Ion Substitution Experiments Research Articles

Monovalent cation conductance in Xenopus laevis oocytes expressing hCAT-3

hCAT-3 (human cationic amino acid transporter type three) was investigated with both the two-electrode voltage clamp method and tracer experiments. Oocytes expressing hCAT-3 displayed less negative membrane potentials and larger voltage-dependent currents than native or water-injected oocytes did. Ion substitution experiments in hCAT-3-expressing oocytes revealed a large conductance for Na + and K +. In the presence of l-Arg, voltage-dependent inward and outward currents were observed. At symmetrical (inside/outside) concentrations of l-Arg, the conductance of the transporter increased monoexponentially with the l-Arg concentrations; the calculated V max and K M values amounted to 8.3 μS and 0.36 mM, respectively. The time constants of influx and efflux of [ 3H] l-Arg, at symmetrically inside/outside l-Arg concentrations (1 mM), amounted to 79 and 77 min, respectively. The flux data and electrophysiological experiments suggest that the transport of l-Arg through hCAT-3 is symmetric, when the steady state of l-Arg flux has been reached. It is concluded that hCAT-3 is a passive transport system that conducts monovalent cations including l-Arg. The particular role of hCAT-3 in the diverse tissues remains to be elucidated.

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26+/-0.04 (without HCO3-) or 7.24+/-0.03 (with HCO3-). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl- or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25+/-0.15 (without HCO3-) or 6.53+/-0.10 (with HCO3-). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3-. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl- and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48+/-0.07. Analysis of recovery data showed that there was a progressive decrease in DeltapH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load.

Zinc Potentiates an Uncoupled Anion Conductance Associated with the Dopamine Transporter

Binding of Zn(2+) to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux. To elucidate the molecular mechanism for this dual effect, we expressed the DAT and selected mutants in Xenopus laevis oocytes and applied the two-electrode voltage clamp technique together with substrate flux studies employing radiolabeled tracers. Under voltage clamp conditions we found that Zn(2+) (10 mum) enhanced the current induced by both DA and amphetamine. This was not accompanied by a change in the uptake rate but by a marked increase in the charge/DA flux coupling ratio as assessed from concomitant measurements of [(3)H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn(2+) facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn(2+) by itself activated such a conductance in a previously described mutant (Y335A). This signifies that the conductance is not strictly dependent on an active transport process. Ion substitution experiments in Y335A, as well as in WT, indicated that the uncoupled conductance activated by Zn(2+) was mainly carried by Cl(-). Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn(2+) could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl(-) conductance, Zn(2+) is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.

Acid and base secretion in the Calu-3 model of human serous cells.

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.

Secretin controls anion secretion in the rat epididymis in an autocrine/paracrine fashion.

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3- ion transport.

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na(+)/H(+) exchange, Cl(-)/HCO(3)(-) exchange and Na(+)/HCO(3)(-) co-transport, and Na(+)-K(+)-2Cl(-) cotransport, respectively. Ion substitution experiments with Na(+)-free, Cl(-)-free, and HCO(3)(-)-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl(-)-free, or HCO(3)(-)-free solutions did not affect restitution. In Na(+)-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na(+)-dependent HCO(3)(-) transport. Because HCO(3)(-)-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in HCO(3)(-) ion transport but essential for cell migration after injury and restitution in the gastric mucosa.

Ca2+ -activated nonselective cation channels in rat neonatal atrial myocytes.

A nonselective cation channel activated by intracellular Ca(2+) was identified in inside-out membrane patches taken from cultured rat atrial myocytes. Ca(2+) (0.01-1.00 m M) reversibly activated the channel in a concentration-dependent manner. The channel often showed a quick and irreversible rundown within a few minutes after patch excision. The I-V relationship of the channel was linear between -100 and +100 mV. The single channel conductance was 26.0 +/- 0.5 pS and its open probability was weakly voltage-dependent. Ion-substitution experiments showed that the channel was permeable to monovalent cations (P(x)/P(Cs): Li(+) (1.5) = K(+) (1.5) > Na(+) (1.2) > Rb(+) (1.1) > Cs(+) (1.0)) but not to Cl(-) (P(Cl)/P(Cs) < 0.01) and Ca(2+) (P(Ca)/P(Cs) = 0.02 +/- 0.01).

Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide.

Whole cell voltage-clamp recordings from Aplysia mechanosensory neurons obtained from the pleural ganglion were used to investigate the actions on membrane currents of the neuropeptides SCP(B) and FMRFamide. At the start of whole cell recording, SCP(B) typically evoked an inward current at a holding potential of -40 mV, due to the cAMP-mediated closure of the S-type K+ channel, whereas FMRFamide evoked an outward current, due to the opening of the S-type K+ channels mediated by 12-lipoxygenase metabolites of arachidonic acid. However, after several minutes of whole cell recording with a high concentration of chloride in the whole cell patch pipette solution, the responses to SCP(B) and FMRF-amide at -40 mV were inverted; SCP(B) evoked an outward current, whereas FMRFamide and YGGFMRFamide evoked inward currents. Ion substitution experiments and reversal potential measurements revealed that these responses were due to the opposing regulation of a Cl(-) current, whose magnitude was greatly enhanced by dialysis with the high Cl(-) - containing pipette solution. SCP(B) inhibited this Cl(-) current through production of cAMP and activation of PKA. YGGFMRFamide activated this Cl(-) current by stimulating a cGMP-activated phosphodiesterase that hydrolyzed cAMP. Thus a cAMP-dependent Cl(-) current undergoes antagonistic modulation by two neuropeptides in Aplysia sensory neurons.

Electrophysiological, mechanosensitive responses of Xenopus laevis oocytes to direct, isotonic increase in intracellular volume

An intra-oocyte injection method for obtaining the electrophysiological response of follicle-enclosed Xenopus laevis oocytes to an increase in intracellular volume (i.e. stretch) without changing the extracellular medium is described. The response comprised a ‘stretch-activated’ (SA) current which was evoked by injection of an isotonic 14–70 nl droplet and had a transient, smooth profile. Ionic substitution experiments revealed that the current was carried mainly by Na +, K + and Cl − and had a reversal potential of about −2 mV. A similar result was obtained from experiments in which the holding potential was varied between −40 and +10 mV whilst repeatedly inducing the SA current. On average, the channel was blocked 60% by 10 μM gadolinium chloride, 58% by 50 μM amiloride, 11% by 50 μM 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid and 63% by 50 μM 4-acetamido-4′-isothiocyanato-stilbene-2-2′-disulfonic acid. Maturation of the oocytes with 100 μM progesterone reduced the mechanosensitivity 12-fold. This injection technique is compared with other methods of eliciting mechanosensitive (MS) currents in X. laevis oocytes. These observed characteristics of the SA current are discussed in relation to the oocytes’ endogenous MS cation and anion channels.

Effects of dissolved metals and other hydrominerals on in vivo intestinal zinc uptake in freshwater rainbow trout

For aquatic organisms, zinc is both an essential nutrient and an environmental contaminant. The intestine is potentially the most important route of zinc absorption, yet little is known regarding this uptake pathway for zinc in fish. A recently developed in vivo perfusion system was used to investigate the effect of luminal composition upon intestinal zinc uptake in freshwater rainbow trout ( Oncorhynchus mykiss). Perfusate cadmium and copper had specific, yet distinct, antagonistic effects upon lumen to tissue zinc movement. Copper significantly reduced the proportion of zinc taken up from the perfusate, and concomitantly limited the passage of zinc into the circulation and beyond. Conversely, cadmium decreased subepithelial zinc accumulation, with rates falling to 29 nmol g −1 h −1 from the control (zinc alone) values of 53 nmol g −1 h −1. Calcium had a similar action to copper, also reducing post-intestinal zinc accumulation from 0.06 to 0.02 nmol g −1 h −1, an effect attributed to interactions between calcium and the zinc uptake pathway. In addition to these effects, luminal composition also had a marked influence upon epithelial response to zinc. Calcium, copper and magnesium all greatly reduced zinc-induced mucus secretion. Cadmium, a toxic metal, significantly increased mucus secretion. It is proposed that these modifications were related to the essentiality of each element, and their potential mechanisms of uptake. Despite changes at the epithelium, the post-epithelial accumulation of zinc was dependent mainly upon the nature of the competing cation. Intestinal saline ion substitution experiments suggested a potential link of potassium ion efflux to zinc uptake. The effect of pH buffering of luminal solutions was also investigated.

