Pedro López-Sánchez

Role of peripheral and spinal 5-HT3 receptors in development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia

The role of peripheral and spinal 5-HT(3) receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia in both paws. In experiments where the test drug was anticipated to augment or antagonize the response, 0.5 or 1% formalin, respectively, was used for injection. Peripheral ipsilateral, but not contralateral, pre-treatment (-10 min) with serotonin (5-HT, 10-100 nmol/paw) and the selective 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG, 10-300 nmol/paw) increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. Moreover, spinal pre-treatment with m-CPBG (10-300 nmol/rat) increased 0.5% formalin-induced secondary hyperalgesia but not allodynia in both paws. Accordingly, peripheral ipsilateral (30-300 nmol/paw), but not contralateral (300 nmol/paw), and spinal (10-100 nmol) pre-treatment with the selective 5-HT(3) receptor antagonist ondansetron prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. The peripheral pronociceptive effects of 5-HT (100 nmol/paw) and m-CPBG (300 nmol/paw) as well as the spinal effect of m-CPBG (300 nmol/rat) were completely prevented by the peripheral (10 nmol/paw) and spinal (1 nmol/rat) injection, respectively, of ondansetron. At these doses, ondansetron did not modify per se formalin-induced nociceptive behaviors. Spinal (30-300 nmol/rat), but not peripheral (300 nmol/paw), post-treatment (on day 6) with ondansetron reversed established formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT(3) receptors participates in the development of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. In addition, our data suggest that spinal 5-HT(3) receptors play an important role during development and maintenance of these evoked long-term behaviors.

Spinal nerve ligation reduces nitric oxide synthase activity and expression: Effect of resveratrol

The effect of resveratrol on activity and expression of nitric oxide synthase (NOS) in the spinal cord of neuropathic rats was assessed. Spinal nerve ligation produced tactile allodynia along with a reduction of catalytic activity of the constitutive Ca(2+)-dependent NOS (eNOS and nNOS isoforms) in the ipsilateral dorsal horn, but not contralateral dorsal or ipsilateral or contralateral ventral, spinal cord at 1, 5, 10 and 15 days after surgery compared to naïve and sham-operated animals. Nerve ligation also induced a reduction of nNOS expression in the ipsilateral dorsal horn spinal cord at 10 and 15 days after surgery. Intrathecal resveratrol reduced allodynia and reversed the reduction of constitutive Ca(2+)-dependent NOS activity in the ipsilateral dorsal spinal cord. Moreover, resveratrol significantly reversed the reduction of nNOS expression in the ipsilateral dorsal horn spinal cord. Results show that spinal nerve ligation leads to development of tactile allodynia along with a reduction in constitutive Ca(2+)-dependent NOS activity and nNOS isoform expression in the ipsilateral dorsal horn. Data suggest that resveratrol may reduce tactile allodynia in neuropathic rats by restoring altered NOS activity and expression.

Renal Angiotensin-II Receptors Expression Changes in a Model of Preeclampsia

The blunted response to angiotensin II (Ang II) during pregnancy is lost in patients by preeclampsia. This impaired response has been attributed to a change in one or both of the Ang II receptors, type 1 (AT1R) and type 2 (AT2R). The ratio of the Ang II receptor types in the kidney has not been studied. We postulated that an imbalance exists between AT1R/AT2R receptors in the renal cortex from rats subjected to an experimental model of preeclampsia, and that this altered ratio can modify the characteristic blunted pressor response to Ang II during pregnancy. The feto-placental units of Wistar rats were made ischemic by subrenal aortic coarctation, thus creating an experimental model of preeclampsia. We measured the AT1R and AT2R protein expression and the presence of the heterodimer AT1R/AT2R in the renal cortex and evaluated the pressor response to Ang II in an isolated kidney preparation from non-pregnant, healthy pregnant, and preeclampsia model rats. Pregnancy increased AT2R and AT1R/AT2R heterodimer expression and decreased the pressor response to Ang II. In contrast, AT1R increased, while AT2R and AT1R/AT2R heterodimer decreased in the preeclampsia model group. Thus, Ang II hypersensitivity observed in preeclampsia might be related to an increased expression of AT1R over AT2R and to a decreased presence of the AT1R/AT2R heterodimer in renal cortex.

