Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 ± 17.3 versus 209 ± 10.9mm Hg in hypertensive controls, p<0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p<0.05). Doxycycline also decreased hypertension-induced oxidative stress (p<0.05), higher MMP activity (p<0.01) and improved NO levels in aortic endothelial cells (p<0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity.     

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 ± 20.8 and 192 ± 17.6 mm Hg, respectively, versus 213 ± 18 mm Hg in hypertensive controls; both p < 0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p > 0.05), prevented the vascular remodeling found in 2K-1C rats (all p < 0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p < 0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p < 0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.     

Modulation of Platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.     

Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis

Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10(-6) M) and the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha-PKC isoform. Both PDBu and Phe caused significant translocation of alpha-PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha-PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha-PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results suggest that one possible mechanism of the pregnancy-associated changes in vascular reactivity may involve changes in the amount and activity of the alpha-PKC isoform.     

Angiotensin II-induced ET and PGI2 release in rat aortic endothelial cells is mediated by PKC.

Angiotensin II (Ang II) has been shown to stimulate the release of immunoreactive endothelin (ET) from cultured bovine ECs. Also, Ang II activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of prostaglandins. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in Ang II-induced release of both ET and prostacyclin (PGI2). The amount of ET and PGI2 produced were determined by radioimmunoassay. Ang II-induced the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine(CL), staurosporine, 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), and calphostin C blocked AII-induced release of both ET and PGI2. In rat aortic endothelial cells that were treated with either AII or PDBu, PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates AII-induced ET and PGI2 release. In summary, AII activates PKC which inhibits rat aortic endothelial cells ET and PGI2 formation, and this inhibition can be overcome by pretreatment with PKC inhibitors.     

Adenosine induced direct negative inotropic effect is abolished during global ischemia: role of protein kinase C and prostacyclin

Adenosine acts as a cardioprotective agent by producing coronary vasodilation, decreasing heart rate and by antagonizing the cardiostimulatory effect of catecholamines; adenosine also exerts a direct negative inotropic effect. Myocardial ischemia is known to be associated with enhanced levels of adenosine, increased protein kinase C (PKC) activity and prostacyclin (PGI2) release. The present study was conducted to determine if myocardial ischemia alters the cardioprotective effect of adenosine by increasing PKC activity and PGI2 release in the isolated rat heart perfused at 10 ml/min with Krebs-Henseleit buffer (KHB; 95% O2+5% CO2). Adenosine (10 mmol/min) reduced myocardial contractility as indicated by a decrease in contractility (dp/dtmax), heart rate (HR) and coronary perfusion pressure (PP). In hearts subjected to 30 min of ischemia (without perfusion) and then reperfused with KHB, adenosine failed to decrease dp/dtmax, HR or PP. However, during infusion of PKC inhibitor H-7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine hydrochloride) (H-7; 6 mmol/min), which commenced 10 min before ischemia and continued throughout reperfusion, adenosine produced a decrease in dp/dtmax, HR and PP, similar to that before ischemia. Infusion of the PKC activator phorbol 12,13-dibutyrate (PDBu; 2 nmol/min) but not an inactive analogue in non-ischemic hearts prevented the adenosine induced decrease in dp/dtmax. During infusion of H-7, PDBu failed to block the direct negative inotropic effect of adenosine in non-ischemic hearts. In addition, pretreatment with H-7 or indomethacin (cyclooxygenase inhibitor) significantly reduced the PGI2 release following ischemia. This data suggest that PKC and PGI2 regulate the direct negative inotropic effect of adenosine, which is abolished during ischemia.     

Effects of inflammatory mediators on canine airway neuromuscular function.

