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CARDIOVASCULAR

Pb²⁺ Inhibition of Sympathetic ₇-Nicotinic Acetylcholine Receptor-Mediated Nitrergic Neurogenic Dilation in Porcine Basilar Arteries

Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois (M.-L.S., T.J.-F.L.); and College of Life Sciences and Neuro-Medical Scientific Center, Tzu Chi University, Hualien, Taiwan (T.J.-F.L.)

Received November 22, 2002; accepted February 25, 2003.

	Abstract

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Chronic exposure to inorganic lead (Pb²⁺) has been shown to facilitate peripheral vasoconstriction causing hypertension. Effect of lead on cerebral vascular function has not been reported. We have suggested in isolated porcine cerebral arteries that ₇-nicotinic acetylcholine receptors (₇-nAChRs) on perivascular sympathetic nerves mediate calcium influx in these neurons, resulting in release of norepinephrine. The released norepinephrine then acts on presynaptic ₂-adrenoceptors located on the neighboring nitrergic nerve terminals, causing nitric oxide (NO) release and vasodilation. Because Pb²⁺ has been shown to inhibit ₇-nAChR-mediated responses in the central nervous system, effects of Pb²⁺ on ₇-nAChR-mediated nitrergic neurogenic dilation in isolated porcine basilar arteries and calcium influx in cultured superior cervical ganglion (SCG) cells of the pig were examined using in vitro tissue bath and confocal microscopic techniques. The results indicated that Pb²⁺ (but not Cd²⁺, Zn²⁺, or Al³⁺) in a concentration-dependent manner blocked relaxation of endothelium-denuded basilar arterial rings induced by nicotine (100 µM) and choline (1 mM) without affecting relaxation induced by sodium nitroprusside or isoproterenol. Furthermore, significant calcium influx in cultured SCG cells induced by choline and nicotine was attenuated specifically by Pb²⁺ with IC₅₀ values comparable with those from tissue bath study. These results provide evidence supporting that lead is a likely antagonist for ₇-nAChRs that are found on postganglionic sympathetic adrenergic nerve terminals of SCG origin. Furthermore, these results indicate that lead can attenuate dilation of cerebral arteries by blocking sympathetic nerve-mediated release of NO from the perivascular nitrergic nerves.

Chronic exposure to lead also has been shown to increase plasma norepinephrine and epinephrine, which may also account for its hypertensive effect (Carmignani et al., 2000). This effect of lead has been suggested to be due to an increased sympathetic activity by central mechanisms (Carmignani et al., 2000; Lai et al., 2002). Direct effect of lead on perivascular postganglionic sympathetic nerves, however, has not been reported.

We have shown recently that nicotine-induced nitric oxide (NO)-mediated neurogenic vasodilation in porcine basilar arteries and feline middle cerebral arteries is dependent on intact perivascular sympathetic, adrenergic innervation originating in the superior cervical ganglion (SCG) (Zhang et al., 1998; Si and Lee, 2002). We have further demonstrated in porcine basilar arteries that nicotine and choline act on ₇-nAChRs located on perivascular postganglionic sympathetic nerve terminals to release norepinephrine, which then acts on presynaptic ₂-adrenoceptors located on the neighboring nitrergic nerve terminals, resulting in release of NO and vasodilation (Lee et al., 2000; Si and Lee, 2001, 2002). Because Pb²⁺ has been shown to inhibit ₇-nAChR-mediated responses in the central nervous system (Mike et al., 2000), effect of Pb²⁺ on perivascular ₇-nAChR-mediated neurogenic vasodilation in porcine basilar arteries was therefore examined in the present study using in vitro tissue bath and calcium image confocal microscopic techniques. Several other metal ions such as zinc (Zn²⁺), cadmium (Cd²⁺), and aluminum (Al³⁺) were examined in parallel because these ions also have been shown to affect the central nerve system and peripheral circulation, possibly through blocking or regulating nicotinic receptors (Gulya et al., 1990; Luoma et al., 1995; Zhao et al., 1996; Palma et al., 1998; Varner et al., 1998). Our results indicated that lead (but not Zn²⁺, Cd²⁺, or Al³⁺) in a concentration-dependent manner blocked ₇-nAChR-mediated calcium influx in cultured SCG neurons and diminished nicotine- and choline-induced sympathetic-dependent nitrergic vasodilation in isolated porcine basilar arteries.

