1 Departamento de Fisiología, Facultad de Medicina, 30100-Murcia, Spain; and 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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The present study evaluated whether
inhibition of guanylyl cyclase (GC) with
1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and
methylene blue (MB) or inhibition of the renal metabolism of
arachidonic acid by cytochrome P-450 (CYP450) enzymes with 1-aminobenzotriazole (ABT) and N-hydroxy-N'-(4
butyl-2-methyl phenyl)formamidine (HET0016) alters the renal tubular
and vascular effects of a nitric oxide (NO) donor in vivo. Intrarenal
infusion of ODQ or MB at a dose of 170 nmol · kg1 · min
1 lowered
renal blood flow (RBF) by 30 and 15%, respectively; glomerular filtration rate (GFR) by 26 and 18%, respectively; and sodium and
water excretion by ~35%. In rats pretreated with
nitro-L-arginine methyl ester (37 nmol · kg
1 · min
1) to block
the endogenous production of NO, intrarenal infusion of the NO donor
S-nitroso-N-acetylcysteine (S-NO-NAC; 50 nmol · kg
1 · min
1) increased
RBF (18%), sodium (73%), and water excretion (61%). ODQ or MB
administration blocked the effect of S-NO-NAC on RBF but not the
diuretic and natriuretic response. Pretreatment of rats with ABT or
HET0016 also abolished the renal vasodilatory response to the NO donor
and reduced its diuretic and natriuretic effect. These results indicate
that both activation of GC and inhibition of CYP450 enzymes contribute
to the renal vascular actions of NO, whereas the natriuretic and
diuretic actions of NO appear to be largely CYP450 dependent.
renal hemodynamics; kidney; guanosine 3',5'-cyclic monophosphate; 20-hydroxytetraenoic acid; epoxyeicosatrienoic acids
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INTRODUCTION |
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RECENT STUDIES HAVE
INDICATED that nitric oxide (NO) plays a central role in the
regulation of renal tubular and vascular function and in the long-term
control of arterial pressure (5, 6). Endothelial-derived
NO affects vascular tone in both afferent and efferent arterioles
(17). Despite the importance of NO in the control of renal
function, its mechanism of action in the kidney is not well understood.
It has generally been assumed that the renal actions of NO are solely
mediated by activation of guanylyl cyclase (GC), which increases the
levels of guanosine 3',5'-cyclic monophosphate (cGMP). This conclusion
is supported by the findings that endothelium-derived NO and NO donors
increase cGMP levels in vascular tissue and that inhibitors of GC
attenuate the vasodilatory response to NO in many vessels
(14). Further support for this hypothesis is provided by
the observation that the effects of N-nitro-L-arginine methyl ester
(L-NAME), an inhibitor of nitric oxide synthase (NOS), on
arterial pressure and renal function can be reversed by
8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP)
(9). However, this scheme for NO-induced vasodilation recently has been questioned because in a variety of vascular beds,
including the renal circulation, NO has been reported to act via both
cGMP-dependent and cGMP-independent mechanisms (3, 29).
Recently, a new route for the metabolism of arachidonic acid (AA) has
been described. This pathway, which is dependent on cytochrome
P-450 (CYP450), NADPH, and molecular oxygen, produces a
series of epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DiHETEs), and hydroxytetraenoic acids (HETEs) from AA. The
primary metabolites formed in the kidney are 20-HETE; 14,15- and
11,12-EETs; and DiHETEs. The available data indicate that CYP450
metabolites of AA play a critical role in the regulation of both
tubular and vascular function (15). CYP450 metabolites of
AA participate in the myogenic response of renal arteries to elevations
in transmural pressure and in the regulation of renal vascular tone
(7, 12). 20-HETE also inhibits
Na+-K+-ATPase and sodium transport in the
proximal tubule (21) and Na+-K+-2Cl transport in the thick
ascending loop of Henle (4). Other studies have
demonstrated that endogenous CYP450 metabolites of AA influence renal
medullary hemodynamics and the excretion of water and electrolytes in
vivo (31) and contribute to the long-term regulation of
arterial pressure (26).
