Nitric oxide synthesis influences the renal vascular response to heme oxygenase inhibition

Francisca Rodriguez, Fan Zhang, Sandra Dinocca, and Alberto Nasjletti

Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the effects of the heme oxygenase (HO) inhibitor stannous mesoporphyrin (SnMP; 40 µmol/kg iv) on renal hemodynamics in anesthetized rats with and without 48-h pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis. SnMP decreased renal blood flow (RBF) and increased renal vascular resistance (RVR) in both groups. The SnMP-induced reduction of RBF in L-NAME-pretreated rats was more prominent than in rats without pretreatment (43 ± 7 vs. 13 ± 3%) as was the SnMP-induced elevation of RVR (87 ± 31 vs. 14 ± 5%). The renal vasoconstrictor effect of SnMP is linked, in part, to amplification of prevailing neurohormonal constrictor mechanisms, since in L-NAME-pretreated rats it was prevented by concurrent administration of prazosin or losartan. However, SnMP (15 µmol/l) also elicits vasoconstriction in isolated, pressurized renal interlobular arteries and the response is more intense in vessels obtained from L-NAME-pretreated rats than from rats without pretreatment. These data indicate that the status of NO synthesis conditions the vascular response to HO inhibition in the rat kidney.

carbon monoxide; renal hemodynamics; angiotensin II; norepinephrine; renal vascular reactivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEME OXYGENASE (HO)-1 and -2 metabolize heme to biliverdin, ferrous iron, and carbon monoxide (CO) (1, 18). The heme-HO system is ubiquitous and relevant to many biological processes that are influenced by heme or by the products of heme metabolism by HO (1, 10, 11, 18, 26). In the kidney, HO-1 and HO-2 are expressed in both vascular and tubular structures, and HO-1 is upregulated in experimental models of renal injury (2, 5, 11, 16, 28). The renal heme-HO system was reported to promote cytoprotective mechanisms (11) and to participate in the regulation of renal function (2, 19, 28) and eicosanoid production (4).

Metalloporphyrins which inhibit HO decrease renal medullary blood flow in normal rats (28) and total renal blood flow in chronically hypoxic rats (19), suggesting the contribution of one or more HO products to renal vasodilatory mechanisms. HO-derived CO is a logical candidate for subserving a renal vasodilatory function, because exogenous CO dilates isolated, perfused rat afferent arterioles (23) and decreases the reactivity of renal interlobar arteries to constrictor agonists (16). CO-induced vasorelaxation and inhibition of agonist-induced vasoconstriction have been linked to activation of soluble guanylyl cyclase (24) and/or stimulation of calcium-activated potassium (KCa) channels (16, 24) in vascular smooth muscle.

Recent studies have identified areas of interaction between the heme-HO and the L-arginine-nitric oxide (NO) synthase systems that may have great functional relevance. On one hand, NO interferes with the ability of CO to stimulate KCa channels in vascular smooth muscle (25), induces HO-1 expression (8, 9), and decreases HO activity (6, 15) as well as ferrochelatase activity (22), the terminal enzyme of the heme synthetic pathway. On the other hand, CO attenuates NO-induced activation of soluble guanylyl cyclase (12) and inhibits NO synthase (23). That CO and NO affect the formation and action(s) of each other raises the possibility that the vasoregulatory functions of the heme-HO and the L-arginine-NO synthase systems are interdependent.

To the extent that NO influences the formation and actions of HO-derived CO, the status of NO synthesis may determine the nature and/or intensity of the regulatory influence of the heme-HO system on the renal vasculature. To test this hypothesis, we contrasted untreated rats and rats undergoing treatment with an inhibitor of NO synthase in terms of the renal hemodynamic response to the administration of stannous mesoporphyrin (SnMP), a nonselective inhibitor of HO isoforms (7). In addition, we compared the effect of SnMP and HO products, CO and biliverdin, on the internal diameter of isolated, pressurized renal interlobular arteries taken from normal untreated rats and rats treated with an inhibitor of NO synthesis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Solutions

SnMP, an inhibitor of HO, was purchased from Frontier Scientific (Logan, UT). SnMP was dissolved in 50 mmol/l Na2CO3, sonicated, and filtered immediately before use. Because of the photosensitivity of porphyrins, SnMP solutions were prepared and experiments were performed under reduced light conditions. CO was purchased from Tech Air (White Plains, NY), and a CO-saturated solution (1 mmol/l) was prepared shortly before use (17). Other chemicals were obtained from Sigma (St. Louis, MO) and diluted in NaCl (0.15 mol/l). The composition of Krebs buffer used in the studies was (in mmol/l) 118.5 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, and 11.1 dextrose.

