Differential patterns of relaxation by atrial natriuretic peptide in major blood vessels of two distantly related teleosts

ELSEVIER

Regulator) Peptldes 53 (1994) 89-101

Differential patterns of relaxation by atrial natriuretic peptide in major blood vessels of two distantly related teleosts A Sverdrup*, K B Helle Department of Phystolog) Umversttt of Bergen .4rstadvewn 19 5009 Bergen, Norwa) Recewed 24 February 1994, rewsed version received and accepted 2 June 1994

Abstract In order to elucidate the role of the atrial hormone m the teleost circulation, the vascular effects of atrial natrmretlc peptlde (ANP) have been compared m major blood vessels of the cod (Gadus morhua) and of Atlantic salmon (Salmo salar) The relaxing effects of ANP from eel (eANP) have been examined in ventral aorta (VA) versus dorsal aorta (DA) m the cod and m VA versus the coellaco mesenterlc artery (CMA), a major branch of DA, in the salmon The vessels were precontracted by acetylchohne (ACh) and adrenaline (A) and by the mammalian endothehum-derlved vasoconstrictor peptlde endothehn-I (ET-1) The role of endothelial integrity for these responses has been assessed m vessels either mechanically probed or chemically impaired by mdomethacln or the L-argmme analogue, NG-monomethyl-L-argmlne (LN MM A ) Adrenaline and noradrenahne (NA) failed to contract the salmon VA In the salmon vessels, eANP was without relaxing effects m ACh-contracted VA, while completely relaxing CMA when precontracted with ACh The eANP was also a relaxant of A-contracted CMA and of ET-l-contracted VA The cod vessels, which were insensitive to ACh were markedly relaxed by eANP when precontracted with either A (VA) or ET-1 (DA) In DA also the resting tension was reversed, an effect of eANP that was highly potentmted in mechanical probed vessels Otherwise the relaxing effects of eANP in these vessels were seemingly independent of endothehal factors In conclusxon ANP ~s not a general relaxant of the precontracted VA whxch m teleosts is the first possible target vessel for the myocardial release of th~s hormone On the arterial s~de ANP serves as a relaxant both m the salmon and the cod vessels, indicating that myocar&al release of ANP m teleosts may have an important role in regulation of blood flow wa dwerse, species-specific effects on major blood vessels on both sides of the gills

Key words Atrial natrmretlc peptxde, Endothehum, Indomethacln, Teleost, Acetylchohne, Adrenahne, Endothehn-1

* Corresponding author 0167-0115/94/$7 00 © 1994 Elsevier Science B V All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 5 0 - 8

90

~ %eJd*tq~

h B Helle

Reeulatml Peptules 5 ~ , 1994J ,~9-i01

1. Introduction The myocardlum of teleosts are lake the atraal m)ocardlum of higher vertebrates abundant in secretory organelles associated with storage of atrial natrluretic peptade precursors [1-5] Sance 1983 It has been establashed that the mammalian heart is an endocrine organ which supplies the circulation with the polypeptlde hormone, commonly referred to as atrial natrluretlc peptide (ANP) [6-10] High levels of immunoreactlve A N P have been measured in the circulation of teleosts and a role for A N P in gill haemodynamlcs has been postulated [ 11 ] Unlike in mammals, the teleost heart pumps only venous blood which enters the gills via the ventral aorta (VA) The blood pressure in the VA is thus higher than in the dorsal aorta (DAy [12-16] which collects the oxygenated blood leaving the gills The venous pressures are also lower in teleosts than m mammals, often negative or close to the ambient pressure [ 17] It is therefore likely that the postulated role of A N P In gill haemodynamlcs may also include A N P regulated blood flow through the major vessels supplying and draining the galls The role of A N P in teleosts has been examined in a few species A N P appears to serve as a potent relaxant in vitro an some larger blood vessels of some marine teleosts, e g . in the VA of the Gulf toadfish (Opsanu~ hera) [11] and In the coehaco mesenterac artery (CMA) of the steelhead trout (Sahno gatrdnerl) [18] Moreover, marked differences in autonomic regulation and effects of A N P on VA versus DA and its major arterial branches were also indicated from in vitro studies of salmon vessels [19] ANP produces hypotension in the perfused VA and DA of the cod [20] and in the perfused DA of the eel, from ~hlch A N P has been isolated and sequenced [21] This eel A N P (eANP) exhibited a 100-fold higher potency than the mammalaan homologue in the teleost vessels [21] On the other hand, lnjectaon of extracts from trout atria and ventricles into DA from ralnbo~ trout (S gatrdnert) increased rather than decreased the blood pressure DA [22]

The present stud) alans at a comparason of effects of a teleost ANP such as eANP. on VA versus DA of the cod (Gadu~ mothua) and VA versus CMA of the Atlanuc salmon (Sahm~ salary In order to assess the role of eANP in regulation of blood flo~ to and from the gills, b) an in vitro approach, the ~essels have been constricted b) parasympathetic and adrenerglc agonlsts and by the mammalian endothehum-derived vasoconstrictor peptlde endothehn-1 (ET-1) The Iole of endothelial lntegrlt? for these responses has been assessed in vessels either mechanically probed or chemically impaired b) lndomethacm or the L-arglnlne analogue. A~'-monometh)l-L-arglnlne (L-NMM ~)

