José Marques-Lopes

Role of superoxide and hydrogen peroxide in hypertension induced by an antagonist of adenosine receptors.

Treatment of Wistar rats for 7 days with 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), an antagonist of adenosine receptors, induces long-lasting hypertension associated with marked changes in vascular structure and reactivity and renin-angiotensin system activation. This study aimed at evaluating the role of oxidative stress in the development of DPSPX-induced hypertension and also at identifying the relative contribution of superoxide radical (O2.-) vs hydrogen peroxide (H2O2). Vascular and systemic prooxidant/antioxidant status was evaluated in sham (saline, i.p., 7 days) and DPSPX (90 microg/kg/h, i.p., 7 days)-treated rats. Systolic blood pressure was determined by invasive and non-invasive methods. The activity of vascular NADPH oxidase, superoxide dismutase (SOD), catalase and glutathione peroxidase was assayed by fluorometric/spectrophotometric methods. H2O2 levels were measured using an Amplex Red Hydrogen Peroxide kit. Plasma thiobarbituric acid reactive substances and plasma antioxidant capacity were also measured. In addition we tested the effects of antioxidants or inhibitors of reactive oxygen species generation on blood pressure, vascular hyperplasia and oxidative stress parameters. DPSPX-hypertensive rats showed increased activity of vascular NADPH oxidase, SOD, catalase and glutathione peroxidase, as well as increased H2O2 generation. DPSPX-hypertensive rats also had increased plasma lipid peroxidation and decreased plasma antioxidant capacity. Treatment with apocynin (1.5 mmol/l, per os, 14 days), or with polyethylene glycol (PEG)-catalase (10,000 U/kg/day, i.p., 8 days), prevented the DPSPX-induced effects on blood pressure, vascular structure and H2O2 levels. Tempol (3 mmol/l, per os, 14 days) failed to inhibit these changes, unless PEG-catalase was co-administered. It is concluded that O2.- generation with subsequent formation of H2O2 plays a major role in the development of DPSPX-induced hypertension.

MICROINJECTION OF ANGIOTENSIN II IN THE CAUDAL VENTROLATERAL MEDULLA INDUCES HYPERALGESIA

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT(1)) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT(1)-immunoreactive neurons occurred in the CVLM, only 3% of AT(1)-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT(1) antagonist losartan. The present study demonstrates that Ang II can act on AT(1) receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT(1)-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.

The hyperalgesic effects induced by the injection of angiotensin II into the caudal ventrolateral medulla are mediated by the pontine A5 noradrenergic cell group

The caudal ventrolateral medulla (CVLM) is a key component of the supraspinal pain modulatory system. Pain modulation from the CVLM is partially relayed by spinally projecting noradrenergic neurons of the pontine A(5) cell group, which leave collateral fibres at the CVLM. The injection of angiotensin II (Ang II) into the CVLM was recently shown to induce hyperalgesia mediated by angiotensin type 1 (AT(1)) receptors, expressed by CVLM neurons that do not project to the spinal cord. The present study evaluates the effects of lesioning the noradrenergic pontine A(5) cell group by the retrograde transport of the selective toxin anti-dopamine beta-hydroxylase-saporin (anti-DBH-SAP) from the CVLM in pain behavioural responses elicited by Ang II injection into the CVLM. The injection of anti-DBH-SAP induced neurodegeneration, identified by the marker Fluoro-Jade B, restricted to the A(5) noradrenergic cell group. These results were confirmed by the decrease in the number of noradrenergic neurons only in the A(5) group. Pain behavioural evaluation using the formalin test showed that Ang II injection into the CVLM induced hyperalgesia, which was partially prevented by lesion of the A(5) noradrenergic cell group with anti-DBH-SAP. Immunostaining of AT(1) receptors in CVLM neurons retrogradely labelled from the A(5) noradrenergic cell group showed that CVLM neurons that project to the A(5) express AT(1) receptors, indicating that Ang II can modulate directly the CVLM-A(5) connection. The results show that Ang II-induced hyperalgesia elicited from the CVLM is mediated by an indirect pathway relayed at the pontine noradrenergic A(5) group.

