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Journal of Physiology (1991), 435, pp. 175-186 With 4 figures Printed in Great Britain
PARTIAL UNCOUPLING OF SALT GLAND BLOOD FLOW AND SECRETION IN THE PEKIN DUCK (ANAS PLATYRHYNCHOS)
BY RUDIGER GERSTBERGER From the Max-Planck-Institut fur Physiologische und Klinische For8chung, W. G. Kerckhoff-Institut, D-6350 Bad Nauheim, Germany
(Received 18 June 1990) SUMMARY
1. The aim of this study was to investigate the relationship between the blood flow through and the secretion by the salt glands of conscious, salt-water-adapted Pekin ducks. 2. Intravenous loading with hypertonic saline induced a steady-state secretion from the salt glands with a concomitant increase in whole-organ blood flow. The distribution of elevated local glandular blood flow was, however, uneven and in addition demonstrated vasomotor patterns that ranged from constant to rhythmic. 3. During on-going salt gland secretion, the infusion of three vasoactive agents, 5Val-angiotensin II (ANG II), 8Arg-vasotocin (AVT) and noradrenaline, via the carotid artery had differential effects on salt gland blood flow and secretion. 4. ANG II (80 pmol min-' (kg body wt)-1) had no effect on mean arterial blood pressure (MABP), produced a transient 30% decrease in glandular blood flow and strongly diminished salt gland secretion (retention of 6-4 mosmol NaCl). 5. AVT (20 pmol min-' (kg body wt)-') had no effect on MABP and did not alter salt gland secretion despite a 35 % reduction in blood flow. 6. Noradrenaline (20 nmol min-' (kg body wt)-1) elevated MABP by 15 mmHg, reduced salt gland blood flow by more than 50 %, but diminished salt gland secretion only slightly (retention of 2 7 mosmol NaCl). 7. Using ANG II, AVT and noradrenaline as hormonal tools, integrated changes in blood flow rate did not correspond with integrated changes in salt gland excretion. The partial dissociation between both parameters shows that control of secretion by the salt gland is more complex than simply being linearly dependent upon blood flow through it. INTRODUCTION
The supraorbital salt-secreting glands ('salt glands') of marine birds respond to an increase in the tonicity or volume of their extracellular fluid compartment with enhanced secretion of a strongly hypertonic fluid rich in sodium chloride (SchmidtNielsen, 1960; Peaker & Linzell, 1975; Simon & Gray, 1989). Efferent control of salt gland activity is achieved by parasympathetic fibres originating from the VIIth cranial nerve (Ash, Pearce & Silver, 1969; Kiihnel, 1972) and utilizing both MS 8571
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acetylcholine and vasoactive intestinal polypeptide (VIP)
as stimulatory neurotransmitters (Gerstberger, 1988; Gerstberger, Sann & Simon, 1988). It has been shown, in a variety of avian species, that actively secreting salt glands require an elevated blood flow (Fiinge, Krog & Reite, 1963; Hanwell, Linzell & Peaker, 1971 a; Kaul, Gerstberger, Meyer & Simon, 1983; Gerstberger et al. 1988) and that a constant 20% of the sodium chloride content of the perfusing glandular blood stream is extracted and secreted against a marked trans-tubular concentration gradient (Kaul et al. 1983). Accordingly it appeared that blood flow and secretion of the avian salt glands are coupled by a simple linear relationship so that augmented basolateral supply of sodium and chloride to the principal secretory cells would lead to increased transport of both ions into the tubular lumen as long as the underlying transport mechanisms are not saturated. However, the recent discovery of atropine-resistant vasodilatation at suppressed secretion (Kaul et al. 1983; Gerstberger et al. 1988) indicates that the control of salt gland function may not be so straightforward and that physiological conditions might exist where blood flow and secretion could be dissociated, as is the case in cat salivary glands (Lundberg, 1981). In order to gain a deeper understanding of the phenomenon of blood flow-secretion coupling in exocrine glands, the relationship between these parameters was investigated in the salt glands of conscious salt-water-adapted ducks. Whole-organ and local glandular blood flow were measured, and the salt gland responses to changes in blood flow induced by the vasoactive hormones 5Val-angiotensin II (ANG II), 5Arg-vasotocin (AVT) and noradrenaline were calculated.
METHODS
Animals The experiments were performed with twenty-six adult Pekin ducks (Anasplatyrhynchos) of both sexes with body weights of 2-2 +0.1 kg (mean+s.E.M.). The animals were reared in flocks under a natural day-night cycle in a thermoneutral environment and fed with commercial dry chicken food, enriched with minerals and vitamins. Over a 6 week period the ducks were gradually adapted to 400 mosmol kg-' saline as drinking and bathing fluid and were kept at this regimen for at least 30 days before experimentation. Hypertrophy of their supraorbital salt glands enabled the saltwater-adapted Pekin ducks to secrete up to 0-8 mosmol min-' of mainly sodium chloride via these glands, thereby maintaining body fluid homeostasis.
Surgery Surgical procedures were performed exclusively under inhalation anaesthesia (halothane, N20, An indwelling Teflon cannula (V/19, Braun, Germany) was implanted into one of the carotid arteries for drug application to the cephalic circulation without impairment of blood perfusion (Kaul et al. 1983). For the recording of systemic blood pressure via an Endevco pressure transducer (N 8510, 5 lbf in2, USA), or the collection of arterial reference blood samples, a polypropylene catheter (PP 90, Portex) was inserted into the left brachial artery. In order to measure organ-specific capillary blood flow with the radioactive microspheres technique (RMT) (Kaul et al. 1983), in five animals a PP 50 catheter was advanced towards the heart from the right brachial artery until its tip protruded into the left ventricle as judged from changes in the pulse pressure. Filling with heparinized saline kept all catheters patent. For the continuous recording of local salt gland blood flow by the laser-Doppler method (LDM), the salt gland ipsilateral to the cannulated carotid artery was freed of connective tissue in fifteen ducks without any damage to the gland after its exposure via a cranial skin incision. A head device made from a metal base-plate was fixed to the skull with stainless-steel wires and acrylic cement
02) and sterile conditions 24-48 h prior to experimentation.
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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to restrain head movements throughout experimentation. The birds showed no apparent signs of stress or discomfort during the experiments, with heart rate and mean arterial blood pressure (MABP) values consistent with this impression.
