Preserved Pressure Autoregulation but Disturbed Cyclo-Oxygenase and Nitric Oxide Effects on Retinal Arterioles during Acute Hypoxia in Diabetic Patients without Retinopathy.

BACKGROUND: Acute hypoxia induces retinal vasodilatation, which depends on cyclooxygenase (COX) products and nitric oxide (NO) in vitro. However, it is unknown whether these mechanisms are active in diabetic patients and may contribute to the development of diabetic retinopathy. METHODS: The Dynamic Vessel Analyzer was used to study the diameter regulation in retinal vessels during hypoxia in type 1 diabetic patients without retinopathy. The influence of NO and COX synthesis inhibition on the diameter of larger retinal vessels was studied during hypoxia, during isometric exercise and during flicker stimulation. RESULTS: Increased arterial blood pressure during L-NMMA infusion and isometric exercise were paralleled by constriction of the retinal arterioles suggesting normal pressure autoregulation. Hypoxia significantly reduced the diameter responses during isometric exercise and during flicker stimulation as compared to normal persons. CONCLUSION: The findings support that changes in metabolic autoregulation develop before changes in pressure autoregulation in diabetic patients.

Retina evokes biphasic relaxations in retinal artery unrelated to endothelium, K(V), K(ATP), K(Ca) channels and methyl palmitate.

Retinal relaxing factor (RRF) is suggested to be released from the retina and to contribute in the maintenance of retinal arterial tone. Herein, we aimed to clarify the effects of retinal tissue in isolated bovine retinal arteries in comparison with choroidal tissue and to evaluate the possible role of endothelium and potassium channels. In parallel, the effects of palmitic acid methyl ester (PAME), a putative vasodilator proposed to be released from the retina, was also examined. A piece of bovine retinal or choroidal tissue was placed within a close proximity on top of retinal arteries mounted in a wire myograph and precontracted with noradrenaline, prostaglandin F(2α), endothelin-1, thromboxane A(2) mimetic, U46619 or potassium (K(+)). To elucidate possible mechanisms in the effects of retinal tissue, retinal arteries were either deendothelized or incubated with inhibitors of endothelial vasodilators, i.e. nitric oxide (NO) and prostaglandins, or K(+) channels. Unlike the choroid, retinal tissue produced rapid, biphasic and complete relaxations in isolated bovine retinal arteries precontracted with various spasmogens acting on distinct receptors. Endothelium removal or preincubation of retinal arteries with inhibitors of NO synthase; L-NOARG (10(-4)M), guanylate cyclase; ODQ (10(-5)M) and cyclooxygenase; indomethacin (10(-5)M), did not cause a significant difference in the relaxation profile. Additionally, retinal relaxations remained unchanged in the presence of respective inhibitors of ATP-sensitive (K(ATP)) (glibenclamide, 10(-5)M), voltage-dependent (K(V)) (4-aminopyridine, 2×10(-3)M), and calcium-activated (K(Ca)) (tetraethylammonium 10mM; charybdotoxin, 10(-7)M; and apamin, 5×10(-7)M) K(+) channels. Thus, our results provide novel evidence regarding the biphasic relaxing profile of RRF in the retinal artery which was unrelated to endothelium and K(+) channels (K(ATP), K(V) and K(Ca)). Interestingly, PAME (10(-14)-10(-5)M) did not provoke a relaxation in bovine retinal artery suggesting no association with RRF.

Hypertension induced by vascular endothelial growth factor signaling pathway inhibition: mechanisms and potential use as a biomarker.

Drugs that inhibit the vascular endothelial growth factor (VEGF) signaling pathway are a rapidly growing chemotherapy class for treatment of solid tumors. This targeted therapy is more specific than traditional chemotherapy, causing fewer side effects. However, VEGF-targeted therapies cause hypertension in 30% to 80% of patients. Unlike traditional off-target side effects, hypertension is a mechanism-dependent, on-target toxicity, reflecting effective inhibition of the VEGF signaling pathway rather than nonspecific effects on unrelated signaling pathways. In this article, we review current understanding of the mechanisms of VEGF-targeted therapy-induced hypertension, discuss similarities with preeclampsia, review implications for therapy of this increasingly common clinical problem, and discuss the potential use of blood pressure increase as a biomarker for proper drug dosing and effective VEGF pathway inhibition.

