11,12-EET increases porto-sinusoidal resistance and may play a role in endothelial dysfunction of portal hypertension

CYP450-dependent epoxyeicosatrienoic acids (EETs) are potent arterial vasodilators, while 20-hydroxyeicosatatraenoic acid (20-HETE) is a vasoconstrictor. We evaluated their role in the control of portal circulation in normal and cirrhotic (CCl(4) induced) isolated perfused rat liver. Phenylephrine (PE) and endothelin-1 (ET-1) increased portal perfusion pressure, as did arachidonic acid (AA), 20-HETE, and 11,12-EET. Inhibition of 20-HETE with 12,12-dibromododecenoic acid (DBDD) did not affect basal pressure nor the responses to PE, ET-1, or AA. However, inhibition of epoxygenase with miconazole caused a significant reduction in the response to ET-1 and to AA, without affecting neither basal pressure nor the response to PE. Hepatic vein EETs concentration increased in response to ET-1, and was increased in cirrhotic, compared to control, livers. 20HETE levels were non-measurable. Miconazole decreased portal perfusion pressure in cirrhotic livers. In conclusion, 20HETE and EETs increase portal resistance; EETs, but not 20-HETE, mediate in part the pressure response to ET-1 in the portal circulation and may be involved in pathophysiology of portal hypertension.     

Role of cytochrome P450-dependent arachidonic acid metabolites in liver physiology and pathophysiology

Arachidonic acid (AA) can undergo monooxygenation or epoxidation by enzymes in the cytochrome P450 (CYP) family in the brain, kidney, lung, vasculature, and the liver. CYP-AA metabolites, 19- and 20-hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs) and diHETEs have different biological properties based on sites of production and can be stored in tissue lipids and released in response to hormonal stimuli. 20-HETE is a vasoconstrictor, causing blockade of Ca(++)-activated K(+) (KCa) channels. Inhibition of the formation of nitric oxide (NO) by 20-HETE mediates most of the cGMP-independent component of the vasodilator response to NO. 20-HETE elicits a potent dilator response in human and rabbit pulmonary vascular and bronchiole rings that is dependent on an intact endothelium and COX. 20-HETE is also a vascular oxygen sensor, inhibits Na(+)/K(+)-ATPase activity, is an endogenous inhibitor of the Na(+)-K(+)-2Cl(-)cotransporter, mediates the mitogenic actions of vasoactive agents and growth factors in many tissues and plays a significant role in angiogenesis. EETs, produced by the vascular endothelium, are potent dilators. EETs hyperpolarize VSM cells by activating KCa channels. Several investigators have proposed that one or more EETs may serve as endothelial-derived hyperpolarizing factors (EDHF). EETs constrict human and rabbit bronchioles, are potent mediators of insulin and glucagon release in isolated rat pancreatic islets, and have anti-inflammatory activity. Compared with other organs, the liver has the highest total CYP content and contains the highest levels of individual CYP enzymes involved in the metabolism of fatty acids. In humans, 50-75% of CYP-dependent AA metabolites formed by liver microsomes are omega/omega-OH-AA, mainly w-OH-AA, i.e. 20HETE, and 13-28% are EETs. Very little information is available on the role of 19- and 20-HETE and EETs in liver function. EETs are involved in vasopressin-induced glycogenolysis, probably via the activation of phosphorylase. In the portal vein, inhibition of EETs exerts profound effects on a variety of K-channel activities in smooth muscles of this vessel. 20-HETE is a weak, COX-dependent, vasoconstrictor of the portal circulation. EETs, particularly 11,12-EET, cause vasoconstriction of the porto-sinusoidal circulation. Increased synthesis of EETs in portal vessels and/or sinusoids or increased levels in blood from the meseneric circulation may participate in the pathophysiology of portal hypertension of cirrhosis. CYP-dependent AA metabolites are involved in the pathophysiology of portal hypertension, not only by increasing resistance in the porto-sinusoidal circulation, but also by increasing portal inflow through mesenteric vasodilatation. In patients with cirrhosis, urinary 20-HETE is several-fold higher than PGs and TxB2, whereas in normal subjects, 20-HETE and PGs are excreted at similar rates. Thus, 20-HETE is probably produced in increased amounts in the preglomerular microcirculation accounting for the functional decrease of flow and increase in sodium reabsorption. In conclusion, CYP-AA metabolites represent a group of compounds that participate in the regulation of liver metabolic activity and hemodynamics. They appear to be deeply involved in abnormalities related to liver diseases, particularly cirrhosis, and play a key role in the pathophysiology of portal hypertension and renal failure.     

Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.     

Role of nitric oxide in the vascular effects of local warming of the skin in humans.