Experimental procedure On the morning of the experiment, the duck was placed in a canvas sling that allowed free movement of the legs. A catheter (Braunula G-20, Braun, Germany) was placed in a leg vein by percutaneous venipuncture for continuous infusion of 04 ml min-' of 1000 mosmol kg-1 NaCl to induce steady-state salt gland secretion at a rate and osmotic concentration equal to the given load. The clear, NaCl-rich salt gland secretion was collected at 5-10 min intervals (Hammel, SimonOppermann & Simon, 1980) and its volume was determined to an accuracy of + 10 ,ul. Osmotic concentrations of salt gland secretion as well as plasma samples obtained at the beginning and end of the experiment were measured by vapour pressure osmometry (Wescor 5100 C, Wescor Inc., USA). Series I. The effect of intravenous hypertonic salt loading upon salt gland secretion, glandular blood flow as well as MABP were monitored until constant salt gland secretion was established. Series II. Under defined conditions of steady-state salt gland activity, the influence of vasoactive agents upon MABP, salt gland secretion and blood flow were determined. Noradrenaline (Sigma, Germany), 8Arg-vasotocin (AVT) and 5Val-angiotensin II (ANG II) (Bissendorf, Germany) were dissolved in sterile isotonic saline and infused over 5 min via the carotid at a rate of 200 ,1 minin concentrations of 20 nmol min-1 (kg body wt)'1, 20 and 80 pmol min-' (kg body wt)-', respectively. Radioactive microspheres technique (RMT) To measure capillary blood flow in the salt glands and various other organs of the salt-wateradapted Pekin duck, microspheres 15 ,um in diameter (NEN DuPont, USA) labelled with 14'Ce, 51Cr, 113Sn, 103Ru, 95Nb and 4"Sc were used. For each measurement 0 8-2 0 x 106 microspheres were injected in a volume of 0 6 ml over 20 s into the left ventricle immediately after starting collection of reference blood for 1 min from the left brachial artery. The availability of six y-emitting tracers allowed consecutive determinations of organ-specific blood flow values in the same animal. After the experiment, the duck was quickly killed with an i.v. injection of sodium pentobarbitone. Tissue samples of known wet weight were dissected and prepared for the measurements of photopeak radioactivity (RI240 y-spectrometer, Strahlentechnik, Germany). The data were converted with a multichannel analyser (ND 60, Nuclear Data, Germany). Compton and background corrections were performed with a Digital computer (VAX-780, USA) and blood flow values are presented in ml min-' (g tissue wet wt)-1.
Laser-Doppler method (LDM) Local salt gland blood flow was monitored continuously throughout the experiment with a PeriFlux PF 2 laser-Doppler flowmeter (Perimed, Sweden) using a PF-103 probe. The base-plate attached to the animal's skull was fixed to a cover-lid as described recently (Gerstberger et al. 1988). The laser probe was positioned 0-5-1-0 mm away from the salt gland surface, which had been freed of connective tissue. Relative values of local blood flow were recorded using signals from red blood cell movements over an area of about 4 mm2 to a depth of 1-2 mm (Tenland, 1982). The of movement artifact filter systems with a frequency bandwidth of 20-12 x 103 Hz, and the inherent linearity of the flowmeter, however, allowed the comparison of flux values obtained during various physiological conditions. All flux values were normalized to gain 1 with a 3 0 s time constant. Histology The supraorbital salt glands of three ducks, which had received an intraventricular injection of non-radiolabelled microspheres during steady-state salt gland secretion, were dissected immediately after the animals were killed with an overdose of intravenously administered sodium pentobarbitone. After submersion fixation of the tissue in 4% paraformaldehyde for 48 h with subsequent incubation in 10 % sucrose overnight at 2 °C, 32 ,um sections of salt gland slices were cut in a cryostat (Frigocut 2700, Reichard-Jung, Germany) at -15°C, thaw-mounted onto
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gelatine-alum-coated slides, and stained with cresyl violet. The numbers of trapped microspheres in all secretory lobes were determined. Data evaluation All data are presented as means + S.E.M. The Wilcoxon matched-pairs sign rank test was employed with 2P < 005 being considered the limit of significance in order to compare altered parameter values after hormone application with control conditions in the same experimental animal. Changes of salt gland secretion were calculated as the number of milliosmoles that were retained, integrated over time of hormone action (20 min) and compared to the secreted milliosmoles during control periods of equal length. Alterations in salt gland blood flow measured by the LDM were calculated as the percentage decrease of flux integrated over the time of hormone action (20 min), compared to the flux integrated over corresponding control periods of equal length. The method of 'repeated measures designs' (Cody & Smith, 1987) was used to relate the secretion and blood flow data during hormonal treatment to each other. RESULTS
Salt gland blood flow during on-going glandular secretion To induce steady-state salt gland secretion, conscious salt-water-adapted Pekin ducks received a continuous systemic infusion of 0-4 ml min-' of 1000 mosmol kg-' NaCl. With a time delay of less than 10 min, the salt glands responded to the salt load with enhanced osmolal excretion, and after 20-30 min of the NaCl application, the output of both salt and water matched the infusion to 102 + 3 and 97+4% (n = 7), respectively (Fig. 1). Local blood flow, as measured by the LDM, increased in parallel with osmotic excretion (n = 7), reaching an elevated plateau of red blood cell fluxes 15-20 min after the start of the NaCl challenge as is shown for a single representative experiment in Fig. 1. In experiments with stable blood flow readings, drop counting of the aspirated secretory fluid revealed tight congruency with the laser-Doppler signal on a 30 s time base. General circulatory parameters such as MABP or heart rate (not shown) remained unchanged with values of 118 +5 mmHg and 185 + 13 beats min-' (n = 12), respectively. To confirm the blood flow data obtained by the LDM, the capillary blood perfusion was determined simultaneously in five experiments using the RMT, which is well established for the Pekin duck. Microspheres bearing different, randomly mixed radioactive tracers were injected intracardially (i.c.) into birds at the threshold of salt gland secretion, before application of the salt load, as well as 20 min after having reached steady-state secretion with stable and high LDM flow recordings. In all organs tested (brain, eyes, harderian glands, salt glands, heart, lung, gut, pancreas, liver, gonads, kidneys, breast muscle, web skin), specific blood flow values were unaltered by salt loading with the exception of the supraorbital salt glands in which flow values increased twelvefold from 1-3 + 02 to 15-1 + 1-4 ml min-' (g tissue wet wt)-1. Thus, enhancement in the LDM signals was accompanied by augmented capillary blood perfusion. In a few experiments, however, local salt gland blood flow exhibited rhythmic vasoconstrictions and vasodilatations at varying frequency and amplitude despite high whole-organ blood flow values (RMT) and steady-state secretion at the stimulated level. To elucidate the relationship between total osmolal output and local salt gland blood flow, recordings of red cell fluxes were obtained from various sites of the same osmotically stimulated salt gland of four birds. Figure 2
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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demonstrates the variability of red cell fluxes measured at three recording sites (A-C) 4-5 mm apart during steady-state salt gland secretion at constant arterial pressure and carotid blood flow as determined with surgically implanted electromagnetic flow probes. The pattern of local blood flow varied from stable high-level Salt load 140
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Fig. 1. Parallel increase in salt gland secretion and blood flow at unchanged mean arterial blood pressure (MABP) in conscious Pekin ducks due to systemic saline loading. Local salt gland blood flow measured by the LDM (flux) before and during saline loading (0 4 ml min-' of 1000 mosmol kg-' NaCl) is shown for a single experiment, while salt gland excretion is demonstrated for experiments in seven conscious salt-water-adapted animals.
fluxes (A) to red cell fluxes at a high level with intermittent reductions possibly representing vasoconstriction (B), and even to rapid alternations of high flux values and cessation of blood perfusion (C). The obviously uneven distribution of blood flow to single secretory lobes of the activated avian salt gland could be verified histologically. Intraventricularly injected non-radioactive microspheres caught during just one cycle of the systemic circulation in the capillaries feeding the glandular parenchyma revealed a non-homogenous distribution in the eight to fifteen
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secretory lobes that comprise the duck salt glands, with numbers of intracapillary microspheres in individual lobes varying between three and forty-one in serial tissue sections.
