Voltage dependence of beta-adrenergic modulation of conduction in the canine Purkinje fiber.

Although recent voltage-clamp and microelectrode studies have demonstrated beta-adrenergic modulation of Na+ current (INa) the modulation of conduction by catecholamines and the voltage dependence of that process have not been elucidated. To determine whether voltage-dependent modulation of conduction occurs in the presence of a beta-adrenergic agonist, the effect of 1 mumol/L isoproterenol on impulse propagation in canine Purkinje fibers was examined by using a dual-microelectrode technique. At physiological membrane potentials ([K+]o 5.4 mmol/L), isoproterenol increased squared conduction velocity [theta 2, 0.39 +/- 0.25 (m/s)2 (mean +/- SD)] from 3.46 +/- 0.86 to 3.85 +/- 0.98 (m/s)2 (P < .011), an 11% change, without altering the maximum first derivative of the upslope of phase 0 of the action potential (Vmax, 641 +/- 50 versus 657 +/- 47 V/s, P = NS). At transmembrane potential of -65 mV, produced by 12 mmol/L [K+]o titration, theta 2 declined 79% to 0.73 +/- 0.44 (m/s)2 as Vmax decreased 85% to 95 +/- 43 V/s (P < .02). The addition of isoproterenol further decreased theta 2 to 0.49 +/- 0.33 (m/s)2 (P = .02) in parallel with a further decline in Vmax to 51 +/- 25 V/s (P < .05). Isoproterenol produced a 3-mV hyperpolarizing shift of apparent Na+ channel availability curves generated from both theta 2 and Vmax, used as indexes of the fast inward INa, without changing the slopes of the relation. The relation between normalized theta 2 and Vmax over a range of depolarized potentials was linear and was not appreciably altered by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute ozone exposure on the electrophysiological properties of guinea pig trachea.

Acute ozone (O3) exposures produce an increase in the apparent permeability of the tracheal epithelium, but the mechanism of this response is poorly understood. Comparison of previous studies suggests that qualitative differences may exist between measurements made in vivo or in vitro. To test this possibility we used both in vitro and in vivo electrophysiological techniques to investigate the effects of O3 exposure on guinea pig tracheal epithelium. Male Hartley guinea pigs were exposed to either 1 or 2 ppm O3 or to filtered air for 3 h and were studied 0, 6, or 24 h after exposure. Air-exposed animals had in vitro mean tracheal potential (VT) -32.0 +/- 1.5 mV, conductance (GTL) 2.18 +/- 0.22 mS/cm, short-circuit current (ISCL) 62.6 +/- 3.7 microA/cm, and diameter (D) 2.44 +/- 0.10 mm. In vitro properties after 1 ppm O3 exposure did not differ at any time point from control. Two parts per million O3 increased ISCL, but only at 6 h postexposure. The effect of O3 on ISCL was abolished by amiloride. There were no significant changes in VT, GTL, or D. In vivo tracheal potential under pentobarbital anesthesia was -19.7 +/- 1.7 mV. At 6 h postexposure to 2 ppm O3, but not at 0 or 24 h, in vivo VT was increased. Thus, acute exposure of guinea pigs to a high concentration of O3 caused a delayed increase in Na+ absorption by the trachea with no change in conductance. This indicates that paracellular permeability of guinea pig tracheal epithelium was not substantially increased by acute O3 and suggests that enhanced macromolecular uptake in this species probably occurs transcellularly. Furthermore, the increase of in vivo VT following O3 exposure is consistent with the in vitro response, indicating that in vivo/in vitro differences are not responsible for the discrepancies between previous electrophysiological and "permeability" studies.

Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation.

We used electrorotation measurements to investigate alterations in the plasma membranes of DS19 murine erythroleukaemia cells that accompanied erythropoietic differentiation induced by hexamethylene bisacetamide (HMBA). Following 3 days of HMBA treatment, the mean cell membrane specific capacitance determined from electrorotation spectra of individual, viable cells at physiological tonicity (300 mosmol/kg) fell from 1.74 to 1.53 microF/cm2, in agreement with trends observed earlier by dielectrophoretic measurements on bulk cell populations. Scanning and transmission electron microscopy revealed that the relatively high values found for cell membrane capacitance (> 1 microF/cm2) reflected the large area of plasma membrane associated with complex surface morphology including numerous microvilli. Furthermore, it demonstrated that the fall in membrane capacitance during HMBA treatment correlated with a reduction in the density of these complex surface features. Differences in the mechanical characteristics of the cell membranes of untreated and treated cells were then examined by exposing cells to osmotic stress. The intricacy of membrane morphology intensified with increasing osmolality of the suspending medium and this was reflected in higher specific capacitance values. When the osmolality was increased from 210 to 450 mosmol/kg, the mean membrane capacitance of untreated DS19 cells changed from 1.58 to 2.05 microF/cm2 while that for HMBA-treated cells changed from 1.47 to 1.72 microF/cm2, a significantly smaller response. This demonstrated that cells exposed to 72 h of differentiation treatment had an enhanced mechanical resilience as compared with their untreated counterparts, evidencing the early stages of the development of the membrane skeleton which becomes fully developed in mature erythrocytes. Our findings demonstrate the value of electrorotation measurements as a method for the non-invasive characterisation of viable leukaemic cells and their responses to stimuli and show that the membrane capacitance values so derived reflect membrane morphology.

Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.

Ionic currents evoked by acetylcholine in isolated acinar cells of the guinea pig nasal gland.

The patch-clamp whole cell recording was used to demonstrate activation of membrane conductance to K+, Cl- and cations induced by acetylcholine (ACh) in the isolated acinar cells of the guinea pig nasal gland. A small outward K+ current at 0 mV and a large transient and sustained inward current at -90 mV were evoked by ACh and ACh-evoked reversal potential was about -3 mV nearly to Cl- equilibrium potential in 140 mM KCl in the pipette and physiological saline in the bath. The ionic substitutional experiments indicated that ACh-evoked inward currents were carried by both Cl- and cations. Both outward and inward currents evoked by ACh were almost completely abolished by removal of external Ca2+ and mimicked those evoked by a calcium ionophore A23187. These findings indicate that ACh-evoked membrane conductances are mediated by an increase in intracellular Ca2+.

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+.

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.

beta-Amyloid increases choline conductance of PC12 cells: possible mechanism of toxicity in Alzheimer's disease.

When beta-amyloid-(1-40) is added to PC12 cells, there is an increase in choline conductance that is proportional to the beta-amyloid concentration. If a similar effect occurs in cholinergic brain cells of Alzheimer's disease patients, the intracellular choline concentration would be reduced, leading to a decrease in the production of acetylcholine. This could explain the reduced level of acetylcholine that has been found in post-mortem brain tissue of Alzheimer's disease patients.

Development of a H(+)-selective conductance during granulocytic differentiation of HL-60 cells.

The NADPH oxidase is one of the main microbicidal systems of granulocytes. Stimulation of the oxidase during infection leads to a burst of metabolic acid generation. Potentially deleterious cytosolic acidification is prevented by the simultaneous activation of homeostatic H+ extrusion mechanisms, including a recently described H+ conductance. Studies in granulocytes from chronic granulomatous disease patients have suggested a relationship between the oxidase and the H+ conductive pathway. In this report we compared the expression of the H+ conductance and the NADPH oxidase during granulocytic differentiation of dimethyl sulfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated that the H(+)-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also estimated fluorimetrically, measuring changes in the cytosolic pH of suspended cells. Imposition of an inward protonmotive force failed to induce significant cytosolic acidification. In contrast, a sizable conductive H+ extrusion was detected in acid-loaded differentiated cells, consistent with the rectifying properties of the current measured electrophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulocytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH oxidase. These findings suggest that the H+ extrusion mechanisms required for the maintenance of the intracellular pH during granulocyte activation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conductance and the NADPH oxidase.

Enhancement of gustatory nerve fibers to NaCl and formation of ion channels by commercial novobiocin.

Single fibers of the rat chorda tympani nerve were used to study the mechanism of action of the antibiotic novobiocin on salt taste transduction. In the rat, novobiocin selectively enhanced the responses of sodium-specific and amiloride-sensitive chorda tympani nerve fibers (N type) without affecting more broadly responsive cation-sensitive and amiloride-insensitive fibers (E type). In the presence of amiloride, novobiocin was ineffective at enhancing the response of N-type fibers toward sodium chloride. Novobiocin also increased the conductance of bilayers formed from neutral lipids by forming nonrectifying ion channels with low conductance (approximately 7 pS in 110 mM NaCl), long open times (several seconds and longer), and high cation selectivity. Amiloride did not alter either the conductance or kinetics of these novobiocin channels. These observations suggest that even though novobiocin is able to form cation channels in lipid bilayers, and possibly in cell membranes as well, its action on the salt-taste response is through modulation of existing amiloride-sensitive sodium channels.

Properties of cardiac I(leak) induced by photosensitizer-generated reactive oxygen.

