INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE CONNECTION between the renin-angiotensin system and bird nasal salt glands was discovered by Hammel and Maggert (19), who showed that nasal fluid secretion in domestic ducks decreased by 97% in response to mammalian [Asp¹,Ile⁵]-ANG II delivered at a rate of 80 ng · kg¹ · min¹ during an intravenous infusion of hypertonic saline (1,000 mosmol/kgH₂O). More than 90% of the infused NaCl was secreted by the nasal salt glands within 60 min after the infusion was stopped. Butler (4) confirmed the inhibitory response to ANG II when he showed that a series of single intravenous injections (14, 28, and 42 pmol/kg body wt) of avian [Asp¹,Val⁵]-ANG II (27) each shut off nasal fluid secretion in Pekin ducks within 3 min. The salt glands remained inactive for a further 3 min, whereupon the rate of fluid secretion returned to normal.

It seemed that ANG II was acting centrally because an intracerebroventricular infusion of 1 mmol/ml of ANG II switched off duck nasal salt glands within 5 min. The salt glands remained inactive for a further 30-80 min (13). Other experiments (12) have shown that [Asp¹,Val⁵]-ANG II attenuated nasal secretion during the response to an increased plasma tonicity (1, 2, 14, 18, 31) and/or a decreased extracellular fluid volume (20, 22).

Salt gland secretion may be controlled exclusively by cranial parasympathetic nerves (3, 8, 11). In ducks, branches of the facial nerve and the glossopharyngeal nerve lead to the ganglion ethmoidale, which lies on the anterioventral side of each gland. Postganglionic fibers radiate from the ganglion, enter the gland, and innervate both blood vessels and secretory cells. However, nasal salt glands are also supplied with sympathetic nerve fibers (3, 8, 17). Norepinephrine may regulate blood flow and/or actual secretion by tubular cells within the salt glands. Finally, the renin-angiotensin system may attenuate salt gland activity.

The present experiments were designed to show whether the attenuation of salt gland secretion by [Asp¹,Val⁵]-ANG II is controlled by 1) the central nervous system (CNS) via postganglionic parasympathetic nerves, 2) peripheral sympathetic nerves and/or the chromaffin cells of the adrenals, 3) the direct action of avian [Asp¹,Val⁵]-ANG II on blood vessels and/or secretory cells, or 4) an indirect action of [Asp¹,Val⁵]-ANG II through the release of catecholamines from peripheral sympathetic nerves and/or adrenal chromaffin cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. White Pekin drakes (Anas platyrhynchos) were purchased from King Cole Duck Farms in Aurora, Ontario, when they were 10 wk old. They were housed in the Department of Zoology under a 12:12-h light-dark cycle and fed commercial duck grower food together with 0.9% NaCl drinking water ad libitum for 8 days. The ducks weighed 3,458 ± 77 g when they were used for the experiments.

Experimental groups. Ducks were randomly assigned to three experimental groups: 1) the methacholine experiment, which studied the effect of fowl ANG II [Asp¹,Val⁵]-ANG II (mol wt = 1,031.5) on actively secreting nasal salt glands before and during the infusion of the ganglionic blocker mecamylamine (10 µg/min iv, n = 6); 2) the prazosin experiment, which examined the effect of -adrenergic blockade with prazosin (20 mg iv infusion during 1 h) on the response of actively secreting salt glands to [Asp¹,Val⁵]-ANG II (n = 5); and 3) the propranolol experiment, which examined the effect of -adrenergic blockade with propranolol (4 × 10 mg iv during 1 h) on the response of actively secreting salt glands to [Asp¹,Val⁵]-ANG II (n = 6).

Surgical preparation. General anesthesia was induced with Equithesin (3.0 ml/kg body wt iv). Suitable lengths of heparin-filled PE-50 polyethylene tubing (ID 0.58 mm, OD 0.97 mm; Intramedic, Clay Adams, NJ) were used for blood catheters. An infusion catheter was inserted into the left ulnar vein and pushed forward into the brachial vein. Then a blood pressure catheter was inserted into the left brachial artery. Both catheters were tied into place with size 3-0 surgical silk and heat sealed. Ampicillin (50 mg/ml in isotonic saline; Penbritin-500; Ayherst Laboratories, Montreal, Canada) was dripped onto the wound, which was then closed with three Michel clips. The catheters were coiled, packed with surgical gauze, and taped so the ducks were unable to pull at them. Healing was rapid, and there was never any evidence of infection. Ducks regained consciousness in a recovery cage. They were held overnight in isolation, returned to the flock the following day, and allowed to recover for 2 days before they were used for the salt gland experiments.

