Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

The influence of hindlimb unloading on the effectiveness of modulation of the mediator secretion via the autoreceptor system

In experiments on neuromuscular synapses of rat fast (m. Extensor digitorum longus, EDL) and slow (m. soleus) skeletal muscles, changes in the intensity of spontaneous quantal mediator secretion in response to the activation of presynaptic cholinoreceptors by the nonhydrolyzable acetylcholine analogue carbachol and to an increase in K+ concentration in the control group of animals and in animals subjected to different terms of unloading of hindlimbs have been compared. The intensity of spontaneous secretion of mediator quanta was evaluated from the mean frequency of miniature endplate potentials. In the control group of animals, the frequency of miniature endplate potentials by the action of carbachol increased by 363% in m. EDL and by 62% in m. soleus. The frequency of miniature endplate potentials in the synapses of m. EDL was more sensitive to K(+)-induced depolarization too. The bearing unloading of hindlimbs abolished the sensitivity of spontaneous secretion to carbachol in the synapses of m. EDL, whereas in m. soleus it was unchanged. However, the preservation of sensitivity of nerve endings of fast muscle to K(+)-induced depolarization allows one to assume that the hindlimb unloading leads to a decrease in the number of functioning presynaptic receptors.     

Cholinergic responsiveness of respiratory and vascular tissues in two different rat strains

The airway and systemic arterial smooth muscle responsiveness to cholinergic agents of two strains of rats, Rat Albino (RA) and Brown Norway (BN), was compared in vivo and in vitro. In vivo, we measured the doses of carbachol that induced a 100% increase in lung resistance (PD100 RL), a 50% decrease in dynamic lung compliance (PD50 Cdyn), and the value of systolic blood pressure at the carbachol dose of 10 micrograms (Pa 10 micrograms). In vitro airway smooth muscle and systemic arterial smooth muscle responsiveness was assessed by measuring the maximal response to acetylcholine, the slope of the linear portion of the dose-response curve, and the negative logarithm of the molar concentration of acetylcholine producing 50% of the maximal response (pD2). PD100 and PD50 were about four times greater in BN rats than in RA rats. In contrast, Pa 10 micrograms was 1.5 lower in the BN rats. These differences persisted after bivagotomy. Tracheal pD2 was 25% greater in the RA than in the BN strain. The mean dose-response curve of parenchymal strips of RA rats was situated upward and to the left of the BN curve, but the reverse was observed for aortic smooth muscle dose-response curves. Thus 1) airway smooth muscle responsiveness to cholinergic agents is greater in RA strain than in BN, but the reverse is true for systemic arterial smooth muscle responsiveness; and 2) these differences are not due to factors extrinsic to the smooth muscle, since they occurred in vitro and may depend on different densities of muscarinic receptors.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Electrophysiological investigation of the properties of acetylcholine receptors of frog sympathetic ganglionic neurons

Changes in membrane potential and conductance were studied in neurons of isolated sympathetic ganglia ofRana ridibunda during perfusion with cholinomimetics and cholinolytics. Activation of nicotinic (N) acetylcholine receptors by carbachol, suberyldicholine, and tetramethylammonium led to depolarization with an increase in conductance, whereas activation of muscarinic (M) acetylcholine receptors by perfusion with carbachol or 5-methylfurmethide, led to depolarization with a decrease or (less frequently) an increase in conductance. The M-cholinolytic atropine was shown to cause depolarization with an increase in conductance if perfusion with atropine was preceded by perfusion with carbachol.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 475–482, September–October, 1979.     

Local anesthetic alteration of miniature endplate currents and endplate current fluctuations.