We reported previously that photomodification of single frog cardiac cells by Rose Bengal induces a time-independent current, designated I(leak)++, having a linear current-voltage (I/V) relationship. The purpose of the present study is to better characterize the properties of I(leak)++. Initially, I(leak)++ has a reversal potential (ER) near -70 mV, but with time, ER shifts toward a final value near 0 mV. This shift in ER is accompanied by a marked increase in conductance (slope of I/V relationship). Evidence is presented that the depolarizing shift in ER with time during photomodification results from a loss of membrane selectivity allowing sodium to make an increasing contribution to I(leak)++. Potassium also contributes to I(leak)++, as indicated by marked depolarizing shifts in ER following replacement of intracellular potassium with either cesium or tetraethylammonium. Since these results occur in calcium-free external media, the depolarizing shifts in ER and increased conductance are not related to activation of a calcium-dependent nonselective cation channel. However, I(leak) does have some properties similar to nonselective cation currents recently reported to be activated by membrane breakdown products such as arachidonic acid and lysophosphoglycerides.

Properties of single calcium-activated potassium channels of large conductance in rat hippocampal neurons in culture.

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K+]i/140 mM [K+]o and 140 mM [K+]i/3 mM [K+]o respectively. From the reversal potential the channel was highly selective for K+, the PK+/PNa+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 nM [Ca2+]i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca2+]i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 microM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K+ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 microM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the 'mode switching' seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.

The effect of pH on the block by L-cis-diltiazem and amiloride of the cyclic GMP-activated conductance of salamander rods.

Block by L-cis-diltiazem and amiloride of the cyclic GMP-activated conductance was studied in inside-out patches excised from the salamander rod outer segment. When cytoplasmic pH was varied, the steady-state level of block by L-cis-diltiazem changed in the way that would be predicted if it were the protonated ammonium group that is responsible for effecting block. This is in contrast to the recent results of Haynes (J. gen. Physiol. 100, 783 (1992)) in catfish cones, where no such change was seen. Amiloride was found to block the conductance with a similar voltage dependence to that of L-cis-diltiazem. The dependence of amiloride block on cytoplasmic pH was found to be shifted relative to that of L-cis-diltiazem, consistent with the 1 pH-unit higher pKa value of amiloride and the idea that it is only the charged form of amiloride which can effect block. This suggests that the results seen with L-cis-diltiazem were indeed due to changes in the proportion of blocker in the protonated form, and not to effects of protons on the channel.

Presynaptic inhibition by GABA is mediated via two distinct GABA receptors with novel pharmacology.

Mechanisms of presynaptic inhibition were examined in giant presynaptic terminals of retinal bipolar neurons, which receive GABAergic feedback synapses from amacrine cells. Two distinct inhibitory actions of GABA are present in the terminals: a GABAA-like Cl conductance and a GABAB-like inhibition of voltage-dependent Ca current. Both of the receptors underlying these actions have unusual pharmacology that fits neither GABAA nor GABAB classifications. The GABA-activated Cl conductance was not blocked by the classical GABAA antagonist bicuculline, while the inhibition of Ca current was neither mimicked by the GABAB agonist baclofen nor blocked by the GABAB antagonist 2-hydroxysaclofen. The "GABAC" agonist cis-4-aminocrotonic acid (CACA) both activated the Cl conductance and inhibited Ca current, but the inhibition of Ca current was observed at much lower concentrations of CACA (< 1 microM) than was the activation of the Cl conductance (K1/2 = 50 microM). Thus, by the criterion of being insensitive to both bicuculline and baclofen, both GABA receptors qualify as potential GABAC receptors. However, it is argued on functional grounds that the two GABA receptors coupled to Cl channels and to Ca channels are best regarded as members of the GABAA and GABAB families, respectively.

Junctional uvomorulin/E-cadherin and phosphotyrosine-modified protein content are correlated with paracellular permeability in Madin-Darby canine kidney (MDCK) epithelia.

