Abstract-The present study tested the hypothesis that increasing epoxyeicosatrienoic acids by inhibition of soluble epoxide hydrolase (sEH) would lower blood pressure and ameliorate renal damage in salt-sensitive hypertension. Rats were infused with angiotensin and fed a normal-salt diet or an 8% NaCl diet for 14 days. The sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), was given orally to angiotensin-infused animals during the 14-day period. Plasma AUDA metabolite levels were measured, and they averaged 10Ϯ2 ng/mL in normal-salt angiotensin hypertension and 19Ϯ3 ng/mL in high-salt angiotensin hypertension on day 14 in the animals administered the sEH inhibitor. Mean arterial blood pressure averaged 161Ϯ4 mm Hg in normal-salt and 172Ϯ5 mm Hg in the high-salt angiotensin hypertension groups on day 14. EH inhibitor treatment significantly lowered blood pressure to 140Ϯ5 mm Hg in the normal-salt angiotensin hypertension group and to 151Ϯ6 mm Hg in the high-salt angiotensin hypertension group on day 14. The lower arterial blood pressures in the AUDA-treated groups were associated with increased urinary epoxide-to-diol ratios. Urinary microalbumin levels were measured, and ED-1 staining was used to determine renal damage and macrophage infiltration in the groups. Two weeks of AUDA treatment decreased urinary microalbumin excretion in the normal-salt and high-salt angiotensin hypertension groups and macrophage number in the high-salt angiotensin hypertension group. These data demonstrate that sEH inhibition lowers blood pressure and ameliorates renal damage in angiotensin-dependent, salt-sensitive hypertension. Key Words: kidney Ⅲ inflammation Ⅲ endothelium-derived factors Ⅲ albuminuria A lthough treatment of hypertension has significantly advanced in recent decades, a chronic elevation in blood pressure still results in progressive renal damage, as evidenced by the escalating incidence of end-stage renal disease (ESRD). 1,2 The development of hypertension after long-term administration of angiotensin has many of the same renal and vascular changes that are associated with human essential hypertension. 3,4 Likewise, animal models of angiotensindependent hypertension demonstrate a further elevation in blood pressure when fed a high-salt diet or salt sensitivity. [3][4][5] High dietary salt also increases the susceptibility to kidney damage in hypertensive patients and in angiotensindependent hypertensive rats. 3,4 These parallels between patients with essential hypertension and the angiotensin infusion model make this an extremely useful model to evaluate early changes that occur in the kidney that ultimately result in ESRD.Cytochrome P450 epoxygenase metabolites are involved in the long-term regulation of blood pressure and in the response of the kidney to a high-salt diet. 6 -8 Similarly, salt-sensitive hypertension is associated with an inability of the kidney to properly increase epoxygenase levels. 8 -10 Recent studies have provided evidence that increasing epoxygenase levels have renal-and c...
Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.
Malignant ascites is a major source of morbidity and mortality in ovarian cancer patients. It functions as a permissive reactive tumor-host microenvironment and provides sustenance for the floating tumor cells through a plethora of survival/metastasis-associated molecules.
Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by N G -nitro-L-arginine methyl ester (L-NAME, 100 M). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.adenosine-5Ј triphosphate; red blood cell EXTRACELLULAR ATP has been suggested to play an important role in the regulation of vascular resistance in a number of vascular beds, including the kidney (24, 26), mesentery (5, 28), heart (17, 20), and lung (8,10,11,12,16,31). The spacial relationship between the cell that is the source of extracellular ATP and vascular smooth muscle is an important determinant of the vascular response to ATP. Thus ATP released from nerve terminals adjacent to vascular smooth muscle would be expected to activate purinergic receptors that produce contraction of that muscle (18,21). In contrast, ATP released from formed elements in the circulation such as red blood cells (RBCs) (6,9,31,33) or ATP released from the endothelium itself (27) would interact with purinergic receptors present on the endothelium. The stimulation of such receptors has been shown to result in the synthesis of endothelium-derived relaxing factors, including nitric oxide (NO) (5,8,12,11,16). Thus extracellular ATP released from nerve terminals would be expected to increase vascular resistance, whereas ATP released into the vascular lumen could be an important mechanism for decreasing vascular resistance.We reported previously that 1) ATP is released from rabbit RBCs as they traverse the pulmonary circulation (29) and 2) RBCs that are capable of releasing ATP were a requisite component in the perfusate of isolated rabbit lungs to demonstrate the participation of NO as a determinant of vascular resistance (31, 33). In the work presented here, we present evidence that ATP, in the absence of RBCs, is capable of promoting NO synthesis in the pulmonary circulation of the rabbit. Thus we determined the effect of ATP infused into the circulation of isolated rabbit lungs on vascular resistance. Moreover, we present evidence that the major mechanism by which ATP ac...
release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins G s and G i are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased PO 2 or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of G i. Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 M, n ϭ 7) or iloprost (100 nM, n ϭ 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or G s, respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 m, P Ͻ 0.05, n ϭ 5), a specific activator of G i. Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n ϭ 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G i. red blood cell; vascular control; adenine nucleotides; pulmonary circulation THE ERYTHROCYTE contains millimolar concentrations of ATP, a portion of which is released in response to decreased PO 2 and/or mechanical deformation (1,6,26). Once released from the circulating erythrocyte, ATP can regulate vascular resistance via stimulation of nitric oxide (NO) synthesis from the vascular endothelium (2). The NO that is released abluminally results in relaxation of the underlying vascular smooth muscle and an increase in vascular caliber (8). However, NO is also released luminally, where it can interact directly with formed elements in the blood, including the erythrocyte. We reported that NO liberated from the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) inhibited ATP release from rabbit erythrocytes in response to decreased PO 2 as well as mechanical deformation (21). These data demonstrate that NO could function in a negative feedback manner to inhibit ATP release from circulating erythrocytes. However, the mechanism by which NO inhibits ATP release has yet to be elucidated.ATP release from erythrocytes has been reported to require the activation of a signal transduction pathway. Components of this signal transduction pathway that have been identified include the heterotrimeric G proteins, G s and G i , adenylyl cyclase, PKA, and the cystic fibros...
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