Collection of nasal fluid samples. Each duck was placed on a holding board in the prone position and held in place with rubber tubing. When noise was kept to a minimum the duck remained calm and there was little, if any, struggling. First, a bolus injection of 10 ml/kg body wt of a 1,000 mosmol/kgH₂O NaCl solution was injected into the left brachial vein to initiate nasal gland secretion. It was followed by a continuous infusion of 1,000 mosmol/kgH₂O NaCl solution at a rate of 0.32 ml · kg¹ · min¹ for the entire experiment. Responses to the various drugs were not measured until the rate of nasal fluid secretion had become more or less constant. Fluid dripped from the beak into clean 100-ml Pyrex beakers during successive 5-min collection periods, which continued through the entire experiment. Fluid from each 5-min collection was withdrawn into preweighed 2.0-ml hypodermic syringes, which were subsequently reweighed. Sample volumes were determined by the difference in weight. Fluid samples were then stored at 20°C in 1.5-ml Eppendorf tubes.

Measurement of arterial blood pressure. Brachial mean arterial pressure was only measured to show that - and -adrenergic blockade were complete in experiments 1 and 2. First, the pressure catheter was flushed with heparinized saline and connected to an RP-1500 pressure transducer (Narco Bio-Systems, Chicago, IL) and a Linear Model 1200 single-pen recorder (Linear Instrument, Reno, NV). The undamped frequency of the pressure-measuring system was 30 Hz, and the damping ratio was 0.1. Zero pressure was adjusted to the level of the duck's heart. The system was then calibrated against a static heparinized saline reservoir before and after each pressure measurement. Mean arterial pressure was calculated as the sum of the diastolic pressure and one-third of the pulse pressure.

Experimental procedures. Each duck was placed on a holding board in the prone position. Nasal fluid secretion started after the injection of 10 ml/kg body wt of a 1,000 mosmol/kgH₂O NaCl solution into the brachial vein. Secretion was sustained by a continuous intravenous infusion of 1,000 mosmol/kg body wt NaCl solution at a rate of 0.32 ml · kg¹ · min¹. Five-minute nasal fluid samples (from 9 to 13) were collected seriatim until the rate of secretion was more or less constant. Then drugs were tested in experiments 1, 2, and 3. Five ducks were used for each experiment.

Mecamylamine experiment. Two micrograms of [Asp¹,Val⁵]-ANG II in 0.2 ml of 0.9% saline were injected intravenously 60 min after the start of the intravenous infusion of 1,000 mosmol/kgH₂O NaCl solution to test the peptide's effectiveness as an inhibitor of nasal fluid secretion. Thirty minutes later, when nasal flow rates had returned to normal, 40 mg of mecamylamine chloride (in 0.5 ml of isotonic saline) were injected intravenously to block parasympathetic and sympathetic ganglia, including the ganglion ethmoidale. An infusion of methacholine bromide (10 µg/min iv dissolved in 1,000 mosmol/kgH₂O NaCl solution) was started 15 min later and continued for 90 min. This was done to activate the salt glands and to maintain secretion. Finally, 2 µg of [Asp¹,Val⁵]-ANG II were injected intravenously 60 min after the start of the methacholine bromide infusion to show whether [Asp¹,Val⁵]-ANG II would inhibit the nasal salt glands directly and apart from any neural inputs.

Prazosin experiment. Two micrograms of [Asp¹,Val⁵]-ANG II in 0.2 ml of isotonic saline were injected into the left brachial vein 45 min after the start of an infusion of 1,000 mosmol/kgH₂O NaCl solution. After a 35-min interval, 1 mg of methoxamine chloride in 0.2 ml of 0.9% saline was injected intravenously to test the pressor response to a pure ₁-adrenergic agonist. After a further interval of 20 min, ₁-adrenergic blockade was induced by the intravenous infusion of 20 mg of prazosin in 2.0 ml of isotonic saline during the next 60 min. Then, 5 min after the prazosin infusion ended, a second dose of 1 mg of methoxamine was injected intravenously to show whether the ₁-adrenergic blockade was complete. After a further 5 min, the duck was given a final intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II.

Brachial arterial blood pressure was measured to assess the pressor response or lack of pressor response to methoxamine before and after -adrenergic blockade with prazosin.