Animals

All protocols employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of New York Medical College. We used male Sprague-Dawley rats (300-325 g body wt; Charles River, Wilmington, DE) with access to a standard chow and tap water ad libitum. Studies were conducted in animals treated and not treated with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (21). L-NAME was provided in the drinking water at a daily dose of 70 mg/kg body wt commencing 48 h before experimentation.

Experimental Design

Protocol to contrast the effect of SnMP on renal hemodynamics in rats treated and not treated with L-NAME. Animals were anesthetized with thiobutabarbital (100 mg/kg ip) and placed on a thermostatically controlled board to maintain body temperature at 36-37°C. Polyethylene cannulas were placed in the trachea (PE-205) to aid ventilation, the bladder (PE-60) for urine collection, the left femoral vein (PE-50) for administration of fluid and drugs, and the left femoral artery (PE-50) for blood sampling and the measurement of mean arterial pressure by means of a pressure transducer (model p23 ID, Oxnard, CA) coupled to a polygraph (model 7D, Grass Instruments, Quincy, MA). Next, the left kidney was exposed through a midline abdominal incision and a flow probe (model EP102, 2.0-mm circumference, Carolina Medical Instruments, King, NC) was placed around the left renal artery for measurement of renal blood flow using a square-wave electromagnetic flowmeter (model FM 501, Carolina Medical Instruments). After completion of the cannulation and instrumentation procedures, an infusion of 0.15 mol/l NaCl (2.5 ml/h iv) was initiated and maintained throughout the study. In some experiments, [3H]inulin (American Radiolabeled Chemicals, St. Louis, MO) was included into the infusion (1 µCi/ml) for measurement of glomerular filtration rate. To this end, timed urine collections were made before and during experimental interventions, and arterial blood samples (200 µl) were collected at the midpoint of urine collections. The concentration of [3H]inulin in urine and plasma was determined by liquid scintillation counting and the clearance of inulin, calculated using the standard formula, was taken to reflect the glomerular filtration rate. Renal vascular resistance was calculated by dividing the value of mean arterial pressure by the value of renal blood flow. The filtration fraction was calculated by the formula: glomerular filtration rate/renal blood flow (1 - hematocrit). Data collection was initiated after a 60-min equilibration period.

Control data on mean arterial pressure and renal hemodynamics were collected over a 30-min control period immediately before the administration of SnMP (40 µmol/kg iv) to rats pretreated (n = 8) and not pretreated with L-NAME (n = 14); data were collected again over a 30-min experimental period commencing 45 min after the onset of SnMP treatment. An identical protocol was used to collect data on renal function before and during administration of drug vehicle only (50 mmol/l Na2CO3) in rats pretreated (n = 8) and not pretreated (n = 12) with L-NAME.

Inhibition of NO synthesis fosters the activity of constrictor mechanisms involving the sympathetic nervous and the renin-angiotensin systems (3). This may condition the effect of SnMP on renal hemodynamics, because the response of small arterial vessels to constrictor stimuli is amplified by HO inhibition (16). To examine this possibility, the effect of SnMP (40 µmol/kg iv) on mean arterial pressure, renal blood flow, and renal vascular resistance was also investigated in rats pretreated with L-NAME along with either prazosin (1 mg/kg sc; n = 6) or losartan (10 mg/kg iv; n = 7), respectively, to block alpha 1-adrenergic and angiotensin AT1 receptors (20). In these experiments, rats undergoing L-NAME pretreatment for 48 h were injected with prazosin or losartan followed, 45 min later, by examination of the effects of SnMP using a protocol similar to that described above.

A complementary study was conducted in rats without L-NAME pretreatment to determine whether SnMP (40 µmol/kg iv) affects the renal circulatory response to norepinephrine and ANG II. Rats were instrumented with a cannula in the left carotid artery to measure mean arterial pressure and a flow probe on the left renal artery to measure renal blood flow. Norepinephrine (2 nmol · kg-1 · min-1; n = 5) or ANG II (5 pmol · kg-1 · min-1; n = 8) was administered by infusion over a 15- to 20-min period via a polyethylene cannula made of stretched PE-10 tubing that was inserted into the left femoral artery and advanced through the abdominal aorta into the left renal artery. After discontinuation of constrictor agonist infusion, a 30-min recovery period was allowed, SnMP (40 µmol/kg iv) was injected, and 45 min later the infusion of constrictor agonist was repeated.

Renal HO activity was measured in a limited number of control untreated rats, rats undergoing treatment with L-NAME for 48 h, and rats 1.0-1.5 h after administration of SnMP (40 µmol/kg iv). Kidneys were homogenized, the homogenate was centrifuged, and the 10,000-g supernatant was analyzed for HO activity using [14C]heme (Leeds Radioporphyrins, Leeds, UK) as substrate (16). HO activity is expressed as picomoles of bilirubin generated per milligram of protein per hour.

