Effects of estrogen and progestin on the CO2 sensitivity of hemispheric cerebral blood volume.

OBJECTIVE: High CO2 sensitivity is one of the major characteristics of the cerebrovascular bed. It has been shown to be influenced by many differrent factors (eg, sex hormones). DESIGN: The effect of ovariectomy and subsequent female sexual hormone treatment on the steady-state hemispheric cerebral blood volume and CO2 responsiveness of the hemispheric blood vessels was studied on anesthetized, ventilated, normotensive, normoxic rats. Cerebral blood volume was measured with Tomita's photoelectric method with Sandor's modification. RESULTS: Steady-state cerebral blood volume values in ovariectomized rats did not differ from those found in control animals. The CO2 responsiveness of hemispheric blood vessels was higher in ovariectomized and progestin-treated, but not estrogen-treated, animals compared with controls. CONCLUSIONS: Our results demonstrate that the CO2 sensitivity of the hemispheric vessels is sex hormone dependent. Estrogen and progestin treatment have opposite effects on this cerebral circulatory parameter.

Contribution of poly(ADP-ribose) polymerase to postischemic blood-brain barrier damage in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood-brain barrier (BBB) disruption and edema formation after ischemia-reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia-reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170+/-9, saline: 760+/-95, PJ34: 472+/-61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction

Somatosensory pain does not affect total cerebral blood volume.

The effect of somatosensory pain on the total cerebral blood volume was investigated in anesthetized rats. Our results show for the first time that total cerebral blood volume remains unaltered in both brain hemispheres during 2.5 min noxious stimulation of the sensory C-fibres of the sciatic nerve. Regional cerebral blood flow was increased by 97% in the thalamus and by 47% in the hypothalamus at the same time. Blockade of the L-arginine-nitric oxide system reduced significantly the steady-state control level of total cerebral blood volume (i.l.: from 5.7+/-1.3 to 4.58+/-1.6 vol%, c.l.: from 5.0+/-0.6 to 4.24+/-0.9 vol%). Nitric oxide synthase blockade, however, did not affect either the stimulation induced increase of regional cerebral blood flow or the steadiness of total cerebral blood volume during the stimulation.

Poly(ADP-Ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity.

BACKGROUND: We have previously shown that endothelial function is impaired not only in diabetes but also in subjects at risk of developing type 2 diabetes. We hypothesized that changes in the expression or activity of the endothelial isoform of nitric oxide synthase (eNOS), the receptor for advanced glycation end products (RAGE), and poly(ADP-ribose) polymerase (PARP) are related to this impairment. METHODS AND RESULTS: We included a control group of 21 healthy subjects, a group of 22 healthy individuals with parental history of type 2 diabetes, a group of 23 subjects with impaired glucose tolerance, and a group of 21 type 2 diabetic patients. Two 2-mm forearm skin biopsies were taken from each participant and used for measurements. The percentage of PARP-positive endothelial nuclei was higher in the group with parental history of type 2 diabetes and diabetic patients compared with the controls (P<0.001). Immunoreactivity for nitrotyrosine (a marker of reactive nitrogen species) was higher in the diabetic group compared with all other groups (P<0.01). No differences in the expression of eNOS and RAGE were found among all 4 groups. The polymorphism of the eNOS gene was also studied and was not found to influence eNOS expression or microvascular functional measurements. CONCLUSIONS: PARP activation is present in healthy subjects at risk of developing diabetes as well as in established type 2 diabetic patients, and it is associated with impairments in the vascular reactivity in the skin microcirculation.

Role of poly(ADP-ribose) polymerase activation in diabetic neuropathy.

Oxidative and nitrosative stress play a key role in the pathogenesis of diabetic neuropathy, but the mechanisms remain unidentified. Here we provide evidence that poly(ADP-ribose) polymerase (PARP) activation, a downstream effector of oxidant-induced DNA damage, is an obligatory step in functional and metabolic changes in the diabetic nerve. PARP-deficient (PARP(-/-)) mice were protected from both diabetic and galactose-induced motor and sensory nerve conduction slowing and nerve energy failure that were clearly manifest in the wild-type (PARP(+/+)) diabetic or galactose-fed mice. Two structurally unrelated PARP inhibitors, 3-aminobenzamide and 1,5-isoquinolinediol, reversed established nerve blood flow and conduction deficits and energy failure in streptozotocin-induced diabetic rats. Sciatic nerve immunohistochemistry revealed enhanced poly(ADP-ribosyl)ation in all experimental groups manifesting neuropathic changes. Poly(ADP-ribose) accumulation was localized in both endothelial and Schwann cells. Thus, the current work identifies PARP activation as an important mechanism in diabetic neuropathy and provides the first evidence for the potential therapeutic value of PARP inhibitors in this devastating complication of diabetes.

