AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 275: H1313-H1321, 1998;
0363-6135/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Asano, Y.
Right arrow Articles by Bucci, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asano, Y.
Right arrow Articles by Bucci, E.
Vol. 275, Issue 4, H1313-H1321, October 1998

Effect of cross-linked hemoglobin transfusion on endothelial-dependent dilation in cat pial arterioles

Yoshio Asano1, Raymond C. Koehler1, John A. Ulatowski1, Richard J. Traystman1, and Enrico Bucci2

1 Department of Anesthesiology/Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore 21287; and 2 Department of Biological Chemistry, The University of Maryland at Baltimore School of Medicine, Baltimore, Maryland 21201

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We determined whether addition of hemoglobin to the plasma would inhibit endothelial-dependent dilation in brain where tight endothelial junctions limit hemoglobin extravasation. Pial arteriolar diameter was measured by intravital microscopy through closed cranial windows in anesthetized cats either without transfusion (hematocrit = 32%) or after exchange transfusion with an albumin or sebacyl-cross-linked human hemoglobin solution (hematocrit = 18%). Dilation of small, medium, and large arterioles to acetylcholine and ADP was not significantly altered by hemoglobin transfusion. The dilatory responses were inhibited by the nitric oxide synthase inhibitor NG-nitro-L-arginine, although significant dilation to 30 µM acetylcholine persisted in small arterioles in the control and albumin-transfused group but not in the hemoglobin-transfused group. The dilatory response to the nitric oxide donor 3-morpholinosydnonimine was unaffected by albumin or hemoglobin transfusion, but the response to nitroprusside was reduced by one-third after hemoglobin transfusion. When cross-linked hemoglobin was superfused through the cranial window, the acetylcholine response became inhibited at a hemoglobin concentration of 0.1 µM and was completely blocked at 10 µM. Because this concentration is substantially less than the 500 µM hemoglobin concentration in plasma after transfusion when there was no inhibition of the acetylcholine response, hemoglobin permeation of the blood-brain barrier was considered negligible. We conclude that exchange of red cell-based hemoglobin with plasma-based hemoglobin does not produce a more effective sink for endothelial-derived nitric oxide evoked by agonist receptor-mediated activation. Furthermore, decreased hematocrit does not affect agonist-evoked endothelial-dependent dilation.

acetylcholine; adenosine 5'-diphosphate; anemia; cerebral blood vessels; nitric oxide

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE USE OF STROMA-FREE hemoglobin as a plasma volume expander with O2-carrying capacity has been of clinical interest for many years (3). Use of intramolecular and intermolecular cross-links to prevent dimerization of tetrameric hemoglobin and to maintain an O2 affinity similar to red cell-based hemoglobin has led to the development of several products that have undergone clinical trials (17, 32). However, detailed knowledge of the physiological effects of cell-free hemoglobin transfusion is rather limited. Transfusion of tetrameric cross-linked hemoglobin produces hypertension in a variety of species including humans (32). The hypertension has been attributed to scavenging of nitric oxide (NO) in peripheral vascular beds (35, 39), such as kidney and intestines which do not have tight endothelial junctions, and to increased endothelin activation (14). However, transfusion of polymeric hemoglobin, which is expected to extravasate slowly, also produces a rapid increase in arterial pressure (1), thereby implying that scavenging of NO by hemoglobin may occur in the plasma space.

The issue of whether erythrocytic or plasma-based hemoglobin scavenges physiologically significant amounts of endothelially derived NO is unclear. Kinetic modeling indicates that hemoglobin is capable of influencing the concentration gradient of freely diffusible NO at a distance of several cell diameters (25). However, nitrosohemoglobin is not normally detectable in red cell-based hemoglobin, but this may be related to conversion of nitrosohemoglobin to methemoglobin and to the low molar concentration of NO relative to hemoglobin. Moreover, millimolar concentrations of glutathione in endothelium may limit loss of NO to either red cell- or plasma-based hemoglobin, and glutathione in red blood cells may further limit access of NO to heme or cysteine groups on erythrocytic hemoglobin (27). Alternatively, cell-free hemoglobin comes into more intimate contact with the endothelial glycocalyx and could provide a more effective sink for NO than erythrocytic hemoglobin. In support of this possibility, both polymeric and unmodified free hemoglobin inhibit acetylcholine-induced relaxation of aortic strips at a lower hemoglobin concentration than red cell- or liposome-encapsulated hemoglobin (30). Thus it is unclear whether plasma-based hemoglobin acts as a sink for NO sufficient to impair endothelial-dependent dilation in vivo.

In the present study, we tested the hypothesis that exchange of erythrocytic hemoglobin with plasma-based hemoglobin inhibits dilation of pial arterioles to acetylcholine and ADP. Pial arterioles offer the advantage of tight endothelial junctions to limit hemoglobin extravasation. Moreover, acetylcholine and ADP are known to be both endothelial-dependent (5, 33, 34) and NO-dependent (28, 43) dilators in cerebral vessels. Responses to acetylcholine and ADP were measured before and after application of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) to demonstrate NO dependency. Responses to the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside also were measured to determine if any attenuation of the acetylcholine and ADP responses was due to an inability to fully activate the guanylate cyclase pathway. Because reduction of hematocrit could reduce the viscous influence on basal shear wall stress and basal NO production, the acetylcholine response was evaluated after exchange transfusion with an albumin solution. Finally, the minimum concentration of cross-linked hemoglobin in the cerebrospinal fluid (CSF) required for inhibiting the acetylcholine response was measured to demonstrate inhibitory efficacy of the modified hemoglobin.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

