Kinetics of acetylcholine-activated cation channel blockade by the calcium antagonist D-600 inAplysia neurons

The effects of the calcium channel blocker D-600 on the cation channels activated by acetylcholine (ACh) was studied in voltage-clamped Aplysia neurons by voltage-jump relaxation analysis. D-600 blocked the steady-state ACh current in a highly voltage-dependent manner, the degree of antagonism increasing with membrane hyperpolarization. In the presence of D-600 the current relaxations following hyperpolarizing command steps became biphasic. The time constants of ACh-induced current relaxations (tau f), which approximate the mean channel lifetime, were reduced in a voltage-dependent manner, the degree of reduction of tau f increasing with increasing membrane potential. In addition to the acceleration of tau f, a slow, inverse kinetic component (tau s) of the relaxation appeared in the presence of D-600. The rate of this inverse kinetic component was accelerated either by increasing the agonist or antagonist dose or by increasing the membrane potential. These results suggest that D-600 acts to antagonize the acetylcholine response through a blockade of the open state of the transmitter-activated cation channel. Possible kinetic schemes for this interaction are discussed.     

Photoactivation and dissociation of agonist molecules at the nicotinic acetylcholine receptor in voltage-clamped rat myoballs.

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.     

Reconstruction of ionic currents in a molluscan photoreceptor.

Two-microelectrode voltage-clamp measurements were made to determine the kinetics and voltage dependence of ionic currents across the soma membrane of the Hermissenda type B photoreceptor. The voltage-dependent outward potassium currents, IA and ICa(2+)-K+, the inward voltage-dependent calcium current, ICa2+ and the light-induced current, IIgt, were then described with Hodgkin-Huxley-type equations. The fast-activating and inactivating potassium current, IA, was described by the equation; IA(t) = gA(max)(ma infinity[1-exp(-t/tau ma)])3 x (ha infinity [1-exp(-t/tau ha)] + exp(-t/tau ha)) (Vm-EK), where the parameters ma infinity, ha infinity, tau ma, and tau ha are functions of membrane potential, Vm, and ma infinity and ha infinity are steady-state activation and inactivation parameters. Similarly, the calcium-dependent outward potassium current, ICa(2+)-K+, was described by the equation, ICa(2+)-K+ (t) = gc(max)(mc infinity(VC)(1-exp[-t/tau mc (VC)]))pc (hc infinity(VC) [1-exp(-t/tau hc)] + exp(-t/tau hc(VC)])pc(VC-EK). In high external potassium, ICa(2+)-K+ could be measured in approximate isolation from other currents as a voltage-dependent inward tail current following a depolarizing command pulse from a holding potential of -60 mV. A voltage-dependent inward calcium current across the type B soma membrane, ICa2+, activated rapidly, showed little inactivation, and was described by the equation: ICa2+ = gCa(max) [1 + exp](-Vm-5)/7]-1 (Vm-ECa), where gCa(max) was 0.5 microS. The light-induced current with both fast and slow phases was described by: IIgt(t) = IIgt1 + IIgt2 + IIgt3, IIgti = gIgti [1-exp(- ton/tau mi)] exp(-ton/tau hi)(Vm-EIgti) (i = 1, 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine-induced current in perfused rat myoballs

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current- voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.     

A study of the cholinolytic actions of strychnine using the technique of concentration jump relaxation analysis

The blocking actions of strychnine on excitatory acetylcholine (ACh) responses in isolated, voltage clamped Aplysia neuronal cell bodies has been studied using a rapid drug application technique. Rapid microperfusion of strychnine (10-50 microM) produced a reduction of the steady-state ACh-induced inward current in Aplysia neurons which decayed exponentially with a highly dose-dependent time constant. At the cessation of strychnine perfusion the ACh-induced current recovered to its original value with an exponential time course which was not sensitive to the dose of strychnine previously applied. The calculated association (k1) and dissociation (k-1) constants for a pseudo-first-order reaction between strychnine and its binding site were k1 = 1.2 X 10(4) M-1. sec-1 and k-1 = 0.12 sec-1 (KD = 1 X 10(-5) M-1). These results demonstrate that concentration jump relaxation experiments can be performed on isolated neurons for the study of voltage-independent antagonists by the use of rapid microperfusion systems and provide the first direct estimates to date of the rate constants of the cholinolytic effect of strychnine.     

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.     

Calcium entry induced by acetylcholine action on snail neurons

A study was made of excitatory and inhibitory responses elicited by acetylcholine (ACh) in neurons of the snail Eobania vermiculata. At resting potential, ACh evoked a depolarizing inward current in some neurons (D-cells) and a hyperpolarizing current in others (H-cells). The currents elicited by ACh were nonlinearly dependent on membrane potential. After either D- or H-cells were equilibrated in chloride-free isotonic calcium, ACh evoked a depolarizing inward current which reversed sign at about -55 mV. These results suggest that ACh causes an influx of Ca2+ in both types of neurons.     

A molecular scheme for the reaction between acetylcholine and nicotinic channels.

In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.     