Pharmacologic inhibition of poly(adenosine diphosphate-ribose) polymerase may represent a novel therapeutic approach in chronic heart failure.

OBJECTIVES: We investigated the effects of a novel ultrapotent poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor, PJ34, on cardiac and endothelial dysfunction in a rat model of chronic heart failure (CHF). BACKGROUND: Overactivation of the nuclear enzyme PARP importantly contributes to the development of cell dysfunction and tissue injury in various pathophysiologic conditions associated with oxidative stress, including myocardial reperfusion injury, heart transplantation, stroke, shock, and diabetes. METHODS: Chronic heart failure was induced in Wistar rats by chronic ligation of the left anterior descending coronary artery. Left ventricular (LV) function and ex vivo vascular contractility and relaxation were measured 10 weeks after the surgery. Nitrotyrosine (NT) formation and PARP activation were detected by immunohistochemistry. RESULTS: Chronic heart failure induced increased NT formation and PARP activation in the myocardium and intramural vasculature, depressed LV performance, and impaired vascular relaxation of aortic rings. PJ34 significantly decreased myocardial PARP activation but not NT formation, and improved both cardiac dysfunction and vascular relaxation. CONCLUSIONS: Poly(ADP-ribose) polymerase inhibition represents a novel approach for the experimental treatment of CHF.

Poly (adp-ribose) polymerase inhibitors as potential therapeutic agents in stroke and neurotrauma.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding protein that is primarily activated by nicks in the DNA molecule. It regulates the activity of various enzymes - including itself- that are involved in the control of DNA metabolism. Upon binding to DNA breaks, activated PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins including histones, transcription factors and PARP itself. Poly(ADP-ribosylation) contributes to DNA repair and to the maintenance of genomic stability. Evidence obtained with pharmacological PARP inhibitors of various structural classes, as well as animals lacking the PARP-1 enzyme indicate that PARP plays an important role in cerebral ischemia/reperfusion, stroke and neurotrauma. Overactivation of PARP consumes NAD+ and ATP culminating in cell dysfunction and necrosis. PARP activation can also act as a signal that initiates cell death programs, for instance through AIF (apoptosis inducing factor) translocation. PARP has also been shown to associate with and regulate the function of several transcription factors. Of special interest is the enhancement by PARP of NF-kappaB-mediated transcription, which plays a central role in the expression of inflammatory cytokines, chemokines, adhesion molecules and inflammatory mediators. Via this mechanism, PARP is involved in the up-regulation of numerous pro-inflammatory genes that play a pathogenetic role in the later stage of stroke and neurotrauma. Here we review the roles of PARP in DNA damage signaling and cell death, and summarize the pathogenetic role of PARP in stroke and neurotrauma.

Blood-brain barrier changes during compensated and decompensated hemorrhagic shock.

Dysfunction of the blood-brain barrier (BBB) can be associated with a large number of central nervous system and systemic disorders. The aim of the present study was to determine BBB changes during different phases of hemorrhagic shock. The experiments were carried out on male Wistar rats anaesthetized with urethane. To produce compensated or decompensated hemorrhagic shock, mean arterial pressure was decreased from the normotensive control values to 40 mmHg by a standardized method of blood withdrawal from the femoral artery. Cerebral blood flow changes were followed by laser-Doppler flowmetry, and arterial blood gas values were monitored over the whole procedure. Cortical blood flow was significantly reduced in compensated and in decompensated hemorrhagic shock compared with the normotensive rats. As the shock shifted to the decompensated phase, the blood flow reduction was more pronounced. BBB permeability studies using sodium fluorescein (molecular weight of 376) and Evan's Blue albumin (molecular weight of 67,000) have revealed a significant increase of the BBB permeability for sodium fluorescein in the decompensated stage of hemorrhagic shock. Western blot analysis of brain capillaries showed that the expression of the transmembrane tight junction protein occludin was reduced in response to hemorrhagic shock, and the decrease of occludin was more pronounced in the decompensated phase. A similar expression pattern was shown by the transmembrane adherens junction protein cadherin as well. Our results suggest that the decompensated phase of hemorrhagic shock is associated with disturbances of the BBB, which may be explained by the dysfunction of interendothelial junctions caused by decreased occludin and cadherin levels.

