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Am J Physiol Heart Circ Physiol 279: H808-H816, 2000;
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Vol. 279, Issue 2, H808-H816, August 2000

Presynaptic beta 2-adrenoceptors mediate nicotine-induced NOergic neurogenic dilation in porcine basilar arteries

Tony J. F. Lee, W. Zhang, and S. Sarwinski

Department of Pharmacology, School of Medicine, Southern Illinois University, Springfield, Illinois 62794-9629


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that nicotine-induced nitric oxide (NO)-mediated cerebral neurogenic vasodilation was dependent on intact sympathetic innervation. We hypothesized that nicotine acted on sympathetic nerve terminals to release norepinephrine (NE), which then acted on adrenoceptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO, resulting in vasodilation. The adrenoceptor subtype in mediating nicotine-induced vasodilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that propranolol and preferential beta 2-adrenoceptor antagonists (ICI-118,551 and butoxamine), in a concentration-dependent manner, blocked the relaxation induced by nicotine (100 µM) without affecting the relaxation elicited by transmural nerve stimulation (TNS, 8 Hz). In contrast, preferential beta 1-adrenoceptor antagonists (atenolol and CGP-20712A) did not affect either nicotine- or TNS-induced relaxation. Results of double-labeling studies indicated that beta 2-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries. These findings suggest that NE, which is released from sympathetic nerves upon application of nicotine, acts on presynaptic beta 2-adrenoceptors located on the NOergic nerve terminals to release NO, resulting in vasodilation. In addition, nicotine-induced relaxation was enhanced by yohimbine, an alpha 2-adrenoceptor antagonist, which, however, did not affect the relaxation elicited by TNS. Prazosin, an alpha 1-adrenoceptor antagonist, on the other hand, did not have any effect on relaxation induced by either nicotine or TNS. The predominant facilitatory effect of beta 2-adrenoceptors in releasing NO may be compromised by presynaptic alpha 2-adrenoceptors.

nitric oxide; norepinephrine; porcine cerebral arteries


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MORPHOLOGICAL STUDIES have demonstrated that the close apposition between the adrenergic nerve terminal and the nonadrenergic or nitric oxide synthase (NOS) immunoreactive nerve terminal has frequently been found in large cerebral arteries at the base of the brain in several species (1, 11, 13). The axo-axonal distance between these different types of nerve terminals is substantially closer than the synaptic distance between the adventitial nerve terminals and the outermost layer of smooth muscle in the media (11, 13, 18). This morphological feature suggests that a functional axo-axonal interaction between nerve terminals is more likely to occur than that between the nerve and muscle. Thus transmitters released from one nerve terminal may modulate the release of transmitters from the neighboring nerve terminals and therefore the neurogenic vasomotor responses (5, 13).

We have recently demonstrated that nicotine-induced nitric oxide (NO)-mediated neurogenic vasodilation is dependent on intact sympathetic, adrenergic innervation in porcine basilar arteries. 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 (6-OHDA), although these treatments do not affect transmural nerve stimulation (TNS)-elicited NO-mediated neurogenic vasodilation in the same preparations. Furthermore, relaxation induced by exogenous norepinephrine (NE) in porcine basilar arterial rings was blocked by Nomega -nitro-L-arginine (L-NNA; see Ref. 34). Accordingly, it was hypothesized that nicotine acts on nicotinic receptors located on sympathetic nerve terminals, resulting in release of NE or a related substance that then diffused to act on its receptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO and therefore induce vasodilation (34). The exact nature of the receptors located on NOergic nerves mediating nicotine-elicited NO release in cerebral arteries has not been clarified. Our preliminary results indicated that nicotine-induced neurogenic vasodilation in porcine basilar arteries was blocked by propranolol, suggesting the possible involvement of beta -adrenoceptors in nicotine-induced neurogenic vasodilation. This latter finding further supported the notion that NE or a related substance was the mediator released from sympathetic nerve terminals to cause NO release from NOergic nerves. The present study, therefore, was designed to pharmacologically and immunohistochemically characterize, in large cerebral arteries of the pig, the presynaptic adrenoceptors presumably located on NOergic nerves, which mediate nicotine-induced NOergic vasodilation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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% O2-5% CO2 at room temperature. The composition of the Krebs solution was as follows (in mM): 122.0 NaCl, 5.16 KCl, 1.2 CaCl2, 1.22 MgSO4, 25.6 NaHCO3, 0.03 EDTA, 0.1 L-ascorbic acid, and 11.0 glucose (pH 7.4). Basilar and middle cerebral arteries were dissected and cleaned of 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 was 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) for isometric recording of changes in force, as described in our previous report (20). The temperature of the Krebs solution equilibrated with 95% O2-5% CO2 was maintained at 37°C. Tissues were equilibrated in the Krebs solution for an initial 30 min and then were mechanically stretched to a resting tension of 750 mg (34).

