Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23.

The orphan receptor ChemR23 is a G-protein coupled receptor (GPCR) with homology to neuropeptide and chemoattractant receptors. Tazarotene, a synthetic retinoid activating retinoic acid receptor (RAR), up-regulates tazarotene-induced gene-2 (TIG2). The function and molecular target of this protein are now described. By means of reverse pharmacology screening using a peptide library generated from human hemofiltrate, we have isolated and identified TIG2 as the natural ligand of ChemR23 and report the specific molecular form of the bioactive, circulating TIG2, representing the amino-acid residues 21 to 154 of the 163 amino acid-containing prepropeptide. Based on the expression pattern of ChemR23 and TIG2, the physiological role in bone development, immune and inflammatory responses and the maintenance of skin is now being investigated.

Modulation of K(+)-induced synaptosomal calcium influx by gabapentin.

Gabapentin, a drug useful in several neurological and psychiatric disorders, decreased K(+) (15 mM)-induced [Ca(2+)](i) increase in Fura-PE3-loaded rat neocortical synaptosomes (IC(50)=9.7 microM; submaximal inhibition of 28.6%). This effect may indicate a selective modulation of presynaptic Ca(2+) influx in response to depolarizing (pathological) conditions causing excessive neurotransmitter release.

Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects.

The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6 microM, Hill coefficient =1.5). The mean peak current induced by 30 microM 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC50 values=95 microM and 127 microM, Hill coefficient = -1.0 and -1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380 microM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 approximately 500 microM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (> or = 300 microM) were necessary to induce moderate (< or = twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits hS-HT3A receptor-mediated currents at anaesthetic concentrations (approximately 90 microM).

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices.

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.

Calcium channels involved in K+- and veratridine-induced increase of cytosolic calcium concentration in human cerebral cortical synaptosomes.

Human cerebral cortical synaptosomes were used to study voltage-dependent Ca(2+) channels mediating calcium influx in human axon terminals. Synaptosomes were depolarized by elevation of the extracellular K(+) concentration by 30 mM or by the addition of veratridine (10 microM). Increase in cytosolic concentration of calcium [Ca(2+)](i) induced by either stimulus was abolished in the absence of extracellular Ca(2+) ions. omega-Agatoxin IVA inhibited the K(+)-induced [Ca(2+)](i) increase concentration-dependently (IC(50): 113 nM). omega-Conotoxin GVIA (0.1 microM) inhibited K(+)-induced [Ca(2+)](i) increase by 20%. omega-Conotoxin MVIIC (0.2 microM) caused an inhibition by 85%. Nifedipine (1 microM) had no effect on K(+)-induced [Ca(2+)](i) increase. Veratridine-induced increase in [Ca(2+)](i) was inhibited by omega-conotoxin GVIA (0.1 microM) and omega-Agatoxin IVA (0.2 microM; by about 25 and 45%, respectively). Nifedipine inhibited the veratridine-evoked [Ca(2+)](i) increase concentration-dependently (IC(50): 4.9 nM); Bay K 8644 (3 microM) shifted the nifedipine concentration-response curve to the right. Mibefradil (10 microM) abolished the increase in [Ca(2+)](i) evoked by K(+) and reduced the increase evoked by veratridine by almost 90%. KB-R7943 (3 microM) an inhibitor of the Na(+)/Ca(2+) exchanger NCX1, decreased the increase in [Ca(2+)](i) evoked by veratridine by approximately 20%. It is concluded that the increase in [Ca(2+)](i) after K(+) depolarization caused by Ca(2+) influx predominantly via P/Q-type Ca(2+) channels and after veratridine depolarization via N- and P/Q-type, but also by L-type Ca(2+) channels. The toxin- and nifedipine-resistant fraction of the veratridine response may result both from influx via R-type Ca(2+) channels and by Ca(2+) inward transport via Na(+)/Ca(2+) exchanger.

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes.

