Changes in extracellular potassium accumulation produced by opioids and naloxone in frog spinal cord: relation to changes of Na-K pump activity

Superfusion of the isolated spinal cord of the frog with Ringer solution containing ouabain or naloxone elevated resting [K]e and depolarized the dorsal roots, while the application of enkephalins or morphine decreased [K]e and hyperpolarized the dorsal roots. During repetitive electrical stimulation (10-100 Hz) ouabain and naloxone increased the transient changes of [K]e and enhanced dorsal root potentials. When stimulation stopped, the clearance of K+ was slowed, the poststimulation K+ undershoot disappeared and poststimulation hyperpolarization of dorsal roots was diminished. The opposite effects were found during the application of enkephalins or morphine. Our results imply that the activity of the membrane Na-K pump is reduced after application of naloxone, while opioids enhance it.     

Evidence that transmitter can be released from regions of the nerve cell other than presynaptic axon terminal: axonal release of acetylcholine without modulation

Release of acetylcholine from isolated preganglionic axons of sympathetic nerve trunk (cervical preganglionic sympathetic branch) of the cat was studied. In response to depolarization (KCl, 48.4 mM) acetylcholine was released into the eserinized Krebs solution. This release was shown to be dependent on extracellular Ca2+. Electrical stimulation (1 Hz) enhanced the release of acetylcholine from the isolated axonal preparation. The release by stimulation proved to be tetrodotoxin-sensitive and Ca2+-dependent. Evidence has been obtained that the acetylcholine released from sympathetic nerve trunks originates from the axon and not from Schwann cells: 5 days after section of the nerve, there was no release in response to stimulation. The release of acetylcholine from the axon is unlike that from axon terminals in that the rate of release cannot be enhanced by the inhibition of Na, K-adenosine 5'-triphosphatase (ouabain 2 X 10(-5) M) and cannot be modulated by noradrenaline (10(-6) M) or by morphine. Furthermore, although isolated nerve trunks took up [3H]choline by a hemicholinium-sensitive process, no radioactivity could be released upon electrical stimulation. It is suggested that the release of acetylcholine is not confined to axon terminals, but that it can be non-synaptically released by depolarization from axons provided Ca2+ is present.     

Potassium outward current dependent on extracellular calcium in snail neuronal membrane

In experiments on isolated intracellularly dialysed neurons of the snail Helix pomatia a component of delayed inactivating potassium outward current depending on the presence of Ca2+ ions in the extracellular medium has been distinguished, which differs from the already known potassium current sensitive to intracellular calcium ions. This component decreases with a decrease in extracellular calcium (in the range of 10(-2) - 10(-5) M); it is not affected or even increased by intracellular introduction of ethyleneglycolbis(aminoethylether)tetra-acetate (10 mM) or fluoride ions (77 mM) and can be blocked by addition of 1.5 mM cobalt ions to the extracellular solution. Contrary to the slow rising potassium current dependent on intracellular calcium, this current has a fast rising phase (several milliseconds) and time-dependent inactivation. The inactivation depends on extracellular potassium ions: it slowed down when [K+]out is increased in the range of 1-10 mM. Extracellular application of calmodulin blockers calmidazolium (6.5 X 10(-7) M) and chlorpromazine (2.5 X 10(-6) M) selectively inhibits the potassium current dependent on intracellular calcium but does not affect that dependent on external calcium. Tetraethylammonium (10 mM) depresses the latter current on both intra- and extracellular application, the former being more effective. The existence of a special type of potassium channel sensitive to extracellular calcium ions is postulated.     

Intracellular Ca antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca²⁺ than in its presence in perfused cat adrenal glands. The intracellular Ca²⁺ antagonist, TMB-8 (10⁻⁴ M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10⁻⁴ M) in the presence of hexamethonium (10⁻³ M) during perfusion with Ca²⁺-free Locke solution containing EGTA (10⁻⁵ M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca²⁺ from an intracellular pool just as caffeine does.     

