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1 Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794; and 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Abstract
Introduction
Procedures
Results
Discussion
References
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The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in the porcine sphenopalatine ganglia (SPG), pial veins, and the anterior cerebral arteries was examined using immunohistochemical and histochemical staining techniques. NOS-immunoreactive (I), ASS-I, and ASL-I fibers were found in pial veins and the anterior cerebral arteries. NOS, ASS, and ASL immunoreactivities were also found in neuronal cell bodies in the SPG. Almost all neuronal cell bodies in the SPG and nerve fibers in pial veins and the anterior cerebral arteries that were reactive to ASS, ASL, and NOS were also stained positively with NADPHd, suggesting that ASS, ASL, and NOS were colocalized in the same neurons in the SPG and perivascular nerves. With the use of in vitro tissue bath techniques, L-citrulline but not D-citrulline reversed inhibition of neurogenic vasodilation in isolated porcine pial veins produced by NOS inhibitors such as NG-nitro-L-arginine methyl ester. In the presence of L-aspartate, L-arginine was synthesized from L-citrulline in homogenates of SPG and endothelium-denuded cerebral arteries and pial veins. These results provide evidence indicating that perivascular nerves in pial veins like cerebral arteries can convert L-citrulline to L-arginine for synthesizing nitric oxide. The conversion is most likely via an argininosuccinate pathway.
argininosuccinate lyase; argininosuccinate synthetase; reduced nicotinamide adenine nucleotide phosphate-diaphorase; nitric oxide synthase; colocalization; cerebral arteries and veins; porcine
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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IT IS WELL ESTABLISHED that cerebral blood vessels from several species receive dense nitric oxidergic innervation and that neurogenic vasodilation of isolated large cerebral arteries at the base of the brain is mediated predominantly by nitric oxide (NO) (9, 12, 17). NO is synthesized from L-arginine with L-citrulline as a byproduct catalyzed by NO synthase (NOS) in various types of cells (2, 4, 31). Based on biochemical and pharmacological studies, we have proposed that L-citrulline is actively converted to L-arginine, which is then converted to L-citrulline and NO in perivascular nerves in cerebral arteries (6, 20). This active citrulline-arginine cycle in cerebral perivascular nerves provides not only a means to maintain an adequate supply of NO during neurovascular transmission, but also the first biochemical evidence indicating that NO is synthesized in perivascular nitric oxidergic nerves (5). Therefore, NO can be released from cerebral perivascular nerves and has been considered a transmitter substance.
The known pathway of L-citrulline conversion to L-arginine in the urea cycle is via the production of argininosuccinate (AS) (13, 32). In this pathway, AS synthetase (ASS) catalyzes the formation of AS from L-citrulline and aspartate. AS is then cleaved to produce L-arginine and fumarate by argininosuccinate lyase (ASL). Our preliminary studies using sequential double-labeling techniques, i.e., immunoperoxidase or immunofluorescence combined with NADPH-diaphorase (NADPHd) histochemical staining, have demonstrated that dense NOS-immunoreactive (I), ASS-I, ASL-I, and NADPHd fibers in porcine middle cerebral arteries, basilar arteries, and the circle of Willis are completely coincident fibers (40). These results indicate the presence of enzymes catalyzing the conversion of L-citrulline to L-arginine and that these enzymes are colocalized with NOS and NADPHd in the same perivascular neurons in cerebral arteries. Thus conversion of L-citrulline to L-arginine is likely mediated by the AS pathway.
Pial veins have also been shown to receive NADPHd fibers (7). Our preliminary results from in vitro pharmacological studies indicated that transmural nerve stimulation (TNS) elicited an exclusive relaxation in isolated porcine pial veins. The relaxation was blocked by inhibition of NO synthesis, and the inhibition was reversed by L-citrulline, suggesting that L-citrulline is also converted to L-arginine in perivascular nerves in porcine pial veins. It is possible that NOS, ASS, and ASL are colocalized in perivascular nerves in pial veins.
The sphenopalatine ganglia (SPG) have been shown to be one major source of NOS-I fibers innervating cerebral arteries of the rat and cat (15, 26, 28). Although this has not been shown in the pig, if neurons in the porcine SPG are also found to cocontain NOS, ASS, ASL, and NADPHd, it will provide evidence supporting the colocalization of NOS, ASS, ASL, and NADPHd in perivascular nerves in pial veins and arteries. The present study was designed to determine by immunohistochemistry, histochemistry, in vitro tissue bath techniques, and thin-layer chromatography (TLC) whether conversion of L-citrulline to L-arginine in porcine SPG and perivascular nerves in pial veins like those in pial arteries is via the AS pathway.
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EXPERIMENTAL PROCEDURES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fresh heads of adult pigs (n = 12) of either sex were obtained at a local slaughterhouse. The entire brain was removed, and cerebral arteries (anterior cerebral, middle cerebral and basilar arteries, and the circle of Willis) and pial veins were dissected under a dissecting microscope in oxygenated (95% O2-5% CO2) Krebs bicarbonate solution (pH 7.4) containing (in mM) 196.87 NaCl, 5.16 KCl, 1.33 CaCl2, 25.56 NaHCO3, 1.22 MgSO4 · 7H2O, 1.01 dextrose, 0.34 EDTA, and 0.28 ascorbic acid (18) at room temperature. The SPG were also removed under a dissecting microscope.
Immunohistochemistry
The SPG, anterior cerebral arteries, and pial veins were fixed in either periodate-lysine-paraformaldehyde fixative (24) or periodate-paraformaldehyde-picric acid-formaldehyde-lysine fixative (25) for 4 h. The anterior cerebral arteries and pial veins were stored in phosphate-buffered saline (PBS) and the SPG in PBS containing 30% sucrose at 4°C for 24 h. The SPG were sectioned at 8-µm thickness with a cryostat microtome and placed on Vectabond (Vector Labs)-coated slides and air-dried for 2 h. Immunohistochemistry for whole-mount specimens (arteries and veins) and sections of SPG was carried out using indirect immunofluorescence method (38, 39). Specific antisera to purified ASS and ASL were raised in the rabbits. The procedures of enzyme purification, characterization, and specificity of antisera were described elsewhere (1, 27, 29, 30). The fixed arteries, veins, and SPG sections were washed in 0.01 M PBS and blocked with 1% normal goat serum diluted in 0.05% Triton X-100/PBS for 30 min at room temperature. After being washed in PBS, the specimens were incubated in rabbit antiserum against ASS in a dilution of 1:2,000 (27, 30), ASL at 1:2,000 (27, 30), or brain NOS at 1:1,000 (Affinity BioReagents) for 24-48 h at 4°C. After another wash in PBS, specimens were incubated in affinity-purified biotinylated goat anti-rabbit immunoglobulin G antibody (1:200 diluted in PBS, Vector Labs) for 4 h at 4°C, washed with PBS, and incubated for 4 h at 4°C with fluorescein avidin D (Vector Labs). The specimens were then rinsed and coverslipped with Vectashield mounting medium (Vector Labs) for photography under a fluorescence microscope fitted with proper filters. After the immunofluorescence fibers and ganglionic cells were photographed, the specimens were washed and processed for NADPHd histochemistry as described below.NADPHd Histochemical Staining
After fixation, incubation with specific antibodies in demonstrating immunofluorescence labeling, and photographing as described above, the PBS-washed specimens were incubated in 0.1 M phosphate buffer (pH 8.0) containing 0.5 mg/ml NADPH (reduced form), 0.1 mg/ml nitro blue tetrazolium, and 0.3% Triton X-100 at 37°C for 1 h (5). The specimens were rinsed with PBS and mounted with Gel Mount (Biomeda, Foster City, CA) and examined under a Zeiss light microscope.Controls
The specificity of the immunolabeling was determined by omitting ASS, ASL, or NOS antiserum as well as by using nonimmunized normal rabbit serum or nonimmunized normal goat serum. No labeling was found in these control specimens. For control of NADPHd activity, NADPH was omitted from the incubation medium. This resulted in elimination of NADPHd reaction product in perivascular nerves and ganglionic cells (6).Measurement of Vascular Tone
