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J Appl Physiol 98: 772-779, 2005. First published November 5, 2004; doi:10.1152/japplphysiol.00638.2004
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20-HETE-mediated vasoconstriction by hemoglobin-O2 carrier in Sprague-Dawley but not Wistar rats

Andrew D. Baines and Patrick Ho

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Submitted 22 June 2004 ; accepted in final form 3 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Hypothetically either decreased nitric oxide (NO) or increased O2 could initiate 20-HETE-mediated vasoconstriction associated with hemoglobin-based blood substitutes (HBOC). To test this hypothesis, we infused Tm-Hb, an HBOC with low O2 affinity, into isoflurane-anesthetized Wistar (W) and Sprague-Dawley (SD) rats after exchanging 20% of their blood with Ringer lactate. For comparison we infused an equal amount of BSA or BSA with NG-nitro-L-arginine methyl ester (BSA+NAME). Tm-Hb increased blood pressure (BP) and renal vascular resistance (RVR) equally in W and SD rats. Renal blood flow (RBF; Doppler ultrasound) decreased. BSA decreased RVR and raised glomerular filtration rate. BSA+NAME raised BP, RVR, and GFR. HET0016, an inhibitor of 20-HETE production, blunted BP and RVR responses to Tm-Hb and BSA+NAME in SD but not W rats. Arterial O2 content with BSA was lower than with Tm-Hb but O2 delivery was 60% higher with BSA because of higher RBF. BSA raised PO2 (Oxylite) in cortex and medulla and reduced RVR. Tm-Hb decreased PO2 and increased RVR. Switching rats from breathing air to 100% O2 raised intrarenal PO2 two- to threefold and increased BP and RVR. HET0016 did not alter hyperoxic responses. In conclusion, 20-HETE contributes to vasoconstriction by Tm-Hb in SD but not in W rats, and increased 20-HETE activity results primarily from decreased NO.

blood substitutes; kidney; nitric oxide; N{omega}-nitro-L-arginine methyl ester; autoregulation; blood pressure

IF THEY DID NOT INCREASE vascular resistance, relatively small amounts of cell-free hemoglobin could effectively replace blood in hemorrhaged rats (29). Unfortunately increased vascular resistance has limited the effectiveness of most hemoglobin-based oxygen carriers (HBOC). Two primary triggers for HBOC-induced vasoconstriction have been identified: nitric oxide (NO) scavenging and O2-mediated autoregulation. The potent vasconstrictor 20-HETE (1, 6, 28) could be involved in the vascular response to either of these stimuli, because it is known to mediate vasconstriction in response to increased PO2 (12) and decreased NO (16).

The vascular response to NO scavenging by HBOC has been recognized for many years. More recently O2-mediated autoregulatory vasoconstriction by HBOC with low O2 affinity has been postulated (19). By releasing O2 in small arteries and arterioles, low-affinity HBOCs could stimulate 20-HETE production, which is linearly related to PO2 between 20 and 140 mmHg, with half-maximal production at ~60 mmHg in renal arterioles (5). Increased 20-HETE production plays an important role in the response to hyperoxia (20) and accounts for up to 80% of the vasoconstrictor response to raised tissue PO2 in cremaster muscles (4). 20-HETE production is also increased when tonic inhibition by NO and its by-products is reduced by inhibiting NO synthase (1, 6). 20-HETE contributes significantly to the renal vascular response after L-NAME inhibition of NO synthase in Sprague-Dawley rats (6).

The following experiments were designed to examine the role of 20-HETE in the response to an HBOC with low oxygen affinity. This 64-kDa HBOC was found to produce sustained hypertension in Sprague-Dawley rats, whereas a similar HBOC with high oxygen affinity did not (19). Rohlfs et al. (19) concluded that the difference in vascular response was not due to NO scavenging but was more likely to result from differences in O2 delivery. In the following experiments we used Sprague-Dawley and Wistar rats because Wistar rats have less of the cyp4A proteins that produce 20-HETE (23) and lower NO synthesis (9, 14, 17). We anticipated that 20-HETE-mediated vascular responses to scavenging of NO by Tm-Hb were more likely to occur in Sprague-Dawley rats.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Hemosol (Mississauga, ON, Canada) provided the cross-linked tetrameric hemoglobin (prepared in lactated Ringer solution). Endotoxin was <0.06 EU/ml. Tm-Hb [trimesoyl-Hb ({beta}1-{beta}82'); P50 34 mmHg, Hill 2.5] (10, 11, 22) contains 33% {alpha}2{beta}-(Val1)-Tm-(Lys82)-{beta} and 67% {alpha}2{beta}-(Val1,Lys82)-Tm-(Lys82)-{beta}. Tm is the cross-linker trimesic acid. O2 affinity was measured with a Hemox analyzer at 37°C; the parameters measured in this lot differ slightly from those described in our previous publication, where P50 was 35 mmHg. Rohlfs et al. (19) report values of 39 and 2.8. We chose to use Rohlfs' abbreviations in this paper. In our previous publication we used the abbreviation Hb35.

