Calcium homeostasis in a clonal pituitary cell line of mouse corticotropes.

Calcium homeostasis was studied following a depolarization-induced transient increase in [Ca2+]i in single cells of the clonal pituitary cell line of corticotropes, AtT-20 cells. The KCl-induced increase in [Ca2+]i was blocked in (i) extracellular calcium-deficient solutions, (ii) external cobalt (2.0 mM), (iii) cadmium (200 microM), and (iv) nifedipine (2.0 microM). The mean increase in [Ca2+]i in single cells in the presence of an uncoupler of mitochondrial function [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 1 microM] was 54 +/- 13 nM (n = 9). The increase in [Ca2+]i produced by FCCP was greater either during or following a KCl-induced [Ca2+]i load. However, FCCP did not significantly alter the clearance of calcium during a KCl-induced rise in [Ca2+]i. Fifty percent of the cells responded to caffeine (10 mM) with an increase in [Ca2+]i (191 +/- 24 nM; n = 21) above resting levels; this effect was blocked by ryanodine (10 microM). Thapsigargin (2 microM) and 2,5 di(-t-butyl)-1,4 hydroquinone (BuBHQ, 10 microM) produced increases in [Ca2+]i (47 +/- 11 nM, n = 6 and 22 +/- 4 nM, n = 8, respectively) that increased cell excitability. These results support a role for mitochondria and sarco-endoplasmic reticulum calcium stores in cytosolic [Ca2+]i regulation; however, none of these organelles are primarily responsible for the return of [Ca2+]i to resting levels following this KCl-induced [Ca2+]i load.

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes.

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.

Activation of potassium and chloride channels by tumor necrosis factor alpha. Role in liver cell death.

Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K(+) and Cl(-) currents, respectively, in HTC cells. These increases occurred within 5-10 min after TNF exposure and were inhibited either by K(+) or Cl(-) substitution or by K(+) channel blockers (Ba(2+), quinine, 0.1 mm each) or Cl(-) channel blockers (10 microm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K(+) and Cl(-) currents were each inhibited by intracellular Ca(2+) chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 micrometer) or PKC 19-36 peptide (1 micrometer). In contrast, currents were not attenuated by the calmodulin kinase II 281-309 peptide (10 micrometer), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 micrometer), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K(+) and Cl(-) channels is an early response to TNF signaling and that channel opening is Ca(2+)- and PKC-dependent. Our findings further suggest that K(+) and Cl(-) channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.

Sites and mechanisms of antibiotic-induced neuromuscular block: a pharmacological analysis using quantal content, voltage clamped end-plate currents and single channel analysis.

Since the original observation of Vital Brazil and Corrado (1957) concerning the antibiotic induced neuromuscular block produced by streptomycin, there has been considerable interest in the mechanisms responsible for not only neuromuscular block but also the effects of antibiotics on different systems. We used the voltage clamped end-plate of transacted skeletal muscle to examine the concentration-dependent actions of several groups of antibiotics. The aminoglycoside antibiotics, neomycin and streptomycin, were both more effective at reducing quantal release of acetylcholine (ACh) than interacting with the postjunctional ACh receptor-channel complex. Neomycin was approximately 10 X more potent prejunctionally than streptomycin and the prejunctional effects of each antibiotic were reversed competitively by raising extracellular calcium. Both neomycin and streptomycin also had postjunctional actions at higher concentrations. Neomycin interacted with the open state of the ACh receptor ion channel complex while streptomycin blocks the ACh receptor. The lincosamide antibiotics, lincomycin and clindamycin produced their neuromuscular block postjunctionally by interacting with the open state of the ACh-receptor channel complex. Clindamycin is approximately 20 X more effective at blocking the open channel than was lincomycin. Using cell attached patch clamp recordings in cultured rat myotubes, we demonstrated a lincosamide-induced block of open ion channels with clindamycin having a much slower unblocking rate than lincomycin. Using epimers of the lincosamides, we demonstrated that lipophilicity of the molecule, rather than stereochemical considerations, is important for open channel blockade affecting primarily the "off" rate of channel blocking. This mechanism appears important for not only the lincosamide antibiotics but also for the postjunctional actions of the aminoglycoside antibiotics, particularly neomycin.