Protocols to investigate the effects of CO and the HO inhibitor SnMP in isolated renal interlobular arteries taken from rats treated and not treated with L-NAME. Animals were anesthetized (60 mg/kg ip pentobarbital sodium), the kidneys were removed and placed on a dish containing ice-cold Krebs buffer, and interlobular arteries were dissected free of surrounding tissue. Vascular segments 1- to 2-mm in length, with an internal diameter of ~40 µm in the absence of flow and transmural pressure, were mounted between two micropipettes in the chamber (1 ml) of a pressure-myograph (Living System Instrumentation, Burlington, VT) filled with Krebs buffer gassed with 95% O2-5% CO2, which was exchanged at a rate of 1 ml/min (27). To measure vascular diameter, the vessel chamber was placed on the stage of a microscope fitted with a video camera (Javelin, Newburgh, NY) linked to a video caliper (Texas A & M, College Station, TX) and a recorder (27). Intraluminal pressure was increased slowly to 100 mmHg using a pressure servo-controller, and this level of pressure was maintained throughout the study unless indicated otherwise. Experiments were conducted after a 60-min equilibration period in vessels that developed a spontaneous tone while pressurized to 100 mmHg. The internal diameter of vessels was measured continuously before and after inclusion of SnMP (15 µmol/l), CO (0.1 and 1.0 µmol/l), or biliverdin (1 µmol/l) into the Krebs buffer flowing into the myograph chamber.

To gain information on whether NO synthesis remains inhibited during ex vivo superfusion of vessels taken from L-NAME-treated rats, a limited number of experiments were conducted to contrast the effect of L-arginine (10 µmol/l) on the diameter of pressurized renal interlobular arteries taken from rats with and without L-NAME pretreatment. The vessels were superfused with Krebs buffer for 90-120 min before the experimental intervention. The inclusion of L-arginine in the superfusion buffer increased (P < 0.05) the internal diameter of arteries taken from rats without L-NAME pretreatment from 69.7 ± 3.1 to 77.5 ± 5.0 µm (n = 4). In contrast, L-arginine did not change the diameter of arteries taken from rats pretreated with L-NAME (68.0 ± 3.9 vs. 68.0 ± 3.9 µm; n = 4). That the dilatory effect of L-arginine is blunted in vessels from rats pretreated with L-NAME implies that NO synthesis in such vessels remains inhibited even after ex vivo superfusion for up to 90-120 min.

Additional experiments were conducted to contrast the effect of SnMP (15 µmol/l) on the intraluminal pressure-internal diameter relationship in renal interlobular arteries taken from rats treated and not treated with L-NAME. After equilibration at 100 mmHg for 60 min, intraluminal pressure was decreased to ~0 mmHg and, after 10 min, it was increased in 20-mmHg steps until it reached 100 mmHg. The pressure was maintained for ~5-10 min at each pressure step so that the vessels could reach a steady-state diameter. Finally, the vascular preparation was superfused with calcium-free Krebs buffer containing 1 mmol/l EGTA and the pressure-diameter relationship was examined again to obtain the passive diameter of the vessels at each level of intraluminal pressure. The internal diameter during superfusion of the vessel with calcium-containing buffer (absolute diameter) and with calcium-free buffer (passive diameter) is expressed in micrometers. The normalized diameter refers to the absolute diameter expressed as a percentage of the passive diameter.

Data Analysis

Results are expressed as means ± SE. Data on renal hemodynamics in vivo are the average of two consecutive 15-min observation periods. Data were analyzed by unpaired Student's t-test and by one- or two-way ANOVA followed by the Newman-Keuls post hoc test or the Fisher test. The null hypothesis was rejected at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of SnMP on Renal Hemodynamics