Inflammatory mediators can both enhance or inhibit canine airway reactivity. PGE2 and PGI2 in general are inhibitory, interfering with release of acetylcholine and with responses to bronchoconstrictors. These prostaglandins may be more effective against agonists that open voltage-dependent Ca2+ channels to induce Ca2+ influx and contraction compared with those agonists that release internal Ca2+. Other mediators are excitatory: histamine, PGD2, thromboxane A2 (TxA2), and leukotrienes (LT) C4, D4, and E4. In canine airway only histamine and TxA2 have effects in the absence of indomethacin, i.e., in the presence of the large amounts of PGE2 and PGI2 produced in vitro LTs are ineffective. Effects of TxA2 and histamine may be potentiated if the synthesis of these inhibitory PGs is inhibited. Whether histamine or TxA2 normally promote synthesis and release of PGE2 and PGI2 in a kind of homeostasis remains to be explored. It is also unclear whether pre- as well as post-junctional TxA2 receptors exist and have different pharmacological sensitivities to antagonists. LTC4 and LTD4 also constrict canine bronchi but only when PGE2 and PGI2 synthesis is blocked and, again, whether this is a result of LT-induced release of inhibitory mediators is unknown. The concept that airway responsiveness can be caused by turning off PGE2 and PGI2 production and turning on TxA2 or LT production (or unmasking their actions) needs further exploration. Our recent data suggest that such a mechanism may explain ozone-induced responsiveness in dogs.     

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Vasoconstrictor mechanisms in the cerebral circulation are unaffected by insulin resistance

Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 +/- 8%; ZO: 33 +/- 3% at 10(-8) M). Inhibition of the ET(A) receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 +/- 1%) and ZO (5 +/- 3%) rats. Expressions of the ET(A) receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A(2) (TxA(2)) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10(-6) M PDBu: ZL: 33 +/- 2%; ZO: 32 +/- 4%; and 10(-7) M U-46619: ZL: 23 +/- 1%; ZO: 19 +/- 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 +/- 4%; ZO: 38 +/- 2% at 10(-5) M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10(-6) M: 10 +/- 3%) compared with ZLs (29 +/- 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA(2), PKC, and Rho-kinase are not affected by IR.     

Increase in uterine prostaglandin E2, F2 alpha, prostacyclin and stability in thromboxane A2 production during late pregnancy in streptozotocin-induced diabetic rats

This experiment was conducted to determine the effect of diabetes on uterine prostanoids production in near-term rats. The incidence of an insulin therapy was also studied. On the 21st day of pregnancy, uterine PGE2, PGF2 alpha and PGI2 levels showed a significant increase (respectively p less than 0.05, p less than 0.01 and p less than 0.05) in diabetic rats compared to controls whereas TxA2 production remained unchanged. The insulin therapy restored PGE2 levels, the most potent stimulatory factor of the myometrial fiber at control values, whereas it enhanced significantly PGI2 concentrations (p less than 0.05) and had no effect on PGF2 alpha production; TxA2 levels remaining always unchanged. It is suggested that the increase in uterine protanolds production during diabetes could induce a myometrial hypertonicity and play a role in the disturbances of the fetal development. The maintenance of PGE2 levels to control values by the insulin therapy might contribute to a normal delivery.     

Current concepts for a drug-induced inhibition of formation and action of thromboxane A2