The dissected pial venous rings (4 mm long; 250-400 µm OD) were cleaned of surrounding tissue under a dissecting microscope. The pial venous ring was cannulated with a stainless steel rod and a platinum wire and was mounted horizontally in a plastic bath containing 5 ml Krebs solution at 37°C and gassed with 95% O2-5% CO2 (21). Changes in isometric tension were measured by Gould Statham UC-2 transducers and recorded on a Grass polygraph. A resting tension of 75 mg was applied, and the tissues were equilibrated for an additional 60 min. An active muscle tone of ~0.6 mg in each ring segment was then elicited by U-46619 (a thromboxane A2 analog, 0.3-1 µM). TNS at various frequencies (19) with a pair of platinum electrodes through which 100 biphasic square-wave pulses of 0.2 ms in duration and 180 mA in intensity was applied. The neurogenic origin of the TNS-induced response was verified by its complete blockade by tetrodotoxin (TTX; 0.9 µM). Guanethidine (1 µM) was present in the bath throughout the entire experiment to eliminate potential contribution from adrenergic component (18). The magnitude of a vasodilator response was expressed as a percentage of the maximum response induced by 300 µM papaverine (18).TLC Analysis of Conversion of L-Citrulline to L-Arginine
Radiolabeled L-[ureido-14C]citrulline (55.9 mCi/mM) was used to assay the conversion of L-citrulline to L-arginine (6, 35). Porcine cerebral arteries (endothelium intact or denuded), pial veins (endothelium denuded), and SPG (3 pairs) were homogenized in 500 µl of ice-cold 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4) containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, and 5 glucose. The homogenate (0.5 mg/ml protein) was incubated for 3 h at 37°C in a solution containing 10 µM L-[ureido-14C]citrulline (55.9 mCi/mM), 1 mM ATP and 1 mM L-aspartate. Incubations were terminated by adding ice-cold methanol. Sample solution (25 µl) was spotted onto TLC plates (Whatman silica gel 60 Å). The plates were developed in the solvent system chloroform-methanol-ammonium hydroxide-water, 0.5:4.5:2.0:1.0 (vol/vol), over a distance of 16 cm. After drying, the plates were scanned with Bio-Rad model GS-250 molecular imager system. The Rf values for L-[14C]arginine (0.6) and L-[14C]citrulline (0.3) were identical to those of authentic standards.Drugs and Solutions
L-Arginine, L-citrulline, NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), HEPES, tetrodotoxin, NADPH, nitro blue tetrazolium (all from Sigma Chemical, St. Louis, MO), D-citrulline (from Research Plus), Triton X-100 (from Amersham), L-[ureido-14C]citrulline (55.9 mCi/mM; from New England Nuclear, Charlotte, NC), U-46619 (from Upjohn, Kalamazoo, MI), and guanethidine (from CIBA, Summit, NJ) were used.| |
RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Colocalization of NOS, ASS, and ASL Immunoreactivities With NADPHd in Porcine SPG
Numerous neuronal cell bodies in the SPG (n = 10 sections from each of 8 ganglia) expressed NOS, ASS, and ASL immunoreactivities (Fig. 1, A-C). All NOS-I, ASS-I, and ASL-I neurons in the SPG were also stained positively for NADPHd (Fig. 1, a-c).
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NOS-I, ASS-I, ASL-I, and NADPHd Fibers Are Coincident Fibers in Pial Veins and Anterior Cerebral Arteries
Anterior cerebral arteries. The whole-mount anterior cerebral arteries (n = 8 from 4 animals) were densely innervated by NOS-I, ASS-I, ASL-I, and NADPHd fibers (Fig. 2). They were composed of bundles of various sizes and fine fibers, which ran longitudinally and spirally along the walls of the arteries. The density and distribution pattern of NOS-I, ASS-I, and ASL-I fibers were very similar (Fig. 2, A-C). NOS-I, ASS-I, or ASL-I fibers were not observed after omitting primary antibody or incubating tissues with normal serum from nonimmunized rabbits or goats (data not shown). Results from sequential "double labeling," i.e., immunofluorescence labeling combined with NADPHd histochemical staining, showed that all NOS-I, ASS-I, and ASL-I fibers were coincident with NADPHd fibers (Fig. 2, a-c). These results are consistent with those found in the basilar and middle cerebral arteries and the circle of Willis of the pig (40).
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