2. Materials and methods

2 1 ~tmmah Atlantic salmon (S salad) were reared from the two year old smolt stage to 1-1 5 kg for 5-8 months Cod (G morhua) were obtained as 2-21 2 years old welghang 1 5-3 5 kg from a fish farm and kept for 2 weeks in the Bergen High Technology Centre in 7 m ~ tanks (1 m deep) with running sea-water (10 + 1 : C) Both species had free access to a commercial maxture of wet and dry pellets The fish ~ere lifted out of the water one b) one and killed by the Japanese '1kl jlme" method [23], by quickly piercing the scull wath a sharp, arrow shaped scalpel before immersing the fish in ice for transport to the laboratory

2 2 Dt ~set non Within less than 10 mln, the cardiac cavity was opened VA was dissected free and cut proxamal to the bulbus arterlosus From both species ~ 4 mm long segments were taken The inner diameter uas 1 mm in the salmon V/k and ~ 2 mm an the cod VA The cod DA was dassected free and a ~ 6 mm long segment was cut approximately 2 rain caudal to ~.~uh~s ~ephah~us (inner diameter ~ 1 ram) A lon-

A Sverdrup, K B Helle / Regulator) Pepttdes53 (1994) 89-101

gltudinal elastic ligament in the salmon DA, analogous to a ligaments found in herring and trout [24,25], was observed, which prevented its use for tension recordings The salmon C M A was therefore chosen as a major arterial branch of DA CMA was dissected free and was cut distal to the arborlzatlon of the 3rd and 4th efferent branchial arteries and the subclavian artery, as a 6 mm long segment (inner diameter ~ 0,5-1 mm) as close to DA as possible The vessel segments were immediately placed in oxygenated fish Ringer (4°C) before carefully removing connective tissue adhering to the adventltm 2 3 Thefish Rmger

The fish Ringer solution [26] contmned in mM, NaC1, 110, KC1,4 8, N a H C O 3, 15, NaH2PO 4 H20, 2 5, MgSO 4 7H20, 1 25, CaC12, 1 5, glucose, 5 55 and ascorblc acid 14 # M The solution was oxygenated with 99~o 02 and 1~o CO2, thermostatically controlled at 10 _ 1 ° C The p H was finally adjusted to 7 55-7 60 with 1 ~o N a O H For depolarlsatlon of the vessel segments, K + in the medium was increased to 80 m M by substituting 76 2 m M of the NaC1 with equivalent amounts of KC1 2 4 Tension recordings

Each vessel segment was divided in two ( ~ 2 - 2 5 m m long), one with intact endothehum ( + E) and the other with probed endothehum ( - E) The segments were mounted in organ baths (2 5 ml) which were thermostatically controlled at 10-11°C The rings were placed over two parallel platinum wires connected at right angels to the tip of stainless steel bars, one stationary and the other connected to a Grass Force Displacement Transducer (FT 03C) coupled to a Grass polygraph (RPS 7A 8A) with Amplifiers (model 7P 122) [27] The time from stabbing the fish until the vessels were mounted was < 30 mln A preload of 24 m N (2 4 g) was found to be optimal for both VA and C M A of the salmon and was applied in three steps of 8 m N every 30 mln There-

91

after these vessels were equilibrated for 30 mln before onset of experiments At this preload the developed tension of the unprobed segments ( + E) to depolansatlon in 80 m M K + was markedly lower in VA(60+03mN, n=6) thanlnCMA(135+l 1 mN, n = 6) The response to high K + was used as an internal reference for expression of the relative effect of the agonlsts in each segment The segments were washed by 4 changes of medium, returning tension to baseline, usually within 90 mln for VA and 40 mln for C M A A preload of 16 m N was found to be optimal for both VA and DA segments in the cod, giving reference responses to 80 m M K + of the unprobed vessels ( + E ) of 5 0 + 0 5 m N ( n = 6 ) in VA and 5 5 + 0 5 m N (n = 6) in DA The preload was applied In two steps of 8 m N at 30 mln intervals and equilibrated for 30 mln before the response to 80 mM K + was determined The medium was changed five times and the baseline tension was re-estabhshed within 90 mln 2 5 Addttlon ofagomsts

Addition of agonlsts was begun > 3 h after the blood vessel segments had been mounted Each segment was routinely exposed to one vasoconstrictor added stepwlse in increasing concentrations In order to relax the maximally constricted vessels increasing concentrations of eANP was added every 10 mln (in salmon) and every 15 mln (in cod) Acetylchohne (ACh) was added every 6 mln, adrenaline (A) every 4 mln and ET-1 was added at 15 mln Intervals to the salmon VA and C M A In the cod, A was added every 15 mln and ET-1 every 20 mm to the VA and DA ACh (70/~M) was without effect in either of the cod vessels The same protocols for agonlst addition were carried out on the paired segments of unprobed ( + E) and probed ( - E) salmon and cod vessels The protocols were also repeated for unprobed vessel segments, one series being exposed to the cyclooxygenase inhibitor, indomethacln (2 4/~M) and another series to L-NMMA (40 #M)