Acetylcholine responses of identified neurons in Helix pomatia--II. Pharmacological properties of acetylcholine responses

A pharmacological separation of depolarizing and hyperpolarizing mechanisms involved in the generation of acetylcholine (ACh) depolarizations was attempted in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia. The selectivity of the drugs employed was assayed in non-identified buccal neurons in which ACh increased a hyperpolarizing Cl- conductance. Voltage clamp techniques were used. Under control conditions the depolarizing ACh currents increased non-linearly with more negative membrane potentials. The hyperpolarizing ACh currents showed a linear potential dependence. The buffer substance Tris (5 mmol/l) depressed the depolarizing ACh currents. The effect was accentuated with more negative membrane potentials. Tris failed to affect hyperpolarizing ACh responses. HEPES (5 mmol/l) did not change depolarizing or hyperpolarizing ACh responses. d-Tubocurarine (0.02-0.2 mmol/l), hexamethonium (0.5-5.0 mmol/l) and atropine (0.1 mmol/l) blocked the depolarizing and hyperpolarizing ACh responses. Arecoline (0.1 mmol/l) had neither an agonistic nor an antagonistic effect on the identified and on the non-identified neurons. It displayed an anticholinesterase activity. Anthracene-9-carbonic acid (0.5 mmol/l) depressed selectively the hyperpolarizing ACh responses. In the neurons B1 and B3 no pharmacologically separable hyperpolarizing ACh responses were detected to be superimposed on the ACh depolarizations.     

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.     

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

IRK1 inward rectifier K(+) channels exhibit no intrinsic rectification

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg(2+) and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K(+)-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K(+) channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump-induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.     

Voltage dependence of acetylcholine-activated membrane conductance in sympathetic ganglion neurons

Acetylcholine-induced membrane conductance was investigated in superior cervical ganglion neurons using a patch-clamp technique. It was found that hyperpolarization and depolarization produce an increase and a reduction in acetylcholine (ACh) conductance. This reduction was unconnected with either reversal of the current induced by iontophoretic ACh application or the presence of Ca ions in the external solution. The time constant of relaxation (_r) of this current, produced by a jump in membrane potential, was found to increase e-fold when the membrane was hyperpolarized by 70 mV, matching the voltage dependence of ACh conductance. This led to the hypothesis that voltage-dependent ACh-induced conductance is entirely determined by the voltage dependence of nicotinic receptor channel gating kinetics.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 167–171, March–April, 1988.     

Correlating Charge Movements with Local Conformational Changes of a Na+-Coupled Cotransporter

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na⁺-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na⁺, providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.     

Correlating Charge Movements with Local Conformational Changes of a Na-Coupled Cotransporter

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na⁺-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na⁺, providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.     

Simulation of the voltage dependence of the Na, K pump applied to cardiac cells

We use simulation to study the dependence of the Na, K pump on membrane potential. Two consecutive mechanisms for the Na, K pump, based on a reduced Post-Albers scheme, are examined: one with six steps called GV3 and one with seven steps called MGV3. In GV3, a single voltage-dependent step combines both Na+ translocation and Na+ release into the extracellular medium. In MGV3, these two processes are allocated to two separate consecutive steps, but only the Na+ translocation step is voltage-dependent. Using the optimization software SCoPfit, numerical values of rate coefficients, symmetry factor (beta), and pump site density were found by fitting the models to published experimental data so that both GV3 and MGV3 could quantitatively reproduce steady-state current-voltage relationships for both forward and backward running of the pump, as well as [Na+]in and [K+]out activation curves. Using the rate coefficient values found by SCoPfit, we simulated a voltage-clamp experiment with both models running under their Na(+)-Na+ exchange mode, and we computed the transient currents generated following voltage steps in both depolarizing and hyperpolarizing directions from a basic potential of -40 mV. The voltage dependence of the rate constant (1/tau) of decay of the transient currents could qualitatively be reproduced when beta = 0.884 for GV3, and 0.932 for MGV3. The quantitative discrepancy between published experimental data and the theoretical curve generated by GV3 at potentials more negative than -20 mV was considerably reduced by using model MGV3. This finding alone suggests that a more detailed mechanism containing a single voltage-dependent step may reproduce all major steady-state and transient characteristics of the Na, K pump without the need of a second voltage sensitive step. However, the quantitative discrepancy between published experimental data and the theoretical curve generated by MGV3 at potentials more negative than -60 mV may be fully removed if either beta itself is voltage-dependent, or if a second voltage-dependent step is included in the model.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.     

Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus

Intracellular recordings have been made from salivary gland cells of the pond snail Planorbis corneus. Gland cells produced a dose-dependent biphasic response to the bath application of acetylcholine (ACh), an initial depolarization being followed by a hyperpolarization. Nicotine and the nicotinic agonist tetramethylammonium had an excitatory action on the gland cells. The muscarinic agonists acetyl-beta-methyl choline and arecoline were also stimulants, but muscarine, bethanechol and pilocarpine produced no response from gland cells at 10(-3) M. A number of cholinergic antagonists, including atropine, hexamethonium and curare, effectively blocked the response to ACh. The depolarizing phase of the ACh response resulted from an increased membrane permeability to Na+ ions, though the participation of other ionic species cannot be ruled out. The hyperpolarizing phase of the ACh response was produced by the activity of an electrogenic Na+/K+ pump.     

Electrophysiological study of frog eggs at different stages of development. III) Ionic currents activated by intense hyperpolarization and depolarization of the oocyte at the final stage of vitellogenesis

In addition to K-selective channels, two kinds of voltage-dependent channels are present in the membrane of full-grown frog oocytes: 1) non-selective channels, activated by hyperpolarizing steps to potentials more negative than -80, -90 mV; 2) cationic channels, activated by depolarizing steps to potentials more positive than +30, +40 mV. The former are related to the inward rectification; the latter could contribute to the outward rectification of the I/V relationship. Therefore the membrane potential in full-grown oocytes tends to be maintained at a constant value due to the changes of permeability that follow possible potential variations occurring in both directions.