Strains I and II of Madin-Darby canine kidney (MDCK) cells, which differ markedly in transepithelial resistance (RT) and paracellular permeability, have been used to investigate whether differences in the cellular content of uvomorulin/E-cadherin and phosphotyrosine may be correlated with junctional properties. Using immunocytochemistry, the strain I "tight" epithelia showed significantly stronger uvomorulin staining at regions of cell-cell contact compared with strain II "leaky" MDCK epithelia. In contrast, strain I MDCK cells showed a relatively faint phosphotyrosine staining, distributed evenly throughout the cytoplasm, while strain II MDCK cells displayed intense staining for phosphotyrosine residues in the junctional region and the lateral cell membrane with additional labelling of the cytoplasm. Exposure to vanadate in conjunction with H2O2 (which are potent inhibitors of protein tyrosine phosphatases) resulted in a dramatic increase in phosphotyrosine staining at the intercellular area and, concomitantly, induced changes in cell morphology, a significant decrease in RT, increase in paracellular inulin permeability, and time-dependent disappearance of uvomorulin from the cell-cell contact sites. Moreover, the effects of vanadate/H2O2 treatment were more dramatic in strain II compared with strain I cells, consistent with greater generation of tyrosine-modified protein in strain II cells. An inverse relationship was demonstrated between membrane-associated uvomorulin/E-cadherin and cellular phosphotyrosine content, which varied between the two strains of MDCK cells and when phosphotyrosine was directly manipulated. These data support the hypothesis that regulation of paracellular permeability may result from specific tyrosine phosphorylation of protein components of the junctional complex.

Adenosine decreases action potential duration by modulation of A-current in rat locus coeruleus neurons.

The possibility that adenosine modulates voltage-dependent conductances in locus coeruleus neurons was investigated in current-clamp and voltage-clamp experiments in a totally submerged rat brain slice preparation. Adenosine (100 microM) reduced the duration of control action potentials and action potentials prolonged by 1 mM barium. Adenosine (100 microM) also reduced the amplitude and slightly reduced the duration of TTX-resistant "calcium" action potentials. Action potential duration was also reduced by the adenosine receptor agonist 2-chloroadenosine in a concentration-dependent manner and the adenosine-induced reduction of action potential duration was blocked by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline, indicating that this action of adenosine is mediated by an adenosine receptor. The adenosine-induced reduction of action potential duration persisted in the presence of externally applied tetraethylammonium ion (6 mM) and cesium (3 mM). By contrast, adenosine did not reduce the duration of the action potential in the presence of 500 microM 4-aminopyridine (4-AP). Furthermore, 4-AP (30 microM) blocked the adenosine-induced reduction of action potential duration recorded in the presence of 1 mM barium. These data suggested that adenosine may be acting on the voltage-dependent, 4-AP-sensitive potassium current, IA. Single-electrode voltage clamp was used to study IA directly. IA was activated by depolarizing voltage pulses from a hyperpolarized holding potential and was blocked by 1 mM 4-AP. Adenosine (300 microM) enhanced IA by shifting the steady-state inactivation curve in the depolarizing direction.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium channel subtypes in the rat display functional differences in the presence of veratridine.

The alkaloid neurotoxin veratridine has the unique property of functionally distinguishing sodium channel subtypes in the rat through differences in single channel conductances, channel substates and probability of channel opening. Veratridine-activated cardiac sodium channels from rat ventricular muscle displayed a single channel conductance of 8.4 pS with no evidence of subconductance states or channel subtypes. Rat skeletal muscle sodium channels displayed both high (8.5 pS) and low conductance (4.7 pS) openings as well as a lower probability of opening (approximately 50%) at depolarized potentials than shown with brain or cardiac sodium channels (90-95%). Rat brain veratridine-activated sodium channels displayed primarily subconductance states at depolarized potentials (3-6 pS) and full conductance of approximately 9.5 pS at hyperpolarized potentials.

Comparative effects of MgCl2 and MgSO4 on the ionic transfer components through the isolated human amniotic membrane.

The effects of MgCl2 and MgSO4 are different on the total transfer through the human amniotic membrane: MgCl2 at low concentration (1 mM) decreases the total conductance Gt and increases it at high concentration (4 mM) on the fetal side (FS) and on the maternal side (MS), while MgSO4 has no effect on the MS and increases Gt on the FS. Moreover, whatever the concentration, MgCl2 increases the flux ration F1/F2 while MgSO4 decreases it to reach a value near to 1. Gt is the sum of various components: three paracellular components (Gp) and nine cellular components (Gc constituted from channels, exchangers, antiporters and cotransporters). All components of Gt, on the two faces, are decreased by 1 mM MgCl2 and increased at 4 mM. MgCl2 also has an effect on all ionic exchangers across the membrane. In contrast, on the MS, MgSO4 (1 mM) decreases GpNa, increases GpK and the antiport Na/H component and has no effect on any of the other components, while at 4 mM, MgSO4 has no effect. On the FS, MgSO4 (1 mM) increases GpNa and GpK, but does not modify the other components. At 4 mM, the effect is the same, except for an increase of GpCl. These data show the importance of the anion-cation association in the ionic exchanges through a membrane: MgCl2 and MgSO4 have a different action--MgCl2 interacts with all the exchangers, while the effect of MgSO4 is limited to paracellular components without interaction with cellular components excepted the antiport Na/H.