	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, Beards-town, IL; 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 long) 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 Instrument Systems Inc., 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).

The basilar arterial ring segments were then precontracted with U-46619 (0.3–3 µM) to induce an active muscle tone of 0.5 to 0.75 g. Transmural nerve stimulation (TNS) at 8 Hz, nicotine (100 µM), and choline (1 mM) were applied to induce a relaxation. After relaxation induced by TNS, 100 µM nicotine, or 1 mM choline, the arteries were washed with prewarmed Krebs' solution. A similar magnitude of active muscle tone was induced with U-46619 again, and TNS was repeated (to serve as a control compared with the relaxation elicited by TNS before the wash). Effects of different concentrations of PbCl₂, ZnCl₂, CdCl₂, and AlCl₃ (1–10 µM) were then administered, and TNS and nicotine/choline at the same concentration before the wash were repeated. To avoid possible development of tachyphylaxis upon repeated applications of nicotinic agonists, at least 90 min with six washes (every 15 min) was allowed before the next application of nicotinic agonists (Zhang et al., 1998; Lee et al., 2000; Si and Lee, 2002). Experimental drugs were added at least 30 min before TNS and application of nicotinic agonists. After this, the arteries were washed with prewarmed Krebs' solution again. A similar magnitude of active muscle tone was induced with U-46619 again, and TNS and nicotine/choline were repeated (to serve as a second control compared with the relaxation elicited by TNS and nicotine before the drug application).

For TNS, tissues were electrically, transmurally stimulated with a pair of electrodes through which 100 biphasic square-wave pulses of 0.6 ms in duration and 200 mA in intensity were applied at various frequencies (Zhang et al., 1998). Stimulation parameters were continuously monitored on a Tektronix oscilloscope. The neurogenic origin of this TNS-induced response was verified by its complete blockade by tetrodotoxin (0.3 µM). At the end of each experiment, papaverine (100 µM) was added to induce a maximum relaxation. The magnitude of a vasodilator response was expressed as a percentage of the maximum response induced by papaverine (Zhang et al., 1998).

For examining effects of experimental drugs on relaxation induced by isoproterenol or sodium nitroprusside, concentration-response relationships for these two vasodilators were obtained by a cumulative technique in arteries without endothelial cells in the presence of active muscle tone induced by U-46619. After the arterial rings were washed with prewarmed Krebs' solution, a similar magnitude of active muscle tone was again induced by U-46619. The experimental drugs were then added, and 15 min later, concentration-response relations for isoproterenol or sodium nitroprusside were repeated. EC₅₀ values (the concentration that produces 50% of the maximum relaxation) were determined for each arterial ring. From these values, the geometric means EC₅₀ values with 95% confidence intervals (Fleming et al., 1972) were calculated.

The endothelial cells of all arterial ring segments were mechanically removed by a standard brief gentle rubbing of the intimal surface with a stainless steel rod having a diameter (25–30 gauge) equivalent to the lumen of the arteries (Zhang et al., 1998; Lee et al., 2000). A complete removal of endothelial cells was verified by lack of effect of nitro-L-arginine in increasing basal tone (Zhang et al., 1998; Lee et al., 2000).

SCG Cell Culture. Freshly dissected SCG from animals were placed in cold Hibernate A (Invitrogen, Carlsbad, CA) 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-Aldrich, St. Louis, MO), collagenase D (1.2 mg/ml; Roche Diagnostics, Indianapolis, IN), and dispase (4.8 mg/ml; Invitrogen), and were incubated for 50 min at 37°C. Cells were released by gentle trituration 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 (Invitrogen) containing B27 (1:50 dilution; Invitrogen), 0.5 mM L-glutamine, 25 µM L-glutamate, and nerve growth factor (50 ng/ml; Alomone Labs, Jerusalem, Israel) (Brewer, 1997). All media and Hanks' balanced salt 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-Aldrich) glass coverslip (12 mm diameter; Fisher Scientific Co., Fair Lawn, NJ) in each well and incubated with air containing 5% CO₂ at 37°C. The growth medium was changed every 6 days. The SCG cells were stained with anti-rabbit neurofilament 200 (Sigma-Aldrich) as a marker of neuronal cells (Liu et al., 2000).