Diabetes-induced overexpression of endothelin-1 and endothelin receptors in the rat renal cortex is mediated via poly(ADP-ribose) polymerase activation.

We hypothesize that poly (ADP-ribosyl)ation, that is, poly (ADP-ribose) polymerase (PARP)-dependent transfer of ADP-ribose moieties from NAD to nuclear proteins, plays a role in diabetic nephropathy. We evaluated whether PARP activation is present and whether two unrelated PARP inhibitors, 3-aminobenzamide (ABA) and 1,5-isoquinolinediol (ISO), counteract overexpression of endothelin-1 (ET-1) and ET receptors in the renal cortex in short-term diabetes. The studies were performed in control rats and streptozotocin-diabetic rats treated with/without ABA or ISO (30 and 3 mg x kg(-1) x day(-1), intraperitoneally, for 2 weeks after 2 weeks of diabetes). Poly (ADP-ribose) immunoreactivity was increased in tubuli, but not glomeruli, of diabetic rats and this increase was corrected by ISO, whereas ABA had a weaker effect. ET-1 concentration (ELISA) was increased in diabetic rats, and this elevation was blunted by ISO. ET-1, ET(A), and ET(B) mRNA (ribonuclease protection assay), but not ET-3 mRNA (RT/PCR), abundance was increased in diabetic rats, and three variables were, at least, partially corrected by ISO. ABA produced a trend towards normalization of ET-1 concentration and ET-1, ET(A), and ET(B) mRNA abundance, but the differences with untreated diabetic group were not significant. Poly(ADP-ribosyl)ation is involved in diabetes-induced renal overexpression of ET-1 and ET receptors. PARP inhibitors could provide a novel therapeutic approach for diabetic complications including nephropathy, and other diseases that involve the endothelin system.

Endothelial dysfunction in aging animals: the role of poly(ADP-ribose) polymerase activation.

Recent work has demonstrated the production of reactive oxygen and nitrogen species in the vasculature of aging animals. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). Here we studied whether the loss of endothelial function in aging rats is dependent upon the PARP pathway within the vasculature. Young (3 months-old) and aging (22 months-old) Wistar rats were treated for 2 months with vehicle or the PARP inhibitor PJ34. In the vehicle-treated aging animals there was a significant loss of endothelial function, as measured by the relaxant responsiveness of vascular rings to acetylcholine. Treatment with PJ34, a potent PARP inhibitor, restored normal endothelial function. There was no impairment of the contractile function and endothelium-independent vasodilatation in aging rats. Furthermore, we found no deterioration in the myocardial contractile function in aging animals. Thus, intraendothelial PARP activation may contribute to endothelial dysfunction associated with aging.

Inhibition of poly (ADP-ribose) polymerase attenuates acute lung injury in an ovine model of sepsis.

It is known that in various pathophysiological conditions, reactive oxidants cause DNA strand breakage and subsequent activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). Activation of PARP results in cellular dysfunction. We hypothesized that pharmacological inhibition of PARP reduces the damage in the ovine model of acute lung injury (ALI). After smoke inhalation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into both lungs. All of the animals were mechanically ventilated with 100% O2. The infusion of the PARP inhibitor (INO-1001, n = 6) began 1 h after the injury and thereafter through 24 h (3 mg bolus + 0.3 mg/kg/h, i.v.). Control animals (n = 6) were treated with saline. Sham injury animals (n = 8) received sham smoke and were mechanically ventilated in the same fashion. One-half of those sham animals (n = 4) were given the same dose of INO-1001. PaO2/FiO2 ratio at 24 h in saline and in the INO-1001-treated groups were 95 +/- 22 and 181 +/- 22, respectively (P < 0.05). Peak airway pressure at 24 h in the saline- and INO-1001-treated groups was 32.6 +/- 3.0 and 24.4 +/- 2.2, respectively (P < 0.05). Pulmonary shunt fraction was also significantly attenuated. INO-1001 treatment reduced pulmonary histological injury and attenuated poly (ADP-ribose) accumulation in the lung. In conclusion, inhibition of PARP improved the ALI after smoke inhalation and pneumonia. The results suggest that the activation of PARP plays a role in the pathophysiology of ALI in sheep.

