ACh-evoked Currents Research Articles

Acetylcholine contractures of skeletal muscles: Inhibition by chlorpromazine and diltiazem

The maximum contractures evoked by 100μM ACh of mouse soleus muscles denervated for 3–7 days are completely inhibited by a 10 min exposure to 100 μM chlorpromazine (CP). Recovery on washout of CP takes more than 1 hr to complete. ACh evoked contractures are also inhibited by diltiazem (D); washout of D is immediately followed by recovery. Electrically evoked twitches and K evoked contractures are largely unaffected by CP, caifeine evoked contractures are decreased but not abolished. Fast mouse and (non-denervated) frog tonic muscles behave similarly. Depolarization by ACh and ACh-evoked whole cell currents show enhanced desensitization at low, and block at high [CP] and [D]; more than 50% recovery is achieved by less than l min washout of CP and D. Currents carried by Na + and Mg 2+ ions behave similarly. It is concluded that activation of ACh evoked contractures may be blocked by CP independent of ionic currents through nicotinic ACh receptors and that it depends on intracellular processes linked to these receptors.

Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia.

Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh-evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5-6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.

Inhibition by cyclothiazide of neuronal nicotinic responses in bovine chromaffin cells

1. The desensitizing acetylcholine (ACh) response of bovine chromaffin cells maintained in culture was examined using rapid agonist applications (of 2 s duration) which imposed nominal drug concentrations within 50 ms. This study was aimed, firstly, at identifying which of the alpha 3, alpha 4 and alpha 7 subunits known to be present in these cells is predominant in the ACh-evoked response and secondly, on the effects on these neuronal nicotinic ACh receptors (AChR) of cyclothiazide (CT), an agent acting as a modulator of a gating desensitization site on other ligand-gated channels. 2. Locally applied 100 microM ACh evoked peak currents (IACh) of -1.5 +/- 0.1 nA (n = 83) at a holding potential of -60 mV. The ACh dose-response curve yielded an estimated EC50 of 60 microM. This current was not sustained but desensitized during the application period; it displayed strong inward rectification, but desensitized equally whether the evoked current was inward or outward going. The latter observation excludes alpha 4 as a major contributor to the recorded current. Because the response was almost insensitive to a 1 microM alpha-bungarotoxin pretreatment (IACh = -1.2 +/- 0.1 nA; n = 6), and because 1, 1-dimethyl-4-phenylpiperazinium (DMPP) works as a potent agonist (peak current = -1.9 nA, n = 2 for 100 microM DMPP), the alpha 7 subunit is also a minor contributor to the response. Taken together, these observations suggest a dominant alpha 3 type of response. 3. Triple exponential fits were used to describe the characteristics of the ACh-evoked currents; one component to fit the rising phase, with 2 components to describe the decay phase. The decay times were 100 ms and 4 s for the fast and slow components respectively. The rate of the slow decay component increased systematically with recording time, approximately doubling from its initial value within 20-40 min. Furthermore there was a gradual rundown of the response, seen first as a loss of the late component of the current, measured at 2 s, with the peak current amplitude decreasing later in the recording.4. CT, when coapplied with ACh, produced a dose-dependent inhibition of the ACh-evoked peak current. The effect showed little voltage-dependency with 100 microM CT producing 46 +/- 5% (s.d.; n = 3)and 47 +/- 8% (s.d.; n = 7) inhibition at -100 and -60 mV respectively. At + 60 mV, inhibition was estimated to be 26 +/- 7% (s.d.; n = 3).5. After pre-exposure of the cells to CT by bath application, 10 and 30 microM CT produced poorly reversible 20 +/- 9% (n = 7) and 42 +/- 5% (n = 4) inhibitions of the peak current respectively. There were no discernible effects on the fitted decay constants at any CT concentration tested, although an increased inhibitory effect of CT was observed at higher concentrations (100 microM) on the amplitude of the late component measured at 2 s.6. Similar effects were observed in conditions chosen to isolate the alpha 3 type of receptor: namely when using DMPP as an agonist, or after a-bungarotoxin pretreatment.7. The 2,3-benzodiazepine, GYKI 53655, is known to antagonize the action of CT on AMPA receptors.Coapplication of 50 microM GYKI 53655 with ACh (100 microM) produced a 29 +/- 4% inhibition of the peak ACh-evoked current and 44 +/- 6% inhibition of its amplitude at 2 s (n = 4). This response was fully reversible. Brief applications of both CT (100 microM) and GYKI 53655 (50 microM) with ACh via the microperfusion system produced a fully reversible inhibition that was not significantly different from the values obtained with either CT or GYKI 53655 alone, with 37 +/- 6% inhibition of peak and 61 +/- 9%inhibition of the amplitude at 2 s (n = 3).8. The results obtained suggest that the CT effect is to impede recovery from a slow desensitization,with a more pronounced effect with longer CT applications. Globally, CT favours the 'rundown state' of the neuronal nicotinic AChR.

