Introduction

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Molecular biological studies have demonstrated that nAChRs in the central nervous system are composed of a diverse array of subunits (₂-₉, ₂-₄) and have a range of pharmacological properties. This has led to increased interest in presynaptic nAChRs that act to modulate the release of transmitter from presynaptic terminals (McGehee and Role, 1995; Lindstrom et al., 1995; Wonnacott, 1997; Kaiser and Wonnacott, 2000). It has long been known that sympathetic neurons have prejunctional nAChRs on their nerve terminals in target tissues, where nicotinic agonists cause the release of NE (Su and Bevan, 1970; Starke, 1977; Haass et al., 1991). In contrast to the central nervous system, the biological significance and subtype of these prejunctional nAChRs in the peripheral nervous system are less defined (Kristufek et al., 1999).

We have demonstrated that nicotine-induced nitric oxide (NO)-mediated neurogenic vasodilation is dependent on intact sympathetic, adrenergic innervation in porcine basilar arteries and cat middle cerebral arteries (Zhang et al., 1998; Lee et al., 2000). This is based on the observations that nicotine-induced NO-mediated cerebral neurogenic vasodilation is abolished by guanethidine, a specific sympathetic neuronal blocker, and by chemical denervation of sympathetic nerves with 6-hydroxydopamine. These treatments, however, do not affect transmural nerve stimulation (TNS)-elicited NO-mediated neurogenic vasodilation in the same preparations. Furthermore, relaxation induced by exogenous NE in porcine basilar arterial rings was blocked by nitro-L-arginine (L-NNA) (Zhang et al., 1998). Accordingly, it was hypothesized that nicotine acted on nicotinic receptors located on sympathetic nerve terminals, resulting in release of NE, which then diffused to act on ₂-adrenoceptors located on the neighboring NOergic nerve terminals to release NO and therefore vasodilation (Zhang et al., 1998; Lee et al., 2000).

The exact nature of the nAChR receptors located on presynaptic sympathetic nerves mediating release of NE, which then elicits NO release, in cerebral arteries has not been clarified. The present study, therefore, was designed to pharmacologically and immunohistochemically characterize in isolated porcine basilar arteries and cultured superior cervical ganglia (SCG), the origin of cerebrovascular sympathetic nerves (Lee, 1981), the presynaptic nAChR subtype located on postganglionic sympathetic nerves, which mediate nicotine-induced NOergic vasodilation.

	Materials and Methods

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General Procedure. Fresh heads of adult pigs (60-100 kg) of either sex were collected at local packing companies (Excel, Beardstown, IL, and Y.T., Springfield, IL). The entire brain, with dura matter attached, was removed and placed in Krebs' bicarbonate solution equilibrated with 95% O₂ and 5% CO₂ at room temperature. The composition of the Krebs' solution was as follows: 122.0 mM NaCl, 5.16 mM KCl, 1.2 mM CaCl₂, 1.22 mM MgSO₄, 25.6 mM NaHCO₃, 0.03 mM ethylenediamine-tetraacetic acid, 0.1 mM L-ascorbic acid, and 11.0 mM glucose, pH 7.4. Basilar and middle cerebral arteries were dissected and cleaned off surrounding tissue under a dissecting microscope.

In Vitro Tissue Bath Studies. The ring segment (4 mm in length) was cannulated with a stainless steel rod (30-gauge hemispherical section) and a short piece of platinum wire and mounted horizontally in a plastic tissue bath containing 6 ml of Krebs' bicarbonate solution. The platinum wire was bent into a U shape and anchored to a gate. The stainless steel rod was connected to a strain gauge (UC2; Gould, Cleveland, OH) for isometric recording of changes in force, as described in our previous report (Lee et al., 1976). The temperature of the Krebs' solution equilibrated with 95% O₂ and 5% CO₂ was maintained at 37°C. Tissues were equilibrated in the Krebs' solution for an initial 30 min and then mechanically stretched to a resting tension of 750 mg (Zhang et al., 1998).