Intracellular Calcium Imaging. Between 3 and 7 days in culture, the SCG cells were used to examine effects of nicotine and choline on calcium influx in these cells by confocal microscopy. The cells were washed with physiological buffer (130 mM NaCl, 5 mM KCl, 10 mM HEPES, 5 mM glucose, 2 mM CaCl₂, and 2 mM MgCl₂, pH 7.3) and were loaded with 3 µM fluo-4 AM in physiological buffer and incubated at room temperature for 30 min. The cells were washed with calcium indicator-free buffer to remove any dye that is nonspecifically associated with the cell surface, and then incubated for a further 30 min to allow complete de-esterification of intracellular AM esters. Nicotine (100 µM) or choline (1 mM) was then applied, and the calcium influx was measured. PbCl₂, ZnCl₂, CdCl₂, and AlCl₃ at 1 to 10 µM were added 15 min before application of nicotine, choline, or KCl (50 mM). Calcium fluorescence images were examined with a Fluoview confocal microscope (Olympus, Melville, NY). Fluo-4 was excited at 488 nm, and emitted fluorescence was filtered with a 535 ± 25-nm bandpass filter and read into a computer running Fluoview software and quantified using this software (Si and Lee, 2002).

Drugs and Statistical Analysis. The following drugs were used: (-)-nicotine, acetylcholine, choline chloride, N-nitro-L-arginine, tetrodotoxin, papaverine, isoproterenol, sodium nitroprusside, PbCl₂, ZnCl₂, CdCl₂, and AlCl₃ (all from Sigma-Aldrich); U-46619 (Upjohn, Kalamazoo, MI); and Fluo-4 AM (Molecular Probes, Eugene, OR). All drugs, unless 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 were expressed as means ± S.E.M. Statistical analysis was evaluated by analysis of variance, and Student's t test for paired or unpaired samples as appropriate. The p < 0.05 level of probability was accepted as significant.

	Results

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Choline-, Nicotine-, and TNS-Induced Neurogenic Vasodilation in Porcine Basilar Arteries. Consistent with our previous reports (Zhang et al., 1998; Lee et al., 2000; Si and Lee, 2002), the porcine basilar arteries without endothelial cells, which in the presence of active muscle tone induced by U-46619 (0.3 µM), were relaxed exclusively upon TNS (8 Hz), and applications of nicotine (100 µM) or choline (1 mM) (Figs. 1 and 2). The relaxation induced by nicotine and choline was significantly blocked by tetrodotoxin (0.3 µM; n = 7) and was abolished by N-nitro-L-arginine (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 (18K): [in this window] [in a new window]   Fig. 1. Effects of Pb2+ on relaxation of porcine basilar arteries without endothelial cells induced by nicotine (Nic) and TNS. A, representative tracing showing effect of Pb2+ on relaxation elicited by nicotine (100 µM) and TNS at 8 Hz in an endothelium-denuded basilar arterial ring in the presence of active muscle tone induced by U-46619 (0.3 µM). PbCl2 at 10 µM almost abolished the relaxation (estimated as a percentage of papaverine/PPV-elicited maximum relaxation) induced by nicotine without affecting that elicited by TNS at 8 Hz. The results were summarized in B, indicating that PbCl2 blockade of nicotine-induced relaxation was fully recovered after the arteries were washed with fresh prewarmed Krebs' solution (n = 5). The blockade by PbCl2 of nicotine-induced relaxation was concentration-dependent (C; n = 6). Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n, number of experiments; *, p < 0.05 indicates significant difference from respective controls.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effects of Pb2+ on relaxation of porcine basilar arteries without endothelial cells induced by choline and TNS. A, representative tracing showing effect of the Pb2+ on relaxation elicited by choline (1 mM) and TNS at 8 Hz in an endothelium-denuded basilar arterial ring in the presence of active muscle tone induced by U-46619 (0.3 µM). PbCl2 at 10 µM almost abolished the relaxation (estimated as a percentage of papaverine/PPV-elicited maximum relaxation) induced by choline without affecting the relaxation elicited by TNS at 8 Hz. The results were summarized in B, indicating that PbCl2 blockade of choline-induced relaxation was fully recovered after the arteries were washed with fresh prewarmed Krebs' solution (n = 5). The blockade by PbCl2 of choline-induced relaxation was concentration-dependent (C; n = 6). Arrowheads in A indicate repeated washings. Values are means ± S.E.M. n, number of experiments; *, p < 0.05 indicates significant difference from respective controls.