Hg2+ increases the open probability of carbachol-activated Cl- channels in Aplysia neurons.

Carbachol and acetylcholine (ACh)-activated whole-cell chloride currents were recorded in identified neurons of Aplysia californica. Application of 0.1-10 microM HgCl2 potentiated the chloride response and reduced the rate of desensitization. To investigate the underlying mechanism we recorded single chloride channels in a cell-attached patch configuration. Recordings were performed successively on the same neuron with 1 microM carbachol and with 1 microM carbachol + 1 microM HgCl2, respectively. The slope conductance did not change significantly, but there was a manyfold increase in the open probability, Po. This may underlie the Hg(2+)-induced enhancement of carbachol- and ACh-evoked chloride currents in Aplysia neurons and may serve as a model for the neurotoxic effect of Hg2+ on the mammalian ACh receptor.

Nicotinic and muscarinic activation of motoneurons in the crayfish locomotor network.

1. We investigated the effects of acetylcholine (Ach) on identified motoneurons (MNs) using an in vitro preparation of the crayfish thoracic nervous system. Discontinuous current-clamp and single electrode voltage-clamp recordings from 50 MNs were performed along with micropipette pressure ejection of Ach (or agonists) close to the recording electrode. 2. Localized ejections of relatively large volumes (500-2,500 pl) of Ach (10(-2) M) or of the muscarinic agonist oxotremorine (Oxo, 10(-2)M) onto the MN neuropile region, usually (90% of the cases) induced a slow, alternating rhythmic activity in antagonistic MNs. In other cases (4 experiments), with similar deliveries of Ach or Oxo, MNs developed the ability to fire rhythmically but only when depolarized by sustained current injection. Pressure ejections of smaller volumes (50-200 pl) of Ach (10(-2)M) close to the recorded MN could give rise to a fast (1-2 s) large amplitude (< or = 20 mV) membrane depolarization (12%), a long-lasting (10 s to several minutes) and small (2-5 mV) depolarization (14%), and a combination of the two (74%). These responses appeared to involve different regions of the neurite because they changed when the drug-ejection pipette was displaced in the neuropile. Moreover, fast and long-lasting depolarizing components resulted from a direct effect of Ach onto the MNs because they persisted under tetrodotoxin (TTX, 10(-6)M) and cobalt (Co2+, 5 x 10(-3) M) superfusion. 3. Whereas the membrane resistance decreased during the fast Ach-induced depolarization, it increased during the long-lasting depolarization. The increase in membrane resistance was more pronounced at depolarized potentials more than -55 mV and involve a reduction in K+ conductance. 4. Superfusion with nicotinic and muscarinic antagonists revealed that the fast Ach-induced depolarization involved nicotinic receptors, muscarinic receptors, or both, whereas the slow depolarization was exclusively muscarinic. 5. The Ach-evoked inward currents were studied under voltage clamp. The fast nicotinic component (Inic) increased with hyperpolarizing holding potentials and decreased with depolarizing potentials, reversing at between 10 and 30 mV. The fast muscarinic current (Ifmus) displayed similar characteristics and reversed at about -10 mV. Whereas both fast components were voltage independent, the long-lasting muscarinic component (Ismus) was voltage dependent. The response grew with membrane depolarization, but when the holding potential was hyperpolarized below resting level, the response declined to disappear at about -60 mV and beyond.(ABSTRACT TRUNCATED AT 400 WORDS)

Presence of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat olfactory bulb neurons.

The presence of a functional nicotinic acetylcholine receptor (nAChR) in rat olfactory bulb (OB) neurons was investigated using the whole-cell patch-clamp technique. Acetylcholine (ACh) or other nicotinic agonists elicited fast-decaying whole-cell currents in cultured OB neurons. ACh-evoked currents could be blocked by the highly selective competitive nicotinic antagonists alpha-bungarotoxin (alpha-BGT, 10 nM) and methyllycaconitine (MLA, 1 nM) and exhibited strong inward rectification in the presence of intracellular Mg2+. The properties of the nicotinic currents in OB neurons were similar to those of type IA currents elicited by nicotinic agonists in rat hippocampal neurons. The present results suggest that the fast-decaying nicotinic currents evoked in OB neurons are subserved by nAChRs containing alpha 7 subunits.