Functional and molecular evidence for Na(+)-HCO cotransporter in porcine vas deferens epithelia.

This study focused on the role of sodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated ion transport in porcine vas deferens epithelium. Ion substitution experiments in modified Ussing chambers revealed that cAMP-mediated stimulation was dependent on the presence of Na(+), HCO, and Cl(-) for a full response. HCO-dependent current was unaffected by acetazolamide, bumetanide, or amiloride but was inhibited by basolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Na(+)-driven, HCO-dependent, stilbene-inhibitable anion flux was observed across the basolateral membrane of selectively permeabilized monolayers. Results of radiotracer flux studies suggest a 4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 base equivalents per Na(+). Antibodies raised against rat kidney NBC epitopes (rkNBC; amino acids 338-391 and 928-1035) identified a single band of ~145 kDa. RT-PCR detected NBC1 message in porcine vas deferens epithelia. These results demonstrate that vas deferens epithelial cells possess the proteins necessary for the vectoral transport of HCO and that these mechanisms are maintained in primary culture. Taken together, the results indicate that vas deferens epithelia play an active role in male fertility and have implications for our understanding of the relationship between cystic fibrosis and congenital bilateral absence of the vas deferens.

Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP.

We used the trinitrobenzenesulphonic acid (TNBS) rat model of experimental colitis to study the alterations in electrogenic ion transport in the inflamed distal colon. The distal colon exhibited decreased basal transport and reduced short-circuit current responses to carbachol and isobutylmethylxanthine (IBMX). The concentration/response curve for IBMX was also shifted to the right. Ion substitution experiments indicated that electrogenic transport was attributable chiefly to Cl(-) secretion. The mucosal layer of the inflamed distal colon (devoid of the submucosa) exhibited normal maximal responses to carbachol and IBMX, although the concentration/response curve for the latter was again shifted to the right. Tetrodotoxin markedly increased the response of the normal distal colon to both secretagogues and nullified the inhibition of the response to carbachol, but not that to IBMX, in the inflamed colon. The response of the mucosal preparation to 8-bromoadenosine 3',5'-cyclic monophosphate was similar in the normal and inflamed intestine, while the G protein activator NaF had a greater effect in the latter. The expression of the cystic fibrosis transmembrane conductance regulator (CFTR), as assessed by Northern blotting, was unchanged. cAMP levels in isolated colonocytes were markedly reduced by inflammation. We conclude that colonic inflammation produces disturbances of the enteric nervous system resulting in defective mucosal cAMP production and inhibition of ionic secretion.

Hypoosmotic stimuli activate a chloride conductance in rat taste cells.

The oral cavity is subjected to a wide range of osmotic conditions, yet little is known about how solution osmolarity affects performance of the gustatory system. In order to elucidate the mechanism by which hypoosmotic stimuli affect the peripheral taste system, I have attempted to characterize the effects of hypoosmotic stimuli on individual rat taste receptor cells (TRCs) using whole-cell patch clamp recording. Currents elicited in response to voltage ramps (-90 to +60 mV) were recorded in control saline and in solutions varying only in osmolarity (-30, -60 and -90 mOsm). In roughly two-thirds of cells, hypoosmotic solutions (230 mOsm) caused a 15% increase in cell capacitance and activated a reversible conductance that exhibited marked adaptation in the continued presence of the stimulus. Similar responses could be elicited in taste cells from taste buds in the foliate and vallate papillae, the soft palate, the nasopharynx and the epiglottis. Ion substitution experiments were consistent with the interpretation that the predominant ion carried through these apparent volume- or stretch-activated channels was Cl(-) under normal conditions. Reversal potentials for the hypoosmotic-induced current closely matched those predicted by the Goldman-Hodgkin-Katz constant field equation for a Cl(-) conductance. The relative permeability sequence of the hypoosmotic-activated current in TRCs was thiocyanate(-) > or = l(-) > or = Br(-) > Cl(-) > or = F(-) > or = isethionate(-) > gluconate(-). Pharmacological experiments revealed that this Cl(-) conductance was inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and 5-nitro-3-(3-phenyl-propylamino)benzoic acid (EC(50) = 1.3 and 4.6 microM, respectively), but not by CdCl(2) (300 microM) nor GdCl(3) (200 microM). I hypothesize that this hypoosmotic-activated Cl(-) conductance, which is similar to the well-characterized swelling-activated Cl(-) current, may contribute to volume regulation and could represent the transduction mechanism by which the presence of hypoosmotic stimuli, including water, may be signaled in taste receptor cells.