In vitro pharmacological study of femoral artery vascular reactivity after inferior canine hindlimb ischemia/reperfusion: effects of in vivo nitric oxide blocker infusion.

The aim of the investigation was to study the possible effects of in vivo infusion of nitric oxide (NO) blockers upon the in vitro endothelium-dependent femoral reactivity. The experimental model tested herein was the inferior canine hindlimb global ischemia induced by infrarenal abdominal aortic cross-clamping followed by reperfusion. The NO blockers employed in the tests were N(G)-nitro-l-arginine methyl ester (L-NAME), aminoguanidine (AMG), and methylene blue (MB), which were infused immediately after the anesthesia induction. The research protocol was standardized in two main experimental groups, control and ischemia/reperfusion (I/R) injury, randomized in eight subgroups including controls and NO blockers. The femoral artery vascular reactivity was studied in vitro with the aid of a setup consisting of eight organ chambers, where segments of 4-5 mm were suspended and connected to force transducers in the presence of indomethacin to block the cyclooxygenase pathway. The NO-release pathway was evaluated by using specific pharmacological agonists in the in vitro experiments. The L-NAME in vivo infusion led to in vitro endothelium dysfunction in both groups and was associated with high mortality in the animals submitted to I/R. AMG and MB, two clinically used drugs, did not cause in vitro endothelium dysfunction in either of the two groups, which gives evidence that these drugs are not deleterious in the milieu of I/R injury. Nitrite/nitrate plasma levels were not significant except for the L-NAME groups, which presented significant NO decrease.

New aspects in the pathophysiology of preeclampsia.

Preeclampsia, the de novo occurrence of hypertension and proteinuria after the 20th week of gestation, continues to exert an inordinate toll on mothers and children alike. Recent clinical trials, new physiologic insights, and novel observations on pathogenesis have altered the thinking about preeclampsia. The mechanisms surrounding relaxin and its effects on the circulation and on matrix metalloproteinases have been elucidated. The growth factor's receptor, fms-like tyrosine kinase 1, has been shown to exist in a soluble form that is able to inactivate vascular endothelial-derived growth factor and human placental growth factor. Compelling evidence has been brought forth suggesting that fms-like tyrosine kinase 1 is a circulating factor that can cause preeclampsia. Preeclamptic women have high circulating levels of asymmetric dimethyl arginine that could account for the generalized endothelial dysfunction observed in preeclampsia. Preeclamptic women also produce novel autoantibodies that may serve to activate angiotensin receptors. These new observations raise the possibility that the treatment of preeclamptic women will soon be improved.

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger.

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.

[Effects of the endothelial relaxing factor on the orthostatic reaction of systemic hemodynamics in rats]. / Vliianie éndotelial'nogo relaksiruiushchego faktora na ortostaticheskuiu reaktsiiu sistemnoi gemodinamiki u krys.

Experiments with unconscious rats showed that blocking of NO secretion intensifies orthostatic hypotension (mean arterial pressure drops by 72%) and reduces proportionally the calculated total peripheral resistance. A supposition has been made concerning involvement of the endothelial relaxing factor in orthostatic hypotension development, since in orthostasis cardiac output (systemic blood circulation) losses its influence on the endothelial NO secretion.

Myogenic constriction is increased in mesenteric resistance arteries from rats with chronic heart failure: instantaneous counteraction by acute AT1 receptor blockade.