Ionic Currents Evoked by Acetylcholine in Isolated Acinar Cells of the Guinea Pig Nasal Gland

The patch-clamp whole cell recording was used to demonstrate activation of membrane conductance to K +, Cl − and cations induced by acetylcholine (ACh) in the isolated acinar cells of the guinea pig nasal gland. A small outward K + current at 0 mV and a large transient and sustained inward current at −90 mV were evoked by ACh and ACh-evoked reversal potential was about −3 mV nearly to Cl − equilibrium potential in 140 mM KCl in the pipette and physiological saline in the bath. The ionic substitutional experiments indicated that ACh-evoked inward currents were carried by both Cl − and cations. Both outward and inward currents evoked by ACh were almost completely abolished by removal of external Ca 2+ and mimicked those evoked by a calcium ionophore A23187. These findings indicate that ACh-evoked membrane conductances are mediated by an increase in intracellular Ca 2+.

Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells.

1 The effects of the local anaesthetics QX-222 and procaine on nicotinic acetylcholine (ACh)-evoked currents in cultured parasympathetic cardiac neurones of the rat were investigated by use of the whole-cell, perforated-patch, and outside-out recording configurations of the patch clamp method. 2 QX-222 and procaine, applied to the extracellular surface, reversibly inhibited the peak amplitude of the whole-cell nicotinic ACh-evoked current in a concentration-dependent manner, with half-maximal inhibitory concentrations (IC50) of 28 microM and 2.8 microM, respectively, at -80 mV. In these neurones, the sustained inward current mediated by M1 muscarinic receptor activation was unaltered by QX-222, and neither local anaesthetic affected the adenosine 5'-triphosphate (ATP)-evoked current. 3 QX-222 and procaine block of nicotinic ACh-evoked inward current was voltage-dependent and enhanced by hyperpolarization. An e-fold change in their dissociation equilibrium constants (Kd) resulted from a 62 mV and a 122 mV change in membrane potential, respectively. 4 Both local anaesthetics produce a concentration-dependent increase in the half-time of decay of the nicotinic ACh-evoked inward current. 5 Measurements of unitary currents in outside-out patches showed that QX-222 reversibly increased the mean burst duration and closed time and reduced the mean channel open time and open-state probability of the nicotinic ACh receptor-channel (AChR) in a concentration-dependent manner. 6 The Kd and voltage sensitivity of local anaesthetic block of the nicotinic AChR in rat intracardiac neurones suggests that the pore-forming region of this channel differs from that of the AChR in frog and rat skeletal muscle and from the neuronal alpha 4 beta 2 ACh receptor-channel.

Emptying and refilling of Ca2+ store in tracheal myocytes as indicated by ACh-evoked currents and contraction

Membrane currents and contractions evoked by acetylcholine (ACh) in freshly dissociated canine tracheal myocytes were investigated using the nystatin perforated-patch recording technique. In cells held at -60 mV in the presence of nifedipine, ACh evoked inward current (IACh) and contraction. Caffeine mimicked the effects of ACh. IACh and contractions could be evoked 3-4 min after removing external Ca2+ but were abolished by prolonged exposure to Ca(2+)-free media. Both responses were restored within minutes of reintroduction of Ca2+, even though the cells were held at -60 mV in the presence of nifedipine. IACh and ACh-evoked contractions were also reversibly abolished by continued exposure to caffeine. Cyclopiazonic acid (CPA), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced IACh by > 95% within 15 min but had little or no effect on the contractile responses evoked by ACh. IACh was restored after washout of CPA even though cells were held at -60 mV. After depleting the Ca2+ store with the use of CPA, depolarization of the membrane to +10 mV immediately before application of ACh led to a partial restoration of IACh. This restorative effect of depolarization was potentiated by Bay K 8644 and antagonized by nifedipine. In conclusion, IACh and contractions in canine tracheal myocytes are mediated by Ca2+ released from an internal store that can be depleted by prolonged removal of extracellular Ca2+, prolonged exposure to caffeine, or by blockade of the SR Ca(2+)-ATPase. At least two Ca2+ influx pathways appear to contribute to refilling of the internal store: one pathway that is not activated by depolarization or ACh and a second involving dihydropyridine-sensitive voltage-activated Ca2+ channels that may be in direct contact with the SR (i.e., conduct extracellular Ca2+ directly into the SR, bypassing the cytosol).

