Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cerebral blood vessels are well innervated by sympathetic nerves; however, the relative importance of these nerves in regulating cerebral blood flow remains an issue (Bill and Linder, 1976; Bevan, 1981; Busija and Heistad, 1984; Buchholz et al., 1999). Although adrenergic nerve stimulation may not play a major role in the control of cerebral blood flow under normal conditions, when arterial pressure is elevated sympathetic nerves cause a marked blunting of the increase in cerebral blood flow (Bill and Linder, 1976). Thus, sympathetic nerve activity may serve to protect the individual organism against environmental stress (Busija, 1984; Faucher et al., 1991).

Since the discovery of the gas nitric oxide (NO) in mammalian cells, it appears that NO is omnipresent in mammalian biology (Nathan, 1992; Michel and Feron, 1997). Nerves containing NO synthase (NOS) have been shown to innervate canine and porcine cerebral arteries, suggesting a role for NO beyond that of relaxation of vascular smooth muscle (Lee and Sarwinski, 1991; Toda and Okamura, 1991). For example, transmural nerve stimulation has been shown to promote the release of NO from canine middle cerebral arteries, an effect abolished by tetrodotoxin (Toda and Okamura, 1990). Similar results have been demonstrated in endothelial-denuded canine cerebral artery strips that relaxed in response to nerve stimulation. This effect was inhibited by an NOS inhibitor and abolished with tetrodotoxin, suggesting that it is due to release of NO from nerves (Toda and Okamura, 1991).

During the late 1980s, several reports have suggested that NO may modulate adrenergic nerve activity; however, the results are mixed. In rabbit carotid arteries, it was reported that stimulation-evoked NE release is inhibited by endothelium-derived NO (Cohen and Weisbrod, 1988; Tesfamariam et al., 1989). In rat medial basal hypothalamus, the NO donor sodium nitroprusside inhibited, whereas an NOS inhibitor enhanced K⁺-evoked norepinephrine (NE) release (Seilicovich et al., 1995). NE release from heart sympathetic nerves was also shown to be enhanced by inhibition of NOS (Schwarz et al., 1995). These studies all suggest that NO inhibits NE release from adrenergic nerves.

In contrast, there also are a number of studies demonstrating the opposite: that NO enhances NE release from adrenergic nerves. Inhibition of NOS decreased stimulation-evoked NE release in rat atria and mesenteric arteries, effects reversed by L-arginine (Yamamoto et al., 1993, 1997; Gironacci et al., 1997). In the anesthetized opossum, NE release induced by hypogastric nerve stimulation was also attenuated by NOS inhibition, and this effect was reversed by L-arginine (Prakash et al., 1993). In rat cerebral cortex, N-methyl-D-aspartate-induced NE release was inhibited by an NOS antagonist (Montague et al., 1994). In similar studies, the direct application of NO enhanced K⁺-evoked NE release from rat cerebral cortex synaptosomes and hippocampal slices (Stout and Woodward, 1994; Martire et al., 1998). Thus, it is still not clear how to explain these conflicting results.

Given that the cerebral vasculature contains both adrenergic and NO-producing nerves and that there are no studies on possible interactions between these nerve types in this vascular bed, a study of this issue was initiated. Stimulation-evoked release of both NE and NO was measured in middle cerebral arteries from adult female sheep. Using the facial artery as a peripheral arterial comparison, we tested the following two hypotheses: 1) in the cerebral vasculature, stimulation that simultaneously activates both NOS-containing nerves and adrenergic nerves will result in increased overflow of both transmitters, NO and NE. Nerve stimulation will not evoke NO release in the facial artery; and 2) release of NO from NOS-containing nerves will augment stimulation-evoked NE release from adrenergic nerves in cerebral arteries.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Adult nonpregnant ewes were obtained from a single supplier (Nebeker Ranch, Lancaster, CA) and were sacrificed with an i.v. injection of pentobarbital sodium (100 mg/kg). The facial arteries and the brain were removed and immediately placed in separate beakers containing ice-cold Krebs' solution with subsequent dissection of middle cerebral arteries. The Krebs' solution contained 118 mM NaCl, 4.8 mM KCl, 1.6 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 0.3 mM ascorbic acid, and 11.5 mM glucose.

