Endogenous ACh enhances striatal NMDA‐responses via M1‐like muscarinic receptors and PKC activation

Cortical glutamatergic fibres and cholinergic inputs arising from large aspiny interneurons converge on striatal spiny neurons and play a major role in the control of motor activity. We have investigated the interaction between excitatory amino acids and acetylcholine (ACh) on striatal spiny neurons by utilizing intracellular recordings, both in current- and in voltage-clamp mode in rat brain slices. Muscarine (0.3–10 μm ) produced a reversible and dose-dependent increase in the membrane depolarizations/inward currents induced by brief applications of N-methyl-d -aspartate (NMDA), while it did not affect the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced responses. These concentrations of muscarine did not alter the membrane potential and the current-voltage relationship of the recorded cells. Neostigmine (0.3–10 μm ), an ACh-esterase inhibitor, mimicked this facilitatory effect. The facilitatory effects of muscarine and neostigmine were antagonized either by scopolamine (3 μm ) or by pirenzepine (10–100 nm ), an antagonist of M1-like muscarinic receptors, but not by methoctramine (300 nm ), an antagonist of M2-like muscarinic receptor. Accordingly, these facilitatory effects were mimicked by McN-A-343 (1–10 μm ), an agonist of M1-like muscarinic receptors, but not by oxotremorine (300 nm ), an agonist of M2-like receptors. Tetrodotoxin (TTX) did not block the facilitatory effect produced by the activation of muscarinic receptors suggesting that this effect is postsynaptically mediated. The action of neostigmine was prevented either by the intracellular calcium (Ca²⁺) chelator BAPTA (200 mm ) or by preincubating the slices with inhibitors of protein kinase C (PKC) (staurosporine 100 nm or calphostin C 1 μm ). McN-A-343 did not alter the excitatory post synaptic potentials (EPSPs) evoked by corticostriatal stimulation in the presence of physiological concentration of magnesium (Mg²⁺ 1.2 mm ), while it enhanced the duration of these EPSPs recorded in the absence of external magnesium. Our data show that endogenous striatal ACh exerts a positive modulatory action on NMDA responses via M1-like muscarinic receptors and PKC activation.     

Modulation of big K+ channel activity by ryanodine receptors and L‐type Ca2+ channels in neurons

As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca²⁺ channels and ryanodine receptors (RyR, 1 ) in cerebellar granule cells, we examined if such a coupling could activate a Ca²⁺-sensitive K⁺ channel, the big K⁺ (BK) channel, in cultured cerebellar granule cells. We observed that (±)-1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)-α-carboxycyclopropyl-glycine (L-CCG-I) and l -(+)-2-amino-4-phosphonobutyrate (L-AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that was independent of L-type Ca²⁺ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca²⁺ channels.     

Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca²⁺/high Mg²⁺ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinicreceptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the γ-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.     

Nicotinic and muscarinic acetylcholine responses in differentiated PC12 cells

Nicotinic and muscarinic acetylcholine (ACh) responses were investigated in PC12 cells using the conventional whole-cell and nystatin perforated patch techniques. With the nystatin perforated patch, ACh induced three kinds of ionic currents: a rapid transient inward current, a subsequent transient outward current and a long-lasting slow inward current, whereas only a transient inward current was recorded by conventional whole-cell patch. The transient rapid inward current was mimicked by nicotine, but not by muscarine. On the contrary, the transient outward current and the long-lasting slow inward current were mimicked by muscarine but not by nicotine. Both nicotinic and muscarinic antagonists inhibited the transient inward current and the subsequent outward current in a concentration-dependent manner. The current-voltage relationship for the nicotine-induced transient current showed an inward rectification and the reversal potential was close to the Na⁺ equilibrium potential. The ACh-, muscarine-, CCh- and oxotremorine-M induced outward currents increased in a sigmoidal fashion with an increase in the concentration. Neither McN-A-343, an M1 agonist, nor oxotremorine, an M2 agonist, mimicked the muscarinic response. The reversal potential of the muscarinic response was close to the K⁺ equilibrium potential. The muscarinic response was not affected by pre-treatment with pertussis toxin but was enhanced by pre-treatment with Li⁺. In the cells perfused with Ca²⁺-free external solution, only the first application of ACh induced the muscarinic response. Calmodulin antagonists reversibly blocked the muscarinic response in a concentration-dependent manner. Neither protein kinase C inhibitor (H-7), protein kinase A inhibitor (H-8), nor Ca-calmodulin dependent kinase II inhibitor (KN-62) affected the muscarinic response. It was concluded that the ACh-induced rapid inward current was passing through non-selectivecation channels coupled with nicotinic ACh receptors. On the other hand, the muscarinic response is mediated by the activation of M3 receptors coupled to IAP-insensitive G-protein which stimulates the phosphatidylinositol pathway through phospholipase C. Consequently, Ca²⁺ was released by the increase in IP₃. Finally, Ca²⁺-calmodulin binding may lead to opening of the K⁺ channels.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Antagonists‐resistant calcium currents in rat embryo motoneurons