P-324 - Pharmacological profile of ACh-evoked cationic currents in guinea-pig ileal smooth muscle.

P-324 - Pharmacological profile of ACh-evoked cationic currents in guinea-pig ileal smooth muscle.

A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

1. Acetylcholine-induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the 'whole-cell' and 'outside-out' configurations of the patch clamp technique. In standard salines, ACh evoked whole-cell currents of -38 pA to -1 nA at -60 mV, which had a reversal potential (EACh) of -7.1 +/- 0.6 mV. The ACh current-voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a -42 mV shift of EACh for a 10-fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/- 0.02 for Cs+ (n = 11), 1.03 +/- 0.02 for Li+ (n = 8) and 0.18 +/- 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)-tubocurarine and hexamethonium caused dose-dependent reductions in the amplitude of ACh-evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at -60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose-dependent reduction in the amplitude of ACh-induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh-evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)-tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current-voltage relationship resembles that described for other types of 'neuronal' nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroids inhibit nicotinic acetylcholine receptors

Application of progesterone to Xenopus oocytes expressing a cloned neuronal nicotinic acetylcholine (nAChR) revealed two effects. The first effect was a fully reversible reduction of the current induced by acetylcholine (ACh), its onset being nearly instantaneous. The second effect, which developed in a few hours, was an irreversible suppression of ACh-evoked currents. The transient inhibition had an apparent Ki of 7 microM when tested with 50 nM ACh, but the percentage of inhibition was positively correlated to the ACh concentration. A reduction of ACh-induced currents which appeared immediately upon progesterone application was also observed with muscle nAChR expressed in oocytes and with nAChR on membrane patches isolated from ciliary ganglion neurons. Thus nAChRs are modulated by progesterone and steroids may play an important role in nicotinic cholinoception.

Acetylcholine‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation. 2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 approximately 1 microM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 microM). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction. 4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (PX/PNa) followed the ionic selectivity sequence Cs+ (1.06) greater than Na+ (1.0) greater than Ca2+ (0.93). Anion substitution experiments showed a relative anion permeability, PCl/PNa less than or equal to 0.05. 5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.

Bicuculline blocks nicotinic acetylcholine response in isolated intermediate lobe cells of the pig

1. The effect of bicuculline on nicotinic acetylcholine (ACh) responses in isolated intermediate lobe (IL) cells of the pig was investigated by use of patch-clamp techniques. Bicuculline was found to reduce ACh-evoked whole-cell currents (IACh) in all cells tested (n = 40). 2. The blocking effect of bicuculline on IACh was dose-dependent, the concentration producing half-maximal blockade being 43.8 microM. 3. The blockade of IACh by bicuculline was not voltage-dependent at membrane potentials above -60 mV, but a slight voltage-dependence was observed at holding potentials (HP) of -80 and -100 mV. 4. The inhibitory effect of bicuculline on IACh was partially competitive at a HP of -60 mV. 5. Neither SR 95531, a pyridazinyl gamma-aminobutyric acid derivative, nor t-butylbicyclophosphorothionate (TBPS) blocked IACh in IL cells. 6. It is concluded that bicuculline interacts directly with the ACh receptor-ionophore complex on porcine IL cells.

Nicotinic acetylcholine receptors in porcine hypophyseal intermediate lobe cells.

1. Acetylcholine (ACh) was found to depolarize isolated porcine intermediate lobe cells maintained in primary cells culture. We investigated the ACh-induced responses in both whole-cell and cell-attached configurations of the patch-clamp technique. 2. From noise analysis of ACh-evoked whole-cell currents, we estimated an elementary conductance of 20 pS and a channel open duration of about 1.7 ms at -60 mV. From single-channel recordings, we obtained a slope conductance of 26 pS and a mean open time of 1.8 ms at membrane potentials between -60 and -80 mV. 3. ACh-evoked responses were blocked by d-tubocurarine (d-TC), hexamethonium and mecamylamine, but were insensitive to alpha-bungarotoxin. These characteristics define a neuronal type of nicotinic receptors. 4. The whole-cell current induced by ACh showed a strong inward rectification with no outward current being obtained. This phenomenon was observed when the intracellular ion is either sodium or caesium, and even when Ca2+ and Mg2+ were totally removed from the intracellular medium. 5. ACh-gated channels in intermediate lobe cells were cation selective and were permeable to Na+ and Cs+. In Ca2(+)-free extracellular solution, single-channel conductances were much larger (46 pS) than in the presence of 2 mM-Ca2+ (26 pS). 6. The possibility of an excitatory cholinergic control of intermediate lobe cells is discussed.

