The Cold Case of Metabotropic Glutamate Receptor 6: Unjust Detention in the Retina?

It is a common opinion that metabotropic glutamate receptor subtype 6 (mGluR6) is expressed exclusively in the retina, and in particular in the dendrites of ON-bipolar cells. Glutamate released in darkness from photoreceptors activates mGluR6, which is negatively associated with a membrane non-selective cation channel, the transient receptor potential melanoma-related 1, TRPM1, resulting in cell hyperpolarization. The evidence that mGluR6 is expressed not only in the retina but also in other tissues and cell populations has accumulated over time. The expression of mGluR6 has been identified in microglia, bone marrow stromal and prostate cancer cells, B lymphocytes, melanocytes and keratinocytes and non-neural tissues such as testis, kidney, cornea, conjunctiva, and eyelid. The receptor also appears to be expressed in brain areas, such as the hypothalamus, cortex, hippocampus, nucleus of tractus solitarius, superior colliculus, axons of the corpus callosum and accessory olfactory bulb. The pharmacological activation of mGluR6 in the hippocampus produced an anxiolytic-like effect and in the periaqueductal gray analgesic potential. This review aims to collect all the evidence on the expression and functioning of mGluR6 outside the retina that has been accumulated over the years for a broader view of the potential of the receptor whose retinal confinement appears understimated.

Palmitoylethanolamide reduces pain-related behaviors and restores glutamatergic synapses homeostasis in the medial prefrontal cortex of neuropathic mice.

BACKGROUND: Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. RESULTS: At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. CONCLUSIONS: Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states.

Antinociceptive effects of tetrazole inhibitors of endocannabinoid inactivation: cannabinoid and non-cannabinoid receptor-mediated mechanisms.

BACKGROUND AND PURPOSE: Tetrazoles were recently developed as inhibitors of the cellular uptake of the endocannabinoid anandamide or of its hydrolysis by fatty acid amide hydrolase (FAAH), but were proposed to act also on non-endocannabinoid-related serine hydrolases. EXPERIMENTAL APPROACH: We tested, in a model of inflammatory pain induced in mice by formalin, five chemically similar inhibitors: (i) OMDM119 and OMDM122, two potent carbamoyl tetrazole FAAH inhibitors with no effect on anandamide uptake; (ii) LY2183240, a carbamoyl tetrazole with activity as both FAAH and uptake inhibitor; (iii) OMDM132, a non-carbamoyl tetrazole with activity only as uptake inhibitor and iv) OMDM133, a non-carbamoyl tetrazole with no activity at either FAAH or uptake. RESULTS: All compounds (2.5-10 mg kg(-1), i.p.) inhibited the second phase of the nocifensive response induced by intraplantar injection of formalin. The effects of OMDM119, OMDM122 and OMDM133 were not antagonized by pretreatment with cannabinoid CB(1) receptor antagonists, such as rimonabant or AM251 (1-3 mg kg(-1), i.p.). The effects of LY2183240 and OMDM132 were fully or partially antagonized by rimonabant, respectively, and the latter compound was also partly antagonized by the CB(2) receptor antagonist, AM630. CONCLUSIONS AND IMPLICATIONS: (i) non-FAAH hydrolases might be entirely responsible for the antinociceptive activity of some, but not all, tetrazole FAAH inhibitors, (ii) the presence of a carbamoylating group is neither necessary nor sufficient for such compounds to act through targets other than FAAH and (iii) inhibition of anandamide uptake is responsible for part of this antinociceptive activity, independently of effects on FAAH.

The antinociceptive effect of 2-chloro-2'-C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), a highly selective adenosine A1 receptor agonist, in the rat.

