Dependence of the acid-sensitive ion channel, ASIC1a, on extracellular Ca(2+) ions

The early H⁺-induced current in the embryonic spinal cord neurone depends on extracellular Ca²⁺ for its function. We have studied the Ca²⁺-dependence of homo- and heteromultimeric acid-sensing ion channels (ASICs) expressed in Cos cells. It was found that single-channel conductance of both the ASIC2a and the ASIC1a channel is reduced at membrane potentials more negative than −40 mV by elevated extracellular Ca²⁺. Due to this effect on unitary currents, the macroscopic ASIC2a peak current at −60 mV decreases gradually with rising extracellular Ca²⁺ concentration. In addition, the macroscopic ASIC1a current is very small at low extracellular Ca²⁺ and increases with rising Ca²⁺ up to 5 mM before decreasing again at still higher concentrations of extracellular Ca²⁺.     

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn²⁺, verapamil (40 μM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg²⁺, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca²⁺ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca²⁺ (nominally Ca²⁺-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn²⁺ and Mg²⁺, as well as Ca²⁺, enter the nerve terminal through Ca²⁺ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca²⁺ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na⁺ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.     

Potassium and sodium channels in human malignant glioma cells

Human malignant glioma cells from 5 different cell lines were voltage clamped and examined for the presence of depolarization-activated ion channels. Outward K-currents were elicited at membrane potentials > 40 mV, which had two main components, one which was delayed and blocked by externally applied tetraethylammonium (TEA, 10 mM), and another which was instantaneous and insensitive to TEA in the outside solution. The proportion of the two K-current components varied between cell lines. An increase in [Ca²⁺] in the range 0–4 mM, decreased the leak conductance and shifted the activation of the instantaneous outward K-current towards more positive potenttials. Mg²⁺, Zn²⁺ and Co²⁺ had qualitatively similar effects. Patch recordings with 150–160 mM K⁺-solution on both sides of the membrane revealed that the delayed outward K-current was carried through large conductance (250–300 pS) channels. Changes in free [Ca²⁺]_i from 0 to 2 × 10⁻⁸ M increased the activation of the large conductance K-channel. Small Na-currents were identified in cells from one cell line (Tp-378MG). The Na-conductance rangedfrom 0.5 to 7.5 nS in 25% of the cells, and was less than 0.5 nS in 75%. The Na-channels were activated and inactivated at 30–40 mV more positive potentials than in the mammalian peripheral nerve. Tetrodotoxin (100 mM) blocked g_Na almost completely.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Different spatial distributions of sodium channels in the slowly and rapidly adapting stretch receptor neuron of the crayfish

Inward Na⁺ currents were studied, using a two-microelectrode intracellular voltage-clamp technique, in the slowly adapting (SA) and rapidly adapting (RA) stretch receptor neurons of the crayfish after the axons were cut at different distances from the soma. In the SA neuron, inward Na⁺ currents were recorded in the soma even when the axon was cut as close as 100 μm from the center of the soma, indicating the presence of Na⁺ channels in these parts. Also, two populations of Na⁺ channels seem to exist in the SA neuron. In the RA neuron, only minute Na⁺ currents were observed if the axon was shorter than 250 μm. The results strongly indicate that the voltage-gated Na⁺ channels in the SA and RA neurons have different distributions and that the difference in the spatial distribution of Na⁺ channel types may be important for the difference in firing properties in the two types of neurons.     

Fe2+ decreases the taurine-induced Cl- current in acutely dissociated rat hippocampal neurons

The effects of ferrous ions (Fe²⁺) on taurine-induced Cl⁻ current (I_tau) recorded from single neurons, which was freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Using standard pharmacological approaches, we found that the currents gated by concentrations of taurine (≤10 mM), which existed in about 90% of the hippocampal neurons tested, were predominantly mediated by strychnine-sensitive glycine receptors. When co-applied with taurine, Fe²⁺ effectively depressed I_tau in a concentration-dependent manner, with an IC₅₀ of 3.76 mM and Hill coefficient of 1.01, while preincubation with 1 mM Fe²⁺ alone did not affect the following membrane currents elicited by taurine. The result suggests that resting taurine-gated channels are insensitive to Fe²⁺. Since internal cell dialysis with 3 mM Fe²⁺ failed to modify I_tau, it was deduced that the site of action of Fe²⁺ is extracellular. Furthermore, the Lineweaver–Burke double reciprocal plot of normalized response to taurine against the concentration of taurine illustrated that the depression of I_tau was noncompetitive, therefore Fe²⁺ may act on the glycine receptor–chloride ionophore complex at a site distinct from where taurine binds. Various concentrations of Fe²⁺ ranging from 0.1 to 20 mM depressed I_tau and this extracellular depression was independent of membrane voltage. These results indicate that Fe²⁺ decreases I_tau in acutely dissociated rat hippocampal neurons and the inhibition of glycine receptors by Fe²⁺ might be one possible approach through which Fe²⁺ induces seizures.     

