Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Neuroprotective and antioxidative effects of pioglitazone in brain tissue adjacent to the ischemic core are mediated by PI3K/Akt and Nrf2/ARE pathways.

The present study elucidates the neuroprotective mechanisms of the PPARγ (peroxisome proliferator-activated receptor γ) agonist pioglitazone in survival of ischemic neurons following middle cerebral artery occlusion with reperfusion (MCAO). Intracerebroventricular infusion of pioglitazone over 5 days before and 24 or 48 h after MCAO alleviated neurological impairments, inhibited apoptosis 24 h, and activated the PI3K/Akt pathway along with increased phosphorylation of Akt (ser473) and GSK-3ß (ser9) in the peri-infarct cortical areas 48 h after MCAO. In primary cortical neurons, pioglitazone suppressed the glutamate-induced release of lactate dehydrogenase by a PPARγ-dependent mechanism. This protective effect was reversed after co-treatment with PI3K and Akt inhibitors, LY294002 and SH-6, respectively. Pioglitazone enhanced the expression of the antioxidative transcription factor Nrf2 and its target gene protein, heme oxidase-1, in the peri-infarct area. Pioglitazone also increased activation of the antioxidant response element (ARE) in neuronal PC12 cells transfected with the pNQO1-rARE plasmid. We demonstrate in primary cortical neurons from Nrf2 knockout mice that the lack of Nrf2 completely abolished the neuroprotective effects of pioglitazone against oxidative and excitotoxic damage. Our results strongly suggest that the neuroprotective effects of PPARγ in peri-infarct brain tissues comprise the concomitant activation of the PI3K/Akt and Nrf2/ARE pathways. KEY MESSAGES: Pioglitazone inhibits apoptosis in ischemic brain tissue.  Pioglitazone acting on PPARγ activates PI3K/Akt pathway in ischemic brain tissue. Pioglitazone activates via Nrf2 the antioxidant defense pathway in injured neurons. Pioglitazone activates the antioxidant response element in neuronal PC12 cells. Pioglitazone fails to protect primary neurons lacking Nrf2 against oxidative damage. Activation of PPARγ supports the survival of viable neurons in peri-infarct regions.

PPAR-gamma: therapeutic target for ischemic stroke.

The peroxisome proliferator activated receptors (PPARs), which belong to the nuclear receptor superfamily, are key regulators of glucose and fat metabolism. The PPAR-gamma isoform is involved in the regulation of cellular glucose uptake, protection against atherosclerosis and control of immune reactions. In addition, the activation of PPAR-gamma effectively attenuates neurodegenerative and inflammatory processes in the brain. Here, we review a novel aspect of beneficial and clinically relevant PPAR-gamma actions: neuroprotection against ischemic injury mediated by intracerebral PPAR-gamma, which is expressed in neurons and microglia. Together with the recent observation that the PPAR-gamma ligand pioglitazone reduces the incidence of stroke in patients with type 2 diabetes, this review supports the concept that activators of PPAR-gamma are effective drugs against ischemic injury.

Peroxisome proliferator-activated receptorsgamma (PPARgamma) differently modulate the interleukin-6 expression in the peri-infarct cortical tissue in the acute and delayed phases of cerebral ischaemia.