This study was undertaken in order to investigate the effect of 2-chloro-2'-C-methyl-N(6)-cyclopentyladenosine (2'-Me-CCPA), a potent and highly selective adenosine A(1) receptor agonist, on nociceptive responses and on the ongoing or tail flick-related changes of rostral ventromedial medulla (RVM) ON- and OFF-cell activities. Systemic administrations of 2'-Me-CCPA (2.5-5 mg/kg, i.p.) reduced the nociceptive response in the plantar and formalin tests, in a way prevented by DPCPX (3 mg/kg, i.p.), a selective A(1) receptor antagonist. Similarly, intra-periaqueductal grey (PAG) 2'-Me-CCPA (0.5-1-2 nmol/rat) reduced pain behaviour in the plantar and formalin tests, in a way inhibited by DPCPX (0.5 nmol/rat). Moreover, when administered systemically (2.5-5 mg/kg, i.p.) or intra-PAG (0.5-1 nmol/rat) 2'-Me-CCPA increased the tail flick latencies, delayed the tail flick-related onset of the ON-cell burst and decreased the duration of the OFF-cell pause in a dose dependent manner. Furthermore, it decreased RVM ON-cell and increased OFF-cell ongoing activities. The in vivo electrophysiological effects were all prevented by DPCPX (0.5 nmol/rat). This study confirms the role of adenosine A(1) receptors in modulating pain and suggests a critical involvement of these receptors within PAG-RVM descending pathway for the processing of pain.

Effects of (S)-3,4-DCPG, an mGlu8 receptor agonist, on inflammatory and neuropathic pain in mice.

In this study, the effect of (S)-3,4-dicarboxyphenylglycine (DCPG), a selective mGlu8 receptor agonist, has been investigated in inflammatory and neuropathic pain models in order to elucidate the role of mGlu8 receptor in modulating pain perception. Inflammatory pain was induced by the peripheral injection of formalin or carrageenan in awake mice. Systemic administration of (S)-3,4-DCPG, performed 15 min before formalin, decreased both early and delayed nociceptive responses of the formalin test. When this treatment was carried out 15 min after the peripheral injection of formalin it still reduced the late hyperalgesic phase. Similarly, systemic (S)-3,4-DCPG reduced carrageenan-induced thermal hyperalgesia and mechanical allodynia when administered 15 min before carrageenan, but no effect on pain behaviour was observed when (S)-3,4-DCPG was given after the development of carrageenan-induced inflammatory pain. When microinjected into the lateral PAG (RS)-alpha-methylserine-O-phoshate (MSOP), a group III receptor antagonist, antagonised the analgesic effect induced by systemic administration of (S)-3,4-DCPG in both of the inflammatory pain models. Intra-lateral PAG (S)-3,4-DCPG reduced pain behaviour when administered 10 min before formalin or carrageenan; both the effects were blocked by intra-lateral PAG MSOP. (S)-3,4-DCPG was ineffective in alleviating thermal hyperalgesia and mechanical allodynia 7 days after the chronic constriction injury of the sciatic nerve, whereas it proved effective 3 days after surgery. Taken together these results suggest that stimulation of mGlu8 receptors relieve formalin and carrageenan-induced hyperalgesia in inflammatory pain, whereas it would seem less effective in established inflammatory or neuropathic pain.

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands.

BACKGROUND AND PURPOSE: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. EXPERIMENTAL APPROACH: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. KEY RESULTS: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CONCLUSIONS AND IMPLICATIONS: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

Metabotropic glutamate and cannabinoid receptor crosstalk in periaqueductal grey pain processing.

Metabotropic glutamate (mGlu) and cannabinoid receptors are G-protein coupled receptors which have shown synaptic co-operation through small lipid messengers in the central nervous system (CNS). A functional interaction between these two receptor families could have a relevant potential in the treatment of CNS disorders, including chronic pain. Indeed, both mGlu and cannabinoid receptors play a crucial role in the neurobiology of pain and their simultaneous manipulation could lead to novel strategies in pain management. In particular, as both mGlu and cannabinoid receptors have been found in the periaqueductal gray (PAG), a crucial station in the pain modulatory system, these receptors could be a substrate for producing analgesia at this level. In this review we aim to briefly illustrate the role of mGlu and can-nabinoid receptors in controlling nociceptive processes, some points of convergence, and their functional interaction in pain processing. Further insights into this functional linkage between the mGlu and cannabinoid receptors could pave the way to a new strategy for pain relief, such as a drug cocktail acting on cannabinoid/metabotropic glutamate receptors.

