Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Presynaptic muscarinic modulation of nicotinic excitation in the rat neostriatum

In rat neostriatal slices, cholinergic agents were tested for their effects on endogenous ACh release and on electrical activity. ACh release was evoked by 25 mM K⁺ during two 5-min periods between which a slice was allowed to rest for 20 min; drugs were present during the second stimulation period. In the absence of a cholinesterase inhibitor, only Ch outflow was monitored. For the recording of electrical activity, intrastriatal stimulation evoked field potentials which were monitored in the absence and presence of drugs in the perfusate.Atropine (1–100 μM) increased endogeneous ACh release by 32–91% and effective doses were 10-fold lower in the presence of a cholinesterase inhibitor. Atropine also increased the amplitudes of synaptic population spikes in the field potentials.The muscarinic agonists muscarine (100 μM) and oxotremorine (25 and 100 μM) decreased endogenous ACh release. Atropine (10 μM) blocked the depressant effect of muscarine (100 μM). Muscarine (100 μM–1 mM) and oxotremorine (10–100 μM) decreased the electrically evoked excitation in the rat neostriatal slices, and their effects were reversed by atropine.Only higher concentrations of nicotine (1 and 5 mM) decreased the synaptic population spikes, but potassium-stimulated Ch outflow was not affected.It is concluded that in the neostriatum presynaptic muscarinic receptors modulate nicotinic excitation since potassium-stimulated ACh release and intrinsically evoked synaptic excitation are influenced by muscarinic drugs in the same way.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Stimulative effect of glia maturation factor and acetylcholine on active amino acid uptake and Ca, Mg-ATPase activity in nodose ganglia excised from adult rats

During aerobic incubation at 37°C, active uptake of labeled non-metabolizable α-aminoisobutyric acid (AIB) into isolated nodose ganglia (NG) excised from adult rats was accelerated to nearly twice that of the control, by the addition of glia maturation factor (GMF, 5 μg/ml) in a dose-dependent manner. A similar but moderate stimulative effect on ganglionic AIB uptake was caused by the addition of acetylcholine (ACh, 1 mM) plus eserine (0.1 mM). This effect, however, was not antagonized by nicotinic (hexamethonium, C₆, 0.1 mM) or muscarinic (atropine, 0.1 mM) blockers. The GMF-induced amino acid uptake seemed to be inhibited by further addition of ACh. On the other hand, ganglionic Ca²⁺, Mg²⁺-ATPase activity was greatly stimulated by either GMF or ACh. These results suggest that the increase in AIB uptake induced by GMF or ACh is possibly linked to Ca²⁺, Mg²⁺-ATPase activity in NG cell membranes.     

Deramciclane inhibits N-methyl-D-aspartate receptor function

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca²⁺ ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 μM) significantly inhibited [³H]D-aspartate release and transmembrane Ca²⁺ flux to N-methyl-D-aspartate in the absence of Mg²⁺. By contrast, inhibition of [³H]D-aspartate release and transmembrane Ca²⁺ flux evoked by 0.1 mM (S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg²⁺ and 10 μM cyclothiazide by 10 μM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 μM) did not inhibit [³H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca stores in NG108-15 cells

Following mobilization with the inositol 1,4,5-trisphosphate (IP₃)-generating agonist bradykinin, Ca²⁺ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca²⁺ to refill. The process by which this store refills with Ca²⁺ was characterized by recording bradykinin-induced intracellular free Ca²⁺ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca²⁺ ATPase inhibitor, reversibly depleted intracellular Ca²⁺ stores in these cells, but did not recruit detectable Ca²⁺ influx, suggesting that these cells lack substantial capacitative Ca²⁺ entry. The paucity of voltage-sensitive Ca²⁺ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca²⁺ entry in nonexcitable cells might assist refilling IP₃-sensitive Ca²⁺ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca²⁺ entry in nonexcitable cells might inhibit the refilling of the IP₃-sensitive store in NG108-15 cells was explored. The IP₃-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca²⁺ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca²⁺ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca²⁺ stores by a plasmalemmal Ca²⁺ leak, this leak shares a pharmacology similar to the capacitative Ca²⁺ entry pathway described for nonexcitable cells.     

Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca²⁺ ions ([Ca²⁺]_i) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca²⁺]_i. The ACh concentration-response curve indicated an apparent dissociation constant (K_d) of 8 μM and a Hill coefficient of 1.0. Removing extracellular free Ca²⁺ did not suppress the ACh-induced Ca²⁺ responses suggesting an intracellular Ca²⁺-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca²⁺ response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca²⁺ responses similar to those observed with ACh, with an apparent K_d of 10 μM and a Hill coefficient of 1.0. The cholinergic-induced Ca²⁺ response was reversibly blocked by muscarinic antagonists with the following order of potency, atropine>4-DAMP>methoctramine>pirenzepine. Nicotine (10 to 100 μM) did not evoke Ca²⁺ responses and the nicotinic antagonist curare (10 μM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Ca and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations

Synaptosomal tubulin was shown to be the major substrate for a Ca²⁺-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca²⁺ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca²⁺-dependent activation of the tubulin kinase system was mediated by the Ca²⁺ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [³²P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca²⁺ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 μM and 0.3 μM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca²⁺-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca²⁺-calmodulin stimulated phosphorylation of tubulin, and α- and β-tubulin were identified as major components of previously designated synaptic phosphoprotein bands DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca²⁺-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca²⁺ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca² on synaptic function.     

Effects of GABA on presumed presynaptic Ca entry in hippocampal slices

Evoked field potentials and changes in [Ca²⁺]_o were measured in the ‘in vitro’ hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/comissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca²⁺]_o. In contrast to findings in normal medium these decreases in [Ca²⁺]_o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca²⁺ in low Ca²⁺ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca²⁺ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

A pharmacological model for studying the role of Na gradients in the modulation of synaptosomal free [Ca]i levels and energy metabolism

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Inhibition by neuroprotective drug NS-7 of nicotine-induced Na influx, Ca influx and catecholamine secretion in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀=15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na⁺,K⁺-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of ²²Na⁺ without altering the EC₅₀ value of nicotine. Also, NS-7 diminished nicotine-induced ⁴⁵Ca²⁺ influx via nicotinic receptors and voltage-dependent Ca²⁺ channels (IC₅₀=14.1 μM) and catecholamine secretion (IC₅₀=19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

The effects of trimethyltin on the Ca, Mg and Ca + Mg-dependent ATPases of human neuroblastoma GM 3320

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca⁺²-dependent, Mg⁺²-independent, Mg⁺²-dependent, Ca⁺²-independent and Ca⁺² + Mg⁺²-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 μM), trimethyltin preferentially stimulated the Ca⁺²-dependent, Mg⁺²independent ATPase activity while inhibiting the Ca⁺² + Mg⁺²-ATPase activity approximately 70%. At 75 μM trimethyltin, the Ca⁺² + Mg⁺²-dependent ATPase activity is inhibited greater than 95% while the Ca⁺²-dependent, Mg⁺²-independent activity is essentially unchanged from control activity and the Mg⁺²-dependent, Ca⁺²-independent activity is inhibited approximately 50%. At concentrations greater than 75 μM, trimethyltin significantly inhibits the Ca⁺²-dependent, Mg⁺²independent ATPase activity. Thus, at trimethyltin concentrations of 50–75 μM, preferential inhibition of the Mg⁺²-dependent, Ca⁺²-independent and Ca⁺² + Mg⁺²-dependent ATPase activities of neuroblastoma GM 3320 is achieved.     

Histamine H1 receptors in UC-11MG astrocytes and their regulation of cytoplasmic Ca

Experiments were carried out on UC-11MG human astrocytoma cells, a continuous cell line that expresses a broad range of the biochemical and electrophysiological properties found in well-differentiated astrocytes. Because of a number of recent reports that astrocytes may express receptors for a variety of neuro-active substances, we measured the effects of 12 different neurotransmitters on intracellular free Ca²⁺ (Ca_i²⁺) in UC-11MG cells. Of these neurotransmitters only histamine was found to have a significant effect. Further characterization of the nature of the histamine response showed that UC-11MG cells express mepyramine-sensitive H₁ receptors the activation of which causes both mobilization of Ca²⁺ from intracellular stores and entry of Ca²⁺ from the extracellular solution. No evidence was found for the presence of H₂ receptors. The Ca_i²⁺ response was maximal at 300 μM histamine and was attenuated by increasing cell density. We suggest that this neurotransmitter may play a role in astrocytic function in the human CNS.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Spontaneous miniature hyperpolarizations of presynaptic nerve terminals in the chick ciliary ganglion

Intracellular recordings from presynaptic nerve terminals in the chick ciliary ganlion revealed the presence of spontaneous miniature hyperpolarizations in virtually all (86%) nerve terminals examined. These spontaneous events appeared as small, brief hyperpolarizations at resting potential and were observed to increase or decrease as the membrane potential was depolarized or hyperpolarized from rest, respectively. The hyperpolarizing potentials were sensitive to blockade by tetraethylammonium and Ba²⁺, while caffeine increased then abolished these events. The voltage fluctuations were unaffected by tetrodotoxin, low Ca²⁺ external solution or the synaptic blockers, picrotoxin and strychnine. These spontaneous, transient, miniature hyperpolarizations may be due to the brief and co-ordinated activation of between 15–60 Ca²⁺-dependent K⁺ channels following the release of Ca²⁺ from internal stores.