INTRODUCTION

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ACETYLCHOLINE (ACh) dilates blood vessels by a guanylyl cyclase-dependent mechanism mediated by nitric oxide (NO) and related metabolites generated in the endothelium by NO synthase (eNOS, type III) (20). In many species, including the mouse (6, 7, 20, 23, 31), NOS inhibitors such as N^G-nitro-L-arginine methyl ester or N^G-nitro-L-arginine block the ACh response. In fact, aortic ring segments of eNOS null mice do not dilate in response to ACh (16). However, the response in small brain vessels seems to be more complex in eNOS null mice as ACh dilates pial arterioles, and the dilation can be blocked by N^G-nitro-L-arginine (23). Moreover, tetrodotoxin, an inhibitor of voltage-dependent sodium channels, attenuated the ACh response only in eNOS mutants and not in wild-type strains (23).

To test the hypothesis that neuronally derived NO plays a compensatory role after deletion of the eNOS gene, we evaluated the response to 7-nitroindazole sodium (7-NI), a relatively selective and water-soluble inhibitor of neuronal NOS (nNOS), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor (3, 11, 27). Whereas ODQ attenuated the ACh response in both mutant and wild-type strains, we found that 7-NI attenuated ACh-induced vasodilation in eNOS null mice only. Our results confirm a compensatory role for nNOS-guanosine 3',5'-cyclic monophosphate (cGMP)-mediated pathways in cerebrovascular regulation in eNOS null mice.

	MATERIALS AND METHODS

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Materials. ACh (Sigma), sodium nitroprusside (SNP) (Sigma), 7-NI (Calbiochem), and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, Research Biochemicals, Natick, MA) were dissolved in artificial cerebrospinal fluid (aCSF) for superfusion. ODQ (Tocris, Cookson, MO) was dissolved in dimethyl sulfoxide and then diluted in aCSF.

General preparation. Wild-type (SV129 and C57 Black/6) and mutant mice lacking either nNOS (type I NOS) or eNOS (type III NOS), weighing 20-27 g, were housed under diurnal lighting conditions and allowed food and tap water ad libitum. eNOS and nNOS mutant mice were constructed on SV129 and C57 background and hence we used both wild-type strains in our control experiments. The deficiency of eNOS or nNOS expression was previously verified by Southern and Western blot, NOS immunostaining, NADPH-diaphorase staining, and NOS activity (13, 15, 16, 19).

Closed cranial window preparation. A stainless steel ring (8.0 mm in inner diameter, 2.0 mm in height) containing three ports was embedded into the skull as described (23). A craniotomy (2 × 1.5 mm) was made in the left parietal bone within the ring of the window. The dura was opened while being superfused with aCSF, and a cover glass was placed. The window was fixed, and the ports were attached to inflow and outflow connections. The volume under the window was ~0.1 ml. The composition of aCSF was as described (23). The pH value of aCSF was kept at 7.35-7.45 by equilibration with 6.5% CO₂, 10% O₂, and balance N₂, and was monitored continuously with a pH meter (Corning, Corning, NY), as were temperature (36.5-37.0°C) and intracranial pressure (5-8 mmHg). The aCSF was superfused by an infusion pump (0.4 ml/min). Pial vessels were visualized by an intravital microscope (Leitz, Germany) equipped with a video camera (C2400, Hamamatsu Photonics, Hamamatsu, Japan). The diameter of a single pial arteriole (20-30 µm) was continuously measured by a video width analyzer (C3161, Hamamatsu, Japan) and recorded on line.

NOS activity assay. NOS activity was measured by the conversion of [³H]arginine to [³H]citrulline (2) with minor modifications (13, 19).