The basilar arterial ring segments were then precontracted with U-46619 (0.3-3 µM) to induce an active muscle tone of 0.5-0.75 g. TNS at 8 Hz and a single concentration of nicotine (100 µM) were applied to induce a relaxation. The arteries were then washed with prewarmed Krebs solution. A similar magnitude of active muscle tone was induced with U-46619 again, and TNS at 8 Hz was repeated (to serve as a control compared with the relaxation elicited by TNS before the wash). Experimental drugs were then administered, and TNS at 8 Hz and nicotine at the same concentration before the wash were repeated. To avoid possible development of tachyphylaxis upon repeated applications of nicotine (34), at least 90 min with six washes (every 15 min) were allowed before the next application of nicotine (34). Experimental drugs were added at least 30 min before TNS and application of nicotine.

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 (17). Stimulation parameters were continuously monitored on a Tektronix oscilloscope. The neurogenic origin of this TNS-induced response was verified by its complete blockade by TTX (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 (17).

For examining effects of experimental drugs on relaxation induced by NE, concentration-response relations for NE 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 NE were repeated. EC50 values were determined for each arterial ring. From these values, the geometric means EC50 with 95% confidence intervals (8) 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 (17). A complete removal of endothelial cells was verified by the lack of effect of L-NNA in increasing basal tone (19).

Double-labeling immunohistochemistry. Fresh porcine basilar and middle cerebral arteries obtained from local slaughterhouses were dissected and placed into periodate-parafomaldehyde-picric acid-formaldehyde-lysine fixative (22) 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 goat serum in 0.25% Triton X-100-PBS for 30 min at room temperature. The arteries were incubated with primary antibodies (anti-rabbit beta 2-adrenocepter, 1:100; Affinity BioReagents, Golden, CO) at 4°C for 24-48 h or at room temperature for 4 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 FITC-labeled goat anti-rabbit IgG (1:40; Sigma Chemicals, St. Louis, MO; see Ref. 33). 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 (33).

For double staining, after taking photographs of immunofluorescence fibers, the specimens were washed in PBS and processed for NADPH diaphorase (NADPHd) histochemistry (Ref. 4; see below), and NADPHd reactivities in the same regions of the specimens were rephotographed. NADPHd is a reliable marker for neuronal NOS in pig cerebral arteries (33).

NADPHd histochemical staining. After fixation and incubation with specific antibodies for immunofluorescence labeling and photographing as described above, specimens were incubated in 0.1 M phosphate buffer (pH 8.0) containing 1 mg/ml of NADPH (reduced form), 0.1 mg/ml of nitro blue tetrazolium, and 0.2% Triton X-100 at 37°C for 1 h (4). The specimens were rinsed with PBS, mounted with Gel/Mount (Biomedia, Foster City, CA), and examined under a light microscope. For control of NADPHd activity, NADPH was omitted from the incubation medium. This resulted in elimination of NADPHd reaction product in perivascular nerves (4).

Drug used and statistical analysis. The following drugs were used: ±atenolol, butoxamine, CGP-20712A, and yohimbine (RBI, Natick, MA); atropine sulfate (Calbiochem, San Diego, CA); guanethidine sulfate and phentolamine (Ciba, Summit, NJ); dobutamine hydrochloride (Eli Lilly, Indianapolis, IN); prazosin hydrochloride (Pfizer, Brooklyn, NY); L-NNA, DL-propranolol hydrochloride, terbutaline, UTP, NADPH (reduced form), Triton X-100, and nitro blue tetrazolium (Sigma Chemical); ICI-118,551 hydrochloride (Imperial Chemical); and U-46619 (Upjohn, Kalamazoo, MI). All drugs were added directly to the tissue baths. The drug concentrations reported were the final concentrations in tissue bath.