NO has been demonstrated recently to inhibit CYP450 enzymes of the 1A, 2B1, 3C, and 4A families by forming iron-nitrosyl complexes at the catalytic heme binding site in these enzymes (1, 18, 30). NO donors also inhibit the synthesis of 20-HETE by renal tubular and vascular tissue (1, 2), and the inhibition of CYP450 enzymes attenuates the vasodilatory response of renal microvessels to NO in vitro to a much greater extent than GC inhibition (1, 2, 28). In addition, inhibitors of the formation of 20-HETE attenuate the systemic vasodilator response to NO donors (1) in rats in vivo. However, the relative contribution of GC and CYP450 metabolites of AA to the renal vascular and excretory effects of NO in vivo remains to be determined. Therefore, the present study examined the effects of the inhibition of GC with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) or methylene blue (MB) and blockade of CYP450 metabolism of AA with 1-aminobenzotriazole (ABT) or N-hydroxy-N'-(4 butyl-2-methyl phenyl)formamidine (HET0016) on the renal hemodynamic and natriuretic actions of an intrarenal infusion of an NO donor in rats in vivo.
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MATERIALS AND METHODS |
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Experiments were performed on 64 anesthetized male
Sprague-Dawley rats (240-260 g) bred in the Animal Care Facility
at the University of Murcia. All procedures performed were in
accordance with the recommendations from the Declaration of Helsinki
and the guiding principles in the care and use of animals approved by
the American Physiological Society. The rats were anesthetized with an
intramuscular injection of ketamine (30 mg/kg im, Rhône Merieux)
and an intraperitoneal injection of thiopental (Pentothal, 50 mg/kg ip;
Abbott). Cannulas were placed in the right femoral vein for infusions,
in the right femoral artery for measurement of mean arterial pressure
(MAP), and in the left ureter for collection of urine. A heat-pulled
PE-10 catheter was inserted in the aorta and advanced into the left
renal artery for intrarenal infusions (19). Isotonic NaCl
solution was continuously infused at a rate of 50 µl/min to maintain
the patency of the catheter. The left kidney was denervated, and an
electromagnetic flow probe (Skalar) was placed around the left renal
artery for measurement of renal blood flow (RBF). Plasma levels of
vasopressin (0.17 ng · kg1 · min
1),
aldosterone (66 ng · kg
1 · min
1), and
norepinephrine (333 ng · kg
1 · min
1) were fixed
at high physiological levels by an intravenous infusion of a hormone
cocktail at a rate of 1 ml · 100 g
1 · h
1 (22). After
surgery and a 90-min equilibration period, urine flow (UV), sodium
excretion (UNaV), RBF, glomerular filtration rate (GFR),
and MAP were measured before and after intrarenal infusion of an NO
donor, S-nitro-N-acetyl-L-cysteine
(S-NO-NAC) (25), in rats pretreated with vehicle or GC or
CYP450 inhibitors. All drugs (except ABT and HET0016) were administered intrarenally.
Group 1: time course studies. After two 15-min control periods, urine and plasma samples were collected at 30-min intervals for 2 h.
Group 2: renal effects of ODQ.
Experiments were performed to find a dose of ODQ that alters renal
function in vivo. UV, UNaV, GFR, and RBF were measured during two 15-min control periods. Then, three different doses of ODQ
(1.7, 17, and 170 nmol · kg1 · min
1) were
infused into the renal artery to obtain concentrations in renal
arterial plasma of ~0.1, 1, and 10 µmol/l. Each dose was infused
for 30 min, and urine and plasma samples were again collected during
three 15-min experimental clearance periods.
Group 3: renal effects of MB.
These experiments were performed to find a dose of MB that alters renal
function in vivo. UV, UNaV, GFR, and RBF were measured during two 15-min control periods. Then, different doses of MB (17, 170, and 1,700 nmol · kg1 · min
1) were
infused into the renal artery to obtain concentrations in renal
arterial plasma of ~1, 10, and 100 µmol/l. Each dose was infused
for 30 min, and then urine and plasma samples were again collected
during three 15-min experimental clearance periods.
Group 4: renal effects of L-NAME and the NO donor
S-NO-NAC.