SCG Cell Culture. Freshly dissected SCG from animals were placed in cold Hibernate A (Life Technologies, Gaithersburg, MD) solution (Liu et al., 2000). After being cut into smaller pieces, the ganglia were transferred to Mg²⁺/Ca²⁺ free Hanks' balanced salt solution containing papain (2 U/ml; Sigma, St. Louis, MO), collagenase D (1.2 mg/ml; Roche Molecular Biochemicals, Indianapolis, IN), and Dispase (4.8 mg/ml; Life Technologies), and were incubated for 50 min at 37°C. Cells were released by gentle triturating at the end of the incubation. The cell suspension was centrifuged at 300g for 5 min. The pellet was gently resuspended in Neurobasal culture medium (Life Technologies), containing B27 (1:50 dilution; Life Technologies), 0.5 mM L-glutamine, 25 µM L-glutamate, and nerve growth factor (50 ng/ml; Alomome Lab Ltd, Jerusalem, Israel) (Brewer, 1997). All medium and Hanks' balanced slat solution contained 100 U/ml penicillin and 100 U/ml streptomycin. The cell suspension was plated into a four-well culture plate with a poly(D-lysine)-coated (50 µg/ml; Sigma) glass coverslip (12 mm in diameter; Fisher Scientific, St. Louis, MO) in each well and incubated with air containing 5% CO₂ at 37°C. The growth medium was changed every 6 days.

Double-Labeling Immunohistochemistry. Fresh porcine brain arteries obtained from local slaughterhouses were dissected and placed into picric acid-periodate-paraformaldehyde-lysine (PPPL) fixative (Yu et al., 1998) overnight at 4°C. After five washes in PBS (pH 7.4), the arteries were permeabilized and nonspecific sites were blocked with 2.5% normal donkey serum in 0.25% Triton X-100 PBS for 30 min at room temperature. The arteries were incubated with primary antibodies (anti-rabbit ₇ nAChR antibody 1:40, Santa Cruz Biotechnology Inc. Santa Cruz, CA, and anti-mouse TH antibody 1:40, Chemicon International Inc., Temecula, CA) at 4°C for 24 to 48 h. After being rinsed with PBS, pH 8.2, three times, the arteries were incubated with the secondary antibodies for 1 h at room temperature. The secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG, and tetramethylrhodamine isothiocyanate (TRITC)-conjugated donkey anti-mouse IgG (1:40, Jackson Immunoresearch, West Grove, PA) (Liu et al., 2000). After being rinsed with PBS, pH 8.2, each artery was whole-mounted with Vectashield mounting medium on Vectabond coated slides (Vector Laboratories, Burlingame, CA). The labeled specimens were observed and photographed under a fluorescence microscope fitted with proper filters (Olympus BX50 microscope). Negative controls were obtained following the same procedure without the primary antibody (Liu et al., 2000).

Drugs Used and Statistical Analysis. The following drugs were used: ()-nicotine, methyllycaconitine (MLA), -bungarotoxin (-BGTX), mecamylamine, dihydro--erythroidine (DHE), L-NNA, NE, paraformaldehyde, sodium nitroprusside, TTX, papaverine (all from Sigma), Triton X-100 (Amersham Pharmacia Biotech, Arlington Heights, IL) and U46619 (Upjohn, Kalamazoo, MI). All drugs, otherwise stated, were dissolved in deionized water, and added directly to the tissue baths. The drug concentrations reported were the final concentration in the bath.

	Results

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Nicotine- and TNS-Induced Neurogenic Vasodilation in Porcine Basilar Arteries. Consistent with our previous reports (Zhang et al., 1998; Lee et al., 2000), the porcine basilar arteries without endothelial cells, in the presence of active muscle tone induced by U-46619 (0.3 µM), relaxed exclusively upon TNS at various frequencies (2, 4, and 8 Hz), and applications of nicotine (1-100 µM) in a concentration-dependent manner (Fig. 1). The relaxation induced by nicotine was significantly blocked by TTX (0.3 µM, n = 7) and was abolished by L-NNA (30 µM, n = 6) and cold-storage denervation (n = 6, data not shown). These results suggest that the relaxation induced by TNS and nicotinic was due to release of neurogenic NO.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of MLA on relaxation of porcine basilar arteries induced by nicotine and TNS. A, representative tracing showing effect of the preferential 7-nAChR antagonist MLA on relaxation elicited by nicotine (100 µM) and TNS at 2, 4, and 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619 (0.3 µM). MLA at 10 µM almost abolished the relaxation induced by nicotine, without affecting the relaxation elicited by TNS at various frequencies. The results are summarized in B. MLA blockade of nicotine-induced relaxation was fully recovered after the arteries were washed with fresh prewarmed Krebs' solution (B, n = 6). The blockade of MLA on nicotine-induced relaxation was concentration-dependent. The IC50 values were 8.63 (2.34-31.84) × 108 M (C, n = 6). Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n is number of experiments, *p < 0.05 indicates significant difference from respective controls.