Male Wistar or Sprague-Dawley rats (250–400 g) (Charles River) anesthetized with isoflurane (5% in 30% O2-70% N2 for induction and 0.8–1% isoflurane for maintenance) were placed on a warmed table and their rectal temperature was maintained at 37.5 ± 0.5°C. Anesthesia was continued through a tracheostomy. A carotid artery and jugular vein were cannulated, and the right kidney was exposed through a midline incision. The kidney was covered with tissue soaked in saline or mineral oil. A transonic Doppler flow probe was placed about the renal artery. The rats were infused with isotonic saline containing [3H]inulin (0.75 µCi/ml) and 30 mg/ml glycine at 2% body wt/h. Glycine was included to increase renal blood flow (RBF) and make intrarenal oxygenation and blood flow more like that in human kidneys (7). After 60- to 90-min equilibration, two 15- to 20-min urine collections were made with midpoint arterial blood collections (50 µl). Then 12 ml/kg of blood was exchange transfused with an equal volume of warmed Ringer lactate over 5 min. After 10 min for equilibration and another 15-min urine collection, 6 ml/kg of 70 g/l Tm-Hb was infused at 1 ml·kg–1·min–1, and functional measurements continued for 1 h. Alternatively, 6 ml/kg of 70 g/l BSA was infused with or without 10 mg/kg of N{omega}-nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich Chemicals). Finally arterial and renal venous samples were collected for measurement of O2 content (LexO2Con-K, Lexington Instruments) and hemoglobin content, O2 saturation, and methemoglobin (OSM3, Radiometer Copenhagen). O2 delivery was calculated from the product of arterial O2 and RBF; O2 consumption was calculated from the arterial venous O2 difference times RBF. We assumed that all the filtered Na (155 mM in plasma water) was reabsorbed, thus Na reabsorption/O2 consumption was calculated as glomerular filtration rate (GFR) x 155/[(arterial O2 – O2) x RBF]. In some experiments, one OxyLite fluorescent O2-sensitive probe was inserted into the kidney 2 mm (cortex) and another probe was inserted 4–5 mm (outer medulla). The probes provided statistical mean readings of intrarenal PO2 for a volume of ~0.3 mm3 (Oxford Optronix; Ref. 2). Blood pressure and RBF were recorded with a Powerlab/4SP (ADInstruments). Mean blood pressure was calculated as 1/3 systolic + 2/3 diastolic pressure; RBF was equated with arterial blood flow.

20-HETE synthesis was blocked with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016 Taisho Pharmaceutical, Saitama, Japan; 1 mg/kg iv plus 1 mg·kg–1·h–1 iv) (13). HET0016 was mixed with lecithin and injected as a 10% suspension in saline. To examine the role of 20-HETE production in responses to reduced NO, rats were pretreated with either lecithin vehicle or HET0016 before infusion of L-NAME alone or before bleeding and subsequent infusion of BSA with L-NAME.

The effect of changing PO2 was examined by beginning anesthesia with air instead of 30% O2. After functional measurements were made, the carrying gas was switched to 100% O2 for 30 min and then reverted to air for 30 min. HET0016 was infused and the sequence of air-100% O2-air was repeated.

Sigma Stat and SPSS were used for statistical analysis.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Tm-Hb.   Bleeding dropped hematocrit from 44 ± 1% to 37 ± 1% in Sprague-Dawley rats and from 42 ± 1% to 35 ± 1% in Wistar rats. Tm-Hb infusion decreased hematocrit further (34 ± 1% and 32 ± 0%). All the rats survived to the end of the experiments. At the end of the experiment, plasma hemoglobin was 0.95 ± 0.14 g/l and methemoglobin was 9.1 ± 1.0%. After bleeding, the blood pressure and RBF of the Sprague-Dawley rats were lower than in Wistar rats (Table 1). GFR/RBF was used as an index of efferent arteriolar resistance.