These experiments were performed to determine the highest dose of the
NO donor that modifies renal hemodynamics and UNaV without altering arterial pressure. After a basal clearance period,
L-NAME (37 nmol · kg1 · min
1 for 30 min followed by 0.37 nmol · kg
1 · min
1) was
infused intrarenally to suppress the endogenous formation of NO. After
a 30-min equilibration period, urine and plasma samples were collected
during a 10-min clearance period. Then, the NO donor S-NO-NAC was
infused intrarenally at doses of 0.5, 5, and 50 nmol · kg
1 · min
1 for 15 min, and urine and plasma samples were again collected during three
additional 15-min clearance periods.
Group 5: effects of ODQ on renal response to L-NAME
and an NO donor.
These experiments examined the effects of blocking GC activity
with ODQ on the renal responses to an intrarenal infusion of the NO
donor S-NO-NAC. These experiments were similar to those described in
protocol 4 except that the rats received an intrarenal infusion of ODQ (170 nmol · kg1 · min
1) along
with L-NAME (37 nmol · kg
1 · min
1 for 30 min followed by 0.37 nmol · kg
1 · min
1)
throughout the experimental period. Then, S-NO-NAC at doses of 0.5, 5, and 50 nmol · kg
1 · min
1
was sequentially infused intrarenally as described above.
Group 6: effects of MB on renal response to L-NAME
and an NO donor.
These experiments examined the effects of MB on the renal responses to
intrarenal infusion of the NO donor S-NO-NAC. These experiments were
similar to those described in protocol 5 except that the
rats received an intrarenal infusion of MB (170 nmol · kg1 · min
1) and
L-NAME (37 nmol · kg
1 · min
1 for 30 min followed by 0.37 nmol · kg
1 · min
1)
throughout the experimental period. Then, S-NO-NAC at doses of 0.5, 5, and 50 nmol · kg
1 · min
1
was infused intrarenally as described in protocol 4.
Group 7: renal effects of L-NAME and an NO donor in rats pretreated with ABT. These rats were pretreated with an intraperitoneal injection of ABT (50 mg/kg ip) to block the renal metabolism of AA by CYP450 enzymes, as previously described (27). Thirty-six hours later, the effects of an intrarenal infusion of S-NO-NAC were studied as described in protocol 4. At the end of each experiment, the kidneys were removed, and the degree of inhibition of the renal metabolism of AA by CYP450 enzymes produced by ABT was determined in microsomes prepared from the renal cortex using an HPLC-based radiochemical assay, as previously described (1, 2). Briefly, microsomes (500 µg protein) were incubated at 37°C for 30 min in 1 ml of potassium phosphate buffer (0.1 M) containing sodium isocitrate (1 mM), isocitrate dehydrogenase (0.0016 U), [14C]AA (1 µCi), and NADPH (1 mM). The pH of the reaction was acidified to 3.5 with 1 M formic acid, samples were extracted twice with ethyl acetate, and the products were separated by reverse phase-HPLC. Products were monitored using a radioactive flow detector, and formation of 20-HETE, EETs, and DiHETEs was measured and expressed as picomoles per minute per milligram protein.
Group 8: renal effects of L-NAME and an NO donor in rats pretreated with HET0016. After a control clearance period, the rats received an intravenous injection of HET0016 (10 mg/kg; Taisho Chemical, Satamia, Japan) to block the renal metabolism of AA by CYP450 enzymes (16). Forty-five minutes later, a second clearance period was performed to assess the effects of HET0016 on renal function. After that, the effects of an intrarenal infusion of L-NAME together with S-NO-NAC were evaluated as described in protocol 4.
Recent studies have indicated that HET0016 is a highly selective competitive inhibitor of the formation of 20-HETE (16). To confirm this in our rats, we evaluated the ability of HET0016 (1 µM) to inhibit the renal metabolism of AA in microsomes prepared from the renal cortex of rats. Renal microsomes were prepared and incubated with [14C]AA as described above. Vehicle or 1 µM HET0016 was added to the incubation. The formation of 20-HETE, EETs, and DiHETEs was measured as previously described (1, 2).Statistical methods. Data are presented as means ± SE. The significance of differences in the measured values within and between groups was analyzed using an analysis of variance for repeated measures followed by a Fisher's least significant difference test. P < 0.05 (2-tailed test) was considered statistically significant.
