Khalid Benamar

Effect of central and peripheral administration of a nitric oxide synthase inhibitor on morphine hyperthermia in rats

The effect of central and peripheral administration of a nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on morphine hyperthermia was studied in male Sprague-Dawley rats. The first series of experiments examined the effect of subcutaneous (s.c.) administration of L-NAME on the hyperthermia induced by morphine given s.c. in doses of 4 and 15 mg/kg. L-NAME, at a s.c. dose of 50 mg/kg, per se, had no influence on body temperature (T(b)). Coadministration of L-NAME (50 mg/kg, s.c.) with the higher dose of morphine (15 mg/kg, s.c.) caused a significant suppression of morphine hyperthermia during the first 30 min and then produced hypothermia. In contrast, s.c. injection of L-NAME (50 mg/kg, s.c.) failed to alter the hyperthermic response induced by the lower dose of morphine (4 mg/kg). In the second series of experiments, we investigated the effect of intracerebroventricular (i.c.v.) administration of L-NAME on the hyperthermia induced by morphine given s.c. L-NAME, itself, given i.c.v. at a dose of 1 mg did not evoke any change in T(b). Intracerebroventricular administration of L-NAME (1 mg) blocked the hyperthermia induced by 15 mg/kg morphine during the first 30 min and induced a slight hypothermia but did not alter the hyperthermia induced by 4 mg/kg morphine. The results indicate that either central or peripheral NO synthesis is required for the production of hyperthermia induced by 15 mg/kg of morphine. However, NO synthesis does not seem to be involved in the hyperthermic process induced by 4 mg/kg of morphine.

Blockade of lipopolysaccharide-induced fever by a μ-opioid receptor-selective antagonist in rats

The endogenous opioid system has been found to be involved in fever caused by pyrogens. In the present study, we have investigated the role of the mu-opioid receptor in the brain in fever induced by lipopolysaccharide. Rats were microinjected with 1 microg of the mu-opioid receptor-selective antagonist, cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), into the preoptic anterior hypothalamus. Thirty minutes later, lipopolysaccharide (50 microg/kg) was injected intraperitoneally (i.p.). CTAP reduced by 1 degrees C the fever induced by lipopolysaccharide. However, it did not affect lipopolysaccharide fever when it was given 3 h after lipopolysaccharide injection. These data indicate that mu-opioid receptors within the preoptic anterior hypothalamus mediate the initiation of lipopolysaccharide fever and suggest that the opioid system is involved in the pathogenesis of fever in rats.

Aldosterone increases cardiac vagal tone via G protein-coupled oestrogen receptor activation.

•  Faster cellular effects of aldosterone incompatible with the genomic effects mediated by mineralocorticoid receptors have been proposed for 40 years but the receptors remained elusive. •  Recently, aldosterone has been shown to activate the G protein‐coupled oestrogen receptor (GPER) in the vasculature. •  Our results indicate that aldosterone activates the GPER in cardiac vagal neurons of nucleus ambiguus leading to an increase in cytosolic Ca2+ concentration and depolarization; in addition, in vivo studies indicate that microinjection of aldosterone in nucleus ambiguus produces bradycardia in conscious rats. •  In summary, our results identified a new role for aldosterone in the modulation of cardiac vagal tone via GPER activation in nucleus ambiguus.

First in Vivo Evidence for a Functional Interaction between Chemokine and Cannabinoid Systems in the Brain

Growing evidence supports the idea that in addition to their well established role in the immune system, chemokines might play a role in both normal and pathological brain function, and the chemokine network could interact with other neuromodulators. The chemokine stromal cell-derived growth factor (SDF)-1α/CXCL12, a member of the CXC chemokine family, was tested for its possible effect on the analgesic responses of the cannabinoid receptor agonist aminoalkylindole 4,5-dihydro-2-methyl-4-(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo-[3,2,1ij]quinolin-6-one [(+)-WIN 55,212-2, hereafter WIN 55,212-2] at the level of the periaqueductal gray (PAG), a brain region critical to the processing of pain signals, and a primary site of action of many analgesic compounds. The administration of WIN 55,212-2 (0.1-0.4 μg/μl) into the PAG resulted in antinociception in a dose-dependent manner. The selective cannabinoid (CB)1 antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A; 1-10 μg) given into the PAG blocked the WIN 55,212-2-induced antinociception. In contrast, the selective CB2 antagonist N-[(1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 10 μg) did not alter the WIN 55,212-2-induced antinociception. Pretreatment with SDF-1α/CXCL12 (100 ng) caused a reduction in antinociceptive responses of WIN 55,212-2. The inhibitory effect of SDF-1α/CXCL12 on WIN 55,212-2-induced antinociception was reversed by 1,1′-[1,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane] octohydrobromide dihydrate (AMD 3100) (10-50 ng), an antagonist of the SDF-1α/CXCL12, acting at its receptor, CXCR4. This study reports the first in vivo evidence of a functional interaction between chemokine and cannabinoid systems in the brain, showing that the activation of SDF-1α/CXCL12 receptors (CXCR4) in the PAG interferes with the analgesic effects of WIN 55212-2.