Results were expressed as means ± SE. Statistical analysis was evaluated by Student's t-test for paired or unpaired samples as appropriate. The P < 0.05 level of probability was accepted as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nicotine- and TNS-induced neurogenic vasodilation in porcine basilar arteries. In the presence of active muscle tone induced by U-46619 (0.3 µM), the basilar arteries without endothelial cells relaxed exclusively upon TNS at various frequencies (2, 4, and 8 Hz) and application of nicotine (1-100 µM). A complete removal of endothelial cells was verified by the lack of effect of L-NNA in increasing basal tone (19). Because TNS at 8 Hz and nicotine at 100 µM induced maximum relaxation, these parameters, which have previously been used by us and many others (17, 29, 34), 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 (34). 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 (34). Furthermore, the relaxation elicited by repeated TNS at 8 Hz, like other reports in the porcine cerebral arteries (17, 19), was reproducible and not different (34).

The relaxation induced by both nicotine (100 µM) and TNS (8 Hz) was significantly blocked by L-NNA (30 µM, n = 6), TTX (0.3 µM), and cold storage denervation for 7 days (data not shown). Furthermore, the relaxation induced by nicotine (100 µM) was diminished by guanethidine (1-10 µM) in a concentration-dependent manner. Blockade of nicotine-induced relaxation by guanethidine (1-10 µM) was fully recovered after the arteries were washed with fresh prewarmed Krebs solution (n = 5). On the other hand, guanethidine at similar concentrations never affected the TNS-elicited relaxation. These results were similar to those reported previously (34).

Blockade of nicotine-induced neurogenic vasodilation by beta -adrenoceptor antagonists but not by alpha -adrenoceptor antagonists. In the presence of active muscle tone induced by U-46619 (0.3 µM) in basilar arteries without endothelial cells, nicotine (100 µM)-induced relaxation was significantly inhibited by propranolol (0.1-10 µM) in a concentration-dependent manner (Fig. 1, A and B). At 10 µM, propranolol abolished the nicotine-induced relaxation without any effect on the TNS-elicited relaxation in the same preparations (Fig. 1, A and B).


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  		Fig. 1.   A: representative tracing showing relaxation of a basilar arterial ring without endothelial cells induced by transmural nerve stimulation (TNS) at 8 Hz and 100 µM nicotine (Nic). B: propranolol blockade of relaxation induced by nicotine but not by TNS. Relaxation was estimated as a percentage of the respective control (C). Values are means ± SE; n, no. of experiments. * P < 0.05, significant difference from controls. # Complete blockade of nicotine-induced relaxation by propranolol. Brackets denote concentration. PPV, papaverine.

The relaxation induced by nicotine in basilar arteries was not significantly affected by prazosin (1-10 µM, n = 8; Fig. 2A) but was significantly enhanced by yohimbine (0.1-1 µM, n = 4; Fig. 2B). Neither prazosin nor yohimbine at the concentrations tested affected the TNS-elicited relaxation (Fig. 2, A and B).


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  		Fig. 2.   Bar graphs showing effects of prazosin (A), yohimbine (B), ICI-118,551 (C), and butoxamine (D) on relaxation of isolated basilar arterial rings without endothelial cells induced by nicotine (100 µM) and TNS at 8 Hz. Relaxation was estimated as a percentage of the respective control. Values are means ± SE; n, no. of experiments. * P < 0.05, significant difference from the respective controls. # At 10 µM, butoxamine almost completely blocked nicotine-induced relaxation.

beta 2-Adrenoceptor but not beta 1-adrenoceptor antagonists blocked nicotine-induced neurogenic vasodilation. In the presence of active muscle tone induced by U-46619 (0.3 µM) in basilar arteries without endothelial cells, relaxation induced by nicotine (100 µM) was diminished by ICI-118,551 and butoxamine (selective beta 2-adrenoceptor antagonists) in a concentration-dependent manner (0.01-10 µM; Fig. 2, A and B). The nicotine-induced relaxation, however, was not appreciably affected by beta 1-adrenoceptor antagonists such as atenolol (0.01-10 µM, data not shown) and CGP-20712A (0.01-10 µM, data not shown). At similar concentrations, ICI-118,551, atenolol, butoxamine, and CGP-20712A did not affect the TNS-elicited relaxation in the same preparations (data not shown).