Sites and mechanisms of antibiotic-induced neuromuscular block: a pharmacological analysis using quantal content, voltage clamped end-plate currents and single channel analysis

Since the original observation of Vital Brazil and Corrado (1957) concerning the antibiotic induced neuromuscular block produced by streptomycin, there has been considerable interest in the mechanisms responsible for not only neuromuscular block but also the effects of antibiotics on different systems. We used the voltage clamped end-plate of transacted skeletal muscle to examine the concentration-dependent actions of several groups of antibiotics. The aminoglycoside antibiotics, neomycin and streptomycin, were both more effective at reducing quantal release of acetylcholine (ACh) than interacting with the postjunctional ACh receptor-channel complex. Neomycin was approximately 10 X more potent prejunctionally than streptomycin and the prejunctional effects of each antibiotic were reversed competitively by raising extracellular calcium. Both neomycin and streptomycin also had postjunctional actions at higher concentrations. Neomycin interacted with the open state of the ACh receptor ion channel complex while streptomycin blocks the ACh receptor. The lincosamide antibiotics, lincomycin and clindamycin produced their neuromuscular block postjunctionally by interacting with the open state of the ACh-receptor channel complex. Clindamycin is approximately 20 X more effective at blocking the open channel than was lincomycin. Using cell attached patch clamp recordings in cultured rat myotubes, we demonstrated a lincosamide-induced block of open ion channels with clindamycin having a much slower unblocking rate than lincomycin. Using epimers of the lincosamides, we demonstrated that lipophilicity of the molecule, rather than stereochemical considerations, is important for open channel blockade affecting primarily the [quot ]off[quot ] rate of channel blocking. This mechanism appears important for not only the lincosamide antibiotics but also for the postjunctional actions of the aminoglycoside antibiotics, particularly neomycin.

Sites and mechanisms of antibiotic-induced neuromuscular block: a pharmacological analysis using quantal content, voltage clamped end-plate currents and single channel analysis.

Since the original observation of Vital Brazil and Corrado (1957) concerning the antibiotic induced neuromuscular block produced by streptomycin, there has been considerable interest in the mechanisms responsible for not only neuromuscular block but also the effects of antibiotics on different systems. We used the voltage clamped end-plate of transacted skeletal muscle to examine the concentration-dependent actions of several groups of antibiotics. The aminoglycoside antibiotics, neomycin and streptomycin, were both more effective at reducing quantal release of acetylcholine (ACh) than interacting with the postjunctional ACh receptor-channel complex. Neomycin was approximately 10 X more potent prejunctionally than streptomycin and the prejunctional effects of each antibiotic were reversed competitively by raising extracellular calcium. Both neomycin and streptomycin also had postjunctional actions at higher concentrations. Neomycin interacted with the open state of the ACh receptor ion channel complex while streptomycin blocks the ACh receptor. The lincosamide antibiotics, lincomycin and clindamycin produced their neuromuscular block postjunctionally by interacting with the open state of the ACh-receptor channel complex. Clindamycin is approximately 20 X more effective at blocking the open channel than was lincomycin. Using cell attached patch clamp recordings in cultured rat myotubes, we demonstrated a lincosamide-induced block of open ion channels with clindamycin having a much slower unblocking rate than lincomycin. Using epimers of the lincosamides, we demonstrated that lipophilicity of the molecule, rather than stereochemical considerations, is important for open channel blockade affecting primarily the [quot ]off[quot ] rate of channel blocking. This mechanism appears important for not only the lincosamide antibiotics but also for the postjunctional actions of the aminoglycoside antibiotics, particularly neomycin.

Intracellular pH and chemoresponse to NH4+ in Paramecium.