In vitro muscarinic ACh receptor autoradiography. Mice were anesthetized with halothane and decapitated. The brains were quickly removed, frozen in liquid N₂, and kept at 80°C until used. Sections (10 µm) were cut with a cryostat-microtome and thaw-mounted onto gelatin-coated slides. The slides were brought from 80°C to room temperature 30 min before the autoradiographic experiments. Slides were preincubated for 15 min at room temperature in 50 mM sodium phosphate buffer (pH 7.4) and they were then incubated for 90 min at 25°C in buffer containing 0.7 nM [³H]quinuclidinyl benzilate (QNB, 43.5 Ci/mmol, NEN, Boston, MA). Nonspecific binding was assessed by the addition of 1 µM atropine. Slides were washed 2 × 5 min in ice-cold buffer, dipped in ice-cold distilled water, dried under a stream of cold air, and exposed to ³H-Hyperfilms (Amersham) for 3 wk. Autoradiograms were analyzed by comparing the optical density of the film over specific brain regions with that over tritiated standards (Amersham) using a computerized system (M4, Imaging Research, St. Catharines, Ontario). Data are given in nanocurie bound radioligand per milligram of tissue.

Experimental design. Before drug treatment, aCSF was superfused for 20-40 min until a stable baseline diameter was achieved. To determine the effect of nNOS or guanylate cyclase inhibition on the ACh response, ACh (1 and 10 µM) was superfused for 4 min followed by washout with aCSF for an additional 10-15 min. 7-NI (100 µM) or ODQ (10 µM) was then superfused for 30 min, after which ACh (1 and 10 µM) plus 7-NI (100 µM) or ACh plus ODQ (10 µM) was superfused for 4 min. The specificity of ODQ was tested in wild-type mice by comparing the response of pial arterioles to SNP (0.1, 1, and 10 nM) or 8-BrcGMP (10, 50, and 100 µM). After this, ODQ (10 µM) was applied for 30 min followed by both SNP and ODQ or 8-BrcGMP plus ODQ. The response of pial arterioles to ACh (1 and 10 µM) was tested in another series of experiments before and after atropine (10 µM).

Data analysis. The maximum change in vessel diameter was used to calculate percent maximum dilation. Changes in vessel caliber of <5% (~1 µm) were below the limits of resolution in this preparation. All data are expressed as means ± SE. Two-way analysis of variance for repeated measurements followed by Student-Newman-Keuls multiple comparison test was used to compare ACh dose responses among the four mouse strains. Student's t-test was used to compare two groups. P < 0.05 was considered statistically significant.

	RESULTS

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Arterial blood CO₂ tension and arterial blood pH did not differ between groups and were within ranges previously reported by us (Table 1) (5, 23). The initial mean arterial blood pressures (MABP) were consistent with previous studies (10, 19, 23). Significantly lower MABP in C57 Black/6 mice was reported previously under halothane anesthesia and distinguishes this strain from others investigated (10). This lower MABP in C57 Black/6 mice probably dilated basal vessel diameter, which by itself could not account for the enhanced response to ACh described below.

Baseline vessel diameter, similar in all groups (Table 2), did not change over time (data not shown). ACh dilated pial arterioles in both SV129 and eNOS mutant mice to the same extent (Table 2). The response was larger in C57 Black/6 mice and, as previously reported, nNOS null mice (23). The enhanced ACh response in C57 Black/6 mice was observed even in a subgroup of mice with MABP of 80 ± 2 mmHg (23 ± 6 and 47 ± 11% increase above baseline, at 1 and 10 µM ACh, respectively, n = 4). In fact, low concentrations of ACh (0.1 µM) significantly relaxed pial arterioles in C57 Black/6 mice (16 ± 3% increase), whereas the increase above baseline was <5% in SV129 and eNOS null mice. The response did not differ (<10%, n = 3, P > 0.05) after repeated ACh applications in C57 Black/6 mice. Atropine completely blocked pial vessel dilation in C57 Black/6 (30 ± 8 and 62 ± 17% vs. 1 ± 6 and 1 ± 8% increase at 1 and 10 µM of ACh, respectively, P < 0.05, n = 3), as previously reported in SV129, eNOS, and nNOS mutant mice (23).