Periaqueductal grey CB1 cannabinoid and metabotropic glutamate subtype 5 receptors modulate changes in rostral ventromedial medulla neuronal activities induced by subcutaneous formalin in the rat.

This study was undertaken to analyze the involvement of periaqueductal gray (PAG) cannabinoid or group I metabotropic glutamate receptors in the formalin-induced changes on the rostral ventromedial medulla (RVM) ON- and OFF-cells activities. S.c. injection of formalin into the hind paw produced a transient decrease (4-6 min) followed by a longer increase (25-35 min) in tail flick latencies. Formalin also increased basal activity in RVM ON-cells (42+/-7%) and decreased it in OFF-cells (35+/-4%). Intra-PAG microinjection of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2 nmol/rat), a cannabinoid receptor agonist, prevented the formalin-induced changes in RVM cell activities. Higher dosages of WIN 55,212-2 (4-8 nmol/rat) increased the tail flick latencies, delayed the tail flick-related onset to ON-cell burst, and decreased the duration of OFF-cell pause. Furthermore, WIN 55,212-2 at a dosage of 8 nmol/rat decreased RVM ON-cell (57+/-7%) and increased OFF-cell ongoing activities (26+/-4%). These effects were prevented by N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide SR141716A, (1 pmol/rat), a CB1 cannabinoid receptor antagonist, or by 2-methyl-6-(phenylethynyl)pyridine (MPEP 20 nmol/rat), a selective mGlu5 glutamate receptor antagonist. T7-(hydroxyimino) cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCOOE/50 nmol/rat) and (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, 20 nmol/rat), selective mGlu1 glutamate receptor antagonists, were ineffective in preventing the WIN-induced effects. This study suggests that s.c. injection of formalin modifies RVM neuronal activities and this effect is prevented by PAG cannabinoid receptor stimulation. Moreover, the physiological stimulation of PAG mGlu5, but not mGlu1 glutamate receptors, seems to be required for the cannabinoid-mediated effect.

Type I and II metabotropic glutamate receptors modulate periaqueductal grey glycine release: interaction between mGlu2/3 and A1 adenosine receptors.

In this study we investigated the effects of type I and II mGlu receptors ligands in glycine extracellular concentrations at the periaqueductal gray (PAG) level by using in vivo microdialysis, in conscious rats. An agonist of type I mGlu receptors, (S)-3,5-DHPG (1 and 5 mM), but not a selective agonist for mGlu5 receptors, CHPG (3 and 5 mM), was noticed to increase the dialysate glycine levels in a concentration-dependent manner (60+/-15% and 136+/-13%, respectively). CPCCOEt (1mM), a selective mGlu1 receptor antagonist, perfused in combination with (S)-3,5-DHPG, counteracted the effect induced by (S)-3,5-DHPG, but did not change per se the extracellular PAG glycine values, even at the highest dosage used (2 mM). MPEP (1 and 2 mM), a selective antagonist of mGlu5 receptor, did not modify extracellular glycine level. An agonist of type II mGlu receptors, 2R,4R-APDC (25 and 50 microM), decreased the dialysate glycine in a concentration-dependent manner (-26+/-4% and -54+/-6%, respectively). The 2R,4R-APDC-induced decrease in extracellular glycine was prevented by EGlu (0.5 mM), a selective type II mGlu receptors antagonist. EGlu (0.5 and 1 mM), per se, led to a significant decrease (-56+/-7% and -57+/-2%, respectively) in extracellular PAG glycine too. This effect was prevented by DPCPX (100 microM), a selective antagonist for A1 adenosine receptors, but was not affected by CPA (1 mM), a selective A1 adenosine receptors agonist. Intra-PAG perfusion of CPA (0.1-1 mM) decreased the extracellular PAG glycine values (-47+/-13%) with 1 mM concentration. The CPA-induced effect was prevented by DPCPX (100 microM), and resulted to be additive with the 2R,4R-APDC-induced decrease in glycine values. DPCPX (1 mM) increased per se extracellular glycine (48+/-7%) at the highest dose used. Dipyridamole (100 microM), an inhibitor of both adenosine reuptake and phosphodiesterases, decreased extracellular glycine (-28+/-7%). Extracellular concentrations of glutamine never changed throughout this study. These data show opposing effects of type I and II mGlu receptors in the regulation of PAG glycine values. Moreover, functional interaction between type II mGlu and adenosine A1 receptors, which possibly operate through a common transductional pathway, may be relevant in the physiological control of glycine release in awake, freely moving rats at the periaqueductal gray matter.