Propranolol experiment. The inhibitory response to 2 µg [Asp¹,Val⁵]-ANG II was measured 65 min after the start of a continuous intravenous infusion of a 1,000 mosmol/kgH₂O NaCl solution at a rate of 0.32 ml · kg¹ · min¹. After a further 35 min, 25 µg of the -agonist isoproterenol was injected intravenously in 0.2 ml isotonic saline. Twenty minutes later there followed a series of four 10-mg intravenous injections of the -blocker propranolol administered at 5-min intervals. After an additional 25 min, the completeness of the -adrenergic blockade was confirmed with a second intravenous injection of 25 µg of isoproterenol. Ten minutes after that, the inhibitory effect of 2 µg of [Asp¹,Val⁵]-ANG II was tested in the -blocked duck.

Brachial arterial blood pressure was measured to assess the vasodepressor response or lack of vasodepressor response to isoproterenol before and after -adrenergic blockade with propranolol.

Drugs and hormones. The drugs and hormones used were [Asp¹,Val⁵]-ANG II (mol wt = 1,031.5; Peninsula Laboratories, Belmont, CA), heparin sodium (10,000 USP units/ml; Hepalean; Organon Teknika, Toronto, ON), ampicillin (Penbritin-500; Ayherst Laboratories, Montreal, PQ), mecamylamine hydrochloride (25 mg/ml isotonic saline; Merck-Frosst, Dorval, PQ), isoproterenol chloride (200 µg/ml isotonic saline; Isuprel; Sanofi Winthrop, Markham, ON), methoxamine HCl (20 mg/ml isotonic saline; Vasoxyl; Burroughs Wellcome, Kirkland, PQ), propranolol hydrochloride (1 mg/ml in water and citric acid; Inderall Wyeth-Ayherst Canada, North York, ON), and prazosin (10 mg/ml propylene glycol; Minipress; Pfizer Canada, Dorval, PQ).

Analytical methods. Nasal fluid samples were thawed, and Na⁺ concentrations were measured in diluted samples with an IL model 943 flame photometer.

Statistical methods. Values in Figs. 1, 2, and 3 are means ± SE for each 5-min collection period. A repeated-measures ANOVA was used to test for within-group differences, followed by Duncan's multiple range test for comparison of individual sample means. Comparisons were made primarily before and after the administration of drugs. The fiducial limit was set at P = 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mecamylamine experiment. The rate of nasal fluid secretion decreased significantly (P < 0.05) after an intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II (P < 0.05; Fig. 1). The time to switch off was 2.80 ± 0.15 min, and the salt glands remained inactive for 5.72 ± 0.41 min, at which time they began to secrete again. After 15 min nasal fluid secretion returned to the normal rate. Then the nasal salt glands stopped secreting fluid 3.24 ± 0.23 min after an intravenous injection of 40 mg of the ganglionic blocker mecamylamine. There was still no evidence of secretion 15 min after the drug was injected (Fig. 1). Secretion was restored 1.35 ± 0.28 min after the beginning of a continuous intravenous infusion of methacholine bromide (10 µg/min). A second intravenous dose of 2 µg of [Asp¹,Val⁵]-ANG II was injected during the methacholine bromide infusion. It did not attenuate the rate of secretion. Nasal fluid Na⁺ concentrations never changed significantly during the course of the experiments, but significant changes in the rate of nasal fluid secretion led to a significant 10-min decrease in Na⁺ excretion after the first injection of [Asp¹,Val⁵]-ANG II. No fluid and therefore no Na⁺ was excreted during the mecamylamine block (Fig. 1), but the rate of Na⁺ excretion returned to normal during the infusion of methacholine bromide.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of ganglionic blocker mecamylamine on secretion by salt glands in Pekin ducks (Anas platyrhynchos). Full secretion was reestablished with intravenous infusion of cholimimetic methacholine during ganglionic blockade. [Asp1,Val5]-ANG II switched off salt glands before, but not after, ganglionic blockade. Values are means ± SE for 5-min collection periods; n = 6 ducks.