All procedures were approved by the Animal Care and Use Committee of The Johns Hopkins University. Forty-four male cats (2.1-4.0 kg) were anesthetized with pentobarbital sodium (40 mg/kg ip), orally intubated, and mechanically ventilated with ~25% O2. Femoral arteries and veins were catheterized, and pentobarbital sodium (6 mg · kg-1 · h-1) and lactated Ringer solution (4 ml · kg-1 · h-1) were continuously infused intravenously. Pancuronium bromide (0.1 mg/kg) was injected intravenously to provide muscle relaxation. A cranial window was constructed over the parietal cortex (8). A circular craniotomy (12 mm) was made, and a plastic ring with three side ports and a thermistor was cemented to the skull around the craniotomy. The ring was filled with artificial CSF, and the dura mater was carefully incised and retracted. A glass coverslip was cemented to the ring. Pressure and temperature of fluid in the window, arterial blood pressure, and rectal temperature were continuously monitored. Rectal temperature was maintained at ~38.5°C with a warm water-perfused water blanket underneath the cat. Window temperature was maintained at ~37.5°C with a heat lamp. Drugs dissolved in warmed, artificial CSF were infused through one side port of the window at a rate of 1 ml/min while the height of the outflow tubing was adjusted to maintain a constant intracranial pressure of 6 mmHg. The artificial CSF was bubbled with 6% O2-6% CO2-88% N2. The constituents of the CSF were (in mM) 151 Na+, 3 K+, 1.3 Ca2+, 0.6 Mg2+, 134 Cl-, 25 HCO-3, 6 urea, and 3.7 glucose.

Internal diameter of pial arterioles was measured by intravital microscopy using a Zeiss axiohead microscope, a Hamamatsu charge-coupled device camera, a Panasonic super videocassette recorder, and a Sony high-resolution monochromatic video display. At each measurement time, images were recorded at several different sites. During playback, diameter was measured at 10-15 arteriolar segments in each cat. Segments were grouped by initial diameters of <50 µm (small), 50-100 µm (medium), and >100 µm (large). The percent change in diameter with each intervention was calculated individually for each arteriolar segment. The percent responses of all arterioles within a particular size group were averaged for each cat. Statistics were performed on each size group using a single value for each cat (i.e., n = number of cats).

Lysine residues of human hemoglobin were intramolecularly cross-linked by the reagent bis(3,5-dibromosalicyl)sebacate to produce a stabilized tetrameric hemoglobin with a partial pressure of O2 at 50% O2 saturation of 34 mmHg as previously described (6).

Experiment 1: dose-dependent effect of abluminal cross-linked hemoglobin superfusion on acetylcholine dilation. In one group of cats (n = 6), the diameter was measured at baseline and 5 min after superfusion with 30 µM acetylcholine for 5 min. After washout of acetylcholine, the window was superfused with 10-8 M cross-linked hemoglobin for 5 min followed by 30 µM acetylcholine in artificial CSF containing 10-8 M hemoglobin. Changes in baseline diameter and in the response to acetylcholine were repeated with stepwise increasing concentrations of cross-linked hemoglobin (10-7, 10-6, and 10-5 M). Artificial CSF without hemoglobin was superfused after each acetylcholine challenge to restore baseline diameter. To control for any potential effect of time or tachyphylaxis on repeated acetylcholine challenges, an additional time control group (n = 4) was studied. After an initial superfusion with 30 µM acetylcholine, artificial CSF was superfused four times followed each time with 30 µM acetylcholine in CSF without hemoglobin over a time period equivalent to the experimental group.

Experiment 2: acetylcholine dilation after hemoglobin transfusion. After baseline measurements of diameter and blood gases were obtained, vasodilatory reactivity to CO2 was tested by ventilating with 5% CO2 for 3 min. Cats then were divided into three groups: a control group (n = 7), an albumin-transfused group (n = 7), and a hemoglobin-transfused group (n = 8). The control group received a continuous intravenous infusion of lactated Ringer solution (4 ml · h-1 · kg-1) throughout the experiment. The albumin and hemoglobin groups underwent an isovolumetric exchange transfusion with either a 5% albumin or 6% cross-linked hemoglobin solution. These solutions had equivalent oncotic pressure. The solutions were infused intravenously at a rate of 1.7 ml/min while arterial blood was withdrawn simultaneously until hematocrit was reduced to ~17-18%. The exchange transfusion required ~45 min. Thereafter, a small maintenance infusion was used to keep hematocrit constant. New baseline measurements were obtained 30 min after the end of the exchange transfusion (75 min after the CO2 challenge in the control group). Acetylcholine (30 µM) was superfused for 5 min, and the diameter was measured 5 min later. After acetylcholine was washed out, the diameter response to the NO donor SIN-1 (1 µM) was measured. Next, L-NNA (300 µM) was superfused in the window for 5 min, and a new baseline was obtained 30 min later. The diameter responses to 30 µM acetylcholine and 1 µM SIN-1 were repeated.

Experiment 3: ADP dilation after hemoglobin transfusion. Cats were divided into two groups: a control group (n = 6) and a hemoglobin-transfused group (n = 6). The experimental protocol was identical to that of experiment 2 except that ADP (100 µM) was used in place of acetylcholine, and sodium nitroprusside (10 µM) was used in place of SIN-1. A group with albumin transfusion was not added to this experiment because no differences in the dilator responses were found among groups in experiment 2.

Statistical analysis. In experiment 1, changes in diameter over time and with increasing hemoglobin concentration were determined by ANOVA with repeated measures and the Newman-Keuls multiple-range test. In experiments 2 and 3, percent changes in diameter for each intervention were analyzed by two-way ANOVA with transfusion groups as a between-subject factor and vessel size as a within-subject factor. If the group treatment effect or the group-size interaction was significant, comparisons were made among groups for each size vessel by one-way ANOVA and the Newman-Keuls test. If the size effect or the group-size interaction was significant, percent changes among different size vessels within each group were compared by ANOVA and the Newman-Keuls test. A significance level of 0.05 was used in all tests. Values are presented as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Hemoglobin superfusion. Superfusion of cross-linked hemoglobin caused a small, dose-dependent constriction of pial arterioles over the 10-8 to 10-5 M range (Fig. 1). Higher concentrations of hemoglobin in the CSF interfered with imaging of the vessels. The percent changes in diameter were greatest in the small arterioles. Constriction became significant in small and medium arterioles at 10-8 M hemoglobin and in large arterioles at 10-5 M hemoglobin. In time controls, repeated superfusion with artificial CSF had no significant effect on diameter.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   Percent change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles during 4 periods of artificial cerebrospinal fluid (CSF) superfusion in a time control group (left, n = 4 cats) and during 4 periods of superfusion of artificial CSF containing stepwise increasing concentrations of sebacyl-cross-linked hemoglobin (Hb) (right, n = 6 cats).