Paramecium are attracted to ammonium chloride solutions relative to sodium chloride control solutions, but little is known about the mechanisms by which attraction is evoked. A known effect of ammonium solutions in other cell types is an alteration of intracellular pH. We show here that intracellular pH is elevated upon initial exposure to 5 mM NH4Cl, but appears to decline within 10 minutes, both in wild type cells and in two mutants which do not show sustained attraction to NH4Cl using the standard behavioral assay, the T-maze. We also present quantitative values of swimming parameters that underlie the response to NH4Cl.

Muscarinic transmission decreases the number of SIF cells demonstrating catecholamine histofluorescence in rat superior cervical ganglia.

Preganglionic electrical stimulation of the cervical sympathetic trunk to the rat superior cervical ganglia produced a mean reduction in the number of visible small intensely fluorescent (SIF) cells demonstrating catecholamine histofluorescence to 32% of the unstimulated contralateral control. The reduction in the number of catecholamine-positive SIF cells required the presence of specific blockers of catecholamine uptake and synthesis and was dependent on normal synaptic transmission. No change in the number of catecholamine-positive SIF cells was observed when ganglionic transmission occurred in solutions containing both hexamethonium and atropine or with atropine alone (97% of the unstimulated control). Furthermore, preganglionic stimulation in the presence of high magnesium/low calcium solutions, which effectively blocked synaptic transmission, prevented the stimulation-induced decrease in the number of catecholamine-positive SIF cells. Prolonged antidromic stimulation of the internal carotid nerve only reduced the number of catecholamine-positive SIF cells to 75% of the unstimulated contralateral control. These results suggest that preganglionic synaptic impulses can induce the release of catecholamines from SIF cells via muscarinic receptor activation. Furthermore, the necessity for pharmacological intervention of uptake and synthesis blockers of catecholamines in order to detect the synaptically-induced reduction in the number of catecholamine-positive SIF cells, suggests that synaptic transmission also modulates the synthesis of catecholamines in SIF cells within the rat superior cervical ganglia.

Spontaneous transients of [Ca2+]i depend on external calcium and the activation of L-type voltage-gated calcium channels in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes.

Spontaneous transients of [Ca2+]i were recorded from single nonstimulated cells of a clonal pituitary cell line of corticotropes, AtT-20/D16v. The spontaneous [Ca2+]i transients were dependent on calcium entry from the extracellular solution because they were abolished both in the absence of extracellular calcium and with the addition of cobalt to the calcium-containing extracellular solution. Calcium entry occurred through voltage-gated (VGCC) L-type calcium channels because the [Ca2+]i transients were blocked by L-type calcium channel antagonists, e.g. nifedipine, and were unaffected by the addition of tetrodotoxin. Bay K 8644 (1 microM) induced transient increases in [Ca2+]i which were also blocked reversibly by either the absence of extracellular calcium or the addition of an L-type calcium channel antagonist (e.g. nifedipine). The resting levels of [Ca2+]i and the frequency, but not the amplitude or duration, of the spontaneous [Ca2+]i transients increased as the concentration of extracellular calcium was elevated in concentrations ranging from 1.8-7.2 mM. Potassium depolarization reversibly elevated resting levels of [Ca2+]i and initiated the spontaneous calcium transients. These results indicate that extracellular calcium modulates the frequency of spontaneous [Ca2+]i transients in AtT-20 cells which are caused by the activation of L-type calcium channels by a spontaneous increase in the permeability of the cell membrane to calcium.

Serotonin-induced changes in membrane potential and cytosolic free calcium in a clonal pituitary cell line (AtT-20) of cultured mouse corticotropes.

