Plasma catecholamines in the lamprey: Intrinsic cardiovascular messengers?

Camp. Biochem. Physiol. Vol. 82C, No. 1, pp. 119-122, 1985 Printedin Great Britain

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0306~4492/U $3.00 + 0.00 1985 Pergamonpress Ltd

PLASMA CATECHOLAMINES IN THE LAMPREY: INTRINSIC CARDIOVASCULAR MESSENGERS? LARRY DASHOW and AUGUST EPPLE* Jefferson Medical College, Daniel Baugh Institute of Anatomy, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA. Telephone: (215) 928-7820

(Received 2 January 1985)

Abstract-l.

The widely scattered cardiovascular chromaffin cells of Perromyzopl rnarinus appear to form an intrinsic control system of circulatory function. 2. In response to blood-borne stimuli, a checkpoint-like accumulation of epinephrine cells in the heart releases its hormone; epinephrine, in turn, stimulates the release of norepinephrine, and probably also of dopamine, from other cardiovascular chromaffin cells. 3. The myocardium seems to be a major target of norepinephrine. On the other hand, high disappearance rates of epinephrine and dopamine in the gills point to these organs as possible major targets of the latter two secretions. 4. Carbon dioxide and hypovolemia are strong stimuli of catecholamine release.

INTRODUCTION

The cyclostomes possess a unique system of chromaffin cells which in most species are scattered over the cardiovascular system (FHnge, 1972; Falkmer et al., 1974; Coupland, 1979; Hardisty, 1979; Mazeaud and Mazeaud, 1981; Laurent et al., 1983; Epple et al., 1985). These cells produce epinephrine (E), norepinephrine (NE) and probably also dopamine (DA). However, their functions are not clearly understood. Fast and strong increases of plasma catecholamines (CAs) in stressed lampreys (Mazeaud, 1969; Plisetskaya and Prozoroveskaya, 1971; Dashow et al., 1982) suggest that the cardiovascular chromaffin cells may be the phylogenetic precursors of the adrenal medulla. On the other hand, these cells resemble, both in their lack of innervation and in their distribution, paraganglia, structures which are particularly well developed in prenatal mammals (Bock, 1982; Phillippe, 1983). Recently, we found that in the lamprey (a) contrary to the situation in higher vertebrates (Epple et al., 1982), the bulk of all three circulating CAs must be released outside the brain (Dashow et al., 1982) and (b) E causes a very fast dose-related increase of plasma DA and NE, while the other two CAs have no catecholaminotropic effects (Dashow and Epple, 1983). We here present data, obtained from prespawning Petromyzon marinus, which strongly support the postulate that the chromaffin cells constitute an intrinsic control system of cardiovascular functions. In concert with the peculiar, stimulatory inne~ation of the lamprey heart (cf. Laurent et al., 19833, the latter seems to work as follows: the E cells, which are restricted to the subendothelium of the heart region, occupy the ideal checkpoint for the composition of the spent, prebranchiai blood. Acting co1lectively as a sensor, they

respond to changes in certain plasma constituents by release of their hormone. E then acts locally on the myocardium and subsequently in high, “portal” concentrations on the gills; finally, it stimulates in lower postbranchial concentrations the release of DA and NE from other chromaffin cells. The latter two hormones then also affect cardiovascular and respiratory activities (Epple, 1982; Dashow and Epple, 1983; Macey et al., 1984). MATERIALS AND METHODS

All lampreys belonged to a land-locked population of Petromyzon marinus. Animals of both sexes (average weight approx. 230g) were trapped during their spawning migration near Lake Cayuga, New York State and transported to Philadelphia, where the experiments took place. Unless indicated otherwise, the techniques of anesthesia, cardiac cannulation, maintenance, radioenzymatic CA assay and enzymatic glucose assay were the same as in previous studies (Dashow et al., 1982; Dashow and Epple, 1983). Studies on regionat CA titers Preliminary experiments had revealed that a hypovolemic reaction will occur when approx. 1.5 ml of blood is withdrawn within 5min. This reaction is characterized by an abrupt reduction of the rate of the heart beat, strongly reduced blood Row and a jump-like increase of the plasma CAs in all regions of the cardiovascular system. Therefore, in addition to the measurements under normovolemic conditions, hypovolemia was used as a further means to obtain information on the sites of CA release. A total of six blood samples was withdrawn from individual animals, i.e. three before and three during the hypovolemic response. Two different sampling sequences (Table 1, Series I and II) were applied in order to avoid a possible impact of regional factors on blood withdrawal. Blood was collected by puncture of the sites indicated in Fig. 2, using a 23 gauge needle fitted to a 1 ml syringe. Studies on the impact ofC0,