Ca²⁺ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5–20 mm Ba²⁺, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≈ 20% L-, ≈ 45% N- and ≈ 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20–200 nm ω-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nm in 5 mm Ba²⁺), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca²⁺ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 μm ω-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α_1E channel. The current activates around ?20 mV in 20 mm Ba²⁺; inactivates slowly and independently of Ca²⁺; is blocked by low [Cd²⁺] and high [Ni²⁺]; and is larger with Ba²⁺ than Ca²⁺. The uncovered R-type calcium current can account for part of the presynaptic Ca²⁺ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP- and ω-CTx-GVIA, and displays anomalous sensitivity to ω-Aga-IVA and ω-CTx-MVIIC ( 1 ) J. Physiol. (Lond.), 482, 283–290; 2 ) Eur. J. Neurosci., 9, 817–823].     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature γ-aminobutyric acid_A (GABA_A) receptor-mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm ) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca²⁺-containing to a nominally Ca²⁺-free medium (in which Ca²⁺ was substituted with Mg²⁺, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca²⁺-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm ), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm ) reversibly reduced the amplitude of the current induced by GABA (100 μm ) indicating a clear postsynaptic effect on GABA_A receptor. Superfusion of ryanodine (30 μm ), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine-sensitive stores facilitates GABA release.     

Vasoactive intestinal peptide potentiates and directly stimulates catecholamine secretion from rat adrenal chromaffin cells

The actions of vasoactive intestinal polypeptide (VIP) on catecholamine secretion and changes in [Ca²⁺]_i in single rat chromaffin cells were studied using amperometry and Indo-1. Application of VIP prior to acetylcholine (ACh) or co-application of VIP and ACh enhanced secretion by 94% and 153% respectively, compared to ACh alone. [Ca²⁺]_i was increased by 17% when VIP was preapplied and by 73% upon co-application. Exposure to VIP before stimulation with 60 mM K⁺ enhanced secretion by 68%, but not [Ca²⁺]_i. VIP application prior to DMPP and nicotine had no effect on [Ca²⁺]_i, but increased [Ca²⁺]_i signals to muscarine by 18%. VIP co-application potentiated only [Ca²⁺]_i responses to muscarine, by 28%. The effect of VIP on muscarine-induced [Ca²⁺]_i signals was mimicked by 8-Br-cAMP, and both were blocked by H-89, a protein kinase A inhibitor. Long-lasting increases in secretion accompanied by a sustained rise in [Ca²⁺]_i to VIP alone were seen in 55% of cells. Removal of Ca²⁺ or addition of La³⁺ inhibited both responses, while L-, N- and P-type Ca²⁺ channel blockers were ineffective. SK&F 96365 inhibited VIP-induced secretion completely and rises in [Ca²⁺]_i by 75%. Neither 8-Br-cAMP nor 8-Br-cGMP evoked responses similar to VIP alone. Thus in rat chromaffin cells, VIP acts both directly as a neurotransmitter in provoking sustained catecholamine secretion in a cAMP-independent manner, and also by enhancing ACh-induced secretion, via a cAMP-dependent action involving muscarinic receptors.     

High‐affinity copper block of GABAA receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

The actions of Cu²⁺ ions on GABA_A receptor-mediated currents in acutely isolated Purkinje cells from rat cerebellum were studied using the whole-cell patch-clamp technique and a rapid perfusion system. Bath application of Cu²⁺ reduced currents induced by 2 μm γ-aminobutyric acid (GABA) in a concentration-dependent manner with an IC₅₀ of 35 n m . The Cu²⁺-induced block of GABA responses was not voltage-dependent. Increasing the GABA concentration (from 2 to 50 μm ) decreased the blocking effect of Cu²⁺. Dose–response analysis for activation of GABA_A receptors revealed a twofold decrease in apparent affinity for GABA in the presence of 0.1 μm Cu²⁺. Recovery from the block required several minutes after removal of Cu²⁺ from the medium. The block was removed by histidine, which preferentially forms complexes with Cu²⁺, or by other chelating substances. Application of 10 μm histidine immediately before application of 2 μm GABA completely relieved the block of GABA responses produced by 0.1 μm Cu²⁺. The effect of histidine was concentration-dependent with an EC₅₀ of 0.75 μm . The results demonstrate that Cu²⁺ is a potent inhibitor of GABA-evoked responses in rat Purkinje cells. Copper may be an endogenous synaptic modulating factor. Cu²⁺ toxicity, notably in Wilson’s disease, could result to some extent from chronic GABA_A receptor blockade.     