Pb²⁺ Inhibition of Nicotine- and Choline-Induced Neurogenic Vasodilation. Because TNS at 8 Hz, nicotine at 100 µM, and choline at 1 mM 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; Si and Lee, 2002), were used in the subsequent studies. As reported previously by many investigators, neurogenic vasodilation induced by nicotinic agonists diminished upon repeated applications of this agonist with short time intervals (Zhang et al., 1998; Si and Lee, 2002). Accordingly, in the present study, a 90-min interval with six washes was allowed before repeating each application of nicotine and choline. Three consecutive, reproducible relaxations induced by nicotine (100 µM) or choline (1 mM) were obtained, which were not significantly different (Zhang et al., 1998; Si and Lee, 2002). 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 was not different.

In basilar arteries (without endothelial cells) in the presence of active muscle tone induced by U-46619 (0.3 µM), relaxation induced by nicotine (100 µM) and choline (1 mM) was blocked by PbCl₂ (10 µM; n = 5; Figs. 1, A and B, and 2, A and B) in a concentration-dependent manner (Figs. 1C and 2C; n = 6). These concentrations of PbCl₂ did not affect the TNS-elicited relaxation (Figs. 1, A and B, and 2, A and B). The IC₅₀ values for PbCl₂ against nicotine- and choline-induced relaxation were 5.63 (2.12–15.81) x 10^-6 M and 4.76 (1.83–13.12) x 10^-6 M, respectively, which were not significantly different (p > 0.05). The PbCl₂ blockade of relaxation induced by nicotine and choline was reversible after washing off PbCl₂ (Figs. 1, A and B, and 2, A and B). On the other hand, ZnCl₂, CdCl₂, and AlCl₃ at 10 µM did not affect relaxation induced by choline, nicotine, or TNS (Fig. 3; n = 5). At concentrations higher than 10 µM, these heavy metals decreased basal tone of the arteries without endothelial cells, and inhibited vasodilation induced by nicotine, choline, and TNS (data not shown), suggesting possible nonspecific effects of these metals at high concentrations.

View larger version (36K): [in this window] [in a new window]   Fig. 3. ZnCl2, CdCl2, and AlCl3 at 10 µM did not affect choline- and nicotine-elicited relaxation of porcine endothelium-denuded basilar arterial rings in the presence of active muscle tone induced by U-46619 (0.3 µM). Relaxation was estimated as percentage of papaverine (PPV)-induced maximum relaxation. Values are means ± S.E.M. n, number of experiments.

Pb²⁺ Had No Effect on Sodium Nitroprusside- and Isoproterenol-Induced Relaxation in Basilar Arteries. In the presence of active muscle tone induced by U-46619 (0.3 µM), porcine basilar arteries without endothelial cells relaxed upon application of sodium nitroprusside and isoproterenol in a concentration-dependent manner (Fig. 4), a result similar to that reported previously (Lee et al., 2000). Pb²⁺ (10 µM) did not affect relaxation induced by sodium nitroprusside (10 nM–0.1 mM) or isoproterenol (1 nM–3 µM) in arteries denuded of endothelial cells (Fig. 4). The EC₅₀ values for sodium nitroprusside in control and in the presence of PbCl₂ were 5.76 (2.53–13.12) x 10^-6 M and 5.06 (2.78–15.32) x 10^-6 M, respectively (n = 6; p > 0.05), and those for isoproterenol were 2.78 (0.91–8.54) x 10^-7 M and 3.01 (1.25–8.94) x 10^-7 M, respectively (n = 7; p > 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of Pb2+ on relaxation in basilar arteries induced by sodium nitroprusside (SNP) (A) and isoproterenol (B). In the presence of active muscle tone induced by U-46619 (0.3 µM), porcine basilar arterial rings without endothelial cells relaxed upon application of SNP and isoproterenol in a concentration-dependent manner. The relaxation (estimated as percentage of papaverine/PPV-elicited maximum relaxation) induced by either dilator substance was not affected by Pb2+. Values are means ± S.E.M. n, 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 dense cell "islands" and left other areas nearly blank. 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 formed around cell soma, and the cells began to detach from the coverslips (data not shown). Therefore, cells between 3 and 7 days in culture were used for calcium influx study. 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).