The percent increase in diameter to 30 µM acetylcholine was calculated from the new baseline at each concentration of cross-linked hemoglobin superfusion. The dilator response to acetylcholine became attenuated in all size arterioles with coadministration of 10-7 M hemoglobin (Fig. 2). Dilation was completely blocked at 10-5 M hemoglobin. In time controls, the dilatory response was not attenuated with five exposures to acetylcholine.


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2.   Percent change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured at 5 min of 30 µM acetylcholine (ACh) superfusion. Responses measured during 5 ACh exposures in time control group (left, n = 4 cats) and with increasing concentrations of hemoglobin in superfusate (right, n = 6 cats).

Acetylcholine and SIN-1 responses after hemoglobin transfusion. Arterial hematocrit was 32 ± 1.3% in the time control group and was decreased to 18 ± 0.2 and 18 ± 0.3% after albumin and cross-linked hemoglobin exchange transfusion, respectively. Total arterial hemoglobin concentration and O2 content decreased after transfusion, but the decreases were less in the group transfused with cell-free hemoglobin (Table 1). The percent of methemoglobin in arterial blood was 3.4 ± 0.2% after transfusion of the hemoglobin solution. Arterial blood gases and pH were maintained at similar levels among groups. There were no differences among the control, albumin, and hemoglobin groups in arterial glucose concentration (7.3 ± 0.6, 9.4 ± 2.0, and 7.6 ± 0.9 mM, respectively), plasma osmolarity (304 ± 5, 321 ± 3, and 322 ± 7 mosM, respectively), or urine osmolarity (587 ± 39, 555 ± 44, and 508 ± 64 mosM, respectively). Mean arterial blood pressure increased by 20 mmHg after hemoglobin transfusion and was unchanged in the other groups (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Arterial blood gas and blood pressure measurements during acetylcholine and SIN-1 reactivity assessment after transfusion

Exchange transfusion with albumin resulted in ~10% dilation of pial arterioles, whereas hemoglobin transfusion resulted in approximately a 10% constriction (Fig. 3). There were no significant changes in the time control group.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 3.   Percent change in baseline diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured 30 min after exchange transfusion with an albumin (n = 7 cats) or hemoglobin (n = 8 cats) solution and at an equivalent time without transfusion (control group, n = 7 cats) used in experiment 2 (left) and in another control group (n = 6 cats) used in experiment 3 (right). * P < 0.05 from respective control group. dagger  P < 0.05 between albumin and hemoglobin groups.

Diameter measured 30 min after topical application of 300 µM L-NNA decreased in all size vessels in the control group (Fig. 4). Unexpectedly, size-dependent dilation occurred in the albumin and hemoglobin-transfused groups. The percent dilation was greatest in the small arterioles. Most of the changes in diameter in all groups were present at 10 min after L-NNA application, and there were no differences between the 20- and 30-min values. There were no changes in blood pressure or blood gases during this time that would account for differences in the response to L-NNA among groups (Table 1). Because diameter values in the albumin group were greater than those in the hemoglobin group before L-NA application, the actual diameter values in the albumin group remained greater than those in the hemoglobin group after L-NNA application.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4.   Change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured 30 min after topical application of 300 µM NG-nitro-L-arginine (L-NNA). Left: reference baseline was obtained after washout and recovery from first 3-morpholinosydnonimine (SIN-1) application (experiment 2) in a control group that did not undergo transfusion (n = 7 cats) and in albumin-transfused (n = 7 cats) and hemoglobin-transfused (n = 7 cats) groups. Right: reference baseline was obtained after washout and recovery from first nitroprusside application (experiment 3) in a nontransfused control group (n = 6 cats) and in a hemoglobin-transfused group (n = 6 cats). * P < 0.05 from respective control group.

Application of 30 µM acetylcholine before L-NNA administration in the control group produced greater dilation in small arterioles than in medium and large arterioles (Fig. 5). This size-dependent dilation was not significantly different between the control group and the albumin- or hemoglobin-transfused groups. After application of L-NNA, dilatory responses to acetylcholine were attenuated by 73 and 67% compared with the pre-L-NNA responses in the control and albumin groups, respectively. Significant dilatory responses persisted in the small and medium arterioles. In the hemoglobin group, the dilator response was completely blocked in all size arterioles, and the responses in the small and medium arterioles were significantly less than those in the control and albumin groups.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 5.   Change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured at 5 min of superfusion with 30 µM acetylcholine in a nontransfused control group (n = 7 cats) and in albumin-transfused (n = 7 cats) and hemoglobin-transfused (n = 8 cats) groups. Values are percent change from baseline obtained after transfusion (left) and from baseline obtained 30 min after topical application of 300 µM L-NNA (right). * P < 0.05 from control group. dagger  P < 0.05 from albumin-transfused group.

Application of 1 µM SIN-1 produced greater dilation of small arterioles than large arterioles (Fig. 6). Responses were not different among the three groups. Responses to SIN-1 were not significantly altered by administration of L-NNA in any group.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 6.   Change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured at 5 min of superfusion with 1 µM SIN-1 in a nontransfused control group (n = 7 cats) and in albumin-transfused (n = 7 cats) and hemoglobin-transfused (n = 8 cats) groups. Values are percent change from baseline obtained after transfusion (left) and from baseline obtained 30 min after topical application of 300 µM L-NNA (right).