Topical 7-NI (100 µM) did not change MABP or baseline diameter, or block the dilation to ACh in SV129, C57 Black/6 (Fig. 1) or nNOS null mice (data not shown). The baseline vessel diameters in SV129, C57 Black/6, eNOS null mice, and nNOS null mice were 25.1 ± 1.3, 23.8 ± 2.0, 26.0 ± 2.1, and 22.2 ± 1.1 µm, respectively, before 7-NI, and 24.4 ± 1.6, 23.4 ± 1.6, 26.9 ± 2.2, and 21.9 ± 1.7 µm, respectively, after 7-NI. 7-NI attenuated the ACh-induced dilation in eNOS mutant by 66 (1 µM ACh) and 25% (10 µM ACh) without altering MABP or baseline diameter (P < 0.05, Fig. 1). NOS activity in subjacent cortex was inhibited by 40% (1.25 ± 0.09 vs. 0.75 ± 0.09 fmol · mg tissue¹ · min¹) in 7-NI-treated SV129 mice (P < 0.05, n = 4).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   7-Nitroindazole sodium (7-NI) superfusion (100 µM for 30 min, solid bars) attenuated ACh-induced dilation of pial arterioles in -chloralose-anesthetized endothelial nitric oxide synthase (eNOS) null mice (C), but not in SV129 (A) and C57 Black/6 (B) mice as measured in closed cranial window. Response in C57 Black/6 mice was greater than SV129 and eNOS null mice at 1 and 10 µM. Baseline diameters were 24 ± 1 (SV129, n = 5), 24 ± 2 (C57 Black/6, n = 5), and 25 ± 2 (eNOS null, n = 6). Stippled bars, artificial cerebrospinal fluid (aCSF). All values are means ± SE. * P < 0.05 vs. before 7-NI treatment.

Superfusion with ODQ alone did not change MABP or baseline vessel diameter significantly (25.2 ± 0.7, 23.5 ± 2.0, and 23.0 ± 0.8 µm before ODQ and 24.8 ± 0.5, 23.8 ± 0.9, and 25.5 ± 1.4 µm after ODQ in SV129, C57 Black/6, and eNOS null mice, respectively). ODQ inhibited ACh-induced dilation in eNOS null and SV129 mice (Fig. 2), and in C57 Black/6, in preliminary experiments (~40% inhibition at 1 µM ACh, baseline diameter 24 ± 1 µm, n = 3). As expected, ODQ superfusion significantly attenuated SNP-induced dilation (11 ± 2 vs. 4 ± 2%, 16 ± 3 vs. 4 ± 4%, and 22 ± 4 vs. 6 ± 6% at 0.1, 1, and 10 nM SNP, respectively, SV129 mice, P < 0.05 vs. before ODQ treatment, n = 3). The dilation to 8-BrcGMP (5 ± 2, 8 ± 4, and 18 ± 1% at 10, 50, and 100 µM, respectively, n = 3, SV129 mice) was not inhibited by ODQ. Baseline diameters were 26 ± 3 (SNP group) and 25 ± 1 µM (8-BrcGMP group).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Superfusion with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 µM for 30 min, solid bars) significantly attenuated ACh-induced pial arteriolar dilation in both SV129 (A) and eNOS null (B) mice. Baseline diameters were 24 ± 0 (SV129, n = 5) and 23 ± 0 µm (eNOS null, n = 6), respectively. Stippled bars, aCSF. Values represent means ± SE. * P < 0.05, ** P < 0.01 vs. before ODQ treatment.

The density and distribution of [³H]QNB binding sites in the coronal brain sections did not differ among wild-type (SV129, C57 Black/6), eNOS, or nNOS null mice (n = 6 in each group, data not shown).