Hormonal actions on salt gland secretion and blood flow The effects of ANG II, AVT and noradrenaline upon MABP, glandular blood flow, salt gland secretion rate and osmolality of the secreted fluid for single experiments are shown in Fig. 3A-C. 60 :4 c
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With regard to general cardiovascular parameters, avian-specific ANG II administered to the cephalic circulation at a dose of 80 pmol min-' (kg body wt)-1 did not influence arterial pressure or heart rate in five out of seven experiments, while in two animals mean arterial blood pressure was slightly elevated by 8-10 mmHg
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
181
for the time of peptide application. The antidiuretic hormone AVT infused at 20 pmol min-' (kg body wt)-' proved to be without any effect on MABP and heart rate. Noradrenaline (20 nmol min-' (kg body wt)-1), however, significantly (2P < 005) increased MABP from 117+6 to 131+8 mmHg (n = 6) with sharp A
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Fig. 3. Effects of 5Val-angiotensin vasotocin (AVT; 20 pmol min-' (kg body wt)-') (B), and noradrenaline (20 nmol min-' (kg body wt)-') (C) infused for 5 min i.C. on mean arterial blood pressure (MABP) and salt gland function. MABP, local salt gland blood flow measured by the LDM (flux), as well as flow rate and osmolality of salt gland secretion are shown for representative single experiments in conscious ducks receiving a continuous systemic infusion of 0-4 ml minof 1000 mosmol kg-' NaCl.
onset immediately after beginning the i.c. infusion and rapid decline to control values about 5 min after cessation of the i.c. application. Heart rate was reduced from 195 + 21 to 165+17 beats min-' (2P < 0-05), suggestive of reflex bradycardia. The infusion of ANG II resulted in a reduction of blood flow through the gland from 15-1 + 1-4 to 10-8+ 1-8 ml min-1 (g tissue wet wt)-' (2P . 0-05) as determined
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by the RMT. The laser-Doppler signal clearly demonstrated a partial inhibition of local salt gland blood flow with maximal flow attenuation observed towards the end of the short-term i.c. application of the peptide hormone (Fig. 3A). In parallel with the diminished blood flow, salt gland secretion markedly decreased from 0 43 + 0-02 H H
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n = 6) and noradrenaline (20 nmol min' (kg body wt)', n = 6) infused I.C. for 5 Fi. Values represent means +.E.M. Statistical differences at the m e 2P 005 level of significance are represented as (1) stars below bars for comparison with steady-state control conditions, and (2) stars between bars for comparison of the various hormonal treatments. Changes in local blood perfusion did not correspond with changes in salt gland secretion.
to a minimum of 0-04 + 001 ml min-' (2P < 0 05) with a reduced osmolality of the secreted fluid from 1080 + 25 to 690 + 89 mosmol kg-1 (2P < 0 05). The time courses of both parameters are shown for a single experiment in Fig. 3A. Lower concentrations of ANG II tested revealed dose-dependent effects on both salt gland secretion and blood flow. The avian antidiuretic hormone AVT caused a significant (2P < 0 05) fall in salt gland blood perfusion of 25 % to 11.2 + 1.7 ml min-' (g tissue wet wt)-' as determined with radioactive microspheres. Similarly, the laser-Doppler signal response to AVT revealed a reduction in local glandular blood flow, which although not reaching the minima observed with ANG II was of much longer duration (Fig. 3B). Salt gland secretion, however, was not influenced by the avian antidiuretic hormone even at the high concentration of 20 pmol min-' (kg body wt)-' with regard to either the secretion rate or the osmolality of the secreted fluid. AVT concentrations lower than 10 pmol min-' (kg body wt)-1 did not reduce salt gland blood perfusion significantly. A possible adrenergic influence on salt gland function, independent of whether the route of action be hormonal or neurotransmitter-like, was mimicked by the i.C. infusion of noradrenaline. At an elevated MABP, capillary blood flow was reduced during drug application by half to 6-9 + 1 1 ml min-' (g tissue wet wt)-1 (2P < 0 05). The laser-Doppler signal responded to noradrenaline infusion with a rapid drop to
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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low red cell flux values with the inhibition lasting for some 8-12 min (Fig. 3C). In contrast to the marked decline in both local and whole-organ glandular blood perfusion, the change in salt gland activity, although significant (2P < 0 05), was proportionally much less, with secretion rates being reduced for 10 min to 025 + 0 05 as compared to 0 39 + 0'03 ml min-' during steady-state control periods. The osmolality of the secreted fluid during noradrenaline infusion was 958 + 24 compared to 1070+27 mosmol kg-1 without the catecholamine. Noradrenaline concentrations lower than 10 nmol min-' (kg body wt)-' produced no effect on salt gland secretion, while reducing blood flow slightly. A comparison of the red cell flux and glandular osmolal excretion values integrated over the time of action of ANG II, AVT and noradrenaline with those for a preceding control period of equal length is given in Fig. 4. ANG II reduced local salt gland blood flow to 69+ 5 % of the control level and decreased osmolal excretion so that NaCl elimination fell by 6-4 +10 mosmol, at unchanged systemic loading. AVT lowered blood flow to 63+11 % of the control but had no effect upon salt gland secretion. Noradrenaline caused a reduction in local blood flow to 49 + 7 % of control levels; however, salt gland secretion was only slightly reduced with osmolal excretion being diminished by 2-7 + 0-6 mosmol. DISCUSSION
There is a large body of evidence to show that secretory function of the avian salt gland is associated with an elevated blood supply to the gland. The use of polarographic oxygen electrodes (Fainge et al. 1963) or the fabrication of microvascular corrosion castings (Hossler & Olson, 1990) revealed a high arterial blood supply when salt gland tissue had undergone functional hypertrophy due to salt adaptation (Peaker & Linzell, 1975), and clearly indicated increased arteriolar perfusion at enhanced secretory activity. Blood flow through the duck carotid arteries, as measured with surgically implanted magnetic flow probes (Burford & Bond, 1968; Bech, Rautenberg, May & Johansen, 1982), indicated an almost 50% diversion of carotid blood to the glands at maximal salt gland secretion. Specific salt gland blood flow can increase more than tenfold during intravenous salt loading in the Pekin duck with values of up to 35 ml min-1 g-' of salt gland tissue (Kaul et al. 1983; Butler, Siwanowicz & Puskas, 1989). Comparable data have been obtained for the goose (Hanwell et al. 1971 a) and the analogous shark rectal gland (Shuttleworth, 1983). Despite this generally tight linear relationship between whole-organ blood flow and secretion rate (Hanwell et al. 1971 a, b; Kaul et al. 1983), the same authors demonstrated that atropine treatment fully abolished the NaCl excretion by, but not the blood flow to, the salt glands of ducks and geese, respectively. Investigations carried out in other exocrine gland systems in various mammalian species and elasmobranchs suggest some degree of uncoupling of both parameters under various physiological and pharmacological conditions. Thus, Solomon and co-workers (Solomon, Taylor, Rosa, Silva & Epstein, 1984; Solomon, Taylor, Dorsey, Silva & Epstein, 1985) demonstrated augmented blood flow to the shark rectal gland during isotonic intravascular volume expansion at unchanged arterial pressure, with