Tissue Preparation. Segments of middle cerebral and facial arteries (3-4 cm) were cannulated at both ends with polyethylene tubing and mounted in a low-volume perfusion system as previously described (Buchholz and Duckles, 1992). The middle cerebral artery segment used was the main branch from the circle of Willis. Middle cerebral artery diameters ranged from 0.8 to 1.1 mm, and facial artery diameters ranged from 1 to 1.4 mm. Arteries were perfused at a rate of 1.0 ml/min, creating a perfusion pressure of approximately 55 to 65 mm Hg in both artery types. The entire perfusion assembly was immersed in a circulating water bath and kept at 37°C. Tissues were perfused with aerated (95% O₂, 5% CO₂) Krebs' solution throughout the experiment, containing 10⁵ M deoxycorticosterone and 10⁵ M cocaine to inhibit extraneuronal and neuronal uptake of NE, respectively. In all experiments, a Grass S-48 model stimulator (Grass Instruments, Quincy, MA) delivered electrical field stimulation to perivascular nerves through a pair of platinum electrodes. The parameters for stimulation were 8 Hz, 60 V, 1-ms duration, and 480 pulses (1-min stimulation).

Effect of N-Nitro-L-arginine Methyl Ester (L-NAME) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP) on Stimulation-Evoked NE Release. In Fig. 1, actual data from one pair of middle cerebral and facial arteries illustrate the protocol used to test the effects on NE release of NOS inhibition with L-NAME. Perivascular nerves in the control arteries (Fig. 1, A and C) were activated three consecutive times (S1-S3) for 1 min with a 45-min equilibration between each stimulation. Perivascular nerves in the treatment tissues (Fig. 1, B and D) were activated one time (S1) followed by a 45-min equilibration period. After S1, tissues were exposed for 20 min to 10 µM L-NAME and activated for 1 min followed by an additional 45-min equilibration period. Finally, tissues were exposed for 15 min to 10 µM L-NAME and 25 µM SNAP, and again the nerves were activated for 1 min. Basal NE release was monitored by taking a 5-ml sample before each stimulation in both time control and treatment arteries.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Representative data to illustrate experimental protocol for measurement of stimulation-evoked NE release in middle cerebral and facial arteries. Tissues were exposed throughout the experiment to deoxycorticosterone and cocaine (105 M). Perivascular nerves were activated three consecutive times [stimulation (S1-S3)], each for 1 min, with a 45-min equilibration separating each stimulation train. In every experiment, one artery served as a time control (A and C). In treated tissues (B and D) after S1, tissues were exposed to 10 µM L-NAME for 20 min and activated again for 1 min. After S2, tissues were exposed to 10 µM L-NAME and SNAP for 15 min and activated again for 1 min.

Simultaneous Assay of NE and NO Release. Nerves were activated for 1 min two consecutive times with a 45-min period separating S1 and S2. Perfusate was collected during the stimulation period until a total of 5 ml was collected. A 900-µl aliquot was obtained and immediately snap frozen in liquid nitrogen and stored at 80°C until NO analysis was carried out. The remaining 4.1-ml sample was used for the measurement of NE. Basal release was monitored by collecting a 5-ml sample before each stimulation.

Measurement of NE. The NE in the perfusates was extracted with alumina at pH 8.6 and quantified with dihydroxybenzylamine (DHBA) as an internal standard (400 pg) using a previously described protocol (Buchholz and Duckles, 1992). A 100-µl sample of extracted amines was injected into an ESA Coulochem II high-performance liquid chromatograph (ESA, Bedford, MA) and separated on an ESA reversed phase C18 column with ESA MD-TM aqueous mobile phase. The mobile phase contained 75 mM Na₂H₂PO₄, 0.5 M SDS, 0.025 mM EDTA, 20% acetonitrile, and 5% methanol. The following formula was used to calculate the amount of NE in the injected sample. Recovery varied from 85 to 99%.

(1)

where Ht is height. To quantify tissue NE content, arteries were taken at the end of each experiment and homogenized in 3 ml of 0.1 N perchloric acid, followed by centrifugation. A 300-µl aliquot of the supernatant was taken, and NE was extracted in a similar manner as the perfusate. NE content was used to calculate fractional NE release.

(2)

Measurement of NO. Frozen aliquots of perfusates were thawed, and NO was measured as previously described (Xiao et al., 1999). Briefly, NO was measured by injection of a 100-µl aliquot of the perfusate into a Sievers 270B NO analyzer (Sievers Instruments, Boulder, CO) in which stable nitrate and nitrite are reduced to NO gas. NO is measured by reaction with ozone, yielding light detected by a photomultiplier tube. Calibration curves were run during each analysis, and individual standard curves (0-200 pmol) were used to convert subsequent NO signals into pmol of NOx.

Statistical Analysis. Effects of L-NAME and SNAP treatment on stimulation-evoked NE release relative to control arteries were analyzed by paired Student's t test. Comparison of stimulation-evoked NO release to basal was also determined by paired t test.

Drugs Used. Cocaine hydrochloride, deoxycorticosterone, L-NAME, and SNAP were all obtained from Sigma Chemical Co. (St. Louis, MO).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

NE and NO Release. Activation of perivascular nerves significantly increased fractional NE release in both the middle cerebral and facial arteries (Fig. 2, A and C). In middle cerebral arteries, stimulation-evoked release of NO was significantly increased compared with basal release (Fig. 2B). In contrast, in facial arteries, stimulation-evoked release of NO and basal release were not significantly different (Fig. 2D).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Measurement of stimulation-evoked NE and NO release from perivascular nerves in middle cerebral and facial arteries. Tissues were exposed throughout to deoxycorticosterone and cocaine (105 M). Perivascular nerves were activated two consecutive times [stimulation (S1-S2)], each for 1 min, with a 45-min equilibration between each stimulation train. Both stimulation-evoked NE and NO release were measured in each sample. Columns represent the mean ± S.E., *, significantly different from basal release (P < .05; n = 7 middle cerebral and facial arteries).

Effects of NO Synthase Inhibition and NO Donor SNAP. Effects of the NOS inhibitor L-NAME and the NO donor SNAP on stimulation-evoked NE release are shown in Fig. 3. Stimulation-evoked NE release from adrenergic nerves in middle cerebral and facial arteries was consistent over the experimental time course (time controls, Fig. 3, A and C). In the presence of L-NAME, stimulation-evoked NE release declined by 48% in middle cerebral arteries (Fig. 3B). Furthermore, the inhibitory effect of L-NAME was significantly reversed by addition of the NO donor SNAP (Fig. 3B).

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Effect of L-NAME and the NO donor SNAP on stimulation-evoked NE release in middle cerebral and facial arteries. In each experiment, one tissue served as a time control (A and C) to demonstrate the consistency of NE release. Tissues were exposed throughout the experiment to deoxycorticosterone and cocaine (105 M). Treatment with L-NAME and SNAP was carried out as described in the text and illustrated in Fig. 1. Columns represent the mean ± S.E., **, significantly different from two other treatments (i.e., S1 and S3 or S1 and S2) by paired t test (P < .05; n = 5-10 arteries).

View this table: [in this window] [in a new window]   TABLE 1 Effect of time and treatments on basal NE release from middle cerebral and facial arteries Values represent the mean ± S.E. (n = 5-10 arteries). All tissues were treated with deoxycorticosterone (105 M) and cocaine (105 M).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The discovery that the gas NO released from the endothelium modulates vascular tone ranks as one of the most significant recent findings in physiology and pharmacology (Michel and Feron, 1997). The sheer number of publications on the subject of NO suggests that NO plays almost a universal role in the modulation of cellular function. However, the regulation of autonomic nerve activity by NO is a relatively novel and understudied topic.

The most important and interesting finding in this study is that stimulation-evoked NE release from sympathetic nerve terminals in cerebral arteries is augmented by NO released from NOS-containing nerves. Blockade of NOS with L-NAME significantly inhibited stimulation-evoked NE release in middle cerebral arteries. In addition, the effect of L-NAME was fully reversed by application of the NO donor SNAP. In contrast, L-NAME had no effect on stimulation-evoked NE release in the facial arteries; however, SNAP significantly elevated stimulation-evoked NE release. These data suggest that NO facilitates adrenergic nerve transmission. Furthermore, although facial arteries are not known to be innervated by NOS-containing nerves and L-NAME had no effect, the addition of an NO donor still enhanced NE release in this tissue, suggesting that the adrenergic nerves still possess mechanisms for response to NO. This study confirms our previous suggestion that NOS nerves serve to augment adrenergic nerve activity in the sheep cerebral vasculature (Buchholz et al., 1999).

The present report clearly shows that NO augments NE release in cerebral arteries, and this result is supported by a number of studies in other tissues. In the rat atria, mesentery, and central nervous system, NO appears to augment NE release from adrenergic nerves (Yamamoto et al., 1993, 1997; Montague et al., 1994; Stout and Woodward, 1994; Gironacci et al., 1997; Martire et al., 1998). However, our results and those of others showing that NO augments NE release stand in distinct contrast to several other reports. For example, in rabbit carotid arteries and rat hypothalamus and heart, NO has been suggested to inhibit NE release (Cohen and Weisbrod, 1988; Tesfamariam et al., 1989; Schwarz et al., 1995; Seilicovich et al., 1995).

There could be many possible reasons for these different results; we suggest at least three. The first possibility rests on the knowledge that arterial endothelium releases numerous substances in addition to NO, including prostaglandins, endothelins, and endothelium-derived hyperpolarizing factor (Hasunuma et al., 1990; Nakashima and VanHoutte, 1993; Koller and Huang, 1995; Koller et al., 1998). Given the number of different substances released from the endothelium, there is the possibility that any one of these endothelial factors may have prejunctional effects. Thus, reports cited earlier demonstrating that endothelial removal modulates NE release may not necessarily be related to prejunctional effects of NO per se. In addition, it is possible that contrasting results may reflect that NO exerts a different effect in similar models in different species, suggesting that indeed NO may have broader regulatory functions.

Another possibility for these contrasting results may be more technical in nature. In all of the reports that have shown that NO inhibits NE release, NE was quantified by HPLC with electrochemical detection using a single oxidizing electrode. However, the direct application of increasing concentrations of NO itself or the NO donor sodium nitroprusside has been shown to decrease the amount of NE quantified by a single oxidizing electrochemical electrode (MacArthur et al., 1995). Furthermore, using spectroscopic methods, it was shown that NO can oxidize NE to its quinone product. NE in this oxidized (quinone state) cannot be detected by a single oxidizing electrode, and this could lead to data suggesting that NO actually decreases NE release. In addition, the biological significance of the oxidation of NE by NO and the consequence for vascular function must be explored.

In contrast, in the current report and two others suggesting that NO augments NE release, HPLC was linked to an ESA "redox" detector (Yamamoto et al., 1993, 1997). This detector type uses two high surface area electrodes in series. The first electrode (reduction electrode) reduces any NE that has been oxidized, whereas the second electrode oxidizes NE, producing the recorded signal. Thus, using this redox method of NE quantification, oxidation of NE by NO would be of no consequence, and this method would detect all NE released. In agreement with studies using HPLC with a redox detector, studies using ³H-labeled NE also show that NO augments NE release (Montague et al., 1994; Stout and Woodward, 1994; Gironacci et al., 1997; Martire et al., 1998). ³H-labeled NE, even if oxidized, would still be measured because reduced and oxidized forms of NE would all be quantified as total tritium released.

A mechanism for the enhancement of NE release by NO has been proposed. In superior cervical ganglion cells, NO donors such as SNAP have been shown to increase Ca²⁺ current amplitude. Furthermore, the application of cGMP also increased the amplitude of Ca²⁺ currents, thus mimicking the effects of NO donors on Ca²⁺ channels (Chen and Schofield, 1995; Fig. 1). These results suggest that NO modulates the sensitivity of Ca²⁺ channels to depolarization. Facilitation of Ca²⁺ current is at least one rational explanation for the positive modulation of NE release by NO found in our study.

The coexistence of NOS-containing nerves and adrenergic nerves in cerebral blood vessels has been demonstrated. In canine cerebral arteries, the activation of vasodilator nerves releases NO; this is inhibited by L-NAME and abolished by tetrodotoxin. These data suggest that the origin of the NO is neuronal (Toda and Okamura, 1990, 1991). Indeed, when we measured both NE and NO release in the same samples, activation of perivascular nerves significantly increased both NE and NO release in middle cerebral arteries. In contrast, activation of perivascular nerves in facial arteries only elevated NE release, with no effect on NO release. These data provide another piece of evidence that the cerebral vasculature contains both adrenergic and NOS-containing nerves.

There is evidence that alterations in flow or shear stress induce the release of NO from the endothelium (Koller and Huang, 1995; Corson et al. 1996; Koller et al., 1998). The present finding of the ample release of NO in the absence of electrical stimulation (basal NO release; Fig. 2, B and D) suggests that NO is released from the endothelium in isolated, perfused middle cerebral and facial arteries. Furthermore, at identical flow rates and similar perfusion pressures (1.0 ml/min; 55-65 mm Hg), basal NO release was similar in the two arteries. However, a significant elevation in NO release when perivascular nerves are activated was seen only in cerebral arteries. This result further suggests a neuronal source of stimulation-evoked NO release in cerebral arteries.

In conclusion, we have shown that adrenergic nerves in blood vessels are regulated by NO and that NO augments NE release in adrenergic nerves in both cerebral and facial arteries. Furthermore, our data suggest that the cerebral arteries are unique in containing both adrenergic nerves and a source of NO that appears to be derived from NOS-containing nerves. This report underscores the idea that NO has diverse regulatory functions, including the regulation of vascular adrenergic nerves. This study also implies that there is another layer of regulation of adrenergic nerves in addition to other well-studied neuromodulatory mechanisms.