Effect of ICI-118,551, atenolol, and CGP-20712A on NE-induced relaxation in basilar arteries. In the presence of phentolamine (1 µM) and active muscle tone induced by U-46619 (0.3 µM), porcine basilar arteries without endothelial cells relaxed upon application of NE in a concentration-dependent manner, a result similar to that reported previously (17). The relaxation was blocked by ICI-118,551 (Fig. 3A), atenolol (Fig. 3B), and CGP-20712A (Fig. 3C), as indicated by parallel shift of the concentration-response curves to the right. CGP-20712A and atenolol appeared to be more potent than ICI-118,551. Both CGP-20712A and atenolol at 0.1 µM already significantly shifted the concentration-response curve to the right, whereas ICI-118,551 at same concentration was without any significant effect on NE-induced relaxation (Fig. 3). These beta -adrenoceptor antagonists did not affect relaxation induced by sodium nitroprusside (10 nM-10 mM) in arteries denuded of endothelial cells (data not shown).


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  		Fig. 3.   Concentration-response curves showing effects of ICI-118,551 (A), atenolol (B), and CGP-2017A (C) on norepinephrine (NE)-induced relaxation of isolated basilar arterial rings without endothelial cells. ICI-118,551 (0.1-10 µM), atenolol (0.1-10 µM), and CGP-20712A (0.1-3 µM) in a concentration-dependent manner inhibited the relaxation induced by NE (0.01-100 µM), as indicated by the rightward shift of the NE concentration-response curves. Although ICI-118,551 at 0.1 µM did not significantly affect NE-induced relaxation, both atenolol and CGP-20712A at similar concentrations significantly shifted the concentration-response curves, with CGP-20712A being more potent. CGP-2017A at 3 µM almost abolished the maximum relaxation induced by NE. Relaxation was estimated as a percentage of maximum relaxation induced by papaverine (PPV; 300 µM). Values are means ± SE; n, no. of experiments.

Immunocytochemistry. Results from double-staining studies, i.e., single immunolabeling followed by histochemical staining for NADPHd, which is a reliable marker for neuronal NOS (33), indicated the presence of beta 2-adrenoceptor immunoreactive (Fig. 4A) and NADPHd reactive fine fibers and bundles (Fig. 4B) in the same whole-mount basilar and middle cerebral arteries. In both arteries, almost all NADPHd reactive fibers were coincident with beta 2-adrenoceptor immunoreactive fibers, but not all beta 2-adrenoceptor immunoreactive fibers were NADPHd reactive. These NADPHd reactive and NADPHd negative fibers were frequently found to run close to each other. It appeared that beta 2-adrenoceptor immunoreactive bundles were composed of NADPHd reactive and NADPHd negative fibers. Therefore, beta 2-adrenoceptor immunoreactive bundles were frequently found to be thicker than the corresponding NADPHd reactive bundles (Fig. 4, A and B). For negative controls by omitting primary antibodies, no immunoreactivities of beta 2-adrenoceptors were observed (data not shown).


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  		Fig. 4.   Photomicrographs showing immunofluorescence labeling of beta 2-adrenoceptor immunoreactive (I) bundles and fine fibers (a) followed by histochemical staining of NADPHd (b) in the same area in a whole-mount middle cerebral artery. Almost all NADPH diaphorase (NADPHd) reactive fibers (arrowheads in b) were coincident with beta 2-adrenoceptor immunoreactive fibers (arrowheads in a). However, not all beta 2-adrenoceptor immunoreactive fibers (open arrow in a) were NADPHd reactive. The beta 2-adrenoceptor immunoreactive bundles were frequently found to be thicker than the corresponding NADPHd reactive bundles, indicating that the beta 2-adrenoceptor immunoreactive bundles are composed of NADPHd reactive and NADPHd negative (such as adrenergic) fibers. A small vessel branch (v with arrow in a and b) was used as a marker for repeating photographing. beta 2-Adrenoceptor antibodies label whole blood vessel, including adventitia. On the other hand, NADPHd histochemistry only stains endothelial cells in this size of vessel, which generally does not receive NOS-containing nerve. Accordingly, "vessel" diameter in b appears to be smaller than that in a. Scale bar, 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of the present study was that nicotine-induced neurogenic vasodilation in porcine basilar arteries was blocked by preferential beta 2-adrenoceptor antagonists but not by preferential beta 1-adrenoceptor antagonists. The preferential beta 1-adrenoceptor antagonists, however, are more potent than preferential beta 2-adrenoceptor antagonists in blocking the postsynaptic beta -adrenoceptor-mediated relaxation induced by exogenously applied NE. beta 2-Adrenoceptor immunoreactivities and NADPHd reactivities were found to colocalize in the same nerve fibers. Because NADPHd is a reliable marker for neuronal NOS in porcine cerebral arteries (33), these results support our hypothesis that nicotine acts on presynaptic nicotinic receptors located on the adrenergic nerve terminals to release NE, which then acts on the presynaptic beta 2-adrenoceptors located on the neighboring NOergic nerves to cause release of NO and therefore induces vasodilation (Fig. 5).