Metabotropic and NMDA glutamate receptors participate in the cannabinoid-induced antinociception.

The purpose of this study was to evaluate the possible contribution of metabotropic glutamate receptors (mGluRs) to cannabinoid-induced antinociception in the periaqueductal grey (PAG) matter of rats. Intra-PAG microinjection of WIN 55,212-2, a cannabinoid receptor agonist, increased the latency of the nociceptive reaction (NR) in a dose-dependent fashion in the plantar test. This effect was prevented by pretreatment with SR141716A, a selective antagonist of CB1 receptors. When injected alone, SR141716A produced, with the highest dosage used, a significant reduction in the latency of the NR. CPCCOEt, a selective mGlu1 receptor antagonist, was unable to prevent the analgesia produced by WIN 55,212-2. On the contrary, MPEP, a selective mGlu5 receptor antagonist, completely antagonized the effect of WIN 55,212-2. However, the analgesia induced by CHPG, a selective mGlu5 receptor agonist, was blocked by MPEP but not by SR141716A. When injected alone, CPCOOEt produced no effect, whereas MPEP produced, with the highest dosage used, a significant reduction in the latency of the NR. These data emphasize that mGlu5 receptors, but not mGluR1, may modulate nociception in the PAG. Similarly, a pretreatment with either 2-(S)-alpha-EGlu or (RS)-alpha-MSOP, selective antagonists for group II and III mGluRs, respectively, prevented the WIN 55,212-2-induced analgesia. When the higher dosage of (RS)-alpha-MSOP was used a decrease in the latency of the NR was observed. This was not the case for 2-(S)-alpha-EGlu. Pretreatment with DL-AP5, a selective antagonist of N-methyl-D-aspartate (NMDA) receptors, blocked the effect of WIN 55,212-2, and by increasing the dosage strongly reduced per se the latency of the NR. This study suggests that endogenous glutamate could tonically modulate nociception through mGlu and NMDA receptors in the PAG matter. In particular, the physiological stimulation of these receptors seems to be required for the cannabinoid-induced analgesia in this midbrain area.

Effects of persistent nociception on periaqueductal gray glycine release.

Glycine is a candidate nociception inhibitory transmitter in specific brain regions, like for example the spinal cord, the thalamic nuclei and the periaqueductal gray matter. However, quantitative changes in glycine released in these brain regions during peripheral inflammation episodes have not been characterized in awake animals. To address this issue, an in vivo microdialysis study was carried out in freely moving rats in order to analyse periaqueductal gray matter extracellular glycine concentration following unilateral formalin injection into the dorsal skin of the right hind-paw. The extracellular concentration of glutamine was also evaluated in order to analyse whether or not a non-neurotransmitter amino acid was equally modified. Intra-periaqueductal gray matter tetrodotoxin perfusion reduced extracellular glycine concentration (-44+/-5%), but did not change the glutamine dialysate values. Peripheral injection of formalin reduced the glycine release during the early phase (-62+/-8%) and the late phase (-36+/-6%) of hyperalgesia, although not during the analgesic period. Perfusion with naloxone (300microM) neither prevented the formalin-induced decreases in extacellular glycine concentration, nor modified the perfusate basal values of glycine and glutamine. These results show that, contrary to what has been recognized on the interactive role of opioids and GABA into the periaqueductal gray matter (i.e. opioid disinhibition), endogenous opioids seem not to modulate the activity of glycinergic neurons in the same midbrain area. In the light of these preliminary data, it is reasonable to suppose that GABA and glycine are probably not co-released at the level of periaqueductal gray matter of the rat.