Prazosin experiment. There was a significant decrease in the average nasal fluid secretion rate after the first intravenous injection of 2 µg/kg body wt of [Asp¹,Val⁵]-ANG II. The nasal salt glands stopped secreting 3.42 ± 0.25 min after administration of the drug. They remained inactive for 4.95 ± 0.78 min then started to secrete fluid. The rate of secretion gradually returned to normal in ~25 min (Fig. 2). An intravenous injection of 1 mg of methoxamine was followed by a clear pressor response and a brief, 5-min decrease in nasal fluid secretion rate. However, the rate quickly returned to normal and then gradually decreased by ~25% during the infusion of the ₁-adrenergic blocker prazosin. The rate had returned to normal, however, 150 min after the start of the intravenous infusion of 1,000 mosmol/kgH₂O NaCl solution. Prazosin blocked the ₁-adrenergic receptors because the second and final injection of 1 mg of intravenous methoxamine was not followed by a pressor response, nor did it change the rate of nasal fluid secretion. The nasal salt glands stopped secreting fluid 3.26 ± 0.32 min after the final intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II. The glands remained inactive for 4.00 ± 0.5 min and then switched on and began to secrete fluid. Flow returned to the normal rate within 15 min. This experiment showed that the attenuated rate of secretion was not the result of an [Asp¹,Val⁵]-ANG II-induced release of norepinephrine. There were no significant changes in the nasal fluid Na⁺ concentration after the administration of drugs. Rates of Na⁺ excretion followed the pattern in rates of nasal fluid secretion, that is, a significant decrease in the rate of Na⁺ excretion after the first and second doses of [Asp¹,Val⁵]-ANG II, and a suggested decrease after the first but not the second injection of methoxamine and during the entire perfusion of the adrenergic blocker prazosin (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Salt gland secretion in Pekin ducks (A. platyrhynchos) in response to intravenous injections of 2 µg of [Asp1, Val5]-ANG II before and after 1-adrenergic blockade with prazosin. Values are means ± SE for 5-min collection periods; n = 6 ducks.

Propranolol experiment. Nasal fluid secretion ceased after the first intravenous injection of 2 µg [Asp¹,Val⁵]-ANG II (Fig. 3). The glands stopped secreting in 2.63 ± 0.32 min and remained inactive for 7.88 ± 1.03 min. Thirty-five minutes later there was a slight but not statistically significant decrease in the rate of nasal fluid secretion after an intravenous injection of 25 µg of the -agonist isoproterenol. The injection of four 10-mg doses of propranolol blocked -adrenergic receptors and prevented the vasodepressor response to a second injection of 25 µg of isoproterenol. The second injection of 2 µg of [Asp¹,Val⁵]-ANG II, administered during -blockade, was followed by the total inhibition of nasal fluid secretion (Fig. 3) within 3.63 ± 0.21 min. The salt glands started to secrete again after a further 5.88 ± 0.72 min, but it took an additional 20 min for the rate of flow to return to normal (Fig. 3). There were no measurable changes in nasal fluid Na⁺ concentrations during the entire experiment, but rates of Na⁺ excretion did change depending on the adjustments in flow rate (Fig. 3). Therefore, the rate of Na⁺ secretion decreased significantly after the first and second injections of [Asp¹,Val⁵]-ANG II and during the four successive intravenous injections of 10 mg of propranolol (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Salt gland secretion in Pekin ducks (A. platyrhynchos) in response to intravenous injections of 2 µg of [Asp1,Val5]-ANG II before and after -adrenergic blockade with propranolol. Values are means ± SE for 5-min collection periods; n = 6 ducks.