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  		Table 1. Effect of HET0016 on responses to bleeding and Tm-Hb

 
Tm-Hb rapidly increased blood pressure, renal vascular resistance (RVR), and GFR, whereas RBF decreased (Table 1, Fig. 1, left).



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  		Fig. 1. Effect of trimesoyl-Hb (Tm-Hb) on blood pressure and renal vascular resistance (RVR) expressed as percent of baseline reading at the end of the period after bleeding. Continuous recordings were averaged for 1-min intervals for each experiment. Baseline was established as the mean of 1-min recordings for 6 min before beginning the Tm-Hb injection. Left: {blacksquare}, Sprague-Dawley with Tm-Hb (n = 11); {triangleup}, Wistar with Tm-Hb (n = 7); {circ}, Wistar with BSA (n = 6). Right: results after pretreatment with HET0016: {blacksquare}, Sprague-Dawley with Tm-Hb (n = 7); {triangleup}, Wistar with Tm-Hb (n = 6). Means ± SE.

 
BSA with and without L-NAME.   Infusion of BSA (n = 6) instead of Tm-Hb into Wistar rats increased blood pressure by 4 ± 3% (Fig. 1, left), decreased hematocrit (34 ± 0% to 31 ± 1%), and decreased RVR (11 ± 4%; P = 0.06; n = 6; Fig. 1). BSA increased GFR by 20 ± 8% (P = 0.03), which was not different from the change produced by Tm-Hb. BSA did not alter GFR/RBF. Rats were infused with BSA + L-NAME to reduce NO production as blood volume was expanded with a protein solution. This raised blood pressure and RVR dramatically, whereas RBF fell equally in both Sprague-Dawley and Wistar rats (Table 2; Fig. 2).


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  		Table 2. Effect of HET0016 on responses to bleeding and BSA + L-NAME

 


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  		Fig. 2. Comparison of HET0016 effects on responses to Tm-Hb and BSA+N{omega}-nitro-L-arginine methyl ester (L-NAME). Bars show the percent change in blood pressure, RVR, renal blood flow (RBF), and glomerular filtration rate (GFR)/RBF in the 15-min period 10 min after beginning the intravenous infusion of Tm-Hb. Baseline values were obtained for the 15-min period beginning 10 min after completion of the exchange transfusion of Ringer lactate for blood (1.2% body wt). Number of experiments shown in Tables 1 and 2. Results for Sprague-Dawley rats shown on left and Wistar rats on right. First 2 columns for each rat strain show the response to Tm-Hb and the second 2 columns show the response to BSA+L-NAME. Superscripts indicate the results of ANOVA and unpaired t-tests comparing control with HET0016-treated rats. Means ± SD.

 
HET0016.   To explore the role of 20-HETE in responses to TM-Hb we used the potent and selective inhibitor HET0016. This inhibitor blocks cytochrome P-4504A, which produces 20-HETE in rat renal microsomes, with an IC50 of 35 nM. HET0016 has much less effect on production of epoxyeicosatetrienoic acids (IC50 2,800 nM) or cyclooxygenase activity (13). Table 1 compares the effect of HET0016 on the responses to bleeding and Tm-Hb. HET0016 blunted the effect of Tm-Hb on blood pressure and RVR, GFR, and GFR/RBF in Sprague-Dawley rats (Table 1, Fig. 1, right, and Fig. 2). In contrast, HET0016 did not significantly alter the effect of Tm-Hb on blood pressure of Wistar rats but exaggerated the fall in RBF (P = 0.03) and the rise in RVR (P = 0.07). HET0016 decreased the Tm-Hb-induced rise in GFR/RBF of Sprague-Dawley rats, whereas it tended to increase the effect on GFR/RBF in Wistar rats. Two-way ANOVA indicates significant interaction between HET0016 and rat strain for GFR/RBF (P = 0.008), GFR (P = 0.003), and possibly for blood pressure as well (P = 0.08).

HET0016 had a strain-specific effect on blood pressure after infusion of BSA+L-NAME (Table 2, Fig. 2; ANOVA P = 0.004). Inhibiting 20-HETE production reduced the effect on blood pressure and perhaps also on RVR in Sprague-Dawley rats but not in Wistar rats. However, the small changes in RBF and GFR/RBF were not significant.