	DISCUSSION

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Our study demonstrated that inhibition of nNOS or soluble guanylyl cyclase attenuated the ACh-induced pial arteriolar dilation in mice lacking the gene for type III NOS. In agreement with results in rats and rabbits (8, 32, 30) and in mice (28), we found that inhibition of soluble guanylyl cyclase reduced the ACh response in wild-type mice, whereas inhibition of nNOS activity was ineffective. Together these results support the importance of endothelium-derived NO (or related products) in ACh-induced pial arteriolar dilation of wild-type mice. The data also suggest that nNOS-cGMP-dependent mechanisms compensate or upregulate after deletion of the eNOS gene in eNOS null mice.

The source of NO-mediated dilation remains to be determined but could be derived from nNOS-containing parasympathetic perivascular nerves (12, 26, 29) or from nNOS within parenchymal neurons (25). Although NOS could be synthesized within the endothelial cells, the membrane-associated NOS activity in the aorta of eNOS null mice was <5% of wild-type (16). Previous studies showed that the density and distribution of ³H-labeled N^G-nitro-L-arginine brain binding sites remain unchanged after deletion of eNOS gene (14). As in wild-type mice, the ACh response in eNOS mutants was atropine sensitive, and we did not detect obvious differences in [³H]QNB binding between wild-type and eNOS null mice. We have not ruled out, however, an increase in the density or distribution of vascular muscarinic receptors that were not selectively visualized in our preparation. Furthermore, alterations in muscarinic receptor-coupled signal transduction may have taken place in the presence of constant receptor density.

The relatively selective nNOS inhibitor 7-NI has been widely used to characterize the role of nNOS in various cerebrovascular responses. We topically applied a water-soluble form of 7-NI to eliminate systemic effects. Consistent with previous reports (8, 30, 32), topically applied 7-NI did not attenuate the ACh response in the pial vessels of wild-type or nNOS null mice, indicating that eNOS was not inhibited at the concentration used in this study (100 µM). However, the same concentration significantly attenuated the ACh response in eNOS mutant mice, suggesting a compensatory role for nNOS after deletion of the eNOS gene. The inhibition was less than complete, suggesting that other candidate mechanisms (e.g., endothelium-derived hyperpolarizing factor, prostaglandins) may upregulate as well. We have previously demonstrated that NO generated from nNOS augments blood flow during whisker stimulation or hypercapnia (1, 21, 22). Moreover, nNOS-containing neurons may be important for cerebral blood flow augmentation by stimulation of nucleus basalis magnocellularis via muscarinic receptors (25). Therefore, nNOS is a likely candidate to compensate in the presence of eNOS deficiency.

The soluble guanylyl cyclase-cGMP pathway is a major target for NO in vascular smooth muscle (18). ODQ inhibits NO-stimulated activity of purified soluble guanylyl cyclase (11), as well as the vasodilation induced by ACh and other NO donors (24, 28) without affecting relaxation by NO-independent substances such as isoproterenol or adenosine (4, 28). ODQ also inhibits N-methyl-D-aspartate-induced cGMP increase in cerebellum, in vivo and in vitro (9, 11), and in hippocampus, in vivo (9), indicating that cGMP formation from neuronal NO sources is also sensitive to ODQ. In our study ODQ (10 µM) inhibited SNP-induced vasodilation by >70% and in a previous report inhibited cGMP production by NO donors in vitro (27). There was no further inhibition of cGMP production by 100 µM ODQ over 10 µM (3, 27). As expected, we also showed that the vasodilation by 8-BrcGMP superfusion was not altered by ODQ (24).

ACh-induced vasodilation was greater in pial vessels of C57 Black/6 than SV129 mice. These unexplained differences underscore the advantages of using wild-type littermate controls or obtaining a single background strain by breeding homozygous mutant mice backcrossed to a wild-type mouse (e.g., C57 Black/6) for at least 10 generations. Unfortunately, neither controls were available because the colony was maintained by breeding homozygote mice null for the eNOS gene. This shortcoming notwithstanding, our studies point to the importance of nNOS upregulation in ACh-induced dilation in eNOS knockout mice.