ADP and nitroprusside responses after hemoglobin transfusion. As in the previous experiment, hemoglobin transfusion resulted in an increase in arterial pressure with no change in arterial pH or blood gases (Table 2), glucose concentration (7.2 ± 1.3 to 7.6 ± 0.9 mM), plasma osmolarity (324 ± 4 to 337 ± 9 mosM), or urine osmolarity (553 ± 83 to 503 ± 6 mosM). Hematocrit was 17 ± 0.1% compared with 31 ± 0.9% in the control group. The changes in arteriolar diameter after hemoglobin transfusion (Fig. 3) and after 300 µM L-NNA application (Fig. 4) were similar to those in the previous experiment.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Arterial blood gas and blood pressure measurements during ADP and nitroprusside reactivity assessment after transfusion

Application of 100 µM ADP produced greater percent dilation in small arterioles than in medium arterioles which, in turn, was greater than that in large arterioles (Fig. 7). The dilatory response in the hemoglobin-transfused group was equivalent to that in the time control group for each size arteriole. After 300 µM L-NNA exposure, responses to ADP were significantly reduced in both groups; ADP did not produce significant dilation except in small arterioles in the control group (5.0 ± 1.8%).


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7.   Change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured at 5 min of superfusion with 100 µM ADP in a nontransfused control group (n = 6 cats) and in a hemoglobin-transfused group (n = 6 cats). Values are percent change from baseline obtained after transfusion (left) and from baseline obtained 30 min after topical application of 300 µM L-NNA (right).

Application of 10 µM sodium nitroprusside elicited greater dilation in small arterioles than in large arterioles in both groups (Fig. 8). The magnitude of dilation in small and large arterioles in the hemoglobin-transfused group was less than the corresponding dilation in the control group. After L-NNA exposure, nitroprusside caused greater dilation of small arterioles than before L-NNA exposure in the control group. In the hemoglobin group, there were no differences between the pre- and post-L-NNA responses.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 8.   Change in diameter of small (<50 µm), medium (50-100 µm), and large (>100 µm) pial arterioles measured at 5 min of superfusion with 10 µM nitroprusside (NTP) in a nontransfused control group (n = 6 cats) and in a hemoglobin-transfused group (n = 6 cats). Values are percent change from baseline obtained after transfusion (left) and from baseline obtained 30 min after topical application of 300 µM L-NNA (right). * P < 0.05 from control group.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The major findings of this study with tetrameric cross-linked hemoglobin solutions are 1) submicromolar concentrations of cross-linked hemoglobin in the CSF were capable of inhibiting pial arteriolar dilation to acetylcholine; 2) near-millimolar concentrations of cross-linked hemoglobin in the plasma had no effect on pial arteriolar dilation to the endothelial-dependent dilators acetylcholine and ADP or to the NO donor SIN-1 and had only a modest attenuation of the nitroprusside response; 3) a L-NNA-resistant dilatory response to acetylcholine was inhibited by plasma-based hemoglobin; and 4) reducing hematocrit with a cross-linked hemoglobin solution resulted in constriction relative to reducing hematocrit with an albumin solution. This study also showed that the percent dilation to endothelial-dependent and -independent agonists was greater in smaller pial arterioles (<50 µm) than in large arterioles (>100 µm).

Hemoglobin has a high affinity for NO that is related to slow dissociation from heme (2). In addition, NO can bind to the beta -93 cysteines on hemoglobin with an affinity that is greater in the oxygenated state (36). In the first experiment, we demonstrated that sebacyl-cross-linked hemoglobin is capable of inhibiting the NO-dependent dilation to acetylcholine when the hemoglobin was placed on the abluminal surface of pial arterioles in vivo. These results are consistent with those showing that fumaryl-cross-linked hemoglobin has a high NO affinity (2) and attenuates acetylcholine-induced relaxation of isolated renal arteries (7). Thus intramolecular cross-linking between the alpha -99 lysines does not interfere with the ability of hemoglobin to inhibit NO-dependent dilation. Moreover, our results indicate that as little as 10-7 M abluminal cross-linked hemoglobin attenuated the acetylcholine response and that 10-5 M concentration completely blocked the response. These concentrations are comparable to the concentrations of non-cross-linked hemoglobin used to attenuate acetylcholine and calcium ionophore relaxation of large arteries in vitro (16, 20, 30).

Our results with cross-linked hemoglobin also show that abluminal application causes dose-dependent constriction of small arterioles in vivo with as little as 10-8 M hemoglobin. In addition to scavenging basal levels of NO, hemoglobin has been postulated to cause contraction of basilar artery by a prostanoid-dependent mechanism at 10-8 M concentration (37). In cell culture, hemoglobin acts directly on smooth muscle to mobilize calcium stores (40). Our observation that abluminal 10-8 M hemoglobin causes constriction without inhibiting acetylcholine dilation is consistent with the possibility that abluminal hemoglobin elicits constriction by a mechanism independent of NO. However, the magnitude of the constriction is small (3% at 10-8 M hemoglobin). Furthermore, acetylcholine-evoked dilation of pial arterioles may involve transfer of NO between endothelium and smooth muscle via a nitrosothiol (24, 41). In this case, a higher concentration of hemoglobin (10-7 M) may be required for lowering interstitial free NO activity sufficiently to drive bound NO from the transferrable thiol group than to simply lower basal NO activity sufficiently to increase basal tone. Whether or not the mechanism of basal vasoconstriction involves NO scavenging, the present results indicate that rather dilute concentrations of hemoglobin in the CSF are capable of causing modest constriction of small pial resistance arterioles in vivo within 5 min of application.