Interleukin-6 (IL-6) exerts neuroprotective effects after cerebral ischaemia but can also exacerbate inflammation and induce neuronal death. The current study investigates the role of cerebral peroxisome proliferator-activated receptor(s) gamma (PPARgamma) in the regulation of IL-6 expression in the peri-infarct cortical tissue in rats exposed to focal cerebral ischaemia. Pioglitazone, a high-affinity PPARgamma ligand, was infused intracerebroventricularly (i.c.v.) via osmotic minipumps over a 5-day period before, during and 24 h or 48 h after middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. The expression of PPARgamma and IL-6 in cortical tissue adjacent to the ischaemic core was studied 24 h and 48 h after MCAO. Pioglitazone augmented the ischaemia-induced upregulation of PPARgamma at both time points. Cerebral ischaemia substantially increased IL-6 expression in the peri-infarct cortical tissue. Twenty-four hours after MCAO, the majority of microglial cells/macrophages showed an intense IL-6 immunoreactivity. IL-6 was also localized in neurons, but the distribution of neurons positively stained for IL-6 at the border of the infarct was very heterogeneous. Pioglitazone effectively decreased the number of IL-6-immunoreactive cells and IL-6 protein levels at 24 h but not at 48 h after MCAO. Pioglitazone treatment reduced the infarct size and improved neurological functions. The present study demonstrates that cerebral PPARgamma suppresses the expression of IL-6 in ischaemic brain tissue during the initial phase of ischaemic stroke, in which the overproduction of IL-6 may aggravate neuronal damage, but not at later time points, when IL-6 promotes neuroprotection and inhibits neuronal death.

Comparison between early and delayed systemic treatment with candesartan of rats after ischaemic stroke.

OBJECTIVE: The effects of candesartan treatment starting early (3 h) and delayed (24 h) after middle cerebral artery occlusion (MCAO) with reperfusion was investigated in normotensive rats. METHODS: Subcutaneous treatment with candesartan (0.3 and 3 mg/kg) or vehicle was initiated 3 or 24 h after the onset of MCAO and continued for seven consecutive days (n=20 per group and timepoint). Neurological outcome was evaluated daily using two different scoring systems. Infarct and oedema volumes were determined in rats 2 or 7 days after MCAO. Mean arterial, systolic and diastolic blood pressures were recorded before and after the application of candesartan. RESULTS: Mean arterial, systolic and diastolic blood pressures were markedly decreased with the high dose, but only moderately decreased with the low dose of candesartan. Vehicle-treated rats showed marked neurological deficits 24 h after MCAO, which gradually improved with time. Candesartan improved neurological outcomes at all timepoints only when treatment was started 3, but not 24 h after MCAO. The infarct volume was reduced on days 2 and 7 after MCAO in rats treated with the low but not the high dose of candesartan. CONCLUSION: The present study demonstrates that only an early but not a delayed onset of treatment with candesartan exerts neuroprotection after focal ischaemia. The degree of neurological impairments did not correlate with the infarct volume, which was reduced only after the low dose of candesartan. The high dose of candesartan failed to reduce the infarct volume, probably because of an excessive blood pressure decrease.

Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats.

Up-regulation of cyclooxygenase (COX)-2 exacerbates neuronal injury after cerebral ischemia and contributes to neuronal cell death. The present study clarifies the function of cerebral peroxisome-proliferator-activated receptor(s) gamma (PPARgamma) in the expression of COX-2 in neurons of the rat brain after middle cerebral artery occlusion (MCAO) with reperfusion by immunohistochemistry, Western blot, and immunofluorescence staining. In peri-infarct cortical areas the PPARgamma was located in both microglia and neurons, whereas COX-2 was almost exclusively expressed in neurons. PPARgamma immunolabeling reached the peak 12 h after MCAO, whereas the number of COX-2 immunostained cells gradually rose and reached its peak at 48 h. Intracerebroventricular infusion of pioglitazone, an agonist of the PPARgamma, over a 5-day period before and 2 days after MCAO, reduced the infarct size, the expression of tumor necrosis factor alpha (TNF-alpha), COX-2, and the number of cells positively stained for COX-1 and COX-2 in the peri-infarct cortical regions. COX-2 induction was also attenuated in the ipsilateral but not in the contralateral hippocampus. In primary cortical neurons expressing the PPARgamma, pioglitazone suppressed COX-2 expression in response to oxidative stress. This protective effect was reversed after cotreatment with GW 9662, a selective antagonist of the PPARgamma, clearly demonstrating a PPARgamma-dependent mechanism. Our data provide evidence that activation of neuronal PPARgamma considerably contributes to neuroprotection by prevention of COX-2 up-regulation in vitro and in peri-infarct brain areas.

Neuroprotective effects of AT1 receptor antagonists after experimental ischemic stroke: what is important?