Urinary and plasma metabolites of thromboxane A2 (TxA2) indicate an increased TxA2 synthesis in a number of diseases, whereby TxA2 is assumed to contribute to the underlying pathomechanisms by its profound effects on platelet aggregation and smooth muscle contraction. In some clinical situations the increment in TxA2 biosynthesis is accompanied by an increased formation of prostacyclin (PGI2) which is one of the most potent inhibitors of platelet activation and smooth muscle contraction. Therefore, drugs are being developed which suppress the formation or action of TxA2 without interfering with its functional antagonist PGI2. Low doses of acetylsalicyclic acid (ASA) preferentially inhibit cyclooxygenase activity in platelets and the synthesis of TxA2 in vivo. However, neither low doses (approximately 300 mg/day) nor very low doses spare the formation of PGI2 completely. Despite its limited selectivity, very low dose ASA (approximately 40 mg/day) provides an attractive perspective in TxA2 pharmacology. Although thromboxane synthase inhibitors selectively suppress TxA2 biosynthesis PGH2 can accumulate instead of TxA2 and substitute for TxA2 at their common TxA2/PGH2 receptors. Thromboxane synthase inhibitors can only exert platelet-inhibiting and vasodilating effects if PGH2 rapidly isomerizes to functional antagonists like PGI2 that can be formed from platelet-derived PGH2 by the vessel wall. TxA2/PGH2 receptor antagonists provide a specific and effective approach for inhibition of TxA2. These inhibitors do not interfere with the synthesis of PGI2 and other prostanoids but prevent TxA2 and PGH2 from activating platelets and inducing smooth muscle contractions. Most of the available TxA2/PGH2 receptor antagonists produce a competitive antagonism that can be overcome by high agonist concentrations. Since in certain disease states very high local TxA2 concentrations are to be antagonized, non-competitive receptor antagonists may be of particular interest. Some recent TxA2/PGH2 receptor antagonists produce such a non-competitive type of inhibition due to their low dissociation rate constant. As a consequence, agonists like TxA2 or PGH2 only reach a hemiequilibrium state at their receptors, previously occupied by those antagonists. A combination of a thromboxane synthase inhibitor with a TxA2/PGH2 receptor antagonist presents a very high inhibitory potential that utilizes the dual activities of the synthase inhibitor to increase PGI2 formation and of the receptor antagonist to antagonize PGH2 and TxA2. Such combinations or dual inhibitors, combining both moieties in one compound, prolong the skin bleeding time to a greater extent than thromboxane synthase inhibitors and even more than low dose ASA or TxA2/PGH2 receptor antagonists.     

The mechanism of cytoprotective effect of CHINOIN-127.

According earlier, investigations nitrogen bridgehead compounds make a representative group of non-prostaglandin type gastroprotective agents. One member of this group is CHINOIN-127 (1,6-dimethyl-4-oxo-1, 6, 7, 8, 9, 9a-hexahydro-4H-pyrido-(1, 2a)-pyrimidine-3-carbox-amide). CHINOIN-127 is a potent non-narcotic analgesic and antiinflammatory agent and has a remarkable protective effect on indomethacin induced ulcer (ED50 = 25 mg/kg p.o.) and on acidified ethanol induced ulcer (ED50 = 26 mg/kg p.o.). In this study we examined the mechanism of action of cytoprotective effect of this drug and we made a comparison between the cytoprotective effect of 20% ethanol and 25 mg/kg CHINOIN-127. In the gastric mucosa of control rats we observed a balance between TxA2 and PGI2 (PGI2/TxA2 = 3.8) and between the cytoprotective prostaglandins (PGI2 and PGE2) and ulcerogen eicosanoids (TxA2 and leukotrienes) (PGI2 + PGE2/TxA2 + LTs = 3.9). 100% ethanol treatment causes disintegration of this balance, shifting the synthesis towards the ulcerogen eicosanoids production. CHINOIN-127 and 20% ethanol pretreatment improves the deranged balance between cytoprotective prostaglandins and ulcerogen eicosanoids. Our results demonstrate that CHINOIN-127 and 20% ethanol have a similar mechanism of cytoprotective action on ethanol induced ulcer in rats.     

Effect of phorbol ester on contractile response of aorta from endotoxic rats

The influence of phorbol ester on the isometric contractile response of aorta from endotoxic rats was examined. In endotoxic rat aorta, the contractile responses to KCl and phorbol 12,13-dibutyrate (PDBu) were both remarkably diminished, compared to those in control rat aorta. Preincubation with PDBu augmented the aortic contractile response to KCl in both control and endotoxic rats. This augmentative effect of PDBu was significantly more pronounced in endotoxic rats than in controls. When the contractile response to 80 mM KCl reached a plateau after PDBu pretreatment, addition of 5 mM CaCl2 (final concentration) to the organ bath completely reversed the diminished contractile response of endotoxic rat aorta to the control level. These results suggest that the hyporesponsiveness of endotoxic rat aorta to KCl may be caused by decreases in both protein kinase C mediated response and calcium sensitivity of vascular smooth muscle cells.     