Table 1 shows data on renal hemodynamics before and after the administration of SnMP or drug vehicle only to rats pretreated and not pretreated with the NO synthesis inhibitor L-NAME. Relative to data in rats without L-NAME pretreatment, rats pretreated with L-NAME displayed elevated mean arterial pressure, decreased renal blood flow, and increased renal vascular resistance, filtration fraction, and hematocrit, without significant changes in glomerular filtration rate. Estimates of renal HO activity did not differ significantly in rats with (96 ± 16 pmol · mg-1 · h-1; n = 4) and without (125 ± 14 pmol · mg-1 · h-1; n = 4) L-NAME pretreatment; renal HO activity was decreased (15 ± 1 pmol · mg-1 · h-1; n = 8; P < 0.05) 1.0-1.5 h after the administration of the HO inhibitor SnMP (40 µmol/kg iv). In rats without L-NAME pretreatment, the administration of SnMP did not change mean arterial pressure but decreased renal blood flow and increased renal vascular resistance without affecting the glomerular filtration rate or filtration fraction. In rats pretreated with L-NAME, the administration of SnMP lowered arterial pressure slightly, decreased renal blood flow, and increased renal vascular resistance; the treatment did not affect glomerular filtration but greatly increased the filtration fraction. The SnMP-induced reduction of renal blood flow was greater (P < 0.05) in rats pretreated with L-NAME (43 ± 7%) than in rats without L-NAME pretreatment (13 ± 3%). The SnMP-induced increase of renal vascular resistance was also greater (P < 0.05) in L-NAME-pretreated rats (87 ± 31%) than in rats without pretreatment (14 ± 5%). Thus the administration of the HO inhibitor SnMP produces renal vasoconstriction and this effect is intensified in rats pretreated with an inhibitor of NO synthesis. At variance with the pronounced effects of SnMP on renal hemodynamics, the administration of the drug vehicle alone affected renal hemodynamics in neither rats with or without L-NAME pretreatment.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of SnMP on renal hemodynamics

The possibility that SnMP elicits renal vasoconstriction by amplifying the renal vascular actions of endogenous vasoconstrictors was examined by contrasting the effects of SnMP on renal hemodynamics in rats pretreated with L-NAME alone, L-NAME plus prazosin to block adrenergically mediated vasoconstriction, and L-NAME plus losartan to block ANG II-induced vasoconstriction. Relative to corresponding data in rats pretreated with L-NAME alone, before the administration of SnMP, rats pretreated with L-NAME and prazosin displayed decreased mean arterial pressure (112 ± 8 vs. 130 ± 3 mmHg, P < 0.05) and renal vascular resistance (19.4 ± 3.1 vs. 26.2 ± 1.4 mmHg · ml-1 · min · g-1, P < 0.05), whereas renal blood flow showed a slight tendency to increase (6.2 ± 0.6 vs. 5.1 ± 0.4 ml · min-1 · g-1, P = 0.14). Rats pretreated with L-NAME and losartan exhibited unchanged mean arterial pressure (129 ± 6 mmHg), diminished renal vascular resistance (15.8 ± 2.1 mmHg · ml-1 · min · g-1, P < 0.05), and increased renal blood flow (8.7 ± 0.7 ml · min-1 · g-1, P < 0.05). In rats pretreated with L-NAME alone, SnMP decreased mean arterial pressure, reduced renal blood flow, and increased renal vascular resistance but had no effect on any of these parameters in rats pretreated with L-NAME and prazosin (Fig. 1). In rats pretreated with L-NAME and losartan, SnMP slightly reduced mean arterial pressure and renal blood flow but was without effect on renal vascular resistance (Fig. 1). Thus the renal vasoconstriction induced by the HO inhibitor, in rats pretreated with L-NAME, is blunted in animals undergoing pharmacological blockade of alpha 1-adrenergic or angiotensin AT1 receptors.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) before (open bars) and after (filled bars) treatment with stannous mesoporphyrin (SnMP; 40 µmol/kg iv) in rats pretreated with NG-nitro-L-arginine methyl ester (L-NAME) alone (n = 8) and concurrently with prazosin (n = 6) or losartan (n = 7). Results are means ± SE. *P < 0.05 relative to control data before SnMP; dagger P < 0.05 relative to the changes in hemodynamic function induced by SnMP in rats pretreated with L-NAME alone.

Figure 2 illustrates the result of experiments comparing the effect of renal arterial infusion of norepinephrine (2 nmol · kg-1 · min-1), or ANG II (5 pmol · kg-1 · min-1), on renal hemodynamics in rats before and after treatment with SnMP. Both constrictor agonists decreased renal blood flow and increased renal vascular resistance without altering mean arterial pressure. The renal vasoconstrictor effect of norepinephrine and ANG II was significantly magnified after the administration of SnMP. Thus inhibition of HO amplifies the renal vasoconstrictor action of norepinephrine and ANG II.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   MAP and RBF before (open bars) and during (filled bars) renal arterial infusion of norepinephrine (NE; n = 5) or ANG II (n = 8), both before and after administration of SnMP (40 µmol/kg iv). Results are means ± SE. *P < 0.05 relative to control data before the infusion of NE or ANG II; dagger P < 0.05 relative to the change in hemodynamic function elicited by the constrictor agonist before the administration of SnMP.