*Author to whom correspondence

and 0, piasm~ CAs

In these experiments, the lampreys were housed in spe-

should be addressed. 119

LARRY DASHOW and AUGUST EPPLE

120 Table I. Local plasma concentrations

of catecholamines

Perromyzon murinus before and during

(pg/ml) and glucose (mg”/,) in anesthetized the hypovolemic response

Before hypovolemia

Response

to hypovolemia

Swres 1

DA NE E GI

Ventral

DOKdl

aorta

aorta

206 13,175 5515 76

-I: 40 +4967 Jo 2008 i 8

123 29,483 1502 72

f + k f

62 8058 229 6

Ventral aorta

Cardinal vein 382 31,664 1301 77

i + * +

I 14b 9810 255 4

2228 + 32,152 f 38,931 f 795

Dorsal GXNtd

412’ 19,037 15,061 13

147 74,243 2688 83

+ ; + f

Cdrdindl

vein

25E.d 91 59b 71 I 7

442i81’ 42,838 f 5010e 1855 f 338 89 & 3

Series II

DA NE E GI The

Cardinal vein

Sinus venous

350 + 67 22,602 f 4022 I898 f 425 51+4

592 + 174 25,990 + 4330 3519 f 1595 51&4

Cardinal vein

Ventral aorta 1188+716 8010 + 1422b,C 7433 + 3391 53 *4

1063 70,856 6817 63

k f + +

Sinus W”OS”S

20‘ 2236c 2139‘ 5

1416 48,052 6601 65

f i k f

Ventral aorta

481 12,433 2667 1

7982 + 4185 16,929 + 6077”h 28 323 + 6045d,‘,h ’ 53+5

two sets of data are arranged according to the blood flow. Series I: ventral aorta + (gills) + mid-dorsal aorta --t (systemic capillaries) + cardinal vein; series II: cardinal vein + sinus venosus + (heart) + ventral aorta. Statistical evaluation with Student’s I test; capital letters: P < 0.001, small letters P < 0.05. The letters indicate differences from the following: ventral aorta (A, a); dorsal aorta (B, b); cardinal vein (C, c); sinus venosus (C. g); all before hypovolemic response. Ventral aorta (D, d); dorsal aorta (E, e); cardinal vein (F, f); sinus venosus (H, h); all during hypovolemic response. N = 5 for all groups except the “Before” group of the second set

(N = 7).

cially designed PVC tubes (Dashow and Epple, 1983). The first blood sample was withdrawn 30 min after the insertion of the cardiac cannula, which took place under light MS 222 anesthesia. At the time of the first sampling, the animals had recovered from the anesthesia. In both sets of the present experiments, the initial titers of the CAs and glucose were higher than the preinjection levels of the unanesthetized, unstressed lamprey (Dashow and Epple, l983), which was probably due to the combined

impact of the preceding anesthesia and surgery. RESULTS

Table 1 shows the regional concentrations of the plasma CAs and glucose before and during the response to hypovolemia. The most consistent result in all four experimental situations was the strong increase of plasma E between the affluent and effluent poles of the heart, while the opposite occurred with NE (compare cardinal vein with ventral aorta; see also Fig. 2). In Series I, the transcardiac net increase of E amounted to 324 and 1999% before and during the hypovolemic response, respectively, while the transcardiac drop of NE was 140 and 25%. In Series II, the corresponding values for E are 291 and 316x, and for NE 65 and 76%, respectively. The highest titers of NE were found in the larger blood vessels, from the dorsal aorta to the sinus venosus. The situation with DA was less clear-cut, though the highest titers of this substance occurred in three out of four situations in the ventral aorta. Both DA and E dropped considerably between the ventral and the dorsal aorta, before, as well as during, the hypovolemic response. The differences were 40 and 66% for DA; and 73 and 93% for E, respectively (Table 1, Series I). The second set of experiments (Fig. 1) was designed to provide further evidence that the release of DA and NE is stimulated by E. The specific predictions were: (1) because of the fast catecholaminotropic effect of E and the short half-life of the CAs (Dashow and Epple, 1983), the plasma titers of all three CAs will undergo fast, parallel fluctuations when short-term stress and “antistress” are applied