The effect of the local anesthetic QX222 on the kinetics of miniature endplate currents and acetylcholine induced endplate current fluctuations was studied in voltage clamped cutaneous pectoris muscle of Rana pipiens. Both the endplate current fluctuation spectra and the miniature endplate current decay consisted of two or three components depending upon the holding potential and local anesthetic concentration. The cutoff frequency of each spectral component was equal to the decay rate of its corresponding constituent of the miniature endplate current. Comparison of the relative amplitudes of the spectral and miniature endplate components indicated that QX222 did not act by creating two kinetically distinct populations of acetylcholine receptors. QX222 action could be explained by alteration of the acetylcholine receptors such that they sequentially change conformation form one open state to another. A specific case in which QX222 binds to the open state of the acetyl-choline receptor creating a blocked state, was found to account for the observed relationship between the relative amplitudes of the miniature endplate current and spectral components, as well as the previously observed voltage and concentration sensitivity of the decay rates of endplate current components.     

Effects of ammonium ions on endplate channels

Miniature endplate currents, recorded from voltage-clamped toad sartorius muscle fibers in solutions containing ammonium ions substituted for sodium ions, were increased in amplitude and decayed exponentially with a slower time constant than in control (Na) solution. The peak conductance of miniature endplate currents was also greater in ammonium solutions. The acetylcholine null potential was - 2.8 +/- 0.8 mV in control solution, and shifted to 0.9 +/- 1.6 mV in solutions in which NH4Cl replaced half the NaCl. In solutions containing NH4Cl substituted for all the NaCl, the null potential was 6.5 +/- 1.3 mV. Single channel conductance and average channel lifetime were both increased in solutions containing ammonium ions. The exponential relationship between the time constant of decay of miniature endplate currents or channel lifetime and membrane potential was unchanged in ammonium solutions. A slight but consistent increase in peak conductance during miniature endplate currents and single channel conductance was seen as membrane potential became more positive (depolarized) in both control and ammonium solutions. Net charge transfer was greater in ammonium solutions than in control solution, whether measured during a miniature endplate current or through a single channel. The results presented here are consistent with an endplate channel model containing high field strength, neutral sites.     

Cholinergic transmission from mechanosensory afferents to an identified nonspiking interneuron in the crayfish Procambarus clarkii girard

Pharmacological properties of excitatory synaptic transmission from mechanosensory afferents to an identifiable nonspiking interneuron of crayfish were studied by drug perfusion experiments using acetylcholine (ACh) agonists and antagonists. Application of carbachol, a general agonist of ACh, caused sustained depolarization of the interneuron and a decrease in the peak amplitude of its excitatory synaptic response to sensory stimulation on the soma side. Similar depolarization was observed during application of carbachol under the low-Ca²⁺, high-Mg²⁺ condition. The peak amplitude was also reduced by application of nicotine and tetramethylammonium, both of which also caused sustained depolarization of the inter-neuron. By contrast, perfusion of muscarinic agonists, muscarine, oxotremorine and pilocarpine, reduced the peak amplitude without affecting the membrane potential of the interneuron. Perfusion of nicotonic antagonists of ACh, d-tubocurarine and hexamethonium, caused reduction of the peak amplitude without any change in the membrane potential. A muscarinic antagonist atropine was also effective in blocking the synaptic transmission but at higher concentration than d-tubocurarine. The results suggest that the ACh receptors on the nonspiking interneuron belong to a previously characterized class of crustacean cholinergic receptors resembling the nicotinic subtype of vertebrates.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart

These experiments employ the photoisomerizable compound, 3,3'-bis- [alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans- Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.     

Properties of an endogenous steady current in rat muscle

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.     

Enzymatic Detachment of Endplate Acetylcholinesterase from Muscle

AT the vertebrate neuromuscular junction acetylcholinesterase catalyses the hydrolysis of the transmitter, acetylcholine, which is released from presynaptic nerve terminals^1,2. The enzyme is present in high concentration at the endplate, where it can be located by histochemical³ and autoradiographic⁴ methods. Electron microscopic studies of the endplate region show most of the histochemical reaction product to be in the synaptic cleft or associated with the nerve and muscle membranes^5–9. We report here that enzymatic treatment of intact muscle causes the detachment of active endplate acetylcholinesterase from the muscle into the bathing fluid.     

Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques

When solutions containing agonists are applied to the innervated face of an Electrophorus electroplaque, the membrane's conductance increases. The agonist-induced conductance is increased at more negative membrane potentials. The "instantaneous" current-voltage curve for agonist-induced currents is linear and shows a reversal potential near zero mV; chord conductances, calculated on the basis of this reversal potential, change epsilon-fold for every 62-mV change in potential when the conductance is small. Conductance depends non-linearly on small agonist concentrations; at all potentials, the dose-response curve has a Hill coefficient of 1.45 for decamethonium (Deca) and 1.90 for carbamylcholine (Carb). With agonist concentrations greater than 10(-4) M Carb or 10(-%) M Deca, the conductance rises to a peak 0.5-1.5 min after introduction of agonist, then declines with time; this effect resembles the "desensitization" reported for myoneural junctions. Elapid alpha-toxin, tubocurarine, and desensitization reduce the conductance without changing the effects of potential; the apparent dissociation constant for tubocurarine is 2 X 10(-7) M. By contrast, procaine effects a greater fractional inhibition of the conductance at high negative potentials.     

The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.     

Reaction of tetraethylammonium with the open and closed conformations of the acetylcholine receptor ionic channel complex

The effect of tetraethylammonium (TEA) bromide on the neurally and iontophoretically evoked endplate current (EPC) of frog sartorius muscle was investigated using voltage-clamp and noise analysis techniques, and its binding to the acetylcholine (ACh) receptor ionic channel complex was determined on the electric organ of Torpedo ocellata. TEA (250-500 microM) produced an initial enhancement followed by a slow decline in the amplitude of the endplate potential and EPC, but caused only depression in the amplitude of the miniature endplate potential and current. In normal ringer's solution, the EPC current-voltage relationship was approximately linear, and the decay phase varied exponentially with membrane potential. Upon addition of 50-100 microM TEA, the current-voltage relationship became markedly nonlinear at hyperpolarized command potentials, and with 250-2000 microM TEA, there was an initial linear segment, an intermediate nonlinear segment, and a region of negative conductance. The onset of nonlinearity was dose-dependent, undergoing a 50 mV shift for a 10-fold increase in TEA concentration. The EPC decay phase was shortened by TEA at hyperpolarized but not depolarized potentials, and remained a single expotential function of time at all concentrations and membrane potentials examined. These actions of TEA were found to be independent of the sequence of polarizations, the length of the conditioning pulse, and the level of the initial holding potential. TEA shifted the power spectrum of ACh noise to higher frequencies and produced a significant depression of single channel conductance. The shortening in the mean channel lifetime agreed closely with the decrease in the EPC decay time constant. At the concentrations tested, TEA did not alter the EPC reversal potential, nor the resting membrane potential, and had little effect on the action potential duration. TEA inhibited the binding of both [3H] ACh (Ki = 200 microM) and [3H]perhydrohistrionicotoxin (Ki = 280 microM) to receptor-rich membranes from the electric organ of Torpedo ocellata, and inhibited the carbamylcholine-activated 22Na+ efflux from these microsacs. It is suggested that TEA reacts with the nicotinic ACh-receptor as well as its ion channel; the voltage-dependent actions are associated with blockade of the ion channel. The results are compatible with a kinetic model in which TEA first binds to the closed conformation of the receptor-ionicchannel complex to produce a voltage-depdndent depression of endplate conductance and sudsequently to its open conformation, giving rise to the shortening in the EPC decay and mean channel lifetime.     

Current-dependent block of endplate channels by guanidine derivatives

Methyl- and ethylguanidine block the endplate current in frog muscle. Both derivatives blocked inward-going endplate currents without affecting outward endplate currents. Repetitive stimulation that evoked several inward endplate currents enhanced the block, which suggests that these agents interact with open endplate channels. The relative conductance vs. potential curve exhibited a transition from a low to a high value near the reversal potential for the endplate current, both in normal and in 50% Na solution. In the latter solution, the reversal potential for endplate current was shifted by a mean value of 16 mV in the direction of hyperpolarization. The results suggest that methyl- and ethylguanidine block open endplate channels in a manner dependent on the direction of current flow rather than on the membrane potential.     

