Phorbol ester blocks the increase of a high affinity GTPase activity induced by δ2-opioid receptor agonist in the mouse spinal cord

The high affinity GTPase activity in the mouse spinal cord was increased in a concentration-dependent manner by a selective δ₂-opioid receptor agonist, [ -Ala²]deltorphin II (0.1-1 μM). This increase of GTPase activity induced by [ -Ala²]deltorphin II was completely blocked by co-incubation with a selective δ₂-opioid receptor antagonist, naltriben (0.1 μM). A protein kinase C activator, phorbol 12,13-dibutyrate (PDB; 0.1-10 μM), which given alone had no effect on basal GTPase activity, blocked dose-dependently the increase of GTPase activity induced by [ -Ala²]deltorphin II (1 μM). Our results indicate the possibility that activation of protein kinase C by phorbol ester uncouples the δ₂-opioid receptor from G-proteins in the spinal cord.     

The neuropeptide FF analogue, 1DMe, acts as a functional opioid autoreceptor antagonist in the rat spinal cord

We assessed the possible influence of a neuropeptide FF analogue, 1DMe ([ -Tyr¹,(NMe)Phe³]neuropeptide FF), on the inhibitory action of endogenous and exogenous ∂-opioid receptor agonists on K⁺-evoked [Met⁵]-enkephalin release from superfused rat spinal cord slices. 1DMe (0.1–10 μM) dose-dependently enhanced the increase in superfusate [Met⁵]-enkephalin content due to the peptidase inhibitors thiorphan (1 μM) and bestatin (20 μM), and prevented the reduction in [Met⁵]-enkephalin release due to stimulation of ∂ receptors by 1 μM deltorphin I. Because it had the same effects as ∂-opioid receptor antagonists, 1DMe might act through the functional blockade of presynaptically located ∂-opioid autoreceptors.     

Is 5-hydroxytryptamine mediating descending inhibition in the neonatal rat spinal cord through different receptor subtypes?

The long-lasting descending inhibition of lumbar segmental reflexes in the neonatal rat spinal cord has been investigated in vitro by recording from lumbar ventral roots on stimulation of a single lumbar dorsal root. Descending inhibition was elicited by a single stimulus to the latero-ventral thoracic cord. A number of strategies were used to clarify the role of 5-hydroxytryptamine (5-HT) in inhibiting the monosynaptic reflex, the ipsilateral polysynaptic response and the contralateral fast response evoked on the opposite side of the lumbar cord. The 5-HT uptake inhibitor, citalopram (10 nM), potentiated both short-interval (0.5–2 s) inhibition and long-interval (5–100 s) inhibition of the monosynaptic reflex, and also inhibition of the polysynaptic response 10–100 s after the thoracic stimulus. Inhibition of the monosynaptic reflex was blocked by ketanserin (1 μM), spiperone (1 μM) and methiothepin (1 μM), but not by spiroxatrine (0.1 μM) or sulpiride (1 μM). Sumatriptan (20 nM) and methysergide (10 nM) enhanced inhibition of the monosynaptic reflex 0.2–1 s after the thoracic stimulus. It was concluded that 5-HT acting through 5-HT_2A/2C receptors is the transmitter responsible for monosynaptic reflex inhibition, at intervals of 0.5–100 s, but a stronger stimulus to the thoracic cord may elicit a non-serotonergic component at intervals of 0.1–2 s. There was no unequivocal evidence that endogenous 5-HT activates 5-HT₁ receptors to produce inhibition. 5-HT acting through 5-HT_2A/2C receptors is also responsible for part of the inhibition of the polysynaptic response, but the transmitter(s) responsible for the ketanserin-resistant component of inhibition and for inhibition of the contralateral fast response has still to be identified.     