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secretion being enhanced subsequently. Treatment with somatostatin or the Na+-K+-Cl- transport blocking agent bumetanide, however, suppressed stimulated secretion, while leaving the glandular blood flow unaffected. Exocrine secretion independent of arterial blood supply has also recently been described for gastric acid secretion in mammalian species under conditions of high blood perfusion, although at low blood flow rates, with possibly insufficient oxygen delivery to the stomach mucosa, acid secretion is linearly dependent on mucosal blood flow (Guth & Leung, 1987). Similarly, in the cat salivary glands, vasoactive intestinal polypeptide (VIP) caused a marked vasodilatation after intra-arterial injection without concomitant secretion (Lundberg, 1981). In the present study, results obtained with the RMT during continuous salt loading support the hypothesis of tight blood flow-secretion coupling in avian salt gland secretion when looking at whole-organ blood flow values. The recordings of rhythmic vasoconstrictions and vasodilatations of local 'single lobe' salt gland blood flow by the LDM, however, in combination with the uneven distribution of trapped microspheres in various secretory lobes at a fixed time during constant on-going secretion, favour the idea of differential uncoupling of both parameters. The vasoactive hormones ANG II, AVT and noradrenaline, which play an important role in avian body fluid homeostasis, were employed as endocrine tools to possibly differentiate between the control of avian salt gland secretion and arteriolar blood supply. ANG II, which is closely linked to the status of the extracellular fluid, induced a pronounced decrease in salt gland blood flow associated with a marked suppression of secretion. This inhibitory action of systemic ANG on salt gland secretion, which has also been shown in other studies (Wilson, Pham & Tan-Wilson, 1985; Gray, Hammel & Simon, 1986), may not be due to a direct effect on the supraorbital salt glands since binding studies have failed to reveal receptors for ANG II in these organs (Gerstberger, Healy, Hammel & Simon, 1987). Rather the inhibitory action of centrally administered ANG II on salt gland function and the labelling of ANG II binding sites in hypothalamic areas of osmoregulatory importance both inside and outside the blood-brain barrier indicate a central nervous target for brain-intrinsic and blood-borne ANG II (Gerstberger, Gray & Simon, 1984b). When ANG II was applied under conditions of ganglionic blockade, neither inhibition of on-going secretion nor vasoconstriction were observed (Bulter et al. 1989). The antidiuretic hormone AVT did not influence on-going steady-state salt gland secretion even at the high concentration of 20 pmol min-' (kg body wt)-', while lowering arteriolar blood perfusion. At unchanged MABP, organ-specific redistribution of blood supply with reductions in salt gland, renal and spleen, but an increase in skin blood flow values (R. Gerstberger, unpublished observation) were induced. Lower doses of AVT failed to influence both salt gland secretion and blood perfusion. Specific binding sites for the antidiuretic hormone could not be demonstrated in the salt gland tissue or its vasculature as revealed by receptor autoradiography using tritiated vasopressin as radioligand (Keil, 1989). The reduction in salt gland blood flow at only slightly reduced secretion during noradrenaline application to the cephalic circulation supports earlier findings of sympathetic innervation of salt gland tissue and arterioles in addition to the main
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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cholinergic-VIPergic innervation (Peaker & Linzell, 1975). With regard to receptor localization and specificity of the receptor system involved, the data published to date are conflicting. While both adrenaline and noradrenaline acting via a-receptors induced vasoconstriction as determined by Fiinge et al. (1963), the fl-agonist isoprenaline caused vasodilatation. On the other hand, the a-antagonist phenoxybenzamine failed to inhibit blood flow during steady-state secretion (Kaul et al. 1983). Data obtained by the in vitro studies of Lowy and co-workers (Lowy & Ernst, 1987) implicated the preferential existence of fl-receptors, although not excluding the existence of cc2-receptors. Transepithelial short-circuit current could also be stimulated by 8-bromo-cyclic AMP and forskolin, indicative of cyclic AMP being involved as second messenger in the cellular response. Preliminary studies using highly specific radiolabelled ac- and f-antagonists (A. Muller & R. Gerstberger, unpublished observation) suggest the presence of ac2-adrenergic receptors in duck salt gland tissue. The prevalence of a2-receptors is supported by findings in the shark rectal gland, where the inhibitory action of noradrenaline on perfusion flow was reversed by phentolamine, but not propranolol (Shuttleworth, 1983). Comparing the marked decline in salt gland blood flow and osmolal excretion during I.C. application of noradrenaline with the strong inhibitory effect of ANG II on secretion at less reduced blood flow or with the long-lasting vasoconstriction of lower magnitude during AVT infusion without changes in secretion, clearly indicates a partial uncoupling of both parameters induced by hormonal action. Taking the demonstrated redistribution of intraglandular blood flow at constant whole-organ blood perfusion during salt loading into consideration, the differentiated control of salt gland blood flow and secretion may enable the avian salt gland to respond quickly to variable demands at altered local transcapillary fluid exchange. The present study could not have been performed without excellent technical assistance in animal surgery, salt gland fluid analysis, figure preparation, plotting routines and the calculation of statistics. The author appreciates the motivated help of Roswitha Bender, Anette Grieb, Regina Lotz and Elisabeth Vieth. The support in laser-Doppler methodology by Dr Holger Sann, and his valuable suggestions, are highly appreciated. The author appreciates the valuable discussions with Dr David A. Gray, and his help in revising the manuscript. REFERENCES
ASH, R. W., PEARCE, J. W. & SILVER, A. (1969). An investigation of the nerve supply to the salt gland of the duck. Quarterly Journal of Experimental Physiology 54, 281-295. BEcH, C., RAUTENBERG, W., MAY, B. & JOHANSEN, K. (1982). Regional blood flow changes in response to thermal stimulation of the brain and spinal cord in the Pekin duck. Journal of Comparative Physiology B 147, 71-77. BURFORD, H. J. & BOND, R. F. (1968). Avian cardiovascular parameters: effect of intravenous osmotic agents, relation to salt gland secretion. Experientia 24, 1086-1088. BUTLER, D. G., SIWANOWICZ, H. & PUSKAS, D. (1989). A re-evaluation of experimental evidence for the hormonal control of avian nasal salt glands. In Progress in Avian Osmoregulation, ed. HUGHES, M. & CHADWICK, A., pp. 127-141. The Leeds Philosophical and Literary Society, Leeds. CODY, R. P. & SMITH, J. K. (1987). Applied Statistics and the SAS Programming Language, pp. 139-152. North-Holland, Amsterdam. FXNGE, R., KROG, J. & REITE, 0. (1963). Blood flow in the avian salt gland studied by polarographic oxygen electrodes. Acta Physiologica Scandinavica 58, 40-47.
