Abstract
Cerebral blood vessels contain both sympathetic and nitric oxide (NO) synthase (NOS)-containing nerves. NO has been proposed to modulate smooth muscle function and adrenergic nerve activity, and the nature of this modulation is controversial: some data show NO inhibits norepinephrine (NE) release, whereas others suggest that NO augments release. To test the hypothesis that in cerebral arteries NO released by NOS-containing nerves augments stimulation-evoked NE release, we used direct measurement of NE and NO release in isolated sheep middle cerebral arteries. The facial artery, which has not been reported to be innervated with NOS-containing nerves, was used as an artery comparison model. HPLC and redox electrochemical detection was used to measure NE, and NO was measured by chemiluminescence. Stimulation-evoked NE release from the middle cerebral artery significantly declined in the presence of the NOS inhibitorNω-nitro-l-arginine methyl ester (l-NAME). The effect ofl-NAME was reversed by the addition of the NO donorS-nitroso-N-acetyl-dl-penicillamine. In contrast, in facial arteries, l-NAME had no effect on stimulation-evoked NE release, whereasS-nitroso-N-acetyl-dl-penicillamine still significantly elevated NE release. Activation of perivascular nerves significantly increased NE release in both the middle cerebral and facial arteries. However, when NO was measured in the same samples, stimulation-evoked release of NO was significantly increased compared with basal release only in middle cerebral arteries. These data support the concept that cerebral arteries in the sheep contain both adrenergic and NOS-containing nerves. Furthermore, this study provides succinct evidence that NO released from NOS nerves augments stimulation-evoked NE release.
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
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 10−5 M deoxycorticosterone and 10−5 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 ofNω-Nitro-l-arginine Methyl Ester (l-NAME) andS-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 μMl-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.
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%.
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
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).
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).
In contrast, l-NAME had no significant effect on stimulation-evoked NE release from adrenergic nerves in facial arteries (Fig. 3D). However, the addition of SNAP significantly elevated stimulation-evoked NE release (Fig. 3D). Basal NE release was consistent during the experimental time course. The addition ofl-NAME or SNAP did not significantly alter basal NE release in either middle cerebral or facial arteries (Table1).
Discussion
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 using3H-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.
Acknowledgments
We thank Charles Hewitt for valuable technical assistance with the experimental protocols used in this study.
Footnotes
-
Send reprint requests to: John Buchholz, Ph.D., Department of Physiology and Pharmacology, Loma Linda University, School of Medicine, Loma Linda, CA 92350. E-mail:jbuchholz{at}som.llu.edu
-
↵1 This work was supported in part by National Institute of Child Health and Human Development, National Institutes of Health, Grant PO1-HD-31226.
- Abbreviations:
- NO
- nitric oxide
- l-NAME
- Nω-nitro-l-arginine methyl ester
- SNAP
- S-nitroso-N-acetyl-dl-penicillamine
- NE
- norepinephrine
- NOS
- NO synthase
- DHBA
- dihydroxybenzylamine
- Received October 1, 1999.
- Accepted January 24, 2000.
- The American Society for Pharmacology and Experimental Therapeutics