The effects of serotonin (5-HT) were examined on the cytosolic free calcium concentration, [Ca2+]i, in fura-2-loaded cells from a clonal cell line derived from the mouse anterior pituitary gland (AtT-20/D16v). Brief applications of 5-HT produced transient increases in [Ca2+]i. The duration and the amplitude of the 5-HT-induced increase in [Ca2+]i depended on the duration of 5-HT application. Longer duration (> 150 msec) applications of 5-HT produced a transient increase followed by a prolonged plateau phase of [Ca2+]i. Oscillations in [Ca2+]i were initiated and maintained in previously quiescent corticotropes following brief exposures to 5-HT. Addition of cobalt (500 microM-3.6 mM) to the extracellular solution abolished oscillations in [Ca2+]i and produced transient but not sustained increases in [Ca2+]i in response to 5-HT. Electrophysiological responses to 5-HT were recorded in separate experiments using the whole-cell patch clamp recording technique. Application of 5-HT to single cells produced a depolarization which was accompanied by a decrease in membrane conductance when measured under voltage clamp. The duration of the depolarizing response also increased with the duration of the 5-HT application. With longer duration 5-HT applications, the 5-HT-induced depolarizing response resembled the spontaneous action potential responses recorded in the same cell. However, the 5-HT-induced depolarizations were unaltered in the presence of extracellular cobalt. This suggests that the transient increase in [Ca2+]i recorded when 5-HT was applied in the presence of cobalt may represent intracellular release of calcium. These results demonstrate that 5-HT activates single cultured corticotropes directly by producing an increase in [Ca2+]i and cell depolarization.

VX enhances neuronal excitability and alters membrane properties of Rana catesbeiana sympathetic ganglion neurons.

1. The effects of VX (10 microM) were examined on sympathetic ganglion neurons from the bullfrog using intracellular recording techniques. 2. VX significantly increased the amplitude of the residual EPSP from 4.8 +/- 0.86 mV (n = 4) to 13.7 +/- 1.23 mV (n = 4). 3. VX significantly decreased the membrane potential 5.2 +/- 0.75 mV (n = 6). The input resistance and the duration of the spike afterhyperpolarization (AHP) were also reduced 69.8% and 69.6% of control, respectively. 4. VX increased neuronal excitability greater than 200% (n = 5) of control. 5. The VX-induced neuronal excitability may result from a reduction in the duration of the AHP and contribute to the CNS toxicity.

Comparison of atropine pre- and post-treatment in ganglion neurons exposed to soman.

The prevention and reversal of the effects of soman (pinacoloxymethylphosporyl fluoride) have been examined on the electrical properties of sympathetic ganglion neurons in vitro from the adult bullfrog Rana catesbeiana. Atropine (10 microM) pre-treatment (before soman) blocked the soman-induced decrease in the membrane potential, membrane resistance, and afterhyperpolarization (AHP) duration. Atropine post-treatment (after soman exposure) restored the soman-induced decrease in the membrane potential but was ineffective in reversing either the membrane resistance or the duration of the AHP. These results demonstrate: 1) that the effects of soman on the electrical properties of these neurons are mediated by the activation of muscarinic receptors, 2) that following receptor activation different cellular mechanisms may be involved in the regulation of the electrical properties of the neuron, and 3) pre-treatment rather than post-treatment with atropine is more effective in blocking these direct effects of soman. These results are discussed in reference to the increased effectiveness of atropine pretreatment in the protocols for soman-induced neurotoxicity.

Compound 48/80 blocks transmission and increases the excitability of ganglion neurons.

Compound 48/80 (5.0-50 micrograms/ml) significantly and reversibly decreased (1) the amplitude, but not the shape of the compound action potential, (2) the amplitude and duration of the acetylcholine potential and (3) the residual fast excitatory postsynaptic potential recorded from neurons of the 9th and 10th paravertebral ganglia of the bullfrog Rana catesbeiana. The excitability of B-type ganglion neurons in the presence of nicotinic and muscarinic receptor antagonists was increased by compound 48/80 without altering the input resistance or membrane potential. In addition, compound 48/80 (10-50 micrograms/ml) significantly decreased the duration of the spike afterhyperpolarization (AHP). The amplitude but not the decay rate of the current underlying the slow component of the spike AHP was decreased by compound 48/80. Compound 48/80 did not, however, alter either the amplitude or the duration of calcium-dependent spikes. Intracellular recordings from dissociated sympathetic neurons also demonstrated a compound 48/80-induced increase in neuronal excitability. These results suggest that compound 48/80 interacts with the nicotinic receptor/channel complex to decrease ganglionic transmission, and also has a direct action to increase neuronal excitability by blocking potassium channels mediating the duration of the spike AHP.

