Dora Pinho

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.

Ground state modulation in nickel(III) chemistry by controlling axial ligation in complexes with N3O2 pentadentate ligands

The accessibility of the +3 oxidation state for nickel(II) complexes with N3O2 pentadentate Schiff base ligands prepared by condensation of naphthaldehyde or 3,5-dichlorosalicylaldehyde with triamines (Scheme 1) has been assessed in several solvents, by combining cyclic voltammetry and EPR spectroscopy of the oxidised metal complexes. The data show that trien/Metrien-based ligands act in pentadentate fashion and allow for the stabilisation of electrochemically and chemically generated nickel(III) complexes in all solvents used. The complexes, formulated as [NiIIIL(solv)]+, are low-spin and have a 2A1, [a dz + b dxy], ground state, with a ⩾ b. For dien-based ligands, steric constraints prevent coordination of the amine nitrogen atom of the pentadentate ligand to the metal centre, thus enforcing a fourfold coordination on the ligand. The resulting complexes can be oxidised to nickel(III) species, but only when using iodine as oxidant, in DMF or (CH3)2SO. The complexes are formulated as [NiIIIL(solv)2]+, and are low-spin and have a 2A1, [a dz + b dxy], ground state, with a >> b. The binding of pyridine and cyanide ion to NiIII trien/Metrien-based complexes was studied by EPR. The data obtained show that these polydentate ligands provide a flexible coordination sphere, and that by varying the ratio equatorial/axial ligand field it is possible to obtain NiIII in three different ground states. For pyridine adducts, the same ground state as the parent complex was observed with Metrien-based ligands, while for trien-based ligands an inverted ground state (2A1, [a dz + b dxy], with a > b, is observed.

Angiotensin II contributes to glomerular hyperfiltration in diabetic rats independently of adenosine type I receptors.

Increased angiotensin II (ANG II) or adenosine can potentiate each other in the regulation of renal hemodynamics and tubular function. Diabetes is characterized by hyperfiltration, yet the roles of ANG II and adenosine receptors for controlling baseline renal blood flow (RBF) or tubular Na(+) handling in diabetes is presently unknown. Accordingly, the changes in their functions were investigated in control and 2-wk streptozotocin-diabetic rats after intrarenal infusion of the ANG II AT1 receptor antagonist candesartan, the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or their combination. Compared with controls, the baseline blood pressure, RBF, and renal vascular resistance (RVR) were similar in diabetics, whereas the glomerular filtration rate (GFR) and filtration fraction (FF) were increased. Candesartan, DPCPX, or the combination increased RBF and decreased RVR similarly in all groups. In controls, the GFR was increased by DPCPX, but in diabetics, it was decreased by candesartan. The FF was decreased by candesartan and DPCPX, independently. DPCPX caused the most pronounced increase in fractional Na(+) excretion in both controls and diabetics, whereas candesartan or the combination only affected fractional Li(+) excretion in diabetics. These results suggest that RBF, via a unifying mechanism, and tubular function are under strict tonic control of both ANG II and adenosine in both control and diabetic kidneys. Furthermore, increased vascular AT1 receptor activity is a contribution to diabetes-induced hyperfiltration independent of any effect of adenosine A1 receptors.

Inhibition of nociceptive responses of spinal cord neurones during hypertension involves the spinal GABAergic system and a pain modulatory center located at the caudal ventrolateral medulla

The mechanisms of hypertension‐induced hypoalgesia were studied in a model of hypertension induced by adenosine receptors blockade with the non‐selective antagonist 1,3‐dipropyl‐8‐sulfophenylxanthine (DPSPX) during 7 days. Based on the positive correlation between pain thresholds and noxious‐evoked expression of the c‐fos protooncogene in spinal cord neurones, we used this marker of nociceptive activation of spinal neurones to evaluate the involvement of the spinal GABAergic system and the caudal ventrolateral medulla (VLM), an important inhibitory component of the supraspinal endogenous pain modulatory system. In DPSPX‐treated animals, a 20% increase in blood pressure was achieved along with a decrease in Fos expression in the superficial (laminae I–II) and deep (laminae III–VII) dorsal horn. In these animals, lower percentages of neurones labeled for GABAB receptors that expressed Fos were obtained in the superficial dorsal horn. Lesioning the VLMlat with quinolinic acid prevented the decrease in Fos expression at the spinal cord of DPSPX‐hypertensive rats whereas in normotensive animals, no changes in Fos expression were detected. The present results support previous findings that hypertension is associated with a decrease of nociceptive activation of spinal cord neurones, through descending inhibition exerted by the VLMlat. This study further shows that during hypertension a decrease in the expression of GABAB receptors in nociceptive spinal neurones occurs, probably due to changes in the local GABAergic inhibitory system.