Ionic currents in cultured supraoptic neurons: Actions of peptides and transmitters

The hypothalamo-neurohypophysial system has proved an excellent model for peptidergic neurons in the central nervous system. Electrophysiological studies using in vivo and in vitro preparations with extracellular and intracellular recording techniques have determined some of the intrinsic and extrinsic mechanisms that generate the striking firing patterns that the neurons exhibit. We have developed a dissociated cell preparation of these neurons and used patch clamp recording techniques to enable detailed studies of membrane properties underlying such activities. Cultured neonatal supraoptic neurons fired spontaneous action potentials which in some cells were distinctively patterned. Under voltage clamp, voltage-activated Na⁺, K⁺, and Ca²⁺ currents were recorded. K⁺ and Ca²⁺ currents were modulated by application of -adrenergic agonists, and Ca²⁺ currents were also modulated by κ-opioid agonists. The neurons were also sensitive to γ-aminobutyric acid which acted directly on Cl-channels. Spontaneous, patterned activity, the presence of functional receptors for neurotransmitters and the ability to study the neurons under voltage clamp suggest that this is an excellent model system for studying these peptidergic neurons.     

The antiepileptic drug losigamone decreases the persistent Na current in rat hippocampal neurons

The tetronic acid derivative losigamone is a new anticonvulsant drug with a mechanism of action that was previously unknown. The drug decreases the frequency of spontaneous action potentials and suppresses repetitive firing of neurons. Here we tested the hypothesis that losigamone suppresses the persistent Na⁺ current (I_NaP) in hippocampal neurons of rat brain slices and in cultured hippocampal neurons. Whole-cell voltage clamp recordings from neurons of juvenile rats (P15–P25) were performed with pipettes filled with Cs-gluconate or CsF. After pharmacological block of K⁺ and Ca²⁺ currents I_NaP was revealed by applying slow depolarizing voltage ramps from −70 to 0 mV. Losigamone (100–200 μM) was dissolved in DMSO (0.1%) and was applied by bath application or local pressure application. Losigamone induced a decrease in amplitude of I_NaP at depolarized membrane potentials which was reversible in cultured neurons. When tetrodotoxin (TTX) was added to the bath, I_NaP was blocked and only a residual non-specific outward cation current (I_cat) remained. Losigamone had no obvious effect on responses to voltage ramps under these conditions. Thus, losigamone did not affect I_cat or induce any additional currents. The data suggest that losigamone decreases neuronal excitability via a decrease in I_NaP.     

Ionic mechanism of generation of receptor potential in response to quinine in frog taste cell

The ionic mechanism of generation of the receptor potential in a frog taste cell elicited by quinine-HCl (Q-HCl) was studied with an intracellular recording technique by replacing the superficial and interstitial fluids of the tongue with various saline solutions. The taste cells whose receptor membranes were adapted to normal saline and deionized water generated depolarizing receptor potentials at Q-HCl concentrations higher than 2 and 0.01 mM, respectively. The input resistance of taste cell during Q-HCl stimulation scarcely changed. The receptor potential did not change even when the membrane potential level was broadly changed. The magnitude of the receptor potential was increased by reducing the concentration of superficial Cl⁻ on the taste receptor membrane, but was independent to the concentration of superficial Na⁺. Injection of Cl⁻ into a taste cell increased the receptor potential to 170%. The magnitude of receptor potential was decreased to 20–30% by removing interstitial Na⁺ or Cl⁻ or both surrounding the basolateral membrane of taste cell. Furosemide (1 mM) added to the interstitial fluid decreased the receptor potential to 15%, while interstitial ouabain (0.1 mM) and superficial SITS (0.1 mM) did not influence it. From these results, we conclude: (1) an electroneutral Na⁺/Cl⁻ cotransport occurs through the basolateral membrane of a taste cell in the resting state, so that Cl⁻ accumulates inside the cell. (2) Q-HCl stimulation induces the active secretion of Cl⁻ across the taste receptor membrane, resulting in a depolarizing receptor potential.     