Intracellular calcium ion concentrations ([Ca2+]i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K+ concentration or by addition of veratridine, which opens voltage-dependent Na+-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K+ (up to 60 mM) and veratridine (1-100 microM) increased the [Ca2+]i in a concentration-dependent manner. In the absence of extracellular Ca2+, the K+- and veratridine-induced increases in [Ca2+]i were abolished, indicating that the increase in [Ca2+]i was due to an influx of extracellular Ca2+. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na+ channel, inhibited the veratridine-induced (10 microM) Ca2+ influx by more than 80%, while the K+-evoked (30 mM) increase of [Ca2+]i was TTX-resistant. Both the K+- and the veratridine-induced Ca2+ influx were not reduced by nifedipine (1 microM), a blocker of L-type Ca2+ channels. Blockade of the voltage dependent N-type Ca2+ channels with omega-conotoxin GVIA (omega-CTx GVIA; 0.1 microM) and of the voltage-dependent P/Q-type channels with omega-agatoxin IVA (omega-AgaTx IVA; 0.2 microM) inhibited the K+-induced increase in [Ca2+]i by about 30 and 55%, respectively; these effects were additive. Omega-conotoxin MVIIC (omega-CTx MVIIC) at a concentration of 0.2 microM, which may be assumed to block predominantly the Q-type Ca2+ channel, inhibited the K+-induced increase in [Ca2+]i by 50%. The veratridine-induced increase in [Ca2+]i was reduced by about 25% by omega-CTx GVIA (0.1 microM), but was resistant to omega-AgaTx IVA (0.2 microM) and omega-CTx MVIIC (0.2 omegaM). Mibefradil (former designation Ro 40-5967), a Ca2+ antagonist which blocks all types of voltage-dependent Ca2+ channels including the T and R channels, led to a concentration-dependent inhibition of the K+- and veratridine-induced increase in [Ca2+]i (abolition at 10 microM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca2+ channels, also inhibited the K+- and veratridine-induced increase in [Ca2+]i in concentration-dependent manner and abolished it at 320 microM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 microM), a highly potent and selective inhibitor of the Na+/Ca2+ exchanger (NCX1), failed to inhibit the K+- and veratridine-induced increase in [Ca2+]i. It is concluded that the K+-induced increase in free cytosolic Ca2+ results from Ca2+ influx through voltage-dependent N- and, above all, Q-type Ca2+ channels. N-type Ca2+ channels also play a minor role in the veratridine-induced increase in [Ca2+]i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, omega-CTx GVIA- and omega-AgaTx IVA-resistant increase in [Ca2+]i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca2+ influx via the R-type Ca2+ channel and/or another so far uncharacterized Ca2+ channel may substantially contribute to the veratridine-induced increase in [Ca2+]i.

Cultured chick sympathetic neurons: prostanoid EP1 receptor-mediated facilitation of noradrenaline release.

Prostanoid EP receptor-mediated modulation of noradrenaline release from cultured chick sympathetic neurons was investigated. Transmitter release from dissociated cell cultures of embryonic paravertebral ganglia, loaded with [3H]-noradrenaline, was elicited either by electrical field stimulation (36 pulses/3 Hz) or by elevating the extracellular concentration of K+ (to 30 mM; for 2 min). Prostaglandin E2 (PGE2; 0.01-3 microM) enhanced electrically evoked [3H]-noradrenaline release in a concentration-dependent manner with a maximal increase by about 50% at 1 microM. Also iloprost (0.1-3 microM) increased transmitter release concentration- dependently, whereas misoprostol (0.1-3 microM) had no effect. Indometacin (10 microM) influenced neither evoked release per se nor the enhancement caused by PGE2- AH6809 (3 microM), a selective EP1 receptor antagonist, blocked the enhancement caused by both PGE2 and iloprost K(+)-evoked noradrenaline release, which was virtually insensitive to tetrodotoxin (0.3 microM), was increased by PGE2 to an extent comparable to that observed after electrical stimulation. In summary, the present data indicate that PGE2 facilitates noradrenaline release from cultured chick sympathetic neurons by a receptor which shows the pharmacological profile of the EP1 subtype and is probably located at the processes of the neuron.