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Am J Physiol Heart Circ Physiol 283: H658-H663, 2002. First published April 25, 2002; doi:10.1152/ajpheart.00073.2002
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Vol. 283, Issue 2, H658-H663, August 2002

Functional NOS 1 in the rat mesenteric arterial bed

Jennifer C. Sullivan1, Ararat D. Giulumian1, David M. Pollock1,2, Leslie C. Fuchs1,3, and Jennifer S. Pollock1,3

1 Vascular Biology Center, 2 Department of Physiology, and 3 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previously we have demonstrated functional nitric oxide synthase (NOS) 1 in large arteries. Because resistance arteries largely determine blood pressure, this study examined whether functional NOS 1 also exists in resistance arteries. Phenylephrine (PE) contraction was measured in the absence and presence of the NOS 1 inhibitor N5-(1-imino-3-butenyl)-L-ornithine (VNIO) in isolated mesenteric resistance arteries (endothelium intact and denuded) from Sprague-Dawley rats. For NOS 1 activity and expression, the mesenteric arterial bed was separated into cytosolic and particulate fractions. NOS activity was assayed by measuring the conversion of [3H]arginine to [3H]citrulline inhibited by a nonselective NOS inhibitor or VNIO. VNIO increased PE sensitivity in endothelium-intact and -denuded arteries. In cytosolic and particulate fractions of the arterial bed, ~40% of NOS activity was inhibited by VNIO. Immunoprecipitation and Western blot analysis revealed two NOS 1 immunoreactive bands. One band corresponded to the rat brain isoform, whereas the second was of a slightly lower molecular mass. The cytosolic fraction contained both isoforms; however, the particulate fraction had only the lower molecular mass form. These studies demonstrate the existence of functional NOS 1 in resistance arteries.

neuronal nitric oxide synthase activity; resistance arteries; vascular reactivity; subcellular fractionation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) synthase (NOS) is the enzyme that catalyzes the conversion of L-arginine and molecular oxygen to NO and L-citrulline. Three NOS isoforms have been identified and characterized based on distinct cellular distribution and regulation. NOS 1 (neuronal NOS), initially characterized in the brain (6, 39), is localized in skeletal and cardiac muscle (2, 16, 18, 24), epithelial cells, pancreatic islets, and kidney macula densa cells. NOS 2 (inducible NOS) has been identified in macrophages, hepatocytes, vascular smooth muscle, endothelial cells, and mesangial cells. NOS 3 (endothelial NOS, eNOS) was first identified in endothelial cells (35) and has subsequently been described in skeletal and cardiac muscle (16, 18). Classically, NOS 1 and NOS 3 are known to be Ca2+/calmodulin dependent and constitutively expressed and release NO immediately upon stimulation. Recent studies, however, have demonstrated that NOS 3 is also able to act in a Ca2+-independent manner after phosphorylation (13, 17, 26). NOS 2 activation is Ca2+ independent, transcriptionally regulated, and can be induced by stimulation with cytokines and/or endotoxin (11, 15, 30).

NOS 3-derived NO is known to play a central role in the modulation of vascular tone and blood pressure. Recent findings, however, have led investigators to examine the role of other NOS isoforms in the vasculature, particularly NOS 1. Recently, our laboratory has demonstrated the presence of functional NOS 1 in the smooth muscle layer of bovine carotid arteries (9). NOS 1 activity has also been shown in the rat aorta (40) and sheep uterine arteries (37). It has been suggested (4) that vascular NOS 1 may serve to attenuate increases in blood pressure. Boulanger et al. (4) reported that NOS 1 activity was increased in endothelium-denuded carotid arteries from spontaneously hypertensive rats compared with normotensive rats. In both sheep uterine arteries (37) and carotid arteries from spontaneously hypertensive rats (4), ~60% of total NOS activity has been attributed to the endothelium and NOS 3, whereas the remaining 40% has been localized to the vascular medial layer and mediated by NOS 1.

The purpose of this study was to examine the subcellular localization and function of NOS 1 in resistance arteries. A majority of the work that has been done in examining the role of NOS 1 in the vasculature has been performed using conduit arteries. However, it is the smaller caliber arteries that play a role in the determination and modulation of blood pressure. We hypothesize that there is functional NOS 1 in resistance-sized arteries that acts to antagonize vasoconstriction.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (200-225 g body wt, Harlan Laboratories; Indianapolis, IN) were used in all studies. All animal protocols were in accordance with National Institutes of Health guidelines and were approved by the Medical College of Georgia Committee for Animal Use in Research and Education. Rats were housed in temperature- and humidity-controlled, light-cycled quarters.