L-NAME, given without prior bleeding or protein infusion, increased blood pressure and RVR to a similar extent in Sprague-Dawley and Wistar rats, whereas it decreased GFR in Sprague-Dawley (P = 0.01) but not Wistar rats (Table 3). HET0016 removed the effect of L-NAME on the GFR of Sprague-Dawley rats and significantly diminished the effect on their blood pressure and RVR. HET0016 did not alter the response to L-NAME significantly in Wistar rats. After L-NAME, GFR/RBF increased proportionately less in Sprague-Dawley rats (ANOVA P = 0.02) but HET0016 did not alter the change.


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  		Table 3. Effect of HET0016 on responses to L-NAME

 
Oxygen delivery and PO2.   To examine the effect of arterial PO2 on renal function, we switched rats from breathing air to breathing 100% O2. This immediately increased outer cortical PO2 from 10 ± 2 to 29 ± 8 mmHg and outer medullary PO2 from 30 ± 8 to 56 ± 20 mmHg (Sprague-Dawley n = 4). Blood pressure and RVR increased and RBF decreased (Table 4). GFR/RBF increased significantly in Sprague-Dawley rats but not in Wistar rats. ANOVA revealed that RBF decreased (P = 0.026) and RVR increased (P = 0.013) more in Sprague-Dawley than in Wistar rats.


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  		Table 4. Effect of HET0016 on changes in Renal hemodynamics produced by hyperoxia

 
The role of increased O2 delivery in responses to Tm-Hb was examined by measuring intrarenal PO2 and O2 consumption. During infusion of Tm-Hb, PO2 fell in the cortex and outer medulla and then gradually increased (Fig. 3). The decrease was more pronounced in the outer medulla of Wistar rats. BSA infusion raised PO2 in the cortex (43%) and medulla (20%) (n = 4) of Wistar rats. For the same time period, Tm-Hb had no effect on cortical PO2 (–1%) in Wistar rats and decreased their medullary PO2 by 33%. Arterial and renal venous blood was drawn at the end of each experiment for calculation of O2 delivery and consumption. Arterial O2 content was significantly lower after BSA than after Tm-Hb administration (Table 5, P = 0.02). Nonetheless, because RBF increased with BSA and decreased with Tm-Hb, renal O2 delivery was 60% greater in Wistar rats that received BSA than in those that received Tm-Hb (P = 0.04). O2 consumption was not different, nor was Na transport/O2 consumption. HET0016 did not alter O2 delivery or O2 consumption.



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  		Fig. 3. Effect of Tm-Hb on PO2 measured with Oxylite probes inserted 2 mm (Cortex) and 4 mm (outer medulla) into the kidney. Results are expressed as percent of baseline readings at the end of the period after bleeding. {blacksquare}, Sprague-Dawley (n = 5); {triangleup}, Wistar (n = 7); {circ}, Wistar BSA (n = 6). Means ± SD. Continuous recordings were averaged for 1-min intervals for each experiment. Baseline was established as the mean of 1-min recordings for 6 min before beginning the Tm-Hb injection.

 

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  		Table 5. Oxygen delivery and consumption

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
{omega}-Hydroxylases that produce the vasoconstrictor 20-HETE are stimulated by PO2 between 20 and 140 mmHg (5) and inhibited by NO (1). HBOCs such as Tm-Hb can scavenge NO and raise O2 in small arteries and arterioles that produce 20-HETE (8, 19, 27). Increased O2 and decreased NO acting either alone or synergistically could stimulate the conversion of arachidonate to 20-HETE. This poses two questions. First, does increased 20-HETE production contribute to the vasoconstrictor response to Tm-Hb? Second, is 20-HETE-mediated vasoconstriction initiated by increased PO2 or decreased NO? The first answer is yes; 20-HETE did contribute to the systemic and renal vasoconstrictor responses to Tm-Hb, but only in Sprague-Dawley rats (Table 1, Figs. 1 and 2). The answer to the second question is more complex.