Periaqueductal gray matter metabotropic glutamate receptors modulate formalin-induced nociception.

The role played by periaqueductal gray (PAG) matter metabotropic glutamate receptors (mGluRs) in the modulation of persistent noxious stimulation was investigated in mice. The formalin test was used as a model of persistent pain. Intra-PAG microinjections of (S)-3, 5-DHPG (25 and 50 nmol/mouse) and L-CCG-I (30 and 60 nmol/mouse), agonists of group I and group II mGluRs, respectively, decreased the nociceptive response (-92+/-6% and -89+/-8%, respectively) during the late phase. No change of the early nociceptive phase was observed after (S)-3,5-DHPG or L-CCG-I treatments. These effects were antagonized by a pretreatment with CPCCOEt (40 nmol/mouse) and (2S)-alpha-EGlu (30 nmol/mouse). CPCCOEt is a selective antagonist of group I mGlu receptors, while (2S)-alpha-EGlu is an antagonist of group II. Intra-PAG microinjections of L-SOP (60 and 120 nmol/mouse), a selective agonist of group III mGluRs, induced an increase of the nociceptive response (+95+/-7%) during the late hyperalgesic phase. (R,S)-alpha-M-SOP (70 nmol/mouse), a selective antagonist of group III mGluRs, completely antagonized the L-SOP-induced effect. These results show that PAG mGluRs participate in modulating the late hyperalgesic behaviours induced by formalin. It seems, therefore, possible that group I and group II mGluRs positively modulate PAG antinociceptive descending pathway following a persistent noxious stimulation, while group III mGluRs modulate it negatively.

Periaqueductal gray matter glutamate and GABA decrease following subcutaneous formalin injection in rat.

Glutamate and GABA are important nociception modulating transmitters in specific brain regions, i.e. the spinal cord, the thalamic nuclei and the periaqueductal gray (PAG). However, quantitative and topographical changes in glutamate and GABA release in these brain regions during peripheral inflammation episodes have not been characterized in awake animals. To address this issue, an in vivo microdialysis study was carried out in freely moving rats in order to analyze PAG extracellular glutamate and GABA concentrations following unilateral formalin injection into the dorsal skin of the right hind-paw. Both glutamate and GABA release decreased after the injection of formalin during phase I and phase II of hyperalgesia. Because naloxone prevented the decrease of GABA and glutamate release induced by formalin, this study shows that, in vivo, a nociceptive stimulation may activate opioidergic fibres into the PAG. The increased release of endogenous opioids may, in turn, inhibit the activity of the GABAergic neurons (i.e. opioid disinhibition). Formalin injection also decreased extracellular glutamate concentration. However, we found that intra-PAG perfusion with tetrodotoxin only decreased GABA, but not glutamate dialysate values. Although it should be reasonable to speculate that opioids also inhibit glutamate fibres, further investigation is needed to clarify whether or not the dialysate glutamate we measured reflects change in the metabolism or neurotransmitter pool of this amino acid.

The role of A3 adenosine receptors in central regulation of arterial blood pressure.