Vascular reactivity. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and a portion of the mesenteric bed was removed and placed in cold modified Krebs-Ringer bicarbonate solution [composed of (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 dextrose]. With the use of a dissecting microscope, a single artery (200-300 µm diameter) was isolated, cleared of fat and connective tissue, transferred to a vessel chamber, and mounted between two glass micropipettes (100-µm-diameter tips). The chamber was transferred to the stage of an Olympus inverted light microscope coupled to a monitor and video dimension analyzer (Living Systems Instrumentation; Burlington, VT). Intraluminal diameter was monitored continuously on a Grass recorder.

Oxygenated (20% O2-5% CO2-balance N2), warmed (37°C) Krebs-Ringer solution was continuously circulated through the tissue bath. The lumen of the vessel was filled with Krebs-Ringer solution and maintained at a constant pressure of 40 mmHg. The vessel was allowed to equilibrate for 1 h. Dose-response curves to phenylephrine (PE, 1 × 10-8-3 × 10-5 M) were performed in endothelium-intact and -denuded mesenteric arteries in the absence and presence of the NOS 1-specific inhibitor N5-(1-imino-3-butenyl)-L-ornithine (VNIO, 1 µM). The inhibitory constants of VNIO for NOS 1, NOS 2, and NOS 3 are 0.1, 60, and 12 µM, respectively (1). VNIO was added to the vessel bath 20 min before performance of the PE dose-response curve. Endothelium denudation was accomplished by first rubbing the vessel lumen with a human hair and then passing air bubbles through the lumen (34, 43). Denudation was verified by the absence of a vasodilator response to acetylcholine (1 × 10-5 M) in a vessel preconstricted with PE. After the denudation protocol, if an artery failed to have a robust constriction to PE or had any residual dilation to acetylcholine, it was not used in the study. Only one dose-response curve was performed per vessel.

Isolation of mesenteric arterial bed. Rats were euthanized with pentobarbital sodium (50 mg/kg ip), and a thoracotomy was performed. The mesenteric bed including arteries and veins was cut away from the intestinal wall and placed in a dissecting dish that contained ice-cold homogenization buffer (50 mM Tris · HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 250 mM sucrose, and 10% glycerol) in the presence of protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µM pepstatin A, 2 µM leupeptin, and 0.1% aprotinin]. The fat was carefully removed from the vessels and the veins were removed using an Olympus dissecting microscope. The remaining arteries (~100-600 µm intraluminal diameter) were placed in a tube containing 1 ml of homogenization buffer and then snap-frozen in liquid nitrogen. To obtain sufficient amounts of protein, a slightly larger size range of resistance arteries was used for the biochemical assays: ~20% by mass of the total was larger arteries. The isolation process from harvesting the arteries to freezing took ~5 min. Slightly thawed arteries were homogenized on ice in the presence of fresh protease inhibitors with a glass-glass homogenizer for 10 strokes. The homogenate was centrifuged at 4°C at 100,000 g for 30 min to separate it into cytosolic and particulate fractions. The particulate fraction was then resuspended in 0.5 ml of homogenization buffer. Protein concentrations were determined by the Bradford assay (Bio-Rad; Hercules, CA) using bovine serum albumin as the standard.

Measurement of NOS activity by conversion of [3H]arginine to [3H]citrulline. Aliquots of cytosolic and particulate fractions were incubated with [3H]arginine (10 µM final arginine, 71 Ci/mmol) in the presence of 1 mM NADPH, 30 nM calmodulin, 3 µM tetrahydrobiopterin, 2 mM CaCl2, 1 µM FAD, and 1 µM flavin mononucleotide (FMN) in a final volume of 50 µl. Additional aliquots were incubated with the nonselective NOS inhibitor Nomega -nitro-L-arginine (L-NNA, 1 mM) or VNIO (1 µM). The remainder of the assay was done as previously described (9). In brief, after a 30-min incubation at room temperature, the reaction was terminated by the addition of 1 ml of 50 mM HEPES, pH 5.5, containing 2 mM EDTA and 2 mM EGTA. The reactions were applied to 1 ml Dowex AG 50WX-8 columns (Na form, Bio-Rad) and the [3H]citrulline was eluted with water. The eluted radioactivity was quantitated by liquid scintillation counting (Beckman 6500, Beckman-Coulter Instruments).