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  		Fig. 5.   Summary diagram showing close apposition of an adrenergic and a nitric oxidergic (NOergic) nerve terminal in porcine large cerebral arteries at the base of the brain. The axo-axonal distance between these two different nerve terminals is closer than that between the nerves and the smooth muscle. In porcine basilar arteries, for example, nicotine (NIC) acts on presynaptic nicotinic receptors located on the adrenergic nerve terminal, causing (+) release of NE (NE), which then acts on presynaptic beta 2-adrenoceptors located on the adjacent NOergic nerve terminal. This effect of NE results in stimulating (+) NO release, which activates guanylate cyclase (GC), increases cGMP synthesis from GTP, and relaxes the smooth muscle. This nerve-released NE can also stimulate alpha 2-adrenoceptors on NOergic nerve terminals, resulting in inhibition (-) of NO release. NE released from sympathetic nerves, however, is a weak postsynaptic transmitter (as indicated by a question mark), although postsynaptic beta 1-(the predominant receptor subtype) and beta 2-adrenergic receptors on smooth muscle have been demonstrated. However, stimulation of these receptors by exogenously applied NE activates adenylate cyclase (AC), resulting in increasing cAMP synthesis from ATP and relaxation. alpha -Adrenoceptors on the smooth muscle cells (SMC) play a negligible role in vasoconstriction in porcine large cerebral arteries. NO, which is not stored in vesicles, is synthesized from L-arginine (L-Arg) in the presence of nitric oxide synthase (NOS). L-Citrulline (L-Cit), the by-product of NO synthesis, is actively converted to L-Arg (4). This L-Cit-L-Arg cycle provides evidence for the neuronal source of NO.

It is well established that nicotine releases NE by acting on nicotinic receptors located on sympathetic adrenergic nerve terminals (10, 27). Accordingly, nicotine was assumed to act directly on NOergic nerve terminals to release NO, resulting in NO-mediated cerebral neurogenic vasodilatation in many species (28). This assumption, however, was questioned, since our recent studies demonstrated for the first time that nicotine-induced NO-mediated relaxation in porcine cerebral arteries was dependent exclusively on intact sympathetic innervation (34). After a complete blockade of sympathetic transmission with guanethidine, or chemical denervation of sympathetic nerves with 6-OHDA, 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 after treatment with 6-OHDA (34). These results indicate that in cerebral 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 the neighboring NOergic nerves and causes the release of NO from these nerves (34). The morphological evidence of close apposition (25 nm) between the adrenergic nerve terminals and the nonadrenergic nerve terminals for such axo-axonal interaction has been demonstrated to be a characteristic of cerebral vessel innervation in several species (1, 5, 11, 13).

Evidence that NE or a related catecholamine mediated nicotine-induced NOergic vasodilation was supported by results of the present study that nicotine-induced relaxation was blocked by propranolol (a nonspecific beta -adrenoceptor antagonist). Further studies demonstrated that this effect of nicotine was mediated specifically by presynaptic beta 2-adrenoceptors, since nicotine-induced relaxation was blocked by preferential beta 2-adrenoceptor antagonists in a concentration-dependent manner, but was not affected by preferential beta 1-adrenoceptor antagonists. Both preferential beta 1- and preferential beta 2-adrenoceptor antagonists, like propranolol, however, significantly blocked postsynaptic beta -adrenoceptor-mediated relaxation induced by exogenous NE, a result consistent to those reported previously (17, 23, 31, 32). Furthermore, propranolol and all preferential beta 1- and preferential beta 2-adrenoceptor antagonists at concentrations examined in the present studies did not affect TNS-elicited relaxation. This result is also consistent to those reported previously (17, 31, 32), suggesting that blockade of nicotine-induced relaxation by propranolol and beta 2-adrenoceptor antagonists was not due to any possible local anesthetic effects of these agents at the concentrations used. Together, these results suggest that presynaptic beta 2-adrenoceptors on the NOergic nerves mediate NE-induced NO release. Evidence has been presented that NE but not dopamine or epinephrine is found in cerebral arteries, including basilar and middle cerebral arteries from different species (14, 17), further indicating that NE is the most likely transmitter released by nicotine from sympathetic nerves to cause release of NO from the neighboring NOergic nerves.