(1) Increased vascular resistance in chronic heart failure (CHF) has been attributed to stimulated neurohumoral systems. However, local mechanisms may also importantly contribute to set arterial tone. Our aim, therefore, was to test whether pressure-induced myogenic constriction of resistance arteries in vitro--devoid of acute effects of circulating factors--is increased in CHF and to explore underlying mechanisms. (2) At 12 weeks after coronary ligation-induced myocardial infarction or SHAM-operations in rats, we studied isolated mesenteric arteries for myogenic constriction, determined as the active constriction (% of passive diameter) in response to stepwise increase in intraluminal pressure (20 - 160 mmHg), in the absence and presence of inhibitors of potentially involved modulators of myogenic constriction. (3) We found that myogenic constriction in mesenteric arteries from CHF rats was markedly increased compared to SHAM over the whole pressure range, the difference being most pronounced at 60 mmHg (24+/-2 versus 4+/-3%, respectively, P<0.001). (4) Both removal of the endothelium as well as inhibition of NO production (L-N(G)-monomethylarginine, 100 micro M) significantly increased myogenic constriction (+16 and +25%, respectively), the increase being similar in CHF- and SHAM-arteries (P=NS). Neither endothelin type A (ET(A))-receptor blockade (BQ123, 1 micro M) nor inhibition of perivascular (sympathetic) nerve conduction (tetrodotoxin, 100 nM) affected the myogenic response in either group. (5) Interestingly, increased myogenic constriction in CHF was fully reversed after angiotensin II type I (AT(1))-receptor blockade (candesartan, 100 nM; losartan, 10 micro M), which was without effect in SHAM. In contrast, neither angiotensin-converting enzyme (ACE) inhibition (lisinopril, 1 micro M; captopril, 10 micro M) or AT(2)-receptor blockade (PD123319, 1 micro M), nor inhibition of superoxide production (superoxide dismutase, 50 U ml(-1)), TXA(2)-receptor blockade (SQ29,548, 1 micro M) or inhibition of cyclooxygenase-derived prostaglandins (indomethacin, 10 micro M) affected myogenic constriction. (6) Sensitivity of mesenteric arteries to angiotensin II (10 nM - 100 micro M) was increased (P<0.05) in CHF (pD(2) 7.1+/-0.4) compared to SHAM (pD(2) 6.2+/-0.3), while the sensitivity to KCl and phenylephrine was not different. (7) Our results demonstrate increased myogenic constriction in small mesenteric arteries of rats with CHF, potentially making it an important target for therapy in counteracting increased vascular resistance in CHF. Our results further suggest active and instantaneous participation of AT(1)-receptors in increased myogenic constriction in CHF, involving increased sensitivity of AT(1)-receptors rather than apparent ACE-mediated local angiotensin II production.

4-Aminopyridine inhibits the neuromuscular effects of nitric oxide and 8-Br-cGMP.

The effects induced by nitric oxide (NO) in different tissues depend on direct and/or indirect interactions with K+ channels. The indirect interaction of NO is produced by activation of guanylyl cyclase which increases the intracellular cGMP. Since NO, cGMP and 4-aminopyridine alone induce tetanic fade and increase amplitude of muscular contractions in isolated rat neuromuscular preparations, the present study was undertaken to determine whether or not the neuromuscular effects of NO and 8-Br-cGMP can be modified by 4-aminopyridine. Using the phrenic nerve and diaphragm muscle isolated from male Wistar rats (200-250 g), we observed that L-arginine (4.7 mM) and 8-Br-cGMP (18 M), in contrast to D-arginine, induced an increase in the amplitude of muscle contraction (10.5 0.7%, N = 10 and 8.0 0.7%, N = 10) and tetanic fade (15 2.0%, N = 8 and 11.6 1.7%, N = 8) at 0.2 and 50 Hz, respectively. N G-nitro-L-arginine (4 mM, N = 8 and 8 mM, N = 8) antagonized the effects of L-arginine. 4-Aminopyridine (1 and 10 M) caused a dose-dependent increase in the amplitude of muscle contraction (15 1.8%, N = 9 and 40 3.1%, N = 10) and tetanic fade (17.7 3.3%, N = 8 and 37.4 1.3%, N = 8). 4-Aminopyridine (1 M, N = 8) did not cause any change in muscle contraction amplitude or tetanic fade of preparations previously paralyzed with d-tubocurarine or stimulated directly. The effects induced by 4-aminopyridine alone were similar to those observed when the drug was administered in combination with L-arginine or 8-Br-cGMP. The data suggest that the blockage of K+ channels produced by 4-aminopyridine inhibits the neuromuscular effects induced by NO and 8-Br-cGMP. Therefore, the presynaptic effects induced by NO seem to depend on indirect interactions with K+ channels.

Effect of arginine vasopressin on the canine epicardial coronary artery: experiments on V1-receptor-mediated production of nitric oxide.