Total NOS activity was defined as the [3H]arginine to [3H]citrulline conversion that was inhibited by L-NNA. Therefore, NOS activity was calculated using the following formula: total NOS activity = (picomoles citrilline in the absence of L-NNA) - (picomoles citrilline in the presence of L-NNA). The percent inhibition of total NOS activity by VNIO was calculated using the following formula: percent inhibition = [(total NOS activity - picomoles of NOS activity in the presence of VNIO)/total NOS activity] × 100. NOS activity was expressed as picomoles of NOS activity per 30 minutes per total protein in the mesenteric arterial bed.

Immunoprecipitation of NOS 1. The mesenteric arterial bed was isolated as described (see Isolation of mesenteric arterial bed) and homogenized on ice in radioimmunoprecipitation assay (RIPA) buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 µM aprotinin, 1 mM PMSF, and 1 mM Na3VO4). Homogenates were incubated with 1 mM PMSF for 30 min at 4°C and then centrifuged at 10,000 g for 25 min at 4°C to remove insoluble material. Lysates were incubated for 30 min at 4°C with the appropriate agarose conjugate (40 µl, sc-2345, Santa Cruz Biotechnology; Santa Cruz, CA) and centrifuged at 1,000 g for 5 min at 4°C. The supernatant was transferred to a fresh tube and incubated with a polyclonal NOS 1 antibody (BioMol; Plymouth Meeting, PA) for 1 h at 4°C. The BioMol NOS 1 antibody recognizes the COOH terminus of NOS 1 at amino acid sequence 1,414-1,434. Protein G Plus-Agarose (20 µl, sc-2002, Santa Cruz Biotechnology) suspension was added and incubated overnight at 4°C. The beads were pelleted, washed four times with 1 ml of RIPA buffer, resuspended in 40 µl of sample buffer, and then boiled for 5 min. The samples were then subjected to gel electrophoresis on 4-20% Express Gels (ISC BioExpress; Kaysville, UT), and NOS 1 proteins were detected by Western blotting (1:1,000 dilution, Transduction Laboratories; Franklin Lakes, NJ). The Transduction NOS 1 antibody recognizes the COOH terminus of NOS 1 at amino acid sequence 1,095-1,289. The primary antibody was stripped using ReBlot Plus Mild Antibody Stripping Solution (Chemicon International; Temecula, CA) and a second primary antibody to NOS 3 (1:500 dilution, Transduction Laboratories) was applied followed by a secondary antibody to the mouse (1:2,000 dilution, Amersham; Piscataway, NJ) to ensure that there was no antibody cross-reactivity between NOS 1 and NOS 3 proteins (data not shown).

Western blotting of NOS 1. The Western blotting protocol was as previously described (9). The primary antibody was a monoclonal anti-NOS 1 (1:1,000 dilution, Transduction Laboratories), and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse antibody (1:2,000 dilution, Amersham). Specific bands were detected with enhanced chemiluminescence (SuperSignal Chemiluminescent Substrate, Pierce; Rockford, IL). Densitometry was performed using a digital imaging system (Alpha Innotech; Staffordshire, UK). Kaleidoscope prestained standards (Bio-Rad) were used as molecular mass markers. The molecular mass of the lower-mass NOS 1 variant was determined using Rf vs. molecular mass plot; mass was calculated on four blots and averaged.

Materials. Buffer reagents, PE, L-NNA, FMN, FAD, leupeptin, pepstatin A, aprotinin, sodium orthovanadate, and PMSF were purchased from Sigma Chemical (St. Louis, MO). VNIO was purchased from Cayman Chemicals (Ann Arbor, MI). NADPH and tetrahydrobioptein were purchased from Alexis Biochemicals (San Diego, CA). [3H]arginine was obtained from Amersham. Nonidet P-40 and sodium deoxycholate were purchased from Calbiochem (La Jolla, CA).