Effect of moderate exercise on IgA levels and lymphocyte count in mouse intestine.

The aim of the present study was to determine the effect of moderate exercise on the production and secretion of IgA in mouse duodenum, on lymphocyte levels in the lamina propria, and on gene expression encoding for cytokines that regulate the synthesis of α-chain of IgA and the expression of pIgR in the lamina propria. Two groups of young Balb/c mice were fed ad libitum, one sedentary and the other with an exercise program (swimming) for 16 weeks. IgA levels in the duodenum were quantified by ELISA; the number of IgA containing cells as well as B cells, CD4+ and CD8+ T cells in the duodenal mucosa was determined by immunohistochemistry; gene expression was analyzed by real-time PCR, and the expression of proteins by Western blotting. Because of physical training, in the duodenum there was a decrease in the number of IgA producing cells, but an increase in the levels of IgA. Additionally, exercise increased the expression of the genes encoding for IL-4, IL-6, IL-10, TNF-α and TGF β, cytokines that regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine. Thus, the increased IgA found in the duodenal lumen is probably due to the increased production of IgA in the LP and the increased transport of the pIgA-pIgR complex across epithelial cells. Possibly the increased S-IgA levels in the bile also contribute to the change in IgA levels.

Effect of pregnancy on the roles of nitric oxide and prostaglandins in 5-hydroxytryptamine-induced contractions in rat isolated thoracic and abdominal aorta.

1. Vascular resistance and sensitivity to circulating pressor and vasoconstrictor substances are blunted during pregnancy. This has been attributed mainly to an increased production of endothelium‐derived mediators. The aim of the present study was to determine whether pregnancy changes the relative participation of nitric oxide (NO) and prostaglandins (PG) in the modulation of the contractile response to 5‐hydroxytryptamine (5‐HT) in two anatomically distint segments of the rat aorta.

Angiotensin-II-Dependent Changes in Alpha-1 Adrenoceptor Vascular Expression in Pregnant Rats

Vessels from pregnant animals show a blunted response to adrenergic agonists. In this work, we explored if pregnancy reduces α₁-adrenergic receptor (α₁-AR)-mediated vascular smooth muscle protein expression as well as agonist-induced contraction in rat aorta, and if angiotensin II (Ang II) levels during pregnancy are related to these changes. Female Wistar rats were divided randomly into nonpregnant (NP) and pregnant groups (first week: P1, second week: P2, third week: P3). Subsets of animals were treated with captopril (5 mg kg^–1 day^–1 for 7 days; n = 6). Phenylephrine (PHE) concentration-response curves were constructed (1 × 10^–9 to 3.1 × 10^–5 mol/l) and α_1A-, α_1B- and α_1D-AR were measured in the aorta by immunoblot. Captopril decreased α-AR expression in the NP group. In contrast, pregnancy decreased α_1A-, α_1B- and α_1D-AR levels and captopril prevented this reduction. PHE sensitivity was decreased in the thoracic and abdominal aorta in the P2 group, with no changes in E_max, and E_max was decreased in the abdominal aorta in all experimental groups. Our results suggest that Ang II levels during pregnancy are related to a decrease in aortic α₁-AR expression. The physiological meaning of this finding remains to be established.

Increased alpha-1 Adrenoceptor Expression in Pregnant Rats with Subrenal Aortic Coarctation

The progression of pregnancy is associated with attenuation in vasopressor response to adrenergic agonists. In pregnancy-induced hypertension this attenuation is reverted. It is not known if this reversion involves alpha-1 adrenoceptor expression. Objective. In this work we propose that in pregnant rats with subrenal aortic coarctation there are changes in the expression of alpha-1 adrenergic receptors in the thoracic and abdominal aorta during pregnancy. Methods. We used non-pregnant, normal pregnant and pregnant with subrenal aortic coarctation female Wistar rats. Pregnancy-induced hypertension indicators, systolic blood pressure, 24 hours proteinuria, pup weight and maternal weight were measured. Dose response curves to phenylephrine were carried out to determine vascular reactivity along pregnancy. Alpha 1-adrenoceptors were detected from thoracic and abdominal aorta using immunoblot. Results. Results show significant increases in arterial pressure and proteinuria in pregnant rats with SRAC at the end of the third week. Pregnancy reduces alpha-1-A, -B and -D adrenoceptor expression and this event is reverted by subrenal aortic coarctation. This phenomenon is more apparent in the abdominal segment of the aorta. Conclusions. These findings suggest that subrenal aortic coarctation is a good animal model of pregnancy-induced hypertension and that α1-adrenoceptors participate in its physiopathology increasing their expression in a segment-dependent manner.