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GERSTBERGER, R. (1988). Functional VIP system in salt glands of the Pekin duck (Anas platyrhynchos). Cell and Tissue Research 252, 39-48. GERSTBERGER, R., GRAY, D. A. & SIMON, E. (1984). Circulatory and osmoregulatory effects of angiotensin II perfusion of the third ventricle in a bird with salt glands. Journal of Physiology 349, 167-182. GERSTBERGER, R., HEALY, D. P., HAMMEL, H. T. & SIMON, E. (1987). Autoradiographic localization and characterization of circumventricular angiotensin II receptors in duck brain. Brain Research 400, 165-170. GERSTBERGER, R., SANN, H. & SIMON, E. (1988). Vasoactive intestinal peptide stimulates blood flow and secretion of avian salt glands. American Journal of Physiology 255, R575-582. GRAY, D. A., HAMMEL, H. T. & SIMON, E. (1986). Osmoregulatory effects of angiotensin II in a bird with salt glands. Journal of Comparative Physiology B 156, 315-321. GUTH, P. H. & LEUNG, F. W. (1987). Physiology of the gastric circulation. In Physiology of the Gastrointestinal Tract, second edn, ed. JOHNSON, L. R., pp. 1031-1053. Raven Press, New York. HAMMEL, H. T., SIMON-OPPERMANN, C. & SIMON, E. (1980). Properties of body fluids influencing salt gland secretion in Pekin ducks. American Journal of Physiology 239, R489-496. HANWELL, A., LINZELL, J. L. & PEAKER, M. (1971 a). Salt gland secretion and blood flow in the goose. Journal of Physiology 213, 373-387. HANWELL, A., LINZELL, J. L. & PEAKER, M. (1971 b). Cardiovascular response to salt-loading in conscious domestic geese. Journal of Physiology 213, 389-398. HOSSLER, F. E. & OLSON, K. R. (1983). Microvasculature of the nasal salt gland of the duckling, Anas platyrhynchos: quantitative responses to osmotic adaptation and deadaptation studied with vascular corrosion casting. Journal of Experimental Zoology 254, 237-247. KAUL, R., GERSTBERGER, R., MEYER, J.-U. & SIMON, E. (1983). Salt gland blood flow in saltwateradapted Pekin ducks: microsphere measurement of the proportionality to secretion rate and investigation of controlling mechanisms. Journal of Comparative Physiology B 149, 457-462. KEIL, R. (1989). Physiologische und biochemische Untersuchungen zur interstitiellen Volumensensitivitiit beider salzadaptierten Pekingente (Anas platyrhynchos). Diploma Thesis, University of Tiibingen, Germany. W. (1972). On the innervation of the salt gland. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 134, 435-438. LOWY, R. J. & ERNST, S. A. (1987).,6-Adrenergic stimulation of ion transport in primary cultures of avian salt glands. American Journal of Physiology 252, C670-676. LUNDBERG, J. M. (1981). Evidence for co-existence of vasoactive polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Acta Physiologica Scandinavica 112, suppl. 496, 1-57. PEAKER, M. & LINZELL, J. L. (1975). Salt Glands in Birds and Reptiles. Cambridge University Press, Cambridge (Monographs of the Physiological Society, no. 32). SCHMIDT-NIELSEN, K. (1960). The salt-secreting gland of marine birds. Circulation 21, 955-967. SHUTTLEWORTH, T. J. (1983). Haemodynamic effects of secretory agents on the isolated elasmobranch rectal gland. Journal of Experimental Biology 103, 193-204. SIMON, E. & GRAY, D. A. (1989). Control of salt glands as a second-line defence system in body fluid homeostasis of birds. In Progress in Avian Osmoregulation, ed. HUGHES, M. & CHADWICK, A., pp. 23-40. The Leeds Philosophical and Literary Society, Leeds. SOLOMON, R. J., TAYLOR, M., DORSEY, D., SILVA, P. & EPSTEIN, F. H. (1985). Atriopeptin stimulation of rectal gland function in Squalus acanthias. American Journal of Physiology 249, R348-354. SOLOMON, R. J., TAYLOR, M., RoSA, R., SILVA, P. & EPSTEIN, F. H. (1984). In vivo effect of volume expansion on rectal gland function. II. Hemodynamic changes. American Journal of Physiology 246, R67-71. TENLAND, T. (1982). On laser-Doppler flowmetry: methods and microvascular applications. Ph.D. Thesis, Linkbping University, Sweden. WILSON, J. X., PHAM, D. V. & TAN-WILSON, H. I. (1985). Angiotensin and converting enzyme regulate extrarenal salt excretion in ducks. Endocrinology 117, 135-140.
KUHNEL,
Journal of Physiology (1991), 435, pp. 175-186 With 4 figures Printed in Great Britain
PARTIAL UNCOUPLING OF SALT GLAND BLOOD FLOW AND SECRETION IN THE PEKIN DUCK (ANAS PLATYRHYNCHOS)
BY RUDIGER GERSTBERGER From the Max-Planck-Institut fur Physiologische und Klinische For8chung, W. G. Kerckhoff-Institut, D-6350 Bad Nauheim, Germany
(Received 18 June 1990) SUMMARY
1. The aim of this study was to investigate the relationship between the blood flow through and the secretion by the salt glands of conscious, salt-water-adapted Pekin ducks. 2. Intravenous loading with hypertonic saline induced a steady-state secretion from the salt glands with a concomitant increase in whole-organ blood flow. The distribution of elevated local glandular blood flow was, however, uneven and in addition demonstrated vasomotor patterns that ranged from constant to rhythmic. 3. During on-going salt gland secretion, the infusion of three vasoactive agents, 5Val-angiotensin II (ANG II), 8Arg-vasotocin (AVT) and noradrenaline, via the carotid artery had differential effects on salt gland blood flow and secretion. 4. ANG II (80 pmol min-' (kg body wt)-1) had no effect on mean arterial blood pressure (MABP), produced a transient 30% decrease in glandular blood flow and strongly diminished salt gland secretion (retention of 6-4 mosmol NaCl). 5. AVT (20 pmol min-' (kg body wt)-') had no effect on MABP and did not alter salt gland secretion despite a 35 % reduction in blood flow. 6. Noradrenaline (20 nmol min-' (kg body wt)-1) elevated MABP by 15 mmHg, reduced salt gland blood flow by more than 50 %, but diminished salt gland secretion only slightly (retention of 2 7 mosmol NaCl). 7. Using ANG II, AVT and noradrenaline as hormonal tools, integrated changes in blood flow rate did not correspond with integrated changes in salt gland excretion. The partial dissociation between both parameters shows that control of secretion by the salt gland is more complex than simply being linearly dependent upon blood flow through it. INTRODUCTION
The supraorbital salt-secreting glands ('salt glands') of marine birds respond to an increase in the tonicity or volume of their extracellular fluid compartment with enhanced secretion of a strongly hypertonic fluid rich in sodium chloride (SchmidtNielsen, 1960; Peaker & Linzell, 1975; Simon & Gray, 1989). Efferent control of salt gland activity is achieved by parasympathetic fibres originating from the VIIth cranial nerve (Ash, Pearce & Silver, 1969; Kiihnel, 1972) and utilizing both MS 8571
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acetylcholine and vasoactive intestinal polypeptide (VIP)
as stimulatory neurotransmitters (Gerstberger, 1988; Gerstberger, Sann & Simon, 1988). It has been shown, in a variety of avian species, that actively secreting salt glands require an elevated blood flow (Fiinge, Krog & Reite, 1963; Hanwell, Linzell & Peaker, 1971 a; Kaul, Gerstberger, Meyer & Simon, 1983; Gerstberger et al. 1988) and that a constant 20% of the sodium chloride content of the perfusing glandular blood stream is extracted and secreted against a marked trans-tubular concentration gradient (Kaul et al. 1983). Accordingly it appeared that blood flow and secretion of the avian salt glands are coupled by a simple linear relationship so that augmented basolateral supply of sodium and chloride to the principal secretory cells would lead to increased transport of both ions into the tubular lumen as long as the underlying transport mechanisms are not saturated. However, the recent discovery of atropine-resistant vasodilatation at suppressed secretion (Kaul et al. 1983; Gerstberger et al. 1988) indicates that the control of salt gland function may not be so straightforward and that physiological conditions might exist where blood flow and secretion could be dissociated, as is the case in cat salivary glands (Lundberg, 1981). In order to gain a deeper understanding of the phenomenon of blood flow-secretion coupling in exocrine glands, the relationship between these parameters was investigated in the salt glands of conscious salt-water-adapted ducks. Whole-organ and local glandular blood flow were measured, and the salt gland responses to changes in blood flow induced by the vasoactive hormones 5Val-angiotensin II (ANG II), 5Arg-vasotocin (AVT) and noradrenaline were calculated.