1. Pharmacological studies have suggested that A3 receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed. 2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N6-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A3 adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A1 adenosine receptors. 3. Anaesthetized rats, after DPCPX (12 microg(-1) kg i.c.v.), were treated with APNEA (0.4-4 microg kg(-1) i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 microg kg(-1). In contrast, pretreatment 48 h before, with 4 microg kg(-1) i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 microg kg(-1) i.c.v.), after DPCPX, induced a decrease in ABP of -66+/-5.4 mmHg and after 3 min a decrease in heart rate of -62+/-6.0 beats min(-1). Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR. 4. These results suggest that a population of A3-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

Characterisation of mGluRs which modulate nociception in the PAG of the mouse.

The contribution of metabotropic glutamate receptors (mGluRs) to the modulation of nociception by the periaqueductal gray (PAG) matter was investigated in mice. Intra-PAG microinjection of (IS,3R)-ACPD, an agonist of groups I and II mGluRs, as well as (S)-3,5-DHPG, a selective agonist of group I mGluRs, increased the latency of the nociceptive reaction (NR) in the hot plate test. (RS)-AIDA, an antagonist of group I mGluRs, antagonized the effect of (S)-3,5-DHPG, but changed the effect induced by (1S,3R)-ACPD in that a decrease in the latency for the NR could now be observed. L-CCG-I and L-SOP, which are agonists of groups II and III mGluRs respectively, decreased the latency of the NR. (2S)-alpha-EGlu and (RS)-alpha-MSOP, which are antagonists of groups II and III mGluRs, respectively, antagonized the effect of L-CCG-I and L-SOP. (RS)-AIDA and (RS)-alpha-MSOP alone decreased and increased, respectively, the latency of the NR with the highest doses used. (2S)-alpha-EGlu alone did not change significantly the latency of the NR. Intra-PAG microinjection of LH, an agonist of ionotropic glutamate receptors, induced a dose-dependent analgesia which was blocked by pretreatment with DL-AP5, a selective antagonist of NMDA receptors. No mGluRs antagonists were able to prevent LH-induced analgesia. These results emphasize the possible involvement of mGluRs in the modulation of nociception. It seems that activation of group I mGluRs potentiates, while groups II and III mGluRs decrease, the activity of the PAG for the modulation of nociception.

Nitric oxide regulatory role in sensitized guinea pig trachea.

Nitric oxide (NO) has been cited to play an important regulatory role in airway function. Moreover, the NO synthase expression in models of inflammation is documented. The aim of this study was to investigate, in vitro, the NO modulation of cholinergic responses in sham-sensitized and ovalbumin-sensitized guinea pig trachea by using L-arginine (L-ARG), a precursor of NO synthesis, and L-Ng-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase. Our results showed that NO's ability to modulate cholinergic responses in ovalbumin-sensitized guinea pig trachea is lost. Indeed L-ARG and L-NAME modify acetylcholine sensitivity in sham-sensitized guinea pig but not in ovalbumin-sensitized guinea pig.

Evidence for non-adrenergic non-cholinergic contractile responses in bovine and swine trachea.