Pharmacological characterization of mechanisms involved in the vasorelaxation produced by rosuvastatin in aortic rings from rats with a cafeteria-style diet

The present study aimed to investigate the possible influence of several inhibitors and blockers on the vascular effect produced by the acute in vitro application of rosuvastatin to phenylephrine‐precontracted aortic rings from rats with a semi‐solid, cafeteria‐style (CAF) diet. It also aimed to examine the effects of rosuvastatin on the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase in aortic rings from rats with a CAF diet. From comparisons of the effect on phenylephrine‐precontracted aortic rings extracted from rats with two different diets (a standard and a CAF diet), it was found that 10−9–10−5‐mol/L rosuvastatin produced lower concentration‐dependent vasorelaxation on rings from the CAF diet group. The vasorelaxant effect was unaffected by the vehicle, but it was significantly attenuated by 10−5‐mol/L NG‐nitro‐l‐arginine methyl ester, 10−2‐mol/L tetraethylammonium, 10−3‐mol/L 4‐aminopyridine, 10−7‐mol/L apamin plus 10−7‐mol/L charybdotoxin, 10−5‐mol/L indomethacin, or 10−5‐mol/L cycloheximide. Moreover, in aortic rings from rats with a CAF diet, rosuvastatin enhanced the expression of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase. The acute in vitro application of rosuvastatin to phenylephrine‐precontracted aortic rings from rats with a CAF diet had a vasorelaxant effect. Overall, the present results suggest that the stimulation of eNOS, the opening of Ca2+‐activated and voltage‐activated K+ channels, the stimulation of prostaglandin synthesis and enhanced protein levels of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase are involved in this relaxant effect.

GR-127935-sensitive mechanism mediating hypotension in anesthetized rats: are 5-HT5B receptors involved?

Abstract: The 5-HT1B/1D receptor antagonist, GR-127935, inhibits hypotensive responses produced by the 5-HT1A, 5-HT1B/1D and 5-HT7 receptor agonist, and 5-HT5A/5B receptor ligand, 5-carboxamidotryptamine (5-CT), in rats. This work further characterized the above mechanism using more selective 5-HT1B and 5-HT1D receptor antagonists. Also, expression of 5-HT5A and 5-HT5B receptor mRNAs in blood vessels was searched by reverse transcription polymerase chain reaction. Decreases in diastolic blood pressure induced by 5-CT (0.001–10 &mgr;g/kg, intravenously) were analyzed in anesthetized rats that had received intravenous vehicle (1 mL/kg), SB-224289 (5-HT1B antagonist; 0.3 and 1.0 mg/kg), BRL15572 (5-HT1D antagonist; 0.3 and 1.0 mg/kg), SB-224289 + BRL15572 (0.3 mg/kg, each), or SB-224289 + BRL15572 (0.3 mg/kg, each) + GR-127935 (1 mg/kg). Because only the latter treatment inhibited 5-CT–induced hypotension, suggestive of a mechanism unrelated to 5-HT1B/1D receptors, the effects of antagonists/ligands at 5-HT5A (SB-699551, 1 mg/kg), 5-HT6 (SB-399885, 1 mg/kg), and 5-HT1B/1D/5A/5B/7 receptors (ergotamine, 0.1 mg/kg) on 5-CT-induced hypotension were tested. Interestingly, only ergotamine blocked 5-CT–induced responses; this effect closely paralleled that of SB-224289 + BRL-15572 + GR-127935. Neither did ergotamine nor GR-127935 inhibit hypotensive responses induced by the 5-HT7 receptor agonist, LP-44. Faint but clear bands corresponding to 5-HT5A and 5-HT5B receptor mRNAs in aorta and mesenteric arteries were detected. Results suggest that the GR-127935-sensitive mechanism mediating hypotension in rats is unrelated to 5-HT1B, 5-HT1D, 5-HT5A, 5-HT6, and 5-HT7 receptors. This mechanism, however, resembles putative 5-HT5B receptors.