successively. (2) Due to simultaneous stimulation of their release by E, upward changes of the titers of DA and NE may be particularly closely related. (3) Regardless of the baseline levels, this mechanism must work when vital factors of the cardiovascular functions (0, and/or COJ are involved. Therefore, we used unanesthetized lampreys 30 min after cardiac cannulation, whose plasma CAs showed the expected stress-related differences (Dashow et al., 1983) from “baseline values”: an increased titer of all three CAs, and a reversed NE:E ratio (N > E). As predicted, CO* was a strong stimulus of CA increase, while O2

ng/ ml

WJ

n 0 100

I

q q

DA NE E glucose

A

:t

10000

VI

Y

3 s = =:

=: z

1000 z

10

z Z

P 100

1

AIR

o*

co*

o*

co*

Fig. I. Effect of O2 and CO, on plasma catecholamines of marinus. After surgery, the animals were exposed, via an airstone, first to 30min of air, then to successive 5 min treatments of O2 and CO,. Blood samples were withdrawn at the end of each treatment. Note the parallel changes of the three catecholamines throughout the experiment. A and a indicate statistically significant differences from the preceding sampling (P < 0.001 and P < 0.05, respectively; f test). Pefromyzon

Lamprey catecholamines

121

S. venosus Fig. 2. Origin, proposed interactions and sampling sites of catecholamines in the cardiovascular system of Petromyzon marinus. A checkpoint type accumulation of E cells in the cardiac region responds to changing blood parameters (e.g. CO?, hypovolemia) by release of their hormone. E acts on the myocardium, gills and cells producing NE and DA. Widely scattered NE cells act locally in the cardiovascular system, with the myocardium as a major target. DA, released in the cardiac region, may affect gills. Another fraction of DA may act locally in the venous drainage. D. aorta: dorsal aorta; V. aorta: ventral aorta; C. vein: cardinal vein; S. venosus: sinus venosus.

reversed or abolished this effect; and there was a good correlation between the CA titers through the experiment (E vs DA and E vs NE: P < 5%; DA vs NE: P < 1%; Spearman’s correlation test). CO, also caused an unusually fast increase of plasma glucose (within 5 min), which obviously was superimposed on a slow, continued increase seen in controls (N = 5), for whom 0, and CO, were replaced by air. In the latter animals there was a 15% increase of glycemia during the duration of the experiment (P < 0.05in Student’s t test; not shown in Fig. 1). DISCUSSION

The present investigations further substantiate the idea that the cardiovascular chromaffin cells of the lamprey form an intrinsic control system of the circulation (Epple, 1982). The previous evidence for this system may be summarized as follows: (1) E stimulates the release of DA and NE, while DA and NE have no catecholaminotropic effect (Dashow and Epple, 1983); (2) the DA and NE response to E is dose-related and occurs at physiological levels (Dashow and Epple, 1983): (3) the E cells are restricted to the ideal checkpoint for the state of the spent blood, i.e. the affluent regions of the heart (for literature, see Introduction); (4) all chromaffin cells are located immediately underneath the endothelium (cf. Paiement and McMillan, 1975) i.e. as close to the blood as might be expected of endocrine cells responding to blood-borne messages; (5) the chromaffin cells are noninnervated (Caravita and Coscia, 1966; Beringer and Hadek, 1972; Lignon and Le Douarin, 1978) and hence respond most likely to humoral (and perhaps also pressure) signals. To these data, the present paper adds the following evidence: (1) epinephrine is released mainly or exclusively from the expected checkpoint in the affluent heart region, as evidenced by its transcardiac increase (Table I); (2) there is a substantial drop of NE between the cardinal vein/sinus venosus region and