Lobeline inhibits nicotine-evoked [H]dopamine overflow from rat striatal slices and nicotine-evoked Rb efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [³H] overflow from [³H]dopamine ([³H]DA)-preloaded rat striatal slices and ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(−)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(−)-nicotine was determined. Both S(−)-nicotine (0.1–1 μM) and lobeline (>1.0 μM) evoked [³H] overflow from superfused [³H]DA-preloaded striatal slices. However, lobeline-evoked [³H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 μM) which did not evoke [³H] overflow, lobeline inhibited S(−)-nicotine (0.1–10 μM)-evoked [³H] overflow, shifting the S(−)-nicotine concentration–response curve to the right. S(−)-Nicotine (30 nM–300 μM) increased (EC₅₀ VALUE=0.2 μM) ⁸⁶Rb⁺ efflux from thalamic synaptosomes. In contrast, lobeline (1 nM–10 μM) did not evoke ⁸⁶Rb⁺ efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC₅₀ VALUE=0.7 μM) ⁸⁶Rb⁺ efflux evoked by 1 μM S(−)-nicotine, a concentration which maximally stimulated ⁸⁶Rb⁺ efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(−)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.     

Diisopropylfluorophosphate-induced depression of segmental monosynaptic transmission in neonatal rat spinal cord is also mediated by increased axonal activity

Involvement of dorsal and ventral root activity for the depressant action of diisopropylfluorophosphate (DFP) on synaptic transmission was examined using in vitro spinal cord/root preparations. Superfusion of DFP produced a dose-dependent depression of monosynaptic reflex (MSR) and maximal depression of about 80% occurred at 1000 μM. The concentration to produce 50% of the maximal inhibition was about 100 μM of DFP. The DFP (100 μM)-induced depression of MSR was reversed by atropine (0.5 μM) but not by mecamylamine (0.5 μM). Contrary to the action on MSR, DFP potentiated the ventral root potential and 1st peak of dorsal root potential. The maximal potentiation was about 25% of control in both the root potentials at 100 μM of DFP. However, the second peak of dorsal root potential was slightly depressed (10–20% of control) by DFP (1–1000 μM). Further, the cords treated with DFP (100 μM) showed significant decrease in the cholinesterase (ChE) activity (27% of control). Results suggest that the DFP-induced depression was mediated at least by two different mechanisms, one through the inhibition of ChE activity and the other through the activation axonal activity having inhibitory inputs to the segmental synaptic transmission. These inputs mediate their action through muscarinic receptors.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

Effects of L- and N-type Ca channel antagonists on excitatory amino acid-evoked dopamine release

In thc present study we tested the effect of dihydropyridine (DHP) Ca²⁺ channel antagonists and of ω-conotoxin GVIA on [³H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca²⁺ channels in these EAA-mediated processes. Micromolar concentrations (10–30 μM) of DHP L-type Ca²⁺ channel antagonists inhibited [³H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [³H]DA release evoked by the L-type Ca²⁺ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1–1 μM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, ( + )-PN 200-110, was more potent than ( − )-PN 200-110 in inhibiting [³H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca²⁺ channels is able to evoke [³H]DA release. However activation of L-type channels is not involved in EAA-induced [³H]DA release and therefore inhibition of EAA-induccd [³H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca²⁺ channels. ω-Conotoxin GVIA (3 μM) had no effect on [³H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation. ω-Conotoxin GVIA (3 μM) partially inhibited [³H]DA release evoked by NMDA or kainate, suggesting that N-type Ca²⁺ channels could possibly play a role in FAA-mediated responses in these cells.     

Effects of opioid drugs on capsaicin-sensitive neurones in guinea-pig atria

Transmural nerve stimulation of isolated guinea-pig atria in the presence of atropine induced a biphasic positive inotropic effect but only a slow increase in contractility (NANC response) in atria obtained from 6-hydroxydopamine-pretreated animals. The latter effect disappeared after exposure of the preparations to capsaicin. The effects of some opioid peptides were investigated on NANC responses. [D-Ala²,D-Leu⁵]enkephalin (DADLE) and [D-Ala²,N-Me-Phe⁴ Gly⁵-ol]enkephalin (DAGO, 0.1–10 μM) inhibited the cardiac response to transmural nerve stimulation in a dose-dependent and naloxone-sensitive manner. Dynorphin-(1–13) and morphine, at 10-fold higher concentrations (1–10 μM), reduced the response in a naloxone-sensitive manner. Naloxone alone however did not affect the response. Opioid peptides were not able to reduce the positive inotropic effect induced by calcitonin gene-related peptide (CGRP), or the increase in cardiac contractility produced by capsaicin. These results suggest that opioid receptors exert a modulatory role on peripheral terminals of capsaicin-sensitive sensory nerves.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

Biphasic effects of mianserin and desipramine on serotonin-evoked current and Cl- efflux in Xenopus oocytes.