Data analysis. All values are expressed as means ± SE. PE dose-response curves are expressed as percent constriction from baseline. PE concentration-response curves were analyzed using nonlinear regression of sigmoidal dose-response curves (GraphPad Prism; San Diego, CA), which was used to calculate the EC50 (concentration of agonist that elicited 50% of the maximum response) and slope. The negative log EC50 values (pD2) were compared using a two-way ANOVA. Individual comparisons were then performed using a Student-Newman-Keuls test. For all comparisons, P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NOS 1 inhibition increases PE sensitivity. PE dose-response curves in endothelium-intact (A) and endothelium-denuded (B) isolated mesenteric arteries are shown in Fig. 1. In both arteries, the addition of the NOS 1-selective inhibitor VNIO (1 µM) significantly increased arterial sensitivity to PE (pD2 values: intact, -5.8 ± 0.04; VNIO, -6.6 ± 0.12; P = 0.001; denuded, -6.1 ± 0.07; VNIO, -6.4 ± 0.06; P = 0.006). There were no differences in maximum response or slope. In control arteries, denudation of the vascular endothelium significantly increased arterial sensitivity to PE compared with endothelium-intact arteries (P = 0.02).


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  		Fig. 1.   Phenylephrine (PE) dose-response curves in endothelium-intact (A) and -denuded (B) isolated mesenteric arteries in the absence and presence of the neuronal nitric oxide synthase (NOS 1)-specific inhibitor N5-(1-imino-3-butenyl)-L-ornithine (VNIO, 1 µM). Numbers in parentheses represent number of animals in each group. Values are means ± SE.

NOS activity. NOS activity was calculated by measuring the conversion of [3H]arginine to [3H]citrulline. NOS activity is expressed as picomoles of NOS activity per total protein from the arterial bed (pmol · 30 min-1 · arterial bed-1). As shown in Fig. 2A, there was significantly more total NOS activity in the particulate fraction compared with the cytosolic fraction (P = 0.02). When normalized to milligrams of protein, NOS activity was 50.4 ± 6 pmol · 30 min-1 · mg protein-1 in the cytosolic fraction and 91.3 ± 20 pmol · 30 min-1 · mg protein-1 in the particulate fraction (cytosolic and particulate protein concentrations were 0.56 ± 0.05 and 1.48 ± 0.2 mg/ml, respectively). In both the particulate and cytosolic fractions, VNIO inhibited ~40% of total NOS activity (Fig. 2B).


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  		Fig. 2.   NOS activity in mesenteric arteries. A: total NOS activity in the arterial bed. B: percent inhibition of total NOS activity by VNIO (1 µM). Values are means ± SE; n = 11.

NOS 1 immunoprecipitation. Immunoprecipitations were performed to verify the presence of NOS 1 in the mesenteric arterial bed. With the use of a NOS 1-specific antibody, two NOS 1 bands were immunoprecipitated. One of the bands corresponds to NOS 1 found in the rat brain, whereas the second band is of a slightly lower molecular mass (Fig. 3). The molecular mass of the lower band is ~136 kDa.


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  		Fig. 3.   Immunoprecipitation of NOS 1 from the mesenteric arterial bed. Western blot using NOS 1-specific antibody is shown. Numbers represent molecular mass markers (in kDa). Lane 1 is rat brain standard; lane 2 is immunoprecipitate (IP).

NOS 1 protein expression in mesenteric arteries. Western blot analysis was performed to examine NOS 1 distribution in the cytosolic and particulate subcellular fractions of mesenteric arteries. NOS 1 protein expression in mesenteric arteries is shown in Fig. 4. There is a single band in the particulate fraction, which corresponds to the lower molecular mass band. There are two bands in the cytosolic fraction: one band corresponding to brain NOS 1 and another band corresponding to the lower molecular mass variant.


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  		Fig. 4.   NOS 1 protein expression in mesenteric arteries. Molecular mass (in kDa) of NOS 1 is represented by 155. Lane 1 is the rat brain standard, lanes 2 and 3 are the particulate (P) and cytosolic (C) fractions from one rat, and lanes 4 and 5 are the particulate and cytosolic fractions from a second rat. Protein loaded per lane: 25 µg.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we found that there is functional NOS 1 present in mesenteric resistance arteries from male Sprague-Dawley rats. Sensitivity to the alpha 1-agonist PE was significantly increased in both endothelium-intact and -denuded isolated mesenteric arteries, which suggests that the source of NOS 1 is nonendothelial. There are two immunoreactive NOS 1 bands present in the mesenteric arterial bed; one corresponds to brain NOS 1, whereas the other is of a lower molecular mass. Furthermore, we observed a differential subcellular distribution of the two NOS 1 bands: the lower molecular mass band is localized in both the cytosolic and particulate fractions, whereas the "normal" NOS 1 is found only in the cytosolic fraction.