Cholinergic pharmacology of mammalian hippocampal pyramidal cells

Responses of CAl pyramidal cells to cholinergic compounds were recorded with intracellular microelectrodes in guinea-pig hippocampal slices. Perfusion of slices with medium containing the muscarinic antagonists atropine or scopolamine (10(-7)-10(-6)M) blocked all actions of acetylcholine. Properties of control neurons and those from separate populations of neurons impaled in slices exposed to muscarinic blocking agents were compared. 1-2 h of perfusion with atropine-containing media significantly decreased membrane input resistance from 37.6 +/- 8.7 (S.D.) M omega (n = 74) to 21.9 +/- 7.7 (S.D.) M omega (n = 24) without producing significant changes in membrane potential. Muscarinic antagonists also reduced or eliminated the anomalous inward rectification normally seen in hippocampal pyramidal neurons. Exposure of slices to 10(-5)-10(-6)M eserine for about 1 h produced changes in neuronal membrane input resistance and potential and slow after hyperpolarizations similar to those elicited by application of acetylcholine. Bethanechol mimicked the actions of acetylcholine but was effective at lower concentrations and had longer lasting effects on afterhyperpolarizations. Nicotine produced an excitatory response in only one of 7 neurons. These experiments demonstrate that the actions of acetylcholine on hippocampal CAl neurons result from interaction with muscarinic receptors. Acetylcholine has modulatory effects on cell membrane properties which may be mediated through tonic release mechanisms.     

Differential involvement of enkephalins in analgesic tolerance, locomotor sensitization, and conditioned place preference induced by morphine

In this study, the authors investigated the role of enkephalins in morphine-induced conditioned place preference, locomotor sensitization, and analgesic tolerance. Both preproenkephalin wild type (ppENK [+/+]) and knockout (ppENK [-/-]) mice showed similar preference for the morphine-paired chamber over the vehicle-paired chamber, indicating morphine induced comparable conditioned place preference in ppENK (+/+) and ppENK (-/-) mice. Sensitization developed to the motor stimulatory action of morphine after its repeated administration, but the magnitude of this response was not altered in ppENK (-/-) mice. However, as shown previously, ppENK (-/-) mice displayed blunted morphine analgesic tolerance. Taken together, the results suggest that enkephalins may be important for the development of analgesic tolerance but not for conditioned place preference or behavioral sensitization induced by morphine.     

A study of the effects of indomethacin,flufenamate and salicylate on the localization of intracellular calcium in rabbit neutrophil polymorphs using an antimonate staining method.

An antimonate staining technique was used to localize intracellular calcium in rabbit peritoneal neutrophil polymorphs. When calcium was added to a previously calcium-free extracellular medium neutrophils showed a slight loss of intracellular stainable calcium after 1 h incubation. There was a significant increase in the number of damaged cells and a slight increase in degranulation. Indomethacin (5 X 10(-5)M and 8 X 10(-4)M), both in the absence and the presence of extracellular calcium, enhanced the loss of stainable calcium, and at 37 degrees, increased the amount of damage, but increased degranulation only at the higher concentration and in the presence of calcium. Indomethacin may act as a membrane labilizer. Salicylate (2.5 X 10(-3)M and 1 x 10(-2)M at 37 degrees) slightly decreased the loss of intracellular calcium in the presence of extracellular calcium and, in the absence and presence of calcium and at the higher concentration, decreased degranulation and decreased the amount of damage. Salicylate may act as a membrane stabilizer. Flufenamate (3 x 10(-5)M and 2.4 x 10(-4)M) showed variable effects.     

BK channel openers inhibit migration of human glioma cells

Large-conductance Ca(2+)-activated K(+) channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500 nM free Ca(2+) and external application of the BK channel opener phloretin (100 micro M) shifted the activation threshold of BK channel currents toward more negative voltages of about -30 mV, which is close to the resting potential of the cells. The concentration of intracellular Ca(2+) in fura-2-loaded 1321N1 cells was measured to be 235+/-19 nM and was increased to 472+/-25 nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100 micro M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100 micro M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5 micro M) and iberiotoxin (100 nM). The phloretin-induced increase in intracellular Ca(2+) was unaffected by the removal of extracellular Ca(2+) and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca(2+).     

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.     

The action of neurotransmitter hormones and analogues and cyclic nucleotides and theophylline on electrolyte transport by the excretory duct of the rabbit mandibular gland

The effects of autonomic drugs on electrolyte transport by the main duct of the rabbit mandibular gland were studied in vitro. Acetylcholine, in concentrations as low as 10(-10) M, reduced nett Na+ reabsorption and partially depolarized the transepithelial potential difference (P.D.); the effects were blocked by atropine. Acetylcholine was relatively ineffective applied from the luminal rather than the interstitial surface of the duct. Noradrenaline and isoproterenol produced similar effects to acetylcholine but the minimum effective concentrations were much higher (10(-7) M and 10(-3) M, respectively). It was concluded that the duct cells possessed specific muscarinic receptors on the basal cell membrane but that adrenergic receptors, particularly beta receptors, were either scanty or lacking altogether. Theophylline (10(-5) M) could mimic the effects of the autonomic drugs and produced a dramatic potentiation of the action of acetylcholine. Both cAMP and cGMP (4 times 10(-5) M) mimicked the acetylcholine response but cAMP was only effective when applied to the luminal cell membrane. It is proposed that cAMP is the intracellular mediator of the acetylcholine response and that it produces some of its effects by acting on an enzyme system close to the apical cell membrane.     