Non-adrenergic non-cholinergic (NANC) contraction of airway smooth muscle has been observed in some but not all animal species. The aim of this study was to investigate the NANC-contractile responses in bovine and swine trachea. Proximal and distal bovine and swine trachea were cut in strips and placed in 10 ml organ baths equilibrated in Krebs Henseleit (KH) solution and electrically stimulated (10 sec, 60 V, 2 ms, 4, 10 and 30 Hz). Contractile frequency response curves performed in the presence of the muscarinic antagonist, atropine (100 mM), the angiotensin converting enzyme inhibitor, captopril (1 microM) and the neutral endopeptidase inhibitor, thiorphan (1 microM), added 30 min prior to electrical field stimulation (EFS). In some tissues, incubated with atropine thiorphan and captopril, were also evaluated the effects of a pretreatment with capsaicin (10 microM) or a selective NK1 receptor antagonist, SR 14033 (100 nM) added to the baths 30 min prior to EFS. Bovine and swine proximal and distal tracheal preparations contracted in a frequency-dependent manner to EFS (4, 10 and 30 Hz). Some experiments were also performed with substance P (0.1 nM to 1 microM) in absence or in presence of SR 14033 (10 nM or 100 nM). At the maximum frequency tested (30 Hz), the contractile response elicited in bovine proximal and distal preparations was 194.5 +/- 17.1% and 229.7 +/- 24.1%, of ACh (100 microM), respectively. Similarly, the contractile response elicited by EFS (30 Hz) in swine proximal and distal preparations was 187.2 +/- 12.1% and 181.6 +/- 9.2% of ACh (100 microM), respectively. In tissues incubated with atropine, a significant decrease in smooth muscle sensitivity to EFS was observed (P < 0.05). When tissues were pretreated with captopril and thiorphan, a significant increase in the contractile response to EFS (30 Hz) was observed in all tested tissue preparations (bovine, proximal 210.1 +/- 14.4%, distal 264.3 +/- 16.2%; swine, proximal 199.3 +/- 14.9%, distal 206.3 +/- 16.2%, P < 0.05). In the presence of atropine, captopril and thiorphan a significant increase in the contractile response was observed in bovine and swine distal preparations compared with tissues incubated with atropine only (P < 0.05). These effects were antagonized by a pretreatment with a selective NK1 receptor antagonist, SR 14033. A pretreatment with capsaicin statistically (P < 0.05) enhanced EFS-induced contraction in all tested preparations respect to tissues incubated with atropine, thiorphan and captopril. Substance P induced a concentration dependent contraction of bovine and swine isolated tracheal preparations which was antagonized by a pretreatment with a selective NK1 receptor antagonist, SR 14033. No significant difference in the contractile potency (EC50) nor in maximum response (Emax) was observed to exogenously administered substance P between proximal and distal tracheal preparations. These data suggest that NANC contractile responses are present in bovine and swine trachea and are more evident in distal airways.

N-arylsubstituted-1-aryl-3,4-diphenyl-5-pyrazole amines with analgesic and antiarrhythmic properties.

A series of N-aryl substituted-1-aryl-3,4-diphenyl-5-pyrazole amines was synthesized and evaluated for antiinflammatory, antihypertensive and platelet antiaggregating activities. Several of them were found to exhibit a significant analgesic and antiarrhythmic activity.

[Problems associated with the use and monitoring of cyclosporin. Experience at a Clinical Pharmacology Service]. / Problematiche connesse all'uso e al monitoraggio della ciclosporina. Esperienza di un Servizio di Farmacologia Clinica.

BACKGROUND: A study on cyclosporine A (CyA) monitoring in the January 1992-December 1995 period is reported. The aim of this work was to give epidemiological data on the use of CyA, to verify the progressive increase of CyA determinations and to evaluate the use in other diseases as well as to compare the different technics of CyA assay in blood samples, to stress the timing of blood samples and to underline the CyA monitoring importance. METHODS: The CyA dosage was evaluated by fluorescence polarization immunoassay (FPIA) and high-performance liquid chromatography (HPLC). RESULTS: The study showed that 70% of CyA determinations come from patients undergone to renal, bone marrow and liver transplantations; the remaining 30% was associated to other diseases (psoriasis, uveitis, diabetes, rheumatoid arthritis). CONCLUSIONS: The results obtained showed a progressive and constant increase of CyA determinations. Moreover, the use of drug was increased in autoimmune diseases. It is stressed that CyA monitoring in blood samples is essential to optimize the therapeutic efficacy of drug and minimizing its toxicity.

2-Aryl-6-methyl-3-phenylamino-6,7-dihydropyrano[4,3-c]pyrazol-4(2H)-ones with analgesic activity.

A series of 2-aryl-6-methyl-3-phenylamino-6,7-dihydropyrano[4,3-c]pyrazol-4(2H )-ones were prepared and tested for antiinflammatory, analgesic, antipyretic, antiarrhythmic, antihypertensive and platelet antiaggregating activities. All of them showed an appreciable level of analgesic activity in mice.