The present study conducted in rats defines the requirements for neuroprotective effects of systemically administered AT1 receptor blockers (ARBs) in acute ischaemic stroke. The inhibition of central effects to angiotensin II (ANG II) after intravenous (i.v.) treatment with candesartan (0.3 and 3 mg/kg) or irbesartan and losartan (3 and 30 mg/kg) was employed to study the penetration of these ARBs across the blood-brain barrier. Verapamil and probenecid were used to assess the role of the transporters, P-glycoprotein and the multidrug resistance-related protein 2, in the entry of losartan and irbesartan into the brain. Neuroprotective effects of i.v. treatment with the ARBs were investigated after transient middle cerebral artery occlusion (MCAO) for 90 min. The treatment with the ARBs was initiated 3 h after the onset of MCAO and continued for two consecutive days. Blood pressure was continuously recorded before and during MCAO until 5.5 h after the onset of reperfusion. The higher dose of candesartan completely abolished, and the lower dose of candesartan and higher doses of irbesartan and losartan partially inhibited the drinking response to intracerebroventricular ANG II. Only 0.3 mg/kg candesartan improved the recovery from ischaemic stroke, and 3 mg/kg candesartan did not exert neuroprotective effects due to marked blood pressure reduction during reperfusion. Both doses of irbesartan and losartan had not any effect on the stroke outcome. An effective, long-lasting blockade of brain AT1 receptors after systemic treatment with ARBs without extensive blood pressure reductions is the prerequisite for neuroprotective effects in ischaemic stroke.

Lack of change in serum angiotensin-converting enzyme activity during the menstrual cycle.

INTRODUCTION: The Deletion (D) rather than Insertion (I) variant of the angiotensin-converting enzyme (ACE) gene is associated with higher circulating ACE activity. Meanwhile, coronary risk rises with the menstrual nadir in oestrogen levels, exogenous oestrogen reduces serum ACE activity (with a greater reduction the higher the baseline ACE activity), and pharmacological reduction in ACE activity is cardioprotective. Alterations in coronary risk associated with the menstrual cycle may thus be mediated through (genotype-dependent) changes in ACE activity. We have examined this hypothesis. MATERIALS AND METHODS: Twenty-three healthy female subjects (12 II, 11 DD genotype) were studied. None were taking oral contraceptive agents. Blood was assayed for oestrogen, follicle stimulating hormone (FSH), luteinising hormone (LH), progesterone and ACE activity every three days throughout their menstrual cycle. RESULTS: ACE activity was unrelated to oestrogen, FSH or LH during the menstrual cycle, irrespective of ACE genotype. CONCLUSIONS: The increase in myocardial ischaemia during low oestrogen phases of the menstrual cycle does not appear mediated through a fall in serum ACE activity.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Low-dose lithium combined with captopril prevents stroke and improves survival in salt-loaded, stroke-prone spontaneously hypertensive rats.

OBJECTIVE: A number of potential interactions between angiotensin-converting enzyme inhibitors and lithium have been described in the literature. In the present study, we investigated the effects of a low-dose combination treatment with lithium and captopril on survival and stroke prevention in salt-loaded, stroke-prone spontaneously hypertensive rats (SHRSP). METHODS: Eight-week-old saline-drinking SHRSP (n = 21 per group) were treated with vehicle, LiCl (1 mmol/kg per day), captopril (25 mg/kg per day) and captopril plus LiCl for up to 37 weeks. Body weight, salt water intake blood pressure and mortality were recorded throughout the experimental period. Plasma renin activity, plasma lithium concentration and urinary excretion of albumin, sodium and potassium were measured at different time points. RESULTS: Captopril treatment doubled the life expectancy when compared with vehicle-treated rats. Lithium alone had minor effects on survival but led to a dramatic increase in survival when added to captopril (mean survival time > 237 versus 147 days, P < 0.001). Systolic blood pressure increased with age in all treatment groups but was comparable in the captopril-treated and the captopril-plus-lithium-treated groups. Plasma renin activity as well as urinary sodium and potassium excretion did not differ between both groups. In the captopril group a striking fivefold increase of albuminuria occurred between 14 and 26 weeks of age, while this progression was completely abolished by the addition of lithium. CONCLUSIONS: Our results demonstrate that the addition of lithium to captopril dramatically prolong the effects of the angiotensin-converting enzyme inhibitor on survival in salt-loaded SHRSP. This effect was independent of a reduction in blood pressure.