Local warming of skin induces vasodilation by unknown mechanisms. To test whether nitric oxide (NO) is involved, we examined effects of NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation induced by local warming of skin in six subjects. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for delivery of L-NAME and sodium nitroprusside. Skin blood flow was monitored by laser-Doppler flowmetry (LDF) at microdialysis sites. Local temperature (Tloc) of the skin at both sites was controlled with special LDF probe holders. Mean arterial pressure (MAP; Finapres) was measured and cutaneous vascular conductance calculated (CVC = LDF/MAP = mV/mmHg). Data collection began with a control period (Tloc at both sites = 34 degrees C). One site was then warmed to 41 degrees C while the second was maintained at 34 degrees C. Local warming increased CVC from 1.44 +/- 0.41 to 4.28 +/- 0.60 mV/mmHg (P < 0.05). Subsequent L-NAME administration reduced CVC to 2.28 +/- 0.47 mV/mmHg (P < 0.05 vs. heating), despite the continued elevation of Tloc. At a Tloc of 34 degrees C, L-NAME reduced CVC from 1.17 +/- 0.23 to 0.75 +/- 0.11 mV/mmHg (P < 0.05). Administration of sodium nitroprusside increased CVC to levels no different from those induced by local warming. Thus NOS inhibition attenuated, and sodium nitroprusside restored, the cutaneous vasodilation induced by elevation of Tloc; therefore, the mechanism of cutaneous vasodilation by local warming requires NOS generation of NO.     

VEGF(121)- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production

The angiogenic proteins basic fibroblast growth factor (bFGF; FGF-2) and vascular endothelial growth factor 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). Growth factors were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic growth factors.     

Comparative haemodynamic studies of resting and active skeletal muscle in anaesthetised rats: role of nitric oxide.

Our previous studies have indicated that nitric oxide takes part in the basal regulation of vascular tone in skeletal muscle. The purpose of this study was to investigate whether nitric oxide has a role in the active hyperaemic response of a working muscle in a resting subject. Haemodynamic effects of nitric oxide synthase (NOS) inhibition (L-NAME, 10 mg/kg/30 min i.v. infusion) were determined simultaneously in the resting m. quadriceps femoris and in the working (breathing) m. rectus abdominis in anaesthetised rats (86Rb accumulation technique). L-NAME increased blood pressure and total peripheral resistance (TPR) while it decreased cardiac output. Blood flow (BF) decreased and vascular resistance (VR) increased both in resting (BF: 8.91+/-1.97-->5.92+/-2.59 ml/min/100 g, p<0.05: VR: 106+/-29.9-->212+/-113 R, p<0.01) and working (BF: 17.0+/-4.78-->6.93+/-2.15 ml/min/100 g, p<0.001; VR: 57.0+/-18.5-->160+/-56.7 R, p<0.01) muscle following NOS inhibition, but the percentile change of BF was higher in the working muscle (59%) than in the resting one (34%, p<0.001). There was a positive correlation between the cardiac output and the blood flow of the resting muscle with or without L-NAME administration, but blood flow of the working muscle failed to have any correlation with the cardiac output in control animals. However, L-NAME administration decreased both the cardiac output and the blood flow and similarly to the resting muscle a positive correlation was found. In conclusion, the haemodynamic effects of NOS inhibition are higher in working muscle than in the resting one: the nitric oxide may have important role in vasodilatation during muscle activity.     

Renal blood flow in normal dogs and in dogs with experimental liver cirrhosis following the acute continuous infusion of endotoxin

The purpose of this study was to determine if the renal circulation of normal and cirrhotic dogs behave similarly in response to an acute endotoxin infusion. Endotoxin was administered as a slow continuous infusion (13-26 micrograms/min) to a total of 20 normal dogs through the femoral vein, portal vein, or into the left renal artery. In each case, there was an initial increment in renal blood flow, of the order of 46%, while arterial blood pressure was actually declining. After 8-20 min, blood flow fell as perfusion pressure declined further. The initial increment in renal perfusion was not due to a hyperthermic response following the endotoxin. When similar doses were given to five dogs with chronic biliary cirrhosis and ascites, the biphasic response in renal perfusion was not observed, rather blood flow declined as perfusion pressure declined. When normal dogs were infused with bilirubin, bile salts, noradrenaline, and angiotensin in pressor doses, the subsequent infusion of endotoxin still produced the usual biphasic response in renal perfusion. Chronic elevation of portal pressure (but not acute elevation), volume contraction by diuresis or hemorrhage, and the infusion of bile intravenously, all abolished the biphasic response in renal perfusion and reproduced in normal dogs the response to endotoxin observed in cirrhotic dogs. Investigation of the factors causing the initial decrease in intrarenal vascular resistance in normal dogs following the endotoxin infusion implicated a role for histamine, kinins, and prostaglandins. We conclude there is a fundamental difference in the response of the renal circulation of normal and cirrhotic dogs to an endotoxin infusion, which may depend on failure of this latter group to release one or more humoral agents. This difference may be due to elevated portal pressure, a decreased effective arterial blood volume, or the products of bile having access to the circulation in cirrhotic dogs.     