METHODS
Animals The experiments were performed with twenty-six adult Pekin ducks (Anasplatyrhynchos) of both sexes with body weights of 2-2 +0.1 kg (mean+s.E.M.). The animals were reared in flocks under a natural day-night cycle in a thermoneutral environment and fed with commercial dry chicken food, enriched with minerals and vitamins. Over a 6 week period the ducks were gradually adapted to 400 mosmol kg-' saline as drinking and bathing fluid and were kept at this regimen for at least 30 days before experimentation. Hypertrophy of their supraorbital salt glands enabled the saltwater-adapted Pekin ducks to secrete up to 0-8 mosmol min-' of mainly sodium chloride via these glands, thereby maintaining body fluid homeostasis.
Surgery Surgical procedures were performed exclusively under inhalation anaesthesia (halothane, N20, An indwelling Teflon cannula (V/19, Braun, Germany) was implanted into one of the carotid arteries for drug application to the cephalic circulation without impairment of blood perfusion (Kaul et al. 1983). For the recording of systemic blood pressure via an Endevco pressure transducer (N 8510, 5 lbf in2, USA), or the collection of arterial reference blood samples, a polypropylene catheter (PP 90, Portex) was inserted into the left brachial artery. In order to measure organ-specific capillary blood flow with the radioactive microspheres technique (RMT) (Kaul et al. 1983), in five animals a PP 50 catheter was advanced towards the heart from the right brachial artery until its tip protruded into the left ventricle as judged from changes in the pulse pressure. Filling with heparinized saline kept all catheters patent. For the continuous recording of local salt gland blood flow by the laser-Doppler method (LDM), the salt gland ipsilateral to the cannulated carotid artery was freed of connective tissue in fifteen ducks without any damage to the gland after its exposure via a cranial skin incision. A head device made from a metal base-plate was fixed to the skull with stainless-steel wires and acrylic cement
02) and sterile conditions 24-48 h prior to experimentation.
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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to restrain head movements throughout experimentation. The birds showed no apparent signs of stress or discomfort during the experiments, with heart rate and mean arterial blood pressure (MABP) values consistent with this impression.
Experimental procedure On the morning of the experiment, the duck was placed in a canvas sling that allowed free movement of the legs. A catheter (Braunula G-20, Braun, Germany) was placed in a leg vein by percutaneous venipuncture for continuous infusion of 04 ml min-' of 1000 mosmol kg-1 NaCl to induce steady-state salt gland secretion at a rate and osmotic concentration equal to the given load. The clear, NaCl-rich salt gland secretion was collected at 5-10 min intervals (Hammel, SimonOppermann & Simon, 1980) and its volume was determined to an accuracy of + 10 ,ul. Osmotic concentrations of salt gland secretion as well as plasma samples obtained at the beginning and end of the experiment were measured by vapour pressure osmometry (Wescor 5100 C, Wescor Inc., USA). Series I. The effect of intravenous hypertonic salt loading upon salt gland secretion, glandular blood flow as well as MABP were monitored until constant salt gland secretion was established. Series II. Under defined conditions of steady-state salt gland activity, the influence of vasoactive agents upon MABP, salt gland secretion and blood flow were determined. Noradrenaline (Sigma, Germany), 8Arg-vasotocin (AVT) and 5Val-angiotensin II (ANG II) (Bissendorf, Germany) were dissolved in sterile isotonic saline and infused over 5 min via the carotid at a rate of 200 ,1 minin concentrations of 20 nmol min-1 (kg body wt)'1, 20 and 80 pmol min-' (kg body wt)-', respectively. Radioactive microspheres technique (RMT) To measure capillary blood flow in the salt glands and various other organs of the salt-wateradapted Pekin duck, microspheres 15 ,um in diameter (NEN DuPont, USA) labelled with 14'Ce, 51Cr, 113Sn, 103Ru, 95Nb and 4"Sc were used. For each measurement 0 8-2 0 x 106 microspheres were injected in a volume of 0 6 ml over 20 s into the left ventricle immediately after starting collection of reference blood for 1 min from the left brachial artery. The availability of six y-emitting tracers allowed consecutive determinations of organ-specific blood flow values in the same animal. After the experiment, the duck was quickly killed with an i.v. injection of sodium pentobarbitone. Tissue samples of known wet weight were dissected and prepared for the measurements of photopeak radioactivity (RI240 y-spectrometer, Strahlentechnik, Germany). The data were converted with a multichannel analyser (ND 60, Nuclear Data, Germany). Compton and background corrections were performed with a Digital computer (VAX-780, USA) and blood flow values are presented in ml min-' (g tissue wet wt)-1.
Laser-Doppler method (LDM) Local salt gland blood flow was monitored continuously throughout the experiment with a PeriFlux PF 2 laser-Doppler flowmeter (Perimed, Sweden) using a PF-103 probe. The base-plate attached to the animal's skull was fixed to a cover-lid as described recently (Gerstberger et al. 1988). The laser probe was positioned 0-5-1-0 mm away from the salt gland surface, which had been freed of connective tissue. Relative values of local blood flow were recorded using signals from red blood cell movements over an area of about 4 mm2 to a depth of 1-2 mm (Tenland, 1982). The of movement artifact filter systems with a frequency bandwidth of 20-12 x 103 Hz, and the inherent linearity of the flowmeter, however, allowed the comparison of flux values obtained during various physiological conditions. All flux values were normalized to gain 1 with a 3 0 s time constant. Histology The supraorbital salt glands of three ducks, which had received an intraventricular injection of non-radiolabelled microspheres during steady-state salt gland secretion, were dissected immediately after the animals were killed with an overdose of intravenously administered sodium pentobarbitone. After submersion fixation of the tissue in 4% paraformaldehyde for 48 h with subsequent incubation in 10 % sucrose overnight at 2 °C, 32 ,um sections of salt gland slices were cut in a cryostat (Frigocut 2700, Reichard-Jung, Germany) at -15°C, thaw-mounted onto
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gelatine-alum-coated slides, and stained with cresyl violet. The numbers of trapped microspheres in all secretory lobes were determined. Data evaluation All data are presented as means + S.E.M. The Wilcoxon matched-pairs sign rank test was employed with 2P < 005 being considered the limit of significance in order to compare altered parameter values after hormone application with control conditions in the same experimental animal. Changes of salt gland secretion were calculated as the number of milliosmoles that were retained, integrated over time of hormone action (20 min) and compared to the secreted milliosmoles during control periods of equal length. Alterations in salt gland blood flow measured by the LDM were calculated as the percentage decrease of flux integrated over the time of hormone action (20 min), compared to the flux integrated over corresponding control periods of equal length. The method of 'repeated measures designs' (Cody & Smith, 1987) was used to relate the secretion and blood flow data during hormonal treatment to each other. RESULTS
Salt gland blood flow during on-going glandular secretion To induce steady-state salt gland secretion, conscious salt-water-adapted Pekin ducks received a continuous systemic infusion of 0-4 ml min-' of 1000 mosmol kg-' NaCl. With a time delay of less than 10 min, the salt glands responded to the salt load with enhanced osmolal excretion, and after 20-30 min of the NaCl application, the output of both salt and water matched the infusion to 102 + 3 and 97+4% (n = 7), respectively (Fig. 1). Local blood flow, as measured by the LDM, increased in parallel with osmotic excretion (n = 7), reaching an elevated plateau of red blood cell fluxes 15-20 min after the start of the NaCl challenge as is shown for a single representative experiment in Fig. 1. In experiments with stable blood flow readings, drop counting of the aspirated secretory fluid revealed tight congruency with the laser-Doppler signal on a 30 s time base. General circulatory parameters such as MABP or heart rate (not shown) remained unchanged with values of 118 +5 mmHg and 185 + 13 beats min-' (n = 12), respectively. To confirm the blood flow data obtained by the LDM, the capillary blood perfusion was determined simultaneously in five experiments using the RMT, which is well established for the Pekin duck. Microspheres bearing different, randomly mixed radioactive tracers were injected intracardially (i.c.) into birds at the threshold of salt gland secretion, before application of the salt load, as well as 20 min after having reached steady-state secretion with stable and high LDM flow recordings. In all organs tested (brain, eyes, harderian glands, salt glands, heart, lung, gut, pancreas, liver, gonads, kidneys, breast muscle, web skin), specific blood flow values were unaltered by salt loading with the exception of the supraorbital salt glands in which flow values increased twelvefold from 1-3 + 02 to 15-1 + 1-4 ml min-' (g tissue wet wt)-1. Thus, enhancement in the LDM signals was accompanied by augmented capillary blood perfusion. In a few experiments, however, local salt gland blood flow exhibited rhythmic vasoconstrictions and vasodilatations at varying frequency and amplitude despite high whole-organ blood flow values (RMT) and steady-state secretion at the stimulated level. To elucidate the relationship between total osmolal output and local salt gland blood flow, recordings of red cell fluxes were obtained from various sites of the same osmotically stimulated salt gland of four birds. Figure 2
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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demonstrates the variability of red cell fluxes measured at three recording sites (A-C) 4-5 mm apart during steady-state salt gland secretion at constant arterial pressure and carotid blood flow as determined with surgically implanted electromagnetic flow probes. The pattern of local blood flow varied from stable high-level Salt load 140
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Fig. 1. Parallel increase in salt gland secretion and blood flow at unchanged mean arterial blood pressure (MABP) in conscious Pekin ducks due to systemic saline loading. Local salt gland blood flow measured by the LDM (flux) before and during saline loading (0 4 ml min-' of 1000 mosmol kg-' NaCl) is shown for a single experiment, while salt gland excretion is demonstrated for experiments in seven conscious salt-water-adapted animals.