₇-nAChR Antagonists Blocked Nicotine-Induced Neurogenic Vasodilation. Since TNS at 8 Hz and nicotine at 100 µM induced maximum relaxation, these parameters, which have previously been used by us and many others (Toda and Okamura, 1998; Zhang et al., 1998; Lee et al., 2000), were used in the subsequent studies. As reported previously by many investigators, neurogenic vasodilation induced by nicotine diminished upon repeated applications of this agonist with short time intervals (Zhang et al., 1998). Accordingly, in the present study, a 90-min interval with six washes was allowed before repeating each application of nicotine. Three consecutive, reproducible relaxations induced by nicotine (100 µM) were obtained, which were not significantly different (Zhang et al., 1998). Furthermore, the relaxation elicited by repeated TNS at 8 Hz, like other reports in the porcine basilar arteries (Zhang et al., 1998; Lee et al., 2000), was reproducible and not different.

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View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of -BGTX on relaxation of porcine basilar arteries induced by nicotine and TNS. A, representative tracing showing effect of a preferential 7-nAChR antagonist -BGTX on relaxation elicited by nicotine (100 µM) and TNS at 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619 (0.3 µM). -BGTX at 1 µM almost abolished the relaxation induced by nicotine without affecting the relaxation elicited by TNS. The results are summarized in B. -BGTX blockade of nicotine-induced relaxation was fully recovered after the arteries were washed with fresh prewarmed Krebs' solution (B, n = 5). The blockade of -BGTX on nicotine-induced relaxation was concentration-dependent. The IC50 values were 8.80 (5.62-13.88) × 109 M (C, n = 5). Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n is number of experiments, *p < 0.05 indicates significant difference from respective controls.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of mecamylamine on relaxation of porcine basilar arteries induced by nicotine and TNS. A, representative tracing showing effect of mecamylamine on relaxation elicited by nicotine (100 µM) and TNS at 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619 (0.3 µM). Mecamylamine at 10 µM almost abolished the relaxation induced by nicotine without affecting the relaxations elicited by TNS. The results are summarized in B. Mecamylamine blockade of nicotine-induced relaxation was fully recovered after the arteries were washed with fresh prewarmed Krebs' solution (B, n = 4). The blockade of mecamylamine on nicotine-induced relaxation was concentration-dependent. The IC50 values were 9.48 (3.21-27.99) × 108 M (C, n = 4). Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n is number of experiments; *p < 0.05 indicates significant difference from respective controls.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Failure of DHE in affecting relaxation in porcine basilar arteries induced by nicotine and TNS. A, representative tracing showing effect of the preferential 4-nAChR antagonist DHE on relaxation elicited by nicotine (100 µM) and TNS at 8 Hz in a basilar artery in the presence of active muscle tone induced by U-46619 (0.3 µM). DHE at 10 µM had no effect on the relaxation induced by nicotine or TNS. The results are summarized in B. Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n is number of experiments.

In Vitro Growth of Porcine SCG Neurons. Isolated SCG cells started to adhere to the poly(D-lysine)-coated surface of glass coverslips 2 to 3 h after incubation. At this stage, they were spherical with various sizes. Some of the cells started to extend processes within 24 to 48 h of incubation. After a week, the processes of the cells were well developed and formed networks at places where the cell density was high. Growing cells always stayed close to each other to form several high-density cell "islands" and left other areas nearly blank. Generally, there were two types of cells that could be visually distinguished in the culture: cells with small spindle-liked soma (the majority) and those with large round soma. Most large soma cells were monopolar or bipolar cells, while most small soma cells were bipolar or tripolar cells. Cells survived in culture at least for 4 weeks. When cultured for a longer time (>4 weeks), the individual cell became ambiguous with a membrane-like substance that appeared around cell soma, and the cells began to detach from the coverslips (data not shown). Therefore, cells between 7 and 10 days in culture were used for immunocytochemical study.

