms, respectively. The voltage dependencies of activation and inactivation were well fit-ted by Boltzmann distributions, giving values of membrane potential at half-maximal activation (V50) equal to -56.5 mV and an equivalent gating charge of n = 3.9 for activation and V50 = -86.7 mV and n = 3.4 for inactivation. In current-clamp recordings, 4-AP reversibly reduced the cell's normal adaptation by lowering the threshold for action potentials, but did not affect the amplitude or duration of single action potentials. These results indicate that IA plays a role in short-term adaptation by opposing membrane depolarization and reducing the spike frequency during maintained stimulation.