Attenuation of nasal fluid secretion after injection of [Asp¹,Val⁵]-ANG II. In most cases the histograms for experiments 1, 2, and 3 indicate that the nasal fluid secretion rate decreased greatly but did not stop. Fluid secretion did stop in almost every duck, but the time between the injection of the peptide and the complete cessation of flow varied slightly. Moreover, the gland switched off for less than a full 5-min collection period. These two factors led to average flow rates that were very low but always greater than zero for one or two 5-min collection periods (Figs. 1, 2, and 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mecamylamine experiment was designed to show whether ganglionic blockade, including the ganglion ethmoidale (3), would cut the link between the secretory nerve and the nasal salt glands and abolish secretion. Nasal fluid secretion stopped immediately after an intravenous injection of 40 mg of mecamylamine chloride (Fig. 1). If salt gland blood flow (10) and secretion (9, 12, 23, 25) are both controlled by the local release of acetylcholine from parasympathetic nerves, the inactive salt glands should respond to a cholimimetic drug. Figure 1 shows that a continuous intravenous infusion of methacholine bromide (10 µg/min) rapidly and fully reestablished normal rates of fluid secretion and fluid Na⁺ concentrations. It was then possible to show whether [Asp¹,Val⁵]-ANG II shuts off the salt glands directly or indirectly. If the peptide acted directly, an intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II would have been expected to shut off secretion by the ganglion-blocked, methacholine-supported salt glands. Such a direct action may be brought about by the vasoconstriction of blood vessels supplying the secretory tubules or by the attenuation of secretory cell activity. On the other hand, if the effect of the peptide was indirect, for example via the binding of [Asp¹,Val⁵]-ANG II in the area postrema (26) and the CNS regulation of parasympathetic efferents, one would not expect to observe a response by the nasal salt glands. Figure 1 shows that neither the rate of fluid secretion nor the fluid Na⁺ concentration changed after the second intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II, providing strong evidence for the CNS-mediated action of the peptide, that is, an indirect action. In freshwater Pekin ducks (A. platyrhynchos) the plasma concentration of ANG II is 35.3 ± 3.9 pg/ml plasma (16), so the test dose of 2 µg iv of [Asp¹,Val⁵]-ANG II used for the present experiments was relatively large, but it stopped nasal fluid secretion quickly. Lower doses of the hormone were effective but did not suit our experimental protocol. For example, an intravenous infusion of [Asp¹,Val⁵]-ANG II given at a much smaller dose of 15 and 77 ng · kg¹ · min¹ together with NaCl solution (250 mmol/kg body wt at 1.6 ml/min) in salt-adapted ducks led to a continuous attenuation of salt gland secretion for a 2-h period (15).

Mecamylamine blocked the nicotinic receptors of both sympathetic and parasympathetic autonomic ganglia. However, full secretion was reestablished by methacholine bromide in mecamylamine-blocked salt glands (Fig. 1), which tended to exclude the possibility that the salt glands are regulated by the ANG II-dependent release of catecholamines. However, verification would depend on subsequent experiments using adrenergic antagonists.

Prazosin blocks ₁-receptors and reduces blood pressure by dilating both resistance and capacitance vessels. It has a plasma half-life of 3-4 h. Specific ₁-antagonists cause less tachycardia than other nonselective -antagonists because they do not increase the discharge of norepinephrine from sympathetic nerve terminals. It has been shown that the pressor response to [Asp¹,Val⁵]-ANG II in ducks was caused not by the direct effect of the peptide on vascular smooth muscle (6, 35, 36) but to the release of norepinephrine from peripheral sympathetic nerves and/or the adrenal chromaffin cells. [Asp¹,Val⁵]-ANG II may have shut off the salt glands by binding to a circumventricular organ such as the subfornical organ (26) and via CNS-directed pathways, increasing norepinephrine release from peripheral sympathetic nerves (17) that supply the salt glands. In geese the majority of the adrenergic fibers which supply the salt glands are found in the walls of blood vessels, but "fibers with varicosities pass along the secretory tubules" (McLean and Hebb, cited in Ref. 29). The local release of norepinephrine may lead to vasoconstriction and a reduced rate of blood flow to the secretory tubules, because there is a direct correlation between salt gland blood flow and secretion in both geese (21) and ducks (5, 24). It is likely that -adrenergic receptors are present in avian nasal salt glands, because norepinephrine, epinephrine, and stimulation of the cervical sympathetic chain each decreased blood flow to the salt glands and inhibited secretion (10, 11).

Instead of acting via the CNS, [Asp¹,Val⁵]-ANG II may have released norepinephrine directly from peripheral sympathetic nerve endings near or within the salt glands, and/or the peptide may have released norepinephrine and epinephrine from the duck adrenal chromaffin (medulla) cells.

Figure 2 shows clearly the inhibitory effect of an intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II. Within 15 min the rate of fluid secretion returned to normal. There was a brief statistically significant (P < 0.05) 5-min decrease in the rate of fluid secretion by the salt glands and a clear pressor response after an intravenous injection of 1 mg of the ₁-agonist methoxamine chloride. It was assumed that this rapid and briefly attenuated secretion was the result of the interplay between arteriolar vasoconstriction within the nasal salt glands and the overall increase in systemic arterial pressure.