fluxes (A) to red cell fluxes at a high level with intermittent reductions possibly representing vasoconstriction (B), and even to rapid alternations of high flux values and cessation of blood perfusion (C). The obviously uneven distribution of blood flow to single secretory lobes of the activated avian salt gland could be verified histologically. Intraventricularly injected non-radioactive microspheres caught during just one cycle of the systemic circulation in the capillaries feeding the glandular parenchyma revealed a non-homogenous distribution in the eight to fifteen
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secretory lobes that comprise the duck salt glands, with numbers of intracapillary microspheres in individual lobes varying between three and forty-one in serial tissue sections.
Hormonal actions on salt gland secretion and blood flow The effects of ANG II, AVT and noradrenaline upon MABP, glandular blood flow, salt gland secretion rate and osmolality of the secreted fluid for single experiments are shown in Fig. 3A-C. 60 :4 c
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With regard to general cardiovascular parameters, avian-specific ANG II administered to the cephalic circulation at a dose of 80 pmol min-' (kg body wt)-1 did not influence arterial pressure or heart rate in five out of seven experiments, while in two animals mean arterial blood pressure was slightly elevated by 8-10 mmHg
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
181
for the time of peptide application. The antidiuretic hormone AVT infused at 20 pmol min-' (kg body wt)-' proved to be without any effect on MABP and heart rate. Noradrenaline (20 nmol min-' (kg body wt)-1), however, significantly (2P < 005) increased MABP from 117+6 to 131+8 mmHg (n = 6) with sharp A
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Fig. 3. Effects of 5Val-angiotensin vasotocin (AVT; 20 pmol min-' (kg body wt)-') (B), and noradrenaline (20 nmol min-' (kg body wt)-') (C) infused for 5 min i.C. on mean arterial blood pressure (MABP) and salt gland function. MABP, local salt gland blood flow measured by the LDM (flux), as well as flow rate and osmolality of salt gland secretion are shown for representative single experiments in conscious ducks receiving a continuous systemic infusion of 0-4 ml minof 1000 mosmol kg-' NaCl.
onset immediately after beginning the i.c. infusion and rapid decline to control values about 5 min after cessation of the i.c. application. Heart rate was reduced from 195 + 21 to 165+17 beats min-' (2P < 0-05), suggestive of reflex bradycardia. The infusion of ANG II resulted in a reduction of blood flow through the gland from 15-1 + 1-4 to 10-8+ 1-8 ml min-1 (g tissue wet wt)-' (2P . 0-05) as determined
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by the RMT. The laser-Doppler signal clearly demonstrated a partial inhibition of local salt gland blood flow with maximal flow attenuation observed towards the end of the short-term i.c. application of the peptide hormone (Fig. 3A). In parallel with the diminished blood flow, salt gland secretion markedly decreased from 0 43 + 0-02 H H
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to a minimum of 0-04 + 001 ml min-' (2P < 0 05) with a reduced osmolality of the secreted fluid from 1080 + 25 to 690 + 89 mosmol kg-1 (2P < 0 05). The time courses of both parameters are shown for a single experiment in Fig. 3A. Lower concentrations of ANG II tested revealed dose-dependent effects on both salt gland secretion and blood flow. The avian antidiuretic hormone AVT caused a significant (2P < 0 05) fall in salt gland blood perfusion of 25 % to 11.2 + 1.7 ml min-' (g tissue wet wt)-' as determined with radioactive microspheres. Similarly, the laser-Doppler signal response to AVT revealed a reduction in local glandular blood flow, which although not reaching the minima observed with ANG II was of much longer duration (Fig. 3B). Salt gland secretion, however, was not influenced by the avian antidiuretic hormone even at the high concentration of 20 pmol min-' (kg body wt)-' with regard to either the secretion rate or the osmolality of the secreted fluid. AVT concentrations lower than 10 pmol min-' (kg body wt)-1 did not reduce salt gland blood perfusion significantly. A possible adrenergic influence on salt gland function, independent of whether the route of action be hormonal or neurotransmitter-like, was mimicked by the i.C. infusion of noradrenaline. At an elevated MABP, capillary blood flow was reduced during drug application by half to 6-9 + 1 1 ml min-' (g tissue wet wt)-1 (2P < 0 05). The laser-Doppler signal responded to noradrenaline infusion with a rapid drop to
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
183
low red cell flux values with the inhibition lasting for some 8-12 min (Fig. 3C). In contrast to the marked decline in both local and whole-organ glandular blood perfusion, the change in salt gland activity, although significant (2P < 0 05), was proportionally much less, with secretion rates being reduced for 10 min to 025 + 0 05 as compared to 0 39 + 0'03 ml min-' during steady-state control periods. The osmolality of the secreted fluid during noradrenaline infusion was 958 + 24 compared to 1070+27 mosmol kg-1 without the catecholamine. Noradrenaline concentrations lower than 10 nmol min-' (kg body wt)-' produced no effect on salt gland secretion, while reducing blood flow slightly. A comparison of the red cell flux and glandular osmolal excretion values integrated over the time of action of ANG II, AVT and noradrenaline with those for a preceding control period of equal length is given in Fig. 4. ANG II reduced local salt gland blood flow to 69+ 5 % of the control level and decreased osmolal excretion so that NaCl elimination fell by 6-4 +10 mosmol, at unchanged systemic loading. AVT lowered blood flow to 63+11 % of the control but had no effect upon salt gland secretion. Noradrenaline caused a reduction in local blood flow to 49 + 7 % of control levels; however, salt gland secretion was only slightly reduced with osmolal excretion being diminished by 2-7 + 0-6 mosmol. DISCUSSION
There is a large body of evidence to show that secretory function of the avian salt gland is associated with an elevated blood supply to the gland. The use of polarographic oxygen electrodes (Fainge et al. 1963) or the fabrication of microvascular corrosion castings (Hossler & Olson, 1990) revealed a high arterial blood supply when salt gland tissue had undergone functional hypertrophy due to salt adaptation (Peaker & Linzell, 1975), and clearly indicated increased arteriolar perfusion at enhanced secretory activity. Blood flow through the duck carotid arteries, as measured with surgically implanted magnetic flow probes (Burford & Bond, 1968; Bech, Rautenberg, May & Johansen, 1982), indicated an almost 50% diversion of carotid blood to the glands at maximal salt gland secretion. Specific salt gland blood flow can increase more than tenfold during intravenous salt loading in the Pekin duck with values of up to 35 ml min-1 g-' of salt gland tissue (Kaul et al. 1983; Butler, Siwanowicz & Puskas, 1989). Comparable data have been obtained for the goose (Hanwell et al. 1971 a) and the analogous shark rectal gland (Shuttleworth, 1983). Despite this generally tight linear relationship between whole-organ blood flow and secretion rate (Hanwell et al. 1971 a, b; Kaul et al. 1983), the same authors demonstrated that atropine treatment fully abolished the NaCl excretion by, but not the blood flow to, the salt glands of ducks and geese, respectively. Investigations carried out in other exocrine gland systems in various mammalian species and elasmobranchs suggest some degree of uncoupling of both parameters under various physiological and pharmacological conditions. Thus, Solomon and co-workers (Solomon, Taylor, Rosa, Silva & Epstein, 1984; Solomon, Taylor, Dorsey, Silva & Epstein, 1985) demonstrated augmented blood flow to the shark rectal gland during isotonic intravascular volume expansion at unchanged arterial pressure, with