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RESULTS |
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Baseline values of blood pressure, RBF, GFR, UV, and
UNaV for the rats in all treatment groups are presented in
Table 1. Basal levels of blood pressure,
RBF, UV, and UNaV were similar in all groups except that
baseline GFR was slightly lower in rats pretreated with ABT. Intrarenal
infusion of saline had no effect on baseline renal function in
groups 1-3. Blockade of the formation of NO with an
intrarenal infusion of L-NAME at a threshold pressor dose
reduced RBF, UV, and UNaV, whereas GFR was unaltered
(group 4). Blockade of GC with ODQ or MB augmented the renal
vasoconstrictor actions of L-NAME, but they had no effect
on the ability of L-NAME to lower UV or UNaV
(groups 5 and 6). Pretreatment of rats with ABT,
which blocks the formation of both EETs and 20-HETE, did not block the
vasoconstrictor response to L-NAME, but it diminished the
fall in UV and UNaV. Administration of the more selective inhibitor of the formation of 20-HETE, HET0016, attenuated the renal
vasoconstrictor response to L-NAME and completely prevented the fall in UV and UNaV.
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Group 2: renal effects of ODQ.
The effects of different doses of intrarenal infusion of ODQ on renal
function of rats are presented in Fig. 1.
The absolute values for the control periods in Fig. 1 correspond to the
values in columns 4, 6, 8, and
10 of Table 1. ODQ had no significant effects on MAP.
Intrarenal infusion of increasing doses of ODQ produced graded
reductions in RBF, GFR, and sodium and water excretion. The largest
effects were seen after administration of the
170-nmol · kg1 · min
1 dose
(30, 26, 34, and 36% decreases, respectively).
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Group 3: renal effects of MB.
The effects of different doses of intrarenal infusion of MB on renal
function of rats are also presented in Fig. 1. The absolute values for
the control periods in Fig. 1 correspond to the values in columns
4, 6, 8, and 10 of Table 1. MB
had no significant effects on MAP. Intrarenal infusion of increasing
doses of MB had effects similar to ODQ and produced graded reductions
in RBF, GFR, and sodium and water excretion. The largest effects were seen after administration of the
1,700-nmol · kg1 · min
1
dose (decreases of 57, 79, 55, and 53%, respectively).
Groups 4, 5, and 6: effects of ODQ or MB on renal response to
L-NAME and an NO donor.
A comparison of the effects of intrarenal infusion of the NO donor in
L-NAME-treated rats receiving vehicle, ODQ, or MB is presented in Fig. 3. The absolute values
for the control periods in Fig. 3 correspond to the values in
columns 4, 6, 8, and 10 of
Table 1. Intrarenal infusion of the NO donor S-NO-NAC at 5 and 50 nmol · kg1 · min
1 produced
significant increases in RBF (+14 and +18%), UNaV (+49 and
+73%), and UV (+52 and +61%) in rats pretreated with
L-NAME and vehicle (group 4). S-NO-NAC had no
significant effect on arterial pressure or GFR when infused
intrarenally at these doses. Intrarenal infusion of ODQ or MB blocked
the effects of the NO donor on RBF, but the diuretic and natriuretic
responses to S-NO-NAC were significantly enhanced.
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Groups 4, 7, and 8: effect of ABT or HET0016 on renal response to
L-NAME and an NO donor.
A comparison of the effects of intrarenal infusion of the NO donor in
L-NAME-treated rats receiving vehicle (group 4),
ABT (group 7), or HET0016 (group 8) is presented
in Fig. 4. The absolute values for the
control periods in Fig. 4 correspond to the values in columns
4, 6, 8, and 10 of Table 1.
Inhibition of CYP450 metabolism of AA with ABT or HET0016 also blocked
the renal vasodilatory response to the NO donor and markedly attenuated
the diuretic and natriuretic responses to intrarenal infusion of
S-NO-NAC. The effects of ABT on the renal production of CYP450 products of AA are presented in Fig. 2B. The generation of DiHETEs,
20-HETE, and EETs by renal cortical microsomes fell by 75, 84, and
85%, respectively, 36 h after pretreatment of the rats with ABT.
Similarly, a high concentration of HET0016 (1 µM), 100 times greater
than the reported IC50 for this compound (16),
selectively reduced the formation of 20-HETE by renal cortical
microsomes from 96 ± 3.9 to 0.86 ± 0.7 pmol · min1 · mg
1 protein
(n = 4), but it had no effect on the formation of EETs or DiHETEs (Fig. 2C).