Haemodynamic effects of angiotensin II in conscious, non-ascitic cirrhotic rats

The effect of angiotensin II (AII) on systemic and regional haemodynamics was studied in 18 control and 18 cirrhotic, non-ascitic conscious rats (CCl4/phenobarbital model). Cirrhotic rats were found to retain sodium and to have normal plasma renin and plasma aldosterone concentrations when compared with control animals. Cirrhotic rats showed an enhanced cardiac output (34.4 +/- 0.5 vs. 27.5 +/- 2.0 ml/min in controls) and decreased peripheral resistances (2.96 +/- 0.25 vs. 3.95 +/- 0.31 mm Hg/min/100 g/ml in controls) under basal conditions. When AII was administered cardiac output decreased by 10.7 +/- 1.2% in cirrhotic rats, whereas it increased in control animals (11.2 +/- 2%, p less than 0.005). The AII-induced increase in arterial pressure was lower in cirrhotic than in control rats. The renal blood supply was particularly impaired by AII in cirrhotics, with a maintained flow to other organs (muscle, testes). It is concluded that the response to AII is disturbed in rats with hepatic cirrhosis even in a stage without ascites and with plasma renin and aldosterone concentrations similar to those of control animals.     

Characterization of 5,6- and 8,9-epoxyeicosatrienoic acids (5,6- and 8,9-EET) as potent in vivo angiogenic lipids

The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.     

Effects of propranolol and metoprolol on the peripheral circulation.

The effects of single doses of propranolol and metoprolol on skin temperature and skin and muscle blood flow were compared in 10 normal subjects and four patients with essential hypertension. In normal subjects the mean skin temperature fell by 1.30 +/- 0.62 degrees C 90 minutes after 80 mg propranolol and 0.15 +/- 0.05 degrees C after 100 mg metoprolol. Skin blood flow and resting muscle blood flow were not affected by metoprolol but fell significantly after propranolol. Both drugs reduced post-exercise muscle hyperaemia, propranolol by more than metoprolol. Similar changes were seen in the hypertensive patients. Propranolol should be used with care in patients with known vascular disease.     

Chiral resolution of the epoxyeicosatrienoic acids, arachidonic acid epoxygenase metabolites

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.     

Glucose turnover in compensated hepatic cirrhosis

Glucose turnover and recycling from glucose derived 3-carbon intermediates were examined in overnight fasted patients with compensated hepatic cirrhosis and in age- and weight-matched normal control subjects. Fasting blood concentrations of glucose, lactate and glycerol were similar in both groups but blood pyruvate (60 +/- 10 vs. 80 +/- mumol/l, P less than 0.05), blood alanine (0.23 +/- 0.02 vs 0.34 +/- 0.02 mmol/l, P less than 0.01) were decreased and serum insulin increased (19 [13-24]v 7 [4-11] mU/l, P less than 0.01) in cirrhotic subjects. Absolute glucose turnover, assessed by analysis of decay of [3H]-3-glucose specific activity was decreased in cirrhotic patients (8.1 +/- 0.6 v 12.1 +/- 0.7 mol/kg-1 min-1). Glucose "recycling", assessed by the difference between absolute glucose turnover and that given by [14C]-1-glucose data, was normal in cirrhotic patients suggesting that Cori cycle (glucose-lactate-glucose) activity was normal. These data support previous findings of decreased peripheral glucose utilisation and insulin resistance in cirrhotic patients.     

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.     

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.     

Decreased nitric oxide- and axon reflex-mediated cutaneous vasodilation with age during local heating.

Cutaneous vasodilation is reduced in healthy older vs. young subjects; however, the mechanisms that underlie these age-related changes are unclear. Our goal in the present study was to determine the role of nitric oxide (NO) and the axon reflexes in the skin blood flow (SkBF) response to local heating with advanced age. We placed two microdialysis fibers in the forearm skin of 10 young (Y; 22 +/- 2 yr) and 10 older (O; 77 +/- 5 yr) men and women. SkBF over each site was measured by laser-Doppler flowmetry (LDF; Moor DRT4). Both sites were heated to 42 degrees C for ~60 min while 10 mM N(G)-nitro-L-arginine methyl ester (L-NAME) was infused throughout the protocol to inhibit NO synthase (NOS) in one site and 10 mM L-NAME was infused after 40 min of local heating in the second site. Data were expressed as a percentage of maximal vasodilation (%CVC(max); 28 mM nitroprusside infusion). Local heating before L-NAME infusion resulted in a significantly reduced initial peak (Y: 61 +/- 2%CVC(max) vs. O: 46 +/- 4%CVC(max)) and plateau (Y: 93 +/- 2%CVC(max) vs. O: 82 +/- 5%CVC(max)) CVC values in older subjects (P < 0.05). When NOS was inhibited after 40 min of heating, CVC declined to the same value in the young and older groups. Thus the overall contribution of NO to the plateau phase of the SkBF response to local heating was less in the older subjects. The initial peak response was significantly lower in the older subjects in both microdialysis sites (Y: 52 +/- 4%CVC(max) vs. O: 38 +/- 5%CVCmax; P < 0.05). These data suggest that age-related changes in both axon reflex-mediated and NO-mediated vasodilation contribute to attenuated cutaneous vasodilator responses in the elderly.     