the ventral aorta, which corroborates the data indicating that the powerful myocardium of the ventricle is a major target of NE (cf. Falck et al., 1966; Lignon, 1979; Nilsson, 1983); (3) very high titers of NE occur in the dorsal aorta, which agrees with the postulate that this substance also controls postbranchial vascular activities (note the strong drop between the dorsal aorta and the cardinal vein in Table 1, Series I, during hypovolemic volemia); (4) the release of the CAs is controlled by appropriate stimuli of cardiovascular activities: CO, and 0, cause the expected, respective increases and decreases of plasma CAs (Fig. 1); (5) the changes of the plasma CAs are fast and correlated, and the correlation between the titers of DA and NE is particularly close, as one would expect, from substances which are released by the same stimulus, i.e. E (see under Results). To these findings, we add here the result of a retrospective evaluation of 12 different groups of data from two previous studies (Dashow et al., 1982; Dashow and Epple, 1983) which shows a high correlation of the titers of the three CAs also under baseline (preinjection) conditions (P < 1%; Spearman’s correlation test). This indicates that the proposed intrinsic cardiovascular CA system functions in both “normal” and stress situations. Furthermore, it must be noted that the disappearance of large quantities of DA and E between the ventral and dorsal aorta (Table 1; upper two sets of data) suggests that these CAs may regulate vascular and/or other gill functions of the lamprey, as has been postulated for E in gnathostome fishes (see e.g. Jones and Randall, 1978; Eddy, 1981; Pit and Djabali, 1982; Djabali and Pit, 1982). However, an inactivation and/or secretion of DA and E by the lamprey gills cannot be excluded at this point, either. Of course, the existence of an E controlled catecholaminotropic system does not preclude Eindependent responses of DA and NE cells under special conditions. An example for the latter is the enormous reflex-like release of NE following decapi-

122

LARRY DASHOW and AUGUST EPPLE

tation (Dashow et ul., 1982), which may be caused by hypovolemic shock and/or changes of the O&O, titers. Similarly, our findings in unanesthetized, cannulated lampreys (Dashow et al., 1982; Dashow and Epple, 1983) make it unlikely that CA titers seen during the hypovolemic response (Table 1) will ever be encountered under physiological conditions. Hence, the higher titers of DA and NE during the hypovolemia may be E-independent responses to a dropping pH in very slowly flowing blood. The fast and parallel increases of plasma CAs and glucose during the CO* treatment (Fig. 1) must be independent phenomena since glycemic changes, seen after very high doses of exogenous E (Dashow and Epple, 1983), require an essentially longer time span. Thus, while there is currently no convincing evidence of a direct role of CAs in the intermediary metabolism of the lamprey under physiological conditions, it is clear that these substances affect cardiovascular and respiratory functions in both cyclostomes (Rovainen, 1980, 1982; Macey et al., 1984) and higher vertebrates (Lightman, 1979; Ghosh, 1980; Usdin et al., 1980; Laurent et al., 1983). The proposed interactions of the CAs in the control of cardiovascular and branchial functions of the lamprey are summarized in Fig. 2. Acknowledgements~These studies were supported by PHS grants 5ROl AGO1 148, I T35 HL07497 and 5 SO7 RR05414 and NSF grant PCM 8209263. The excellent technical assistance of B. Nibbio and H. Kahn and the skillful typing of S. Parsons are gratefully acknowledged.

REFERENCES Beringer T. and Hadek R. (1972) Cardiac innervation in the lamprey. Petromyzon murinus. Anat. Rec. 172, 269-270. Back P. (1982) The paraganglia. In Hundbuch mikrosk. Anal. Menschen VI 18 pp. l-31 5. Springer, Berlin. Caravita S. and Coscia L. (1966) Les cellules chromaffines du coeur de la lamproie (Lumpetra zunadreai). Etude au microscope &lCctronique avant et apr&s un traitement g la rtsperpine. Arch. Biol., Paris 77, 723-753. Coupland R. E. (1979) Catecholamines. In Hormones and Evolution (Edited by Barrington E. J. W.), Vol. 1, pp. 309-340. Academic Press, New York. Dashow L. and Epple A. (1983) Effects of exogenous catecholamines on plasma catecholamines and glucose in the sea lamprey, Petromyzon murinus. J. camp. Physiol. 152, 35-41. Dashow L., Epple A. and Nibbio B. (1982) Catecholamine in anadromous lampreys: baseline levels and stressinduced changes, with a note on cardiac cannulation. Gen. camp. Endow. 46, 500&504. Dashow L., Nibbio B. and Epple A. (1983) Circulating catecholamines: evolutionary aspects. Am. Zool. 23, 1011 (abstract). Djabali M. and Pit P. (1982) Effects of a and B adrenoceptors on branchial CAMP level in seawater mullet, Mugil cupito. Gen. camp. Endocr. 46, 193-199. Eddy F. B. (1981) Effects of stress on osmotic and ionic regulation in fish. In Stress and Fish (Edited by Pickering A. D.), pp. 77-102. Academic Press, London. Epple A. (1982) Functional principles of vertebrate endocrine systems. Verh. dtsch Zool. Ges. I 17-126. Epple A., Katz Y. and Dashow L. (1982) Stress effects on circulating steroids and catecholamines in fishes. Gen. camp. Endocr. 46, 410 (abstract).

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. Falck B., Mecklenburg C., Myhrberg H. and Persson H. (1966) Studies on adrenergic and cholinergic receptors in the isolated hearts of LumpetraJluuiutilis (Cyclostomata) and Pleuronectes platessu (Teleostei). Acta physiol. scund. 68, 64-71. Falkmer S., Thomas N. W. and Boquist L. (1974) Endocrinology of the cyclostomata. In Chemical Zoology (Edited by Florkin and Scheer), Vol. 8, pp. 194-257. Academic Press, London. Flnge R. (1972) The circulatory system. In The Biology cf Lampreys (Edited by Hardisty and Potter), Vol. 2, pp. 241-259. Academic Press, London. Ghosh A. (1980) Avian adrenal medulla: structure and function. In Auiun Endocrinology (Edited by Epple and Stetson), pp. 301-318. Academic Press, New York. Hardisty M. W. (1979) Biology of the Cyclostomes. Chapman & Hall, London. Jones D. R. and Randall D. J. (1978) The respiratory and circulatory systems during exercise. In Fish Physiology (Edited by Hoar and Randall), Vol. VII, pp. 425-501. Academic Press, London. Laurent P., Holmgren S. and Nilsson S. (1983) Nervous and humoral control of the fish heart: structure and function. Camp. Biochem. Physiol. 76A, 525-542. Lightman S. (1979) The adrenal medulla. In Comprehensive Endocrinology (Edited by James V. H. T.), pp. 283-307. Raven Press, New York. Lignon J. M. (1979) Responses to sympathetic drugs in the ammocoete heart: probable influence of the small intensely fluorescent (SIF) cells. J. molec. cell. Cardiol. 11, 447-465. Lignon J. M. and LeDouarin G. (1978) Small intensely fluorescent (SIF) cells and myocardial cells in the ammocoete heart: a correlative histofluorescence, light and electron microscopic study with special reference to the action of reserpine. Biol. CeNul. 31, 169-176. Macey D. J.. Epple A., Potter J. C. and Hilliard R. W. (1984) The effect of catecholamines on branchial and cardiac electrical recordings in adult lampreys Geotriu uustrulis. Camp. Biochem. Physiol. 79C, 295-300. Mazeaud M. M. (1969) Adrknalinemie et noradr&nalinemie chez la lamproie marine (Petromyzon murinus L). C.r. Seunc. Sot. Biol. 163, 349-352. Mazeaud M. M. and Mazeaud F. (1981) Adrenergic responses to stress in fish. In Stress and Fish (Edited by Pickering A. D.), pp. 49-70. Academic Press, London. Nilsson S. (1983) Autonomic nerve function in the vertebrates. In Zoophysiology, Vol. 13. Springer, Berlin. Paiement J. M. and McMillan D. B. (1975) The extracardiac chromaffin cells of larval lampreys. Gen. camp. Endocr. 27, 495-508. Phillippe M. (1983) Fetal catecholamines. Am. J. Obstet. Gynecol. 146, 840-855. Pit P. and Djabdli M. (1982) Effects of catecholamines on cyclic AMP in the gill of seawater-adapted mullet, Magi/ cupito. Gen. camp. Endocr. 46, 184-192. Plisetskaya E. M. and Prozorovskaya M. P. (1971) Catecholamines in the blood and cardiac muscle of the lamprey, Lampetra ffuuiutilis, during insulin hypoglycaemia. Zh..Eool. Biokhim. Fisiol. 7, 101-103. Rovainen C. M. (1980) Neurouhvsiologv of lamprevs. Can. J. Fish. Ayuat. Sci. ‘37, 172j%i738. -. _ . Rovainen C. M. (1982) Neurophysiology. In The Biology qf Lampreys (Edited by Hardisty and Potter), Vol. 4a, pp. l-136. Academic Press, London. Usdin E., Kvetnansky R. and Kopin I. J. (1980) Catechoirmines and Stress: Recent Aduunces. Elsevier/North Holland, New York.