Purinergic P2Y2 receptor-induced activation of endothelial TRPV4 channels mediates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. Here, we investigated the cellular mechanisms underlying lung IR-induced activation of endothelial TRPV4 channels, which play a central role in lung edema and dysfunction after IR. In a left lung hilar-ligation model of IRI in mice, we found that lung IRI increased the efflux of ATP through pannexin 1 (Panx1) channels at the endothelial cell (EC) membrane. Elevated extracellular ATP activated Ca2+ influx through endothelial TRPV4 channels downstream of purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro models of IR. Endothelium-specific deletion of P2Y2R, TRPV4, or Panx1 in mice substantially prevented lung IRI-induced activation of endothelial TRPV4 channels and lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a mediator of the pathological sequelae of IRI in the lung and show that disruption of the endothelial Panx1-P2Y2R-TRPV4 signaling pathway could be a promising therapeutic strategy for preventing lung IRI after transplantation.

Purinergic P2Y2 Receptor-Induced Activation of Endothelial TRPV4 Channels Mediates Lung Ischemia-Reperfusion Injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. We recently reported that endothelial cell (EC) TRPV4 channels play a central role in lung edema and dysfunction after IR. However, the cellular mechanisms for lung IR-induced activation of endothelial TRPV4 channels are unknown. In a left-lung hilar ligation model of IRI in mice, we found that lung IR increases the efflux of extracellular ATP (eATP) through pannexin 1 (Panx1) channels at the EC membrane. Elevated eATP activated elementary Ca2+ influx signals through endothelial TRPV4 channels through purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro surrogate models of lung IR. Endothelium-specific deletion of P2Y2R, TRPV4, and Panx1 in mice had substantial protective effects against lung IR-induced activation of endothelial TRPV4 channels, lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a novel mediator of lung edema, inflammation, and dysfunction after IR, and show that disruption of endothelial Panx1-P2Y2R-TRPV4 signaling pathway could represent a promising therapeutic strategy for preventing lung IRI after transplantation.

Endothelial IK and SK channel activation decreases pulmonary arterial pressure and vascular remodeling in pulmonary hypertension.

Endothelial cells (ECs) from small pulmonary arteries (PAs) release nitric oxide (NO) and prostacyclin, which lower pulmonary arterial pressure (PAP). In pulmonary hypertension (PH), the levels of endothelium-derived NO and prostacyclin are reduced, contributing to elevated PAP. Small-and intermediate-conductance Ca2+-activated K+ channels (IK and SK)-additional crucial endothelial mediators of vasodilation-are also present in small PAs, but their function has not been investigated in PH. We hypothesized that endothelial IK and SK channels can be targeted to lower PAP in PH. Whole-cell patch-clamp experiments showed functional IK and SK channels in ECs, but not smooth muscle cells, from small PAs. Using a SU5416 plus chronic hypoxia (Su + CH) mouse model of PH, we found that currents through EC IK and SK channels were unchanged compared with those from normal mice. Moreover, IK/SK channel-mediated dilation of small PAs was preserved in Su + CH mice. Consistent with previous reports, endothelial NO levels and NO-mediated dilation were reduced in small PAs from Su + CH mice. Notably, acute treatment with IK/SK channel activators decreased PAP in Su + CH mice but not in normal mice. Further, chronic activation of IK/SK channels decreased PA remodeling and right ventricular hypertrophy, which are pathological hallmarks of PH, in Su + CH mice. Collectively, our data provide the first evidence that, unlike endothelial NO release, IK/SK channel activity is not altered in PH. Our results also demonstrate proof of principle that IK/SK channel activation can be used as a strategy for lowering PAP in PH.

TRPV4-dependent signaling mechanisms in systemic and pulmonary vasculature.