Pb²⁺ Blockade of Choline- and Nicotine-Induced Calcium Influx in Cultured SCG. Cultured SCG cells, like cerebral perivascular sympathetic neurons in whole-mount arterial preparations, have been shown to contain dense ₇-nAChRs (Si and Lee, 2001), which form membrane cation channels possessing high Ca²⁺ permeability (Sargent, 1993). Using the intracellular calcium imaging indicator Fluo-4 AM to examine calcium influx, as shown in Fig. 5, both nicotine (100 µM) and choline (1 mM) induced a significant increase in calcium image in the SCG cells (1524 of 1780 cells in 10 plates from at least 10 animals, and 1455 of 1636 cells in eight plates from at least eight animals, respectively). Quantitative analysis on single cells indicated that both nicotine (100 µM) and choline (1 mM) significantly increased calcium image in the SCG cells as reported previously (Si and Lee, 2002). Addition of KCl (50 mM) did not further increase intracellular calcium. The nicotine- and choline-induced calcium influx was attenuated in cells pretreated with Pb²⁺ in a concentration-dependent manner (1–10 µM; Figs. 5C and 6), with IC₅₀ values of 4.56 (2.35–9.87) x 10^-6 M and 3.87 (1.94–8.75) x 10^-6 M, respectively (p > 0.05). PbCl₂ alone did not affect calcium influx (Figs. 5C2 and 6). In the presence of blockade of calcium influx by Pb²⁺, KCl (50 mM) still induced a significant calcium influx (Figs. 5C4 and 6), which was comparable with that seen in preparations in the absence of antagonist, Pb²⁺. In this latter study, KCl-induced intracellular calcium increases over the basal concentration before and after Pb²⁺ (10 µM) were 321 ± 25.4 and 328 ± 32.3% (n = 4), respectively, and were not statistically different (p > 0.05). In contrast, ZnCl₂, CdCl₂, and AlCl₃ at 10 µM did not affect calcium influx induced by nicotine or choline (Fig. 7), suggesting the specific blockade of ₇-nAChRs by Pb²⁺.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effects of Pb2+ on nicotine- and choline-induced calcium influx in cultured porcine SCG cells. Cultured SCG cells were loaded with fluo-4 AM (3 µM) in physiological buffer and incubated at room temperature for 30 min (A1, B1, and C1). Nicotine (100 µM) or choline (1 mM) was applied to the medium and changes in the calcium image in the neuronal cells were determined. Both choline (A2) and nicotine (B2) induced a significant calcium influx. Pb2+ (10 µM; C2), which did not affect the basal intracellular calcium concentrations, blocked choline-elicited calcium influx (C3). In the presence of Pb2+ blockade of choline effect, KCl (50 mM) induced significant calcium influx in these cells (C4).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Summary of Pb2+ blockade of choline (1 mM)and nicotine (100 µM)-induced calcium influx in the porcine SCGs. Changes in intracellular calcium was compared with the basal calcium concentration, which served as control (100%). Pb2+ (1–10 µM) inhibited choline- and nicotine-induced calcium influx in a concentration-dependent manner. In the presence of Pb2+ (10 µM), KCl (50 mM) still induced calcium influx that was comparable with that in the absence of Pb2+ (see Results). Values are means ± S.E.M. n, number of experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Summary of effects of ZnCl2, CdCl2, and AlCl3 on choline- and nicotine-induced calcium influx in the porcine SCGs. Changes in intracellular calcium was compared with the basal calcium concentration, which served as control (100%). ZnCl2, CdCl2, or AlCl3 at 10 µM did not affect basal calcium levels. In the presence of these metal ions, calcium influx induced by choline (1 mM) and nicotine (100 µM) was comparable with that found in control preparations in the absence of any inhibitor (see Fig. 6). Addition of KCl (50 mM) did not further increase calcium influx. Values are means ± S.E.M. n, number of experiments.