Let us first consider the connection between NO and 20-HETE. Others have shown that 20-HETE contributes part of the hemodynamic response to lowered NO concentration in Sprague-Dawley rats (16). In our experiments, blocking 20-HETE production blunted the renal and systemic vasoconstrictor effects of L-NAME in Sprague-Dawley but not in Wistar rats (Tables 2 and 3, Fig. 2). Several observations suggest that Sprague-Dawley rats are more likely to increase 20-HETE production when NO concentration is reduced. 20-HETE production by isolated perfused Sprague-Dawley kidneys was 0.9 ng/min but only 0.33 ng/min from Wistar kidneys (17), which is consistent with the presence of more CYP4A in Sprague-Dawley renal microsomes (23). Inhibition of NO synthase increased efflux of 20-HETE from both Wistar and Sprague-Dawley kidneys with peak levels of 3.6 ng/min from Sprague-Dawley rats and but only 1.4 from Wistar rats (17). However, subsequent investigations suggest that the rise in 20-HETE release during isolated perfusion may have been a time-dependent phenomenon unrelated to inhibition of NO synthase (18). Sprague-Dawley alveolar macrophages produce 10 times more NO than Wistar macrophages when stimulated by lipopolysacharide (9) and NO contributes to nonadrenergic noncholinergic relaxation significantly more in Sprague-Dawley intestine than in Wistar intestine (14, 15). These observations suggest that 20-HETE production could be more sensitive to removal of NO in Sprague-Dawley than in Wistar rats. Lower 20-HETE production or less tonic inhibition by NO may explain the lack of a significant renal and systemic response to HET0016 in Wistars.

The response to BSA+L-NAME was qualitatively similar to that seen with Tm-Hb (Fig. 2, Table 2), although the increase in blood pressure and RVR was greater with BSA+L-NAME. Vascular responses were blunted by HET0016 in Sprague-Dawley but not in Wistar rats. A similar strain-specific response to HET0016 was found when L-NAME was used without prior bleeding or BSA infusion (Table 3). The similarity between responses to BSA+L-NAME and Tm-Hb is consistent with reduced NO concentration as a common vasoconstrictor mechanism. The rat strain specificity of HET0016 responses to both BSA+L-NAME and Tm-Hb support this conclusion.

Changes in RVR could be simply an autoregulatory response to altered arterial blood pressure. A rise in blood pressure should trigger an equal rise in RVR, with no change in RBF, if autoregulation is perfect. This was the case with Tm-Hb (Figs. 1 and 2) but the increase in RVR with L-NAME was greater than the rise in arterial pressure (Table 2, Fig. 2). Treeck and Aukland (24) observed a similar exaggerated effect of L-NAME on RVR of Sprague-Dawley rats. By constricting the aorta to maintain constant renal perfusion pressure they revealed that roughly one-half of the rise in RVR was due to preglomerular autoregulation (24). The other one-half appeared to be due to increased postglomerular resistance (RE). They estimated changes in RE by reasoning that it is proportional to glomerular capillary blood pressure (PC) divided by RBF, assuming that venous pressure is ~0. Because GFR is proportional to PC it follows that RE {approx} GFR/RBF. This relationship holds if glomerular surface area x permeability (kf), protein oncotic pressure, and proximal tubular pressure are constant. Applying Treeck and Aukland's analysis to our data showed that in Sprague-Dawley rats BSA+L-NAME increased RE more than L-NAME (54 ± 31% vs. 21 ± 17%; P = 0.02) but there was no significant difference in the effect in Wistar rats (37 ± 20% and 39 ± 28%). HET0016 did not significantly alter the responses to either L-NAME alone or BSA+L-NAME. These results indicate that when NO production is inhibited in Sprague-Dawley rats, 20-HETE contributes to increased systemic blood pressure and increased preglomerular RVR but possibly not to the increase in RE.

Tm-Hb increased RE in Wistar and Sprague-Dawley rats. HET0016 had no effect on RE increases in Wistars but significantly decreased the effect of Tm-Hb on RE in Sprague-Dawley rats (Fig. 2). In contrast, HET0016 did not alter the effect of BSA+L-NAME on RE, which suggests that Tm-Hb increased RE by an HET0016-sensitive mechanism not related to NO concentration. This might be evidence that O2-mediated autoregulation is responsible for some of the increase in the RE of Sprague-Dawley rats.

