INTRODUCTION

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NITRIC OXIDE (NO) is produced enzymatically from the terminal guanidino nitrogen of L-arginine by the action of NO synthase (NOS) (12, 14). There are at least three isoforms of NOS: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS. The adrenal medulla possesses characteristic postganglionic sympathetic neurons, and the presence of nNOS has been demonstrated (7, 9, 11, 15). In vitro studies using NOS inhibitors and NO donors were performed to examine the role of NO in modulating catecholamine secretion from the adrenal medulla but the results remain controversial. It has been reported that the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) inhibits acetylcholine (ACh)-induced catecholamine secretion in bovine chromaffin cells (17) and that the NO donor sodium nitroprusside (SNP) enhances nicotine-induced catecholamine secretion in cultured bovine chromaffin cells (10). These findings suggest that NO may facilitate cholinergic agonist-induced catecholamine secretion. In contrast, it has been reported that the NOS inhibitor L-NAME enhances K⁺-stimulated catecholamine secretion in cultured bovine chromaffin cells (16) and that SNP inhibits ACh-induced catecholamine secretion in bovine chromaffin cells (13). These studies suggest that NO may play an inhibitory role in the control of catecholamine secretion. Moreover, the presence of endothelial cells has been reported to inhibit the K⁺-induced or the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium-induced catecholamine secretion in cultured bovine chromaffin cells (16), suggesting that not only nNOS but also eNOS may play roles in modulating adrenal catecholamine secretion. On the other hand, a few in vivo studies have suggested that NO does not play a role in regulation of adrenal catecholamine secretion (1, 2).

In the present study, we investigated the effects of L-NAME and the NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC 7) on the secretion of catecholamine induced by splanchnic nerve stimulation (SNS) and ACh in anesthetized dogs to elucidate the functional role of NO in the control of adrenal catecholamine secretion. The effects of NOC 7 during treatment with L-NAME were also examined. L-NAME, NOC 7, and ACh were administered intra-arterially into the adrenal gland to eliminate hemodynamic effects on adrenal catecholamine secretion.

	MATERIALS AND METHODS

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Animal preparation. Mongrel dogs of either sex, weighing 10-14 kg, were anesthetized with 30 mg/kg iv of pentobarbital sodium, and a constant level of anesthesia was then maintained by intravenous infusion of pentobarbital sodium at a rate of 6 mg · kg¹ · h¹ with an infusion pump (201B, Atom, Tokyo, Japan). Artificial respiration was performed using a respiration pump (model-607, Harvard Apparatus, Millis, MA), with room air administered at 18 strokes/min (20 ml/kg tidal volume). The surgical procedure used in the present study was described previously (4). The left adrenal gland was exposed by a retroperitoneal flank incision, and a polyethylene cannula was inserted into the left adrenolumbar vein for collection of the venous effluent blood from the adrenal gland. A ligature was placed around the juncture of the adrenolumbar vein with the abdominal vena cava. Adrenal blood samples were obtained by pulling the ligature, thus occluding the adrenolumbar vein and causing retrograde flow of blood. Blood samples of 1 or 2 ml were collected in chilled test tubes containing disodium EDTA. When not being sampled, adrenal venous blood was returned directly to the vena cava. Coagulation of blood was prevented by an initial intravenous injection of sodium heparin (500 U/kg) and hourly intravenous injections of 100 U/kg. Systemic blood pressure and heart rate were measured with a pressure transducer (MPU-0.5, Nihon Kohden, Tokyo, Japan) and a cardiotachometer (RT-5, Nihon Kohden), respectively, and recorded on a heat-writing oscillograph (RJG-4128, Nihon Kohden).

Administration of drugs into the adrenal gland. The procedure for intra-arterial administration of drugs into the adrenal gland was reported previously (5). The left phrenicoabdominal artery was dissected to expose its origin from the abdominal aorta. A needle connected to a Y-shaped polyethylene catheter was inserted into the phrenicoabdominal artery at its origin for intra-arterial infusion of 0.9% saline solution (as a vehicle), L-NAME, NOC 7, and the combination of NOC 7 with L-NAME. These drugs were infused into the adrenal gland using an infusion pump (1140-001, Harvard Apparatus). ACh was injected intra-arterially for 3 s during infusion of saline, L-NAME, NOC 7, and the combination of NOC 7 with L-NAME.

