Manuela Morato

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.

Angiotensin converting enzyme inhibition prevents trophic and hypertensive effects of an antagonist of adenosine receptors

The continuous infusion of 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), a non-selective antagonist of adenosine receptors, causes hypertension and marked cardiovascular structural changes in Wistar rats. Adenosine inhibits noradrenaline and renin release. We investigated the effects of sympathetic denervation, evaluated renin activity and the influence of angiotensin converting enzyme inhibition in DPSPX-treated rats. Captopril was given (30 or 100 mg kg(-l) day(-l); p.o.) from day -l to day 28. On day 0, constant infusions of DPSPX (90 microg kg(-l) h(-l); i.p.) or vehicle were started. On day 28, fragments of the left ventricle, mesenteric and tail arteries were processed for morphological studies. Plasma renin activity was increased in DPSPX-treated animals. Sympathetic denervation delayed and partially prevented blood pressure rise. Angiotensin converting enzyme inhibition prevented DPSPX-induced hypertension and morphological changes. Our results, although pointing to the involvement of the sympathetic system, suggest that other mechanisms are involved. We could not differentiate between the trophic and anti-hypertensive effects of angiotensin converting enzyme inhibition.

Role of H2O2 in hypertension, renin‐angiotensin system activation and renal medullary disfunction caused by angiotensin II

BACKGROUND AND PURPOSE Activation of the intrarenal renin‐angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H2O2) contribute to hypertension. We examined whether H2O2 mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II).

The role of angiotensin II in hypertension due to adenosine receptors blockade.

The renin-angiotensin system may be involved in hypertension induced by adenosine receptors blockade with 1,3-dipropyl-8-sulfophenylxanthine (DPSPX). Contractions of the mesenteric vasculature to angiotensin II, noradrenaline and potassium chloride were studied in DPSPX-induced hypertension. Male Wistar rats received infusions of saline or DPSPX (90 microg kg(-1) h(-1), i.p.) for 3 or 7 days. Blood pressure was determined by the tail-cuff method. On days 3 or 14, concentration-response curves were obtained on mesenteric arteries and veins. Plasma angiotensin II levels, measured by radioimmunoassay, were higher in DPSPX-hypertensive rats. The maximum contractile effect of angiotensin II was lower in vessels from DPSPX-hypertensive rats while that for noradrenaline was higher. Potassium chloride-induced contractions were larger in veins from DPSPX-hypertensive rats but similar in arteries, when compared with control rats. We conclude that raised angiotensin II levels and altered vascular reactivity are consistent with a renin-angiotensin-mediated hypertension.

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.

Purinergic receptors in the splanchnic circulation

There is considerable evidence that purines are vasoactive molecules involved in the regulation of blood flow. Adenosine is a well known vasodilator that also acts as a modulator of the response to other vasoactive substances. Adenosine exerts its effects by interacting with adenosine receptors. These are metabotropic G-protein coupled receptors and include four subtypes, A1, A2A, A2B and A3. Adenosine triphosphate (ATP) is a co-transmitter in vascular neuroeffector junctions and is known to activate two distinct types of P2 receptors, P2X (ionotropic) and P2Y (metabotropic). ATP can exert either vasoconstrictive or vasorelaxant effects, depending on the P2 receptor subtype involved. Splanchnic vascular beds are of particular interest, as they receive a large fraction of the cardiac output. This review focus on purinergic receptors role in the splanchnic vasomotor control. Here, we give an overview on the distribution and diversity of effects of purinergic receptors in splanchnic vessels. Pre- and post-junctional receptormediated responses are summarized. Attention is also given to the interactions between purinergic receptors and other receptors in the splanchnic circulation.

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.