Soman reversibly decreases the duration of Ca2+ and Ba2+ action potentials in bullfrog sympathetic neurons.

Soman, (pinacoloxymethyl-phosphoryl fluoride) (0.1-10 microM) an irreversible cholinesterase inhibitor, reversibly reduced the duration of calcium (Ca2+)- and barium (Ba2+) spikes without significantly affecting spike amplitude in sympathetic postganglionic neurons of the adult bullfrog (Rana catesbeiana). The soman-induced shortening of the spike duration was not prevented by pretreatment with either (+)-tubocurarine (100 microM) or hexamethonium (100 microM) and atropine (10 microM) and was also recorded from acutely-dissociated sympathetic neurons. These results suggest that soman has a direct action to decrease calcium entry through voltage-dependent channels activated during a spike. This effect may contribute to both the decrease in the duration of the spike after-hyperpolarization (AHP) and the enhanced neuronal excitability produced by soman in these neurons.

The effects of soman on the electrical properties and excitability of bullfrog sympathetic ganglion neurones.

1. The effects of soman (0.1-10 microM), an irreversible inhibitor of acetylcholinesterase (AChE), were examined on the electrical properties of ganglion neurones of the paravertebral sympathetic chain of the bullfrog, Rana catesbeiana. 2. Soman (10 microM) depolarized 29 of 35 (83%) ganglion neurones studied by 6.4 +/- 0.65 mV within 10 min of application and reduced the cell input resistance in 9 of 11 neurones examined (82%) to 55 +/- 5.3% of control. 3. Soman (10 microM) significantly reduced the maximum amplitude and the maximum rate of rise of the action potential and the duration, but not the amplitude, of the after-hyperpolarization (AHP) following the action potential elicited by either direct or antidromic stimulation. The maximum rate of fall and the duration of the action potential were not significantly affected by soman. These actions of soman were independent of the agent-induced depolarization. When examined by a single microelectrode voltage clamp, soman reduced the amplitude and the time constant of the current underlying the slow AHP, IAHs. 4. Soman (1-10 microM) produced an increase in neuronal excitability which was evidenced as either an increase in the number of action potentials or a decrease in the interspike interval in response to constant-current depolarizing pulses. The soman-induced increase in excitability occurred independently of both the agent-induced depolarization and the decrease in input resistance, was reversible with washing, was not caused by an inhibition of the M-current and was also recorded in dissociated sympathetic ganglion neurones.5. The effects of soman on the membrane potential, input resistance and the duration of the AHP but not cell excitability were blocked by pretreatment with atropine (10 microM). Pretreatment with dihydro-/J-erythroidine (DHbetalE) (10 microM) was ineffective in blocking or reversing the effects of soman. These results suggest that the direct actions of soman on the electrical properties of these neurones are mediated by activation of muscarinic receptors.

The effects of irreversible acetylcholinesterase inhibitors on transmission through sympathetic ganglia of the bullfrog.

The effects of soman, sarin and VX were examined on ganglionic transmission through paravertebral chain ganglia of the bullfrog, Rana catesbeiana. Low frequency (0.1 Hz), short (2 sec) and long (10 sec) trains of preganglionic stimulation, after exposure to the agents, induced repetitive activity in the extracellularly recorded compound action potential. An irreversible transient depression was observed after exposure to the agents during the first second of short and long stimulus trains. Long stimulus trains of high frequency were required to produce a rundown in the amplitude of the compound action potential, whether recorded in the presence of each agent (10 microM) or following a wash with agent-free solution. The rundown of the compound action potential was use-dependent and not blocked or reversed by atropine (10 microM). Intracellular recordings, in the presence of either soman or VX, demonstrated (1) an increase in the amplitude of the residual excitatory postsynaptic potential or current evoked by synaptic stimulation, (2) an increase in the amplitude and duration of the acetylcholine-induced potential, (3) no increase in either the amplitude or duration of the carbachol-induced potential, (4) repetitive firing with orthodromic but not antidromic stimulation and (5) a concentration- and frequency-dependent depolarization of individual ganglion neurons with orthodromic stimulation which resulted in a decrease in the generation of action potentials. These results suggest that the agent-induced decrease in the compound action potential occurred as a consequence of activity-dependent depolarization of ganglion neurons, which occurs after inhibition of cholinesterase.