The delicate balance between constrictor and dilator mechanisms is a vital determinant of blood pressure and blood flow. The maintenance of this balance requires constant communication between different cell-types in the vascular wall. In this regard, the transient receptor potential vanilloid type 4 (TRPV4) ion channel, a Ca2+-permeable non-selective cation channel, has emerged as a crucial regulator of Ca2+-mediated changes in vascular reactivity. Recent studies suggest that TRPV4 channels regulate vasoconstriction and arterial pressure in the systemic and pulmonary vasculature. New emerging data support a dilatory role of endothelial TRPV4 channels, and both constrictor and dilator roles of smooth muscle TRPV4 channels. Moreover, TRPV4 channel activity has been implicated in physiological functions of vascular support cells, such as fibroblasts and pericytes, to assist the sustenance of vascular reactivity in response to changes in intravascular pressure or external stimulation. Importantly, a growing body of evidence connects abnormal TRPV4 channel activity to multiple vascular disorders. This chapter will review the current literature on the cell-type specific roles of vascular TRPV4 channels in regulating physiological function. Additionally, we summarize our understanding of the contribution of abnormal TRPV4 channel activity to various vascular disorders.

Novel Smooth Muscle Ca2+-Signaling Nanodomains in Blood Pressure Regulation.

BACKGROUND: Ca2+ signals in smooth muscle cells (SMCs) contribute to vascular resistance and control blood pressure. Increased vascular resistance in hypertension has been attributed to impaired SMC Ca2+ signaling mechanisms. In this regard, transient receptor potential vanilloid 4 (TRPV4SMC) ion channels are a crucial Ca2+ entry pathway in SMCs. However, their role in blood pressure regulation has not been identified. METHODS: We used SMC-specific TRPV4-/- (TRPV4SMC-/-) mice to assess the role of TRPV4SMC channels in blood pressure regulation. We determined the contribution of TRPV4SMC channels to the constrictor effect of α1 adrenergic receptor (α1AR) stimulation and elevated intraluminal pressure: 2 main physiologic stimuli that constrict resistance-sized arteries. The contribution of spatially separated TRPV4SMC channel subpopulations to elevated blood pressure in hypertension was evaluated in angiotensin II-infused mice and patients with hypertension. RESULTS: We provide first evidence that TRPV4SMC channel activity elevates resting blood pressure in normal mice. α1AR stimulation activated TRPV4SMC channels through PKCα (protein kinase Cα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Intraluminal pressure-induced TRPV4SMC channel activity opposed vasoconstriction through activation of Ca2+-sensitive K+ (BK) channels, indicating functionally opposite pools of TRPV4SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR-TRPV4SMC and intraluminal pressure-TRPV4SMC-BK channel signaling have opposite effects on blood pressure, with α1AR-TRPV4SMC signaling dominating under resting conditions. Furthermore, in patients with hypertension and a mouse model of hypertension, constrictor α1AR-PKCα-TRPV4 signaling was upregulated, whereas dilator pressure-TRPV4-BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. CONCLUSIONS: Our data identify novel smooth muscle Ca2+-signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

Endothelial Transient Receptor Potential Vanilloid 4 Channels Mediate Lung Ischemia-Reperfusion Injury.