Selective thromboxane synthetase inhibition by picotamide and effects on endotoxin-induced lethality

The efficacy of N,N'-bis-(3-picolyl)-methoxyisophthalamide (picotamide) as an in vitro thromboxane synthetase inhibitor and its effect on endotoxin (LPS)-induced lethality in rats were assessed. Picotamide at 0.5 and 1.0 mM concentrations significantly (P less than 0.05) inhibited basal and LPS-stimulated synthesis of TxA2 measured by its stable immunoreactive (i) metabolite TxB2 in rat peritoneal macrophages. This compound did not inhibit synthesis of i6-keto-PGF1 alpha, the stable metabolite of PGI2, and produced significant shunting to i6-keto-PGF1 alpha. For lethality studies rats were pretreated, by gavage with picotamide, at either 75, 150, 300, or 600 mg/kg 2 hr prior to iv S. enteritidis (LPS, 20 mg/kg). Both 150 and 300 mg/kg doses of picotamide significantly (P less than 0.05) improved survival in endotoxin shock at 48 hr. These studies demonstrate that picotamide is a selective thromboxane synthetase inhibitor, and that it may be useful during disease states characterized by increased TxA2 synthesis.     

Norepinephrine induces lung vascular prostacyclin synthesis via alpha 1-adrenergic receptors

Sympathetic nerve stimulation can cause pulmonary vasoconstriction related to norepinephrine (NE) release. Because of recent reports that NE caused prostacyclin (PGI2) release from systemic arteries, we wondered whether NE caused pulmonary vascular PGI2 release and whether a feedback mechanism existed whereby PGI2 modulated NE-induced vasoconstriction. NE-induced PGI2 synthesis in rat main pulmonary artery rings was larger than that induced by KCl, passive stretch, or a thromboxane analogue, was alpha-adrenergic receptor dependent, and was enhanced by endothelium removal. The NE-induced PGI2 synthesis was not tightly coupled to the magnitude of the pulmonary artery ring contractile response, and inhibition of NE-induced PGI2 production by cyclooxygenase blockade in either the pulmonary artery ring preparation or in isolated rat lungs perfused with a physiological solution did not augment the magnitude of the contractile response. We concluded that NE is a potent stimulus for PGI2 synthesis in the rat main pulmonary artery ring and in the rat lung, yet PGI2 is not important as a modulator of NE-induced vasoconstriction in the rat lung.     

Vascular responses to sodium arachidonate in experimental hypertension

This study characterizes vascular responsiveness to sodium arachidonate (C 20:4) in four models of hypertension [deoxycorticosterone acetate (DOCA) hypertensive rats, two kidney-one clip (2K-1C) renal hypertensive rats, spontaneously hypertensive rats (SHR), and psychosocial hypertensive mice]. Isolated arterial strips (aorta, mesenteric artery, tail artery) were equilibrated under optimal resting tension in physiological salt solution for measurement of isometric force generation. Dose-response curves to arachidonate (10(-10) to 10(-4) g/ml) in arteries from DOCA and 2K-1C hypertensive rats were shifted to the left compared to those in arteries from control rats. In arteries from SHR and psychosocial hypertensive mice, the dose-response relationships were unchanged compared to normotensive values. Arteries from DOCA hypertensive and 2K-1C hypertensive rats developed greater maximal contractile responses to arachidonate than controls; maximal responses in arteries from SHR and psychosocial hypertensive mice were unchanged compared to normotensive values. Contractions to arachidonate were inhibited by indomethacin (0.5 and 5 micrograms/ml) and by aspirin (5 and 50 micrograms/ml). The fatty acid, oleate (C 18:1), had no effect on the contractile state of the arteries, whereas prostaglandin F2 alpha caused contraction. These results indicate altered responsiveness to exogenous arachidonate in arteries from DOCA and 2K-1C hypertensive rats, but not in arteries from SHR and psychosocial hypertensive mice.     