It should be noted that the porcine cerebral vascular smooth muscle, unlike that of other species, contains mainly beta -adrenoceptors, with beta 1-adrenoceptors as the predominant subtype in mediating NE-induced relaxation (23, 31, 32). This is further supported by results of the present study that beta 1-adrenoceptor antagonists were more potent than beta 2-adrenoceptor antagonists in blocking exogenous NE-induced relaxation. In particular, CGP-20712A (a preferential beta 1-adrenoceptor antagonist) appears to be the most potent beta -adrenoceptor antagonist in blocking relaxation induced by exogenous NE. At 3 µM, CGP-20712A almost abolished the maximum relaxation induced by NE but did not appreciably affect nicotine-induced neurogenic vasodilation. A similar result was found with a different beta 1-adrenoceptor antagonist (atenolol). On the other hand, ICI-118,551 at 0.1 µM, which did not significantly affect the NE concentration-relaxation response relationship (Fig. 3C), significantly inhibited the nicotine-induced relaxation (Fig. 2C). These results indicate that neuronally released NE induced by nicotine does not act on the postsynaptic beta -adrenoceptors located on the smooth muscle cells to induce a relaxation. Rather, NE acts as a presynaptic transmitter on presynaptic beta 2-adrenoceptors on the NOergic nerves to cause release of NO and therefore vasodilation. The presence of presynaptic beta 2-adrenoceptors on the NOergic nerves is supported by results from double-labeling studies that NADPHd reactive fibers (markers for NOS-I fibers) are almost coincident with beta 2-adrenoceptor immunoreactive fibers in both basilar and middle cerebral arteries. However, not all beta 2-adrenoceptor imunoreactive fibers were NADPHd reactive. This is an expected result, since beta 2-adrenoceptors are also found in association with other types of nerves such as the noradrenergic neurons (24).

Similar results were found in isolated large cerebral arteries at the base of the cat brain in which nicotine-induced vasodilation, which was sensitive to L-NNA, was blocked by beta 2- but not beta 1-adrenoceptor antagonists (our preliminary data). In these cerebral arteries of the cat, postsynaptic alpha -adrenoceptors are predominant. Accordingly, exogenous NE has been shown to induce a constriction exclusively (16, 22). These findings clearly indicate that nicotine-induced vasodilation in the cat cerebral arteries cannot be due to a direct effect of NE on the postsynaptic smooth muscle cells. Rather, NE as a presynaptic transmitter acts on presynaptic beta 2-adrenoceptors located on NOergic nerves to cause release of NO, which then induces vasodilation. This conclusion is consistent with the reported biochemical findings that neurogenic vasodilation in cerebral arteries from different species induced by either TNS or nicotine is accompanied by an increase in cGMP but not cAMP (9, 15, 29), suggesting that the terminal transmitter acting on the smooth muscle to induce a relaxation is NO (known to increase cGMP synthesis) or a related substance but not NE (known to increase cAMP synthesis by its beta -adrenoceptor activity).

In addition to that found in the cerebral arteries in the present studies, the presence of beta 2-adrenoceptors on NOergic nerves in the pulmonary and mesenteric arteries of the rat has been speculated (21, 25). Adrenergic nerve terminals in several preparations have been shown to contain beta 2-adrenoceptors, and these receptors mediate positive feedback of NE release (24, 30). Blockade of these presynaptic receptors by propranolol and other beta -adrenoceptor antagonists only partially decreases the release of NE or NE-mediated vascular responses induced by nicotine or field electrical nerve stimulation (24, 30). This is different from the present findings that nicotine-induced NE-mediated NOergic vasodilation was abolished by propranolol and preferential beta 2-adrenoceptor antagonists. It is possible that the presynaptic beta 2-adrenoceptors on NOergic nerve terminals are more sensitive than those located on the adrenergic nerve terminals to beta -adrenoceptor antagonists.

It should be pointed out that NE is generally considered to be a weak agonist for beta 2-adrenoceptors in the cardiovascular system (24). The possibility that other receptor subtypes such as the beta 3-adrenoceptors and beta 4-adrenoceptors (12) are involved in NE-mediated NO release remains to be clarified. However, the complete blockade of nicotine-induced relaxation by propranolol, which is not a ligand for beta 3-adrenoceptors (12), and the failure of CGP-20712A, which is a beta 1- and beta 4-adrenoceptor antagonist (12), in blocking nicotine-induced relaxation render this possibility tenuous.