BACKGROUND: Lung ischemia-reperfusion injury (IRI), involving severe inflammation and edema, is a major cause of primary graft dysfunction after transplant. Activation of transient receptor potential vanilloid 4 (TRPV4) channels modulates vascular permeability. Thus, this study tests the hypothesis that endothelial TRPV4 channels mediate lung IRI. METHODS: A left lung hilar-ligation model was used to induce lung IR in C57BL/6 wild-type (WT), Trpv4-/-, tamoxifen-inducible endothelial Trpv4 knockout (Trpv4EC-/-), and tamoxifen-treated control (Trpv4fl/fl) (n ≥ 6 mice/group). WT mice were also treated with GSK2193874 (WT+GSK219), a TRPV4-specific inhibitor (1 mg/kg). Partial pressure of arterial oxygen, edema (wet-to-dry weight ratio), compliance, neutrophil infiltration, and cytokine concentrations in bronchoalveolar lavage fluid were assessed. Pulmonary microvascular endothelial cells were characterized in vitro after exposure to hypoxia-reoxygenation. RESULTS: Compared with WT, partial pressure of arterial oxygen after IR was significantly improved in Trpv4-/- mice (133.1 ± 43.9 vs 427.8 ± 83.1 mm Hg, P < .001) and WT+GSK219 mice (133.1 ± 43.9 vs 447.0 ± 67.6 mm Hg, P < .001). Pulmonary edema and neutrophil infiltration were also significantly reduced after IR in Trpv4-/- and WT+GSK219 mice vs WT. Trpv4EC-/- mice after IR demonstrated significantly improved oxygenation vs control (109.2 ± 21.6 vs 405.3 ± 41.4 mm Hg, P < .001) as well as significantly improved compliance and significantly less edema, neutrophil infiltration, and proinflammatory cytokine production (tumor necrosis factor-a, chemokine [C-X-C motif] ligand 1, interleukin 17, interferon-γ). Hypoxia-reoxygenation-induced permeability and chemokine (C-X-C motif) ligand 1 expression by pulmonary microvascular endothelial cells were significantly attenuated by TRPV4 inhibitors. CONCLUSIONS: Endothelial TRPV4 plays a key role in vascular permeability and lung inflammation after IR. TRPV4 channels may be a promising therapeutic target to mitigate lung IRI and decrease the incidence of primary graft dysfunction after transplant.

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice.

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension.

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.

Role of the purinergic signaling network in lung ischemia-reperfusion injury.

PURPOSE OF REVIEW: Primary graft dysfunction (PGD) is the leading cause of early mortality following lung transplantation and is typically caused by lung ischemia-reperfusion injury (IRI). Current management of PGD is largely supportive and there are no approved therapies to prevent lung IRI after transplantation. The purinergic signaling network plays an important role in this sterile inflammatory process, and pharmacologic manipulation of said network is a promising therapeutic strategy. This review will summarize recent findings in this area. RECENT FINDINGS: In the past 18 months, our understanding of lung IRI has improved, and it is becoming clear that the purinergic signaling network plays a vital role. Recent works have identified critical components of the purinergic signaling network (Pannexin-1 channels, ectonucleotidases, purinergic P1 and P2 receptors) involved in inflammation in a number of pathologic states including lung IRI. In addition, a functionally-related calcium channel, the transient receptor potential vanilloid type 4 (TRPV4) channel, has recently been linked to purinergic signaling and has also been shown to mediate lung IRI. SUMMARY: Agents targeting components of the purinergic signaling network are promising potential therapeutics to limit inflammation associated with lung IRI and thus decrease the risk of developing PGD.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Mechanisms underlying selective coupling of endothelial Ca2+ signals with eNOS vs. IK/SK channels in systemic and pulmonary arteries.