G‐proteins and G‐protein subunits mediating cholinergic inhibition of N‐type calcium currents in sympathetic neurons

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca²⁺ currents: this reduces Ca²⁺-activated K⁺ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M₄ muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M₁ mAChRs ( 1 –536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their α and βγ subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, over-expression of selected constitutively active subunits, and antagonism of endogenously liberated βγ subunits by over-expression of βγ-binding β-adrenergic receptor kinase 1 (βARK1) peptide. The results indicate that: (i) M₄ mAChR-induced inhibition is mediated by G_oA; (ii) α and βγ subunits released from the activated G_oA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M₁ mAChR-induced inhibition is likely to be mediated by the α subunit of G_q. Hence, Ca²⁺ current inhibition results from the concerted, but independent actions of three different G-protein subunits.     

A presynaptic N‐methyl‐d‐aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool

A possible role of the N-methyl-d -aspartate receptor (NMDA-R) as a presynaptic autoreceptor was investigated using Percoll-purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose-dependently (10–100 μm ) and in the absence of Mg²⁺, stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μm ), an open NMDA-R-channel blocker, reduced this effect even below control levels, indicating endogenous NMDA-R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA-induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg²⁺ (1 m m ), was slow in onset and returned to baseline after NMDA-removal. The NMDA-effect was also found in the absence of extracellular Ca²⁺, suggesting that amino acids were released from a non-vesicular (cytoplasmic) pool. Secondly, in KCl-depolarized synaptosomes exposed to 1 m m Mg²⁺, NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl-evoked Ca²⁺-independent release of aspartate and glutamate, but not of GABA. l -trans-PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801-effect, suggesting a coupling between NMDA-Rs and these transporters. These data provide evidence for a presynaptic NMDA autoreceptor in rat hippocampus. We speculate on the role of this NMDA-R to depolarize the presynaptic membrane by Na⁺-entry, which may induce reversal of amino acid transporters and thereby releasing amino acids from a cytoplasmic pool.     

Multiple actions of acetylcholine at a muscarinic receptor studied in the rat hippocampal slice

Responses of hippocampal pyramidal cells to topical application of acetylcholine (ACh) were measured in the in vitro hippocampal slice preparation.ACh but not cyclic GMP produced a short-latency hyperpolarization associated with a decrease in input resistance. This was followed by a long-latency but long duration depolarization associated in some cells with an increase in input resistance. This change in resistance followed the depolarization and outlasted it by 5–20min, until complete recovery. During the depolarization there was a reduction in magnitude of EPSPs produced by activation of the Schaffer collateral excitatory afferents. The reversal potential for the hyperpolarization was about —95 mV, and it was blocked by 4-aminopyridine. The depolarization, but not the hyperpolarization was markedly attenuated in slices maintained in low (25°C) temperature. The responses to ACh were blocked by atropine but not byd-tubocurarine. The hyperpolarization as well as the depolarization were present in slices treated with tetrodotoxin (TTX)., but were reduced in slices superfused with a low Ca²⁺-high Mg²⁺ medium, and in slices treated with Mn²⁺ and Co²⁺ ions. It is suggested that ACh causes a fast increase in gK⁺, followed by along-lasting energy-dependent depolarization associated with action potential discharges, a decrease in conductance and a suppression of EPSPs.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) 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.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Stimulus-secretion coupling in porcine adrenal chromaffin cells: Effect of dexamethasone

Recent studies from this laboratory established that dexamethasone (DEX) potentiates Ca²⁺ current via voltage-gated Ca²⁺ channels (VGCC), and as a consequence potentiates agonist-induced cytosolic Ca²⁺ transients in rat adrenal chromaffin cells. The present study examined whether DEX can also modulate VGCC activity and agonist-induced cytosolic Ca²⁺ transients in porcine adrenal medullary chromaffin (PAMC) cells, and if so whether this results in alterations in catecholamine secretion. Forty-eight-hr exposure to 1 μM DEX significantly increased peak Ca²⁺ current (Δ + 138%; n = 6; P < 0.05) in PAMC cells. DEX treatment also significantly potentiated the increase in cytosolic Ca²⁺ in response to membrane depolarization with KCl (Δ + 20%; n = 29; P < 0.05), but did not affect the amplitude of Ca²⁺ transients elicited by nicotine or acetylcholine. Despite the potentiation of intracellular Ca²⁺, DEX treatment had no effect on KCl-induced secretion of either norepinephrine or epinephrine. These data demonstrate that as in the rat chromaffin cell, DEX can also increase VGCC activity in PAMC cells. However, the subsequent potentiation of selected agonist-induced increases in intracellular Ca²⁺ does not appear to be sufficient to alter catecholamine secretion. J. Neurosci. Res. 49:416–424, 1997. © 1997 Wiley-Liss, Inc.     