	Discussion

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The present study demonstrated that acute administration of PbCl₂ (but not ZnCl₂, CdCl₂, or AlCl₃) blocked nicotine- and choline-induced ₇-nAChR-mediated calcium influx in cultured SCG neurons, and diminished nicotine- and choline-induced sympathetic ₇-nAChR-mediated nitrergic dilation in isolated porcine basilar arteries. These results provide further evidence supporting the presence of functional ₇-nAChRs on cerebral perivascular postganglionic sympathetic adrenergic neurons of SCG origin.

Nicotine has been shown to elicit neurogenic nitrergic vasodilation in peripheral and cerebral arteries in many species (Jiang et al., 1997; Toda et al., 1997; Uchiyama et al., 1997; Zhang et al., 1998; Okamura et al., 1999). Strong evidence indicates that in porcine basilar arteries nicotinic agonists do not act directly on nitrergic nerves to release transmitter NO. Rather, these agonists act on ₇-nAChRs located on sympathetic nerves to release norepinephrine, which then diffuses to act on ₂-adrenoceptors located on the neighboring nitrergic nerves, causing release of NO from these nerves and vasodilation (Zhang et al., 1998; Lee et al., 2000; Si and Lee, 2001, 2002).

This hypothesis that functional ₇-nAChRs located on the sympathetic (but not the parasympathetic) nerve terminals mediating sympathetic-dependent, nitrergic vasodilation in porcine basilar arteries (Si and Lee, 2001) is further supported by results of the present study. The relaxation of porcine endothelium-denuded basilar arteries induced by nicotine and choline, a selective agonist for ₇-nAChR (Si and Lee, 2002), was blocked specifically by lead, which has been shown to be a likely antagonist for ₇-nAChRs in the central nerve system (Mike et al., 2000). Similarly, choline- and nicotine-induced ₇-nAChR-mediated calcium influx in the cultured SCG cells, the origins of cerebral sympathetic nerves, was blocked specifically by lead in a concentration-dependent manner.

The exact nature of the antagonism by lead still remains unknown. We speculate that lead, a cation like calcium, will occupy the nAChR channels and block the calcium influx. The IC₅₀ for lead inhibition is comparable with that by nicotinic receptor antagonists (Zhang et al., 1998; Si and Lee, 2001, 2002). In addition, lead has been suggested to accelerate the rate of nicotinic receptor desensitization (Mike et al., 2000). Furthermore, lead-blocking effect on nicotine- and choline-induced relaxation is readily reversible after washing off, suggesting that the lead effect is not due to an intracellular action. These results favor the notion of direct interaction between lead and the ₇-nAChR.

According to this axo-axonal or sympathetic-nitrergic interaction hypothesis, ₂-adrenoceptors located on the nitrergic nerves play a key role in mediating norepinephrine-induced NO release from these nerves (Zhang et al., 1998; Lee et al., 2000; Si and Lee, 2001, 2002). Any possible effect of lead on ₂-adrenoceptors, therefore, may alter NO-mediated relaxation. Indeed, chronic treatment of animals with lead has been shown to decrease -adrenoceptors in the heart and the central nerve system (Chang et al., 1997; Tsao et al., 2000). In the present study, relaxation of the basilar arteries induced by isoproterenol that acts on both presynaptic and postsynaptic -adrenoceptors, however, was not appreciably affected by lead. Therefore, lead blockade of relaxation induced by nicotine or choline in the present study was not likely due to any effect on presynaptic ₂-adrenoceptors or postsynaptic ₁-adrenoceptors (Lee, 1994).

Furthermore, lead did not directly affect relaxation of the basilar arterial smooth muscle mediated by NO-cGMP pathway either. The relaxation induced by sodium nitroprusside, which is known to release NO upon entering the smooth muscle cells (Ignarro et al., 1981) was not affected by lead, indicating that lead blockade of nitrergic neurogenic vasodilation induced by nicotine and choline was not due to blockade of NO-cGMP pathway in the smooth muscle. Similar results were found in the rat aorta (Purdy et al., 1997).

