pH-dependent inhibition of native GABA(A) receptors by HEPES.

BACKGROUND AND PURPOSE: Artificial buffers such as HEPES are extensively used to control extracellular pH (pH(e) ) to investigate the effect of H(+) ions on GABA(A) receptor function. EXPERIMENTAL APPROACH: In neurones cultured from spinal cord dorsal horn (DH), dorsal root ganglia (DRG) and cerebellar granule cells (GC) of neonatal rats, we studied the effect of pH(e) on currents induced by GABA(A) receptor agonists, controlling pH(e) with HCO(3) (-) or different concentrations of HEPES. KEY RESULTS: Changing HEPES concentration from 1 to 20 mM at constant pH(e) strongly inhibited the currents induced by submaximal GABA applications, but not those induced by glycine or glutamate, on DH, DRG or GC neurones, increasing twofold the EC(50) for GABA in DH neurones and GC. Submaximal GABA(A) receptor-mediated currents were also inhibited by piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid, tris(hydroxymethyl)aminomethane or imidazole. PIPES and HEPES, both piperazine derivatives, similarly inhibited GABA(A) receptors, whereas the other buffers had weaker effects and 2-(N-morpholino)ethanesulfonic acid had no effect. HEPES-induced inhibition of submaximal GABA(A) receptor-mediated currents was unaffected by diethylpyrocarbonate, a histidine-modifying reagent. HEPES-induced inhibition of GABA(A) receptors was independent of membrane potential, HCO(3) (-) and intracellular Cl(-) concentration and was not modified by flumazenil, which blocks the benzodiazepine binding site. However, it strongly depended on pH(e) . CONCLUSIONS AND IMPLICATIONS: Inhibition of GABA(A) receptors by HEPES depended on pH(e) , leading to an apparent H(+) -induced inhibition of DH GABA(A) receptors, unrelated to the pH sensitivity of these receptors in both low and physiological buffering conditions, suggesting that protonated HEPES caused this inhibition.

Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron-keratinocyte coculture model.

We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17beta-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.

Functional modulation of gamma-aminobutyric acid(A) receptors by etifoxine and allopregnanolone in rodents.

We looked for an interaction between etifoxine and the neurosteroid allopregnanolone at central gamma-aminobutyric acid (GABA(A)) receptors. Etifoxine (2 microM) did not affect the affinity of allopregnanolone (IC(50)=108 nM) for its site in preparations of Sprague-Dawley rat cerebral cortex membranes, as determined by the inhibition of [(35)S] t-butylbicyclophosphorothionate binding, a specific ligand of the GABA(A) receptor chloride channel site. Etifoxine and allopregnanolone were anticonvulsants, blocking the clonic convulsions induced by bicuculline (an antagonist of the GABA(A) receptor) in CD1 mice. A combination of subactive doses of the two compounds showed additive anticonvulsant effects. These results suggest that etifoxine and allopregnanolone bind to distinct putative recognition sites at or near the chloride channel site. Functionally, their binding may have an additive effect by enhancing GABA(A) inhibitory transmission.

Synaptically released 5-HT modulates the activity of tonically discharging neuronal populations in the rostral ventral medulla (RVM).

There is substantial evidence for an important modulating role of monoamines (catecholamines and serotonin, 5-HT) in the rostral ventral medulla (RVM), a region which plays an important role in cardiovascular and nociceptive functions. We investigated in slices the role of endogenous monoamines in the synaptic control of the activity of rat RVM neuronal populations using intracellular recordings in the lateral RVM plus lateral aspect of nucleus paragigantocellularis lateralis. A triple-labelling protocol allowed us to identify the location of impaled neurons and their eventual monoaminergic phenotype within the serotonergic and catecholaminergic populations of the RVM. Focal electrical stimulation revealed the existence of a functional monoaminergic input onto RVM neurons which was mediated by endogenous 5-HT acting at inhibitory 5-HT1A receptors but did not involve noradrenergic neurotransmission. The slow 5-HT-mediated inhibitory postsynaptic potential (IPSP) was only observed in the regularly discharging neurons, which were found to be neither catecholaminergic nor serotonergic. The synaptic release of 5-HT was, itself, under an inhibitory control involving GABAA (gamma-aminobutyric acid) receptors. Moreover, we characterized the effect of the 5-HT-releasing agent fenfluramine on this functional 5-HT-mediated synaptic transmission. Our results show that the effect of fenfluramine is biphasic consisting of an initial prolongation of the serotonergic IPSP followed by a decrease in amplitude. Our data provide a basis for the previously reported inhibitory effects of exogenously applied serotonin agonists/antagonists on the autonomic functions controlled by the RVM. This 5-HT pathway, which functionally links the serotonergic and catecholaminergic regions, might play an important role in cardiovascular and nociceptive functions.

Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine.

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABA(A) receptor function. Etifoxine displaced [(35)S]TBPS (t-butylbicyclophosphorothionate) from GABA(A) receptors of rat cortical membranes with an IC(50) of 6.7+/-0.8 microM and [(3)H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC(50) of 27.3+/-1.0 microM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABA(A) receptor-mediated membrane currents elicited by submaximal (5-10 microM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 microM which reflected the post-synaptic potentiation of a small ( approximately 20 pA) tonic and bicuculline-sensitive GABA(A) receptor-gated Cl(-) current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 microM), an antagonist of central-type benzodiazepine sites present at GABA(A) receptors, but were partly inhibited by PK11195 (10 microM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABA(A) receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

Presynaptic P2X receptors facilitate inhibitory GABAergic transmission between cultured rat spinal cord dorsal horn neurons.

The superficial layers of the spinal cord dorsal horn (DH) express P2X2, P2X4, and P2X6 subunits entering into the formation of ionotropic (P2X) receptors for ATP. Using a culture system of laminae I-III from neonatal rat DH, we show that ATP induced a fast nonselective cation current in 38% of the neurons (postsynaptic effect). ATP also increased the frequency of miniature IPSCs (mIPSCs) mediated by GABA(A) receptors or by glycine receptors in 22 and 9%, respectively, of the neurons tested (presynaptic effect) but had no effect on glutamatergic transmission. The presynaptic effect of ATP on GABAergic transmission was not significantly affected by thapsigargin (1 microM) but was completely dependent on Ca(2+) influx. Presynaptic and postsynaptic effects were inhibited by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and reactive blue and were not reproduced by uridine 5'-triphosphate (UTP) or adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S), suggesting the implication of ionotropic P2X rather than of metabotropic P2Y receptors. alphabeta-methylene-ATP (100 microM) did not reproduce the effects of ATP. ATP reversibly increased the amplitude of electrically evoked GABAergic IPSCs and reduced paired-pulse inhibition or facilitation without affecting IPSC kinetics. This effect was preferentially, but not exclusively, observed in neurons coreleasing ATP and GABA. We conclude that in cultured DH neurons, the effects of ATP are mediated by P2X receptors having a pharmacological profile dominated by the P2X2 subunit. The presynaptic receptors might underlie a modulatory action of ATP on a subset of GABAergic interneurons involved in the spinal processing of nociceptive information.

Interactions of etifoxine with the chloride channel coupled to the GABA(A) receptor complex.

This study examined the nature of the interactions of etifoxine, an anxiolytic and anticonvulsant compound, with the GABA(A) receptor/chloride channel complex. In membrane preparations of Sprague-Dawley rat cerebral cortex, etifoxine competitively inhibited the binding of [35S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA(A) receptor chloride channel site. In vivo studies demonstrated an anticonvulsant effect of etifoxine (50 and 75 mg/kg, i.p.) against the clonic convulsions induced by TBPS in CD1 mice. Flumazenil (10 and 40 mg/kg, i.p.), an antagonist of benzodiazepine sites at GABA(A) receptors, had no effect on the action of etifoxine. These findings suggest that etifoxine exerts its effect by interacting with the Cl- channel of GABA(A) receptors and probably by facilitating GABAergic inhibition.

Functional high- and low affinity agonist binding sites at native dorsal horn AMPA receptors.