Acknowledgement to Referees for Pharmacology 2009

www.karger.com/pha J. Ahamed, New York, N.Y., USA Jörg Ahrens, Hannover, Germany Reiko Akagi, Hiroshima, Japan Satoshi Akiba, Kyoto, Japan Y. Akiba, Los Angeles, Calif., USA Naohiko Anzai, Tochigi, Japan H. Aso, Sendai, Japan Jean Bastin, Paris, France Melvin L. Billingsley, Hershey, Pa., USA F. Boulle, Maastricht, Netherlands William Bowen, Rochester, N.Y., USA J. Breslin, Tampa, Fla., USA Keith K. Burkhart, Silver Spring, Md., USA Frederic Canini, La Tronche, France De-Liang Cao, Springfield, Ill., USA Kejiang Cao, Nanjing, China Rong Chu, Little Rock, Ark., USA George Crystal, Chicago, Ill., USA S.J. Czuczwar, Lublin, Poland Sergio De Marchi, Verona, Italy Carina Denny, Piracicaba, Brazil Le Ding, Kansas, Mo., USA Patrick D’Silva, Bangalore, India Michele Emdin, Pisa, Italy Qingfeng Fan, Philadelphia, Pa., USA J. Faridi, Stockton, Calif., USA Silvia Franchi, Milan, Italy Masanori Fujii, Kyoto, Japan M. Fujimuro, Kyoto, Japan Fenghua Fu, Yantai, China Stefano Fumagalli, Firenze, Italy Li Gan, Madison, Wis., USA Chantal Gauthier, Nantes, France Huiming Ge, Nanjing, China A. Gentilini, Firenze, Italy Lisa Giocomo, Palo Alto, Calif., USA Javier González-Maeso, New York, N.Y., USA Joseph Granchelli, Rochester, N.Y., USA Luis Granero, Valencia, Spain Xiu-Li Guo, Jinan, China E. Gussoni, Boston, Mass., USA Ki Baik Hahm, Seongnam, Korea R. Hardeland, Göttingen, Germany Noriyasu Hirasawa, Sendai, Japan Michael Holzer, Graz, Austria Martin Hönigl, Graz, Austria Lai S. Hooi, Johor Bahru, Malaysia Shigeo Horinaka, Tochigi, Japan Longshuang Huang, Chicago, Ill., USA Christian Humpel, Innsbruck, Austria Yoshimi Imura, Fujisawa, Japan N. Inagaki, Gifu, Japan Makoto Inui, Yamaguchi, Japan Keiichi Ishihara, Kyoto, Japan Yoichiro Isohama, Kumamoto, Japan Kazumi Iwata, Kyoto, Japan Yangfu Jiang, Sichuan, China Melanie S. Joy, Aurora, Colo., USA K. Kadoyama, Himeji, Japan Ira Kass, Brooklyn, N.Y., USA H. Katsuki, Kumamoto, Japan Atsufumi Kawabata, Higashiosaka, Japan Naoyuki Kawao, Osaka, Japan Zunji Ke, Shanghai, China Frieder Keller, Ulm, Germany H.R. Kim, Chungnam, Korea Yoshihisa Kitamura, Kyoto, Japan Yasuo Kizawa, Chiba, Japan T. Kobayashi, Tokyo, Japan J. Komano, Osaka, Japan Vasilios Kouloulias, Athens, Greece Toshiaki Kume, Kyoto, Japan Solene Le Douairon Lahaye, Rennes, France M-T. Lin, Tainan, Taiwan Yi-Wen Liu, Chiayi, Taiwan Zhen-Guo Liu, Columbus, Ohio, USA Pamela Lucchesi, Columbus, Ohio, USA Denisa Margina, Bucharest, Romania Xianfang Meng, Wuhan, China Francesca Menniti, Rome, Italy I. Mérida, Madrid, Spain Jing Miao, Philadelphia, Pa., USA S. Minagawa, Kyoto, Japan Keiji Miyata, Tsukuba, Japan Mizuo Miyazaki, Nagaokakyo, Japan Nobuaki Mizutani, Kobe, Japan M.J. Moshi, Dar es Salaam, Tanzania H. Motohashi, Kyoto, Japan Nariya Mukeshkumar, Jamnagar, India T. Murayama, Kanazawa, Japan H. Murota, Osaka, Japan