Effect of SnMP on the Internal Diameter of Isolated, Pressurized Renal Interlobular Arteries

Inclusion of SnMP into the Krebs buffer superfusing pressurized renal interlobular arteries elicited a sustained reduction of internal diameter in vessels taken from rats treated and not treated with L-NAME (Fig. 3). In vessels obtained from rats not treated with L-NAME, SnMP decreased (P < 0.05) the internal diameter by 6.0 ± 0.8, 8.5 ± 1.1, 9.5 ± 1.1, and 9.8 ± 1.1 µm after 5, 10, 20, and 30 min of exposure to the HO inhibitor, respectively. In vessels obtained from rats treated with L-NAME, SnMP decreased (P < 0.05) the internal diameter by 13.1 ± 1.6, 18.1 ± 1.3, 22.4 ± 1.3, and 23.3 ± 1.4 µm after 5, 10, 20, and 30 min of exposure to the HO inhibitor, respectively. Importantly, at all time points, the SnMP-induced reduction of internal diameter was greater (P < 0.05) in vessels obtained from L-NAME-treated rats than in vessels obtained from untreated rats. Thus exposure to SnMP brings about constriction of renal interlobular arteries and this effect is intensified in vessels taken from rats treated with an inhibitor of NO synthesis.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Internal diameter (ID) of pressurized interlobular arteries obtained from rats with and without 48-h pretreatment with L-NAME, before and during superfusion with buffer containing SnMP (15 µmol/l). Results are means ± SE. *P < 0.05 relative to control data before SnMP.

The vasoconstrictor effect of HO inhibitors in isolated, pressurized resistance vessels has been linked to magnification of the prevailing myogenic tone (17). To investigate whether SnMP promotes myogenic behavior and, if so, to determine whether this effect is conditioned by the status of NO synthesis, we contrasted the effect of SnMP on the pressure-diameter relationship in isolated renal interlobular arteries obtained from rats treated and not treated with L-NAME. As shown in Fig. 4, stepwise elevation of intraluminal pressure elicited a pressure-dependent reduction (P < 0.05) of internal diameter in vessels taken from rats treated and not treated with L-NAME. Before exposure to SnMP, vessels from rats treated and not treated with L-NAME did not differ from each other in terms of internal diameter over the pressure range 20 to 100 mmHg. The inclusion of SnMP into the superfusion buffer enhanced (P < 0.05) the pressure-induced reduction of internal diameter in both groups of vessels. In preparations exposed to SnMP, the internal diameter reduction of vessels obtained from L-NAME-treated rats surpassed (P < 0.05) that of vessels taken from rats not treated with L-NAME over the pressure range of 40 to 100 mmHg. Thus SnMP enhances pressure-induced constriction of renal interlobular arteries and this effect is further intensified in vessels obtained from rats treated with a NO synthesis inhibitor.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of stepwise increments in intraluminal pressure on the normalized ID of renal interlobular arteries superfused with buffer containing and not containing SnMP (15 µmol/l). The studies were conducted in vessels obtained from rats with and without 48-h pretreatment with L-NAME. Results are means ± SE. *P < 0.05 relative to control data in vessels not exposed to SnMP.

To the extent that the vascular response to an inhibitor of HO results from decreased vascular formation of an HO product capable of influencing vasomotor function, the increased constrictor effect of SnMP in renal interlobular arteries of L-NAME-treated rats may be linked to alterations in the responsiveness of such vessels to the products of HO activity. This possibility was investigated by comparing the effects of CO and biliverdin on the internal diameter of isolated, pressurized renal interlobular arteries taken from rats with and without L-NAME treatment. As shown in Fig. 5, the inclusion of 1 µmol/l CO into the superfusion buffer elicited a sustained reduction (P < 0.05) of internal diameter in vessels taken from rats not treated with L-NAME. In contrast, 1 µmol/l CO brought about augmentation of internal diameter in vessels taken from rats treated with L-NAME. At 0.1 µmol/l, CO was without effect on the diameter of vessels taken from either rats with and without L-NAME treatment. Biliverdin at 1 µmol/l did not affect the internal diameter of renal interlobular arteries obtained from either untreated rats (n = 4; 68 ± 2 and 69 ± 2 µm before and 30 min after, respectively) or L-NAME-treated rats (n = 4; 67 ± 4 and 68 ± 4 µm before and 30 min after, respectively).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   ID of pressurized renal interlobular arteries from rats with and without 48-h pretreatment with L-NAME, before and during superfusion with buffer containing exogenous carbon monoxide (CO). Results are means ± SE. *P < 0.05 relative to control data before CO treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies provide information on whether the status of NO synthesis influences the renal vascular response to treatment with an inhibitor of HO. In anesthetized rats, we found that the administration of SnMP increases renal vascular resistance and lowers renal blood flow and that both responses are more intense in rats pretreated with L-NAME than in control rats without pretreatment. Intensification after L-NAME pretreatment of the renal vasoconstrictor and blood flow-lowering effects of SnMP was accompanied by a major elevation in filtration fraction, an observation consistent with SnMP-induced vasoconstriction at postglomerular sites and/or increase of the ultrafiltration coefficient. We also found that the exposure of isolated, pressurized renal interlobular arteries to SnMP elicits sustained reduction of internal diameter and that this response is more intense in vessels taken from L-NAME-treated rats than in vessels from untreated controls. Hence, the results of both the in vivo and ex vivo studies indicate that the expression of renal vasoconstriction after treatment with SnMP is magnified in settings in which NO synthesis is inhibited with L-NAME. Inhibition of NO synthesis also facilitates development of vasoconstriction after treatment with an inhibitor of HO in rat hindlimbs in vivo and in isolated gracilis muscle arterioles (13).