A Novel Role of Cannabinoids: Implication in the Fever Induced by Bacterial Lipopolysaccharide

There is continuing interest in elucidating the actions of drugs of abuse on the immune system and on infection. The present study investigated the effects of the cannabinoid (CB) receptor agonist aminoalkylindole, (+)-WIN 55,212-2 [(4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], on fever produced after injection of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, the best known and most frequently used experimental model. Intraperitoneal injection of LPS (50 μg/kg) induced a biphasic fever, with the first peak at 180 min and the second at 300 min postinjection. Pretreatment with a nonhypothermic dose of the cannabinoid receptor agonist WIN 55,212-2 (0.5–1.5 mg/kg i.p.) antagonized the LPS-induced fever. However, pretreatment with the inactive enantiomer WIN 55,212-3 [1.5 mg/kg i.p.; S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthanlenyl)methanone mesylate] did not. The inhibitory effect of WIN 55,212-2 on LPS-induced fever was reversed by SR141716 [N-(piperdin-1-yl)-5-(4-chloropheny)-1-(2,4-dichloropheny)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride], a selective CB1 receptor antagonist, but not by SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-choro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide), a selective antagonist at the CB2 receptor. The present results show that cannabinoids interact with systemic bacterial LPS injection and indicate a role of the CB1 receptor subtype in the pathogenesis of LPS fever.

Elevated level of the proinflammatory chemokine, RANTES/CCL5, in the periaqueductal grey causes hyperalgesia in rats.

The present data provide the first in vivo evidence that the proinflammatory chemokine, Regulated on Activation Normal T cell Expressed and Secreted (RANTES/CCL5) microinjected directly into the periaqueductal grey in rats, a brain region critical to the processing of pain signals, and a primary site of action of many analgesic compounds, induced hyperalgesia. Pretreatment with antibodies against RANTES/CCL5 prevented the hyperalgesic response, indicating that RANTES/CCL5 is able to interfere with the control of hyperalgesia at the level of the periaqueductal grey and suggesting that chemokine blockers could have analgesic properties.

Effect of a μ-opioid receptor-selective antagonist on interleukin-6 fever

The endogenous opioid system has been found to be involved in fever caused by pyrogens. Recent work in our laboratory has demonstrated that the mu-opioid receptor is involved in interleukin-1beta (IL-1beta)- and in lipopolysaccharide (LPS)-induced fevers. In the present study, we have investigated the role of the mu-opioid receptor in the preoptic anterior hypothalamus (POAH) in fever induced by interleukin-6 (IL-6). Following stereotaxic implantation of a guide cannula into the POAH for microinjection, radio transmitters to monitor body temperature (Tb) continuously were inserted intraperitoneally. Adult male Sprague-Dawley rats were microinjected with 0.5 microg of the selective mu-opioid receptor antagonist, cyclic D-phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), into the POAH. Thirty min later, IL-6 (100 ng) was injected into the POAH. CTAP significantly blocked the IL-6 fever. CTAP alone had no effect on Tb during the 390-min recording period. These data indicate that mu-opioid receptors within the POAH mediate IL-6 fever and add to the increasing evidence that the opioid system is involved in the pathogenesis of fever in rats.

Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats

Nesfatin‐1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin‐1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin‐1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin‐1 elicits Ca2+ signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca2+ in cardiac pre‐ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin‐1 increases cytosolic Ca2+ concentration via a Gi/o‐coupled mechanism. The nesfatin‐1‐induced Ca2+ rise is critically dependent on Ca2+ influx via P/Q‐type voltage‐activated Ca2+ channels. Repeated administration of nesfatin‐1 leads to tachyphylaxis. Furthermore, nesfatin‐1 produces a dose‐dependent depolarization of cardiac vagal neurons via a Gi/o‐coupled mechanism. In vivo studies, using telemetric and tail‐cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin‐1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin‐1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus.

The Lysophosphatidylinositol Receptor GPR55 Modulates Pain Perception in the Periaqueductal Gray.

Emerging evidence indicates the involvement of GPR55 and its proposed endogenous ligand, lysophosphatidylinositol (LPI), in nociception, yet their role in central pain processing has not been explored. Using Ca2+ imaging, we show here that LPI elicits concentration-dependent and GPR55-mediated increases in intracellular Ca2+ levels in dissociated rat periaqueductal gray (PAG) neurons, which express GPR55 mRNA. This effect is mediated by Ca2+ release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptors and by Ca2+ entry via P/Q-type of voltage-gated Ca2+ channels. Moreover, LPI depolarizes PAG neurons and upon intra-PAG microinjection, reduces nociceptive threshold in the hot-plate test. Both these effects are dependent on GPR55 activation, because they are abolished by pretreatment with ML-193 [N-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)-phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide], a selective GPR55 antagonist. Thus, we provide the first pharmacological evidence that GPR55 activation at central levels is pronociceptive, suggesting that interfering with GPR55 signaling in the PAG may promote analgesia.

Unresponsiveness of mu-opioid receptor knockout mice to lipopolysaccharide-induced fever.

Recently, we demonstrated that lipopolysaccharide (LPS)‐induced fever could be suppressed by a selective mu‐opioid receptor antagonist, indicating that the mu‐opioid system is involved in the LPS fever. In the present study, to confirm the role of the mu‐opioid system in the pathogenesis of LPS fever, we used mice lacking the mu‐opioid receptor. In the wild type (WT), following intraperitoneal (i.p.) injection of 100 μg kg−1 of LPS, body temperature (Tb) increased approximately 1°C and remained elevated during the 360‐min recording period. In the mu‐opioid receptor knockout (MOR‐KO) mice, the administration of 100 μg kg−1 i.p. of LPS did not induce fever during the recording period. Saline by itself, given i.p., did not alter the Tb, either in WT or MOR‐KO. These results confirm that the mu‐opioid system is involved in LPS‐induced fever.