SNS. The left splanchnic nerves were dissected free from surrounding tissue and cut. A bipolar platinum electrode was placed in contact with the distal ends of the splanchnic nerves. The splanchnic nerves were stimulated with rectangular pulses of 1 ms and 10 V (supramaximal voltage) delivered by an electronic stimulator (SEN-1101, Nihon Kohden) and an isolation unit (SS-101J, Nihon Kohden). Stimuli were applied at 1 Hz for 2 min, subsequently at 2 Hz for 2 min and 3 Hz for 2 min during a 6-min stimulus period.

Experimental protocol. The dogs were divided into eight groups. In group 1 (n = 6), the effects of repeated SNS on increases in catecholamine output were examined without drug treatment. SNS was repeated four times at 30-min intervals. Infusion of 0.9% saline was started 20 min before the start of the first, second, third, and fourth SNS. In group 2 (n = 6), the effects of repeated injection of ACh on increases in catecholamine output were examined. A set of ACh injections (0.75, 1.5, and 3 µg) into the adrenal gland was repeated four times at 35-min intervals. The interval between doses of ACh was 5 min. Infusion of 0.9% saline was started 20 min before the first, second, third, and fourth set of ACh injection. In group 3 (n = 8), the effects of L-NAME on the SNS-induced increases in catecholamine output were examined by the same protocol as used in group 1. The first SNS trial during saline infusion was regarded as a control. L-NAME infusion (0.1, 0.3, and 1 mg/min) was started 20 min before the start of the second, third, and fourth SNS, respectively. In group 4 (n = 6), the effects of L-NAME on the ACh-induced increases in catecholamine output were examined with the same protocol as used in group 2. The first set of ACh injection during saline infusion was regarded as a control. L-NAME infusion was started 20 min before the start of the second, third, and fourth set of ACh injections. The effects of NOC 7 (0.2, 0.6, and 2 µg/min) on increases in catecholamine output induced by SNS (group 5; n = 8) and ACh (group 6; n = 6) were examined by the same protocol as used in groups 3 and 4, respectively. In group 7 (n = 8), after the first SNS trial as a control, L-NAME infusion (1 mg/min) was started 20 min before the second SNS trial. Subsequently, the combined infusion of NOC 7 (2 µg/min) and L-NAME was started 20 min before the third SNS trial. In group 8 (n = 8), the effects of NOC 7 during treatment with L-NAME on increases in catecholamine output induced by ACh were examined with the same protocol as used in group 7.

Blood sampling and determination of adrenal catecholamine output. Adrenal venous blood was sampled before and during SNS and ACh injections to determine basal catecholamine output and stimuli-induced increases in catecholamine output, respectively. Sampling during the basal state (during saline, L-NAME, or NOC 7 infusion or the combined infusion of NOC 7 and L-NAME) was performed 2 min before SNS or sets of ACh injections. The time required to collect 1 (during basal state or SNS) or 2 ml (during ACh injections) of blood served to estimate adrenal venous flow rate.

Adrenal blood samples were centrifuged to obtain plasma samples. Catecholamine was extracted from plasma by the alumina adsorption method, and plasma epinephrine (Epi) and norepinephrine (NE) concentrations were determined by high-performance liquid chromatography with electrochemical detection (LC-4B, Bioanalytical Systems, West Lafayette, IN), as described previously (4). Epi and NE output (ng/min) were calculated by multiplying plasma catecholamine concentration (ng/ml) by adrenal plasma flow rate (ml/min), and the total output of Epi and NE was expressed as catecholamine output. Adrenal plasma flow rate was calculated by multiplying adrenal venous blood flow by 1  hematocrit of adrenal venous blood. The basal catecholamine output was determined from samples collected before SNS or ACh injections. The SNS- or ACh-induced changes in catecholamine output were calculated by subtracting the basal catecholamine output from that obtained during the stimulus state.

Analysis of data. The results are expressed as means ± SE throughout the study. Single-factor ANOVA was used for statistical analysis of data. When ANOVA showed a significant difference, Dunnett's test or Scheffé's test was used to determine significance level. P values <0.05 were considered to be statistically significant.