According to the present study, in vitro estimates of renal HO activity in L-NAME-treated rats do not differ significantly from estimates in untreated control rats. Yet, there are reports that exogenous NO decreases HO activity (6, 15), which prompt expectation of enhanced HO product generation during NO synthesis inhibition. In line with this notion, a preliminary assessment of CO in the headspace of urine samples, using gas chromatography-mass spectroscopy (16), indicates that the urinary excretion of CO in anesthetized rats undergoing pretreatment with L-NAME for 48 h (15.3 ± 4.5 pmol/min; n = 4) surpasses (P < 0.05) the CO excretion rate in untreated controls (2.2 ± 0.5 pmol/min; n = 4) (Rodriguez F, Kemp R, and Nasjletti A; unpublished observations). In rats pretreated with L-NAME, augmentation of HO product generation in vivo may condition intensification of SnMP-induced renal vasoconstriction.

Treatment with SnMP magnifies the reduction of renal blood flow produced by renal arterial infusion of norepinephrine or ANG II. This finding fits well with reports that interventions that attenuate the expression or activity of HO enhance the sensitivity of renal arterial vessels to constrictor agonists (16). It is conceivable, then, that the renal vasoconstrictor effect of SnMP is linked, at least in part, to amplification of prevailing neurohormonal constrictor mechanisms. This seems to be the case in anesthetized rats treated with L-NAME, because concurrent treatment with prazosin or losartan prevents SnMP from increasing renal vascular resistance.

Previous studies indicate that the increase in renal vascular resistance elicited by the systemic administration of an inhibitor of NO synthesis relies, in part, on a constrictor mechanism involving the sympathetic nervous and renin-angiotensin systems (3). In line with this notion, we found that the values of renal vascular resistance in rats treated with L-NAME alone exceed the corresponding values in rats concurrently treated with L-NAME and prazosin or L-NAME and losartan. In view of these findings, consideration should be given to the possibility that intensification of the renal vasoconstrictor effect of SnMP in L-NAME-treated rats is conditioned by the recruitment, after inhibition of NO synthesis, of a constrictor mechanism dependent on the sympathetic nervous and renin-angiotensin systems.

According to the present study, the conditioning influence of L-NAME pretreatment on SnMP-induced vasoconstriction is also demonstrable in renal interlobular arteries, isolated and pressurized, under experimental conditions that preclude participation of the sympathetic nervous and renin-angiotensin systems in vasomotor regulation. The constrictor effect of SnMP in these vessels is attributable to amplification of the prevailing myogenic tone, as documented previously in gracilis muscle arterioles (17, 27), because inclusion of SnMP into the superfusion buffer magnifies the reduction of vessel diameter produced by stepwise elevation of intraluminal pressure. This effect of SnMP enhancing myogenic constrictor responsiveness is prominently expressed in renal arteries taken from rats pretreated with L-NAME, which could explain the greater constrictor effect of the HO inhibitor in such vessels.

A priori, the renal vasoconstrictor effect of SnMP may be expected to result from diminished synthesis of one or more HO products capable of effecting vasodilation. Biliverdin does not dilate pressurized renal interlobular arteries and, therefore, it is unlikely that a decreased production of biliverdin contributes to the vasoconstrictor effect of the HO inhibitor. Because renal arteries taken from rats without L-NAME pretreatment respond to exogenous CO with constriction, like gracilis muscle arterioles not treated with an inhibitor of NO synthesis (14), the constrictor effect of SnMP cannot be explained on the basis of diminished CO production in such vessels, unless it is postulated that exogenous CO and CO manufactured by vascular structures have divergent vasomotor activity. On the other hand, exogenous CO was found to cause prompt dilation of renal arteries taken from rats pretreated with L-NAME, which is in agreement with the notion that the constrictor effect of SnMP in such vessels is a consequence of reduced CO production. On the basis of these observations, intensification of the renal vasoconstrictor effect of SnMP in L-NAME-treated rats may be conditioned by the preeminence of renal vasodilatory mechanisms mediated by endogenous CO in settings in which NO synthesis is impaired.