Next, a solution containing 20 mg of prazosin was infused intravenously during a 1-h period wherein the rate of secretion drifted downward (Fig. 2), possibly because of the dilation of both pressure and capacitance vessels that followed the ₁-adrenergic blockade. The rate of fluid secretion started to increase again during the final 10 min of the prazosin infusion and eventually reached the normal range (Fig. 2). Then the duck was injected intravenously with a second dose of 1 mg methoxamine. The pressor response was completely abolished, showing that ₁-adrenergic blockade was complete. Five minutes later (Fig. 2) a second intravenous injection of 2 µg [Asp¹,Val⁵]-ANG II was followed by a typical and significant (P < 0.05) decrease in nasal fluid secretion lasting for 10 min. This response showed clearly that [Asp¹,Val⁵]-ANG II shut off the salt glands without any ₁-agonistic stimulation from norepinephrine or epinephrine.

Experiment 3 was concerned primarily with -adrenergic receptors, because -adrenergic stimulation via [Asp¹,Val⁵]-ANG II was precluded by the results of the earlier prazosin experiment. It was now important to show whether [Asp¹,Val⁵]-ANG II attenuated salt gland secretion by releasing epinephrine from duck adrenal chromaffin cells directly (30, 34, 35) or indirectly via the CNS and peripheral sympathetic afferents synapsing with chromaffin cells (30, 34). Epinephrine might then be carried in the circulation to -receptors in the heart and blood vessels, including those supplying the nasal salt glands, and even the basolateral surfaces of salt gland secretory cells (35). These cells respond to ₁-stimulation by increasing cellular adenyl cyclase, cyclic AMP that in turn stimulates active Cl transport through the apical membrane and into the lumen of the secretory tubule. Thus it was important to show whether the attenuated salt secretion after an intravenous injection of [Asp¹,Val⁵]-ANG II was caused by the release of epinephrine from duck adrenal chromaffin cells, which in turn might switch off the salt glands.

Figure 3 illustrates the typical inhibitory response of the salt glands to an intravenous injection of 2 µg of [Asp¹,Val⁵]-ANG II. After the rate of fluid secretion returned to normal, the ducks were given an intravenous injection of 25 µg of the ₁-agonist isoproterenol. The heart rate decreased and the systemic arterial blood pressure drifted downward then leveled off. The rate of nasal fluid secretion decreased significantly (P < 0.05) for ~10 min, probably the effect of vasodilation and a drop in blood pressure on rate of delivery of blood to the nasal salt gland secretory tubules. There is a strong positive correlation between blood flow and salt secretion by duck (4, 5, 24) and goose (21) nasal salt glands.

Once the response to isoproterenol was established, a series of four 10-mg injections of the -antagonist propranolol was administered intravenously at 5-min intervals. During this period the rate of fluid secretion by the salt glands decreased significantly (P < 0.05) and only began to return to normal after the final injection of propranolol (Fig. 3). This seems paradoxical, yet the blockage of ₁-receptors in the secretory cells, together with the fact that propranolol may act as a local anesthetic by blocking impulse transmission in the secretory nerves (33), may have somehow contributed to the lowered rate of fluid secretion at the start (Fig. 3). Propranolol antagonizes both ₁- and ₂-adrenergic receptors, so that an intravenous injection of the drug is usually followed by a decrease in systemic blood pressure caused by a decreased heart rate (bradycardia) together with a decreased cardiac output. Eventually, the cardiac output may return to normal, but peripheral vasodilation persists, leading to a decrease in systemic arterial pressure. Propranolol inhibits the ₁-receptors in renal juxtaglomerular cells and thus decreases the release of renin (28), but sensitization to [Asp¹,Val⁵]-ANG II would not be a factor in the present short-term experiment. Figure 3 shows that nasal fluid secretion rates returned to normal ~30 min after the final propranolol injection. However, the -blockade was still in force, because a second intravenous injection of 25 µg of isoproterenol had no measurable effect on heart rate or arterial blood pressure. [Asp¹,Val⁵]-ANG II (2 µg iv) was then tested in fully -blocked ducks (Fig. 3). There followed a rapid and pronounced decrease in rate of fluid secretion by the nasal salt gland. This showed that [Asp¹,Val⁵]-ANG II did not shut off the glands by way of the agonistic effect of epinephrine and/or norepinephrine on putative -receptors in blood vessels or secretory cells in the salt glands.

In all of the experiments, there was a clear dissociation between the sodium chloride concentrations of the salt gland fluid and the rate of fluid secretion. Short- term, nonadaptive adjustments in salt secretion are achieved primarily by adjustments in the flow rate and not the salt concentration. This observation is in line with our earlier work (4-7).