Drugs. The drugs used were L-NAME (Sigma Chemical), NOC 7 (Dojindo, Kumamoto, Japan), and ACh chloride (Daiichi Seiyaku, Tokyo, Japan). NOC 7 was dissolved in 0.01 N NaOH. Other drugs were dissolved in 0.9% saline solution.

	RESULTS

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Increases in catecholamine output in response to SNS and ACh. SNS (1, 2, and 3 Hz) or intra-arterial injection of ACh (0.75, 1.5, and 3 µg) into the adrenal gland produced frequency- or dose-dependent increases in adrenal venous plasma catecholamine concentration (data not shown). The 3-Hz SNS- and ACh-induced increases in catecholamine concentration were accompanied by increases in adrenal plasma flow rate (Tables 1 and 2). SNS at 1 and 2 Hz had no effect on adrenal plasma flow rate. Catecholamine output, calculated from catecholamine concentration and adrenal plasma flow rate, was increased by SNS and ACh injection (Table 1). The increases in catecholamine output induced by SNS and ACh during the four stimulation periods are shown in Table 1. Respective increases in catecholamine output induced by SNS and ACh did not vary during the time course of the experiment. The increases in catecholamine output induced by SNS (3 Hz) and ACh (3 µg) during the control stimulation periods were 438 ± 33 (n = 30) and 570 ± 85 ng/min (n = 26), respectively, in groups 1-8, in which basal catecholamine output during the resting state was 3.0 ± 0.5 ng/min (n = 56).

SNS produced small pressor and bradycardic responses. The increase in blood pressure produced by 3-Hz SNS was 11 ± 3 mmHg (n = 30), and the decrease in heart rate was 16 ± 3 beats/min (n = 30). Injection of ACh decreased blood pressure slightly but did not modify heart rate. The decrease in blood pressure produced by 3 µg of ACh was 8 ± 2 mmHg (n = 26). It is unlikely that baroreflex-mediated catecholamine secretion is involved in the catecholamine response to SNS and ACh, as described previously (8).

Effects of L-NAME on the SNS- and ACh-induced increases in catecholamine output. Intra-arterial infusion of L-NAME (0.1, 0.3, and 1 mg/min) into the adrenal gland inhibited increases in catecholamine output induced by SNS; statistically significant effects were observed with 0.3 or 1 mg/min at 1 Hz and 1 mg/min at 2 Hz (Fig. 1A). On the other hand, L-NAME enhanced increases in catecholamine output induced by ACh, but the degree of enhancement decreased in proportion to increases in the dose of L-NAME (Fig. 1B). Basal catecholamine output was not affected by L-NAME. In groups 3 and 4 (n = 14), basal catecholamine output before and during 0.1, 0.3, and 1 mg/min L-NAME infusion were 2.9 ± 0.8, 2.1 ± 0.5, 1.9 ± 0.5, and 1.8 ± 0.4 ng/min, respectively. Adrenal plasma flow rate was decreased by L-NAME (Table 2). L-NAME produced a small bradycardic response but did not affect blood pressure (Table 3).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of N-nitro-L-arginine methyl ester (L-NAME) on adrenal catecholamine output in response to splanchnic nerve stimulation (SNS) (A) and ACh (B). L-NAME and ACh were infused and injected intra-arterially into the adrenal gland. Histograms and vertical bars indicate means ± SE from 8 and 6 dogs (n) in SNS and ACh experiments, respectively. * P < 0.05; ** P < 0.01, compared with corresponding control values obtained before L-NAME infusion.

Effects of NOC 7. Intra-arterial infusion of NOC 7 (0.2, 0.6, and 2 µg/min) into the adrenal gland inhibited increases in catecholamine output induced by SNS and ACh (Fig. 2, A and B). Basal catecholamine output was not affected by NOC 7. In groups 3 and 4 (n = 14), basal catecholamine output before and during 0.2, 0.6, and 2 µg/min NOC 7 infusion was 3.4 ± 1.0, 3.4 ± 1.2, 4.0 ± 1.0, and 6.0 ± 2.1 ng/min, respectively. Adrenal plasma flow rate was not affected by NOC 7 (Table 2). NOC 7 produced a small depressor response but did not modify heart rate (Table 3).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effects of 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC 7; NOC) on adrenal catecholamine output in response to SNS (A) and ACh (B). NOC 7 and ACh were infused and injected intra-arterially into the adrenal gland. Histograms and vertical bars indicate means ± SE from 8 and 6 dogs (n) in SNS and ACh experiments, respectively. * P < 0.05; ** P < 0.01, compared with corresponding control values obtained before NOC 7 infusion.