Serotonin (5-HT, 1 μM) elicited two phases of Cl⁻ inward current in Xenopus oocytes injected with rat brain mRNA: a transient current (T-current), which was generated rapidly (within 1 min), and a sustained current (S-current), which persisted for 10 min. Each type of 5-HT-evoked response was time-dependent after mRNA injection. The T-current was generated at 20-30 h and the S-current at 30–40 h. Although mianserin at 0.1 μ M completely inhibited the T-current, 10 μ M mianserin was required to suppress the S-current. 5-HT also caused Cl⁻ efflux from oocytes preloaded with ³⁶Cl⁻, Cl⁻ efflux during 1 min, corresponding to the T-current, was inhibited by 0.1 μ M mianserin. A higher concentration of mianserin (10 μ M) was required to block the efflux for 10 min, corresponding to the S-current, as well as the current response. Desipramine selectively inhibited the T-current and Cl⁻ efflux for 1 min. The mechanisms underlying the different sensitivity to mianserin of oocytes injected with rat brain mRNA are discussed.     

Delta(9)-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Δ⁹-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca²⁺]_i spiking by Win55,212-2. In control experiments, 0.1 μM Win55,212-2 inhibited [Ca²⁺]_i spiking by 93 ± 5%. Win55,212-2 produced significantly less inhibition of [Ca²⁺]_i spiking following 18–24 h treatment with 1 μM THC (48 ± 5%) or treatment with 1 μM Win55,212-2 (29 ± 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 μM, remained stable between 1 and 7 days of preincubation and shifted the EC₅₀ of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.     

Thromboxane A(2)-mediated Cl(-) secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides in guinea pig tracheal smooth muscle

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 μM) and phosphoramidon (1 μM) and contracted with histamine (3 μM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4–23) was inactive suggesting the involvement of ANP_A receptors in the relaxant effect of ANP. ODQ (1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one, 10 μM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 μM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 μM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 μM), a selective blocker of ATP-sensitive K⁺ channels, and by apamin (1 μM), a selective blocker of small-conductance Ca²⁺-activated K⁺ channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca²⁺-activated K⁺ channels. In addition, ANP (10 nM) and ANP-(4–23) (100 nM) significantly reduced forskolin (1 μM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP_C receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4–23) or ANP demonstrating that the inhibitory effect of ANP-(4–23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.     

Blockade of 5-HT(3) receptor with MDL 72222 and Y 25130 reduces beta-amyloid protein (25--35)-induced neurotoxicity in cultured rat cortical neurons

The present study was performed to examine neuroprotective effects of 5-hydroxytryptamine (5-HT)₃ receptor antagonists against β-amyloid protein (25–35)-, a synthetic 25–35 amyloid peptide, induced neurotoxicity using cultured rat cortical neurons. β-Amyloid protein (25–35) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca²⁺ channel blocker, and N^G-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor. The 5-HT₃ receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1–10 μM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.05–5 μM), decreased the β-amyloid protein (25–35) (10 μM)-induced neuronal cell death as assessed by a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. MDL72222 and Y25130 inhibited the β-amyloid protein (25–35) (10 μM)-induced elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and glutamate release, generation of reactive oxygen species, and caspase-3 activity. These neuroprotective effects of MDL72222 (10 μM) and Y25130 (5 μM) were completely blocked by the simultaneous treatment with 100 μM 1-phenylbiguanide, a 5-HT₃ receptor agonist, indicating that the protective effects of these compounds were due to 5-HT₃ receptor blockade. These results suggest that the activation of the 5-HT₃ receptor may be partially involved in β-amyloid protein-induced neurotoxicity, by membrane depolarization for Ca²⁺ influx. Therefore, the blockade of 5-HT₃ receptor with MDL72222 and Y25130, may ameliorate the β-amyloid protein-induced neurotoxicity by interfering with the increase of [Ca²⁺]_c, and then by inhibiting glutamate release, generation of reactive oxygen species and caspase-3 activity.     