Receptors on smooth muscle cells: characterization by contraction and specific antagonists

Smooth muscle cells were isolated from the stomach of the guinea pig, and the kinetics, stoichiometry, and specificity of contraction in response to the C-terminal octapeptides of cholecystokinin (CCK-OP), gastrin-17, and acetylcholine were examined. All three agonists elicited dose-dependent peak contraction that did not depend on the presence of extra-cellular calcium. The potencies of CCK-OP and gastrin-17 were equal (D50, 10(-11) M) and 10 times greater than the potency of acetylcholine (D50, 10(-10) M). A combination of low doses of acetylcholine and CCK-OP was synergistic; however, its effect did not exceed the maximal responses to either agonists alone or to high extracellular concentrations of calcium. The specificity of the receptors was established by the use of atropine and the two CCK-receptor antagonists dibutyryl cGMP and proglumide. The span of the dose-response curves was wide, suggesting the existence of receptor heterogeneity. It is concluded that gastric smooth muscle cells of the guinea pig possess distinct, high-affinity receptors for CCK-gastrin and acetylcholine; the receptors mediate contraction that is not immediately dependent on the presence of extracellular calcium.     

Substances which aggregate neutrophils. Mechanism of action.

Several agents which influence calcium fluxes in neutrophils were tested for their influence on human neutrophil aggregation. Formyl-methionyl-leucyl-phenylalanine, a synthetic chemotactic tripeptide, aggregated the cells. Cytochalasin B and high levels of extracellular calcium or phosphate enhanced this effect; 10(-6) M to 10(-5) M lanthanum inhibited it. In addition, the calcium ionophore A23187 aggregated the cells. Aggregation induced by the chemotactic factor and A23187 required extracellular calcium. These results correlate with the known or postulated ability of chemotactic factors, A23187, calcium, phosphate, lanthanum, and cytochalasin B to enhance or inhibit the influx and intracellular accumulation of the calcium ion. Transmembrane fluxes or intracellular levels of calcium may modulate PMN aggregation. Aggregation induced by the chemotactic tripeptide and A23187 also required extracellular magnesium. Since calcium and magnesium cannot substitute for each other in the aggregation response to the chemotactic factor or A23187, each bivalent cation must play a separate role in PMN aggregation. The role of magnesium is unknown. Since magnesium, unlike calcium, is known to be necessary for PMN adherence to glass, it may play a permissive role in PMN aggregation. Thus, magnesium may foster the formation of cell-cell adhesions. In addition to inhibiting chemotactic factor-induced aggregation at concentrations of 10(-6) M to 10(-5) M, lanthanum, at concentrations of 10(-4) M to 10(-3) M, aggregated the cells. Lanthanum-induced aggregation did not require extracellular calcium or magnesium. This aggregation may result from the formation of intercellular adhesions by the lanthanum ion directly.     

Studies on cyclic AMP-cyclic GMP cooperation and on the influence of acetylcholine, adrenaline and thiols in lymphocyte reactivity.

In rat's lymph node lymphocytes, maximal stimulation of the response after suboptimal doses of PHA (1/2 of the optimal dose), measured by the incorporation of 3H-thymidine, after 10(-6) M acetylcholine and after 10 min interval between PHA and acetylcholine application was obtained. In these experiments inhibition of acetylcholine action by its muscarainic antagonists was not observed. It was shown that exogeneous cAMP as well as other factors, which increase cAMP level in lymphocytes had opposite effects to those, which could be caused by increasing of cGMP. Acetylcholine in optimal concentration (10(-6) M) as well as thiols, did not increase the level of endogeneous cAMP in rat lymphocytes.     