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DISCUSSION |
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The present study evaluated the relative contribution of cGMP and CYP450 metabolites of AA to the renal effects of an intrarenal infusion of an NO donor in rats. Infusion of the NO donor S-NO-NAC into the renal artery of rats pretreated with L-NAME to block the endogenous formation of NO increased RBF and sodium and water excretion, but it had no effect on MAP or GFR. These findings are consistent with previous results reported in dogs (13) and rats (20) infused intrarenally with NO donors or endothelium-dependent vasodilators (10, 23, 24). Pretreatment of rats with ODQ or MB blocked the effects of the NO donor on RBF. In contrast, ODQ and MB enhanced, rather than inhibited, the diuretic and natriuretic response to S-NO-NAC. The potentiation of the diuretic and natriuretic effects of NO after blockade of GC was not due to differences in baseline UNaV. As can be seen in Table 1, baseline sodium and water excretion after treatment with L-NAME was similar in rats given vehicle (group 4), ODQ (group 5), or MB (group 6). These results indicate that the renal vasodilatory response to NO is cGMP dependent or that cGMP has an obligatory permissive role in this response. On the other hand, the diuretic and natriuretic actions of NO are likely mediated by a cGMP-independent action of NO. Overall, these results are consistent with previous findings indicating that cGMP contributes to the vascular relaxation induced by endothelium-dependent vasodilators, such as acetylcholine or bradykinin, that are known to increase the endogenous synthesis of NO (11, 23). Indeed, Lahera et al. (9) suggested that the actions of L-NAME on arterial pressure and renal hemodynamics are mediated by decreased availability of cGMP, because exogenous administration of 8-BrcGMP reverses these effects in rats in vivo. However, our finding that GC inhibitors completely block the vasodilatory response to intrarenal infusion of NO donors in vivo conflicts with the other reports indicating that NO elicits both cGMP-dependent and -independent effects on vascular tone in nonfiltering kidneys (29), and that the vasodilator response to NO in isolated renal vessels is largely cGMP independent (2) and mediated by inhibition of 20-HETE formation.
We therefore examined the contribution of CYP450 metabolites of AA to the renal actions of an NO donor. S-NO-NAC was given to rats pretreated with ABT or HET0016 to block the renal metabolism of AA by CYP450 enzymes. In this regard, Su et al. (27) recently reported that ABT (50 mg/kg ip) was a potent inhibitor of the metabolism of AA by CYP450 enzymes that selectively inhibits the formation of 20-HETE and diminishes the expression of CYP4504A protein in the kidney (27). In the present study, a single dose of ABT (50 mg/kg ip) given 36 h before the acute experiment reduced the renal formation of 20-HETE and EETs by >80%. Pretreatment of rats with ABT completely blocked the effects of an intrarenal infusion of the NO donor to increase RBF. ABT also attenuated the diuretic and natriuretic response to the intrarenal infusion of the NO donor. These results are consistent with the view that the renal vasodilator responses to NO are secondary to inhibition of the formation of CYP450 metabolites of AA. They are also in complete agreement with the results of previous in vitro studies performed on renal interlobular arteries (1, 2, 18, 28).
The results obtained with ABT, however, do not allow one to determine which CYP450 metabolite of AA contributes to the renal response to NO, since ABT blocked the formation of both 20-HETE and EETs in the kidney. Therefore, we repeated the study using HET0016, which is a more selective inhibitor of 20-HETE formation (16). In a previous study, we have found that blood levels of HET0016 averaged 2.8 µM after a 10-mg/kg (iv) dose given to rats (8). This concentration is 100 times higher than the reported IC50 needed for inhibition of 20-HETE formation in renal microsomes in vitro (16). In the present study, we verified that HET0016 at a concentration of 1 µM is a highly selective inhibitor that blocks the formation of 20-HETE in rat renal microsomes and had no effect on renal epoxygenase activity. Also in the present study, we found that the effects of HET0016 on the renal response to an NO donor were similar to those obtained with ABT. HET0016 blocked the renal vasodilator response to intrarenal infusion of an NO donor, attenuated the diuretic and natriuretic response to NO, and completely blocked the antinatriuretic response to L-NAME. Together, these results suggest that the renal hemodynamic and natriuretic responses to NO are probably related to inhibition of the renal formation of 20-HETE and not EETs.