KEY POINTS: Endothelial cell TRPV4 (TRPV4EC ) channels exert a dilatory effect on the resting diameter of resistance mesenteric and pulmonary arteries. Functional intermediate- and small-conductance K+ (IK and SK) channels and endothelial nitric oxide synthase (eNOS) are present in the endothelium of mesenteric and pulmonary arteries. TRPV4EC sparklets preferentially couple with IK/SK channels in mesenteric arteries and with eNOS in pulmonary arteries. TRPV4EC channels co-localize with IK/SK channels in mesenteric arteries but not in pulmonary arteries, which may explain TRPV4EC -IK/SK channel coupling in mesenteric arteries and its absence in pulmonary arteries. The presence of the nitric oxide-scavenging protein, haemoglobin α, limits TRPV4EC -eNOS signalling in mesenteric arteries. Spatial proximity of TRPV4EC channels with eNOS and the absence of haemoglobin α favour TRPV4EC -eNOS signalling in pulmonary arteries. ABSTRACT: Spatially localized Ca2+ signals activate Ca2+ -sensitive intermediate- and small-conductance K+ (IK and SK) channels in some vascular beds and endothelial nitric oxide synthase (eNOS) in others. The present study aimed to uncover the signalling organization that determines selective Ca2+ signal to vasodilatory target coupling in the endothelium. Resistance-sized mesenteric arteries (MAs) and pulmonary arteries (PAs) were used as prototypes for arteries with predominantly IK/SK channel- and eNOS-dependent vasodilatation, respectively. Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4EC ) channels played an important role in controlling the baseline diameter of both MAs and PAs. TRPV4EC channel activity was similar in MAs and PAs. However, the TRPV4 channel agonist GSK1016790A (10 nm) selectively activated IK/SK channels in MAs and eNOS in PAs, revealing preferential TRPV4EC -IK/SK channel coupling in MAs and TRPV4EC -eNOS coupling in PAs. IK/SK channels co-localized with TRPV4EC channels at myoendothelial projections (MEPs) in MAs, although they lacked the spatial proximity necessary for their activation by TRPV4EC channels in PAs. Additionally, the presence of the NO scavenging protein haemoglobin α (Hbα) within nanometer proximity to eNOS limits TRPV4EC -eNOS signalling in MAs. By contrast, co-localization of TRPV4EC channels and eNOS at MEPs, and the absence of Hbα, favour TRPV4EC -eNOS coupling in PAs. Thus, our results reveal that differential spatial organization of signalling elements determines TRPV4EC -IK/SK vs. TRPV4EC -eNOS coupling in resistance arteries.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

BACKGROUND: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. METHODS: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. RESULTS: We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. CONCLUSIONS: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Novel Regulators and Targets of Redox Signaling in Pulmonary Vasculature.

Dysregulated redox signaling in pulmonary vasculature is central to the development of pulmonary arterial hypertension (PAH) and lung injury. Modulators of reactive oxygen species (ROS) production and downstream signaling targets are critical for mediating the physiological or pathological effects of ROS. Understanding the complex interactions between the modulators and signaling targets of ROS is essential for developing novel strategies to prevent or attenuate lung pathologies. In this review, we discuss recent studies on the modulators and targets of ROS in pulmonary endothelial and smooth muscle cells, their cellular effects, and the disease conditions associated with dysregulated redox signaling.

Formation of HETE-EAs and dihydroxy derivatives in mouse kidney tissue and analysis by high-performance liquid chromatography tandem mass spectrometry.

The kidneys play an important role in the long-term regulation of blood pressure by control of salt and water balance in the body through various systems including the endocannabinoid system. The endocannabinoid system consists of the two major cannabinoid receptor agonists, anandamide (AEA) and 2-arachidonylglycerol (2-AG), their hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid receptors, CB1 and CB2. AEA can be converted into 12- and 15(S)-hydroperoxyeicosatetraenoic acid ethanolamides by 12-LOX and 15-LOX, respectively and can form epoxyeicosatrienoic acid- (EET-EAs) (5,6-, 8,9-, 11,12-, 14,15-) and hydroxyeicosatetraenoic acid- (HETE) ethanolamides. Furthermore, the EET-EAs produce a secondary metabolism by microsomal epoxide hydrolase to form the corresponding dihydroxyeicosatetraenoic acid-EAs (DiHETE-EA). Reference material was not available for DiHETE-EA. These metabolites were synthesized by incubation of the corresponding EET-EAs with mouse liver cytosol containing epoxide hydrolases. Presented is a solid phase extraction and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the extraction and quantitation of AEA, 2-AG, their metabolites, oleoylethanolamide (OEA), and palmitoylethanolamide (PEA), and the in vivo formation of the DiHETE-EAs in kidney after a single intravenous bolus administration of 20 mg/kg of anandamide in C57BL/6 J and FAAH KO mice.

Diuretic, Natriuretic, and Vasodepressor Activity of a Lipid Fraction Enhanced in Medium of Cultured Mouse Medullary Interstitial Cells by a Selective Fatty Acid Amide Hydrolase Inhibitor.