Biphasic increase of apical Cl− conductance by muscarinic stimulation of ht-29cl.19a human colon carcinoma cell line: Evidence for activation of different cl− conductances by carbachol and forskolin

Summary The modulation of ion transport pathways in filtergrown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments.Basolateral addition of 10^–4 m carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15±1 mV from a resting value of –52±1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22±1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11±1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6±1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4±0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl^– conductance in the apical and basolateral membrane, the second phase by increased K⁺ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl^– conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl^– channels are involved in the carbachol response: one Ca²⁺ dependent and a second which may be PKC sensitive.In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl^– conductance.It is concluded that the short-lasting carbachol/Ca²⁺-dependent Cl^– conductance is different from the forskolin-activated conductance. The increase of the Cl^– conductance in the presence of forskolin by carbachol may be due to activation of different Cl^– channels or to modulation of the PKA-activated Cl^– channels by activated PKC.The authors are grateful to Drs. Laboisse and Augeron for providing the cell clone, and we thank Prof. Dr. F.H. Lopes da Silva for his comments. This work was supported by a grant from the Dutch Organization for Scientific Research, NWO.     

Time course of isolated rat fundus response to muscarinic agonists: a measure of intrinsic efficacy.

The establishment of a dose-response relationship and its quantification is the usual procedure for analysing drug action on an isolated organ. However, the time course of the effect seems to be an inherent characteristic of the agonist which produces it. In our study, we have analyzed the time-response curves of four cholinergic agonists (acetylcholine, methacholine, carbachol and bethanechol) which produce tonic contractions of the isolated rat gastric fundus. The order of affinity of agonists to muscarinic receptors on the rat fundus were carbachol > bethanechol > methacholine > acetylcholine (K(A) values: 46 +/- 12, 84 +/- 21, 380 +/- 110 and 730 +/- 120 nM, respectively). The effective concentrations which produced 60% of the maximal response (EC60) were used for establishing the time-response curves. The time-response curves were also recorded after partial alkylation of muscarinic receptors with phenoxybenzamine, after exposure of the isolated rat fundus to physostigmine and after addition of supramaximal concentrations of the agonists. The experimental time-response curve for acetylcholine was on the extreme left, followed by curves for methacholine, bethanechol and carbachol, respectively. Phenoxybenzamine and supramaximal doses of the agonists did not change the order of response development in time, but supramaximal doses shifted all curves to the left and phenoxybenzamine shifted all time-response curves to the right. Only physostigmine shifted the time-response curve for methacholine to the right. The results of our study suggest that the response rate of the isolated rat gastric fundus to cholinergic agonists depends on the intrinsic activity of these agents, but not on their affinity for muscarinic receptors.     

Ionic mechanisms of nonlinearity of the retinal horizontal cell membrane

Changes in ionic conductance lying at the basis of nonlinearity of the current-voltage characteristic curve of the cell (nonsynaptic) membrane of horizontal cells were studied in experiments on the goldfish and turtle retina. All measurements were made during blocking of synaptic transmission by bright light or Co⁺⁺. An increase in the K⁺ concentration led to depolarization and to a reduction of the steepness of the hyperpolarization branch of the current-voltage curve, whereas a decrease in K⁺ had the opposite effect. Changes in the Cl^– or Na⁺ concentrations had no significant effect on membrane potential or on the shape of the current-voltage curve. The principal potential-forming ion in the horizontal cells is thus K⁺; conductance for Cl^– is absent or very low, and conductance for Na⁺ also is evidently small. In the presence of Ba⁺⁺ (2–5 mM) the steepness of the hyperpolarization branch of the current-voltage curve was increased and the whole curve became more linear. It is concluded that nonlinearity of the current-voltage curve of the horizontal cell membrane is due mainly to potential-dependent potassium channels, whose conductance increases during hyperpolarization; this increase in conductance is blocked by Ba⁺⁺. An increase in the Ca⁺⁺ concentration to 20 mM led to an increase in steepness of the depolarization branch of the current-voltage curve, suggesting that depolarization increases membrane conductance for Ca⁺⁺.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 531–539, September–October, 1981.