The vasodilatory effect of exogenous CO is ascribed to activation of soluble guanylate cyclase (24) and/or stimulation of KCa channels (16, 24) in vascular smooth muscle, whereas the vasoconstrictor effect is attributed to inhibition of NO production by the endothelium (14). That renal arteries from rats without L-NAME pretreatment respond to exogenous CO with constriction rather than dilation implies that CO-induced inhibition of NO synthesis subserves a vasoconstrictor action that prevails over the direct vasodilatory actions of the gas mediator. On the other hand, when the ability of CO to inhibit NO synthesis is rendered inconsequential, as in renal arteries taken from rats pretreated with L-NAME, the vasodilatory actions of CO are unmitigated. The preferential expression of CO-induced dilation of vessels from rats pretreated with L-NAME may also be due to facilitation of CO-induced stimulation of KCa channels in vascular smooth muscle, an action that is inhibited by NO (25).

From the preceding discussion, it would appear that the net effect of CO, both exogenous CO and CO manufactured by vascular tissues, is determined by the balance between actions on the smooth muscle, to effect vasodilation, and actions on the endothelium, to inhibit NO synthesis and bring about vasoconstriction. If so, the nature and intensity of the vasomotor response elicited by exogenous CO and CO of vascular origin may be determined not only by the status of NO synthesis but also by the relative access of the gas mediator to sites of actions in the endothelium and smooth muscle. For example, in vessels from rats without L-NAME pretreatment, CO manufactured by the smooth muscle may be expected to preferentially promote vasodilation, reflecting unfettered access to sites of action in smooth muscle coupled to suboptimal access to sites of action in the endothelium, whereas exogenous CO and CO produced by the endothelium are expected to preferentially promote vasoconstriction.

In summary, this study demonstrates that the HO inhibitor SnMP produces renal vasoconstriction and that this effect is greatly magnified in rats pretreated with L-NAME. The study also shows that SnMP amplifies the renal vasoconstriction induced by norepinephrine or ANG II, that pretreatment with either prazosin or losartan prevents the HO inhibitor from increasing renal vascular resistance in L-NAME-treated rats, and that CO elicits dilation and constriction, respectively, of renal interlobular arteries taken from rats with and without L-NAME pretreatment. Thus the status of NO synthesis impacts importantly on the regulatory influence of the heme-HO system on the renal vasculature.


    ACKNOWLEDGEMENTS

We thank J. Brown for secretarial assistance.


    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-18579 and HL-34300 and American Heart Association, New York Affiliate, Grant 0020152T. F. Rodriguez was supported by Fundacion Seneca, Spain.

Address for reprint requests and other correspondence: F. Rodriguez, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: francisca_rodrigues{at}nymc.edu).

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.

10.1152/ajprenal.00435.2002

Received 19 December 2002; accepted in final form 28 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, NG, Drummond GS, Lutton JD, and Kappas A. The biological significance and physiological role of heme oxygenase. Cell Physiol Biochem 247: 725-732, 1997.

2.   Aizawa, T, Ishizaka N, Taguchi J, Nagai R, Mori I, Tang SS, Ingelfinger JR, and Ohno M. Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats: possible role in renoprotection. Hypertension 35: 800-806, 2000[Abstract/Free Full Text].

3.   Baylis, C, and Qiu C. Importance of nitric oxide in the control of renal hemodynamics. Kidney Int 49: 1727-1731, 1996[ISI][Medline].

4.   Botros, FT, Laniado-Schwartzman M, and Abraham NG. Regulation of cyclooxygenase- and cytochrome p450-derived eicosanoids by heme oxygenase in the rat kidney. Hypertension 39: 639-644, 2002[Abstract/Free Full Text].

5.   Da Silva, JL, Zand BA, Yang LM, Sabaawy HE, Lianos E, and Abraham NG. Heme oxygenase isoform-specific expression and distribution in the rat kidney. Kidney Int 59: 1448-1457, 2001[ISI][Medline].

6.   Ding, Y, McCoubrey WK, and Maines MD. Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular "sink" for NO? Eur J Biochem 264: 854-861, 1996.

7.   Drummond, GS, Galbraith RA, Sardana MK, and Kappas A. Reduction of the C2 and C4 vinyl groups of Sn-protoporphyrin to form Sn-mesoporphyrin markedly enhances the ability of the metalloporphyrin to inhibit in vivo heme catabolism. Arch Biochem Biophys 255: 64-74, 1987[ISI][Medline].

8.   Durante, W, Kroll MH, Christodoulides N, Peyton KJ, and Schafer AI. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth cells. Circ Res 80: 557-564, 1997[Abstract/Free Full Text].