In contrast to abluminal application, results from the second experiment show that exchange transfusion of cross-linked hemoglobin did not inhibit acetylcholine-evoked dilation of pial arterioles. Because plasma-based hemoglobin comes into closer contact with the luminal glycocalyx of the endothelium than red cell-based hemoglobin, we postulated that NO may be scavenged to a greater extent by plasma-based hemoglobin and limit the amount of NO to be transported from the endothelium to the smooth muscle. However, our results imply that plasma-based hemoglobin does not provide a more effective sink for endothelial-derived NO than red cell-based hemoglobin. One explanation for the lack of effect of plasma-based hemoglobin is that activity of free NO in the plasma is already maintained at a low level by red cell-based hemoglobin and that the addition of hemoglobin to the plasma does not substantially increase the NO diffusion gradient from the abluminal-to-luminal endothelial surface. In support of this possibility, switching the perfusate of isolated kidneys from 0 to 1% hematocrit produced an L-NNA inhibitable vasoconstriction that was not augmented by further increases in hematocrit (18). Thus very low hematocrits may be adequate for producing maximum intraluminal NO scavenging consistent with kinetic modeling (25).

If red cell-based hemoglobin normally scavenges most of the NO in blood while permitting acetylcholine-evoked dilation, the question arises of why dilute solutions of topically applied hemoglobin inhibited transfer of NO from endothelium to smooth muscle. Two explanations can be offered. First, a fraction of the topically applied hemoglobin presumably permeates the interstitial space between endothelium and smooth muscle (as assumed for topically applied agonists that act rapidly on the endothelium). Interstitial hemoglobin will impede NO transfer more effectively than intraluminal hemoglobin.

A second explanation is that luminal and abluminal hemoglobin together may scavenge NO more effectively than at either site alone without requiring hemoglobin to permeate the interstitial space. The kinetic analysis of Lancaster (25, 26) indicates that hemoglobin distribution can become more important than hemoglobin concentration when intravascular hemoglobin concentration is in the physiological range. This analysis demonstrates that intravascular concentrations in the micromolar range can theoretically influence the standing concentration gradient of NO in tissue several cell diameters away from the endothelial abluminal surface. However, there was little additional influence of increasing intravascular hemoglobin concentration beyond 0.2 mM, which would explain the lack of effect of exchanging red cell with plasma-based hemoglobin when total hemoglobin is maintained greater than 0.2 mM. Nevertheless, a positive NO gradient from endothelium to smooth muscle persists in the presence of abundant intravascular hemoglobin. Because micromolar concentrations of hemoglobin can influence the NO gradient at a distance of tens of microns, small amounts of hemoglobin distributed at a second locus that is external to the smooth muscle could influence the NO gradient between endothelium and smooth muscle without necessarily permeating the intercellular space between endothelium and smooth muscle. Thus the presence of hemoglobin at both the intraluminal and adventitial surfaces may decrease the endothelium-to-smooth muscle NO gradient more effectively than the presence of hemoglobin at only one surface.

The plasma concentration of hemoglobin achieved by exchange transfusion in this model is ~5 × 10-4 M (39). This concentration is three orders of magnitude greater than that required in the CSF to inhibit the acetylcholine response. Thus the lack of inhibition of the acetylcholine response with near-millimolar concentrations in the plasma indicates negligible permeation of the blood-brain barrier by cross-linked hemoglobin over the acute duration of this experiment. These observations also imply that the hemoglobin solution does not cause acute damage to the barrier. In contrast to the brain, acellular hemoglobin infusion has been reported to shift the coronary acetylcholine response in isolated perfused hearts (29) and in patients undergoing cardiac catheterization (9). This difference is probably related to coronary microvascular permeability of hemoglobin.

Data in the second experiment also indicate that an acute reduction in hematocrit after exchange transfusion with the albumin solution does not alter the response to acetylcholine. Reduction of hematocrit would be expected to decrease endothelial wall shear stress, although increased blood velocity may counteract this effect. If anemia does cause an alteration in shear wall stress, the present data indicate that this alteration does not influence the acetylcholine response. This interpretation is consistent with the concept that shear-sensitive mechanoreceptors and agonist receptors control NOS activity by independent pathways (15, 19).

In addition to testing responses to acetylcholine after hemoglobin transfusion, we evaluated the response to ADP, another endothelial-dependent dilator (5, 33). As with acetylcholine, the pial arteriolar dilator response to ADP was not reduced by hemoglobin transfusion. In the rat, L-NNA inhibits ADP-induced dilation of pial arterioles (28), whereas in the mouse, there are both NOS- and indomethacin-inhibitable components (33). Our results showing near-complete inhibition of the ADP response with 300 µM L-NNA indicate that the ADP response is largely NO dependent in the cat. A cautionary note in this interpretation is that 250 µM L-NNA may inhibit ATP-sensitive potassium channels (23) that may be activated by endothelial-dependent hyperpolarizing factor (4). However, the hyperpolarizing factor does not appear to be prominent in ADP-induced relaxation of rabbit pial arteries (5), thereby suggesting that L-NNA is not acting via direct inhibition of potassium channels. An endothelial and NO-independent component of ADP relaxation has been described at 10-100 µM ADP in isolated rat middle cerebral arteries (44). The lack of a significant L-NNA-resistant component in medium and large arterioles to 100 µM ADP application in the present study might be related to rapid purine uptake in vivo decreasing the effective concentration at smooth muscle purinoreceptors.

Hemoglobin transfusion attenuated pial arteriolar dilation to nitroprusside by ~35% but had no effect on the response to SIN-1. The mechanism for a selective effect on the nitroprusside response is not obvious. Application of receptor agonists such as acetylcholine and ADP may produce localized increases in NO possibly coupled to thiol transport, whereas topical application of nitroprusside and SIN-1, both of which are NO donors that do not require the presence of thiols for guanylate cyclase activation (12, 31), is expected to produce a more diffuse increase in free NO activity. Clearance of NO may depend more heavily on hemoglobin perfusion when there is a diffuse increase in NO throughout the fluid in the window. With hemoglobin in the plasma, the effective surface area for diffusion of NO from the superfusion fluid into capillaries in underlying cortex should increase and could result in increased clearance of NO. Thus NO activity in pial arteriolar smooth muscle after nitroprusside application might be reduced by hemoglobin transfusion. The lack of an effect of hemoglobin transfusion on SIN-1 dilation may be related to either different kinetics of NO release or to the concurrent formation of superoxide anion (13, 21) and possibly peroxynitrite. Superoxide and peroxynitrite cause pial arteriolar dilation via potassium channel activation rather than guanylate cyclase activation (42). Persistent dilation to SIN-1 after hemoglobin transfusion may be related to activation of smooth muscle potassium channels as well as guanylate cyclase activation.