Effects of aldose reductase inhibitors on prostacyclin (PGI2) synthesis by aortic rings from rats with streptozotocin-induced diabetes

The effects of aldose reductase inhibitors (ARIs) on the synthesis of prostacyclin (PGI2) by aortic rings from diabetic rats were examined. The ARIs studied were ONO-2235 and isoliquiritigenin, a new compound extracted from glycyrrhizae radix. The content of sorbitol in the sciatic nerve of diabetic rats induced by streptozotocin was significantly increased as compared with that of controls. This increase was significantly inhibited by the administration of an ARI. On the other hand, there was a marked decrease in the synthesis of PGI2 by the diabetic rats compared with the control rats. The decrease in PGI2 synthesis was significantly reversed by the administration of an ARI. Furthermore, the synthesis of PGI2 by the aortic rings was inversely correlated with the content of sorbitol in sciatic nerves. Those observations suggest that an ARI may have a beneficial effect on the vascular synthesis of PGI2 in diabetes mellitus.     

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.     

The effects of thyroxine and methimazole treatment on the synthesis of prostacyclin (PGI2) in the rat

The effects of thyroxine (T4) and methimazole administration on plasma prostacyclin (PGI2) levels in vivo and on PGI2 release by aortic rings incubated in vitro were investigated in rats. Male rats were given single injection of T4 (200 micrograms/100 g body wt) ip every 24 h for either 3, 7 or 14 days for hyperthyroid rats. For hypothyroid rats, a group of rats were given methimazole (0.01 % in drinking water) for 14 days. PGI2 concentrations were determined in plasma and also in the medium in which aortic rings were incubated. PGI2 was measured as 6-keto-PGF1 alpha by RIA. Plasma PGI2 levels in T4-treated groups were found to be significantly higher than those of control animals. Aortic rings obtained from rats given single injection of T4 for 7 and 14 days showed significant increases in release of PGI2 into the incubation medium. In contrast, rats given methimazole for 14 days showed a significant decrease in the production of PGI2 by aortic rings without any significant changes in plasma levels. Direct addition of T4 into the incubation medium did not cause any significant changes in PGI2 release by aortic rings obtained from control rats. These results suggest the regulatory role of thyroid hormone in PGI2 synthesis in vivo.     

Prostaglandins and thromboxane outflow from the perfused mesenteric vascular bed in spontaneously hypertensive rats

To clarify the metabolism of PGE2, prostacyclin (PGI2) and thromboxane A2 (TxA2) in small vessels in spontaneously hypertensive rats (SHR), we removed superior mesenteric vascular beds from 10 week old SHR and age matched normotensive controls (WKY). The mesenteric artery was perfused with Krebs-Henseleit buffer and samples of effluent collected every 15 minutes during 3 hours perfusion for analysis of PGE2, 6-keto-PGF1 alpha (a stable metabolite of PGI2) and TxB2 (a stable metabolite of TxA2) by specific radioimmunoassays. Levels of all three arachidonic acid (AA) metabolites, PGE2, 6-keto-PGF1 alpha and TxB2, in the mesenteric effluent were significantly reduced in SHR as compared to WKY. TxB2 was detected in all samples throughout the perfusion. 6-keto-PGF1 alpha/PGE2 ratios and TxB2/PGE2 ratios were significantly increased in SHR. 6-keto-PGF1 alpha/TxB2 ratios in the first four samples were significantly decreased in SHR as compared to WKY. These data suggest that there may be reduced availability of PG precusor AA and unbalanced synthesis of PGs in small vessels in SHR. Both may have relevance to the development of hypertension in the animals.