Effects of NOC 7 during treatment with L-NAME. Intra-arterial infusion of L-NAME (1 mg/min) into the adrenal gland inhibited increases in catecholamine output induced by SNS at 1 and 2 Hz but did not affect 3-Hz-induced catecholamine response in the same manner as observed in group 1. During treatment with L-NAME, intra-arterial infusion of NOC 7 (2 µg/min) did not affect the SNS-induced increases in catecholamine output compared with the values obtained with L-NAME (Fig. 3A). On the other hand, L-NAME enhanced increases in catecholamine output induced by ACh in the same manner as observed in group 2. During treatment with L-NAME, NOC 7 inhibited the ACh-induced increases in catecholamine output compared with the values obtained with L-NAME (Fig. 3B).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of NOC 7 (NOC) with L-NAME treatment on adrenal catecholamine output in response to SNS (A) and ACh (B). L-NAME and NOC 7 were infused and ACh was injected intra-arterially into the adrenal gland. Histograms and vertical bars indicate means ± SE from 8 and 8 dogs (n) in SNS and ACh experiments, respectively. * P < 0.05; ** P < 0.01, compared with corresponding control values obtained before L-NAME infusion. P < 0.05, compared with corresponding values obtained with L-NAME infusion.

Basal catecholamine output was not affected by L-NAME or NOC 7 during treatment with L-NAME. In groups 7 and 8 (n = 16), basal catecholamine output before, during L-NAME infusion, and during combined infusion of NOC 7 with L-NAME was 3.6 ± 0.6, 2.4 ± 0.7, and 2.5 ± 0.6 ng/min, respectively. Adrenal plasma flow rate was decreased by L-NAME. During treatment with L-NAME, NOC 7 increased adrenal plasma flow rate under basal conditions and SNS but had no effect after ACh injection (Table 2). L-NAME produced a small bradycardic response but did not affect blood pressure. NOC 7 produced a depressor response and slightly increased heart rate during treatment with L-NAME (Table 3).

	DISCUSSION

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The present study was performed to elucidate the role of NO in regulation of adrenal catecholamine secretion. We assessed the effects of L-NAME, NOC 7 alone, and NOC 7 during L-NAME treatment on catecholamine secretion induced by SNS and exogenous ACh. The changes in catecholamine response to stimuli during treatment with L-NAME suggest that the effect of endogenously released NO on catecholamine secretion is the opposite of that of L-NAME. The changes in catecholamine response during treatment with NOC 7 in the absence or presence of L-NAME suggest that the action of NO on catecholamine secretion is due to NOC 7 under conditions in which endogenously released NO is or is not functional, respectively.

It has been suggested that NO plays an inhibitory role in controlling catecholamine secretion induced by various stimuli in bovine chromaffin cells (9, 13, 16) and PC12 cells (6). However, a few in vivo studies using the dog adrenal gland provided evidence suggesting that NO plays no role in catecholamine secretion in response to SNS (1, 2). In this study, L-NAME infused into the adrenal gland significantly enhanced the secretion of adrenal catecholamine in response to ACh. It can be assumed that ACh acts on endothelial cells and/or chromaffin cells and produces NO through activation of eNOS and/or nNOS, respectively. Therefore, this result suggests that endogenously released NO (basal NO plus NO produced by the ACh-induced activation of NOS) inhibits the ACh-induced secretion of catecholamine. However, no dose-response effect of L-NAME on ACh-induced catecholamine secretion was observed; the enhancement declined at the highest dose of L-NAME examined. Previously, we demonstrated under the same experimental conditions that ACh stimulated the secretion of catecholamine by activating both nicotinic and muscarinic receptors (5). It has been reported that L-NAME, in addition to inhibiting NOS, also functions as an antagonist at muscarinic receptors (3). This may explain the lack of a dose-response effect of L-NAME: the muscarinic component of catecholamine secretion was inhibited by increasing doses of L-NAME. NOC 7 inhibited the secretion of catecholamine in response to ACh, suggesting that further inhibition of catecholamine secretion is produced by NO derived from NOC 7 in addition to the inhibitory effect of endogenously released NO. Furthermore, NOC 7-induced inhibition of ACh-induced secretion of catecholamine was observed when NOC 7 was administered concomitantly with L-NAME. These results suggest that NO has an inhibitory effect on the secretion of catecholamine from the dog adrenal gland in response to exogenous ACh in vivo.