Although 300 µM L-NNA inhibited the response to 30 µM acetylcholine, there was still significant dilation in small and medium-sized arterioles of the control and albumin groups. In contrast, Wei et al. (43) did not observe an L-NNA-resistant component to 0.1 µM acetylcholine in cat pial arterioles of ~100 µm size. Thus the L-NNA-resistant dilator response may be more prominent in small arterioles and may require a higher acetylcholine concentration than that necessary for NOS activation. Potential L-NNA-resistant dilators include an endothelial-dependent hyperpolarizing factor (4) and carbon monoxide (45), which is also scavenged by hemoglobin.

The arteriolar size dependency of endothelial-dependent responses has not been well investigated in pial vessels in vivo. Others have reported no major differences in the percent dilation to acetylcholine or nitroprusside between 60- and 70-µm-diameter arterioles and larger vessels in cat (41) and rat (11). Our results consistently showed greater percent dilation in the small arterioles (<50 µm baseline diameter) for acetylcholine, ADP, SIN-1, and nitroprusside. Similar size dependency to acetylcholine, nitroprusside, and calcium ionophore were observed in halothane-anesthetized cats in this laboratory (8), thereby indicating that the presently obtained results are not specific for pentobarbital sodium anesthesia. These results imply a greater sensitivity of the NOS-guanylate cyclase pathway in small arterioles. Limitations in the total duration of the experimental protocol prevented us from evaluating a full dose-response curve necessary for assessing a 50% effective dose. In addition, we cannot exclude that a diffusion barrier in thicker-walled large arterioles limits bioavailability of topically applied drugs. Nevertheless, our results indicate that small pial arterioles are highly reactive to NO-dependent agonists and are consistent with reactivity of small intracerebral arterioles (10, 22).

With equivalent reductions in hematocrit, we previously found a greater increase in cerebral blood flow after albumin transfusion than after hemoglobin transfusion (38). This difference persisted after systemic administration of nitroarginine methyl ester. The constriction of pial arterioles seen in the present study after hemoglobin transfusion relative to albumin transfusion is consistent with the blood flow results. In addition, the increase in arterial pressure with hemoglobin transfusion requires greater vasoconstriction to maintain a lower level of blood flow. Thus we attribute differences in the diameter response between albumin and hemoglobin transfusion to be related both to greater oxygenation after hemoglobin transfusion and to an autoregulatory response to hypertension, rather than to scavenging of NO.

Although topical application of L-NNA failed to constrict pial arterioles after hemoglobin transfusion, this lack of constriction appears to be unrelated to potential scavenging of endogenous NO by plasma-based hemoglobin because L-NNA failed to constrict arterioles after albumin transfusion. This lack of constriction to L-NNA after both albumin and hemoglobin transfusion suggests that reducing hematocrit decreased basal NO production, possibly by reducing shear stress on the endothelial wall. The paradoxical increase in diameter with L-NNA after albumin and hemoglobin transfusion implies either that NO inhibits another vasodilatory pathway at reduced hematocrit and viscosity or that L-NNA is nonspecific at this dose.

In summary, the present study demonstrates that relatively large amounts of hemoglobin in the plasma space do not interfere with endothelial-dependent dilation in a vascular bed with tight endothelial junctions that limit extravasation. This lack of interference by hemoglobin "wetting" of the endothelial luminal membrane is consistent with the hypothesis that agonist activation of endothelial NOS leads to localized increases in NO that are stored and transferred via thiol interactions distant from the luminal membrane.

    ACKNOWLEDGEMENTS

We thank Lydia Burnett and Candace Berryman for help in preparing the manuscript and Michael O'Hearne for making the hemoglobin solutions.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-48517.

Address for reprint requests: R. C. Koehler, Dept. of Anesthesiology/Critical Care Medicine, The Johns Hopkins University School of Medicine, 600 N. Wolfe St., Blalock 1404, Baltimore, MD 21287-4961.

Received 29 December 1997; accepted in final form 16 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Abassi, Z., S. Kotob, F. Pieruzzi, M. Abouassali, H. R. Keiser, J. C. Frantontoni, and A. I. Alayash. Effects of polymerization on the hypertensive action of diaspirin cross-linked hemoglobin in rats. J. Lab. Clin. Med. 129: 603-610, 1997[Medline].

2.   Alayash, A. I., J. C. Frantontoni, C. Bonaventura, J. Bonaventura, and R. E. Cashon. Nitric oxide binding to human ferrihemoglobins cross-linked between either alpha  or beta  subunits. Arch. Biochem. Biophys. 303: 332-338, 1993[Medline].

3.   Amberson, W. R. Blood substitutes. Biol. Rev. 12: 48-86, 1937.

4.   Brayden, J. E. Membrane hyperpolarization is a mechanism of endothelium-dependent cerebral vasodilation. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H668-H673, 1990[Abstract/Free Full Text].

5.   Brayden, J. E. Hyperpolarization and relaxation of resistance arteries in response to adenosine diphosphate. Distribution and mechanism of action. Circ. Res. 69: 1415-1420, 1991[Abstract/Free Full Text].

6.   Bucci, E., A. Razynska, H. Kwansa, B. Matheson-Urbaitis, M. O'Hearne, J. A. Ulatowski, and R. C. Koehler. Production and characteristics of an infusible oxygen-carrying fluid based on hemoglobin intramolecularly cross-linked with sebacic acid. J. Lab. Clin. Med. 128: 146-153, 1996[Medline].