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secretion being enhanced subsequently. Treatment with somatostatin or the Na+-K+-Cl- transport blocking agent bumetanide, however, suppressed stimulated secretion, while leaving the glandular blood flow unaffected. Exocrine secretion independent of arterial blood supply has also recently been described for gastric acid secretion in mammalian species under conditions of high blood perfusion, although at low blood flow rates, with possibly insufficient oxygen delivery to the stomach mucosa, acid secretion is linearly dependent on mucosal blood flow (Guth & Leung, 1987). Similarly, in the cat salivary glands, vasoactive intestinal polypeptide (VIP) caused a marked vasodilatation after intra-arterial injection without concomitant secretion (Lundberg, 1981). In the present study, results obtained with the RMT during continuous salt loading support the hypothesis of tight blood flow-secretion coupling in avian salt gland secretion when looking at whole-organ blood flow values. The recordings of rhythmic vasoconstrictions and vasodilatations of local 'single lobe' salt gland blood flow by the LDM, however, in combination with the uneven distribution of trapped microspheres in various secretory lobes at a fixed time during constant on-going secretion, favour the idea of differential uncoupling of both parameters. The vasoactive hormones ANG II, AVT and noradrenaline, which play an important role in avian body fluid homeostasis, were employed as endocrine tools to possibly differentiate between the control of avian salt gland secretion and arteriolar blood supply. ANG II, which is closely linked to the status of the extracellular fluid, induced a pronounced decrease in salt gland blood flow associated with a marked suppression of secretion. This inhibitory action of systemic ANG on salt gland secretion, which has also been shown in other studies (Wilson, Pham & Tan-Wilson, 1985; Gray, Hammel & Simon, 1986), may not be due to a direct effect on the supraorbital salt glands since binding studies have failed to reveal receptors for ANG II in these organs (Gerstberger, Healy, Hammel & Simon, 1987). Rather the inhibitory action of centrally administered ANG II on salt gland function and the labelling of ANG II binding sites in hypothalamic areas of osmoregulatory importance both inside and outside the blood-brain barrier indicate a central nervous target for brain-intrinsic and blood-borne ANG II (Gerstberger, Gray & Simon, 1984b). When ANG II was applied under conditions of ganglionic blockade, neither inhibition of on-going secretion nor vasoconstriction were observed (Bulter et al. 1989). The antidiuretic hormone AVT did not influence on-going steady-state salt gland secretion even at the high concentration of 20 pmol min-' (kg body wt)-', while lowering arteriolar blood perfusion. At unchanged MABP, organ-specific redistribution of blood supply with reductions in salt gland, renal and spleen, but an increase in skin blood flow values (R. Gerstberger, unpublished observation) were induced. Lower doses of AVT failed to influence both salt gland secretion and blood perfusion. Specific binding sites for the antidiuretic hormone could not be demonstrated in the salt gland tissue or its vasculature as revealed by receptor autoradiography using tritiated vasopressin as radioligand (Keil, 1989). The reduction in salt gland blood flow at only slightly reduced secretion during noradrenaline application to the cephalic circulation supports earlier findings of sympathetic innervation of salt gland tissue and arterioles in addition to the main
AVIAN SALT GLAND SECRETION AND BLOOD FLOW
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cholinergic-VIPergic innervation (Peaker & Linzell, 1975). With regard to receptor localization and specificity of the receptor system involved, the data published to date are conflicting. While both adrenaline and noradrenaline acting via a-receptors induced vasoconstriction as determined by Fiinge et al. (1963), the fl-agonist isoprenaline caused vasodilatation. On the other hand, the a-antagonist phenoxybenzamine failed to inhibit blood flow during steady-state secretion (Kaul et al. 1983). Data obtained by the in vitro studies of Lowy and co-workers (Lowy & Ernst, 1987) implicated the preferential existence of fl-receptors, although not excluding the existence of cc2-receptors. Transepithelial short-circuit current could also be stimulated by 8-bromo-cyclic AMP and forskolin, indicative of cyclic AMP being involved as second messenger in the cellular response. Preliminary studies using highly specific radiolabelled ac- and f-antagonists (A. Muller & R. Gerstberger, unpublished observation) suggest the presence of ac2-adrenergic receptors in duck salt gland tissue. The prevalence of a2-receptors is supported by findings in the shark rectal gland, where the inhibitory action of noradrenaline on perfusion flow was reversed by phentolamine, but not propranolol (Shuttleworth, 1983). Comparing the marked decline in salt gland blood flow and osmolal excretion during I.C. application of noradrenaline with the strong inhibitory effect of ANG II on secretion at less reduced blood flow or with the long-lasting vasoconstriction of lower magnitude during AVT infusion without changes in secretion, clearly indicates a partial uncoupling of both parameters induced by hormonal action. Taking the demonstrated redistribution of intraglandular blood flow at constant whole-organ blood perfusion during salt loading into consideration, the differentiated control of salt gland blood flow and secretion may enable the avian salt gland to respond quickly to variable demands at altered local transcapillary fluid exchange. The present study could not have been performed without excellent technical assistance in animal surgery, salt gland fluid analysis, figure preparation, plotting routines and the calculation of statistics. The author appreciates the motivated help of Roswitha Bender, Anette Grieb, Regina Lotz and Elisabeth Vieth. The support in laser-Doppler methodology by Dr Holger Sann, and his valuable suggestions, are highly appreciated. The author appreciates the valuable discussions with Dr David A. Gray, and his help in revising the manuscript. REFERENCES
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