Mechanosensitive cation channels in human leukaemia cells: calcium permeation and blocking effect.

Cell-attached and inside-out patch-clamp methods were employed to identify and characterize mechanosensitive (MS) ionic channels in the plasma membrane of human myeloid leukaemia K562 cells. A reversible activation of gadolinium-blockable mechanogated currents in response to negative pressure application was found in 58 % of stable patches (n = 317). I-V relationships measured with a sodium-containing pipette solution showed slight inward rectification. Data analysis revealed the presence of two different populations of channels that were distinguishable by their conductance properties (17.2 +/- 0.3 pS and 24.5 +/- 0.5 pS), but were indistinguishable with regard to their selective and pharmacological properties. Ion-substitution experiments indicated that MS channels in leukaemia cells were permeable to cations but not to anions and do not discriminate between Na(+) and K(+). The channels were fully impermeable to large organic cations such as Tris(+) and N-methyl-D-glucamine ions (NMDG(+)). Ca(2+) permeation and blockade of MS channels were examined using pipettes containing different concentrations of Ca(2+). In the presence of 2 mM CaCl(2), when other cations were impermeant, both outward and inward single-channel currents were observed; the I-V relationship showed a unitary conductance of 7.7 +/- 1.0 pS. The relative permeability value, P(Ca)/P(K), was equal to 0.75, as estimated at physiological Ca(2+) concentrations. Partial or full inhibition of inward Ca(2+) currents through MS channels was observed at higher concentrations of external Ca(2+) (10 or 20 mM). No MS channels were activated when using a pipette containing 90 mM CaCl(2). Monovalent mechanogated currents were not significantly affected by extracellular Ca(2+) at concentrations within the physiological range (0-2 mM), and at some higher Ca(2+) concentrations.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons.

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.

Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na(+) conductance and induces current-noise.

Split gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10 per thousand salinity were mounted in a modified Ussing-type chamber. With NaCl saline on both sides, split gill lamellae generated a short-circuit current (I(sc)) of -301+/-16 microA cm(-2) at a conductance (G(te)) of 40+/-2 mS cm(-2). The net influxes of Na(+) and Cl(-) were 8.3+/-2.6 and 18.2+/-2.7 micromol cm(-2) h(-1), respectively. External amiloride (100 micromol l(-1)) reduced G(te) to approximately 50 % of the original value at unchanged I(sc); Cl(-) fluxes remained unaffected, whereas Na(+) fluxes were markedly reduced by 70-80 %. The I(sc) in the presence of external amiloride was almost completely inhibited by internal ouabain. At a clamp voltage of 50 mV (outside-positive), a positive current was measured at unchanged G(te). Under these conditions, amiloride reduced the current and conductance at half-maximal concentrations of 3.6 and 2.0 micromol l(-1), respectively. At outside-positive voltages, but not under short-circuit conditions, external amiloride induced Lorentzian components in the power density spectra. The amiloride-dependent changes in the corner frequency (linear) and of the low-frequency plateau ('bell-shaped') were as expected for channel blockade by amiloride with pseudo-first-order kinetics. With an outside-positive clamp voltage of 50 mV across isolated cuticles, a positive cuticular current (I(cut)) of 25 188+/-3791 microA cm(-2) and a cuticular conductance (G(cut)) of 547+/-76 mS cm(-2) were measured. External amiloride reduced I(cut) and G(cut) at half-maximal concentrations of 0.7 and 0.6 micromol l(-1), respectively. Amiloride-induced current-noise analysis gave similar results to those observed with split gill lamellae. Ion-substitution experiments with isolated cuticles further support inhibition by external amiloride of the cuticular Na(+) conductance of shore crab gills and not amiloride-sensitive transporters (Na(+) channels or Na(+)/H(+) antiports) in the apical membrane.