Mechanisms of pain-induced local cerebral blood flow changes in the rat sensory cortex and thalamus.

It is a well-known phenomenon that cerebral blood flow is coupled to neural activation induced by non-noxious somatosensory stimulation. However, basic questions related to pain-induced cerebral blood flow changes remain unanswered. In the present study, the sciatic nerve of anesthetized rats was subjected to electric stimulation with noxious and non-noxious parameters. Changes in local cerebral blood flow and neuronal activity were determined simultaneously in the sensory cortex and in the thalamus by laser-Doppler flowmetry and c-fos immunohistochemistry, respectively. The role of different vasoregulatory mechanisms and the pain-induced increase in mean arterial blood pressure (MABP) were examined with specific blocking agents and by means of rapid intra-arterial transfusion. Noxious stimulation resulted in significant enhancement of neuronal activity both in the thalamus and in the somatosensory cortex indicated by marked c-fos expression in these areas. Cortical and thalamic blood flow (cBF and tBF) increased by 47+/-4 and 44+/-3% during the stimulation while the MABP elevated by 35+/-2%. Similar changes in MABP induced by intra-arterial transfusion had no effect on tBF, while cBF increased only by 18+/-5%. Blockade of ATP sensitive potassium channels (K(+)(ATP)) and sympathetic beta-receptors significantly attenuated the pain-induced blood flow increases in both investigated areas, while inhibition of nitric oxide synthase was effective only in the thalamus. The blockade of the sympathetic alpha-receptors, opiate receptors, and the cyclooxygenase enzyme had no effect on the pain-induced cerebral blood flow elevations. These findings demonstrate that during noxious stimulation, cerebral blood flow is adjusted to the increased neural activity by the interaction of vasoconstrictor autoregulatory and specific vasodilator mechanisms, involving the activation of sympathetic beta-receptors, K(+)(ATP)-channels and the release of nitric oxide.

Poly(ADP-ribose) polymerase inhibition improves postischemic myocardial function after cardioplegia-cardiopulmonary bypass.

BACKGROUND: Poly(ADP-ribose) polymerase activation has been shown to contribute to the pathogenesis of myocardial ischemia-reperfusion injury. We hypothesized that a novel poly(ADP-ribose) polymerase inhibitor, INO-1001, provides myocardial protection and improves cardiac function after regional ischemia and cardioplegia-cardiopulmonary bypass (CPB). STUDY DESIGN: Pigs were subjected to 30 minutes of regional ischemia by distal left anterior descending coronary artery ligation followed by CPB (60 minutes) with hyperkalemic cardioplegia (45 minutes). The myocardium then was reperfused post-CPB for 90 minutes. After 15 minutes of ischemia, the treatment group (n = 6) received an INO-1001 bolus (1mg/kg) before a continuous infusion (1mg/kg/hour). Control pigs (n = 6) received vehicle solution. Left ventricular pressure was monitored, from which the maximum, positive first derivative of left ventricular pressure over time (+dP/dt) was calculated. Regional myocardial function in the ischemic area was determined by sonomicrometric analysis. Infarct size was measured as the percent of the ischemic area by tetrazolium staining. Myocardial sections were immunohistochemically stained for poly(ADP-ribose) as a measure of poly(ADP-ribose) polymerase activity and inhibition. RESULTS: Pigs treated with INO-1001 showed improvements in the +dP/dt at 60 and 90 minutes of post-CPB reperfusion (both p = 0.03) and percent segmental shortening at 30, 60, and 90 minutes of post-CPB reperfusion (p = 0.03, 0.009, and 0.03, respectively). Infarct size was decreased in the treatment group (18.5 +/- 5.7% versus 52.0 +/- 7.7%, INO-1001 versus control, p = 0.03). Poly(ADP-ribose) was reduced in myocardial sections from INO-1001-treated animals compared with controls. CONCLUSIONS: These results suggest that INO-1001 provides myocardial protection by reducing the extent of infarction and improves cardiac function after regional ischemia and cardioplegia-CPB.

Significance of endogenous opioids in the maintenance of cerebral and spinal vascular CO2-sensitivity in deep hemorrhagic hypotension.