We addressed the question of Tm-Hb initiating O2 autoregulation mediated by 20-HETE by examining the relationship between intrarenal PO2 and vascular resistance during hyperoxia and after infusions of Tm-Hb or BSA. Hyperoxia, which raised cortical PO2 threefold and medullary PO2 almost twofold, increased RVR in both rat strains (Table 4). The estimated RE increased significantly in Sprague-Dawley but not in Wistar rats. The RVR of Sprague-Dawley rats appears to be more sensitive to hyperoxia. HET0016 had no significant effect on the renal responses to hyperoxia but there was a significant rise in blood pressure of Sprague-Dawley rats. Understanding this unexpected observation would require additional information about cardiac output and systemic vascular resistance, which we do not have. We cannot determine how HET0016 altered systemic vascular responses to hyperoxia because we did not measure cardiac output. In conscious hamsters, hyperoxia reduced cardiac output, microvascular blood flow, and functional capillary density in a skin fold (25). In conscious Wistar rats, hyperoxia had little effect on blood pressure and increased RVR insignificantly (4 ± 5%) in both cortex and outer medulla (3). We can conclude that hyperoxia increases RVR but that the magnitude of the effect is small compared with the rise produced by Tm-Hb. Furthermore the hyperoxic response does not appear to be mediated primarily by 20-HETE in either rat strain.

Although hyperoxia raised arterial and intrarenal PO2 dramatically, it did not substantially increase arterial O2 content because hemoglobin was already >96% saturated and dissolved O2 contributes very little to total O2 content. Tm-Hb increased arterial O2 content relative to that in rats that received BSA (Table 5). Tm-Hb reduced RBF, whereas BSA increased RBF. The net effect was that BSA increased O2 delivery by 60% and also raised cortical and medullary PO2 (Fig. 3). The higher PO2 is not surprising given that O2 consumption, which is linked to GFR, was not different between BSA- and Tm-Hb-treated Wistar rats, although O2 delivery was 60% higher with BSA. Despite higher O2 delivery and higher average intrarenal PO2 RVR decreased with BSA. Clearly neither O2 delivery nor average cortical and outer-medullary PO2 can explain the changes in RVR. However, because Oxylite probes reflect an average PO2 for 0.3 mm3 of tissue, we cannot rule out the possibility that Tm-Hb increased PO2 in arcuate arteries and radial (interlobular) arteries while decreasing delivery to the peritubular capillary network (26).

NO- rather than O2-mediated vasoconstriction probably accounts for the vascular response to Tm-Hb for the following reasons. The vasoconstrictor response to hyperoxia is much smaller than the response to Tm-Hb or L-NAME. HET0016 did not appreciably alter the renal responses to hyperoxia but did alter responses to Tm-Hb and L-NAME. Tm-Hb, which lowered intra-renal PO2, was associated with vasoconstriction. BSA infusions, which greatly increased O2 delivery and intrarenal PO2 were associated with vasodilation.

Our experiments differ from those of Rohlfs et al. (19), who exchanged 50% of conscious rats' blood volume for an equal volume of Tm-Hb. This produced a 20% rise in blood pressure, which increased more during the 30-min observation period. In our experiments, 20% of blood volume was first exchanged with Ringer solution to simulate the effect of hemorrhage and fluid therapy. We then infused Tm-Hb equivalent to 10% of blood volume. Compared with baseline blood pressure before hemorrhage, Tm-Hb increased blood pressure by 23%, which is similar to the rise observed by Rohlfs et al. (19).

In summary, NO scavenging by Tm-Hb was primarily responsible for 20-HETE-mediated vasoconstriction in Sprague-Dawley rats, but we cannot rule out a small role for O2 stimulated 20-HETE production. Nor can we rule out some O2-mediated autoregulation independent from 20-HETE production. 20-HETE did not participate appreciably in the vasoconstrictor responses to either Tm-Hb or L-NAME in Wistars possibly because they have lower NO production and less 20-HETE production. HBOC-induced vasoconstriction is mediated by a number of factors including angiotensin, endothelin, thromboxane, and the sympathetic nervous system. 20-HETE contributes to the vasoconstrictor response to not only NO ablation but also to angiotensin and endothelin in Sprague-Dawley rats. Our observations indicate that the balance among these vasoactive factors differs between Sprague-Dawley and Wistar rats with significant differences in the impact on glomerular hemodynamics.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by a grant from the Canadian Blood Services/Canadian Institutes of Health Research Partnership in Transfusion Science.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Gord Adamson at Hemosol, Toronto, Canada, for providing Tm-Hb and Dr. Mariko Sato at Taisho Pharmaceutical, Saitama, Japan, for providing the HET0016.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Baines, Dept of Laboratory Medicine and Pathobiology, Univ. of Toronto, 100 College St., Rm. 408, Toronto, Ontario M5G 1L5 (E-mail: andrew.baines{at}utoronto.ca)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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