Immunohistochemistry. Results from double-labeling immunostaining studies, i.e., FITC-conjugated second antibody to detect the ₇-nAChR and TRITC-conjugated second antibody to detect tyrosine hydroxylase, indicated the presence of ₇-nAChR- (Figs. 5A and 6A) and tyrosine hydroxylase-immunoreactive fibers (Figs. 5B and 6B) in whole-mount basilar and middle cerebral arteries, and of cultured SCG cells (7-10 days). The neuronal nature of cultured SCG was verified by positive immunoreactivities of both soma and dendrites of SCG for neurofilament 200, a neuron marker (data not shown). Almost all tyrosine hydroxylase-immunoreactive fibers were coincident with ₇-nAChR-immunoreactive fibers in the whole-mount preparations, and ₇-nAChR-immunoreactivities and tyrosine hydroxylase-immunoreactivities were coexpressed in both dendrites and soma of the same SCG cells. ₇-nAChR immunoreactive bundle fibers were frequently found to be slightly thicker than the corresponding tyrosine hydroxylase immunoreactive fibers in whole-mount preparations (Fig. 5), while these two immunoreactive fibers of cultured SCG cells were almost identical (Fig. 6).

View larger version (117K): [in this window] [in a new window]   Fig. 5.   7-nAChR- and tyrosine hydroxylase immunoreactive fibers in a whole-mount porcine basilar artery. Florescence photomicrographs showing almost all 7-nAChR immunoreactive fibers (FITC-labeling, arrowheads in A) were coincident with tyrosine hydroxylase immunoreactive fibers (TRITC-labeling, arrowheads in B). Scale bar, 50 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 6.   Colocalization of 7-nAChR- and tyrosine hydroxylase immunoreactivities in cultured porcine SCG cells (8 days). Florescence photomicrographs showing all 7-nAChR-immunoreactive somas (FITC-labeling, arrows in A) and dendrites (arrowheads in B) were also tyrosine hydroxylase immunoreactive (TRITC-labeling, arrowheads for dendrites and arrows for somas in B). Scale bar, 25 µM.

	Discussion

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Nicotine has been shown to elicit neurogenic NOergic vasodilation in peripheral and cerebral arteries in many species (Jiang et al., 1997; Toda et al., 1997; Uchiyama et al., 1997; Okamura et al., 1999). It was assumed that nicotine acted directly on NOergic nerve terminals to release NO, resulting in NO-mediated neurogenic vasodilatation. This assumption however was questioned. Our recent studies demonstrated for the first time that nicotine-induced NO-mediated relaxation in porcine basilar arteries was dependent exclusively on intact sympathetic innervation (Zhang et al., 1998). Following a complete blockade of sympathetic transmission with guanethidine, or chemical denervation of sympathetic nerves with 6-hydroxydopamine, nicotine never induced a relaxation, although TNS-elicited NO-mediated relaxation in the same preparations remained unchanged. This latter finding was consistent with morphological observations that NOergic innervation remained intact, while adrenergic nerves were completely denervated following treatment with 6-hydroxydopamine (Zhang et al., 1998). These results indicate that in porcine basilar arteries nicotine does not act directly on NOergic nerves to release transmitter NO. Rather, nicotine acts on the nicotinic receptors located on sympathetic nerves to release NE, which then diffuses to act on adrenoceptors located on the neighboring NOergic nerves, causing release of NO from these nerves (Zhang et al., 1998; Lee et al., 2000). The role of NE as the mediator released from the sympathetic nerves was further supported by the findings that ₂-adrenoceptors located on the NOergic nerves mediated nicotine-induced relaxation (Lee et al., 2000). This functional axo-axonal interaction is also supported by morphological evidence that close apposition (25 nm) between the adrenergic nerve terminals and the nonadrenergic nerve terminals is a characteristic of innervation of cerebral arteries at the base of the brain in several species (Iwayama et al., 1970; Edvinsson et al., 1977; Lee, 1981; Barroso et al., 1996). This nicotine-induced, sympathetic-dependent neurogenic vasodilation also has been demonstrated in the mesenteric vascular beds (Shiraki et al., 2000).

Release of NE by nicotine acting on nicotinic receptors located on sympathetic adrenergic nerve terminals is well established (Su and Bevan, 1970; Haass et al., 1991). The exact nature of the nAChR on adrenergic sympathetic nerves mediating transmitter release in regulating vascular function, including cerebral circulation, however, has not been determined. Results of the present study indicated for the first time that ₇-nAChR located on the sympathetic nerve terminals mediated nicotine-induced neurogenic NOergic vasodilation in porcine basilar arteries. This conclusion was based on the findings that nicotine-induced relaxation was blocked by preferential ₇-nAChR antagonists but not by preferential ₄-nAChR antagonists. The findings that preferential ₇-nAChR antagonists (-BGTX and MLA) and nonspecific nAChR antagonist (mecamylamine) almost completely block nicotine-induced relaxation, with the former more potent (-BGTX) than or equally potent (MLA) to the latter, further suggest that ₇-nAChR on sympathetic nerves play a major role in modulating transmitter NE release in porcine basilar arteries.