7.   Cases, A., J. M. Stulak, Z. Katusic, E. Villa, and J. C. Romero. Hemodynamic and renal effects of cross-linked hemoglobin infusion. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R793-R799, 1997[Abstract/Free Full Text].

8.   Clavier, N., J. R. Kirsch, P. D. Hurn, and R. J. Traystman. Effect of postischemic hypoperfusion on vasodilatory mechanisms in cat. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2012-H2018, 1994[Abstract/Free Full Text].

9.   Collins, P., J. Burman, H. I. Chung, and K. Fox. Hemoglobin inhibits endothelium-dependent relaxation to acetylcholine in human coronary arteries in vivo. Circulation 87: 80-85, 1993[Abstract/Free Full Text].

10.   Dacey, R. G., Jr., and J. E. Bassett. Cholinergic vasodilation of intracerebral arterioles in rats. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H1253-H1260, 1987[Abstract/Free Full Text].

11.   Faraci, F. M. Role of endothelium-derived relaxing factor in cerebral circulation: large arteries vs. microcirculation. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1038-H1042, 1991[Abstract/Free Full Text].

12.   Feelisch, M., and E. Noack. Nitric oxide (NO) formation from nitrovasodilators occurs independently of hemoglobin or non-heme iron. Eur. J. Pharmacol. 142: 465-469, 1987[Medline].

13.  Feelisch, M., J. Ostrowski, and E. Noack. On the mechanism of NO release from sydnonimines. J. Cardiovasc. Pharmacol. 14, Suppl. 11: S13-S22, 1989.

14.   Gulati, A., A. C. Sharma, and G. Singh. Role of endothelin in the cardiovascular effects of diaspirin crosslinked hemoglobin. Crit. Care Med. 24: 137-147, 1996[Medline].

15.   Hecker, M., I. Siegle, H. MacArthur, W. C. Sessa, and J. R. Vane. Role of intracellular thiols in release of EDRF from cultured endothelial cells. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H888-H896, 1992[Abstract/Free Full Text].

16.   Hongo, K., H. Ogawa, N. F. Kassell, T. Nakagomi, T. Sasaki, T. Tsukahara, and R. M. Lehman. Comparison of intraluminal and extraluminal inhibitory effects of hemoglobin on endothelium-dependent relaxation of rabbit basilar artery. Stroke 19: 1550-1555, 1988[Abstract/Free Full Text].

17.   Hughes, G. S., E. P. Yancey, R. Albrecht, P. K. Locker, S. F. Francom, E. P. Orringer, E. J. Antal, and E. E. Jacobs, Jr. Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans. Clin. Pharmacol. Ther. 58: 434-443, 1995[Medline].

18.   Imig, J. D., D. Gebremedhin, D. R. Harder, and R. J. Roman. Modulation of vascular tone in renal microcirculation by erythrocytes: role of EDRF. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H190-H195, 1993[Abstract/Free Full Text].

19.   Kanai, A. J., H. C. Strauss, G. A. Truskey, A. L. Crews, S. Grunfeld, and T. Malinski. Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor. Circ. Res. 77: 284-293, 1995[Abstract/Free Full Text].

20.   Kanamaru, K., S. Waga, T. Kojima, K. Fujimoto, and S. Niwa. Endothelium-dependent relaxation of canine basilar arteries. Part 2: inhibition by hemoglobin and cerebrospinal fluid from patients with aneurysmal subarachnoid hemorrhage. Stroke 18: 938-943, 1987[Abstract/Free Full Text].

21.   Kelm, M., R. Dahmann, D. Wink, and M. Feelisch. The nitric oxide/superoxide assay. Insights into the biological chemistry of the NO/O-2· interaction. J. Biol. Chem. 272: 9922-9932, 1997[Abstract/Free Full Text].

22.   Kimura, M., H. H. Dietrich, and R. G. Dacey, Jr. Nitric oxide regulates cerebral arteriolar tone in rats. Stroke 25: 2227-2234, 1994[Abstract].

23.   Kontos, H. A., and E. P. Wei. Arginine analogues inhibit responses mediated by ATP-sensitive K+ channels. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H1498-H1506, 1996[Abstract/Free Full Text].

24.   Kukreja, R. C., E. P. Wei, H. A. Kontos, and J. N. Bates. Nitric oxide and S-nitroso-L-cysteine as endothelium-derived relaxing factors from acetylcholine in cerebral vessels in cats. Stroke 24: 2010-2015, 1993[Abstract/Free Full Text].

25.   Lancaster, J. R., Jr. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc. Natl. Acad. Sci. USA 91: 8137-8141, 1994[Abstract/Free Full Text].

26.   Lancaster, J. R., Jr. Diffusion of free nitric oxide. Methods Enzymol. 268: 31-50, 1996[Medline].

27.   Loscalzo, J. Nitric oxide binding and the adverse effects of cell-free hemoglobins: what makes us different from earthworms (Editorial). J. Lab. Clin. Med. 129: 580-583, 1997[Medline].

28.   Mayhan, W. G. Endothelium-dependent responses of cerebral arterioles to adenosine 5'-diphosphate. J. Vasc. Res. 29: 353-358, 1992[Medline].

29.   Motterlini, R., and V. W. MacDonald. Cell-free hemoglobin potentiates acetylcholine-induced coronary vasoconstriction in rabbit hearts. J. Appl. Physiol. 75: 2224-2233, 1993[Abstract/Free Full Text].

30.   Nakai, K., T. Ohta, I. Sakuma, K. Akama, Y. Kobayashi, S. Tokuyama, A. Kitabatake, Y. Nakazato, T. A. Takahashi, and S. Sadayoshi. Inhibition of endothelium-dependent relaxation by hemoglobin in rabbit aortic strips: comparison between acellular hemoglobin derivatives and cellular hemoglobins. J. Cardiovasc. Pharmacol. 28: 115-123, 1996[Medline].

31.  Noack, E., and M. Feelisch. Molecular aspects underlying the vasodilator action of molsidomine. J. Cardiovasc. Pharmacol. 14, Suppl. 11: S1-S5, 1989.

32.   Przybelski, R. J., E. K. Daily, J. C. Kisicki, C. Mattia-Goldberg, M. J. Bounds, and W. A. Colburn. Phase I study of the safety and pharmacologic effects of diaspirin cross-linked hemoglobin solution. Crit. Care Med. 24: 1993-2000, 1996[Medline].

33.   Rosenblum, W. I., G. H. Nelson, and S. Murata. Endothelial dependent dilation by purines of mouse brain arterioles in vivo. Endothelium 1: 287-294, 1994.

34.   Rosenblum, W. I., G. H. Nelson, and J. T. Povlishock. Laser-induced endothelial damage inhibits endothelium-dependent relaxation in the cerebral microcirculation of the mouse. Circ. Res. 60: 169-176, 1987[Abstract/Free Full Text].

35.   Sharma, A., G. Singh, and A. Gulati. Role of NO mechanism in cardiovascular effects of diaspirin cross-linked hemoglobin in anesthetized rats. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1379-H1388, 1995[Abstract/Free Full Text].

36.   Stamler, J. S., L. Jia, P. Eu, T. J. McMahon, I. T. Demchenko, J. Bonaventura, K. Gernert, and C. A. Piantadosi. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276: 2034-2037, 1997[Abstract/Free Full Text].

37.   Toda, N., M. Kawakami, and K. Yoshida. Constrictor action of oxyhemoglobin in monkey and dog basilar arteries in vivo and in vitro. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H420-H425, 1991[Abstract/Free Full Text].

38.   Ulatowski, J. A., E. Bucci, T. Nishikawa, A. Razynska, M. A. Williams, R. Takeshima, R. J. Traystman, and R. C. Koehler. Cerebral O2 transport with hematocrit reduced by cross-linked hemoglobin transfusion. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H466-H475, 1996[Abstract/Free Full Text].

39.   Ulatowski, J. A., T. Nishikawa, B. Matheson-Urbaitis, E. Bucci, R. J. Traystman, and R. C. Koehler. Regional blood flow alterations after bovine fumaryl-crosslinked hemoglobin transfusion and nitric oxide synthase inhibition. Crit. Care Med. 24: 558-565, 1996[Medline].

40.   Vollrath, B. A. M., B. K. A. Weir, R. L. MacDonald, and D. A. Cook. Intracellular mechanisms involved in the responses of cerebrovascular smooth-muscle cells to hemoglobin. J. Neurosurg. 80: 261-268, 1994[Medline].

41.   Wei, E. P., and H. A. Kontos. H2O2 and endothelium-dependent cerebral arteriolar dilation. Hypertension 16: 162-169, 1990[Abstract/Free Full Text].

42.   Wei, E. P., H. A. Kontos, and J. S. Beckman. Mechanisms of cerebral vasodilation by superoxide, hydrogen peroxide, and peroxynitrite. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H1262-H1266, 1996[Abstract/Free Full Text].

43.   Wei, E. P., R. Kukreja, and H. A. Kontos. Effects in cats of inhibition of nitric oxide synthesis on cerebral vasodilation and endothelium-derived relaxing factor from acetylcholine. Stroke 23: 1623-1629, 1992[Abstract/Free Full Text].

44.   You, J., T. D. Johnson, W. F. Childres, and R. M. Bryan, Jr. Endothelial-mediated dilations of rat middle cerebral arteries by ATP and ADP. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H1472-H1477, 1997[Abstract/Free Full Text].

45.   Zakhary, R., S. P. Gaine, J. L. Dinerman, M. Ruat, N. A. Flavahan, and S. H. Snyder. Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc. Natl. Acad. Sci. USA 93: 795-798, 1996[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 275(4):H1313-H1321
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
X. Qin, H. Kwansa, E. Bucci, S. Dore, D. Boehning, D. Shugar, and R. C. Koehler
Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R498 - R504.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Rebel, S. Cao, H. Kwansa, S. Dore, E. Bucci, and R. C. Koehler
Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin
Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1027 - H1037.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
X. Qin, H. Kwansa, E. Bucci, R. J. Roman, and R. C. Koehler
Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin
J Appl Physiol, January 1, 2006; 100(1): 336 - 342.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Sampei, J. A. Ulatowski, Y. Asano, H. Kwansa, E. Bucci, and R. C. Koehler
Role of nitric oxide scavenging in vascular response to cell-free hemoglobin transfusion
Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1191 - H1201.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
F. M. Faraci, C. Lynch, and K. G. Lamping
Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms
Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2871 - H2876.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
M. Kavdia and A. S. Popel
Contribution of nNOS- and eNOS-derived NO to microvascular smooth muscle NO exposure
J Appl Physiol, July 1, 2004; 97(1): 293 - 301.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Rebel, J. A. Ulatowski, H. Kwansa, E. Bucci, and R. C. Koehler
Cerebrovascular response to decreased hematocrit: effect of cell-free hemoglobin, plasma viscosity, and CO2
Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1600 - H1608.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. M. Bobofchak, T. Mito, S. J. Texel, A. Bellelli, M. Nemoto, R. J. Traystman, R. C. Koehler, W. S. Brinigar, and C. Fronticelli
A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter
Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H549 - H561.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
G. M. T. Hare, C. D. Mazer, W. Mak, R. M. Gorczynski, K. M. Hum, S. Y. Kim, L. Wyard, A. Barr, R. Qu, and A. J. Baker
Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression
J Appl Physiol, May 1, 2003; 94(5): 2058 - 2067.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
B. Matheson, H. E. Kwansa, E. Bucci, A. Rebel, and R. C. Koehler
Vascular response to infusions of a nonextravasating hemoglobin polymer
J Appl Physiol, October 1, 2002; 93(4): 1479 - 1486.
[Abstract] [Full Text] [PDF]