End-plate ion channel block produced by lincosamide antibiotics and their chemical analogs.

Five lincosamide compounds were studied for their effects on end-plate currents (epcs), miniature end-plate currents and acetylcholine-induced current fluctuations in the garter snake costocutaneous nerve-muscle preparation. At high concentrations, lincomycin and clindamycin reduced epc amplitude, but analysis of driving functions showed that only with clindamycin was this due solely to changes in epc quantal content. The effect of lincomycin on epc amplitude was exaggerated by rapid channel block during the rising phase of the epc. Clindamycin produced currents with a single exponential decay and single Lorentzian noise spectra. All the other compounds produced currents which decayed as the sum of two exponential components. For lincomycin and epilincomycin, noise spectra consisted of two Lorentzian components. For epiclindamycin and deoxylincomycin, although epcs and miniature end-plate currents decayed with two components, it was not possible to separate two components in the noise spectra. A kinetic analysis of ion channel blocking actions showed only small differences between the two pairs of stereoisomers studied. End-plate ion channel blocking and unblocking rate constants did not vary greatly among the compounds but the end-plate ion channel unblocking rate constant values for the two lincomycin stereoisomers were larger than those for the two clindamycin stereoisomers. Deoxylincomycin exhibited properties similar to those of the clindamycins. It was concluded that lipid solubility, not stereochemical conformation, plays the greater role in determining the ion channel blocking properties within the series, particularly that of the rate of dissociation of the compound from end-plate ion channels.

Acceleration of desensitization by agonist pre-treatment in the snake.

1. The kinetics of carbachol-induced desensitization have been studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage clamped to +50 mV. 2. Microperfusion of carbachol (162-756 microM) induces a transient outward current which peaks within a few seconds and then slowly decays towards the base line. The time course of current decay estimates the time course of desensitization onset. 3. Brief exposure (30 s) to a 'conditioning' concentration of agonist (10.8 microM) accelerates the desensitization onset produced by exposure to higher 'test' concentrations of agonist (162-756 microM). 4. The acceleration of desensitization by pre-treatment with 10.8 microM-carbachol was independent of the duration of exposure between 15 and 60 s. This observation indicated that the mechanism responsible for the alteration in desensitization kinetics by treatment with 10.8 microM-carbachol differed from that responsible for the time-dependent development of desensitization produced in the presence of higher carbachol concentrations. 5. Pre-treatment with the muscarinic agonists, methylcholine and bethanechol, did not accelerate 216 microM-carbachol-induced desensitization, suggesting that the alteration of desensitization kinetics by pre-treatment was specific for nicotinic agonists. 6. The conditioning concentrations of carbachol (5.4-10.8 microM) produced no measurable outward current in muscle fibres voltage clamped to +50 mV. Further, in patch-clamp recordings it was observed that, with these concentrations of carbachol, there was no channel activity in many successful patches voltage clamped to +50 mV and, when present, the frequency of channel activity was very low. These results demonstrated that the alteration in desensitization was not the consequence of significant amounts of either receptor activation or desensitization produced by the conditioning concentration. 7. Exposure to 10.8 microM-carbachol for periods of up to 150 s did not change the amplitude of miniature end-plate currents recorded at end-plates voltage clamped to +50 mV. These results also demonstrated that the acceleration of desensitization by pre-treatment with conditioning concentrations of agonist was not due to partial desensitization occurring during the pre-treatment period. 8. Our results are consistent with the view that there are distinct populations of agonist binding sites on the acetylcholine receptor which separately regulate desensitization and channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions of edrophonium, physostigmine and methanesulfonyl fluoride with the snake end-plate acetylcholine receptor-channel complex.