The involvement of the presynaptic alpha 2-adrenoceptors in mediating inhibition of NO release from NOergic nerves and NE release from adrenergic nerves in peripheral vascular preparations has been reported (2). This appears to be true also in the porcine cerebral arteries, since nicotine-induced relaxation was potentiated by yohimbine but not by prazosin. This is consistent with the present hypothesis that increased NE release after blocking presynaptic alpha 2-adrenoceptors on the sympathetic nerves by yohimbine can result in increased NO release from the NOergic nerves and enhanced vasodilation. The relative significance of alpha 2-adrenoceptors located on adrenergic sympathetic nerve terminals and NOergic nerve terminals in mediating nicotine-induced NO-mediated relaxation remains to be determined. The presynaptic beta 2-adrenoceptors on NOergic nerve terminals, however, appear to be predominant in cerebral perivascular nerves, since NE-mediated nicotine-induced NOergic vasodilation in the absence of yohimbine was demonstrated.

Results of our previous (34) and present studies indicate that nicotine-induced NO-mediated neurogenic vasodilation in porcine basilar arteries is indirectly mediated by release of NE from sympathetic nerves. Nicotine does not act directly on NOergic nerves to elicit an NO-mediated vasodilation (34). This effect of nicotine in inducing NO-mediated neurogenic vasodilation is different from that by TNS. The latter depolarizes the NOergic and sympathetic nerve terminals simultaneously, resulting in NO-mediated relaxation, although NE also is released upon TNS (our preliminary results). It is possible that direct depolarization of the NOergic nerves by TNS at various frequencies, resulting in NO release, is already at the maximum enzyme stimulating capacity of each frequency. An additional modulatory effect elicited by simultaneous release of NE from the sympathetic nerves may be relatively small and therefore is not detected. This may explain the well-established findings of the failure of guanethidine (a blocker of sympathetic adrenergic transmission), propranolol, preferential beta 2-adrenoceptor antagonists, yohimbine, and other alpha -adrenoceptor antagonists in affecting TNS-elicited NO-mediated neurogenic vasodilation in cerebral arteries (Refs. 16, 17, 32, and 34 and the present results).

In summary, the present study demonstrated that nicotine-induced, NO-mediated relaxation in porcine basilar arteries was inhibited by blocking presynaptic beta 2-adrenoceptors and was enhanced by blocking presynaptic alpha 2-adrenoceptors. Although the distribution of both presynaptic beta 2- and alpha 2-adrenoceptors in adrenergic and NOergic nerves remains to be determined, results from the present studies suggest that these receptors are present on NOergic nerve terminals. Similar results were found in the cat middle cerebral arteries and porcine internal carotid arteries (our preliminary results). These findings together with those from previous studies provide strong evidence that NE is the most likely mediator, released from the adrenergic nerve terminals upon application of nicotine, and acting predominantly on beta 2-adrenoceptors located on NOergic nerve terminals to cause release of NO and the resulting cerebral vasodilation (Fig. 5). NE therefore acts predominantly as a presynaptic transmitter. Accordingly, it is possible that regional vasoconstriction induced by electrical stimulation of the sympathetic nerves in vivo may be offset by immediate vasodilation in the same regions due to NO release from NOergic nerves. This finding may provide an explanation for the reported observations that electrical stimulation of the sympathetic nerves to cerebral circulation in normal experimental animals in general results in a very weak effect or no response in cerebral vascular tone and cerebral blood flow (3, 6, 7). This concept of presynaptic modulation of NOergic nerves by sympathetic adrenergic nerves appears to be supported by reports from some in vivo experimentation that the functional consequence of neuronal NO and NE interaction may play a role in blood pressure regulation (26).


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-27763 and HL-47574, by the American Heart Association/Illinois Heart Affiliate (9807871), and by the Southern Illinois University-Central Research Committee/Excellence in Academic Medicine.


    FOOTNOTES

Address for reprint requests and other correspondence: T. J. F. Lee, Dept. of Pharmacology, School of Medicine, Southern Illinois Univ., P O Box 19629, Springfield, IL 62794-9629 (E-mail: tlee{at}siumed.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. §1734 solely to indicate this fact.

Received 22 December 1999; accepted in final form 25 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(2):H808-H816
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


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