Circulating angiotensin converting enzyme activity is correlated with muscle strength.

PURPOSE: The D-variant of the angiotensin-1 converting enzyme (ACE) gene is associated with higher circulating and tissue ACE activity. Some studies have suggested a similar association of genotype with muscle strength or the gain in strength in response to training. This study has assessed the relationship between circulating ACE activity, strength, and the response to training. METHODS: Eighty-one untrained men were tested for quadriceps muscle strength, and 44 of these performed an 8-wk program of dynamic strength training of the quadriceps muscle group. Venous blood was obtained for assessment of circulating ACE activity before and after the training program. ACE genotype was also determined. RESULTS: At baseline, circulating ACE activity was significantly correlated with isometric (r = 0.25-0.29, P < 0.02) and isokinetic (r = 0.38, P < 0.0005) quadriceps muscle strength. ACE genotype also seemed to be related to pretraining muscle strength. However, circulating ACE activity showed no significant association with the 9-14% mean increases of muscle strength in response to the training intervention. ACE genotype also showed no association with the training-induced change in muscle strength. Circulating ACE activity did not change significantly after the training program. CONCLUSIONS: The data support a role for ACE in the regulation of human skeletal muscle strength, but do not confirm a role in altering the response to short-term training.

Omapatrilat: penetration across the blood-brain barrier and effects on ischaemic stroke in rats.

Omapatrilat (OMA), which simultaneously inhibits the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (neprilysin (NEP)), is widely used in experimental protocols related to hypertension and heart failure. The penetration of OMA across the blood-brain barrier (BBB) and the effects of ACE/NEP inhibition on the recovery from ischaemic stroke have not yet been investigated. Angiotensin (Ang) I injected intracerebroventricularly (ICV) or intravenously (IV) is converted to Ang II by ACE and induces an immediate increase in blood pressure. The pressor responses to OMA administered ICV, orally or IV were studied in male Wistar rats instrumented with an ICV and arterial and venous catheters. OMA infused ICV rapidly appeared in the systemic circulation and more effectively attenuated the systemic than the central pressor responses to Ang I. OMA administered orally (5, 25, 100 µmol/kg body weight) or IV (0.5, 1, 5, 25 µmol/kg body weight) completely abolished increases in blood pressure to IV Ang I up to 2 h after treatment. The pressor responses to ICV Ang I were not altered, indicating that systemically administered OMA does not cross the BBB. To study the effects of ACE and NEP inhibition in the brain on the recovery from ischaemic stroke, OMA was infused ICV over a 5-day period before and 24 h after the occlusion of the middle cerebral artery (MCAO) for 90 min. ICV application of OMA had no effect on infarction volume and marginally improved neurological outcome. We demonstrate for the first time that simultaneous inhibition of ACE and NEP in the brain tissue does not alter the recovery from ischaemic stroke.

Effect of angiotensin AT2 receptor stimulation on vascular cyclic GMP production in normotensive Wistar Kyoto rats.