Expression of tetrodotoxin-sensitive and resistant sodium channels by rat melanotrophs

Rat melanotrophs fire Na+ and Ca2(+)-dependent action potentials. Whereas the molecular identity of Ca2+ channels expressed by these cells is well documented, less is known about Na channels. We characterize the expression of seven sodium channel alpha-subunit and the beta1- and beta2-subunit mRNAs. The tetrodotoxin-resistant Nav1.8 and Nav1.9 alpha subunit mRNAs are detected in the newborn intermediate lobe and in cultured melanotrophs. Electrophysiological recordings further demonstrate the expression of both tetrodotoxin-sensitive and tetrodotoxin-resistant currents by dissociated melanotrophs. Moreover, activated sodium channels are able to elicit intracellular calcium waves, both in the absence or in the presence of tetrodotoxin. This work shows that rat melanotrophs express functional tetrodotoxin-resistant sodium channels, whose activation can lead to the generation of intracellular calcium waves.     

Inhibition of the rat brain sodium channel Nav1.2 after prolonged exposure to gabapentin

Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na⁺-dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat sodium channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000 μM), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30 μM gabapentin, while higher concentrations (100–1000 μM) did not result in greater effects. Prolonged treatment with gabapentin also caused the channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na⁺-dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.     

Does calcium influx regulate melatonin production through the circadian pacemaker in chick pineal cells? Effects of nitrendipine, Bay K 8644, Co, Mn, and low external Ca

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin production and release. Here, we describe the effects of nitrendipine (NTR) (a dihydropyridine ‘antagonist’ of L-type calcium channels), Bay K 8644 (BK) (a dihydropyridine calcium channel ‘agonis’), cobalt and manganese ions (both inorganic calcium channel blockers), and low external calcium concentrations, on the melatonin rhythm. NTR inhibited and BK stimulated melatonin output; they were potent and effective. Co²⁺, Mn²⁺, and low external Ca²⁺ markedly inhibited melatonin output. These results support a role for calcium influx through voltage-dependent calcium channels (L-type) in the regulation of the melatonin production. Four or 8 h pulses of white light or darkness, in otherwise constant red light, cause, in addition to acute effects, phase-dependent phase shifts of the melatonin rhythm in subsequent cycles. Such phase shifts indicate an effect on (proximal to) the pacemaker generating the rhythm. Four or 8 h pulses of NTR, BK, Co²⁺, however, did not appreciably alter the phase of subsequent melatonin cycles. Neither did BK interfere with phase shifts induced by light pulses. Mn²⁺ pulses did induce phase-dependent phase shifts, but, unlike those evoked by light or dark pulses, these were all delays. Such effects of Mn²⁺ in other systems have been attributed to, and are characteristic of, ‘metabolic inhibitors’. On balance, the results fail to support a prominent role for calcium influx in regulating the pacemaker underlying the circadian rhythm in chick pineal cells. Rather, calcium influx appears to regulate melatonin production primarily by acting on the melatonin-synthesizing apparatus, distal to the pacemaker.     

Action potentials produce a long-term enhancement of M-current in frog sympathetic ganglion

M-current is a voltage-gated K⁺ current that can be turned off by the muscarinic action of acetylcholine. We examined the effects of postsynaptic action potential firing on the level of M-current in B-cells of the bullfrog sympathetic ganglion. High frequency stimulation of action potentials induced an approximately two-fold increase in the level of the M-current that could last up to 35 min. The ‘enhanced’ M-current was similar to the ‘resting’ one in its time-dependence, voltage-dependence and sensitivity to neurotransmitters. Experiments were undertaken to examine the functional consequences of the enhanced M-current. Following high frequency stimulation the number of spikes evoked by depolarizing current was reduced. In addition, the excitatory postsynaptic potential (EPSP) evoked by maximal input became subthreshold, thereby blocking information flow through the ganglion cell. These results indicate that the enhancement of M-current by spikes provides a negative feedback mechanism for the control of excitability. It has been reported that postsynaptic stimulation of ganglion cells also produces a long-term increase in the nicotinic EPSP, but we were unable to confirm this observation.     