Our results support other recent reports of functional NOS 1 in large arteries (4, 9, 37, 40); however, this is the first report to identify functional NOS 1 in a resistance arterial bed. Nonselective NOS inhibition has been shown to reverse Mg2+-induced relaxation in the endothelium-denuded rat aorta, rat pulmonary artery, dog femoral vein, and dog mesenteric artery (12). Whereas the authors did not identify NOS 1 in the vasculature, they postulate that there may be a yet-to-be identified NOS isoform located in vascular smooth muscle. Other investigators as well as ourselves have identified this isoform as NOS 1. Boulanger et al. (4) reported that there was an increase in NOS 1 activity and expression in carotid arteries from spontaneously hypertensive rats compared with normotensive rats, which suggests that NOS 1 acts to antagonize increases in blood pressure associated with hypertension. Inhibition of NOS 1 has been shown to increase the contractile responses to a number of agonists including angiotensin II (4), KCl (9, 40), and norepinephrine (40). Our results support the hypothesis that vascular NOS 1 acts to modulate contraction. After incubation with a NOS 1-selective inhibitor, there was a significant increase in PE-induced vasoconstriction, which suggests that there is a NOS 1 source of NO normally acting to oppose increases in vascular tone.

In conjunction with our findings, the presence of NOS 1 in the vasculature would suggest a potential role for NOS 1 in modulating or maintaining blood pressure. Whereas a number of investigators have examined the effects of both acute and chronic in vivo inhibition of NOS 1, there is not a consensus in the literature as to whether NOS 1 antagonism alters blood pressure. Ollerstam et al. (33) examined the effects of chronic NOS 1 inhibition using 7-nitroindazole (7-NI) on blood pressure and kidney function. After 2 wk of NOS 1 inhibition, there was a significant increase in blood pressure, which suggests that chronic but not acute NOS 1 inhibition can influence blood pressure. Xu et al. (45) showed that in rats with cirrhosis, decreases in systemic vascular resistance and mean arterial pressure (MAP) are normalized by chronic treatment with 7-NI, which supports the finding that chronic NOS 1 inhibition alters blood pressure. Additional investigators have also reported that acute NOS 1 blockade using 7-NI does not alter blood pressure (3, 44); in addition, Beierwaltes (3) found that acute 7-NI treatment had no effect on renal blood flow or renal vascular resistance. With the use of a more selective inhibitor of NOS 1, S-methyl-L-thiocitrulline (SMTC), Komers et al. (25) reported that acute NOS 1 inhibition produced dose-dependent increases in MAP in both control and diabetic rats. Using SMTC, Gozal et al. (19) also reported that acute NOS 1 inhibition increases blood pressure; however, in this study, the increase in pressure was transient. In contrast, infusions of either VNIO or Nomega -propyl-L-arginine (L-NPA) have been reported to decrease blood pressure, although renal interstitial NO levels were decreased (22). Finally, bilateral microinjections into the nucleus tractus solitarii of Wistar-Kyoto rats using antisense oligodeoxynucleotides to NOS 1 significantly increases blood pressure (28). Consequently, it appears that the effects of NOS 1 inhibition on blood pressure are dependent on the antagonist, duration of treatment, and method used to induce inhibition. More work remains to be done to fully characterize the physiological or pathological role of NOS 1 in the vasculature.

Vascular NOS 1 has been localized to the smooth muscle layer in rat (4) and bovine carotid arteries (9), ovine uterine arteries (37), rat aortas (40), and throughout the vasculature of male hamsters (41). In the present study, selective NOS 1 inhibition increased sensitivity to vasoconstriction in both endothelium-intact and -denuded mesenteric arteries. These findings demonstrate that NOS 1 is located in vascular tissue other than the endothelium in mesenteric resistance arteries. NOS 1 is known to be localized in nonadrenergic noncholinergic (NANC) neurons that innervate arteries (10, 27, 38). We feel, however, that it is unlikely that we are stimulating the release of neuronal derived NO with PE. Whereas it is known that the release of NANC neurotransmitters can be modulated by alpha 2-adrenergic receptors, there is no evidence of alpha 1-receptors on sensory nerves (20, 23). Therefore, there is no known mechanism for PE to stimulate the depolarization of sensory nerve fibers and the subsequent release of neuronal NO, which suggests that nerves are not the source of NO in our preparation.