In the present study in normotensive Wistar Kyoto rats (WKY), we investigated whether any angiotensin II (ANG II) increases in vascular cyclic GMP production were via stimulation of AT(2) receptors. Adult WKY were infused for 4h with ANG II (30 ng/kg per min, i.v.) or vehicle (0.9% NaCl, i.v.) after pretreatment with (1) vehicle, (2) losartan (100 mg/kg p.o.), (3) PD 123319 (30 mg/kg i.v.), (4) losartan+PD 123319, (5) icatibant (500 microg/kg i.v.), (6) L-NAME (1 mg/kg i.v.), (7) minoxidil (3 mg/kg i.v.). Mean arterial blood pressure (MAP) was continuously monitored, and plasma ANG II and aortic cyclic GMP were measured at the end of the study. ANG II infusion over 4h raised MAP by a mean of 13 mmHg. This effect was completely prevented by AT(1) receptor blockade. PD 123319 slightly attenuated the pressor effect induced by ANG II alone (123.4+/-0.8 versus 130.6+/-0.6) but did not alter MAP in rats treated simultaneously with ANG II + losartan (113+/-0.6 versus 114.3+/-0.8). Plasma levels of ANG II were increased 2.2-3.7-fold by ANG II infusion alone or ANG II in combination with the various drugs. The increase in plasma ANG II levels was most pronounced after ANG II+losartan treatment but absent in rats treated with losartan alone. Aortic cyclic GMP levels were not significantly changed by either treatment. Our results demonstrate that the AT(2) receptor did not contribute to the cyclic GMP production in the vascular wall of normotensive WKY.

Chronic treatment with a low dose of lithium protects the brain against ischemic injury by reducing apoptotic death.

BACKGROUND AND PURPOSE: In vitro and in vivo studies have demonstrated neuroprotective actions of lithium. The present study investigated the effect of a low dose of lithium on infarct volume and neurological outcome as well as on apoptotic and inflammatory processes in rats exposed to focal ischemia. METHODS: Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 90 minutes followed by reperfusion. Lithium (1 mmol/kg) was given subcutaneously daily for 14 days before the onset of MCAO and 2 days thereafter. Blood parameters and cerebral blood flow were assessed before, during, and after MCAO. Rats were examined for neurological deficits 24 and 48 hours after MCAO. Two days after MCAO, the brains were removed for immunohistochemical evaluation of caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), activated microglia, and the expression of AP-1 proteins (c-Fos and c-Jun). Infarct volume was assessed by cresyl violet staining. RESULTS: Pretreatment with lithium did not alter cerebral blood flow or blood parameters. Neurological deficits were significantly decreased in rats treated with lithium at 24 and 48 hours after ischemia. Infarct volume was reduced in rats treated with lithium at 48 hours after ischemia. Lithium significantly decreased the ischemia-induced caspase-3 immunoreactivity and TUNEL staining as well as the AP-1 protein expression in the penumbra of the ischemic cortex. No changes in activated microglia were observed. CONCLUSIONS: The present study demonstrates that chronic treatment with lithium at a low dose exhibits neuroprotection in transient focal cerebral ischemia. Antiapoptotic mechanisms are involved in the lithium-induced neuroprotective effects.

Sustained blockade of brain AT1 receptors before and after focal cerebral ischemia alleviates neurologic deficits and reduces neuronal injury, apoptosis, and inflammatory responses in the rat.

In the present study, we investigate whether a long-term blockade of brain AT1 receptors in male Wistar rats before and after ischemic injury exerts neuroprotective effects and modulates apoptosis and inflammatory responses, which are associated with the post-ischemic progression of brain damage. The AT1 receptor antagonist irbesartan was continuously infused intracerebroventricularly using osmotic minipumps over a 5-day period before and for 3 or 7 days after middle cerebral artery occlusion (MCAO) for 90 minutes. Neurologic status was evaluated daily, starting 24 hours after MCAO. After MCAO (3 and 7 days), brains were removed for the measurement of infarct size and immunohistochemical evaluation of apoptosis and accumulation of reactive microglia and macrophages. Treatment with irbesartan before ischemia improved motor functions, whereas post-ischemic treatment improved sensory functions. Blockade of brain AT1 receptors reduced the infarct size on days 3 and 7 after MCAO. In the peri-infarct cortex, irbesartan treatment decreased the number of apoptotic cells on day 3 and attenuated the invasion of activated microg-lia and macrophages on days 3 and 7 after ischemia. Long-term blockade of brain AT1 receptors improves the recovery from cerebral ischemia. Antiapoptotic mechanisms and inhibition of post-ischemic inflammation are involved in the AT1 receptor blockade-induced neuroprotective effects in ischemic brain tissue.