Inhibitors of asparagine-linked oligosaccharide processing alter the kinetics of the nicotinic acetylcholine receptor.

We used selective inhibitors of the asparagine-linked oligosaccharide processing pathway to study the effect of sugar trimming on the functional properties of the nicotinic acetylcholine (ACh) receptor expressed in clonal mammalian BC3H-1 cells. Inhibitors of initial steps of the processing pathway (1-deoxynojirimycin[DNJ] and castanospermine[CS]) reduced the density of ACh receptors on the cell surface (3- to 5-fold) but their responsiveness to ACh was more reduced (5- to 10-fold). These results suggest that the function of the ACh receptor was altered. When the ACh receptors were expressed in the presence of DNJ or CS, analysis of ACh-evoked single-channel currents (-100 mV and 11 degrees C) revealed an approximate threefold reduction in the opening rate (control: 600-650 s(-1)), treated: 130-250 s(-1)) and an approximate twofold reduction in the rate of agonist dissociation (control: 900-1,000 s(-1), treated: 400-500 s(-1)). In addition, the proportion of brief duration bursts (tau = 50-100 microseconds) was increased (1.5- to 2-fold) by treatments with DNJ or CS. In contrast, an inhibitor of a late processing step (swainsonine) did not produce such alterations. The single-channel conductance was not altered by any of the three inhibitors, and the slopes of log-log dose-response curves at low concentrations and desensitization did not appear to be affected. Each inhibitor altered the electrophoretic mobility of the ACh receptor subunits. We conclude that early sugar trimming can influence the kinetics of the nicotinic ACh receptor in BC3H-1 cells.

Membrane currents of rat satellite cells attached to intact skeletal muscle fibers

Muscle satellite cells play an important role in the postnatal growth of skeletal muscle and in the regeneration of damaged muscle during adult life. Little is known about the physiological properties of satellite cells in their dormant state as they lie adjacent to the intact muscle fibers, underneath the basement membrane. Our recent experiments, using patch clamp techniques, indicate that no tight electrical coupling is present between satellite cells and the muscle fiber dissociated from rat flexor digitorum brevis. Satellite cells possess sodium channels with low sensitivity to tetrodotoxin and at a much lower density than muscle. In addition, satellite cells are insensitive to acetylcholine (ACh) for at least 24 hr after having been removed from the animal, even when detached from their muscle fiber. However, we could measure ACh-evoked currents from satellite cells 48–72 hr in culture, indicating that ACh sensitivity develops with time.

Further characterization of the slow muscarinic responses inXenopus oocytes

In immature follicular oocytes of the frog Xenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29-0.39 microM), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32-56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 microM 4-phorbol 12,13-dibutyrate (PDBu).(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of acetylcholine- and cyclic GMP-induced currents inXenopu oocytes

In Xenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3',5'-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current ("threshold dose") varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 microM) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hypothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.

Modulation of GABAA receptor activity by alphaxalone.

The modulation of the gamma-aminobutyric acidA (GABAA) receptor by alphaxalone has been investigated by use of voltage-clamp recordings from enzymatically isolated bovine chromaffin cells maintained in cell culture. Alphaxalone (greater than 30 nM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by locally applied GABA (100 microM). The potentiation was not associated with a change in the reversal potential of GABA-evoked currents and was not influenced by the benzodiazepine receptor antagonist, Ro15-1788 (300 nM). At relatively high concentrations (greater than 1 microM), alphaxalone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor activation since they were reversibly suppressed by bicuculline (3 microM), dose-dependently enhanced by phenobarbitone (100-500 microM), and had a similar reversal potential (approximately 0 mV) to currents elicited by GABA. Additionally, on outside-out membrane patches, alphaxalone activated single channel currents with amplitudes and a reversal potential similar to those evoked by GABA. Alphaxalone (30 nM-1 microM) had no effect upon the amplitude of membrane currents elicited by locally applied acetylcholine (ACh) (100 microM). However, higher concentrations of alphaxalone (10-100 microM) reversibly suppressed ACh-evoked currents, the IC50 for blockade being 20 microM. The beta-hydroxy isomer of alphaxalone, betaxalone (100 nM-1 microM), did not potentiate GABA-induced currents, nor did higher concentrations of the steroid (10-100 microM) directly evoke a membrane current. However, over the latter concentration range, betaxalone suppressed the amplitude of currents elicited either by GABA or ACh. The relevance of the present results to the anaesthetic action of alphaxalone is discussed together with the broader implications of steroidal modulation of the GABAA receptor.