Calcium Channel Currents in Undifferentiated Human Neuroblastoma (SH-SY5Y) Cells: Actions and Possible Interactions of Dihydropyridines and ω-Conotoxin

Ca²⁺ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba²⁺ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 μMCd²⁺. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca²⁺ channels. Currents were also inhibited by the marine snail peptide ω-conotoxin GVIA (ω-CgTx). At a concentration of 10 nM inhibition by ω-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1μM, supramaximal concentrations reduced the inhibitory actions of ω-CgTx in a concentration-dependent manner. Ca²⁺ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and ω-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca²⁺ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by ω-CgTx.     

Inward rectifying potassium channels in human malignant glioma cells

Human glioma cells obtained from established cell lines (Tp-276MG, Tp-301MG, Tp-378MG, Tp-483MG and U-251MG) were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizing voltages, due to the presence of inward rectifying K-channels in cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibrium potential. The slope conductance for the inward K-currents (g_Ki) was affected both by [K⁺]_i and [K⁺]₀. g_Ki was proportional to [K⁺]₀ raised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K⁺]_i (25mM). The rectification was not significantly different in cells perfused with Mg-free EDTA-buffered internal solution. Tl⁺ was 3.5 times more permaant than K⁺. g_ki was blocked by Cs⁺ (1 mM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na⁺ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the single inward rectifying channel was estimated to be 27 ± 7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-homeostasis in the brain.     

K+ channel properties in cultured mouse Schwann cells: dependence on extracellular K+

In cultured Schwann cells, single-channel and whole-cell K+ currents can be activated by depolarizing the membrane to values more negative than -50 mV. In elevated extracellular K+ concentration ([K+]o), however, single-channel activity and whole-cell currents could be recorded at more negative potentials. Thus, the threshold of current activation was shifted to more negative potentials. This shift in the activation threshold was only observed with normal (50-60 mM) intracellular [K+] levels; it was not apparent when [K+]i was elevated to 145 mM. The control of [K+]o on the gating properties of K+ channels may serve to enhance the capability of the Schwann cell to take up [K+]o and thus may serve for [K+] homeostasis in the peripheral nerve.     

Evidence for a persistent sodium conductance in neurons from the nucleus prepositus hypoglossi

Intracellular recordings were made from 39 neurons in a slice preparation of the prepositus hypoglossi nucleus from guinea pigs. Morphological characteristics were confirmed by dying neurons with Lucifer yellow. The neurons were spontaneously active, firing in the range of 8–50 spikes/s. Spike duration was short (0.32 ms) and the spikes were followed by fast and slow afterhyperpolarizations. The current vs frequency relationship was linear during steady state firing, but showed dual firing ranges corresponding to the first, third and fifth interspike interval. The instantaneous frequency of the first few interspike intervals could reach 500 spikes/s. Depolarizing and hyperpolarizing responses to square pulses displayed initial sag and rebound responses sensitive to extracellular Cs⁺, pharmacologically classifying the responses as a result of a Q-like current. Substitution of Ca²⁺ in the medium with the inorganic calcium blockers Mn²⁺ or Co²⁺ resulted in oscillatory firing, depolarizing excursions being sensitive to tetrodotoxin (TTX). Mn²⁺ or Co²⁺ in combination with extracellular Cs⁺ elicited TTX-sensitive plateau potentials, blocked in Na⁺-free solution. In conclusion, the prepositus neurons displayed spontaneous activity in the slice preparation and active membrane properties above as well as below the threshold of the action potential. In addition, the prepositus neurons possess a persistent sodium conductance that can be uncovered by inorganic calcium blockers. It may be involved in sustaining the spontaneous discharge.     

Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg²⁺ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation - spontaneous and agonist induced.     

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na⁺ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na⁺ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na⁺ clearance in dendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na⁺ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K⁺ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).     

Adenosine Inhibits L- and N-Type Calcium Channels in Pituitary Melanotrophs. Evidence for the Involvement of a G Protein in Calcium Channel Gating

It has been previously demonstrated that activation of A₁ adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A₁ adenosine receptor agonist R-PIA (50 μM each) produced a decrease of the amplitude of the barium current, while the selective A₂ adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTPyS (100 μM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 μg/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 μM) together with the phosphodiesterase inhibitor IBMX (100 μM) to the intracellular solution did not modify the effect of R-PIA on the current.
It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.