Chronic pretreatment with candesartan improves recovery from focal cerebral ischaemia in rats.

OBJECTIVE: In the present study, we investigated whether systemic pretreatment with the AT1 receptor antagonist, candesartan, reduces neuronal injury after cerebral ischaemia in rats. DESIGN AND METHODS: Focal cerebral ischaemia in male, normotensive Wistar rats was induced by 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. Experiment 1: Candesartan was injected intravenously (i.v.) at doses of 0.1 or 0.3 mg/kg, 4 h prior to ischaemic injury. Experiment 2: Rats were treated with candesartan [0.1 mg/kg, subcutaneously (s.c.) twice daily], on 5 consecutive days prior to ischaemia. The last injection was administered 12 h before MCAO. Mean arterial pressure (MAP) was measured before, during and after ischaemic injury. Twenty-four hours after ischaemia, neurological outcome, infarct volume and brain oedema were evaluated. RESULTS: Acute i.v. pretreatment with candesartan, 0.1 and 0.3 mg/kg, dose-dependently decreased MAP before, during and after ischaemic injury but did not improve recovery from brain ischaemia. Systemic long-term s.c. pretreatment with 0.1 mg/kg candesartan, reduced MAP during and after ischaemia to the same extent as did the i.v. dose of 0.1 mg administered 4 h before MCAO, but significantly improved neurological outcome and reduced infarction size and oedema of the ipsilateral hemisphere when compared with the vehicle-treated group. CONCLUSION: Long-term blockade of AT1 receptors improves neurological outcome of focal cerebral ischaemia and protects brain tissue against ischaemic injury.

Effects of orally applied candesartan cilexetil on central responses to angiotensin II in conscious rats.

OBJECTIVE: In the present study, we investigated the ability of the peripherally administered angiotensin II type 1 (AT1) receptor antagonist, candesartan cilexetil, to block central effects of angiotensin II (Ang II) in conscious rats. DESIGN AND METHODS: Candesartan cilexetil was administered orally by gavage at doses of 0.1, 1, 10 and 30 mg/kg. Drinking response, pressor response and release of vasopressin into the circulation following intracerebroventricular (i.c.v.) Ang II (10 or 100 ng) were measured at 0.5, 2, 4 and 24 h following the drug application. The same parameters were measured after chronic treatment with candesartan cilexetil for 1 week. In a separate experiment, the release of vasopressin induced by microinjection of Ang II (100 ng) into the paraventricular nucleus (PVN) was determined 4 h after oral administration of candesartan cilexetil (1 mg/kg) or vehicle. RESULTS: Oral treatment with candesartan cilexetil inhibited all central responses to i.c.v. Ang II in a dose- and time-dependent manner. The Ang II-induced responses were inhibited 4 h after acute or chronic treatment with 0.1 mg/kg candesartan cilexetil, but had returned to control levels 24 h after drug application. In contrast, the highest dose of candesartan cilexetil (30 mg/kg) nearly abolished the central responses to Ang II for 24 h. Candesartan cilexetil completely blocked vasopressin release into the circulation induced by Ang II microinjection into the PVN. CONCLUSIONS: Our results demonstrate that the AT1 receptor antagonist, candesartan cilexetil, very effectively inhibits the centrally mediated effects of Ang II upon peripheral application.

Differential effect of acute angiotensin II type 1 receptor blockade on the vascular and adrenal response to exogenous angiotensin II in humans.