Chronic exposure to lead has been shown to affect neuronal nitric-oxide synthase activity with a decrease or increase in NO concentrations depending on different brain regions (Chen et al., 2000; Weaver et al., 2002). Plasma levels of NO have been shown to be reduced by chronic lead treatment (Carmignani et al., 2000). In the present study, although it blocked NO-mediated relaxation of endothelium-denuded arteries induced by nicotine or choline, lead did not affect NO-mediated relaxation elicited by direct depolarization of nitrergic nerves with TNS in the same arteries. This result suggested that acute application of lead did not affect the nitric-oxide synthase activity or NO release in cerebral perivascular nitrergic nerves. Together, these results found in endothelium-denuded arteries favor the notion that lead blockade of neurogenic nitrergic vasodilation is most likely due to blocking ₇-nAChRs located on the perivascular postganglionic sympathetic nerves (Si and Lee, 2002).

Chronic low-level lead exposure is known to cause hypertension in humans and experimental animals (Harlan, 1988; Pocock et al., 1988; Khalil-Manesh et al., 1993; Schwartz, 1995; Gonick et al., 1997; Bost et al., 1999; Carmignani et al., 1999, 2000; Marques et al., 2001; Vaziri and Ding, 2001). The exact pathophysiology of lead-induced cardiovascular changes is not determined, although several hypotheses have been proposed. Lead exposure has been shown to decrease endothelium-dependent and endothelium-independent relaxation (Khalil-Manesh et al., 1993; Gonick et al., 1997; Marques et al., 2001), although this effect is not universally agreeable (Purdy et al., 1997; Shelkovnikov and Gonick, 2001). Chronic lead-treatment also has been shown to increase release of endothelial vasoconstrictor hormone, endothelin-3, and/or decrease release of endothelial vasodilator hormones (Khalil-Manesh et al., 1993; Gonick et al., 1997). Chronic lead exposure, thus, seems to favor contractile properties in peripheral vascular beds.

Chronic lead treatment also has been shown to increase plasma angiotensin-converting enzyme and kininase II, resulting in increased plasma levels of angiotensin II (a potent constrictor) and decreased plasma levels of bradykinin (an endothelium-dependent dilator). In parallel to alterations in these non-neuronal components, chronic lead treatment has been shown to increase plasma levels of norepinephrine and epinephrine, possibly due to an increased sympathetic nerve activity by central mechanisms (Carmignani et al., 2000; Lai et al., 2002), accompanied by an increased postsynaptic -adrenoceptor-mediated vasoconstriction (Webb et al., 1981). These findings may account for the increased cardiac inotropism, total peripheral resistance, and hypertension after chronic lead treatment (Carmignani et al., 2000).

Direct effects of lead on postganglionic sympathetic neural transmission in the peripheral vascular beds, however, are not clarified. In the rabbit saphenous arteries, increases in perfusion pressure resulting from electrical stimulation of periarterial sympathetic nerves were reduced or abolished by lead in concentrations that did not affect responses to exogenously applied norepinephrine or to direct electrical stimulation of the muscle (Cooper and Steinberg, 1977). These results suggested that lead reduced the response to sympathetic nerve stimulation primarily through an effect on presynaptic nerve terminals. This postganglionic sympathetic inhibitory effect of lead is in contrast to the enhanced plasma norepinephrine.

Our present study also demonstrated that lead exhibited inhibitory effect on sympathetic activities in the cerebral arteries as evidenced by blockade of ₇-nAChR-mediated calcium influx in the SCG cells and sympathetic ₇-nAChR-mediated nitrergic vasodilation. Norepinephrine is a rather weak postsynaptic transmitter in causing cerebral vasoconstriction in the large cerebral arteries at the base of the brain (Lee et al., 1976; Lee, 1994). It, however, is an effective presynaptic transmitter acting on ₂-adrenoceptors located on the neighboring nitrergic nerves to release NO, which is the primary transmitter in the cerebral neurogenic vasodilation (Lee, 1994). Lead blockade of ₇-nAChRs on the perivascular postganglionic sympathetic nerves, therefore, may impair cerebral neurogenic vasodilation resulting in vasoconstriction.