9.   Foresti, R, and Motterlini R. The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis. Free Radic Res 31: 459-475, 1999[ISI][Medline].

10.   Haider, A, Olszanecki R, Gryglewski R, Schwartzman ML, Lianos E, Nasjletti A, Kappas A, and Abraham NG. Regulation of cyclooxygenase by the heme-heme oxygenase system in microvessel endothelial cells. J Pharmacol Exp Ther 300: 188-194, 2002[Abstract/Free Full Text].

11.   Hill-Kapturczak, N, Chang SH, and Agarwal A. Heme oxygenase and the kidney. DNA Cell Biol 21: 307-321, 2002[ISI][Medline].

12.   Ingi, T, Cheng J, and Ronnett GV. Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system. Neuron 16: 835-846, 1996[ISI][Medline].

13.   Johnson, FK, Teran FJ, Prieto-Carrasquero M, and Johnson RA. Vascular effects of a heme oxygenase inhibitor are enhanced in the absence of nitric oxide. Am J Hypertens 15: 1074-1080, 2002[ISI][Medline].

14.   Johnson, RA, and Johnson FK. The heme-heme oxygenase-carbon monoxide system and hypertension. In: Carbon Monoxide and Cardiovascular Functions, edited by Wang R.. Boca Raton, FL: CRC, 2002, p. 149-163.

15.   Juckett, M, Zheng Y, Yuan H, Pastor T, Antholine W, Weber M, and Vercellotti G. Heme and the endothelium. Effects of nitric oxide on catalytic iron and heme degradation by heme oxygenase. J Biol Chem 273: 23388-23397, 1998[Abstract/Free Full Text].

16.   Kaide, JI, Zhang F, Wei Y, Jiang H, Yu C, Wang WH, Balazy M, Abraham NG, and Nasjletti A. Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors. J Clin Invest 107: 1163-1171, 2001[Abstract/Free Full Text].

17.   Kozma, F, Johnson RA, Zhang F, Yu C, Tong X, and Nasjletti A. Contribution of endogenous carbon monoxide to regulation of diameter in resistance vessels. Am J Physiol Regul Integr Comp Physiol 276: R1087-R1094, 1999[Abstract/Free Full Text].

18.   Maines, MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517-554, 1997[ISI][Medline].

19.   O'Donaughy, TL, and Walker BR. Renal vasodilatory influence of endogenous carbon monoxide in chronically hypoxic rats. Am J Physiol Heart Circ Physiol 281: H298-H307, 2001[Abstract/Free Full Text].

20.   Pucci, ML, Lin L, and Nasjletti A. Pressor and renal vasoconstrictor effects of NG-nitro-L-arginine as affected by blockade of pressor mechanisms mediated by the sympathetic nervous system, angiotensin, prostanoids and vasopressin. J Pharmacol Exp Ther 261: 240-245, 1992[Abstract].

21.   Rees, DD, Palmer RM, Schulz R, Hodson HF, and Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 101: 746-752, 1990[Abstract].

22.   Sellers, VM, Johnson MK, and Dailey HA. Function of the [2FE-2S] cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor. Biochemistry 35: 2699-2704, 1996[ISI][Medline].

23.   Thorup, C, Jones CL, Gross SS, Moore LC, and Goligorsky MS. Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS. Am J Physiol Renal Physiol 277: F882-F889, 1999[Abstract/Free Full Text].

24.   Wang, R, Wang Z, and Wu L. Carbon monoxide-induced vasorelaxation and the underlying mechanisms. Br J Pharmacol 121: 927-934, 1997[Abstract].

25.   Wu, L, Cao K, Lu Y, and Wang R. Different mechanisms underlying the stimulation of K(Ca)+2 channels by nitric oxide and carbon monoxide. J Clin Invest 110: 691-700, 2002[Abstract/Free Full Text].

26.   Zhang, F, Kaide JI, Rodriguez-Mulero F, Abraham NG, and Nasjletti A. Vasoregulatory function of the heme-heme oxygenase-carbon monoxide system. Am J Hypertens 14: 62S-67S, 2001[ISI][Medline].

27.   Zhang, F, Kaide JI, Wei Y, Jiang H, Yu C, Balazy M, Abraham NG, Wang W, and Nasjletti A. Carbon monoxide produced by isolated arterioles attenuates pressure- induced vasoconstriction. Am J Physiol Heart Circ Physiol 281: H350-H358, 2001[Abstract/Free Full Text].

28.   Zou, AP, Billington H, Su N, and Cowley AW, Jr. Expression and actions of heme oxygenase in the renal medulla of rats. Hypertension 35: 342-347, 2000[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(6):F1255-F1262
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society