Transmission and processing of nociceptive information in the superficial dorsal horn (DH) of the spinal cord involves activation of AMPA-type glutamate receptors (AMPARs). We have studied the properties of native AMPARs in freshly dissociated laminae I-II neurones from postnatal rats using a modified form of the concentration-clamp technique for fast agonist application. Analysis of kainic acid dose-response curves showed the existence of two types of functional agonist binding sites governing AMPAR activation. These sites differ by their affinity for the agonist. Depending on the neurotransmitter concentration reached in the synaptic cleft, the relative contribution of high affinity and low affinity sites might play an important role in the shaping of AMPAR-mediated postsynaptic currents.

Synaptic corelease of ATP and GABA in cultured spinal neurons.

In the spinal dorsal horn (DH), transmission and modulation of peripheral nociceptive (pain-inducing) messages involve classical neurotransmitters and neuropeptides. We show that approximately half of DH neurons use ATP as a fast excitatory neurotransmitter acting at ionotropic P2X postsynaptic receptors. ATP was not codetected with glutamate but was coreleased with the inhibitory neurotransmitter GABA. Moreover, adenosine, probably generated by extracellular metabolism of ATP, finely tuned GABAergic inhibitory postsynaptic currents. Differential modulation of excitatory versus inhibitory components of this mixed cotransmission may help to explain changes in sensory message processing in the DH during mechanical hyperalgesia and neuropathic pain.

Lability of the pacemaker activity in the rat rostro-ventrolateral medulla: effects of noradrenaline.

Cardiovascular neurons in the Rostro-Ventrolateral Medulla (RVLM) have been shown to display a regular action potential discharge activity in vitro which has been proposed to result either from pacemaker conductances or from the activity of an oscillating network. Using intracellularly recordings in vitro from regularly-discharging RVLM neurons, we observed in young adult rats (> 19 days) that the regular discharge activity in RVLM is labile, as many neurons were or spontaneously became quiescent during the recording. A regular discharge could be induced or restored in quiescent neurons by superfusing an external concentration of K+ ions ([K+]ext) of 6 mM. In order to elucidate how neurotransmitters might influence this discharge activity, we studied the effects of a catecholamine, noradrenaline (present in this brain region). Noradrenaline depolarized or increased the spiking frequency of regularly-discharging neurons. This excitatory effect was sensitive to prazosin and propranolol. In the presence of these two blockers, noradrenaline induced a hyperpolarization sensitive to idazoxan and mimicked by alpha 2-adrenergic agonists. These effects persisted in the presence of tetrodotoxin. In spontaneously active neurons, prazosin plus propranolol abolished the discharge activity. At hyperpolarized potentials, the adrenoceptor blockers reduced the baseline synaptic/oscillatory activity. Our results demonstrate that the regular discharge activity of RVLM neurons is labile and depends on external ionic conditions, such as [K+]ext. This discharge activity can be modulated by catecholamines, acting at the 3 subtypes of adrenoceptors which co-exist on the same neuron. We propose that an endogenous release of catecholamines may condition the discharge activity of RVLM neurons.

Electrophysiological properties of cultured neonatal rat dorsal horn neurons containing GABA and met-enkephalin-like immunoreactivity.

We have developed a culture of neurons dissociated from the most superficial laminae of the neonatal rat spinal cord dorsal horn (DH). By using the perforated patch-clamp technique, we distinguished four types of neurons based on their firing properties in response to intracellular injection of 900 ms lasting current pulses. Type 1 neurons were characterized by a tonic firing. Type 2 neurons displayed marked spike accommodation and fired brief (<500 ms) bursts of action potentials, whereas type 3 neurons fired a single spike. Type 4 neurons exhibited different types of firing patterns, but all of them possessed a time-dependent inwardly rectifying current activated by membrane hyperpolarization. Met-enkephalin-like immunoreactivity (met-ENK-LI) and glutamic acid decarboxylase-like immunoreactivity (GAD-LI) were colocalized in 42% of the neurons (n = 59), which were previously identified electrophysiologically. Type 1-4 neurons represented respectively 4, 64, 20, and 12% of the population of neurons colocalizing met-ENK-LI and GAD-LI. We conclude that the electrophysiological properties of DH neurons present in our cultures are similar to those described in acute slice or hemisected spinal cord preparations and that met-ENK-LI and GABA-LI are preferentially colocalized in type 2 neurons.