OBJECTIVE: To determine whether arginine vasopressin releases endothelium-derived nitric oxide (EDNO) from the epicardial coronary artery. METHODS: We studied segments of canine left circumflex coronary arteries suspended in organ chambers to measure isometric force. The coronary artery segments were contracted with prostaglandin F2alpha (2 x 10-6M) and exposed to a unique, strong arginine vasopressin concentration (10-6M) or titrated concentrations (10-9 a 10-5 M). RESULTS: The unique dose of arginine vasopressin concentration (10-6M) induced transient, but significant (p<0.05), relaxation in arterial segments with endothelium, and an increase, not significant, in tension in arteries without endothelium. Endothelium-dependent relaxation to arginine vasopressin was inhibited by Ng-monomethyl-L-arginine (L-NMMA, 10-5M) or N G-nitro-L-arginine (L-NOARG) (10-4M), 2 inhibitors of nitric oxide synthesis from L-arginine. Exogenous L-arginine (10-4M), but not D-arginine (10-4M), reversed the inhibitory effect of L-NMMA on vasopressin-mediated vasorelaxation. Endothelium dependent relaxation to vasopressin was also reversibly inhibited by the vasopressin V1-receptor blocker d(CH2)5Try(Me) arginine vasopressin (10-6M) (n=6, P<0.05). CONCLUSION: Vasopressin acts through V1 endothelial receptors to stimulate nitric oxide release from L-arginine.

Efeito da vasopressina em artérias coronárias epicárdicas caninas. Experimentos sobre a produção de óxido nítrico mediante a estimulação de receptores V1 / Effect of arginine vasopressin on the canine epicardial coronary artery. Experiments on V1-receptor-mediated production of nitric oxide

OBJECTIVE: To determine whether arginine vasopressin releases endothelium-derived nitric oxide (EDNO) from the epicardial coronary artery. METHODS: We studied segments of canine left circumflex coronary arteries suspended in organ chambers to measure isometric force. The coronary artery segments were contracted with prostaglandin F2alpha (2 x 10-6M) and exposed to a unique, strong arginine vasopressin concentration (10-6M) or titrated concentrations (10-9 a 10-5 M). RESULTS: The unique dose of arginine vasopressin concentration (10-6M) induced transient, but significant (p<0.05), relaxation in arterial segments with endothelium, and an increase, not significant, in tension in arteries without endothelium. Endothelium-dependent relaxation to arginine vasopressin was inhibited by Ng-monomethyl-L-arginine (L-NMMA, 10-5M) or N G-nitro-L-arginine (L-NOARG) (10-4M), 2 inhibitors of nitric oxide synthesis from L-arginine. Exogenous L-arginine (10-4M), but not D-arginine (10-4M), reversed the inhibitory effect of L-NMMA on vasopressin-mediated vasorelaxation. Endothelium dependent relaxation to vasopressin was also reversibly inhibited by the vasopressin V1-receptor blocker d(CH2)5Try(Me) arginine vasopressin (10-6M) (n=6, P<0.05). CONCLUSION: Vasopressin acts through V1 endothelial receptors to stimulate nitric oxide release from L-arginine

The effect of chronic nitric oxide inhibition on vascular reactivity and blood pressure in pregnant rats

CONTEXT: The exact mechanism involved in changes in blood pressure and peripheral vascular resistance during pregnancy is unknown. OBJECTIVE:To evaluate the importance of endothelium-derivated relaxing factor (EDRF) and its main component, nitric oxide, in blood pressure and vascular reactivity in pregnant rats. DESIGN: Clinical trial in experimentation animals. SETTING: University laboratory of Pharmacology. SAMPLE: Female Wistar rats with normal blood pressure, weight (152 to 227 grams) and age (90 to 116 days). INTERVENTION: The rats were divided in to four groups: pregnant rats treated with L-NAME (13 rats); pregnant control rats (8 rats); virgin rats treated with L-NAME (10 rats); virgin control rats (12 rats). The vascular preparations and caudal blood pressure were obtained at the end of pregnancy, or after the administration of L-NAME in virgin rats. MAIN MEASUREMENTS: The caudal blood pressure and the vascular response to acetylcholine in pre-contracted aortic rings, both with and without endothelium, and the effect of nitric oxide inhibition, Nw-L-nitro-arginine methyl-ester (L-NAME), in pregnant and virgin rats. The L-NAME was administered in the drinking water over a 10-day period. RESULTS: The blood pressure decreased in pregnancy. Aortic rings of pregnant rats were more sensitive to acetylcholine than those of virgin rats. After L-NAME treatment, the blood pressure increased and relaxation was blocked in both groups. The fetal-placental unit weight of the L-NAME group was lower than that of the control group. CONCLUSION: Acetylcholine-induced vasorelaxation sensitivity was greater in pregnant rats and that blood pressure increased after L-NAME administration while the acetylcholine-induced vasorelaxation response was blocked.

