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Am J Physiol Regul Integr Comp Physiol 276: R799-R808, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 3, R799-R808, March 1999

Impact of development and chronic hypoxia on NE release from adrenergic nerves in sheep arteries

John Buchholz1, Kim Edwards-Teunissen2, and Sue P. Duckles3

1 Department of Pharmacology and Physiology, School of Medicine, Loma Linda University, Loma Linda 92350; 3 Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697; and 2 Catholic University of Nijmegen, Sophiaweg 104, The Netherlands


    ABSTRACT
Top
Abstract
Introduction
METHODS
Results
Discussion
References

To examine effects of development and chronic high-altitude hypoxia on sympathetic nerve function in sheep, norepinephrine release was measured in vitro from middle cerebral and facial arteries. Capsaicin was used to test the role of capsaicin-sensitive sensory nerves; norepinephrine release was not altered by capsaicin treatment. Nomega -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, decreased stimulation-evoked norepinephrine release in middle cerebral arteries from normoxic sheep with no effect in hypoxic arteries or facial arteries. Thus NO-releasing nerves augmented norepinephrine release. Furthermore, the function of NO-releasing nerves declined after chronic hypoxia. Despite loss of the augmenting effects of NO, stimulation-evoked fractional norepinephrine release was unchanged after chronic hypoxia, suggesting that middle cerebral arteries adapt to hypoxia by increasing stimulation-evoked norepinephrine release. In fetal facial arteries, chronic hypoxia resulted in a decline in stimulation-evoked norepinephrine release, but there was an increase in the adult facial artery. In the adult, adaptation to chronic hypoxia is similar in both cerebral and facial arteries. However, differential adaptation in fetal adrenergic nerves may reflect differences in fetal redistribution of blood flow in the face of chronic hypoxia but could also possibly contribute to increased incidence of fetal morbidity.

nitric oxide synthase; chronic hypoxia; sympathetic nerve function


    INTRODUCTION
Top
Abstract
Introduction
METHODS
Results
Discussion
References

SYMPATHETIC NERVE ACTIVITY serves to protect the individual organism against environmental stress. For example, acute hypercapnia is associated with an increase in plasma catecholamines, reflecting an alteration in sympathetic nerve activity, which is believed to protect vital organs such as the heart and brain from damage (12). In the awake rabbit, cerebrovascular nerves are thought to have a protective effect against damage due to hemorrhagic hypotension. During hemorrhagic hypotension, reflex stimulation of sympathetic nerves dampens cerebral blood flow during hypoxia with very little effect in normoxic animals (4). In lambs, sympathetic stimulation blunts the increase in cerebral blood flow by 25% during hypoxia, with less effect during normoxia (39). Adrenergic innervation may also play an important role in reducing damage to cerebral blood vessels due to hypertension (1, 5).

High-altitude hypoxemia during gestation has been associated with an increase in cerebrovascular morbidity in the fetus and infant. For example, infants exposed to hypoxia during the prenatal period show a higher incidence of intraventricular hemorrhage (14, 21). Effects of acute hypoxia on adrenergic nerve function are not fully understood, but the body of knowledge is much greater compared with the effects of chronic hypoxic exposure. Acute hypoxia has been shown to inhibit stimulation-evoked norepinephrine release in vitro. For example, in thoracic aorta strips from the rabbit, acute hypoxia inhibits stimulation-evoked contractile responses to a much greater extent than inhibition of contractile responses to exogenously applied norepinephrine (24). The implication of these findings has been confirmed by observations that acute hypoxia in vitro inhibits stimulation-evoked norepinephrine release (23). In contrast, in fetal sheep exposed to hypercapnia in the absence of hypoxia, there is an increase in circulating plasma levels of norepinephrine and epinephrine (12). Similarly, in conscious dogs, acute hypoxia causes an increase in circulating norepinephrine and epinephrine (32). Furthermore, combined hypoxia and hypercapnia elevate norepinephrine and epinephrine to a greater extent compared with hypoxia alone (32). In both studies, norepinephrine was the predominant catecholamine released, suggesting that the major source of norepinephrine was from the sympathetic nerve terminals. In the human, exposure to acute hypoxia during exercise has been shown to increase release of cardiac norepinephrine and its cotransmitter, neuropeptide Y, from the heart (19).

Very little is known about the effect of chronic hypoxia on the function of blood vessels or adrenergic nerves. Long-term hypoxia has been shown to increase dopamine, but not norepinephrine, turnover in adult rat sympathetic ganglia (9). Chronic hypoxia in the rat also blunts pressor and vasoconstrictor responses to phenylephrine, angiotensin, and arginine vasopressin (10). There is evidence that chronic hypoxic exposure results in a decrease in contractile response in isolated fetal sheep middle cerebral arteries (26). In fetal and adult sheep middle cerebral arteries, chronic high-altitude hypoxia results in a significant decline in density of alpha 1-adrenergic and inositol 1,4,5-trisphosphate receptors and a parallel depression of norepinephrine-induced increases in smooth muscle inositol 1,4,5-trisphosphate (38, 41). These data suggest that adrenergically mediated contraction in cerebral blood vessels is depressed after exposure to chronic hypoxia.

The current study was undertaken to examine the effects of chronic hypoxia on sympathetic nerve function in fetal and adult sheep middle cerebral and facial arteries given the noted protective effects of adrenergic nerves. We tested whether chronic hypoxia would alter function of adrenergic nerves in cerebral and peripheral arteries from the fetus or adult.

It has been suggested that NO may modulate stimulation-evoked norepinephrine release, but results are controversial. For example, some investigators have suggested that NO released from the endothelium inhibits the release of norepinephrine from peripheral adrenergic nerve endings (8, 15, 34). In contrast, others have shown that selective antagonists of nitric oxide synthase (NOS) decrease the release of norepinephrine (40), suggesting that NO may augment stimulation-evoked norepinephrine release. Therefore, we used Nomega -nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase to test the hypotheses that NO regulates sympathetic nerve function and that this regulation varies with maturation or hypoxia. There is also ample evidence that capsaicin-sensitive vasodilator nerves innervate the cerebral vasculature (16, 17); therefore we used capsaicin to test whether such modulation might vary with maturation or after exposure to chronic hypoxia.


    METHODS
Top
Abstract
Introduction
METHODS
Results
Discussion
References

Eighteen pregnant and fourteen nonpregnant ewes of mixed breed were obtained from a single supplier (Nebeker Ranch, Lancaster, CA). These were randomly separated into control normoxic (9 pregnant and 7 nonpregnant) and long-term hypoxic groups (9 pregnant and 7 nonpregnant). Animals in the control group remained at Nebeker Ranch (718 m). At 30 days gestation, pregnant and nonpregnant animals in the long-term hypoxic group were transported to the Barcroft Laboratory, White Mountain Research Station (Bishop, CA; altitude, 3,820 m). At 138-142 days gestation, pregnant and nonpregnant normoxic and hypoxic animals were transported to the Department of Perinatal Biology at Loma Linda University where they underwent immediate study.

In the case of hypoxic ewes awaiting study, immediately after arrival at the Department of Perinatal Biology, a nonocclusive tracheal catheter was surgically implanted (13) so that N2 gas could be administered to maintain the arterial PO2 at ~60 Torr. Arterial blood gases in the adults were measured in 0.5-ml samples (ABL300; Radiometer, Copenhagen, Denmark). The mean PO2 were 102 ± 2 and 60 ± 2 Torr for normoxic adults and hypoxic animals, respectively. On the experimental day, ewes were killed by administration of 100 mg/kg intravenous pentobarbital sodium, and near-term fetuses were delivered by cesarean section. The arterial PO2 were 23.3 ± 0.5 and 19.3 ± 0.8 Torr for normoxic and hypoxic fetuses, respectively. Fetal weights were unaffected by hypoxia. Weights were 2,728 ± 183 (n = 8) and 3,256 ± 297 g (n = 8) for control and hypoxic fetuses, respectively.

Facial arteries and brains were removed from both adults and fetuses and placed in ice-cold Krebs solution, with subsequent dissection of the middle cerebral arteries. The Krebs solution contained (in mM): 118 NaCl, 4.8 KCl, 1.6 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, 0.3 ascorbic acid, and 11.5 glucose.

Norepinephrine release. Segments of the middle cerebral and facial arteries 3-4 cm in length were cannulated at both ends with polyethylene tubing and mounted in an in vitro low-volume perfusion system as previously described (3). The middle cerebral artery segment used was the main branch from the circle of Willis. Middle cerebral artery diameters ranged from 0.35 to 0.6 mm in the fetus and 0.8 to 1.2 mm in the adult. Facial artery diameters ranged from 0.8 to 1.2 mm in the fetus and from 1 to 1.3 mm in the adult. Arteries were perfused at a rate of 1.0 ml/min, creating perfusion pressures of 40-50 mmHg in facial arteries and 50-75 mmHg in the middle cerebral arteries. The entire perfusion assembly was immersed in a circulating water bath and kept at 37°C. At the beginning of the experiment, tissues were perfused for 1 h with aerated (95% O2-5% CO2) Krebs solution containing 10-5 M cocaine and 10-5 M deoxycorticosterone to inhibit neuronal and extraneuronal norepinephrine uptake, respectively.

Electrical field stimulation was delivered by a Grass S-48 stimulator (Grass Instruments, Quincy, MA) through two platinum electrodes placed at either end of the tissue chamber. The parameters for excitation were 8 Hz, 60 V, 1-ms duration, and 480 pulses (1-min stimulation). In Fig. 1, actual data are shown to illustrate the two experimental protocols. In each experiment, one middle cerebral artery and one facial artery served as time controls. Protocol 1 was designed to test the effect of inhibition of NO synthase with L-NAME on norepinephrine release. Perivascular nerves in the control tissues (Fig. 1, Protocol 1A) were activated four consecutive times [stimulation (S)1-S4] for 1 min, with a 30-min equilibration between each stimulation. Perivascular nerves in the treatment tissues (Fig. 1, Protocol 1B) were activated two consecutive times (S1, S2), with a 30-min equilibration separating S1 and S2. After S2, tissues were exposed for 30 min to L-NAME (10 µM). Nerves were once again activated (S3) for 1 min. After S3, tissues were exposed to L-NAME (10 µM) and L-arginine (100 µM) for 30 min. Once again, nerves were activated (S4) for 1 min. The effects of drug treatments were represented as the ratio of fractional norepinephrine release with each treatment, Sn/S2, where n represents the treatment number.


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  		Fig. 1.   Representative data to illustrate experimental protocol for measurement of stimulation-evoked norepinephrine release in adult middle cerebral arteries (MCA). Protocol for facial arteries was identical. Tissues were exposed throughout to deoxycorticosterone (Doc) and cocaine (Coc; 10-5 M). Perivascular nerves were activated 4 consecutive times [stimulation (S)1-S4], each for 1 min, with at least 30 min of equilibration between each stimulation train. In each experiment, 1 artery served as a time control (A and C). Protocol 1: in treated tissues (B) after S2, tissues were exposed to Nomega -nitro-L-arginine methyl ester (L-NAME; 10 µM) for 30 min and activated again for 1 min. After S3, tissues were exposed to L-NAME (10 µM) and L-arginine (100 µM) for 30 min and activated for 1 min. Protocol 2: in treated tissues (D) after S2, tissues were exposed to capsaicin (1 µM) for 30 min, followed by 30-min washout, and nerves were activated again (S3) for 1 min. Tissues were then treated with tetrodotoxin (TTX; 1 µM) for 30 min, and nerves were activated again (S4) for 1 min.

Protocol 2 was designed to test for possible effects of capsaicin-sensitive nerves on norepinephrine release. Perivascular nerves in the control tissues (Fig. 1, Protocol 2A) were activated four consecutive times (S1-S4) for 1 min, with a 30-min equilibration between each stimulation. Perivascular nerves in the treatment tissues (Fig. 1, Protocol 2B) were activated two consecutive times (S1, S2), with a 30-min equilibration separating S1 and S2. After S2, tissues were exposed for 30 min to capsaicin (1 µM) followed by a 30-min washout, and nerves were once again activated (S3) for 1 min. After S3, tissues were exposed to tetrodotoxin (1 µM) for 30 min, and once again nerves were activated (S4) for 1 min. The effects of drug treatments were represented as the ratio of fractional norepinephrine release with each treatment, Sn/S2, where n represents the treatment number.

Measurement of norepinephrine. The perfusate was collected continuously starting at the beginning of each stimulation period until 5 ml was collected. Basal norepinephrine release was monitored by collection of 5 ml of perfusate before each stimulation period. Perfusates were extracted and quantified with dihydroxybenzylamine (DHBA) as an internal standard (300 pg) after a previously described protocol (2). 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 reverse-phase C18 column with ESA MD-TM aqueous mobile phase. The mobile phase contained (in mM): 75 Na2H2PO4, 0.5 sodium dodecyl sulfate, and 0.025 EDTA, 20% acetonitrile, and 5% methanol. The following formula was used to calculate the amount of norepinephrine (NE) in the injected sample
pg NE = <FR><NU>NE peak Ht sample</NU><DE>NE peak Ht standard</DE></FR> × 100 pg DHBA
× <FR><NU>DHBA peak Ht standard</NU><DE>DHBA peak Ht sample</DE></FR>
where Ht is height. Recovery varied from 85-98%.

To quantitate tissue norepinephrine 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 norepinephrine was then extracted similarly as the perfusate. Norepinephrine content was used to calculate fractional norepinephrine release
fractional NE release 
= <FR><NU>pg NE released</NU><DE>pg NE tissue content × no. of stimulation pulses</DE></FR>

Statistical analysis. The impact of development and hypoxia on norepinephrine content and release was analyzed by two-way ANOVA. The effect of capsaicin treatment on norepinephrine release was analyzed by two-way ANOVA with repeated measures. Individual differences were then analyzed by the Fischer protected least significant difference post hoc test. The effect of L-NAME on norepinephrine release and reversal by L-arginine relative to controls was analyzed by paired t-test.

Drugs used. Cocaine hydrochloride, deoxycorticosterone, tetrodotoxin, 8-methyl-N-vanillyl-nonamide (capsaicin), L-NAME, and L-arginine were obtained from Sigma (St. Louis, MO).


    RESULTS
Top
Abstract
Introduction
METHODS
Results
Discussion
References

Effect of NO synthase inhibition. The effect of the NOS inhibitor L-NAME on stimulation-evoked norepinephrine release in fetal and adult middle cerebral and facial arteries is shown in Fig. 2. In the presence of L-NAME, stimulation-evoked norepinephrine release significantly declined in both fetal and adult normoxic middle cerebral arteries (Fig. 2A). Furthermore, the effect of L-NAME was significantly reversed by addition of L-arginine in both fetal and adult middle cerebral arteries from normoxic animals. In contrast, in middle cerebral arteries from hypoxic animals, either fetal or adult, L-NAME had no significant effect on stimulation-evoked norepinephrine release (Fig. 2B). Furthermore, L-NAME had no significant effect on stimulation-evoked norepinephrine release from facial arteries from either normoxic or hypoxic animals, fetal or adult (Fig. 2, C and D). Basal norepinephrine release was not significantly affected by L-NAME treatment in any of the groups (Table 1).


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  		Fig. 2.   Effect of L-NAME on stimulation-evoked norepinephrine release in fetal and adult normoxic (A) and hypoxic (B) MCA. Effects in fetal and adult normoxic (C) and hypoxic (D) facial arteries are also shown. Treatment with L-NAME and L-arginine was carried out as described in METHODS and illustrated in Fig. 1. Effects of drug treatments were represented as ratio of fractional norepinephrine release with each treatment, Sn/S2, where n represents treatment number. Each bar represents mean ± SE. Open bars, S3/S2 time control; hatched bars, S3/S2 L-NAME; crosshatched bars, S4/S2 time control; filled bars, S4/S2 L-NAME + L-arginine. * Significantly different from normoxic time controls, P < 0.05; + significantly different from L-arginine, P < 0.05; n = 6-8 arteries from each group.

                              
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  		Table 1.   Effect of treatments on basal norepinephrine release in middle cerebral and facial arteries

Effect of capsaicin treatment. Capsaicin treatment had no significant effect on basal norepinephrine release from fetal or adult middle cerebral or facial arteries (Table 1). Similarly, stimulation-evoked fractional norepinephrine release was not significantly changed after treatment with 1 µM capsaicin in normoxic or hypoxic fetal or adult middle cerebral or facial arteries (Fig. 3). Thus capsaicin had no significant effects on basal or stimulation-evoked norepinephrine release in any of the groups studied.


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  		Fig. 3.   Effect of capsaicin treatment on stimulation-evoked fractional norepinephrine release in fetal and adult normoxic (A) and hypoxic (B) MCA are shown. Effects in fetal and adult normoxic (C) and hypoxic (D) facial arteries are also shown. Capsaicin treatment was carried out as described in METHODS and illustrated in Fig. 1. Effects of drug treatments were represented as ratio of fractional norepinephrine release with each treatment, S3/S2. Bars represent means ± SE; n = 6-8 arteries from each group. Open bars, S3/S2 time control; shaded bars, S3/S2 + capsaicin.

Effect of development. As shown in Fig. 4A, in normoxic animals, there were no significant developmental differences in norepinephrine content in either middle cerebral or facial arteries when arteries from normoxic fetal animals were compared with arteries from normoxic adults. In contrast, as shown in Fig. 4B, total or mass of stimulation-evoked norepinephrine released, expressed as picograms norepinephrine released per milligrams tissue, tended to decline with development from fetus to adult. There was a significant developmental decline in mass stimulation-evoked norepinephrine release in both middle cerebral and facial arteries from normoxic animals. When norepinephrine release was expressed as a fraction of norepinephrine content (fractional release), a significant effect of development on norepinephrine release was revealed in both middle cerebral and facial arteries; development resulted in a significant decrease in stimulation-evoked fractional norepinephrine release in both middle cerebral and facial arteries (Fig. 4C). Thus with development from fetus to adult there was a decline in stimulation-evoked norepinephrine release, with no change in norepinephrine content, resulting in a developmental decline in total or mass stimulation-evoked norepinephrine release.


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  		Fig. 4.   Effect of development and chronic hypoxia on norepinephrine content (A), mass stimulation-evoked norepinephrine release (B), and stimulation-evoked fractional norepinephrine release (C) in MCA and facial arteries. Left, fetal; right, adult. Bars represent means ± SE; n = 14-18 arteries from each group. + Significantly different from adult; * significantly different from normoxic, P < 0.05.

Effect of hypoxia. In general, chronic hypoxia caused a decline in norepinephrine content in both middle cerebral and facial arteries from both fetuses and adults (Fig. 4A). However, this decline only reached statistical significance in fetal middle cerebral and adult facial arteries. Hypoxia caused an overall decline in mass of norepinephrine release in both fetal and adult middle cerebral and facial arteries. However, this decline reached statistical significance only in fetal middle cerebral and facial arteries (Fig. 4B). In terms of fractional norepinephrine release, chronic hypoxia caused a significant effect on stimulation-evoked fractional norepinephrine release only in facial arteries (Fig. 4C). Chronic hypoxia produced significant but opposite effects in fetal and adult facial arteries, causing a decrease in norepinephrine release in fetal facial arteries but a significant increase in the facial artery in the adult (Fig. 4C). Chronic hypoxia had no significant effect on stimulation-evoked fractional norepinephrine release in either fetal or adult middle cerebral arteries (Fig. 4C).

Basal norepinephrine release. The impact of development and chronic hypoxia on basal norepinephrine release is shown in Fig. 5. In both middle cerebral and facial arteries, basal norepinephrine release was significantly greater in arteries from normoxic fetal compared with normoxic adult animals. In contrast, chronic hypoxia significantly affected basal norepinephrine release only in fetal middle cerebral and facial arteries where basal norepinephrine release increased in the middle cerebral artery and decreased in the facial artery after chronic hypoxia (Fig. 5A).


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  		Fig. 5.   Effect of development and chronic hypoxia on basal norepinephrine release in MCA and facial arteries from fetal (A) and adult (B) animals. Bars represent means ± SE; n = 14-18 arteries from each group. + Significantly different from adult, P < 0.05; * significantly different from normoxic, P < 0.05.


    DISCUSSION
Top
Abstract
Introduction
METHODS
Results
Discussion
References

One mechanism that allows for a successful adaptive response to environmental stresses such as hypoxemia is a change in the reactivity of the sympathetic nervous system (32). In utero, the normal mammalian fetus adapts to low arterial oxygen tension by high cardiac output and an oxygen consumption about two times that of the adult (27); thus maternal hypoxia provides an additional challenge for the fetus. The model of maternal and fetal hypoxia used in the current study, exposure of pregnant sheep to high altitude throughout gestation, is one of moderate, well-adapted hypoxia. During the time at high altitude (3,820 m), adult and fetal arterial PO2 values decline significantly and arterial pH remains constant. Furthermore, hemoglobin increases in response to hypoxia to increase the extraction of oxygen (20). In hypoxic fetuses, weight increases over the course of gestation in a normal fashion, with near-term fetal weights similar to animals maintained at sea level (20). Furthermore, there is no significant increase in fetal morbidity, mortality, or abortion during the hypoxic exposure period. Animals used in this study were exposed to the same degree of hypoxic stress as in previous studies, and arterial PO2 levels in both adult and near-term fetuses were nearly identical to previous studies mentioned above. These observations justify the labeling of this model as one of moderate chronic hypoxia in which adaptive responses can be successfully studied.

Hypoxia and norepinephrine release in middle cerebral arteries. One of the most significant results of the current study is that chronic hypoxia did not result in any significant change in stimulation-evoked fractional norepinephrine release from either adult or fetal middle cerebral arteries (Table 2). These data would suggest that sympathetic nerves in the middle cerebral artery do not adapt to chronic hypoxia. However, these data need to be interpreted in the context of two other very important findings in this study. The first is that in normoxic adult and fetal middle cerebral arteries, L-NAME, an antagonist of NOS, significantly reduced stimulation-evoked norepinephrine release without affecting basal release. It is well known that cerebral arteries are innervated with nerves that contain NOS and release NO (37). The effect of L-NAME occurred only in the cerebral arteries and not in the facial arteries. These data suggest that in the middle cerebral artery, there are NOS-containing nerves that are coactivated in vitro along with sympathetic nerves, releasing NO, which then augments stimulation-evoked norepinephrine release. However, these data do not rule out the possibility that endothelium-derived NO may be a factor in our observations.

                              
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  		Table 2.   Summary of effect of development and long-term hypoxia on norepinephrine content and basal and stimulation-evoked norepinephrine release in MCA and FA

The second critical finding is that chronic hypoxia eliminated the effect of L-NAME on norepinephrine release in both fetal and adult middle cerebral arteries. Thus, with chronic hypoxia, sympathetic nerves innervating cerebral arteries do adapt to the loss of NO-releasing nerves by increasing stimulation-evoked fractional norepinephrine release. However, in our study, this effect is masked because in normoxic animals nerve stimulation also releases NO from NO-containing nerves and NO augments norepinephrine release.

Others have also shown that inhibitors of NO synthase decrease stimulation-evoked norepinephrine release from sympathetic nerves (40), implying that endogenous NO production augments norepinephrine release. A mechanism for the augmentation of norepinephrine release by NO has been proposed. In isolated superior cervical ganglion cells, NO donors such as sodium nitroprusside have been shown to increase stimulation-evoked calcium influx (6). This increased calcium influx leads to higher calcium levels inside the cell and an augmentation of norepinephrine release. In contrast, it has been hypothesized that NO released from endothelial cells inhibits norepinephrine release from sympathetic nerves innervating blood vessels (8, 15). Thus it appears that NO can act either as an inhibitor or augmentor of norepinephrine release, but, in our studies of sheep cerebral arteries, the predominant effect of NO released from nerves appears to be augmentation of stimulation-evoked norepinephrine release. The mechanism by which the augmentation of norepinephrine release by NO is lost after chronic hypoxia is unknown. This could reflect a loss with hypoxia of the NO-containing nerves innervating cerebral arteries, a decrease in levels of NO synthase, or a decrease in sensitivity to NO. Further studies will be necessary to explore this issue.

Chronic high-altitude hypoxia resulted in a significant decline in norepinephrine content in the fetal middle cerebral artery. In rat sympathetic ganglia, norepinephrine contents in the superior cervical, celiac, and mesenteric ganglia were unaltered after 28 days of hypoxic stress (9). In another study, acute hypoxia did not alter norepinephrine content in the midbrain, brain stem, cerebral cortex, or cerebellum of newborn rats (35). To take into account the decline with chronic hypoxia of norepinephrine content, data for stimulation-evoked norepinephrine release were expressed in two different ways: as mass of norepinephrine release in picograms per milligrams tissue, reflecting the total amount of stimulation-evoked norepinephrine released, and as a fraction of the total content (fractional release). In the middle cerebral artery, mass norepinephrine release tended to mirror the norepinephrine content, suggesting that the fall in mass norepinephrine release with hypoxia is primarily a consequence of the fall in norepinephrine content (Table 2).

In contrast, stimulation-evoked norepinephrine release expressed as fractional release was not significantly affected by chronic hypoxia in the middle cerebral artery of either fetal or adult sheep. However, as discussed above, in this specific case, an effect of hypoxia on the augmentation of norepinephrine release by NO appears to have masked an effect of hypoxia on fractional norepinephrine release. Thus it appears that fractional norepinephrine release from adrenergic nerves innervating the middle cerebral artery may have actually increased with hypoxia, giving a net effect of no overall change with hypoxia when NO synthase is functional. This appears to have occurred in middle cerebral arteries from both fetuses and adults.

Facial arteries and hypoxia. Similar to findings in cerebral arteries, chronic hypoxia tended to decrease norepinephrine content in facial arteries; however, this decrease only reached statistical significance in the adult facial artery. In contrast to the middle cerebral artery, L-NAME had no effect on stimulation-evoked norepinephrine release from facial arteries from normoxic or hypoxic adults or fetuses. In facial arteries from the fetus, chronic hypoxia resulted in a substantial decline in stimulation-evoked norepinephrine release, expressed either as mass or fractional release. In contrast, in the adult facial artery, because norepinephrine content declined with hypoxia, mass norepinephrine release was unaffected by hypoxia, although fractional norepinephrine release was increased (Table 2B). Thus in the adult facial artery, adrenergic nerves appear to adapt to chronic hypoxia with an increase in fractional transmitter release, whereas in the fetus, adrenergic nerve activity does not adapt to hypoxia in the same manner. These findings demonstrate that sympathetic nerves innervating fetal and adult middle cerebral arteries and the adult facial artery respond to chronic hypoxia by increasing fractional norepinephrine release. In contrast, adrenergic nerves in the fetal facial artery decreased fractional norepinephrine release after exposure to hypoxia.

Hypoxia and basal norepinephrine release. In the fetal middle cerebral artery, chronic hypoxia resulted in an increase in basal norepinephrine release, but a decline was seen in the fetal facial artery. In the adult, basal norepinephrine release was unaffected by chronic hypoxia in either the middle cerebral or facial arteries. Thus the effect of chronic hypoxia on basal release in fetal arteries was similar to stimulation-evoked fractional norepinephrine release that increased in the fetal middle cerebral artery (when modulating effects of NO are eliminated) and declined in the facial artery. The maintained robust response of adrenergic nerves to stimulation after long-term hypoxia in both the fetal and adult middle cerebral arteries underscores the idea that increased function of adrenergic nerves may play an important role in the successful adaptation of fetuses and adults to the stress of chronic hypoxia (9, 18, 41). Acute hypoxia in vivo has been shown to result in an increase in circulating catecholamines in fetal sheep, whereas in humans there is evidence for increased norepinephrine release from adrenergic nerves in the heart (12, 19, 29, 30). Our findings with long-term hypoxia are consistent with previous studies suggesting that a high and sustained level of adrenergic nerve activity is a critical component of successful adaptation.

In contrast, in the fetal facial artery, chronic hypoxia resulted in significant declines in both basal and stimulation-evoked fractional norepinephrine release. Together these data point to a substantial decline in function of facial artery adrenergic nerves in the hypoxic fetus. It is interesting that, in the hypoxic fetal sheep, previous reports indicate that, acutely, as well as over the first few days of hypoxia, plasma norepinephrine levels significantly increase (20, 22). Thus, in contrast to fetal and adult middle cerebral arteries and adult facial arteries, it appears that fetal peripheral vascular adrenergic nerves are unable to sustain increased activity in the face of chronic hypoxia. Adrenergic nerves are thought to serve a protective function for the cerebral circulation that is magnified in the presence of hypoxia (4). Therefore it is possible that the failure of the fetus to sustain a rise in peripheral adrenergic nerve function over the course of chronic hypoxia could contribute to a less-robust adaptation to the stress of hypoxia.

Development and norepinephrine release. There is little information in the literature about the impact of development per se on function of adrenergic nerves. In normoxic near-term fetal sheep, stimulation-evoked fractional norepinephrine release was substantially lower in both middle cerebral and facial arteries from adults compared with near-term fetuses (Table 2). Effects of development on stimulation-evoked norepinephrine release were paralleled by changes in basal (or non-stimulation evoked) norepinephrine release. Basal norepinephrine release from adult middle cerebral and facial arteries was significantly lower compared with basal norepinephrine release in the corresponding arteries from normoxic near-term fetuses. Norepinephrine content did not change with development in either middle cerebral or facial arteries, and developmental changes in mass of norepinephrine released parallel changes in fractional release. Thus, in the middle cerebral and facial artery, development appears to have resulted in a decrease in adrenergic nerve function, reflected by decreases in both basal and stimulation-evoked fractional norepinephrine release with no change in norepinephrine content. Overall, these data on the effects of development on adrenergic nerve function are consistent with other studies suggesting that sympathetic nerve activity rises significantly before birth, resulting in increased stimulation-evoked norepinephrine release and increased plasma catecholamine levels in both sheep and rats (7, 11, 36). This gestational increase in adrenergic nerve function appears to decline further with development to adulthood.

Very little is known with regard to the mechanisms of adaptation of adrenergic nerves to acute or chronic hypoxia. One possibility to explain the increase in plasma catecholamine levels would be a decline in uptake of norepinephrine by the nerve terminal. Indeed, in the rat heart, hypoxia lasting 5-21 days results in a decline in norepinephrine uptake (33). Similar results have been reported in isolated rabbit thoracic aortic strips exposed to acute hypoxia, in which norepinephrine reuptake decreased (23). Changes in norepinephrine uptake with hypoxia may contribute to the increases in plasma catecholamine levels seen by other investigators. However, the effects of hypoxia on norepinephrine uptake cannot account for the changes in norepinephrine release that we have seen, because in our study uptake of norepinephrine was blocked throughout the in vitro measurement of norepinephrine release.

The sustained stimulation-evoked fractional norepinephrine release with chronic hypoxia seen in both fetal and adult middle cerebral arteries and the increase in adult facial arteries occurs independently of changes in norepinephrine content or basal norepinephrine release. This could be due to a variety of changes in adrenergic nerve function, including alterations in ion channels and the resting state of the membrane, in calcium handling and sequestration by intracellular mechanisms, or in the docking and release mechanisms of synaptic vesicles. The lack of these adaptive changes in vascular adrenergic nerves of the fetal facial artery could be due to less-fully developed adrenergic nerve function. Further studies will be necessary to define these mechanisms in the near-term fetus.

Chronic hypoxia also causes substantial changes in the function of vascular smooth muscle in both fetal and adult arteries. Chronic high-altitude hypoxia results in a depression of maximum contractile responses to potassium, with an even greater effect on contractile responses to serotonin and norepinephrine in fetal arteries compared with arteries from adults (26). Associated with this depression in contractile response is a decline in density of alpha 1-adrenergic receptors and norepinephrine-induced increases in smooth muscle inositol 1,4,5-trisphosphate (38, 41). These studies stand in contrast to our study of fetal and adult middle cerebral arteries showing sustained norepinephrine release in the absence of the augmentation effects of NO and an increase in norepinephrine release in the adult facial artery. However, these studies complement our data in the fetal facial artery showing a decline in adrenergic nerve function.

There is abundant evidence that capsaicin-sensitive sensory nerves innervate the cerebral vasculature (16, 17). Stimulation parameters that activate adrenergic nerves in vitro will also activate capsaicin-sensitive nerves (31). Thus we tested the hypothesis that these nerves may possibly influence norepinephrine release. Capsaicin was used to selectively deplete sensory nerve transmitters (25, 28); however, we found that capsaicin had no significant effect on stimulation-evoked or basal norepinephrine release in either middle cerebral or facial arteries from normoxic or hypoxic animals. These data suggest that in the middle cerebral artery, capsaicin-sensitive nerves do not normally modulate norepinephrine release. Furthermore, the lack of a role of sensory nerves to modulate norepinephrine release was unaltered after chronic hypoxic stress. This suggests that the influence on the cerebral vasculature of capsaicin-sensitive sensory nerves, if any, must lie in postjunctional effects on the smooth muscle rather than modulation of transmitter release.

In conclusion, we have shown that stimulation-evoked norepinephrine release from adrenergic nerves in sheep middle cerebral and facial arteries is greater in arteries from near-term fetuses compared with adults. Furthermore, chronic hypoxia has similar effects on stimulation-evoked norepinephrine release from sympathetic nerves in the middle cerebral arteries of both the fetus and adult. We have also shown that NOS-containing nerves in the cerebral vasculature augment norepinephrine release, an effect that is lost with chronic hypoxia. Despite the loss of NO nerve function with hypoxia, cerebral arterial adrenergic nerves maintain their function, suggesting adaptation to chronic hypoxia. Sympathetic nerves in the adult facial artery showed a similar adaptation to hypoxia with an increase in norepinephrine release, but this adaptation did not occur in fetal facial arteries. These data support the conclusion that adaptation to chronic hypoxia occurs in both adult adrenergic nerves. However, in the fetus, adrenergic nerves do not adapt in the same way. Differential adaptation in fetal adrenergic nerves may possibly contribute to increased incidence of fetal morbidity or may reflect differences in fetal redistribution of blood flow in the face of chronic hypoxia.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge Charles Hewitt and Jonnie Sephus for technical expertise in the execution of these experiments.


    FOOTNOTES

This work was supported in part by from National Institute of Child Health and Human Development Grant PO1-HD-31226.

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.

Address for reprint requests: J. Buchholz, Dept. of Physiology and Pharmacology, Loma Linda Univ., School of Medicine, Loma Linda, CA 92350 (E-mail: jbuchholz{at}som.llu.edu).

Received 8 May 1998; accepted in final form 23 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
Results
Discussion
References

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Am J Physiol Regul Integr Compar Physiol 276(3):R799-R808
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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