Similar to the ACh-induced secretion of catecholamine, that induced by SNS was inhibited by NOC 7. This could be explained by the inhibitory action of NO on catecholamine secretion. However, the secretion of catecholamine in response to SNS at 1 and 2 Hz was inhibited by L-NAME. Furthermore, no inhibition by NOC 7 of SNS-induced secretion of catecholamine was observed when NOC 7 was administered concomitantly with L-NAME. These results are not consistent with the observations in the ACh experiments suggesting that NO inhibits the secretion of catecholamine. SNS causes the release of ACh from splanchnic nerve terminals, and this released ACh stimulates medullary cells. Exogenously administered ACh stimulates medullary cells directly. Therefore, the discrepant results of the effects of L-NAME and NOC 7 on the SNS-induced secretion of catecholamine might be related to a presynaptic action of NO on ACh release from splanchnic nerve terminals. If NO facilitates the release of ACh presynaptically, ACh release would increase and subsequently the secretion of catecholamine from medullary cells would increase. Moreover, if the presynaptic facilitatory action of NO is dominant rather than its inhibitory action on the secretion of catecholamine, L-NAME would be expected to inhibit the SNS-induced secretion by canceling the presynaptic facilitatory action of NO. However, this hypothesis cannot explain the discrepancy in the effects of NOC 7 in the presence (no effect) or absence (inhibitory effect) of L-NAME. The mechanism underlying the unexplainable effects of L-NAME and NOC 7 on the SNS-induced secretion of catecholamine remains to be resolved.

Basal catecholamine secretion was not affected by L-NAME or NOC 7 in the presence or absence of L-NAME, suggesting that NO plays no role in basal secretion of catecholamine. These results are not consistent with the observations that N^G-monomethyl-L-arginine increases basal efflux of catecholamine from the perfused dog adrenal gland (18) and that pure NO markedly stimulates catecholamine secretion by cultured bovine chromaffin cells (9). The different results may have been due to differences in experimental conditions or in the species examined.

Adrenal plasma flow rate was decreased by L-NAME. This decrease was observed under basal conditions and after ACh injection or SNS. These results indicate that endogenously released NO regulates adrenal blood flow through its vasodilatory action in the adrenal vascular bed, as suggested in the dog adrenal gland (1, 2). NOC 7 had no effect on adrenal plasma flow rate in the absence of L-NAME. In the presence of L-NAME, however, NOC 7 increased the flow rate under basal conditions or SNS, suggesting that NO derived from NOC 7 acts on the vasculature. Therefore, it seems likely that NO derived from NOC 7 is unable to produce vasodilation in the absence of L-NAME because the vasodilatory effect of basal NO has already reached its maximum. No NOC 7-induced increase in adrenal plasma flow rate was observed during ACh even in the presence of L-NAME. As the inhibition by L-NAME of the ACh-induced increases in the flow rate was incomplete (Table 2), remaining NO (probably released by the ACh-induced activation of NOS) may operate under these conditions and thus mask the vasodilatory effect of NO derived from NOC 7.

In conclusion, this study demonstrated that the secretion of adrenal catecholamine induced by ACh was enhanced by L-NAME and inhibited by NOC 7 in the presence or absence of L-NAME. We also found that the secretion induced by SNS was inhibited by L-NAME and by NOC 7. During treatment with L-NAME, no inhibition of SNS-induced secretion by NOC 7 was observed. These results suggest that NO may play an inhibitory role in the regulation of adrenal catecholamine secretion in response to exogenously applied ACh.

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