Scavenging of nitric oxide by an antagonist of adenosine receptors

Chronic treatment of rats with 1,3‐dipropyl‐8‐sulfophenylxanthine (DPSPX), an antagonist of adenosine receptors, causes hypertension, cardiovascular hypertrophy and hyperplasia and impaired endothelium‐dependent vasodilatation. An accelerated degradation of nitric oxide (NO) by scavenging molecules could account for endothelial dysfunction and trophic changes in this hypertension. Our aim was to determine whether DPSPX is a scavenger of NO and if this putative effect is shared by caffeine (1,3,7‐trimethylxanthine) and DPCPX (1,3‐dipropyl‐8‐ciclopentylxanthine), which are also adenosine receptor antagonists but do not induce hypertension in rats. This effect was evaluated by electrochemical and spectrofluorometric assays. Urinary NOx (nitrate + nitrite) excretion was also evaluated in controls and DPSPX‐treated rats as a marker for NO bioavailability. DPSPX behaved as a scavenger of NO in a concentration‐dependent manner in the electrochemical and spectrofluorometric assays. Caffeine and DPCPX had no scavenging effect. DPSPX‐treated rats had decreased excretion of urinary nitrites. We can conclude that: DPSPX has NO scavenging properties that may be involved in the alterations described for DPSPX‐hypertensive rats; this NO‐scavenging effect is not shared by caffeine and DPCPX, which are also xanthine derivatives and adenosine antagonists.

Lesion of the caudal ventrolateral medulla prevents the induction of hypertension by adenosine receptor blockade in rats.

The continuous infusion for 7 days of the adenosine receptor antagonist 1,3-dipropyl-8-sulfophenylxanthine (DPSPX) causes a sustained hypertension in rats, with an enhancement of sympathetic neurotransmission and activation of the renin-angiotensin system. We studied the involvement of the caudal ventrolateral medulla in the establishment of this hypertensive model by evaluating the effect of local lesioning in blood pressure (BP). Male adult Wistar rats received stereotaxic injections of 0.3 mul of saline or quinolinic acid (QA; 180 mM) in the caudal ventrolateral medulla followed by abdominal implant of minipump for infusion of saline or DPSPX (90 microg(-1) kg(-1) h(-1)). BP was measured in conscious animals every 2 days for 12 days. The sustained increase of BP (22.1 mm Hg; P < 0.001) detected in rats infused with DPSPX was reverted (6.7 mm Hg; P > 0.05) from day six onwards in animals with lesion of the lateralmost part of caudal ventrolateral medulla (VLMlat). The present results suggest that the development of hypertension induced by adenosine receptor antagonist involves the participation of the VLMlat. They further add new data as to the functional complexity of this medullary area involved in a variety of functions such as cardiovascular, respiratory, motor and pain control.

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.

Morphine glucuronidation increases its analgesic effect in guinea pigs

AIMS Morphine is extensively metabolized to neurotoxic morphine-3-glucuronide (M3G) and opioid agonist morphine-6-glucuronide (M6G). Due to these different roles, interindividual variability and co-administration of drugs that interfere with metabolism may affect analgesia. The aim of the study was to investigate the repercussions of administration of an inducer (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and an inhibitor (ranitidine) of glucuronidation in morphine metabolism and consequent analgesia, using the Guinea pig as a suitable model. MAIN METHODS Thirty male Dunkin-Hartley guinea pigs were divided in six groups: control, morphine, ranitidine, ranitidine+morphine, TCDD and TCDD+morphine. After previous exposure to TCDD and ranitidine, morphine effect was assessed by an increasing temperature hotplate (35-52.5°C), during 60min after morphine administration. Then, blood was collected and plasma morphine and metabolites were quantified. KEY FINDINGS Animals treated with TCDD presented faster analgesic effect and 75% reached the cut-off temperature of 52.5°C, comparing with only 25% in morphine group. Animals treated with ranitidine presented a significantly lower analgesic effect, compared with morphine group (p<0.05). Moreover, significant differences between groups were found in M3G levels and M3G/morphine ratio (p<0.001 and p<0.0001), with TCDD animals presenting the highest values for M3G, M6G, M3G/morphine and M6G/morphine, and the lowest value for morphine. The opposite was observed in the animals treated with ranitidine. SIGNIFICANCE Our results indicate that modulation of morphine metabolism may result in variations in metabolite concentrations, leading to different analgesic responses to morphine, in an animal model that may be used to improve morphine effect in clinical practice.