Interleukin-2 regulates membrane potentials and calcium channels via mu opioid receptors in rat dorsal root ganglion neurons

Our previous studies revealed that interleukin-2 (IL-2) exerted peripheral antinociception that was partially mediated by μ opioid receptors. No ionic explanations of this effect have yet been reported. The present study was designed to investigate effects of IL-2 on the physiological properties of capsaicin-sensitive small dorsal root ganglion (DRG) neurons, which are predominantly responsible for nociceptive transmission from the periphery to the spinal cord. Intracellualr recordings of DRG neurons were made in DRG/peripheral nerve preparation in vitro. IL-2 (10³ U/ml) produced membrane hyperpolarization of –9.4 ± 3.0 mV and this effect was blocked by β-FNA (5 μM), a μ opioid receptor antagonist. Under whole-cell patch clamp recordings, transient high-threshold Ca²⁺ currents were inhibited by –56.6 ± 11.3% by IL-2. Simultaneous calcium imaging showed that this cytokine also inhibited depolarization-evoked increase in intracellular calcium concentration. All the effects of IL-2 were blocked by naloxone (1 μM). Consistent with previous studies, DAMGO, a selective μ opioid agonist, exerted similar inhibitory effects on membrane potentials and Ca²⁺ currents.

The present results indicated that μ opioid receptors were involved in the regulatory effects of IL-2 on membrane potentials and calcium channels in DRG neurons, which may contribute to IL-2-induced peripheral analgesia.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

Effects of adenosine A2 receptor agonists on the excitation of capsaicin-sensitive afferent sensory nerves in airway tissues

We examined the effects of adenosine analogues on the asthmatic reactions induced by the stimulation of capsaicin-sensitive afferent sensory nerves. Intravenous (i.v.) injection of adenosine A₂ receptor agonists, 5′-(N-ethylcarboxamido)-adenosine (NECA) and 2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamido-adenosine (CGS 21680), dose dependently inhibited capsaicin-induced guinea-pig bronchoconstriction (1–1000 nmol kg⁻¹), whereas i.v. administration of the adenosine A₁ receptor agonist, N⁶-cyclo-hexyladenosine (CHA), did not affect it (1000 nmol kg⁻¹). Intratracheal injection of NECA (0.05–5 nmol site⁻¹) and CGA 21 680 (0.05−5 nmol site⁻¹) also reduced capsaicin-induced constriction in a dose-dependent manner. However, NECA (1000 nmol kg⁻¹) failed to inhibit substance P-induced guinea-pig bronchoconstriction. NECA (1–1000 nmol kg⁻¹) dose-dependently inhibited cigarette smoke-induced rat tracheal plasma extravasation, but not substance P-induced reaction. NECA (0.1–10 μM) and CGS 21 680 (10 μM) significantly blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea-pig lung, whereas CHA (10 μM) had no effect. This evidence suggests that adenosine A₂ receptors modulate negatively the excitation of capsaicin-sensitive afferent sensory nerves and substance P release from their endings in airway tissues.     

Selective effects of [D-Ser(O-t-butyl),Leu]enkephalyl-Thr and [D-Ser(O-t-butyl),Leu]enkephalyl-Thr(O-t-butyl), two new enkephalin analogues, on neurotransmitter release and adenylate cyclase in rat brain slices

The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser²(O-t-butyl), Leu⁵]enkephalyl-Thr⁶) and BUBU ([D-Ser²(O-t-butyl),Leu⁵]enkephalyl-Thr⁶(O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD₂ 7.6–7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [³H]dopamine (DA) release nor cortical [³H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD₂ 8.2–8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain.