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The sources of plasma catecholamines in the American eel, Anguilla rostrata
G E N E R A L A N D COMPARATIVE E N D O C R I N O L O G Y
74, 418-430 (1989)
The Sources of Plasma Catecholamines in the American Eel,
Anguilla rostrata CHARLES B.
H A T H A W A Y AND A U G U S T E P P L E
Daniel Baugh Institute of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Accepted October 24, 1988 The origin of the catecholamines (CAs) in the systemic blood of the American eel, Anguilla rostrata, was studied by three approaches: (1) determination of the CA content of tissues suspected to release large quantities of dopamine, norepinephrine, and/or epinephrine into the circulation; (2) measurement of local CA titers in selected regions of the cardiovascular system; and (3) removal of tissues with high CA concentrations, followed by determination of its impact on stimulated CA release. Large quantities of all three CAs were found in the walls of the posterior cardinal veins, from their caudal origin within the opisthonephric kidney to their termination in the ductus Cuvieri. Near the ductus Cuvieri, the CA concentration was 1-3 orders of magnitude above that in other tissues. In this region, which contains the presumed adrenal medulla equivalent, occur the highest plasma levels of the CAs. Strong CA release also in the opisthonephric kidney region raises the question if these CAs affect locally the kidney functions, and/or via the hepatic portal vein (which originates in this region), the liver. Other organs (especially brain and heart) contain CA concentrations high enough to potentially affect the CA level in the systemic blood, if instantly released. However, neither partial removal of the brain nor hypophysectomy, "adrenomedullectomy," Stanniectomy, or urophysectomy had an appreciable impact on stimulated CA release. Together with previous data, these findings show that in the e d (a) the region of the presumed adrenal medulla equivalent is the most important source of all three CAs in systemic plasma; (b) that strong CA-stimulated CA release also occurs outside this region; and (c) that the pituitary, forebrain, and midbrain are not necessary for the CA-stimulated CA release. 9 1989AcademicPress, Inc.
Probably all vertebrates have three circulating catecholamines (CAs), i.e., dopamine (DA), norepinephrine (NE), and epinephrine (E). Though these substances are known or suspected to control cardiovascular, respiratory, metabolic, osmoregulatory, and endocrine functions, precise information on their sites of production and release is insufficient or lacking for many taxa. The present investigations were designed to identify the major sources of systemically circulating CAs in the American eel, a species ideally suited for many in vivo studies on endocrine/metabolic regulations (see, e.g., Lewis and Epple, 1984; Epple and Kahn, 1985; Epple and Nibbio, 1985; Epple, 1987; Epple and Brinn, 1987). Specifically, we wanted to confirm that in the eel the bulk of circulating DA and NE
comes from the brain, while plasma E comes mainly from extraencephalic sources. This scenario was suggested by the detection of higher levels of DA and NE in head than in body blood, and of more or less equal titers of E in both types of blood (Epple et al., 1982). However, the present studies show that the situation is more complex, and that the bulk of all three CAs in the systemic circulation is released by chromaffin cells (mainly the presumed adrenal medulla equivalent) which occur in the wail of the posterior cardinal veins (PCVs).
MATERIALS AND METHODS Animals. Yellow American eels (Anguilla rostrata) from Atlantic coast rivers and estuaries of the United States were obtained from a commercial distributor. 418
0016-6480/89 $1.50 Copyright 9 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
419
CATECHOLAMINES IN AMERICAN EEL The average body weight was approximately 400 g. Techniques of maintenance and cardiac cannulation have been described previously (Epple et al., 1982). The animals were anesthetized in a 3% solution of ethyl carbamate (urethane; U.S. Biochemical Corporation) for 10 min prior to experimental manipulation; or, depending on the experimental design, kept after surgery and cardiac cannulation for specified times in "eel t u b e s " which were suspended in clear, wellaerated fresh water. Preparation of tissue homogenates. Tissues were removed, weighed, and placed in test tubes containing 0.4 N perchloric acid with 5 mM reduced glutathione (10 ixl acid/mg tissue wet wt). Following homogenization, samples were centrifuged (30 min, 210g, at 6~ and aliquots from the supernatant were stored at - 7 0 ~ These extracts were diluted with double distilled water prior to assay for CAs. Regional CA titers. Each sampling series consisted of two rounds of sequential blood withdrawal from five vascular sites (Fig. 1), i.e., a total of 10 samples/ individual animal. These sites were selected in order to identify CA release in the head kidney (major concentration of chromaffin cells), heart (target of CAs and reported site of their release in the eel: Le Bras, 1984; Pennec and Le Bras, 1984), and the gills (also suspected target; however, see Epple and Kahn, 1985). Series I and II represent reversed sampling sequences from the same five sites. Since blood withdrawal may stimulate CA release due to hypovolemic and/or other stress reactions, this procedure guarded against the impact of the time sequence of sampling. Series III was designed to identify changing CA titers as blood passes from the dorsal aorta to the caudal vein ("peripheral" CA extraction; cf. Davie, 1981), and
from there continues in either one of two ways: (a) to the heart via the PCVs, which receive CAs from the urophysis and corpuscles of Stannius (cf. Hirano and Mayer-Gfstan, 1978) as well as from the kidney (local chromaffin cells?); or (b) to the liver, via collaterals from the renal portal veins to the hepatic portal vein (Anguilla-type of Bertin, 1958). Blood was collected by puncture, using a 27-gauge needle fitted to a 1-ml syringe. Functional evaluations. Tissues selected for surgical removal, recovery periods for unanesthetized animals, and CAs injected are shown in Table 1. Hypophysectomy was performed via a buccal approach (Hirano et al., 1967). Partial removal of the brain was carried out following removal of the overlying skull, and subsequent survival of the animals under continued anesthesia was controlled by observation of the cardiac activity. Corpuscles of Stannius were removed through a ventral incision following displacement of the urinary bladder. The urophysectomy was performed by removal of the tall region in which the urophysis is located, followed by clamping of the caudal artery and vein. Bilateral extirpation of the anterior regions of the PCV/head kidney complex (bulk of chromaffin tissue, probably the adrenomedullary equivalent) involved a ventral incision over the liver, followed by double ligation of the PCVs and excision of the intervening tissue (Admedex). Cardiac cannulation followed each of the procedures described above. Control animals underwent cardiac cannulation only. The impact of organ removal was evaluated using the catecholaminotropic effect of CAs (Epple and Nibbio, 1985); in the present study, we induced the release of DA and NE by E injections, and of NE and E by DA injections. The CAs were injected via the indwelling
~PITUITARY
I
DORSAL AORTA
h ~ GILLS
JUGULAR VEIN
"'
>
UROPHYSIS
q HEPATIC PORTAL VEIN
HEAD KIDNEY
(It ~
POSTERIORCARDINAL VEINS
FIG. 1. Vascularization and sampling sites inAngaiUa rostrata. (1) Anterior region of right posterior cardinal vein; (2) posterior region of fight posterior cardinal vein; (3) caudal vein; (4) dorsal aorta; (5) ventral aorta; (6) sinus vcnosus; (7) anterior region of hepatic portal vein; and (8) posterior region of hepatic portal vein. V, ventricle; A, atrium; SV, sinus vcnosus (not readily distinguishable from ductus Cuvieri); CS, corpuscles of Stannius.
420
HATHAWAY AND EPPLE TABLE 1 SUMMARY OF PROCEDURES FOR FUNCTIONAL EVALUATIONS Surgery
N
Recovery period a
Injection
Hypophysectomy Control Control
8 5 4
7 days 5-7 days 5-7 days
Epinephrine Saline Epinephrine
2 i~g/kg
"Mid-brain X ''b Mid + forebrain X c Mid + forebrain X Control
4 5 4 4
Anesthesia Anesthesia Anesthesia 5-7 days
Epinephrine Epinephrine Dopamine Epinephrine
8 ~g/kg
Urophysectomy Urophysectomy Stanniectomy Stanniectomy Control
5 4 4 4 4
Anesthesia Anesthesia Anesthesia Anesthesia Anesthesia
Epinephrine Dopamine Epinephrine Dopamine Epinephrine
2 2 2 2 2
Head kidney X d Head kidney X Head kidney X Control
4 4 5 4
Anesthesia Anesthesia 1 day Anesthesia
Dopamine Epinephrine Epinephrine Epinephrine
3 ~,g/kg 2 I~g/kg 1 i~g/kg 2 ixg/kg
2 txg/kg 8 ixg/kg 8 ixg/kg 8 i~g/kg ixg/kg ~g/kg i~g/kg ~g/kg i~g/kg
Anesthetized animals were injected 10 min postsurgery. b Two transverse cuts at borders of optic lobes and cerebrum and cerebellum, respectively. c Two transverse cuts at olfactory tract and at border of optic lobe and cerebellum. '~ Bilateral removal of anterior region of head kidney and posterior cardinal vein. cardiac cannula following the designated recovery period in unanesthetized animals or 10 min postsurgery in anesthetized animals (Table 1). DA (dopamine hydrochloride, Elkins-Sinn) and E (epinephrine hydrochloride, Elkins-Sinn) were diluted in saline (0.85% NaC1) and were injected in a final volume of 1 ml/kg body wt. Blood samples (0.3 ml) were withdrawn prior to CA injection and at 3 and 10 min postinjection. Analysis. All blood samples were delivered into test tubes containing the anticoagulant EGTA/glutathione and centrifuged (20 min, 210g, 6~ Supernatants were stored at - 70~ Diluted tissue acid extracts and plasma samples were analyzed for unconjugated DA, NE, and E with a radioenzymatic technique (CAT-A-KIT, Amersham). This assay has a sensitivity of 2-5 pg for E and NE and 15-20 pg for DA. Cross-contamination of the three determinations in the presence of high concentrations of E and DA (as would follow their exogenous administration) ranged from 0 to 7%. Statistical analyses. One-way analysis of variance, followed by the Student-Newman-Keuls test (regional sampling), Dunnett's test, or paired t test (functional evaluations) were done.
are shown in Table 2. In general, tissues contained the three CAs in the proportions of E I> NE >> DA. However, in brain and pituitary, DA and NE concentrations exceeded those of E. Levels of CAs in regions of the posterior cardinal veins (including dorsal opisthonephric kidney regions) exceeded those in other tissues by 1-3 orders of magnitude. With the exception of the pituitary and CS, most of the tissues analyzed contained amounts of CAs potentially capable of altering the plasma level of at least one CA (based on total tissue per organ weight and a 3% blood volume in whole eels: see Thorson, 1961; Holmes and Donaldson, 1969). This assumes, however, the unlikely event of rapid and total movement of stored CAs into the circulation (Table 3).
Regional Levels of Plasma CAs RESULTS Tissue CA Concentrations
The tissue concentrations 0xg/g) of CAs
Tables 4 and 5 show local CA plasma titers at selected vascular sites. In almost all cases the three CAs were present in the ra-
421
CATECHOLAMINES IN AMERICAN EEL
TABLE 2 CATECHOLAMINE CONCENTRATIONS IN TISSUES OF Anguilla rostrata Micrograms per gram wet weight tissue N~
Dopamine
Norepinephrine
Epinephrine
Head kidney (w/PCV b) Anterior ~ Middle d Posterioff
7 4 9
3.85 • 1.22 2.20 • 0.79 0.28 • 0.10
42.38 • 15.17 22.75 • 7.42 6.88 • 2.57
84.83 - 27.44 49.50 --- 17.36 8.35 -+ 1.73
Head kidney (w/o PCV) Anterior Middle Posterior
4 4 4
0.23 • 0.10 0.09 • 0.03 0.02 • 0.003
5.27 • 1.55 2.31 • 0.43 0.12 --- 0.04
6.23 --- 2.30 2.50 --- 1.41 0.18 • 0.04
Opisthonephric kidney r Anterior dorsal Middle dorsal Posterior dorsal Ventral
5 5 5 5
0.04 0.04 0.03 0.01
1.41 1.47 1.10 0.34
1.97 1.82 1.24 0.32
Heart Sinus venosus Atrium Ventricle
4 4 4
0.02 --+ 0.01 0.02 • 0.008 0.03 • 0.01
0.05 --- 0.03 0.11 • 0.05 0.16 --- 0.06
0.33 --- 0.20 0.40 +- 0.11 0.26 -+ 0.08
Whole brain Pituitary 8
9 1
0.14 • 0.02 0.07
0.30 • 0.02
0.02
0.03 • 0.01
0.008
Stannius corpuscles g
2
0.03 • 0.02
0.18 •
0.11
0.24 •
0.11
Liver
4
0.01 • 0.002
0.01 •
0.002
0.01 •
0.003
Gills
4
0.01 • 0.002
0.01 •
0.007
0.06 --- 0.04
• 0.006 -+ 0.009 • 0.009 - 0.002
• --• ---
0.25 0.33 0.34 0.10
-+ • • --+
0.35 0.38 0.30 0.09
a N equals n u m b e r of animals from which tissues were removed. b Posterior cardinal vein contained within head kidney segment. c Two-centimeter region located immediately posterior to ductus Cuvieri. d Two-centimeter region located at level of gall bladder. e Two-centimeter region located immediately anterior to opisthonephros. f Dorsal regions included caudal/cardinal vessels. g N equals batches of pooled samples from five animals.
tit E > DA > NE. Two exceptions, in which DA and NE titers were essentially equal, were seen in caudal vein samples in Series IV (Table 5). As expected, the second round of sampling in each of the three series yielded higher CA values than the first. The two rounds were therefore viewed as representing different levels of stress and were treated separately in statistical analyses. The highest mean plasma CA levels in each round of Series I, II, and III were observed in the anterior region of the right
PCV. Due to very strong individual variations, the statistical analysis yielded few significant differences between sampling sites. DA, NE, and E levels in some anterior PCV samples from Series II and III (Table 4) differed significantly from levels measured at the neighboring upstream and downstream sites. In five animals, however, CA titers in the posterior region of the PCV were greater than those in the anterior PCV (Table 6). The results of Series III and IV show that DA, NE, and E were entering both the PCVs and the hepatic portal vein
422
H A T H A W A Y A N D EPPLE TABLE 3 POTENTIAL CONTRIBUTIONS OF DIFFERENT ORGANS TO PLASMA CATECHOLAMINES IN THE AMERICAN EEL (pg/ml)
PCV/HK anterior a PCV/HK posterior b Opisthonephros Brain Pituitary Stannius corpuscles Heart Gills Liver
Dopamine
Norepinephrine
Epinephrine
18,000 750 3,000 750 15 10 700 2,000 5,000
190,000 18,500 100,000 1,600 4 60 2,700 2,000 5,000
390,000 21,000 130,000 150 2 80 8,000 12,000 5,000
Note. Estimated from catecholamine content (p~g/g wet tissue, Table 2) in a hypothetical 500-g eel with blood volume equal to 3% of body weight. Anterior 5 cm of the right posterior cardinal vein and associated head kidney. b Posterior 2 cm of the right posterior cardinal vein and associated head kidney.
TABLE 4 LOCAL PLASMA CATECHOLAMINE TITERS (pg/ml • SEM) IN Anguilla rostrata BASED ON SEQUENTIAL SAMPLING AT SELECTED VASCULAR SITES Dopamine Series
Round 1
Norepinephrine
Round 2
Round 1
Round 2
Epinephrine Round 1
Round 2
I P-PCV A-PCV SV VA DA
161 • 650 • 193 • 204• 233 •
53
DA VA SV A-PCV P-PCV
271 • 51 3 5 2 • 74 3 1 4 • 54 867 ~ 172"* 453 • 162
55 60 77
336 • 1162 • 496• 657 • 717 •
112 531 223 378 406
75 • 24 277 • 153 5 9 • 21 64• 19 63 • 19
262 • 65 447 • 150 1 2 4 • 35 162 • 62 195 • 57
292 • 1119 • 391 • 415 • 506•
105 622 126 142 181
797 2360 1048 1457 1641
• • • • •
395 • 631• 639• 1364 • 969 •
88 142 145 312 277**
134 • 40 1 7 5 • 49 155 • 44 731 • 168" 354 • 105"*
199 • 48 3 1 0 • 58 3 5 2 • 89 975 • 132" 698 • 131
591 • 797• 691 • 2414 • 1018 •
131 257 176 693** 159"*
1023 • 1533 • 1551• 3928 • 2254 •
251 956 402 851 846
II
III A-PCV P-PCV CV DA PV
3154 2036 519 1746 1171
• • • • •
577 551 130 276 112
15776 7147 1102 3302 1240
• 1655 • 720* • 286* • 913 • 274
1358 946 205 517 257
• 335 • 343 • 46 • 46 • 64
4280 2624 350 668 173
• 477 • 456* • 67* • 128 • 34
6438 3521 1210 3517 2233
• 1988 • 742 • 350 • 408 • 261
31437 14505 2568 6714 1969
320 471 443 983** 163"*
• 3142 • 544* • 517" • 1612 • 155
Note. Sites were sampled in order listed in Series I (N = 5), II (N = 5), and III (N = 4). Round 2 sampling began upon completion of round 1. Location of sampling sites is shown in Fig. 1: posterior region of posterior cardinal vein (P-PCV), anterior region of posterior cardinal vein (A-PCV), sinus venosus (SV), ventral aorta (VA), dorsal aorta (DA), caudal vein (CV), and hepatic portal vein (PV). Statistical evaluation of each round with A N O V A and Student-Newman-Keuls test: * and ** indicate differences (P < 0.01 and P < 0.05, respectively) from previously sampled site. All sampling was done in urethane-anesthetized animals.
423
C A T E C H O L A M I N E S IN A M E R I C A N E E L TABLE 5 LOCAL PLASMA CATECHOLAMINE TITERS (pg/ml • SEM) IN Anguilla rostrata BASED ON SEQUENTIAL SAMPLING AT SELECTED VASCULAR SITES Series IV
Dopamine
Norepinephrine
Epinephrine
Hepatic portal vein (anterior) Caudal vein Dorsal aorta Caudal vein Hepatic portal vein (anterior)
296 102 204 142 288
- 73 -+ 32 +- 43 -+ 61 • 33
126 115 186 102 109
• • • • -
34 52 54 27 51
465 305 590 285 449
Hepatic portal vein (posterior) Caudal vein Dorsal aorta Caudal vein Hepatic portal vein (posterior)
277 159 377 180 410
-+ 39 - 38 -+ 99 -+ 37 -+ 19
142 165 317 160 154
• -+ • -
65 63 100 60 52
648 634 1386 665 842
-• -+ -+ •
94 97 91 94 122
_ 193 -- 266 • 537 -- 234 -+ 299
Note. E a c h animal was s a m p l e d at the 10 sites in the order listed. N = 4 for all sites e x c e p t posterior hepatic portal vein ( N = 3). Statistical evaluation with A N O V A and S t u d e n t - N e w m a n - K e u l s test revealed no significant differences (P < 0.05) b e t w e e n sites; however, trends are consistent.
within the opisthonephric kidney (Tables 4 and 5). Functional Evaluations
Hypophysectomy failed to alter the CA tropic effect of E in unanesthetized eels (Table 7). The possibility existed for the regeneration of hypothalamo-hypophyseal tracts (Moll, 1957) and for the continued release from the pituitary stalk equivalent (Mens et al., 1982), and/or the involvement of circumventricular organ(s) such as the organum vasculosum laminae terminalis (Leonhardt, 1980; Tsuneki, 1986). We therefore removed the diencephalon plus pituitary and finally all brain tissue anterior to the cerebellum prior to CA challenge. However, this also had no impact on the action of the CAs in releasing other CAs (Table 8). Neither urophysectomy nor stanniectomy (Table 9), nor surprisingly, bilateral removal of the anterior PCV/head kidney complexes (Table 10) prevented the CA tropic effects of injected CAs. DISCUSSION The CA concentrations in various tissues ofA. rostrata (Table 2) are generally of the
same order of magnitude as the average levels reported previously for anguillid and other fishes. Le Bras (1984) finds CA levels in the heart and brain of A. anguilla which are lower than those reported here. Technical differences (method of sacrifice ?) may account for the discrepancies. Nevertheless, despite the differences in absolute values, the relative proportions of DA, NE, and E observed in both studies are similar. The significance of the CAs identified in the pituitary cannot be assessed until we know more about the cellular location of these substances; however, the relatively high concentration of DA is compatible with its well-known presence in neurosecretory fibers (Peter, 1986). In contrast to our data, Unsicker et al. (1977) found significantly more NE than E in corpuscle of Stannius extracts from Salmo irideus, though the total levels for NE and E combined are similar in both studies. Whether the lower NE values in the present study indicate an absence of presumed NE-containing chromaffin cells in the corpuscles of Stannius (cf. Wendelaar Bonga and Pang, 1986) of anguillids or a species difference in type of adrenergic neurotransmitter is not known. The CA concentrations in the PCVs and head kidneys presented here are, to the
424
HATHAWAY AND EPPLE
stores are more closely associated with the PCV than the surrounding head kidney (Abrahamsson and Nilsson, 1976); and that (3) the anterior portion of the PCV/head kidney complex (close to the ductus Cuvieri which Mott (1950) found to not be Animal Post-PCV Ant-PCV readily distinguishable from the sinus veno022988-1 (round 1) sus in A . anguilla) contains the densest acNE 1967 1837 cumulation of chromaffin tissue (cf. Nils122387-1 (round 1) son, 1983; Mastrolia et al., 1984). ApparDA 103 93 ently, this is the first report on tissue NE 138 100 concentrations of DA in this region (Table E 169 131 2). Interestingly, they are comparable to (round 2) those in mammalian adrenal glands (CarbalNE 416 325 leira et al., 1987; Pratt et al., 1987). In all 122487-1 (round 2) likelihood, the chromaffin cells close to the NE 323 152 ductus Cuvieri represent the equivalent of 013088-1 (round 2) the adrenal medulla for the following reaDA 1128 988 sons: (a) they are in immediate proximity of E 2175 1972 the interrenal tissue (Giacomini, 1908; 013188-1 (round 1) Nandi, 1962; personal observations); (b) DA 1069 967 t h e y are very strongly i n n e r v a t e d by NE 757 518 E 1312 1129 "preganglionic" fibers (Brinn et al. in prep(round 2) aration); and (c) their location coincides DA 1880 1475 with the highest tissue concentration of NE 985 915 CAs (Table 2). However, in contrast to the E 2882 1958 adrenal medulla cells of "higher vertebrates," their majority, perhaps even all of best of our knowledge, the first ones re- them, are supplied by venous blood (deported in an anguillid species. The diversity rived mainly from the caudal/renal portal of kidney/cardinal vein structures (Ogawa, complex and small veins of the head kidney 1961) and chromaffin cell arrangements region: Mott, 1950; Bertin, 1958). It re(Giacomini, 1908; Nandi, 1962)among the mains to be seen if the chromaffin cells in teleosts makes a comparison with the the posterior head kidney and opisthowidely varying data on tissue CAs in other nephric kidney regions are also innervated, fishes (J6nsson et al., 1983; cf. Nilsson, or perhaps represent the equivalent of the 1983) rather meaningless. Nevertheless, it paraganglia (which are usually not inneris noteworthy that (1) E is the predominant vated: B6ck, 1982). This question is curCA in the PCV/head kidney region of sev- rently under investigation. At any rate, the eral other, though not all, bony fishes (Na- regional CA levels in different regions of kano and Tomlinson, 1967; Stabrovskii, head and opisthonephric kidney (Table 2) 1969; Abrahamsson and Nilsson, 1976; show that significant CA storage occurs Abrahamsson et al., 1979; Balashov et al., along the entire length of the PCVs and also 1981; Nilsson, 1981; JOnsson et al., 1983). in the region of the caudal/renal vein sysIn the rainbow trout, J6nsson et al. (1983) tem (cf. Bertin, 1958). Their tissue concenfound slightly higher NE than E levels in trations match perfectly the regional distrithis region, while Nakano and Tomlinson bution of the chromaffin cells of the eel (1967) report the opposite; that (2) CA (Giacomini, 1908). TABLE 6 INDIVIDUAL VALUES OF PLASMACATECHOLAMINES (pg/ml) FROM THE POSTERIOR REGION OF THE POSTERIOR CARDINAL VEIN (POST-PCV), AS COMPARED WITH VALUES FROM THE ADRENAL MEDULLA EQUIVALENT(ANT-PCV)
425
C A T E C H O L A M I N E S IN A M E R I C A N EEL
TABLE 7 IMPACT OF HYPOPHYSECTOMY ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE CARDIAC-CANNULATED UNANESTHETIZED EEL
Times before and after injections
State of the animal and treatment
N
Plasma CA measured
I
Intact c o n s o l s (saline)
5
DA NE E
135 • 18 • 29 •
25 12 17
II Intact controls (2~g/kgE)
4
DA NE E
98 • 53 • 31 •
51 28 14
6643 • 1925 a 1258 • 478 a 7 7 2 0 • 1984"
III H y p e x eels (2~g/kgE)
5
DA NE E
445 • 139 270 • 135 306 • 72
7429 • 2105 ~ 2341 • 474 a 17697 •
Before
3 min 111 • 8 • 15 •
30 min 12 5 8
149 • 8 • 18 •
40 5 7
308 • 38 • 74•
65 18 24
506 • 113 332 • 99 292 • 69
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. All animals injected 5-7 days after surgery.
It is noteworthy that the presumed adrenal medulla equivalent of the eel (and apparently that of other actinopterygians as well: Nilsson, 1983) is located just up-
stream from the heart; i.e., virtually the same place in which the chromaffin cells of the Southern lamprey, Geotria australis (Epple et al., 1985), the "axillary" bodies
TABLE 8 IMPACT OF PARTIAL BRAIN REMOVAL ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE EEL
State of the animal and treatment I
Unanesthetized intact control (8 txg/kg E) b
II " M i d b r a l n " removed (8 ~g/kg E) C III Fore- and midbrain removed d a. 8 I~g/kg DA
b. 8 ~g/kg E
N
Plasma CA measured
Times before and after injections Before 217 • 36 • 99 •
3 min 45 19 39
25347 • 1356 • 35771 •
10 min
4
DA NE E
3077 a 852 a 5420 a
----
4
DA NE E
7625 • 6335 3537 • 2976 9701 • 8088
99730 • 32946 a 14800 • 6840 ~ 179994 • 52134 a
34901 • 19965 9126 • 6619 60470 • 30901
4
DA NE E
1498 • 643 • 1670 •
664 387 499
247602 • 92453 a 3016 • 1160 a 26748 • 10896 a
33560 • 22363 1877 • 718 7639 • 2624
5
DA NE E
1262 • 559 1279 • 1016 2182 • 984
62993 • 7374 ~ 10297 • 3320 ~ 143528 • 23627 a
20624 • 8742 4656 • 3247 43760 • 20582
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. b Cardiac-cannulated animals, injected 5-7 days after surgery. c Anesthetized animals injected 10 min after cardiac cannulation and removal of midbrain, diencephalon, and pituitary. d Anesthetized animals injected 10 min after cardiac cannulation and removal of midbrain and forebrain (i.e., all tissue anterior to the cerebellum).
426
H A T H A W A Y A N D EPPLE TABLE 9 IMPACT OF STANNIECTOMY AND UROPHYSECTOMY ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE EEL a
State of the animal and treatment I
Anesthetized controls (2 ixg/kg E)
II Stanniectomized eels a. 2 ~g/kg E
b. 2 ~g/kg DA
III Urophysectomized eels a. 2 Ixg/kg E
b. 2 t~g/kg DA
N
Plasma CA measured
Times before and after injections Before
10 min
63 62 87
7974 • 1406 b 620 • 150b 10740 • 2479 b
2829 • 983 507 • 198 3584 • 1102
5094 • 1404 b 1065 • 149b 7400 • 708 b
979 • 634 • 2990 •
202 200 560
3015 • 106 802 •
1092 • 146 • 403 •
448 55 91
4
DA NE E
5
DA NE E
330 • 70 508 • 199 1172 • 536
4
DA NE E
437 • 154 78 • 32 212 • 76
DA NE E
275 • 59 341 • 101 739 • 128
7906 • 1432 b 2 2 4 4 • 653 b 24716 • 3226 b
DA NE E
453 • 89 378 • 36 527 • 150
7477 • 1781 b 468 • 21 b 1745 • 397 ~
5
3
211 • 203 • 311 •
3 min
658 b 45 150 b
1975 • 610 717 • 182 5958 • 1348 1089 • 576 • 873 •
294 34 b 335
Note. All values in pg/ml plasma -+ SEM. a Anesthetized animals injected 10 min after cardiac cannulation (I) and cardiac cannulation plus endocrinectomy (II + III). b Indicates significant difference (P < 0.05) from " b e f o r e " value.
of the elasmobranchs (Abrahamsson, 1979, Nilsson, 1983; Watzka, 1943), and the chromaffin cells of the African lungfish (Abrahamsson et al., 1979) are found. Probably this "fish pattern" indicates a functional relationship (Nilsson, 1983). Our data leave little doubt that the presumed adrenal medulla equivalent is the main source of all three plasma CAs (Tables 2-6). As far as we know, this is also the first demonstration of the major source of systemic plasma DA in a "lower" vertebrate. In mammals, including the human, systemic DA also seems to come mainly from the adrenal medulla (Kvetnanski et al., 1979; Kuchel et al., 1979; Planz and Planz, 1979; Unger et al., 1979). Wahlqvist and Nilsson (1980) found that sectioning of the nerve supply to the head kidney of the Atlantic cod greatly reduced but did not abolish stress-induced increases
in plasma E and NE. They attributed this result to either incomplete denervation or overflow into the plasma from adrenergic nerve endings. Our finding of a lack of an impact of bilateral removal of the presumed adrenal medulla equivalent on the CA tropic effects of DA and E (Table 10) suggests that the chromaffin cells in the opisthonephric kidney region are capable of releasing large quantities of CAs into the systemic circulation, even when the blood flow in the anterior PCVs is interrupted. The peculiar collateral connections between the renal and hepatic portal vein systems of the eel (Mott, 1950; Bertin, 1958) are probably involved. In addition, nonchromaffin cells in the opisthonephric kidney could also be involved; in mammals, DA derived from tubular decarboxylation of 1-DOPA and from intrarenal sympathetic fibers has been implicated in the control of kidney blood flow
C A T E C H O L A M I N E S I N A M E R I C A N EEL
427
T A B L E 10 IMPACT OF " A D R E N O M E D U L L E C T O M Y " ON THE CATECHOLAMINOTROPIC E F F E C T S OF CATECHOLAMINES IN THE E E L
State of the animal and treatment
N
I
Unanesthetized controls (1 ixg/kg E) b
4
II Unanesthetized admedex eels (1 ~g/kg E) C III Anesthetized controls (2 ~g/kg E) a
Plasma CA measured
Times before and after injections Before
3 min 1090 + 56 • 1041 -t-
10 min
DA NE E
37 --- 24 1 -+ 0 3 -+ 3
441 a 27 a
--
644 a
__
5
DA NE E
261 --- 145 49 -+ 13 126 • 35
5431 --- 1216 a 1057 • 272 a 10323 • 2436 a
911 • 168 196 --- 42 1533 • 228
4
DA NE E
211 • 63 203 -+ 62 311 • 87
7974 • 1406 a 620 ----- 150a 10740 -+ 2479 ~
2829 • 983 507 -- 198 3584 • 1102
IV Anesthetized admedex eels d a. 2 Ixg/kg E
4
DA NE E
916 • 160 612 • 155 1665 -+ 296
9671 • 1628 a 1362 • 254 a 16529 • 3788"
3048 -+ 324 866 • 180 6297 - t825
b. 3 ixg/kg DA
4
DA NE E
516 • 203 283 • 56 990 • 273
12775 • 2222 a 367 • 63 2951 ----- 655 a
2114 • 317 • 1387 •
884 74 482
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. b Cardiac-cannulated animals injected 5-7 days after surgery. c Cardiac-cannulated animals injected after overnight recovery from admedex. a Anesthetized animals injected 10 min after cardiac cannulation (III) and cardiac cannulated and admedex (IV).
(Bell, 1987). However, in teleosts the innervation of the kidneys is poorly understood (Braun and Dantzler, 1987). Apart from their possible involvement in renal functions, the CA stores in the opisthonephric kidney region raise another functional question: Is the release of CAs into the portal vein related to the physiological hyperglycemic response to E, seen in the eel (Epple and Nibbio, 1985), but not in the hagfish (Plisetskaya et al., 1984), lamprey (Dashow and Epple, 1983), rat (Epple et al., 1988), and human (Epple et al., 1987, in press)? The high tissue concentrations of CAs in the heart (Tables 2 and 3) corroborate findings of Pennec and Le Bras (1984) who report storage and release of CAs from nerve endings in the isolated heart of the eel. H o w e v e r , a c o m p a r i s o n b e t w e e n the
plasma titers in sinus venosus and ventral aorta in vivo does not reveal a clear picture (Table 4). Since the heart of the eel is a target organ of CAs (Chan and Chow, 1976; Peyraud-Waitzenegger et al., 1980), it is possible that the discrepancy is due to a balanced net effect of uptake of humoral CAs and a release of neuronal CAs in vivo (cf. Laurent et aL, 1983). This question requires further study. The high CA concentrations in the brain (Tables 2 and 3) are in good agreement with the higher titers of DA and NE than E, seen in the head blood of the eel (Epple et al., 1982). Nevertheless, the data in Tables 2, 3, and 4 indicate that the overall contribution of CAs from this source to the systemic CA titers is of minor significance (note the low CA values in the sinus venosus, where the head blood mixes with the body blood).
428
HATHAWAY AND EPPLE
Similar to the situation in the heart, the This would resemble the situation in mamcomparison of the CA titers of the affluent mals where basal (von Euler et al., 1954), and effluent blood of the gills (ventral vs but not stress-induced (Andersson et al., dorsal aorta) does not show a clear gradi- 1982), NE titers depend upon neuronal ent. Again, a balanced net effect between sources. Further studies must prove or disCA uptake and release could be postulated: prove this possibility for the eel. In summary: The bulk of all three CAs in Nekvasil and Olson (1986a,b) show considerable extraction and metabolism of CAs in the systemic blood of the eel derives from the gills of the rainbow trout; on the other the presumed adrenal medulla equivalent, hand, the gills of trout have adrenergic in- which is located in the anterior PCV. Hownervation (Donald, 1984) which may re- ever, after removal of this region, strong lease CAs. However, other factors could CA release can yet be induced by CA inalso be involved; for instance, due to a spe- jections. Chromaffin cells in the veins of cific extraction mechanism, the venous the opisthonephric kidney region are prob"metabolic" blood of the gills could have a ably the major CA source in the latter case. lower CA content than the arterial "respiACKNOWLEDGMENTS ratory" blood measured here (Tables 4 and 5; cf. Wood, 1975). The authors are indebted to Ms. Barbara Nibbio for The tissue concentrations of all three her excellent technical assistance throughout these inCAs in the liver (Table 2) are essentially vestigations. Supported by NSF Grant DCB-8510585. higher than the plasma concentrations of REFERENCES unstressed eels (at least 20 times for DA and 100 times for NE and E: see, Epple et Abrahamsson, T. (1979). Phenylethanolamineal., 1982; Epple and Nibbio, 1985). We N-methyl transferase (PNMT) activity and catecholamine storage and release from chromaffin have no explanation for this finding (though tissue of the spiny dogfish, Squalus acanthias. one may suspect the presence of sympaComp. Biochem. Physiol. C 64, 169--172. thetic fibers); nor do we know its signifi- Abrahamsson, T., Hohngren, S., Nilsson, S., and Petcance for the plasma titer of the CAs. tersson, K. (1979). On the chromaffin system of In addition to the potential sources of the African lungfish, Protopterus aethiopicus. Acta Physiol. Scand. 107, 135-139. plasma CAs discussed in the preceding, contributions from some chromaffin cells in Abrahamsson, T., and Nilsson, S. (1976). Phenylethanolamine-N-methyl transferase (PNMT) activity the anterior cardinal vein (Giacomini, 1908) and catecholamine content in chromaffin tissue and a mammalian-like "synaptic leakage" and sympathetic neurons in the cod, Gadus come to mind. These sources could become morhua. Acta Physiol. Scand. 96, 94-99. important in CA-stimulated CA release af- Andersson, P.-O., Farnebo, L.-O., Fredholm, B. B., Hamberger, B., Holst, J., and J~irhult, J. (1982). ter "adrenomedullectomy" in the eel; howMetabolic and hormonal adjustments during hemever, we have no evidence for either one of orrhage in cats after interference with the symthese mechanisms. patho-adrenal system. Acta Physiol. Scand. 114, Finally, the discrepancy between the CA 111-119. tissue concentration in the presumed adre- Balashov, N. V., F~inge, R., Govyrin, V. A., Leont'eva, G. R., Nilsson, S., and Prozonal medulla equivalent (E > NE >>DA), the rovskaya, M. P. (1981). On the adrenergic system plasma titers of these compounds during of ganoid fish: The beluga, Huso huso (Chonanesthesia (E > DA > NE), and without drostei). Acta Physiol. Scand. 111, 435--440. anesthesia (DA > NE = E) remains to be Bell, C. (1987). Endogenous renal dopamine and control of blood pressure. Clin. Exp. Theory Prac. resolved (compare Tables 2-6 with the data A9, 955-975. in Epple et al., 1982 and Epple and Nibbio, Bertin, L. (1958). Appareil circulatoire. Trait~ Zool. 1985). The simplest explanation appears to 13, 1399-1458. be a neuronally controlled selective release Bfck, P. (1982). "The Paraganglia." Springer-Vedag, of different CAs from different cell types. Berlin.
CATECHOLAMINES IN AMERICAN EEL Braun, E. J., and Dantzler, W. H. (1987). Mechanisms of hormone actions on renal function. In "Vertebrate Endocrinology: Fundamentals and Biomedical Implications," Vol. 2, "Regulation of Water and Electrolytes" (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 189-210. Academic Press, New York. Carballeira, A., Brown, J. W., Fishman, L. M., Trujillo, D., and Odell, D. K. (1987). The adrenal gland of stranded whales (Kogia breviceps and Mesoplodon europaeus): Morphology, hormonal contents, and biosynthesis of corticosteroids. Gen. Comp. Endocrinol. 68, 293-303. Chart, D. K. O., and Chow, P. H. (1976). The effects of acetylcholine, biogenic amines and other vasoactive agents on the cardiovascular functions of the eel, Anguilla japonica. J. Exp. Zool. 196, 1326. Dashow, L., and Epple, A. (1983). Effects of exogenous catecholamines on plasma catecholamines and glucose in the sea lamprey, Petromyzon marinus. J. Comp. Physiol. 152, 35-41. Davie, P. S. (1981). Vascular resistance responses of an eel tail preparation: Alpha constriction and beta dilation. J. Exp. Biol. 90, 65-84. Donald, J. (1984). Adrenergic innervation of the gills of brown and rainbow trout, Salmo trutta and S. gairdneri. J. Morphol. 182, 307-316. Epple, A. (1987). Pancreatic islet hormones. In "Vertebrate Endocrinology: Fundamentals and Biomedical Implications," Vol. 2, "Regulation of Water and Electrolytes" (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 103-119. Academic Press, New York. Epple, A., Bertoni, J., and Hathaway, C. B. (1987). Is epinephrine a hyperglycemic hormone? Amer. Zool. 27, 97A. Epple, A., Bertoni, J., and Hathaway, C. B. Physiological doses of epinephrine in the human: Chronotropic, but not hyperglycemic or catecholaminotropic. J. Exp. Zool., in press. Epple, A., and Brinn, J. E. (1987). "The Comparative Physiology of the Pancreatic Islets." SpringerVerlag, New York. Epple, A., Heim, K., and Vogel, W. (1988). Single doses of catecholamines in the rat: Catecholaminotropic, but not hyperglycemic. Comp. Biochem. Physiol. C 89, 83-86. Epple, A., Hilliard, R. W., and Potter, I. C. (1985). The cardiovascular chromaffin cell system of the southern hemisphere lamprey, Geotria australis Gray. J. Morphol. 183, 225-231. Epple, A., and Kahn, H. A. (1985). Exogenous catecholamines do not affect osmoregulatory parameters in the eel. J. Exp. Zool. 234, 485-488. Epple, A., and Nibbio, B. (1985). Catecholaminotropic effects of catecholamines in a teleost fish, Anguilla rostrata. J. Comp. Physiol. B 155, 285-290.
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Epple, A., Vogel, W., and Nibbio, B. (1982). Catecholamines in head and body blood of eels and rats. Comp. Biochem. Physiol. C 71, 115-118. yon Euler, U. S., Frantisson, C., and Helstr6m, J. (1954). Adrenaline and noradrenaline output in urine after unilateral and bilateral adrenalectomy in man. Acta Physiol. Scand. 31, 1-5. Giacomini, E. (1908). Il sistema interrenale e il sistema cromaffine (sistema feocromo) nelle anguille adulte, nelle cieche e nei leptocefali. Mem. Accad. Sci. Instit. Bologna Cl. Sci. Fis., Ser. 6 5, 407-441. Hirano, T., Karniya, M., Saishu, S., and Utida, S. (1967). Effects of hypophysectomy and urophysectomy on water and sodium transport in isolated intestine and gills of Japanese eel (Anguilla japonica). Endocrinol. Japan. 14, 182-186. Hirano, T., and Mayer-G6stan, N. (1978). Endocrine control of osmoregulation in fish. In "Comparative Endocrinology" (P. J. Gaillard and H. H. Boer, Eds.), pp. 209-212. E l s e v i e r / N o r t h Holland, Amsterdam. Holmes, W. N., and Donaldson, E. M. (1969). The body compartments and the distribution of electrolytes. In "Fish Physiology" (W. S. Hoar and D. J. Randall, Eds.), Vol. 1, pp. 1-89. Academic Press, New York. J6nsson, A.-C., Wahlqvist, I., and Hansson, T. (1983). Effects of hypophysectomy and cortisol on the catecholamine biosynthesis and catecholamine content in chromaffin tissue from rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 51, 278-285. Kuchel, O., Buu, N. T., and Unger, T. (1979). Free and conjugated dopamine; physiological and clinical implications. In "Peripheral Dopaminergic Receptors" (J.-L. Imbs and J. Schwartz, Eds.), pp. 15-27. Pergamon, New York. Kvetnansky, R., Weise, V. K., Thoa, N. B., and Kopin, I. J. (1979). Effects of chronic guanethidine treatment and adrenal medullectomy on plasma levels of catecholamines and corticosterone in forcibly immobilized rats. J. Pharmacol. Exp. Ther. 209, 287-291. Laurent, P., Holmgren, S., and Nilsson, S. (1983). Nervous and humoral control of the fish heart: Structure and function. Comp. Biochem. Physiol. A 76, 525-542. Le Bras, Y. M. (1984). Circadian variations of catecholamine levels in brain, heart, and plasma in the eel, Anguilla anguilla L., at three different times of year. Gen. Comp. Endocrinol. 55, 472-479. Leonhardt, H. (1980). Ependym und circumventricul~ire Organe. In: "Handbuch der mikroskopischen Anatomie des M e n s c h e n " (A. Oksche and L. Vollrath, Eds.), Vol. IV/10, pp. 177-666. Springer-Verlag, Berlin. Lewis, T. L., and Epple, A. (1984). Effects of fasting,
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pancreatectomy, and hypophysectomy in the yellow eel, Anguilla rostrata. Gen. Comp. Endocrinol. 55, 182-184. Mastrolia, L., Gallo, V. P., and La Marca, A. (1984). The adrenal chromaffin cells of Salmo gairdneri Richardson (Teleostei, Salmonidae). J. Anat. 138, 503-511. Mens, W. B. J., van Dam, A. F., van Egmond, M. A. H., Bakker, E. A. D., Legros, J. J., and van Wimersma Greidanus, T. B. (1982). Neurohypophyseal hormones in cerebrospinal fluid. Front. Horm. Res. 9, 119-130. Moll, J. (1957). R e g e n e r a t i o n of the supraoptico-hypophyseal and paraventricular hypophyseal tracts in the hypophysectomized rat. Z. Zellforsch. 46, 686-709. Mott, J. C. (1950). The gross anatomy of the blood vascular system of the eel Anguilla anguilla. Proc. Zool. Soc. London 120, 503-518. Nakano, T., and Tomlinson, N. (1967). Catecholamine and carbohydrate concentrations in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J. Fish. Res. Board Canad. 24, 1701-1715. Nandi, J. (1962). The structure of the interrenal gland in teleost fishes. Univ. Calif. Publ. Zool. 65, 129212. Nekvasil, N. P., and Olson, K. R. (1986a). Extraction and metabolism of circulating catecholamines by the trout gill. Amer. J. Physiol. 250, R526-R531. Nekvasil, N. P., and Olson, K . R . (1986b). Plasma clearance, metabolism, and tissue accumulation of 3H-labeled catecholamines in trout. Amer. J. Physiol. 250, R519-R525. Nilsson, S. (1981). On the adrenergic system of ganoid fish: The Florida spotted gar, Lepisosteus platyrhincus. Acta Physiol. Scand. 111, 447-454. Nilsson, S. (1983). "Autonomic Nerve Function in the Vertebrates." Springer-Verlag, Berlin. Ogawa, M. (1961). Comparative study of the external shape of the teleostean kidney with relation to phylogeny. Sci. Rep. Tokyo Kyoiku Daigaku B10, 61-68. Pennec, J. P., and Le Bras, Y. M. (1984). Storage and release of catecholamines by nervous endings in the isolated heart of the eel (Anguilla anguiUa L). Comp. Biochem. Physiol. C 77, 167-171. Peter, R. E. (1986). Vertebrate neurohormonal systems. In "Vertebrate Endocrinology: Fundamentals and Biomedical Implications, Vol. 1, "Morphological Considerations" (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 57-104. Academic Press, New York. Peyrand-Waitzenegger, M., Barthelemy, L., and Peyrand, C. (1980). Cardiovascular and ventilatory effects of catecholamines in unrestrained eels (Anguilla anguilla L.). A study of seasonal changes in reactivity. J. Comp. Physiol. B 138, 367-375.
Planz, G., and Planz, R. (1979). D o p a m i n e 13-hydroxylase, adrenaline, noradrenaline and dopamine in the venous blood of adrenal gland of man: A comparison with levels in the periphery of the circulation. Experientia 35, 207-208. Plisetskaya, E., Rich, A. A., Dickhoff, W. W., and Gorbman, A. (1984). A study of triiodothyroninecatecholamine interactions: Their effect on plasma fatty acids in Pacific hagfish, Eptatretus stouti. Comp. Biochem. Physiol. A 78, 767-772. Pratt, J. H., Turner, D. A., Bowsher, R. R., and Henry, D. P. (1987). Dopamine in rat adrenal glomerulosa. Life Sci. 40, 811-816. Stabrovski, E. M. (1969). Adrenaline and noradrenaline in the organs of elasmobranch (cartilaginous) and teleost (bony) fish of the Black Sea. J. Evol. Biochem. Physiol. 5, 38-41. Thorson, T. B. (1961). The partitioning of body water in osteichthyes: Phylogenetic and ecological implications in aquatic vertebrates. Biol. Bull. 120, 238-254. Tsuneki, K. (1986). A survey of occurrence of about seventeen circumventricular organs in brains of various vertebrates with special reference to lower groups. J. Hirnforsch. 27, 441-470. Unger, T., Buu, N. T., and Kuchel, O. (1979). Renal and adrenal dopamine balance: Implications for the role of conjugated dopamine. In "Peripheral Dopaminergic Receptors" (J,-L. Imbs and J. Schwartz, Eds.), pp. 357-367. Pergamon, New York. Unsicker, K., Polonius, T., Lindmar, R., Loffelholz, K., and Wolf, U. (1977). Catecholamines and 5hydroxytryptamine in corpuscles of Stannius of the salmonid, Salmo irideus L. A study correlating electron microscopical, histochemical and chemical findings. Gen. Comp. Endocrinol. 31, 121-132. Wahlqvist, I., and Nilsson, S. (1980). Adrenergic control of the cardio-vascular system of the Atlantic cod, Gadus morhua, during "stress." J. Comp. Physiol. 137, 145-150. Watzka, M. (1943). Die Paraganglien. In "Handbuch der mikroskopischen Anatomie des Menschen" (W. v Mtllendorff, Ed.), Vol. VI/4, pp. 262-308. Springer-Verlag, Bedim Wendelaar Bonga, S., and Pang, P. K. T. (1986). Stannius corpuscles. In "Vertebrate Endocrinology: Fundamentals and Biomedical Implications," Vol. 1, " M o r p h o l o g i c a l C o n s i d e r a t i o n s " (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 439--464. Academic Press, New York. Wood, C. M. (1975). A pharmacological analysis of the adrenergic and cholinergic mechanisms regulating branchial vascular resistance in the rainbow trout (Salmo gairdnert). Canad. J. Zool. 53, 15691577.
74, 418-430 (1989)
The Sources of Plasma Catecholamines in the American Eel,
Anguilla rostrata CHARLES B.
H A T H A W A Y AND A U G U S T E P P L E
Daniel Baugh Institute of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Accepted October 24, 1988 The origin of the catecholamines (CAs) in the systemic blood of the American eel, Anguilla rostrata, was studied by three approaches: (1) determination of the CA content of tissues suspected to release large quantities of dopamine, norepinephrine, and/or epinephrine into the circulation; (2) measurement of local CA titers in selected regions of the cardiovascular system; and (3) removal of tissues with high CA concentrations, followed by determination of its impact on stimulated CA release. Large quantities of all three CAs were found in the walls of the posterior cardinal veins, from their caudal origin within the opisthonephric kidney to their termination in the ductus Cuvieri. Near the ductus Cuvieri, the CA concentration was 1-3 orders of magnitude above that in other tissues. In this region, which contains the presumed adrenal medulla equivalent, occur the highest plasma levels of the CAs. Strong CA release also in the opisthonephric kidney region raises the question if these CAs affect locally the kidney functions, and/or via the hepatic portal vein (which originates in this region), the liver. Other organs (especially brain and heart) contain CA concentrations high enough to potentially affect the CA level in the systemic blood, if instantly released. However, neither partial removal of the brain nor hypophysectomy, "adrenomedullectomy," Stanniectomy, or urophysectomy had an appreciable impact on stimulated CA release. Together with previous data, these findings show that in the e d (a) the region of the presumed adrenal medulla equivalent is the most important source of all three CAs in systemic plasma; (b) that strong CA-stimulated CA release also occurs outside this region; and (c) that the pituitary, forebrain, and midbrain are not necessary for the CA-stimulated CA release. 9 1989AcademicPress, Inc.
Probably all vertebrates have three circulating catecholamines (CAs), i.e., dopamine (DA), norepinephrine (NE), and epinephrine (E). Though these substances are known or suspected to control cardiovascular, respiratory, metabolic, osmoregulatory, and endocrine functions, precise information on their sites of production and release is insufficient or lacking for many taxa. The present investigations were designed to identify the major sources of systemically circulating CAs in the American eel, a species ideally suited for many in vivo studies on endocrine/metabolic regulations (see, e.g., Lewis and Epple, 1984; Epple and Kahn, 1985; Epple and Nibbio, 1985; Epple, 1987; Epple and Brinn, 1987). Specifically, we wanted to confirm that in the eel the bulk of circulating DA and NE
comes from the brain, while plasma E comes mainly from extraencephalic sources. This scenario was suggested by the detection of higher levels of DA and NE in head than in body blood, and of more or less equal titers of E in both types of blood (Epple et al., 1982). However, the present studies show that the situation is more complex, and that the bulk of all three CAs in the systemic circulation is released by chromaffin cells (mainly the presumed adrenal medulla equivalent) which occur in the wail of the posterior cardinal veins (PCVs).
MATERIALS AND METHODS Animals. Yellow American eels (Anguilla rostrata) from Atlantic coast rivers and estuaries of the United States were obtained from a commercial distributor. 418
0016-6480/89 $1.50 Copyright 9 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
419
CATECHOLAMINES IN AMERICAN EEL The average body weight was approximately 400 g. Techniques of maintenance and cardiac cannulation have been described previously (Epple et al., 1982). The animals were anesthetized in a 3% solution of ethyl carbamate (urethane; U.S. Biochemical Corporation) for 10 min prior to experimental manipulation; or, depending on the experimental design, kept after surgery and cardiac cannulation for specified times in "eel t u b e s " which were suspended in clear, wellaerated fresh water. Preparation of tissue homogenates. Tissues were removed, weighed, and placed in test tubes containing 0.4 N perchloric acid with 5 mM reduced glutathione (10 ixl acid/mg tissue wet wt). Following homogenization, samples were centrifuged (30 min, 210g, at 6~ and aliquots from the supernatant were stored at - 7 0 ~ These extracts were diluted with double distilled water prior to assay for CAs. Regional CA titers. Each sampling series consisted of two rounds of sequential blood withdrawal from five vascular sites (Fig. 1), i.e., a total of 10 samples/ individual animal. These sites were selected in order to identify CA release in the head kidney (major concentration of chromaffin cells), heart (target of CAs and reported site of their release in the eel: Le Bras, 1984; Pennec and Le Bras, 1984), and the gills (also suspected target; however, see Epple and Kahn, 1985). Series I and II represent reversed sampling sequences from the same five sites. Since blood withdrawal may stimulate CA release due to hypovolemic and/or other stress reactions, this procedure guarded against the impact of the time sequence of sampling. Series III was designed to identify changing CA titers as blood passes from the dorsal aorta to the caudal vein ("peripheral" CA extraction; cf. Davie, 1981), and
from there continues in either one of two ways: (a) to the heart via the PCVs, which receive CAs from the urophysis and corpuscles of Stannius (cf. Hirano and Mayer-Gfstan, 1978) as well as from the kidney (local chromaffin cells?); or (b) to the liver, via collaterals from the renal portal veins to the hepatic portal vein (Anguilla-type of Bertin, 1958). Blood was collected by puncture, using a 27-gauge needle fitted to a 1-ml syringe. Functional evaluations. Tissues selected for surgical removal, recovery periods for unanesthetized animals, and CAs injected are shown in Table 1. Hypophysectomy was performed via a buccal approach (Hirano et al., 1967). Partial removal of the brain was carried out following removal of the overlying skull, and subsequent survival of the animals under continued anesthesia was controlled by observation of the cardiac activity. Corpuscles of Stannius were removed through a ventral incision following displacement of the urinary bladder. The urophysectomy was performed by removal of the tall region in which the urophysis is located, followed by clamping of the caudal artery and vein. Bilateral extirpation of the anterior regions of the PCV/head kidney complex (bulk of chromaffin tissue, probably the adrenomedullary equivalent) involved a ventral incision over the liver, followed by double ligation of the PCVs and excision of the intervening tissue (Admedex). Cardiac cannulation followed each of the procedures described above. Control animals underwent cardiac cannulation only. The impact of organ removal was evaluated using the catecholaminotropic effect of CAs (Epple and Nibbio, 1985); in the present study, we induced the release of DA and NE by E injections, and of NE and E by DA injections. The CAs were injected via the indwelling
~PITUITARY
I
DORSAL AORTA
h ~ GILLS
JUGULAR VEIN
"'
>
UROPHYSIS
q HEPATIC PORTAL VEIN
HEAD KIDNEY
(It ~
POSTERIORCARDINAL VEINS
FIG. 1. Vascularization and sampling sites inAngaiUa rostrata. (1) Anterior region of right posterior cardinal vein; (2) posterior region of fight posterior cardinal vein; (3) caudal vein; (4) dorsal aorta; (5) ventral aorta; (6) sinus vcnosus; (7) anterior region of hepatic portal vein; and (8) posterior region of hepatic portal vein. V, ventricle; A, atrium; SV, sinus vcnosus (not readily distinguishable from ductus Cuvieri); CS, corpuscles of Stannius.
420
HATHAWAY AND EPPLE TABLE 1 SUMMARY OF PROCEDURES FOR FUNCTIONAL EVALUATIONS Surgery
N
Recovery period a
Injection
Hypophysectomy Control Control
8 5 4
7 days 5-7 days 5-7 days
Epinephrine Saline Epinephrine
2 i~g/kg
"Mid-brain X ''b Mid + forebrain X c Mid + forebrain X Control
4 5 4 4
Anesthesia Anesthesia Anesthesia 5-7 days
Epinephrine Epinephrine Dopamine Epinephrine
8 ~g/kg
Urophysectomy Urophysectomy Stanniectomy Stanniectomy Control
5 4 4 4 4
Anesthesia Anesthesia Anesthesia Anesthesia Anesthesia
Epinephrine Dopamine Epinephrine Dopamine Epinephrine
2 2 2 2 2
Head kidney X d Head kidney X Head kidney X Control
4 4 5 4
Anesthesia Anesthesia 1 day Anesthesia
Dopamine Epinephrine Epinephrine Epinephrine
3 ~,g/kg 2 I~g/kg 1 i~g/kg 2 ixg/kg
2 txg/kg 8 ixg/kg 8 ixg/kg 8 i~g/kg ixg/kg ~g/kg i~g/kg ~g/kg i~g/kg
Anesthetized animals were injected 10 min postsurgery. b Two transverse cuts at borders of optic lobes and cerebrum and cerebellum, respectively. c Two transverse cuts at olfactory tract and at border of optic lobe and cerebellum. '~ Bilateral removal of anterior region of head kidney and posterior cardinal vein. cardiac cannula following the designated recovery period in unanesthetized animals or 10 min postsurgery in anesthetized animals (Table 1). DA (dopamine hydrochloride, Elkins-Sinn) and E (epinephrine hydrochloride, Elkins-Sinn) were diluted in saline (0.85% NaC1) and were injected in a final volume of 1 ml/kg body wt. Blood samples (0.3 ml) were withdrawn prior to CA injection and at 3 and 10 min postinjection. Analysis. All blood samples were delivered into test tubes containing the anticoagulant EGTA/glutathione and centrifuged (20 min, 210g, 6~ Supernatants were stored at - 70~ Diluted tissue acid extracts and plasma samples were analyzed for unconjugated DA, NE, and E with a radioenzymatic technique (CAT-A-KIT, Amersham). This assay has a sensitivity of 2-5 pg for E and NE and 15-20 pg for DA. Cross-contamination of the three determinations in the presence of high concentrations of E and DA (as would follow their exogenous administration) ranged from 0 to 7%. Statistical analyses. One-way analysis of variance, followed by the Student-Newman-Keuls test (regional sampling), Dunnett's test, or paired t test (functional evaluations) were done.
are shown in Table 2. In general, tissues contained the three CAs in the proportions of E I> NE >> DA. However, in brain and pituitary, DA and NE concentrations exceeded those of E. Levels of CAs in regions of the posterior cardinal veins (including dorsal opisthonephric kidney regions) exceeded those in other tissues by 1-3 orders of magnitude. With the exception of the pituitary and CS, most of the tissues analyzed contained amounts of CAs potentially capable of altering the plasma level of at least one CA (based on total tissue per organ weight and a 3% blood volume in whole eels: see Thorson, 1961; Holmes and Donaldson, 1969). This assumes, however, the unlikely event of rapid and total movement of stored CAs into the circulation (Table 3).
Regional Levels of Plasma CAs RESULTS Tissue CA Concentrations
The tissue concentrations 0xg/g) of CAs
Tables 4 and 5 show local CA plasma titers at selected vascular sites. In almost all cases the three CAs were present in the ra-
421
CATECHOLAMINES IN AMERICAN EEL
TABLE 2 CATECHOLAMINE CONCENTRATIONS IN TISSUES OF Anguilla rostrata Micrograms per gram wet weight tissue N~
Dopamine
Norepinephrine
Epinephrine
Head kidney (w/PCV b) Anterior ~ Middle d Posterioff
7 4 9
3.85 • 1.22 2.20 • 0.79 0.28 • 0.10
42.38 • 15.17 22.75 • 7.42 6.88 • 2.57
84.83 - 27.44 49.50 --- 17.36 8.35 -+ 1.73
Head kidney (w/o PCV) Anterior Middle Posterior
4 4 4
0.23 • 0.10 0.09 • 0.03 0.02 • 0.003
5.27 • 1.55 2.31 • 0.43 0.12 --- 0.04
6.23 --- 2.30 2.50 --- 1.41 0.18 • 0.04
Opisthonephric kidney r Anterior dorsal Middle dorsal Posterior dorsal Ventral
5 5 5 5
0.04 0.04 0.03 0.01
1.41 1.47 1.10 0.34
1.97 1.82 1.24 0.32
Heart Sinus venosus Atrium Ventricle
4 4 4
0.02 --+ 0.01 0.02 • 0.008 0.03 • 0.01
0.05 --- 0.03 0.11 • 0.05 0.16 --- 0.06
0.33 --- 0.20 0.40 +- 0.11 0.26 -+ 0.08
Whole brain Pituitary 8
9 1
0.14 • 0.02 0.07
0.30 • 0.02
0.02
0.03 • 0.01
0.008
Stannius corpuscles g
2
0.03 • 0.02
0.18 •
0.11
0.24 •
0.11
Liver
4
0.01 • 0.002
0.01 •
0.002
0.01 •
0.003
Gills
4
0.01 • 0.002
0.01 •
0.007
0.06 --- 0.04
• 0.006 -+ 0.009 • 0.009 - 0.002
• --• ---
0.25 0.33 0.34 0.10
-+ • • --+
0.35 0.38 0.30 0.09
a N equals n u m b e r of animals from which tissues were removed. b Posterior cardinal vein contained within head kidney segment. c Two-centimeter region located immediately posterior to ductus Cuvieri. d Two-centimeter region located at level of gall bladder. e Two-centimeter region located immediately anterior to opisthonephros. f Dorsal regions included caudal/cardinal vessels. g N equals batches of pooled samples from five animals.
tit E > DA > NE. Two exceptions, in which DA and NE titers were essentially equal, were seen in caudal vein samples in Series IV (Table 5). As expected, the second round of sampling in each of the three series yielded higher CA values than the first. The two rounds were therefore viewed as representing different levels of stress and were treated separately in statistical analyses. The highest mean plasma CA levels in each round of Series I, II, and III were observed in the anterior region of the right
PCV. Due to very strong individual variations, the statistical analysis yielded few significant differences between sampling sites. DA, NE, and E levels in some anterior PCV samples from Series II and III (Table 4) differed significantly from levels measured at the neighboring upstream and downstream sites. In five animals, however, CA titers in the posterior region of the PCV were greater than those in the anterior PCV (Table 6). The results of Series III and IV show that DA, NE, and E were entering both the PCVs and the hepatic portal vein
422
H A T H A W A Y A N D EPPLE TABLE 3 POTENTIAL CONTRIBUTIONS OF DIFFERENT ORGANS TO PLASMA CATECHOLAMINES IN THE AMERICAN EEL (pg/ml)
PCV/HK anterior a PCV/HK posterior b Opisthonephros Brain Pituitary Stannius corpuscles Heart Gills Liver
Dopamine
Norepinephrine
Epinephrine
18,000 750 3,000 750 15 10 700 2,000 5,000
190,000 18,500 100,000 1,600 4 60 2,700 2,000 5,000
390,000 21,000 130,000 150 2 80 8,000 12,000 5,000
Note. Estimated from catecholamine content (p~g/g wet tissue, Table 2) in a hypothetical 500-g eel with blood volume equal to 3% of body weight. Anterior 5 cm of the right posterior cardinal vein and associated head kidney. b Posterior 2 cm of the right posterior cardinal vein and associated head kidney.
TABLE 4 LOCAL PLASMA CATECHOLAMINE TITERS (pg/ml • SEM) IN Anguilla rostrata BASED ON SEQUENTIAL SAMPLING AT SELECTED VASCULAR SITES Dopamine Series
Round 1
Norepinephrine
Round 2
Round 1
Round 2
Epinephrine Round 1
Round 2
I P-PCV A-PCV SV VA DA
161 • 650 • 193 • 204• 233 •
53
DA VA SV A-PCV P-PCV
271 • 51 3 5 2 • 74 3 1 4 • 54 867 ~ 172"* 453 • 162
55 60 77
336 • 1162 • 496• 657 • 717 •
112 531 223 378 406
75 • 24 277 • 153 5 9 • 21 64• 19 63 • 19
262 • 65 447 • 150 1 2 4 • 35 162 • 62 195 • 57
292 • 1119 • 391 • 415 • 506•
105 622 126 142 181
797 2360 1048 1457 1641
• • • • •
395 • 631• 639• 1364 • 969 •
88 142 145 312 277**
134 • 40 1 7 5 • 49 155 • 44 731 • 168" 354 • 105"*
199 • 48 3 1 0 • 58 3 5 2 • 89 975 • 132" 698 • 131
591 • 797• 691 • 2414 • 1018 •
131 257 176 693** 159"*
1023 • 1533 • 1551• 3928 • 2254 •
251 956 402 851 846
II
III A-PCV P-PCV CV DA PV
3154 2036 519 1746 1171
• • • • •
577 551 130 276 112
15776 7147 1102 3302 1240
• 1655 • 720* • 286* • 913 • 274
1358 946 205 517 257
• 335 • 343 • 46 • 46 • 64
4280 2624 350 668 173
• 477 • 456* • 67* • 128 • 34
6438 3521 1210 3517 2233
• 1988 • 742 • 350 • 408 • 261
31437 14505 2568 6714 1969
320 471 443 983** 163"*
• 3142 • 544* • 517" • 1612 • 155
Note. Sites were sampled in order listed in Series I (N = 5), II (N = 5), and III (N = 4). Round 2 sampling began upon completion of round 1. Location of sampling sites is shown in Fig. 1: posterior region of posterior cardinal vein (P-PCV), anterior region of posterior cardinal vein (A-PCV), sinus venosus (SV), ventral aorta (VA), dorsal aorta (DA), caudal vein (CV), and hepatic portal vein (PV). Statistical evaluation of each round with A N O V A and Student-Newman-Keuls test: * and ** indicate differences (P < 0.01 and P < 0.05, respectively) from previously sampled site. All sampling was done in urethane-anesthetized animals.
423
C A T E C H O L A M I N E S IN A M E R I C A N E E L TABLE 5 LOCAL PLASMA CATECHOLAMINE TITERS (pg/ml • SEM) IN Anguilla rostrata BASED ON SEQUENTIAL SAMPLING AT SELECTED VASCULAR SITES Series IV
Dopamine
Norepinephrine
Epinephrine
Hepatic portal vein (anterior) Caudal vein Dorsal aorta Caudal vein Hepatic portal vein (anterior)
296 102 204 142 288
- 73 -+ 32 +- 43 -+ 61 • 33
126 115 186 102 109
• • • • -
34 52 54 27 51
465 305 590 285 449
Hepatic portal vein (posterior) Caudal vein Dorsal aorta Caudal vein Hepatic portal vein (posterior)
277 159 377 180 410
-+ 39 - 38 -+ 99 -+ 37 -+ 19
142 165 317 160 154
• -+ • -
65 63 100 60 52
648 634 1386 665 842
-• -+ -+ •
94 97 91 94 122
_ 193 -- 266 • 537 -- 234 -+ 299
Note. E a c h animal was s a m p l e d at the 10 sites in the order listed. N = 4 for all sites e x c e p t posterior hepatic portal vein ( N = 3). Statistical evaluation with A N O V A and S t u d e n t - N e w m a n - K e u l s test revealed no significant differences (P < 0.05) b e t w e e n sites; however, trends are consistent.
within the opisthonephric kidney (Tables 4 and 5). Functional Evaluations
Hypophysectomy failed to alter the CA tropic effect of E in unanesthetized eels (Table 7). The possibility existed for the regeneration of hypothalamo-hypophyseal tracts (Moll, 1957) and for the continued release from the pituitary stalk equivalent (Mens et al., 1982), and/or the involvement of circumventricular organ(s) such as the organum vasculosum laminae terminalis (Leonhardt, 1980; Tsuneki, 1986). We therefore removed the diencephalon plus pituitary and finally all brain tissue anterior to the cerebellum prior to CA challenge. However, this also had no impact on the action of the CAs in releasing other CAs (Table 8). Neither urophysectomy nor stanniectomy (Table 9), nor surprisingly, bilateral removal of the anterior PCV/head kidney complexes (Table 10) prevented the CA tropic effects of injected CAs. DISCUSSION The CA concentrations in various tissues ofA. rostrata (Table 2) are generally of the
same order of magnitude as the average levels reported previously for anguillid and other fishes. Le Bras (1984) finds CA levels in the heart and brain of A. anguilla which are lower than those reported here. Technical differences (method of sacrifice ?) may account for the discrepancies. Nevertheless, despite the differences in absolute values, the relative proportions of DA, NE, and E observed in both studies are similar. The significance of the CAs identified in the pituitary cannot be assessed until we know more about the cellular location of these substances; however, the relatively high concentration of DA is compatible with its well-known presence in neurosecretory fibers (Peter, 1986). In contrast to our data, Unsicker et al. (1977) found significantly more NE than E in corpuscle of Stannius extracts from Salmo irideus, though the total levels for NE and E combined are similar in both studies. Whether the lower NE values in the present study indicate an absence of presumed NE-containing chromaffin cells in the corpuscles of Stannius (cf. Wendelaar Bonga and Pang, 1986) of anguillids or a species difference in type of adrenergic neurotransmitter is not known. The CA concentrations in the PCVs and head kidneys presented here are, to the
424
HATHAWAY AND EPPLE
stores are more closely associated with the PCV than the surrounding head kidney (Abrahamsson and Nilsson, 1976); and that (3) the anterior portion of the PCV/head kidney complex (close to the ductus Cuvieri which Mott (1950) found to not be Animal Post-PCV Ant-PCV readily distinguishable from the sinus veno022988-1 (round 1) sus in A . anguilla) contains the densest acNE 1967 1837 cumulation of chromaffin tissue (cf. Nils122387-1 (round 1) son, 1983; Mastrolia et al., 1984). ApparDA 103 93 ently, this is the first report on tissue NE 138 100 concentrations of DA in this region (Table E 169 131 2). Interestingly, they are comparable to (round 2) those in mammalian adrenal glands (CarbalNE 416 325 leira et al., 1987; Pratt et al., 1987). In all 122487-1 (round 2) likelihood, the chromaffin cells close to the NE 323 152 ductus Cuvieri represent the equivalent of 013088-1 (round 2) the adrenal medulla for the following reaDA 1128 988 sons: (a) they are in immediate proximity of E 2175 1972 the interrenal tissue (Giacomini, 1908; 013188-1 (round 1) Nandi, 1962; personal observations); (b) DA 1069 967 t h e y are very strongly i n n e r v a t e d by NE 757 518 E 1312 1129 "preganglionic" fibers (Brinn et al. in prep(round 2) aration); and (c) their location coincides DA 1880 1475 with the highest tissue concentration of NE 985 915 CAs (Table 2). However, in contrast to the E 2882 1958 adrenal medulla cells of "higher vertebrates," their majority, perhaps even all of best of our knowledge, the first ones re- them, are supplied by venous blood (deported in an anguillid species. The diversity rived mainly from the caudal/renal portal of kidney/cardinal vein structures (Ogawa, complex and small veins of the head kidney 1961) and chromaffin cell arrangements region: Mott, 1950; Bertin, 1958). It re(Giacomini, 1908; Nandi, 1962)among the mains to be seen if the chromaffin cells in teleosts makes a comparison with the the posterior head kidney and opisthowidely varying data on tissue CAs in other nephric kidney regions are also innervated, fishes (J6nsson et al., 1983; cf. Nilsson, or perhaps represent the equivalent of the 1983) rather meaningless. Nevertheless, it paraganglia (which are usually not inneris noteworthy that (1) E is the predominant vated: B6ck, 1982). This question is curCA in the PCV/head kidney region of sev- rently under investigation. At any rate, the eral other, though not all, bony fishes (Na- regional CA levels in different regions of kano and Tomlinson, 1967; Stabrovskii, head and opisthonephric kidney (Table 2) 1969; Abrahamsson and Nilsson, 1976; show that significant CA storage occurs Abrahamsson et al., 1979; Balashov et al., along the entire length of the PCVs and also 1981; Nilsson, 1981; JOnsson et al., 1983). in the region of the caudal/renal vein sysIn the rainbow trout, J6nsson et al. (1983) tem (cf. Bertin, 1958). Their tissue concenfound slightly higher NE than E levels in trations match perfectly the regional distrithis region, while Nakano and Tomlinson bution of the chromaffin cells of the eel (1967) report the opposite; that (2) CA (Giacomini, 1908). TABLE 6 INDIVIDUAL VALUES OF PLASMACATECHOLAMINES (pg/ml) FROM THE POSTERIOR REGION OF THE POSTERIOR CARDINAL VEIN (POST-PCV), AS COMPARED WITH VALUES FROM THE ADRENAL MEDULLA EQUIVALENT(ANT-PCV)
425
C A T E C H O L A M I N E S IN A M E R I C A N EEL
TABLE 7 IMPACT OF HYPOPHYSECTOMY ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE CARDIAC-CANNULATED UNANESTHETIZED EEL
Times before and after injections
State of the animal and treatment
N
Plasma CA measured
I
Intact c o n s o l s (saline)
5
DA NE E
135 • 18 • 29 •
25 12 17
II Intact controls (2~g/kgE)
4
DA NE E
98 • 53 • 31 •
51 28 14
6643 • 1925 a 1258 • 478 a 7 7 2 0 • 1984"
III H y p e x eels (2~g/kgE)
5
DA NE E
445 • 139 270 • 135 306 • 72
7429 • 2105 ~ 2341 • 474 a 17697 •
Before
3 min 111 • 8 • 15 •
30 min 12 5 8
149 • 8 • 18 •
40 5 7
308 • 38 • 74•
65 18 24
506 • 113 332 • 99 292 • 69
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. All animals injected 5-7 days after surgery.
It is noteworthy that the presumed adrenal medulla equivalent of the eel (and apparently that of other actinopterygians as well: Nilsson, 1983) is located just up-
stream from the heart; i.e., virtually the same place in which the chromaffin cells of the Southern lamprey, Geotria australis (Epple et al., 1985), the "axillary" bodies
TABLE 8 IMPACT OF PARTIAL BRAIN REMOVAL ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE EEL
State of the animal and treatment I
Unanesthetized intact control (8 txg/kg E) b
II " M i d b r a l n " removed (8 ~g/kg E) C III Fore- and midbrain removed d a. 8 I~g/kg DA
b. 8 ~g/kg E
N
Plasma CA measured
Times before and after injections Before 217 • 36 • 99 •
3 min 45 19 39
25347 • 1356 • 35771 •
10 min
4
DA NE E
3077 a 852 a 5420 a
----
4
DA NE E
7625 • 6335 3537 • 2976 9701 • 8088
99730 • 32946 a 14800 • 6840 ~ 179994 • 52134 a
34901 • 19965 9126 • 6619 60470 • 30901
4
DA NE E
1498 • 643 • 1670 •
664 387 499
247602 • 92453 a 3016 • 1160 a 26748 • 10896 a
33560 • 22363 1877 • 718 7639 • 2624
5
DA NE E
1262 • 559 1279 • 1016 2182 • 984
62993 • 7374 ~ 10297 • 3320 ~ 143528 • 23627 a
20624 • 8742 4656 • 3247 43760 • 20582
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. b Cardiac-cannulated animals, injected 5-7 days after surgery. c Anesthetized animals injected 10 min after cardiac cannulation and removal of midbrain, diencephalon, and pituitary. d Anesthetized animals injected 10 min after cardiac cannulation and removal of midbrain and forebrain (i.e., all tissue anterior to the cerebellum).
426
H A T H A W A Y A N D EPPLE TABLE 9 IMPACT OF STANNIECTOMY AND UROPHYSECTOMY ON THE CATECHOLAMINOTROPIC EFFECTS OF CATECHOLAMINES IN THE EEL a
State of the animal and treatment I
Anesthetized controls (2 ixg/kg E)
II Stanniectomized eels a. 2 ~g/kg E
b. 2 ~g/kg DA
III Urophysectomized eels a. 2 Ixg/kg E
b. 2 t~g/kg DA
N
Plasma CA measured
Times before and after injections Before
10 min
63 62 87
7974 • 1406 b 620 • 150b 10740 • 2479 b
2829 • 983 507 • 198 3584 • 1102
5094 • 1404 b 1065 • 149b 7400 • 708 b
979 • 634 • 2990 •
202 200 560
3015 • 106 802 •
1092 • 146 • 403 •
448 55 91
4
DA NE E
5
DA NE E
330 • 70 508 • 199 1172 • 536
4
DA NE E
437 • 154 78 • 32 212 • 76
DA NE E
275 • 59 341 • 101 739 • 128
7906 • 1432 b 2 2 4 4 • 653 b 24716 • 3226 b
DA NE E
453 • 89 378 • 36 527 • 150
7477 • 1781 b 468 • 21 b 1745 • 397 ~
5
3
211 • 203 • 311 •
3 min
658 b 45 150 b
1975 • 610 717 • 182 5958 • 1348 1089 • 576 • 873 •
294 34 b 335
Note. All values in pg/ml plasma -+ SEM. a Anesthetized animals injected 10 min after cardiac cannulation (I) and cardiac cannulation plus endocrinectomy (II + III). b Indicates significant difference (P < 0.05) from " b e f o r e " value.
of the elasmobranchs (Abrahamsson, 1979, Nilsson, 1983; Watzka, 1943), and the chromaffin cells of the African lungfish (Abrahamsson et al., 1979) are found. Probably this "fish pattern" indicates a functional relationship (Nilsson, 1983). Our data leave little doubt that the presumed adrenal medulla equivalent is the main source of all three plasma CAs (Tables 2-6). As far as we know, this is also the first demonstration of the major source of systemic plasma DA in a "lower" vertebrate. In mammals, including the human, systemic DA also seems to come mainly from the adrenal medulla (Kvetnanski et al., 1979; Kuchel et al., 1979; Planz and Planz, 1979; Unger et al., 1979). Wahlqvist and Nilsson (1980) found that sectioning of the nerve supply to the head kidney of the Atlantic cod greatly reduced but did not abolish stress-induced increases
in plasma E and NE. They attributed this result to either incomplete denervation or overflow into the plasma from adrenergic nerve endings. Our finding of a lack of an impact of bilateral removal of the presumed adrenal medulla equivalent on the CA tropic effects of DA and E (Table 10) suggests that the chromaffin cells in the opisthonephric kidney region are capable of releasing large quantities of CAs into the systemic circulation, even when the blood flow in the anterior PCVs is interrupted. The peculiar collateral connections between the renal and hepatic portal vein systems of the eel (Mott, 1950; Bertin, 1958) are probably involved. In addition, nonchromaffin cells in the opisthonephric kidney could also be involved; in mammals, DA derived from tubular decarboxylation of 1-DOPA and from intrarenal sympathetic fibers has been implicated in the control of kidney blood flow
C A T E C H O L A M I N E S I N A M E R I C A N EEL
427
T A B L E 10 IMPACT OF " A D R E N O M E D U L L E C T O M Y " ON THE CATECHOLAMINOTROPIC E F F E C T S OF CATECHOLAMINES IN THE E E L
State of the animal and treatment
N
I
Unanesthetized controls (1 ixg/kg E) b
4
II Unanesthetized admedex eels (1 ~g/kg E) C III Anesthetized controls (2 ~g/kg E) a
Plasma CA measured
Times before and after injections Before
3 min 1090 + 56 • 1041 -t-
10 min
DA NE E
37 --- 24 1 -+ 0 3 -+ 3
441 a 27 a
--
644 a
__
5
DA NE E
261 --- 145 49 -+ 13 126 • 35
5431 --- 1216 a 1057 • 272 a 10323 • 2436 a
911 • 168 196 --- 42 1533 • 228
4
DA NE E
211 • 63 203 -+ 62 311 • 87
7974 • 1406 a 620 ----- 150a 10740 -+ 2479 ~
2829 • 983 507 -- 198 3584 • 1102
IV Anesthetized admedex eels d a. 2 Ixg/kg E
4
DA NE E
916 • 160 612 • 155 1665 -+ 296
9671 • 1628 a 1362 • 254 a 16529 • 3788"
3048 -+ 324 866 • 180 6297 - t825
b. 3 ixg/kg DA
4
DA NE E
516 • 203 283 • 56 990 • 273
12775 • 2222 a 367 • 63 2951 ----- 655 a
2114 • 317 • 1387 •
884 74 482
Note. All values in pg/ml plasma • SEM. a Indicates significant difference (P < 0.05) from " b e f o r e " value. b Cardiac-cannulated animals injected 5-7 days after surgery. c Cardiac-cannulated animals injected after overnight recovery from admedex. a Anesthetized animals injected 10 min after cardiac cannulation (III) and cardiac cannulated and admedex (IV).
(Bell, 1987). However, in teleosts the innervation of the kidneys is poorly understood (Braun and Dantzler, 1987). Apart from their possible involvement in renal functions, the CA stores in the opisthonephric kidney region raise another functional question: Is the release of CAs into the portal vein related to the physiological hyperglycemic response to E, seen in the eel (Epple and Nibbio, 1985), but not in the hagfish (Plisetskaya et al., 1984), lamprey (Dashow and Epple, 1983), rat (Epple et al., 1988), and human (Epple et al., 1987, in press)? The high tissue concentrations of CAs in the heart (Tables 2 and 3) corroborate findings of Pennec and Le Bras (1984) who report storage and release of CAs from nerve endings in the isolated heart of the eel. H o w e v e r , a c o m p a r i s o n b e t w e e n the
plasma titers in sinus venosus and ventral aorta in vivo does not reveal a clear picture (Table 4). Since the heart of the eel is a target organ of CAs (Chan and Chow, 1976; Peyraud-Waitzenegger et al., 1980), it is possible that the discrepancy is due to a balanced net effect of uptake of humoral CAs and a release of neuronal CAs in vivo (cf. Laurent et aL, 1983). This question requires further study. The high CA concentrations in the brain (Tables 2 and 3) are in good agreement with the higher titers of DA and NE than E, seen in the head blood of the eel (Epple et al., 1982). Nevertheless, the data in Tables 2, 3, and 4 indicate that the overall contribution of CAs from this source to the systemic CA titers is of minor significance (note the low CA values in the sinus venosus, where the head blood mixes with the body blood).
428
HATHAWAY AND EPPLE
Similar to the situation in the heart, the This would resemble the situation in mamcomparison of the CA titers of the affluent mals where basal (von Euler et al., 1954), and effluent blood of the gills (ventral vs but not stress-induced (Andersson et al., dorsal aorta) does not show a clear gradi- 1982), NE titers depend upon neuronal ent. Again, a balanced net effect between sources. Further studies must prove or disCA uptake and release could be postulated: prove this possibility for the eel. In summary: The bulk of all three CAs in Nekvasil and Olson (1986a,b) show considerable extraction and metabolism of CAs in the systemic blood of the eel derives from the gills of the rainbow trout; on the other the presumed adrenal medulla equivalent, hand, the gills of trout have adrenergic in- which is located in the anterior PCV. Hownervation (Donald, 1984) which may re- ever, after removal of this region, strong lease CAs. However, other factors could CA release can yet be induced by CA inalso be involved; for instance, due to a spe- jections. Chromaffin cells in the veins of cific extraction mechanism, the venous the opisthonephric kidney region are prob"metabolic" blood of the gills could have a ably the major CA source in the latter case. lower CA content than the arterial "respiACKNOWLEDGMENTS ratory" blood measured here (Tables 4 and 5; cf. Wood, 1975). The authors are indebted to Ms. Barbara Nibbio for The tissue concentrations of all three her excellent technical assistance throughout these inCAs in the liver (Table 2) are essentially vestigations. Supported by NSF Grant DCB-8510585. higher than the plasma concentrations of REFERENCES unstressed eels (at least 20 times for DA and 100 times for NE and E: see, Epple et Abrahamsson, T. (1979). Phenylethanolamineal., 1982; Epple and Nibbio, 1985). We N-methyl transferase (PNMT) activity and catecholamine storage and release from chromaffin have no explanation for this finding (though tissue of the spiny dogfish, Squalus acanthias. one may suspect the presence of sympaComp. Biochem. Physiol. C 64, 169--172. thetic fibers); nor do we know its signifi- Abrahamsson, T., Hohngren, S., Nilsson, S., and Petcance for the plasma titer of the CAs. tersson, K. (1979). On the chromaffin system of In addition to the potential sources of the African lungfish, Protopterus aethiopicus. Acta Physiol. Scand. 107, 135-139. plasma CAs discussed in the preceding, contributions from some chromaffin cells in Abrahamsson, T., and Nilsson, S. (1976). Phenylethanolamine-N-methyl transferase (PNMT) activity the anterior cardinal vein (Giacomini, 1908) and catecholamine content in chromaffin tissue and a mammalian-like "synaptic leakage" and sympathetic neurons in the cod, Gadus come to mind. These sources could become morhua. Acta Physiol. Scand. 96, 94-99. important in CA-stimulated CA release af- Andersson, P.-O., Farnebo, L.-O., Fredholm, B. B., Hamberger, B., Holst, J., and J~irhult, J. (1982). ter "adrenomedullectomy" in the eel; howMetabolic and hormonal adjustments during hemever, we have no evidence for either one of orrhage in cats after interference with the symthese mechanisms. patho-adrenal system. Acta Physiol. Scand. 114, Finally, the discrepancy between the CA 111-119. tissue concentration in the presumed adre- Balashov, N. V., F~inge, R., Govyrin, V. A., Leont'eva, G. R., Nilsson, S., and Prozonal medulla equivalent (E > NE >>DA), the rovskaya, M. P. (1981). On the adrenergic system plasma titers of these compounds during of ganoid fish: The beluga, Huso huso (Chonanesthesia (E > DA > NE), and without drostei). Acta Physiol. Scand. 111, 435--440. anesthesia (DA > NE = E) remains to be Bell, C. (1987). Endogenous renal dopamine and control of blood pressure. Clin. Exp. Theory Prac. resolved (compare Tables 2-6 with the data A9, 955-975. in Epple et al., 1982 and Epple and Nibbio, Bertin, L. (1958). Appareil circulatoire. Trait~ Zool. 1985). The simplest explanation appears to 13, 1399-1458. be a neuronally controlled selective release Bfck, P. (1982). "The Paraganglia." Springer-Vedag, of different CAs from different cell types. Berlin.
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