How then can one reconcile the observations that the renal vasodilatory response to an intrarenal infusion of an NO donor is completely blocked by inhibition of both GC and CYP450 enzymes? One possibility is that cGMP and 20-HETE may act on a common effector system, i.e., K+ channels in vascular smooth muscle (VSM) cells. According to this hypothesis, blockade of cGMP synthesis with ODQ or MB should enhance basal renal vascular tone by depolarizing VSM cells by blocking K+ channels. Under these conditions, NO-induced inhibition of 20-HETE formation may be unable to enhance K+ channel activity and reduce vascular tone. Blockade of cGMP formation is also known to enhance the contractile response to intracellular Ca2+. Thus activation of K+ channels and decreases in intracellular Ca2+ concentration after NO inhibition of 20-HETE formation might not be able to reduce vascular tone when cGMP levels are low (3). Similarly, blockade of 20-HETE formation with ABT or HET0016 would be expected to increase K+ channel activity, as is the case with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) (28), and to lower intracellular Ca2+ levels by reducing Ca2+ influx through L-type Ca2+ channels (12). Under these conditions, NO-induced elevations in cGMP levels may have little effect to open K+ channels, reduce intracellular Ca2+ levels, or alter vascular tone. Thus the present findings are consistent with the view that both intact cGMP and 20-HETE systems are probably required to elicit a vasodilator response to NO.
In the present study, inhibition of GC activity with ODQ or MB did not affect the natriuretic and diuretic response to intrarenal infusion of the NO donor. This finding was unexpected, since cGMP is thought to mediate both the hemodynamic and the tubular actions of NO. One possible explanation is that ODQ may not be filtered in the kidney, and, therefore, tubular cGMP formation was not inhibited. However, this possibility seems remote, since we found that the ability of NO to stimulate cGMP production in renal cortical homogenates (largely proximal tubules) was blocked in rats infused with ODQ. Also, MB is rapidly excreted in urine (which rapidly becomes blue during the intrarenal infusion), indicating that it is filtered. Thus the results of the present study suggest that inhibition of tubular sodium reabsorption after intrarenal infusion of an NO donor is probably mediated by a cGMP-independent effect.
The question then arises as to whether the natriuretic response to NO is dependent on the formation of CYP450 metabolites of AA. 20-HETE and EETs inhibit sodium transport in the proximal tubule and thick ascending limb of the loop of Henle (21). Thus NO-induced inhibition of 20-HETE production would be expected to promote sodium retention rather than increase UNaV. Also, the effect of L-NAME to reduce UV and UNaV, as it has previously been reported (5) and was confirmed in the present study (Table 1), is incompatible with the view that this response is mediated by enhanced 20-HETE production, since 20-HETE inhibits sodium reabsorption. However, L-NAME also reduces RBF and medullary blood flow, which lowers renal interstitial pressure and increases proximal tubular reabsorption (5). Thus the renal hemodynamic effects of L-NAME that enhance sodium reabsorption might predominate over any natriuretic effects associated with elevations in tubular 20-HETE levels. This possibility is also consistent with the present finding that HET0016 had a greater ability than ABT to block the renal vasodilator effects of the NO donor. If HET0016 was also able to prevent NO-induced changes in medullary blood flow, this could explain how it attenuated the natriuretic and diuretic response to the NO donor.
In summary, the results of the present study indicate that the renal vasodilator response to exogenous administration of an NO donor is dependent on both the activation of GC and the inhibition of CYP450. However, the natriuretic and diuretic responses to NO appear to be cGMP independent and are mediated in part by inhibition of the formation of CYP450 metabolites of AA.
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ACKNOWLEDGEMENTS |
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This study was supported by the Ministerio de Educación y Ciencia (PM98-0057), the Fundación Séneca (PB/6/FS/97 and 00728/CV/99), and grants from National Heart, Lung, and Blood Institute (HL-29587 and HL-36279).
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. J. Fenoy, Departamento de Fisiología y Farmacología, Facultad de Medicina, Campus de Espinardo, 30100-Murcia, Spain (E-mail: fjfenoy{at}um.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 October 2000; accepted in final form 14 May 2001.
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