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.     

Effect of portacaval surgical anastomosis on systemic and splanchnic hemodynamics in portal hypertensive, cirrhotic rats

The effect of surgical end-to-side portacaval anastomosis (PCSA) on systemic and splanchnic circulation has been studied in cirrhotic rats with portal hypertension (CCl4-phenobarbital method) and in control animals. Hemodynamics have been measured using the microsphere technique, with a reference sample for the systemic hemodynamic measurements, and intrasplenic injection for portal systemic shunting rate measurements. Compared with controls, sham-operated (SO) cirrhotic rats showed a hyperdynamic circulation with increased cardiac output (CO) and decreased mean arterial pressure and peripheral resistances. PCSA in control rats induced only a small change in systemic hemodynamics, with parallel decreases in arterial pressure and peripheral resistances, and a small, nonsignificant increase in CO. In cirrhotic rats, PCSA induced a decrease of CO to values similar to those of control rats, with an increase in total peripheral resistances. PCSA induced an increase in hepatic arterial blood flow in control and in cirrhotic rats, portal pressure becoming in this latter group not different from that of control rats. Blood flow to splanchnic organs was higher in SO cirrhotic than in SO control animals. Thus portal venous inflow was also increased in SO cirrhotic rats. PCSA induced an increase in portal venous inflow in control rats, which was only significant in cirrhotic rats when expressed as a percentage of CO. In SO control animals, a significant correlation was observed between total peripheral resistances and splanchnic arteriolar resistances and between CO and splanchnic blood flow. These correlations were not observed in cirrhotic rats. These results do not support the hypothesis that hyperdynamic circulation shown by cirrhotic rats is based on increases in splanchnic blood flow and (or) massive portal systemic shunting.     

Modulation of V1-receptor-mediated renal vasoconstriction by epoxyeicosatrienoic acids

This study examined the effects of renal arterial infusion of a selective cytochrome P-450 epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 2 mg/kg plus 1.5 mg.kg(-1).h(-1)), on renal hemodynamic responses to infusions of [Phe(2),Ile(3),Orn(8)]vasopressin and ANG II into the renal artery of anesthetized rabbits. MS-PPOH did not affect basal renal blood flow (RBF) or cortical or medullary blood flow measured by laser-Doppler flowmetry (CLDF/MLDF). In vehicle-treated rabbits, [Phe(2),Ile(3),Orn(8)]vasopressin (30 ng.kg(-1).min(-1)) reduced MLDF by 62 +/- 7% but CLDF and RBF were unaltered. In MS-PPOH-treated rabbits, RBF and CLDF fell by 51 +/- 8 and 59 +/- 13%, respectively, when [Phe(2),Ile(3),Orn(8)]vasopressin was infused. MS-PPOH had no significant effects on the MLDF response to [Phe(2),Ile(3),Orn(8)]vasopressin (43 +/- 9% reduction). ANG II (20 ng.kg(-1).min(-1)) reduced RBF by 45 +/- 10% and CLDF by 41 +/- 14%, but MLDF was not significantly altered. MS-PPOH did not affect blood flow responses to ANG II. Formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DiHETEs) was 49% lower in homogenates prepared from the renal cortex of MS-PPOH-treated rabbits than from vehicle-treated rabbits. MS-PPOH had no effect on the renal formation of 20-hydroxyeicosatetraenoic acid (20-HETE). Incubation of renal cortical homogenates from untreated rabbits with [Phe(2),Ile(3),Orn(8)]vasopressin (0.2-20 ng/ml) did not affect formation of EETs, DiHETEs, or 20-HETE. These results do not support a role for de novo EET synthesis in modulating renal hemodynamic responses to ANG II. However, EETs appear to selectively oppose V(1)-receptor-mediated vasoconstriction in the renal cortex but not in the medullary circulation and contribute to the relative insensitivity of cortical blood flow to V(1)-receptor activation [corrected].     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

EET homologs potently dilate coronary microvessels and activate BK(Ca) channels

Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50-140 microm ID) and activating large-conductance Ca2+-activated K+ (BK(Ca)) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50 values of 3-120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BK(Ca) channels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BK(Ca) channels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.