Endothelium-dependent vasodilation in response to Pseudomonas aeruginosa lipopolysaccharide: an in vitro study on canine arteries

Early systemic arterial hypotension is a common clinical feature of Pseudomonas septicemia. To determine if Pseudomonas aeruginosa endotoxin induces the release of endothelium-derived nitric oxide (EDNO), an endogenous nitrovasodilator, segments of canine femoral, renal, hepatic, superior mesenteric, and left circumflex coronary arteries were suspended in organ chambers (physiological salt solution, 95 per cent O2/5 per cent CO2, pH 7.4, 37oC) to measure isometric force. In arterial segments contracted with 2 µM prostaglandin F2a, Pseudomonas endotoxin (lipopolysaccharide (LPS) serotype 10(Habs) from Pseudomonas aeruginosa (0.05 to 0.50 mg/ml)) induced concentration-dependent relaxation of segments with endothelium (P<0.05) but no significant change in tension of arteries without endothelium. Endothelium-dependent relaxation in response to Pseudomonas LPS occurred in the presence of 1 µM indomethacin, but could be blocked in the coronary artery with 10 µM NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthesis from L-arginine. The inhibitory effect of L-NMMA on LPS-mediated vasorelaxation of the coronary artery could be reversed by exogenous 100 µM L-arginine but not by 100 µM D-arginine. These experiments indicate that Pseudomonas endotoxin induces synthesis of nitric oxide from L-arginine by the vascular endothelium. LPS-mediated production of EDNO by the endothelium, possibly through the action of constitutive nitric oxide synthase (NOSc), may decrease systemic vascular resistance and may be the mechanism of early hypotension characteristic of Pseudomonas septicemia.

Óxido nítrico na Isquemia Miocárdica Aguda: estudo experimental sobre a proteção miocárdica no modelo de oclusão/reperfusão, em cães / Nitric Oxide in Acute Myocardial Ischemia: experimental study on myocardial protection in the model of occlusion / reperfusion in dogs

Investigações recentes indicam que tanto a isquemia quanto a reperfusão miocárdicas causam disfunção da vasodilatação mediada pelo endotélio vascular sugerindo que as ações do óxido nítrico (ON) encontrem-se prejudicadas nestas circunstâncias. Com o objetivo de determinar a participação do ON no tamanho do infarto do miocárdio, estudamos no Protocolo A, 51 cães anestesiados que foram submetidos a oclusão da artéria coronária descendente anterior por 90 minutos, e a seguir reperfundidos por 30 minutos. ECG e pressões arteriais foram monitorados. Fluxo coronário foi determinado com microesferas marcadas com 99m Tc. 18 cães controle (Grupo I) receberam solução salina. A sínese de ON foi estimulada pela infusão de L-arginina (200 mg/kg iv) em 14 cães (Grupo II) e bloqueada pela infusão do antagonista L-NAME (20 mg/kg iv) em 12 cães (Grupo III). Outros 7 cães (Grupo IV) foram tratados com L-NAME (20 mg/kg iv) e enalaprilato (5 mg/kg iv) para bloquear a hipertensão arterial induzida por L-NAME. Área de risco (AR) (%VE) foi determinada pela infusão de azul de Evans na raiz da aorta e área de necrose (NA) (%AR) foi obtida através de coloração com cloreto de trifenil tetrazólio. Preservação miocárdica foi definida como % AR não necrosada e expressa através da relação (AR-AN / AR x 100. AR foi similar em todos os cães, sendo em média 33 +- 5% VE (+-DPM). Fluxo coronário na zona isquêmica foi 8,8% do fluxo normal.