The actions of two reversible anticholinesterase agents, edrophonium and physostigmine, were compared with the irreversible agent methanesulfonyl fluoride (MSF) on miniature end-plate currents (MEPCs) and ACh-induced end-plate current fluctuations recorded from twitch fibers of costocutaneous muscles of garter snakes (Thamnophis sp.). Low concentrations of edrophonium (less than 25 microM) produced a concentration-dependent increase in both the MEPC amplitude and the time constant of MEPC decay. MEPCs recorded at all concentrations studied (25-100 microM) decayed as a single exponential function with time. As the concentration of edrophonium was increased, MEPC amplitude was initially increased and then decreased such that, at concentrations above 50 microM, MEPC amplitude was decreased below control values. Concentrations of edrophonium greater than 30 microM produced power density spectra that required the sum of two Lorentzian components--one faster and one slower than control. Single-channel conductance was not significantly altered by edrophonium. Low concentrations of physostigmine (less than 10 microM) increased MEPC amplitude and prolonged MEPC decay. Higher concentrations of physostigmine (10-100 microM) decreased mean peak MEPC amplitude and accelerated MEPC decay in a concentration-dependent manner. The relationship between MEPC decay and membrane potential was reduced, and then reversed, with increasing concentrations of physostigmine. Current fluctuation spectra recorded in physostigmine were described by a single Lorentzian function at all membrane voltages and concentrations studied. Increasing the concentration of physostigmine or membrane hyperpolarization did not alter single-channel conductance but shortened the apparent lifetime of open end-plate channels. MSF increased MEPC amplitude and increased the time constant of MEPC decay without altering the temperature and voltage dependence of both MEPC decay and channel lifetime determined from end-plate current fluctuations. Exposure of MSF-treated end-plates to either edrophonium or physostigmine produced results that were similar to those obtained before MSF treatment. These results demonstrate that edrophonium and physostigmine have direct actions on the end-plate receptor-channel complex that are unrelated to their inhibitory action on junctional acetylcholinesterase.

Concentration-dependent effects of neostigmine on the endplate acetylcholine receptor channel complex.

The concentration-dependent actions of neostigmine, a carbamate anticholinesterase agent, were studied on the acetylcholine receptor channel complex in voltage-clamped twitch fibers of costocutaneous muscles of garter snakes. Low concentrations of neostigmine (10(-6) or 10(-5) M) increased miniature endplate current (MEPC) amplitude and the time constant of MEPC decay without changing the relationship between the MEPC decay time constant and membrane potential. Acetylcholine- or carbachol-induced endplate current fluctuation spectra were well fitted by a single Lorentzian curve with a characteristic frequency and single-channel conductance unaltered by low concentrations of neostigmine. Concentrations of neostigmine greater than 5 X 10(-5) M decreased MEPC amplitude and split the decay of MEPCs into two components, one faster and one slower than the control rate. These effects were both voltage and concentration dependent. Spectra of current fluctuations recorded in concentrations greater than or equal to 5 X 10(-5) M neostigmine required two time constants, one faster and one slower than the control. Two component spectra were also obtained with carbachol-induced current fluctuation spectra, indicating that these effects of neostigmine were direct and not a consequence of acetylcholinesterase inhibition. Similar results were also obtained in muscles pretreated with collagenase to remove junctional acetylcholinesterase. The fast and slow time constants obtained from current fluctuation spectra decreased and increased, respectively, with either increases in the concentration of neostigmine or membrane hyperpolarization when analyzed in the same fiber. The effects of neostigmine on channel lifetime were reversible with washing. These results indicate that the effects of neostigmine are concentration dependent. Concentrations greater than 2.5 X 10(-5) M exhibit direct effects on the endplate receptor channel complex which are unrelated to acetylcholinesterase inhibition. These actions include: a prolongation of the gating kinetics of the endplate receptor channel complex, the production of an altered state of the receptor channel complex evidenced by a high frequency component to current fluctuation spectra, and a direct action to block the acetylcholine receptor.