Decrease in the expression of N‐methyl‐D‐aspartate receptors in the nucleus tractus solitarii induces antinociception and increases blood pressure

N‐methyl‐D‐aspartate receptors (NMDAR) have a role in cardiovascular control at the nucleus tractus solitarii (NTS), eliciting increases or decreases in blood pressure (BP), depending on the area injected with the agonists. In spite of the association between cardiovascular control and pain modulation, the effects of manipulating NMDAR in pain responses have never been evaluated. In this study, we decreased the expression of NMDAR in the NTS using gene transfer to target receptor subunits and evaluate long‐term effects. Seven days after the injection of lentiviral vectors containing the NR1a subunit cDNA of NMDAR, in antisense orientation, into the intermediate NTS of Wistar rats, BP was measured, and the formalin test of nociception was performed. The antisense vector induced a decrease of NR1 expression in the NTS and elicited BP rises and hypoalgesia. Antisense vectors inhibited formalin‐evoked c‐Fos expression in the spinal cord, indicating decreased nociceptive activity of spinal neurons. Using a time‐course approach, we verified that the onset of both the increases in BP and the hypoalgesia was at 4 days after vector injection into the NTS. The injection of NMDA into the NTS reversed the effects of antisense vectors in pain behavioral responses and spinal neuronal activation and decreased BP and heart rate. The present study shows that the NR1 subunit of the NMDAR at the NTS is critical in the regulation of tonic cardiovascular and nociceptive control and shows an involvement of the nucleus in the modulation of sustained pain.

393 ROLE OF ANGIOTENSIN II IN PAINFUL DIABETIC NEUROPATHY AND HYPERTENSION

Recognition memory was tested in a novel-object-recognition paradigm. Animals were killed by transcardiac perfusion and spinal cords were removed, sectioned and stained with FITC-conjugated isolectin B4 (IB4), a marker of primary afferent C-fibres. Sections were visualised using fluorescent microscopy. Results: IB4 staining was decreased in laminae 1–2 of the spinal cord on the ipsilateral side to ligation, compared with the contralateral side and with sham controls, which indicates the ligation was successful. SNL rats developed mechanical allodynia, and expressed thermal hyperalesia. SNL surgery had no effect on spatial learning in the acquisition phase of the MWM or on spatial memory assessed in both the MWM probe trial and the T-maze. Recognition memory was also unaffected in SNLs as compared with shams. Conclusions: SNL surgery modelled symptoms characteristic of neuropathic pain but was not associated with cognitive impairment under these experimental conditions.

198 HYPERTENSIVE MONOARTHRITIC RATS EXHIBIT LOWER INFLAMMATION AND HYPERALGESIA THAN THEIR NORMOTENSIVE CONTROLS

Background and Aims: Tapentadol [(−)-(1R,2R)-3-(3-Dimethylamino-1ethyl-2-methyl-propyl)-phenol] is a novel analgesic with a dual mode of action, m opioid receptor (MOR) agonism and noradrenaline (NA) reuptake inhibition, and a broad analgesic efficacy. Methods: Tapentadol was tested in a range of in vitro and in vivo experiments to characterize its dual mode of action. Results: Tapentadol binds to MOR with a Ki = 0.1 mM, and has an efficacy of 88% in a [35S]GTPgammaS binding assay, relative to morphine. In addition, it has a Ki = 0.5mM for synaptosomal NA reuptake inhibition. The functional relevance of NA reuptake inhibition was supported by in vivo brain microdialysis studies showing that tapentadol, in contrast to morphine, produces dose-dependent increases in extracellular levels of NA in the analgesic dose range (up to 450% at 10mg/kg). A pronounced noradrenergic contribution to the analgesic efficacy was demonstrated in a rat neuropathic pain model, where the analgesic effect of tapentadol (10mg/kg) was strongly antagonized by the a2-NA receptor antagonist yohimbine (2.15mg/kg), but was only weakly affected by the MOR antagonist naloxone (0.3mg/kg), whereas the opposite was the case for morphine (6.81mg/kg). In the mouse writhing model, the analgesic effect of morphine (0.681mg/kg) was much more susceptible to antagonism by naloxone (0.001−1mg/kg) than that of an equipotent dose of tapentadol (3.16mg/kg). Conclusions: Tapentadol’s dual mode of action is based on MOR agonism and NA reuptake inhibition. There is clear evidence that both mechanisms of action contribute to its analgesic effects.