Oxytocin modulates glutamatergic synaptic transmission between cultured neonatal spinal cord dorsal horn neurons.

The functional characteristics of binding sites for the neuropeptide oxytocin (OT) detected by radioautography in laminae I and II of the dorsal horn (DH) and on cultured neonatal DH neurons were studied on the latter using perforated patch-clamp recordings. The neurons were identified by their spike discharge properties and on the basis of the presence of met-enkephalin-like and glutamate decarboxylase-like immunoreactivities. OT (100 nM) never induced any membrane current at a holding potential of -60 mV but increased the frequency of spontaneously occurring AMPA receptor-mediated EPSCs or the mean amplitude of electrically evoked EPSCs in a subset (35%) of neurons. The frequency of miniature EPSCs (m-EPSCs) recorded in the presence of 0.5 microM tetrodotoxin was also increased by OT (100 nM) without any change in their mean amplitude, indicating an action at a site close to the presynaptic terminal. The decay kinetics of any type of EPSC were never modified by OT. The effect of OT was reproduced by [Thr4, Gly7]-OT (100 nM), a selective OT receptor agonist, and blocked by d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH29]-ornithine vasotocin (100 nM), a specific OT receptor antagonist. Reducing the extracellular Ca2+ concentration from 2.5 to 0.3 mM in the presence of Cd2+ (100 microM) reversibly blocked the effect of OT on m-EPSCs. The OT receptors described here may represent the substrate for modulatory actions of descending hypothalamo-spinal OT-containing pathways on the nociceptive system.

Modulation of GABAA receptor-mediated IPSCs by neuroactive steroids in a rat hypothalamo-hypophyseal coculture model.

1. We have used the whole-cell configuration of the patch-clamp technique to investigate the effects of neuroactive steroids on GABAA receptor-mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre- and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor-mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half-maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) reversibly potentiated IGABA in a dose-dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/- 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta-isomer of allopregnanolone, epipregnanolone (5 beta-pregnan-3 beta-ol-20-one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5-pregnen-3 alpha-ol-20-one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of -80 and -60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre- and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

Co-culture of hypothalamic neurons and melanotrope cells: a model to study synaptogenesis between central neurons and endocrine cells.

As a first step towards elucidating mechanisms involved in neuroendocrine synaptogenesis, we developed a model of co-culture based on hypothalamic-intermediate pituitary interactions. Dissociated hypothalamic neurons from fetal rats at embryonic day 15 were cultured in a defined medium together with melanotrope cells of the pituitary intermediate lobe from neonatal rats. In these co-cultures, establishment of synaptic contacts between GABAergic or dopaminergic neurons and an endocrine target cell the melanotrope cell, was studied by morphofunctional approaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and alpha-melanocyte-stimulating hormone, we demonstrated morphological contacts between GABAergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synapsin I, we showed a modification of synapsin I immunoreactivity from diffuse to punctate distribution correlated with the establishment of contacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrophysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic currents, which occurred either spontaneously and/or could be evoked chemically by 50 mM KCl or 100 microM kainate. The proportion of the melanotrope cells receiving functional synaptic inputs increased until 10 days in culture, a stage at which virtually all melanotrope cells in contact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approaches.

Electrophysiological characterization of non-NMDA glutamate receptors on cultured intermediate lobe cells of the rat pituitary.