We found that the rat mesenteric arterial bed has two proteins recognized by a NOS 1-specific antibody: one corresponding to brain NOS 1 and a second of a lower molecular mass. Although our study is the first to examine arterial subcellular localization of NOS 1 isoforms, Nelson et al. (31) reported two NOS 1-specific immunoreactive bands in uterine arteries from women. Whereas they did not address the identity of the bands, NOS 1 is known to have both a number of splice variants and to undergo posttranslational modification. Schwarz et al. (40) reported that the rat aorta possesses both "normal" NOS 1 and the skeletal muscle µNOS 1 in the smooth muscle layer. However, it is unlikely that the second variant in the mesenteric arterial bed is µNOS, because it contains a 102-bp insert that results in a larger molecular mass (42).

beta NOS 1 and gamma NOS 1 are NOS 1 splice variants that were first described in NOS 1 knockout mice (36). NOS 1 knockout mice are viable with normal blood pressure, organ perfusion, and central nervous system (21). This has been attributed to the finding that after deletion of NOS 1 in knockout mice, there is a small amount of residual NOS 1 activity in the brain, which is now attributed to beta NOS 1 and gamma NOS 1. beta NOS 1 is upregulated in NOS 1 knockout mice and is most likely responsible for a majority of the remaining NOS 1 activity in the brain (14). PCR and sequence analysis have revealed that in wild-type mice, beta NOS 1 accounts for ~5% of total NOS 1 mRNA (8). These two splice variants lack exon 2, which encodes the NH2 terminal PDZ domain of NOS 1. beta NOS 1 has been shown to have ~80% activity compared with NOS 1, whereas gamma NOS 1 has 3% (7). Additionally, Eliasson et al. (14) have reported that beta NOS 1 is active in vivo in many brain regions of wild-type mice. Whereas these variants are of similar molecular mass to the second band detected in our preparation (the molecular masses of beta NOS 1 and gamma NOS 1 are 136 and 125 kDa, respectively) in the brain, these variants are solely localized in the cytosolic fraction. It is unlikely that gamma NOS 1 accounts for the lower molecular mass protein in our preparation. It has very low levels of activity and expression, and the molecular mass is lower than the mass of the lower band in our preparation. However, it is possible that the lower band in our preparation is an alternative form of beta NOS 1 present in the mesenteric arterial bed that is able to associate with the membrane. beta NOS 1 is the correct molecular mass and has a reasonable amount of activity. Whereas the physiological role of brain beta NOS 1 is unknown, it may simply serve as an alternative to NOS 1 in times of need. If one of the roles of vascular NOS 1 is to oppose increases in vascular tone, it is possible that under conditions of high tone vascular NOS 1 would increase. Additionally, there may be a yet-to-be-described splice variant localized in the particulate fraction of resistance arteries.

Alternatively, the two immunoreactive NOS 1 bands may represent differences in posttranslational modifications of NOS 1. For example, NOS 1 has been shown to be phosphorylated (5, 29), and phosphorylation of NOS 1 has been demonstrated to be influenced by factors such as tetrahydrobiopterin availability (32). Therefore, it is possible that the two NOS 1 immunoreactive bands that we observed represent phosphorylated and nonphosphorylated NOS 1.

In summary, the mesenteric arterial bed possesses functional NOS 1 that normally acts to antagonize vasoconstriction. There are two immunoreactive NOS 1 bands in the mesenteric arterial bed with different subcellular localization. The particulate fraction possesses a lower molecular mass immunoreactive NOS 1 band of ~136 kDa, whereas the cytosolic fraction has both brain NOS 1 as well as the lower molecular mass variant.


    ACKNOWLEDGEMENTS

The authors acknowledge the excellent technical assistance of Jingdong Xin.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-60653 (to J. S. Pollock), HL-64776 (to D. M. Pollock), and HL-49924 (to L. C. Fuchs), an American Heart Association Scientist Development Grant (to J. S. Pollock), and the American Physiological Society (Postdoctoral Fellowship in Physiological Genomics to J. C. Sullivan).

Address for reprint requests and other correspondence: J. C. Sullivan, Medical College of Georgia, Vascular Biology Center, 1459 Laney-Walker Blvd., Augusta, GA 30912 (E-mail: jsullivan{at}mail.mcg.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 25, 2002;10.1152/ajpheart.00073.2002

Received 28 January 2002; accepted in final form 10 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(2):H658-H663
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