It should be noted that effects of lead on nicotinic receptors on sympathetic nerve terminals in peripheral vascular beds and the axo-axonal interaction-mediated vasodilation in peripheral circulation such as the mesenteric vascular bed (Shiraki et al., 2000) are unknown, because the nicotinic receptor subtype(s) on sympathetic nerves in these vascular beds is practically unavailable. Both ₇-nAChRs and non-₇-nAChRs may be present on the postganglionic sympathetic nerves in the peripheral vascular beds (Si and Lee, 2002), and lead has been shown to exhibit different effects (stimulation or inhibition) on different subtypes of non-₇-nAChRs (Zwart et al., 1995, 1997; Oortgiesen et al., 1997). Obviously, the exact effects of lead on sympathetic regulation of NE release (either increase or decrease) in peripheral vascular beds and its involvement in raising blood pressure cannot be determined until the subtypes of sympathetic presynaptic nAChRs in systemic circulation are fully identified,.

It should be pointed out that CdCl₂ like lead has been shown to block sympathetic activity in rabbit saphenous arteries (Cooper and Steinberg, 1977) and portal veins (Holecyova and Torok, 1990). Zn²⁺ also has been shown to block ₇-nAChRs (Palma et al., 1998), and Al³⁺ to block specific nicotine binding (Gulya et al., 1990). Unlike lead, however, CdCl₂, ZnCl_2, and AlCl₃ at 10 µM did not affect ₇-nAChRs on calcium influx in the SCG cells or sympathetic dependent nitrergic neurogenic dilation in cerebral arteries in the present studies. At concentrations higher than 10 µM as used in the experiments by others (Cooper and Steinberg, 1977; Gulya et al., 1990; Holecyova and Torok, 1990; Palma et al., 1998), these three metal ions did inhibit calcium influx and nitrergic vasodilation in the present study. But at these high concentrations, these metal ions also decreased arterial basal tone and TNS-elicited neurogenic dilation, suggesting the nonspecific effects of these ions at concentrations higher than 10 µM.

Inhibition of cerebral sympathetic-dependent neurogenic nitrergic vasodilation by acute application of lead suggests a possible decrease in cerebral blood flow and a lower intracranial pressure by lead. This, however, is inconsistent with clinical observations that chronic lead exposure increases intracranial pressure (Braun and Gutjahr, 1971; Teo et al., 1997). One possible explanation for the latter findings is that chronic administration of lead may cause blood brain barrier dysfunction (Struzynska et al., 1997), resulting in "leaky" microvessels and increase in membrane permeability.

In summary, the present studies demonstrate that ₇-nAChRs located on cerebral postganglionic sympathetic neurons are specifically blocked by lead, resulting in inhibition of neuronal calcium influx and diminished nicotine- and choline-induced sympathetic-dependent nitrergic dilation in isolated porcine basilar arteries. Because choline is an endogenous agonist and lead a likely antagonist for ₇-nAChRs, the present finding provides further evidence supporting the presence of functional ₇-nAChRs on the sympathetic neurons of SCG origin. This result also reveals that lead can cause "vasoconstriction" such as in the cerebral circulation by blocking ₇-nAChR-mediated neurogenic nitrergic vasodilation. Results of the present study also point out that the exact systemic hemodynamic effects of chronic lead exposure will not be clarified until the subtypes of nicotinic receptors found on sympathetic nerves in peripheral vascular beds are fully determined.

	Acknowledgements

 
We thank Dr. T. Chiu for reading the manuscript and Carol Morgan for preparing the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grants HL27763 and HL47574, Tzu Chi Foundation (Taiwan, Japan), and Southern Illinois University-Central Research Committee/Excellence in Academic Medicine.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.102.046854.

ABBREVIATIONS: NO, nitric oxide; SCG, superior cervical ganglion; nAChR, nicotinic acetylcholine receptors; TNS, transmural nerve stimulation; AM, acetoxymethyl ester; U04661 [GenBank] 9, 9,11-dideoxy-9,11-methanoepoxy prostaglandin F₂

Address correspondence to: Dr. Tony J.-F. Lee, Department of Pharmacology, Southern Illinois University School of Medicine, P.O. Box 19629, Springfield, IL 62794-9629. E-mail: tlee{at}siumed.edu or tlee{at}mail.tcu.edu.tw

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