Muscarinic and purinergic Ca mobilizations in the neural retina of early embryonic chick

Acetylcholine and adenosine triphosphate (ATP) raise intracellular Ca²⁺ concentration via muscarinic receptors and P_2U purinoceptors by releasing Ca²⁺ from intracellular Ca²⁺ stores in the neural retina of early embryonic chick. The signal transduction mechanisms for the muscarinic and purinergic Ca²⁺ responses with fura-2 fluorescence measurements were studied. Li⁺ (1 mM), which inhibits phosphatidylinositol metabolism, enhanced both the Ca²⁺ rises to carbamylcholine (CCh, 30 μM), a muscarinic agonist, and ATP (200 μM). Thapsigargin (250 nM), an inhibitor of Ca²⁺-ATPase of inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores, abolished both the Ca²⁺ rises to CCh (100 μM) and ATP (500 μM). U-73122 (2 μM), an inhibitor of phospholipase C_b, suppressed the Ca²⁺ rise to ATP (500 μM), but its analog U-73343 (2 μM) did not suppress it. In contrast, both U-73122 and U-73343 suppressed the Ca²⁺ rise to CCh (100 μM). Pertussis toxin (250 ng/ml) suppressed the ATP-induced Ca²⁺ rise at least partly, whereas no inhibition was observed on the CCh-induced Ca²⁺ rise. Cross-talk occurred between the muscarinic and purinergic Ca²⁺ mobilizations but they were not occlusive. This study suggests that the muscarinic and purinergic Ca²⁺ mobilizations utilize IP₃-sensitive Ca²⁺ stores, but different signal transduction pathways are involved in between the muscarinic and purinergic Ca²⁺ responses.     

Muscarinic Activation of a Novel Voltage-sensitive Inward Current in Rabbit Prevertebral Sympathetic Neurons

The muscarinic activation of rabbit prevertebral sympathetic neurons was studied in non-dissociated coeliac and superior mesenteric ganglia using whole-cell patch-clamp techniques. In the presence of nicotinic blockers, carbachol, muscarine and oxotremorine-M (1–50 μM) induced tonic firing by activating a persistent inward current. These effects were abolished by atropine. They persisted when the M-current was blocked with Ba²⁺ (1 mM) and intracellular Cs⁺. The muscarinic inward current was found to be time- and voltage-dependent. It peaked at -60 mV, decreased at large hyperpolarizations and was tonically activated between —110 and —20 mV, which gave steady-state I—V curves an N-shape between —96 and —54 mV. The negative slope accounted for the large hyperpolarizing responses generated by current pulses in carbachol-treated cells. The muscarinic current was abolished when Na⁺ was replaced by choline, Tris⁺, sucrose, N-methyl-D-glucamine and Cs⁺ but not Li⁺. It was resistant to tetrodotoxin (3 μM), amiloride (3 μM), benzamil (10 μM) and tetraethylammonium (5–20 mM). No involvement of K⁺ and Cl^- could be detected. We therefore styled it l_{Na, M}, in reference to its ionic selectivity and its coupling to muscarinic receptors. Low Ca²⁺-Mg²⁺ salines enhanced the Na, M-current. The current was blocked by Cd²⁺, Co²⁺, La³⁺ (1 mM) and Ba²⁺ (5 mM) but insensitive to methoxyverapamil hydrochloride, nicardipine, nifedipine and ω-conotoxin MVII A (2–20 μM). These effects were ascribed to the binding of di- and trivalent ions to the Na, M-channels. Spike bursts transiently blocked l_{Na, M}. With high intracellular ethylene glycol bis(b-aminoethyl ether)-N, N'-tetraacetic acid or 1, 2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (20–50 mM), this effect was reduced, whereas l_{Na, M} persisted in long-term recordings and its amplitude increased twofold, indicating that intracellular calcium negatively regulated the Na, M-channels. We conclude that we have described a novel muscarinic receptor-coupled channel which appears to play a major part in regulating the firing behaviour of sympathetic neurons.