Glutamate is the major excitatory neurotransmitter in the central nervous system, yet little is known about its actions on endocrine cells. We have investigated the membrane effects of glutamate in cultured neonatal rat pituitary intermediate lobe (IL) cells using the whole-cell configuration of the patch-clamp technique. In a standard Na(+)-based extracellular solution, glutamate failed to induce a detectable membrane current at a holding potential (HP) of -60 mV (n = 40). However, when cyclothiazide (50 microM), a benzothiazide that blocks desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole-propanoic acid (AMPA)-type receptors, was added to the extracellular solution, glutamate (0.5-1 mM) induced an inward current at a HP of -60 mV in 65% of the cells tested (n = 72). This response was usually small in amplitude (mean amplitude: 28.6 +/- 37.5 pA, n = 47). The glutamate-induced current reversed polarity close to 0 mV and was reversibly blocked when extracellular Na+ was replaced by the impermeant cation N-methyl-D-glucamine, suggesting that this current was a nonselective cation current. The response to glutamate (1 mM) was reproduced by AMPA (50 microM), kainate (200 microM), and quisqualate (200 microM). N-Methyl-D-aspartate (NMDA, 100 microM) in the presence of 10 microM glycine did not induce any membrane current in cells responding to glutamate (n = 8). The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) reversibly inhibited the response to glutamate (0.5 mM) by 85 +/- 14% (n = 7), whereas D(-)-2-amino-5-phosphonopentanoic acid (20 microM), an antagonist of NMDA receptors, had no effect on the glutamate-induced current (n = 3). Moreover, we show that although the amplitude of the glutamate currents was small, the latter induced large (30-mV) membrane depolarizations and triggered the firing of action potentials. Taken together, our results indicate that neonatal rat IL cells possess AMPA-type glutamate receptors that could possibly underlie a fast excitatory glutamatergic synaptic input to these cells.

Characterization of functional GABAergic synapses formed between rat hypothalamic neurons and pituitary intermediate lobe cells in coculture: Ca2+ dependence of spontaneous IPSCs.

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the alpha-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 microM) and SR95531 (5 microM), two competitive antagonists of the GABAA receptor. At a holding potential of -60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast ( < or = 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8-10 GABAA channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

Selective inhibition of high voltage-activated L-type and Q-type Ca2+ currents by serotonin in rat melanotrophs.

1. Whole-cell Ca2+ currents (ICa) from cultured rat melanotrophs were identified by their sensitivity to Ca2+ channel blockers, and their modulation by serotonin (5-HT) was studied. All cells displayed high voltage-activated (HVA; > -30 mV) Ca2+ currents. A low voltage-activated (LVA; > -60 mV) Ca2+ current was detected in 92% of the cells. 2. The whole-cell ICa was insensitive to omega-conotoxin GVIA (0.5-1 microM) indicating the absence of N-type Ca2+ channels. 3. At a holding potential (Vh) of -70 mV, the L-type channel blocker nifedipine reduced ICa in a dose-dependent manner with a half-maximal effective concentration (IC50) of 28 nM. The L-type current represented 39% of the total ICa. 4. omega-Agatoxin IVA (omega-Aga IVA) produced a biphasic dose-dependent inhibition of ICa, with IC50 values of 0.4 and 91 nM, indicating the presence of P-type and Q-type Ca2+ channels, which accounted respectively for 16 and 45% of the total ICa. The P-type current was also blocked by synthetic funnel-web spider toxin (sFTX 3.3; 1-10 microM) and was present only in a subpopulation (60-70%) of cells. 5. All cells possessed a Ca2+ current which was resistant to nifedipine (10 microM) and omega-Aga IVA (50 nM). This current was not affected by Ni2+ (40 microM) but was abolished by a low concentration of Cd2+ (10 microM) and by omega-conotoxin MVIIC (1 microM) indicating that it was a Q-type Ca2+ current. 6. 5-HT (10 microM) inhibited the whole-cell ICa in 70% of the cells tested (n = 120) by activating 5-HT1A and 5-HT2C receptors. 5-HT produced either a kinetic slowing of the activation phase (37% of the cells) or a scaling down (14% of the cells) of ICa. In the majority of cells (49%) both types of inhibition were found to coexist. 7. The effects of 5-HT were voltage dependent, rendered irreversible when GTP-gamma-S (30 microM) was present in the pipette solution and abolished by pretreatment of the cells with pertussis toxin (PTX; 150 ng ml-1, 18 h). 8. Low concentrations of omega-Aga IVA (20 nM), which blocked mainly P-type channels, did not reduce the effect of 5-HT on ICa. The scaling down effect of 5-HT on ICa was eliminated in the presence of nifedipine (10 microM) and the kinetic slowing effect of 5-HT persisted after blockade of L- and P-type channels but was abolished by omega-conotoxin MVIIC (1 microM). 9. We conclude that rat melanotrophs possess functional L-, P- and Q-type Ca2+ channels and that 5-HT inhibits selectively L-type and Q-type Ca2+ currents with different modalities. These effects are voltage dependent and mediated by a PTX-sensitive G-protein.