BACKGROUND: Aldosterone stimulation by angiotensin II may not exclusively be mediated by the angiotensin II type 1 (AT(1)) receptor. We have, therefore, investigated the vascular and adrenal response to angiotensin II infusion without and with pretreatment with the AT(1) receptor antagonist valsartan (160 mg). METHODS: In nine healthy human volunteers, angiotensin II was administered intravenously at doses of 1, 3, and 10 ng/kg/min, each over 45 min. Arterial blood pressure (BP) was measured oscillometrically at 5-min intervals. Blood for the determination of plasma renin activity and aldosterone was taken before the start of the infusion, at the end of each infusion period, and 1 h after the infusion was stopped. RESULTS: Angiotensin II increased systolic and diastolic BP from 121 +/- 3/70 +/- 2 mm Hg to a maximum of 146 +/- 2/97 +/- 1 mm Hg (P <.001) and plasma aldosterone from 39.2 +/- 9.8 to 290.7 +/- 48.3 (P <.001). The increase in BP after exogenous angiotensin II was completely abolished in volunteers pretreated with valsartan, averaging 118 +/- 3/72 +/- 1 mm Hg by the end of the maximum angiotensin infusion dose. In contrast, plasma aldosterone stimulation by angiotensin II was only partially blunted by concomitant AT(1) receptor blockade (98.9 +/- 16.3 pg/mL after the maximal dose of angiotensin II). CONCLUSIONS: These results indicate that although the vascular response to exogenous angiotensin II is exclusively mediated by the AT(1) receptor, the effects of angiotensin II on adrenal aldosterone release may involve other pathways.

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects.

BACKGROUND: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. METHODS: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. RESULTS: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were < or =12% and < or =10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 +/- 0.26 fmol/ml (mean +/- SD). CONCLUSIONS: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1.

Treatment of rats with pioglitazone in the reperfusion phase of focal cerebral ischemia: a preclinical stroke trial.

Thiazolidinediones (TZDs), pioglitazone, rosiglitazone and troglitazone, the synthetic agonists for the PPARγ, administered prior or during ischemic insult improve stroke outcome in rodents, post-occlusion treatments yielded inconsistent results. In the present experiments carried out according to the Stroke Therapy Academic Industry Roundtable (STAIR) guidelines, we studied the effects of post-ischemic pioglitazone treatment on the outcome of focal cerebral ischemia, inflammatory and apoptotic processes, neuronal degeneration and regeneration, blood pressure, heart rate and physiological variables in blood. Male Wistar rats were subjected to a 90 min middle cerebral artery occlusion (MCAO). Subcutaneous (SC) treatment with vehicle or pioglitazone was initiated 90 min after MCAO, i.e. in the post-ischemic, reperfusion phase and continued on 2 (2 day-experiment, protocol 1) or 5 (5-day experiment, protocol 2) consecutive days. In the 2-day experiment, pioglitazone at a dose of 2.5 mg/kg body weight (bw) reduced infarct volume by 31% and oedema by 43% on day 2 after MCAO and attenuated the infiltration of ischemic cortical tissue with activated microglia and macrophages. The slight reduction in infarct volume by approximately 18%, detected in rats treated with 10 mg/kg bw pioglitazone did not reach statistical significance. The neurological scores of sham-operated rats treated with vehicle or 10 mg/kg bw pioglitazone were not significantly different. In rats subjected to cerebral ischemia, post-ischemic treatment with either dose of pioglitazone alleviated particular motor deficits and sensory impairments on day 2 after MCAO. A single injection of 10 mg/kg bw pioglitazone in the reperfusion phase (90 min after the onset of reperfusion) did not modify systolic and diastolic blood pressure, heart rate and physiological variables compared to vehicle-treated rats at any time point after MCAO. In the 5-day experiment, continuous post-occlusion treatment with 2.5 mg/kg body weight pioglitazone significantly reduced cerebral infarction by 29% and improved the partial paralysis of the forelimb and alleviated sensory deficits. In the peri-infarct cortex, pioglitazone effectively suppressed the accumulation of activated microglia/macrophages, inhibited neuronal degeneration and promoted neuroregeneration and formation of neuronal networks. The current results provide evidence that pioglitazone treatment in the post-ischemic, reperfusion phase improves the recovery from ischemic stroke. Neuroprotective effects of pioglitazone are mediated by inhibition of post-ischemic inflammation and neuronal degeneration, protection of neurones against ischemic injury and by promoting of neuronal regeneration. Our data together with previous findings favour the view that pioglitazone is a promising candidate for clinical stroke trials.