High CO(2)-sensitivity, one of the major characteristics of the cerebrovascular bed, has been shown to be influenced by a variety of factors. There are no reports, however, on the involvement of the endogenous opioid peptides in the modulation of the CO(2)-sensitivity of the cerebral and spinal cord vessels, either in normotensive or, in hypotensive conditions. The effect of general opiate receptor blockade (1.0mg/kg naloxone, i.v.) on regional cerebrovascular CO(2)-sensitivity was studied with radiolabeled microspheres in 10 distinct brain and spinal cord regions of the anesthetized cat. The CO(2)-induced flow changes were investigated in normotensive, in moderately hypotensive (MAP=80 mmHg) and in deep hypotensive cats (MAP=40 mmHg). The systemic arterial pressure was lowered by hemorrhage. In the normotensive cats, opiate receptor blockade caused no changes in the vascular CO(2)-sensitivity in the investigated cerebral and spinal cord regions. In moderate hypotension, cerebral and spinal CO(2)-sensitivity was significantly reduced by the hemorrhage itself, but remained unaffected by the naloxone administration. In deep hemorrhagic hypotension, however, general opiate receptor blockade resulted not only in a further reduction of the already impaired CO(2)-sensitivity, but even in a reversal of the effect of CO(2) from flow increase to flow decrease. These results indicate that endogenous opioid peptides, which do not seem to influence cerebrovascular reactions in steady-state, normotensive conditions, may contribute significantly to the maintenance of the normal vasodilatory response of the cerebral and spinal cord vessels to CO(2) during hemorrhage-induced deep arterial hypotension.

Poly(ADP-ribose) polymerase inhibition protect neurons and the white matter and regulates the translocation of apoptosis-inducing factor in stroke.

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP). Apoptosis-inducing factor (AIF) is a flavoprotein that is normally confined to the mitochondria, but translocates to the nucleus, as shown by in vitro models of neuronal injury. Using INO-1001, a novel potent inhibitor of PARP, we determined the role of PARP activation in the process of AIF translocation in a rat model of focal cerebral ischemia. The potency of INO-1001 as a PARP inhibitor and its cytoprotective potential in oxidant-challenged human neuronal SK-N-MC cells was first confirmed in vitro. PARP inhibition markedly reduced infarct size and improved neurological status in both transient and permanent models of MCA occlusion in Sprague-Dawley rats, with a therapeutic window of 6 h and 2 h in the transient and permanent ischemia models, respectively. The PARP inhibitor reduced the accumulation of poly(ADP-ribose) in the ischemic/reperfused hemisphere and reduced the accumulation of APP in the white matter of the affected hemisphere, consistently with protection against neuronal necrosis and axonal damage, respectively. Immunohistochemical analysis showed the appearance of AIF labeling in neuronal nuclei of the border zone ischemic area in the striatum after stroke. Cytoplasmatic (axonal) AIF staining was significantly diminished in the necrotic core of the striatum, while it was somewhat enhanced at the borderline ischemic territories of the white matter. Inhibition of PARP with INO-1001 reshifted the location of the apoptotic marker to the axons in the ipsilateral striatum. Thus, PARP inhibition is neuroprotective and regulates the ischemic nuclear translocation of AIF in stroke.

PARP inhibition improves the effectiveness of neural stem cell transplantation in experimental brain trauma.

Neural stem cell transplantation is a promising new treatment of ischemic or traumatic brain injury. We have now investigated the involvement of the peroxynitrite - poly(ADP-ribose) polymerase (ONOO- - PARP) activation cascade in brain trauma and neural stem cell transplantation. The forelimb motor cortex of adult male rats was exposed to cold lesion (-60 degrees C) and motor function was monitored. Neural stem cells isolated from E14 rat embryos were labeled with brome deoxyuridine (BrDU) and injected into the injured cortex 6 days after the lesion. After another 6 days, the survival and differentiation of the grafted cells were investigated with immunohistochemistry. Increased production of ONOO- revealed by tyrosine nitration was seen in the lesion 2 days after transplantation. Animals treated with the ONOO- decomposition catalyst FP15 or the PARP inhibitor PJ34 had a significantly improved motor score, when compared to vehicle-treated controls. The neurological score further improved following stem cell grafting in the PJ34 treated, but not in the control animals. Six days after transplantation, differentiated BrDU positive cells were found in the cortical penumbra. The majority of these differentiated cells expressed an astrocyte marker and some of the cells expressed oligodendrocyte or neuronal markers. The number of surviving transplanted cells was significantly higher in the PJ34 treated group. Inhibition of the ONOO- - PARP activation cascade significantly improves the effectiveness of neural stem cell transplantation and promotes rapid functional recovery.