Calcium-permeable glutamate receptors in horizontal cells of the mammalian retina

Mechanisms that mediate the calcium influx in mammalian horizontal cells were studied. Horizontal cells (HCs) enzymatically dissociated from the rabbit retina were recorded by the whole-cell configuration of the patch-clamp technique and by calcium image ratioespectrophotometry of Fura-2 loaded cells. AMPA-preferring glutamate receptors were shown to permeate Ca2+ in mammalian HCs by ionic substitution experiments. Furthermore, after blocking the L-type calcium current with nifedipine (100 microM), calcium current through the AMPA-preferring glutamate receptors was measured. Calcium image ratioespectrophotometry was performed on the dissociated HCs in order to determine the changes in the intracellular calcium ([Ca2+]i). Fura-2 microspectrophotometry showed that in HCs, K+-induced cell depolarization promoted an increase in [Ca2+]i, mediated by the L-type calcium channels, since it was abolished in the presence of nifedipine. The increase in [Ca2+], upon cell depolarization was observed throughout each cell: however, it was maximal at the cell soma. Activation of glutamate receptors in dissociated HCs by glutamate, AMPA or kainate promoted an increase in [Ca2+]i. This increase in [Ca2+]i, was abolished in nominally Ca2+-free solution (0 mM Ca2+); in contrast, nifedipine decreased the glutamate-induced influx of calcium in ca. 50%. The present study demonstrates that calcium may permeate through glutamate receptors expressed in HCs of the rabbit retina.

Structure–activity and possible mode of action of S-Iamide neuropeptides on identified central neurons of Helix aspersa

Intracellular recordings were made from identified neurons from the suboesophageal ganglia of Helix aspersa. The inhibitory action of nine S-Iamide peptides was investigated. Structure–activity studies suggest that all act through a common receptor, which normally requires FVRIamide at the C terminal, with a preferred length of seven amino acids. Substitution at the N-terminal with alanine (A), threonine (T), proline (P) or leucine (L) results in little change in potency, suggesting the N-terminal requirements are relatively flexible. Ion substitution experiments suggest that potassium is the main ion involved in the inhibitory response to S-Iamide application. Studies using a range of compounds, which modify second messenger systems, would suggest that S-Iamide peptides may interact with adenylate cyclase. No evidence was found for an interaction with either guanylate cyclase or nitric oxide synthase.

18-beta-Glycyrrhetinic acid (BGA) as an electrical uncoupler for intracellular recordings in confluent monolayer cultures.

In the study of epithelial cell biology, primary cell cultures or cell lines grown to confluency offer considerable advantages compared with isolated cells and cell clusters. This is due mainly to the development of appropriate cell-to-cell contacts that are a prerequisite for cell polarity and thus vectorial solute transport. On the other hand, electrical coupling via gap junctions in most instances significantly hinders the use of voltage-clamp techniques for electrophysiological analysis of transport processes in single cells. In the present study we employed the gap junctional blocker 18-beta-glycyrrhetinic acid (BGA) to reduce electrical cell-to-cell coupling in confluent primary cultures of rat hepatocytes. In current-clamp experiments, 40 micromol/l BGA reversibly increased apparent cell input resistance approximately tenfold. Due to this partial electrical isolation of cells, two-channel voltage-clamp experiments became feasible and, for the first time, the hypertonicity-induced Na+ conductance of rat hepatocytes could be analysed quantitatively. In ion substitution experiments, however, it became obvious that BGA, while leaving Na+ and K+ conductances virtually unchanged, completely blocked cell membrane Cl- conductance. This additional effect of BGA necessitates independent control experiments to ensure that the transport process under consideration is itself not changed by the compound. Nevertheless, BGA may serve as a powerful tool for the quantitative electrophysiological study of epithelial cells that are in quasi physiological contact with their neighbours.