The presence of postganglionic sympathetic innervation of superior cervical ganglionic origin in cerebral circulation in many species is well established (Lee et al., 1976; Lee, 1981). Nicotine-induced relaxation in isolated porcine basilar arteries, therefore, suggests that functional ₇-nAChRs are located on the postganglionic sympathetic neurons and terminals. This conclusion is supported by the immunohistochemical findings that ₇-nAChR- and tyrosine hydroxylase-immunoreactive fibers were almost completely coincident in whole-mount basilar arterial preparations, and that ₇-nAChR- and tyrosine hydroxylase immunoreactivities were completely coexpressed in both dendrites and soma of cultured SCG. These results provide strong evidence for the presence of ₇-nAChRs on tyrosine hydroxylase-containing adrenergic, sympathetic nerves. These findings also suggest the presence of ₇-nAChRs on porcine SCG neurons, a result consistent to that found by electrophysiological study in the rat SCG (Cuevas et al., 2000).

In whole-mount arterial preparations in the present study, most ₇-nAChR-immunoreactive fibers were found thicker than tyrosine hydroxylase immunoreactive fibers, while the coexpressed ₇-nAChR- and tyrosine hydroxylase immunoreactive fibers in cultured SCG are identical. This is consistent with the suggestion that sympathetic and nonsympathetic (parasympathetic) nerves run closely together, suggesting that ₇-nAChRs also are present on nonsympathetic or parasympathetic nerves. The reason for the lack of effect of ₇-nAChRs located on nonsympathetic NOergic nerves in mediating nicotine-induced relaxation (Zhang et al., 1998; Lee et al., 2000) remains to be determined. The present results from immunohistochemical studies, however, support our hypothesis that nicotine acts on presynaptic nicotinic receptors (₇-nAChR) on the adrenergic nerve terminals to release NE, which then acts on the presynaptic ₂-adrenoceptors located on the neighboring NOergic nerves to cause release of NO and therefore vasodilation (Lee et al., 2000).

The superior cervical ganglionic neurons of the rat have been shown to express a variety of nAChR genes, including ₇, and display nicotine-induced responses (Brown and Fumagalli, 1977; Kristufek et al., 1999; Skok et al., 1999; Erkman et al., 2000). The ₇-nAChRs have a high relative permeability to calcium-dependent events in neurons, including release of neurotransmitter from presynaptic sites in the brain and periphery (McGehee et al., 1995; Gray et al., 1996; Broide and Leslie, 1999; Fu et al., 1999). Furthermore, ₇-nAChRs in most instances have been found to develop nicotinic responses that rapidly desensitize and are blocked by -BGTX (Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Zhang et al., 1994; Blumenthal et al., 1997; Cuevas et al., 2000). These findings on characteristics of ₇-nAChRs are consistent with our present and previous observations that nicotine-induced relaxation in isolated porcine basilar arteries develops tachyphylaxis upon repeated applications of nicotine in short time intervals (Zhang et al., 1998; Lee et al., 2000).

The suggestion that ₇-nAChRs on sympathetic nerves mediate NE release in porcine basilar arteries in the present study is consistent with findings in several reports, for examples, in rat dorsal raphe neurons (Li et al., 1998) and rat hippocampus (Fu et al., 1999). Other investigators, however, have demonstrated that non-₇-nAChRs such as ₃₄ subunits on sympathetic nerves in the rat stomach (Yokotani et al., 2000) and rat hippocampal synaptosomes (Luo et al., 1998), and ₃₂ in rat hippocampal slices (Sershen et al., 1997) are involved in regulating nicotine-induced NE release. It appears that regional variations exist in functional subunits of nAChRs on adrenergic neurons in regulating NE release.

In summary, the present study demonstrates for the first time that ₇-nAChRs are present on perivascular postganglionic, sympathetic nerves of SCG origin in porcine basilar arteries. This ₇-nAChR appears to be the main type of nAChR that is functional in mediating nicotine-induced release of NE. This result further supports our hypothesis that nicotine acts on nAChR on sympathetic nerve terminals to release NE, which then diffuses to act on presynaptic ₂-adrenoceptors located on the neighboring NOergic nerves, causing release of NO and therefore vasodilation.