Calcium influx through neuronal-type nicotinic acetylcholine receptors present on the neuroendocrine cells of the porcine pars intermedia.

The properties of neuronal-type nicotinic acetylcholine receptors (nAChRs) present on the neuroendocrine cells of the porcine pars intermedia of the pituitary were studied in intact single cell using measurements of the free intracellular Ca2+ concentration ([Ca]i) with the calcium-sensitive dye fura 2. Local application of an extracellular solution containing 50 mM K+ or of the selective nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) depolarised the cells and induced an elevation in [Ca]i. The effect of DMPP on [Ca]i was dose dependent (EC50 = 6 microM), reversibly blocked by d-tubocurarine and strictly dependent on the concentration of extracellular Ca2+. The calcium channel blocker Cd2+ (100 microM) reversibly blocked 80% of the response induced by 50 mM K+, whereas it reduced the DMPP response by only 50%. In the absence of extracellular Na+, DMPP no longer depolarised the cells but still increased [Ca]i. The rise in [Ca]i under these conditions represented 41% of the control response, i.e. in the presence of external Na+. Thus activation of nAChRs induced an elevation in [Ca]i which was in part independent of cell depolarisation. This was confirmed by recording simultaneously, under whole-cell voltage-clamp, a rise in [Ca]i associated with the inward nicotinic current. During prolonged application of the agonist (50 s), the amplitude of the nicotinic current decayed rapidly to a very low plateau level reflecting nAChR desensitisation. However, photometric experiments performed on intact non-dialysed cells revealed the presence of a slowly decaying phase in [Ca]i throughout the application of DMPP. This suggests the persistence of a substantial Ca2+ influx during prolonged exposure to the agonist. Taken together, our results show that stimulation of nAChRs induces an influx of Ca2+ which elevates [Ca]i. This phenomenon is due to activation of voltage-dependent Ca2+ channels and to Ca2+ entry through the nAChR.

Subpopulations of neonatal rat sensory neurons express functional neurotransmitter receptors which elevate intracellular calcium.

We have attempted to identify which subpopulations of rat sensory neurons possess functional neurotransmitter receptors which elevate the free concentration of intracellular calcium. Subpopulations of sensory neurons were identified using three accepted criteria: (i) the distribution and proportion of neurons with differing somatic diameters; (ii) the expression of substance P-like immunoreactivity; and (iii) the responsiveness of each neuron to capsaicin. The total neuronal population was primarily grouped into three classes according to somatic diameter and defined as small- (< 17 microns), intermediate- (17-25 microns) and large- (> 25 microns) sized neurons. It was not possible to distinguish between small and intermediate-sized neurons since a similar percentage of each class expressed substance P-like immunoreactivity or sensitivity to capsaicin. Large-sized neurons did not possess these characteristics and, therefore, represented a distinct neuronal population. In single, intact neurons of differing diameter, the ability of a variety of receptor agonists to elevate the free concentration of intracellular calcium was determined using the calcium-sensitive indicator, Fura-2. Local application of capsaicin, adenosine, bradykinin, ATP and substance P elevated the resting level of the free concentration of intracellular calcium in small and intermediate-sized neurons. The large-sized neurons were unresponsive to these receptor agonists with the exception of ATP. The response to ATP was relatively transient in nature and did not differ between neurons of differing somatic diameter.(ABSTRACT TRUNCATED AT 250 WORDS)