Hypersensitivity to thromboxane receptor mediated cerebral vasomotion and CBF oscillations during acute NO-deficiency in rats.

BACKGROUND: Low frequency (4-12 cpm) spontaneous fluctuations of the cerebrovascular tone (vasomotion) and oscillations of the cerebral blood flow (CBF) have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO) suppresses constitutively the release and vascular effects of thromboxane A(2) (TXA(2)), NO-deficiency is often associated with activation of thromboxane receptors (TP). In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations. METHODOLOGY/PRINCIPAL FINDINGS: Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats) and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs) both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg i.v.) to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl ester (L-NAME, 100 mg/kg i.v.) resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA(2) synthesis by ozagrel (10 mg/kg i.v.) attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632. CONCLUSION/SIGNIFICANCE: These results suggest that hypersensitivity of the TP-receptor-Rho-kinase signaling pathway contributes to the development of low frequency cerebral vasomotion which may propagate to vasospasm in pathophysiological states associated with NO-deficiency.

Effects of estrogen and progestin on hypothalamic blood flow autoregulation.

OBJECTIVES: The consequences of female sex hormone deficiency and the effects of hormone replacement therapy are controversial because individual hormones and their derivates can result in partially antagonistic activities. This intricate system involving cerebral autoregulatory mechanisms caused by ovariectomy and female sex hormone replacement was studied in rats. METHODS: The lower limit of cerebral blood flow autoregulation was determined by stepwise reduction of systemic arterial pressure while simultaneously measuring the changes of the hypothalamic blood flow (HBF) using the hydrogen gas-clearance method. RESULTS: In ovariectomized rats resting HBF decreased substantially and the threshold of cerebrovascular autoregulation decreased to 40 mm Hg. Estrogen replacement prevents the former change and shifts the latter upwards. Similarly, progestin replacement restores autoregulation to the physiological levels found in control animals, whereas it has no influence on the ovariectomy-induced reduction of resting blood flow. CONCLUSIONS: Steady-state HBF and compensatory changes of regional cerebral vascular autoregulation are altered significantly following ovariectomy. Estrogen or progestin replacement has an opposite effect on these cerebral circulatory parameters. Our observations highlight the essential role of female sex hormones in hypothalamic autoregulation during hypotensive stress.

Angiotensin II-mediated endothelial dysfunction: role of poly(ADP-ribose) polymerase activation.

Angiotensin II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction. AII in cultured endothelial cells induced DNA single-strand breakage and dose-dependently activated PARP, which was inhibited by the AII subtype 1 receptor antagonist, losartan; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Infusion of sub-pressor doses of AII to rats for 7 to 14 d induced the development of endothelial dysfunction ex vivo. The PARP inhibitors PJ34 or INO-1001 prevented the development of the endothelial dysfunction and restored normal endothelial function. Similarly, PARP-deficient mice infused with AII for 7 d were found resistant to the AII-induced development of endothelial dysfunction, as opposed to the wild-type controls. In spontaneously hypertensive rats there was marked PARP activation in the aorta, heart, and kidney. The endothelial dysfunction, the cardiovascular alterations and the activation of PARP were prevented by the angiotensin-converting enzyme inhibitor enalapril. We conclude that AII, via AII receptor subtype 1 activation and reactive oxygen and nitrogen species generation, triggers DNA breakage, which activates PARP in the vascular endothelium, leading to the development of endothelial dysfunction in hypertension.

Effect of poly(ADP ribose) synthetase inhibition on burn and smoke inhalation injury in sheep.

We investigated the role of the nuclear enzyme poly (ADP ribose) synthetase (PARS) in the pathogenesis of combined burn and smoke inhalation (burn/smoke) injury in an ovine model. Eighteen sheep were operatively prepared for chronic study. PARS inhibition was achieved by treatment with a novel and selective PARS inhibitor INO-1001. The PARS inhibitor attenuated 1) lung edema formation, 2) deterioration of gas exchange, 3) changes in airway blood flow, 4) changes in airway pressure, 5) lung histological injury, and 6) systemic vascular leakage. Lipid oxidation and plasma nitrite/nitrate (stable breakdown products of nitric oxide) levels were suppressed with the use of INO-1001. We conclude that PARS inhibition attenuates various aspects of the pathophysiological response in a clinically relevant experimental model of burn/smoke inhalation injury.