Changes in cytosolic calcium during cholinergic and adrenergic stimulation of the parotid salivary gland

Ca-selective microelectrodes were used to examine calcium transport during acetylcholine (ACh) and Epinephrine (Ep) stimulation of amylase secretion in the parotid gland. The cytosolic concentration of free ionized Ca2+ ( [Ca]i) determined in unstimulated cells was 0.44 +/- 0.04 microM. By measuring the induced changes in intracellular electrode potentials (ECa, EM) we were able to demonstrate that ACh at 10(-9), 10(-8), 10(-7), 10(-6), and 10(-5) M increased [Ca]i by 0.20 +/- 0.02, 0.61 +/- 0.04, 0.53 +/- 0.02, 0.30 +/- 0.05, and 0.14 +/- 0.03 microM. Similarly, Ep increased [Ca]i by 0.14 +/- 0.01, 0.42 +/- 0.06, 0.31 +/- 0.04, 0.15 +/- 0.03, and 0.05 +/- 0.04, respectively. Removal of extracellular Ca2+ significantly (P less than 0.001) altered the changes in ECa in response to ACh and Ep stimulation, thereby demonstrating that the induced increases in [Ca]i must be due to a transmembrane movement of Ca2+. Enzyme secretion was found to vary with the concentration of the stimulus used. Maximal secretion occurred during stimulation using 10(-7) M and 10(-8) M Ep with a suppression of release at supramaximal concentrations. The dose-response curve for ACh differed in that there were two concentrations of stimulus (2 X 10(-9) and 1 X 10(-6) M ACh) in which the greatest rate of secretion occurred. Concentrations of stimulus which increase [Ca]i between 0.86 +/- 0.06 microM and 0.74 +/- 0.05 appeared to produce optimal amylase secretion, indicating that salivary secretion in the mouse parotid is regulated within a narrow concentration range of cytosolic Ca2+.     

Characterization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fibre.

1. The effects of alpha-bungarotoxin, nicotine and muscarine on the Schwann cell membrane potential have been studied in the giant nerve fibre of the squid. The external application of alpha-bungarotoxin (10(-6), 10(-8), 10(-9) M) irreversibly blocks the long-lasting Schwann cell hyperpolarizations following the conduction of nerve impulse trains by the axon. It also blocks the Schwann cell hyperpolarizing response to the external application of carbamylcholine (10(-6)M) to the resting nerve fibre. 2. Externally applied D-tubocurarine (10(-5)M) protects against the irreversible action of alpha-bungarotoxin (10(-9)M) on the Schwann cell. Within 10 min of reimmersion in toxin-free sea water there is complete recovery of the Schwann cell hyperpolarizing response to carbamylcholine (10(-6)M) which had been initially abolished. 3. Nicotine (10(-6)M) induces a prolonged hyperpolarization of the Schwann cells in the resting nerve fibre, wheras at the same concentration, muscarine has no appreciable effect on the Schwann cell membrane potential. 4. None of these drugs, at the concnetrations utilized in the present study, had any appreciable effect on the resting and action potentials of the axon. 5. These findings show the presence of acetylcholine receptors of the nicotinic type in the Schwann cell membrane, and give further support to the hypothesis on the role of the acetylcholine system in the genesis of the long-lasting Schwann cell hyperpolarizations caused by the conduction of nerve impulse trains by the axon.     

In vivo evidence for acetylcholine control of serotonin release in the cat caudate nucleus: influence of halothane anaesthesia

Using a push-pull cannula technique and an isotopic method for the estimation of [3H]serotonin continuously synthesized from [3H]tryptophan, the effects of acetylcholine were investigated on the in vivo release of [3H]serotonin in the cat basal ganglia and the dorsal raphe nucleus. The unilateral striatal application of acetylcholine (5 x 10(-5) M) reduced local release of [3H]serotonin. This effect was mimicked by nicotine (5 x 10(-5) M) and prevented by mecamylamine (10(-6) M. Oxotremorine (5 x 10(-5) M) had no effect on the local release of [3H]serotonin. All these treatments failed to modify [3H]serotonin release in the ipsilateral substantia nigra or in the dorsal raphe nucleus. The superfusion of serotonergic nerve terminals of the caudate nucleus with tetrodotoxin prevented the inhibitory acetylcholine-induced effect on serotonin release. Furthermore, bicuculline (5 x 10(-5) M) in the caudate nucleus blocked the effect of nicotine, while gamma-aminobutyric acid (10(-5) M) induced a decrease in local release of [3H]serotonin. These data strongly suggest that the inhibitory control exerted by acetylcholine on serotonergic transmission could involve gamma-aminobutyric acid interneurons. Acetylcholine-induced changes in [3H]serotonin release were only observed in non-anaesthetized "encéphale isolé" cats and not in halothane-anaesthetized animals. The possibility that such a regulation could be presynaptic (direct or through other neurotransmitters) or related to a change in the activity of the serotonergic raphe-striatal neuronal system is discussed.     

Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release

1. The effects of acetylcholine upon the output of amylase, Ca(2+) efflux and membrane potential of pancreatic acinar cells have been measured in segments of mouse pancreas superfused in vitro.2. Amylase output was measured continuously using an on-line automated fluorimetric method; Ca(2+) efflux was monitored by measuring the release of (45)Ca(2+) from pre-labelled tissue; and intracellular recordings of acinar transmembrane potentials were obtained with glass micro-electrodes. In some experiments membrane potentials, and in others (45)Ca(2+) efflux, were measured concomitantly with amylase release.3. Acetylcholine depolarized the acinar cells, increased tissue (45)Ca(2+) efflux and raised amylase output, each with a similar dose-dependence, i.e. a maximal response at 10(-5)M, threshold =/< 10(-8)M, and ED(50) values of 0.7 x 10(-7)M, 0.5 x 10(-7)M, and 2 x 10(-7)M for depolarization, amylase release, and (45)Ca(2+) efflux, respectively.4. In response to acetylcholine both depolarization and (45)Ca(2+) efflux preceded or coincided with the increase in amylase output.5. Acetylcholine 10(-5)M and [K](0) 47 mM were without effect on (45)Ca(2+) efflux in the presence of atropine (3 x 10(-6)M) but pancreozymin (0.3 u./ml.) still elicited a marked increase in (45)Ca(2+) release.6. These results suggest that the stimulatory action of acetylcholine on the pancreatic acinar cell involves, sequentially, a specific receptor-activated increase in membrane permeability, depolarization, Ca(2+) mobilization and amylase release. These events are discussed in relation to the integrated mechanism of stimulus-secretion coupling.     

Calcium fluxes and contractility in isolated guinea pig atrium: effect of A23187.

The Ca2+ ionophore A23187 (10(-6) to 3 X 10(-5) M) increased the force of contraction is isolated guinea pig atria. In individual twitches, peak tension, maximum rate of tension development, time to peak tension, and total twitch duration were all increased by A23187. Tripelennamine, indomethacin, and atropine did not significantly alter the inotropic effect of A23187. Serotonin produced changes in individual twitches that differed qualitatively and quantitatively from those of A23187. Therefore, the inotropic action of A23187 is probably not mediated by release of endogenous histamine, prostaglandins, acetylcholine, or serotonin. 45Ca influx and efflux were increased by A23187. The enhanced 45Ca efflux exceeded that which would be predicted if the ionophore acted only to increase the passive Ca2+ permeability of the myocardial cell membrane. These results suggest that A23187 facilitates the entry of extracellular Ca2+ into the myocardial cell and the release of intracellular Ca2+ stores into the myoplasm. The resultant increase in intracellular Ca2+ activity could account for the positive inotropic action of A23187.     

Excitatory effect of serotonin on pacemaker neurons in spinal cord cell culture

Intracellular recordings were made from fetal mouse spinal cord neurons in primary culture. One type of neuron, with large somata (40-50 microns diameter) and thick neurites exhibited endogenous bursting or beating pacemaker electrical activity. Noradrenaline depolarized this type of neuron by decreasing an M-like conductance. Micropressure application of serotonin (10(-5) M in the delivery pipette) onto the surface of pacemaker neurons evoked a depolarization of the membrane potential in a dose-dependent manner with an increased input resistance. No such response was observed with other types of spinal cord neurons in culture. The response to serotonin was partially voltage-dependent. The serotonin-induced depolarization reversed at holding potential close to -100 mV. However, the input resistance variation evoked by serotonin increased exponentially when membrane potential was depolarized. The reversal potential was modified by increasing extracellular K+ concentration and it was unaltered by increasing the intracellular Cl- concentration. The decrease in K+ conductance induced by serotonin was not suppressed by the application of tetraethylammonium (50 mM) or 4-aminopyridine (10 mM). Furthermore, application of Ba2+ (6 mM) or Cd2+ (0.1 mM) had no effect on this response, suggesting that the depolarization evoked by serotonin application was not calcium-dependent. The serotonin evoked increase in input resistance was mediated by activation of a 5-HT1A-like receptor site. Spiperone, a 5-HT1A antagonist reversibly blocked the response. Methiothepin, a 5-HT1-5-HT2 antagonist (10(-3) M); cocaine, a 5-HT3 antagonist (10(-3) M); ketanserin, a 5-HT2 antagonist (10(-3) M); and prazosin, an alpha 1 antagonist (10(-3) M) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calcium on nicotine-induced current expressed by an atypical α-bungarotoxin-insensitive nAChR2

Two distinct native alpha-bungarotoxin (α-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 μM α-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2.