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not yet understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured, and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase, PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide). Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. High-performance liquid chromatography (HPLC) analyses of lipid extracts of MMIC culture medium revealed a 205-nm absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 area units under the peak/10 min) stimulated diuresis and natriuresis, whereas these parameters returned toward baseline at higher doses but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, the glomerular filtration rate, and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urine parameters. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid that acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Role of Nitric Oxide in the Cardiovascular and Renal Systems.

The gasotransmitters are a family of gaseous signaling molecules which are produced endogenously and act at specific receptors to play imperative roles in physiologic and pathophysiologic processes. As a well-known gasotransmitter along with hydrogen sulfide and carbon monoxide, nitric oxide (NO) has earned repute as a potent vasodilator also known as endothelium-derived vasorelaxant factor (EDRF). NO has been studied in greater detail, from its synthesis and mechanism of action to its physiologic, pathologic, and pharmacologic roles in different disease states. Different animal models have been applied to investigate the beneficial effects of NO as an antihypertensive, renoprotective, and antihypertrophic agent. NO and its interaction with different systems like the renin⁻angiotensin system, sympathetic nervous system, and other gaseous transmitters like hydrogen sulfide are also well studied. However, links that appear to exist between the endocannabinoid (EC) and NO systems remain to be fully explored. Experimental approaches using modulators of its synthesis including substrate, donors, and inhibitors of the synthesis of NO will be useful for establishing the relationship between the NO and EC systems in the cardiovascular and renal systems. Being a potent vasodilator, NO may be unique among therapeutic options for management of hypertension and resulting renal disease and left ventricular hypertrophy. Inclusion of NO modulators in clinical practice may be useful not only as curatives for particular diseases but also for arresting disease prognoses through its interactions with other systems.

Modulation of mean arterial pressure and diuresis by renomedullary infusion of a selective inhibitor of fatty acid amide hydrolase.

The kidneys contribute to the control of body fluid and electrolytes and the long-term regulation of blood pressure through various systems, including the endocannabinoid system. Previously, we showed that inhibition of the two major endocannabinoid-hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, in the renal medulla increased the rate of urine excretion (UV) and salt excretion without affecting mean arterial pressure (MAP). The present study evaluated the effects of a selective FAAH inhibitor, N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidine carboxamide (PF-3845) on MAP and renal functions. Infusion of PF-3845 into the renal medulla of C57BL/6J mice reduced MAP during the posttreatment phases and increased UV at 15 and 30 nmol/min per gram kidney weight (g kwt), relative to the pretreatment control phase. Intravenous PF-3845 administration reduced MAP at the 7.5, 15, and 30 doses and increased UV at the 15 and 30 nmol⋅min-1⋅g-1 kwt doses. PF-3845 treatment elevated sodium and potassium urinary excretion and medullary blood flow. Homozygous FAAH knockout mice were refractory to intramedullary PF-3845-induced changes in MAP, but UV was increased. Both MAP and UV responses to intramedullary PF-3845 in C57BL/6J mice were diminished by pretreatment with the cannabinoid type 1 receptor-selective antagonist, rimonabant (3 mg/kg, ip) but not the cyclooxygenase 2-selective inhibitor, celecoxib (15 mg/kg, iv). Liquid chromatography-tandem mass spectrometry analyses showed increased anandamide in kidney tissue and 2-arachidonoyl glycerol in plasma after intramedullary PF-3845. These data suggest that inhibition of FAAH in the renal medulla leads to both a diuretic and blood pressure-lowering response mediated by elevated anandamide in kidney tissue or 2-arachidonoyl glycerol in plasma.

Stimulation of diuresis and natriuresis by renomedullary infusion of a dual inhibitor of fatty acid amide hydrolase and monoacylglycerol lipase.

The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30, and 60 nmol·min-1·kg-1) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine whether endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide nor IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg·kg-1·30 min-1 iv) nor intramedullary (15 nmol·min-1·kg-1·30 min-1) IDFP pretreatment before intramedullary anandamide (15-30 nmol·min-1·kg-1) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary PGE2 concentrations by HPLC-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE2 These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney.