Background and Purpose Tetrahydrobiopterin (THBP) is an essential cofactor for nitric oxide synthase (NOS), which is responsible for the synthesis of the endothelium-derived relaxing factor (EDRF) responsible for mediating the vasorelaxation produced by acetylcholine (ACh). Previous publications suggest that EDRFACh is continuously synthesized and released from the endothelium of mouse pial arterioles. If so, then one may predict that addition of THBP will increase the local production of EDRFACh and produce an endothelium-dependent relaxation that can be blocked by application of a known inhibitor of NOS. This study tests the prediction.
Methods The pial vessels were observed at a continuously suffused craniotomy site by means of intravital television microscopy. The effects of topically applied THBP on diameter were monitored before and after endothelial damage and before and after local treatment with the NOS inhibitor N-guanidino-l-monomethyl arginine (LNMMA). The endothelial damage was produced by a helium-neon laser in the presence of Evans blue dye.
Results A dose-dependent relaxation was produced by 10−3 and 10−2 mol/L THBP. The response was virtually eliminated by endothelial injury. LNMMA 10−6 mol/L also greatly inhibited dilation.
Conclusions The data are consistent with all reports that THBP is a cofactor for constitutive endothelial NOS. The data are consonant with previous results suggesting that EDRFACh is continually synthesized and released. It appears that THBP increases this synthesis and consequently the local level of released EDRFACh. The continuous spontaneous synthesis/release of EDRFACh modulates basal tone and, according to other studies, helps maintain a platelet-free endothelial surface.
Previous in vivo studies of mouse pial arterioles have provided evidence for basal release of the EDRF that also mediates the dilation produced by ACh.1 2 3 This EDRF is derived from l-arginine through the action of NOS, and in some vascular beds this EDRF may simply be NO.4 However, in pial arterioles there is strong evidence that this EDRF is not simply NO, although it appears to contain NO with nitrogen derived from arginine and may be a nitrosothiol.5 6 Moreover, in pial arterioles, unlike the situation found with cultured endothelial cells or with some intact blood vessels, the EDRF for ACh is different from the EDRFs that mediate the responses to bradykinin or to calcium ionophore.5 7 8 9 For these reasons, I have simply used the term EDRFACh to denote the EDRF released by ACh.5
The evidence for spontaneous release of EDRFACh from pial arterioles in vivo is as follows: First, inhibitors of its synthesis will constrict mouse pial arterioles.1 2 Second, EDRFACh is known to inhibit platelet adhesion and aggregation, and inhibitors of its synthesis enhance that adhesion and aggregation.2 Finally, the tendency of platelets to adhere to and aggregate over a site of focal endothelial damage is mitigated by a prior, transient increase in shear.10 Since increases in shear lead to greater release of EDRFACh,11 12 the continued inhibition of adhesion/aggregation has been interpreted to indicate that the increased release of EDRFACh continues beyond the period of increased shear.10
More recently, a cofactor for NOS and for the synthesis of EDRFACh has been identified. This cofactor is THBP.13 14 15 If a vessel is dilated by THBP, this is interpreted to indicate an increase in basal synthesis and release of EDRFACh by the treated vessel. The following study provides such a demonstration for THBP and pial arterioles in vivo. Moreover, using a well-established technique for producing minor endothelial injury in arterioles, we show that the response to THBP is solely dependent on an intact endothelium. We also show for the first time that an established inhibitor of NOS and of EDRFACh synthesis, one that inhibits dilation caused by ACh, also inhibits dilation caused by THBP. Taken all together, these data offer strong additional evidence for basal (ie, non–agonist-induced) synthesis and release of EDRFACh by endothelial cells of mouse pial arterioles. These data strengthen the conclusion of an earlier report16 which showed that the response to THBP was inhibited by an antisense oligonucleotide to the mRNA for NOS and inferred from this that THBP was working through an NOS-dependent mechanism.
Materials and Methods
All procedures were approved by the institutional animal care and use committee. Male ICR mice (Harlan Sprague Dawley) were anesthetized with urethane. A tracheostomy was performed, and the vessels on the surface of the brain (pial vessels) were exposed by craniotomy with stripping of the dura.17 The mice were maintained at 37°C, and the pial surface was suffused with mock cerebrospinal fluid (Elliott's solution18 , at pH 7.35 and 37°C. One pial arteriole was arbitrarily selected for study in each mouse, based solely on having an internal diameter 25 to 45 μm wide over a straight segment at least 100 μm long. The diameter was monitored with a microscope and television attachment coupled to an image splitter19 and recorder. Image splitting permits accurate measurements of changes less than 0.5 μm in size.20 With our epi-illumination system, 20 consecutive measurements of a 10-μm object were made. This had a standard error of only 0.2 μm. With a 10-μm object as a calibration standard, a putative 5-μm object had a mean diameter (20 measures) within 1% (0.05 μm) of the predicted measurement.
The diameter of the arteriole was altered by placing the dilator of interest in the Elliott's solution at the same pH and temperature as the suffusate itself. THBP dihydrochloride was obtained from ICN. When two doses of a dilator were applied, the doses were given cumulatively for 5 minutes each. Changes in diameter were expressed as a percentage of baseline diameter. In experiments in which LNMMA (Sigma) was used, this inhibitor of NOS4 21 was dissolved in the Elliott's solution and suffused for 10 minutes before and continuing during a 2-minute application of 10−3 mol/L THBP.
One set of studies examined the effect of endothelial damage rather than the effect of LNMMA on the response to THBP. The injury was produced in situ by a well-established light/dye technique in which a 6-mW helium neon laser and intravascular Evans blue dye were used.1 2 7 22 The mechanical characteristics of the setup are described in detail in these previous reports. The width of the laser beam at the focal plane was approximately 36 μm. Neither the dye by itself nor the laser by itself produces endothelial dysfunction. Mice were given Evans blue dye 25 mg/kg IV(0.5% solution in normal saline) 30 minutes before laser challenge. Injury to smooth muscle was avoided by reducing the time of exposure to the laser. With the short time (20 seconds) used here, no denudation or ultrastructural damage was recognized21 22 and no platelet adhesion or aggregation23 was seen over the injured area. Only more prolonged exposure results in local platelet adhesion/aggregation, which is recognized as a “white body” when one directly views the vessel through the microscope. Repeated studies (eg, see References 22 and 2422 24 ) show that with short exposure times, endothelium-independent dilators such as SNP, 2-chloroadenosine, and prostacyclin are unaffected, as is the endothelium-independent constrictor UTP.25 Thus, we know that the vascular smooth muscle is unaffected.
To minimize the damaging action of the laser/dye, this set of studies was performed with the suffusate at 23°C22 rather than 37°C. The core temperature of the mouse was still 37°C. A single dose of THBP (10−2 mol/L, the highest dose we used in the dose-response study) was suffused for 2 minutes, on two successive occasions, 15 minutes apart. The second application was 10 minutes after laser injury. If the response to THBP at the injured site was smaller than the prelaser response at the same site, a third application was suffused 15 minutes later and response was monitored at a site 100 μm away from the site of injury. This third response served as a time control.
The Wilcoxon matched pairs test was used to detect differences between responses within a group in which each mouse served as its own control.26 All group values are expressed as mean±SD.
At the end of the observation period, 100 μL of blood was collected from the carotid artery for analysis of arterial pH, O2, and CO2 levels. These values testified to the general condition of the mice and were similar to those obtained in this laboratory and reported in many previous publications. The mean (±SD) values were as follows and will not be referred to again: Paco2=32±4 mm Hg; pH=7.36±.04; and Pao2=113±7 mm Hg.
Endothelium-Dependent Dilation by THBP
This cofactor of NOS caused slight but definite relaxation of the arterioles, which was dose dependent: on 10 arterioles 33±21-μm-wide dilations (mean±SD) of 5±2% and 10±2% were produced by doses of 10−3 and 10−2 mol/L, respectively (P<.01). The higher dose (10−2 mol/L) was used in experiments testing endothelium dependence. Fig 1⇓ shows that the response to THBP was abolished 10 minutes after the endothelium was injured by laser/dye. Time control, 60 minutes after injury, at a site 100 μm away showed that the response to THBP was identical to the prelaser response. A control experiment in 10 additional mice showed (Fig 1⇓) as usual22 that dilation by endothelium-independent SNP was not impaired by the injury, thus showing that smooth muscle was not impaired. The dose of SNP was chosen because it produces a dilation comparable to that produced by 10−2 mol/L THBP and is well below the maximum effective dose of SNP.
Effect of LNMMA on Dilation by THBP
The endothelium dependence of the response to THBP, a known cofactor for NOS, implies that THBP produced dilation by enhancing basal release of EDRFACh from the endothelium. If this is so, then inhibition of NOS with LNMMA should inhibit or prevent THBP from causing dilation. A low (10−6 mol/L) concentration of LNMMA was used that is known to block dilation by ACh in this preparation without causing narrowing of basal diameter.1 Ten arterioles 36±2 μm wide were tested with the lower dose (10−3 mol/L) of THBP and dilated 5±1%. After 10 minutes of 10−6 mol/L LNMMA, the same arterioles dilated only 1±1% (P<.01, Wilcoxon test) (Fig 2⇓). In fact, 8 of the 10 vessels failed to dilate, and the remaining 2 dilated less than in the control situation. The treatments (control or LNMMA) were randomized so that in half the mice the response in the presence of LNMMA was tested before the control response. Consequently, the inhibition or abolition of the response by LNMMA was not due to passage of time or development of tachyphylaxis to THBP. As expected, at the low dose used here, LNMMA had no effect on the resting diameter of the arterioles.
There are several important new findings:
THBP is a dilator (albeit weak) of mouse pial arterioles in vivo. This dilation is totally endothelium dependent and is inhibited by a low concentration of LNMMA. These data are in keeping with reports that THBP is a cofactor for NOS,13 14 15 that endothelium contains LNMMA-inhibitable NOS,4 and that this NOS produced an EDRF that appears to be basally released from several vessels, including mouse pial arterioles.1 It would appear from the data in the present report that basal release of EDRFACh is enhanced by THBP.
The responses to THBP were very small and required high doses. This may reflect limited accessibility of the endothelium to THBP administered outside the arteriole. In addition, THBP is relatively insoluble in lipid, making its entry into cells difficult. Finally, endogenous THBP levels might already be close to optimal, thus limiting the effect of additional cofactor. Although small, there is no doubt that the effects were real. They were well within the limits of detection, they were dose dependent, and the initial observations reflected in the initial study of dose dependence were made by a “blinded” observer who had no idea of the putative action of THBP. Indeed, the high doses required were found only after negative preliminary trials of lower concentrations. The small effects in no way alter the significance of the data, which is to show that there is basally active NOS capable of increasing its activity when cofactor is added.
In view of a publication27 suggesting that THBP can act not only as a cofactor for NOS but also as an endothelium-derived dilator in its own right, we must consider this possibility as an explanation for some of our data. This can be rejected because THBP did not act as a direct dilator of vascular smooth muscle; rather, its action was totally dependent on an intact endothelium and was inhibited by LNMMA, thus suggesting that it functioned only as a cofactor for NOS within endothelial cells.
THBP may be auto-oxidized and generate superoxide or hydrogen peroxide, which could dilate pial arterioles. However, these dilations are neither endothelium dependent nor inhibited by LNMMA.
Although other pterins were not tested, it seems highly unlikely that the dilation produced by THBP was a nonspecific effect of pterins directed at a target other than NOS because the dilation was both endothelium dependent and inhibited by LNMMA. In fact, a parallel investigation showed that the dilation produced by THBP was inhibited by an antisense oligonucleotide directed against the mRNA for NOS.16 The present data support the inference from the antisense study16 that THBP is working through its action on NOS.
The finding that THBP is an endothelium-dependent, LNMMA-inhibitable dilator of mouse pial arterioles is not only consonant with the evidence that it is a cofactor for NOS but also supports the prior evidence1 2 3 that in pial arterioles, at least of mice, EDRFACh can be released by endothelium without stimulation of the endothelium by ACh or some other humoral agent or neurotransmitter. This is important because the arrival of such agents at the endothelial surface may be limited except in pathological states. On the other hand, shear-stimulated, spontaneous synthesis/release of EDRFACh can provide a compound that continuously modulates vascular tone and helps to maintain a platelet-free endothelial surface.
Selected Abbreviations and Acronyms
|EDRF||=||endothelium-derived relaxing factor|
|NOS||=||nitric oxide synthase|
This study was supported by grant HL-35935 from the National Heart, Lung, and Blood Institute.
Reprint requests to William I. Rosenblum, MD, Medical College of Virginia/Virginia Commonwealth University, Department of Pathology, Division of Neuropathology, PO Box 980018, Richmond, VA 23298.
Reviews of this manuscript were directed by Frank M. Faraci, PhD.
- Received May 28, 1996.
- Revision received August 14, 1996.
- Accepted September 6, 1996.
- Copyright © 1997 by American Heart Association
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THBP is a naturally occurring reducing agent and a cofactor required for NOS activity.1R 2R 3R 4R The concentration of THBP needed for half-maximal stimulation (Km) of NOS is 2×10−8 mol/L.5R The precise role of THBP in regulation of NOS is still not clear, but existing evidence suggests that it functions as both allosteric and redox cofactor.6R
The results of Rosenblum's study demonstrate that exogenous THBP is capable of producing endothelium-dependent relaxations of mouse pial arterioles. This is the first in vivo observation regarding the stimulatory effect of THBP on NO production in cerebral circulation. Two recent reports from our laboratory demonstrated that despite the fact that under in vitro conditions THBP is susceptible to auto-oxidation, causing formation of superoxide anions, it can activate production of NO and augment endothelium-dependent relaxations in the presence of high enzymatic activity of superoxide dismutase.7R 8R The results of the present study suggest that under in vivo conditions, auto-oxidation of exogenously added THBP and subsequent formation of superoxide anions do not occur. The ability of THBP to produce endothelium-dependent relaxations mediated by NO supports the idea that increased availability of THBP may stimulate NOS activity and increase formation of NO.
What are the clinical implications of these findings? A recent study by Higman et al9R demonstrated that smoking-induced impairment of endothelial NOS activity in human veins could be reversed by exogenous THBP. The authors speculated that smoking decreases levels of THBP because the aromatic amines absorbed into the circulation from the combustion of tobacco are potent inhibitors of the enzymes involved in biosynthesis of this important cofactor. In another study on isolated coronary microvessels, Tiefenbacher et al10R demonstrated that ischemia/reperfusion causes impairment of endothelium-dependent relaxations in porcine coronary microvessels. This dysfunction of endothelial cells could be corrected by increasing intracellular levels of THBP. The authors concluded that decreased availability of THBP is responsible for coronary microvascular endothelial dysfunction associated with reperfusion injury. Together with findings reported in the present study, these results suggest that intracellular concentration of THBP is an important determinant of endothelial NOS activity. At the present time it is unknown whether smoking or reperfusion injury may have similar effects on THBP levels in human cerebral blood vessels. More importantly, future studies will determine whether suboptimal intracellular levels of THBP may cause dysfunction of endothelial NOS and contribute to the pathogenesis of other cerebrovascular diseases.
Selected Abbreviations and Acronyms
|EDRF||=||endothelium-derived relaxing factor|
|NOS||=||nitric oxide synthase|
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Kinoshita H, Katusic ZS. Exogenous tetrahydrobiopterin causes endothelium-dependent contractions in isolated canine basilar artery. Am J Physiol.. 1996;271:H738-H743.
Tsutsui M, Milstien S, Katusic ZS. Effect of tetrahydrobiopterin on endothelial function in canine middle cerebral arteries. Circ Res.. 1996;79:336-342.
Higman DJ, Strachan AMJ, Buttery L, Hicks RCJ, Springall DR, Greenhalgh RM, Powell JT. Smoking impairs the activity of endothelial nitric oxide synthase in saphenous vein. Arterioscler Thromb Vasc Biol.. 1996;16:546-552.
Tiefenbacher CP, Chilian WM, Mitchell M, DeFily DV. Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin. Circulation.. 1996;94:1423-1429.