92

4 S~e~drup 1~ B Helle, Regulatori Pepttde~ 53 H994) 89-101

These drugs were added 20 mm before addition of the constrictor agonlst and were present throughout the contraction cycle In a separate experiment with A as vasoconstrictor, propanolol (1 6 /~M) was added to the fish Ringer medium In separate series the salmon and cod vessels were examined for fl-adrenerglc relaxation by noradrenahne (NA) and lsoprenahne in the first and second cycle of K +-evoked contractions, respectively The constrictor responses were measured m mN, with mean values obtained for vessels from 6 fish Relaxation was expressed aS°o dechne m the ma,~lmal tension evoked by the given agonlst

A

unprobed VA

, 8

s mm

...............................................

I i 0 1 03

l 1

l 8

l 74

eANP(nM) 36 120

=lmHHllh

l

l

I

ACh (p.M) eANP (nM)

5~mln probed VA 1o mN

[

i ......

01 i

36

jJ

i .......

: ........

03

1

i.......

8

i ......

74

i .........

i ..........

i~o

I

q

ACh (gM) 5

unprobed CMA

eANP (nM) 36 1 ;~0

8

mm

2 6 Morphology ,o

In order to assess the degree of endothehal lnjur 3, as a result of endothehal probing and of the subsequent tension recording procedures, series of vessel segments were examined before and after the drug experiments [28,29]

i .......

i .......

0 1 03

L........

1

( ......

8

i ......

i .........

I/tll

i

74

ACh (izM) 5 mm probed CMA

eANP (nM)

10mN l'--'~

8

36

1;~0

2 7 Chenucah

Acetylchohne, adrenahne, atropine, noradrenahne and lsoprenahne were purchased from the Norwegian PharmaceuUcal Association (NAF) Propanolol (Inderal) was purchased from ICI Pharmaceutacals, U K Endothehn-1, L - N M M A and synthetic atrial natrmreUc peptlde from eel were obtained from Peninsula Laboratories I n c , USA, and were dissolved in 0 01 M C H 3 C O O H and dduted m destllled H . O Indomethacln (Confomd, Dumex, Denmark) was dissolved in destdled H 2 0 and &luted m the fish Ringer soluUon apphed Stock solutions of all drugs were stored at - 2 0 °C 2 8 Stattsttcs

Significance for difference between data was calculated by Wdcoxon rank test for mdependent and dependent groups Data are expressed as the mean + S E M of (n) observations ( * P < 0 05)

01

03

1

8

74

ACh (gM)

Fig 1 ~tlantlc salmon (A) \ cntral aorta ('V A) and (B) coehaco mesenterlc arter) ( C M ~ ) Both ~essels were contracted b~ mcreasing concentrations of acet~lchohne (ACh) and thereafter relaxed b'~ increasing concentrations of eel atrial natrmreuc pepude (eANP) Concentrauon-response cycles uere compared for unprobed and probed vessel segments

3. Results Relaxation by A N P was examined m precontracted vessels for comparison of the effects and potency of the eANP on the maximal vasocontractlons evoked by ACh, A and ET-1 These agonIsts eltclt both species- and region-specific patterns of vasoconstriction m the vessel segments presentl} under examination [ 19,29] In order to compare the effects of eANP concentraUon response curves were

93

A Sverdrup K B Helle / Regulatory Pepttdes 53 (1994) 89-101

3 1 The salmon VA and CMA

m the intact than m the e n d o t h e h u m - p r o b e d vessel segments (Fig 2), with an EDso value for e A N P of 5 n M in the u n p r o b e d vessel In the presence o f l n d o m e t h a c m there was no change, either m the absence o f e A N P relaxation m VA or in the patterns o f relaxation m C M A when the A C h c o n t r a c t e d vessels were examined (not shown)

AceO'lchohne (A Ch)

Adrenahne (A)

A C h was a forceful v a s o c o n s t r i c t o r o f both vessels (Fig I A and B), the highest potency for A C h being o b t a i n e d in VA The A C h - e v o k e d response was abolished in the presence o f atropine m both vessels In VA e A N P was without relaxing power on A C h - e v o k e d c o n t r a c t i o n s In u n p r o b e d segments, while a slight relaxation was detected after mechanical probing o f the e n d o t h e h u m (Fig 1A and Table 1) The sustained contractions o f VA was finally relaxed s o m e w h a t when xsoprenahne (1 6/~M) was a d d e d to the e A N P exposed segments, 17 + 2°,0, n = 6 in u n p r o b e d vessels versus 15 + 2~o, n = 6 after probing In c o n t r a s t C M A was completely relaxed by e A N P (Fig 1B), with a s o m e w h a t higher potency

In VA the catecholamlnes (A and N A ) were without v a s o c o n s t r i c t o r effects W h e n c o n t r a c t e d by high K + VA was partially relaxed by N A (Table 2) Following N A exposure the r e s p o n s e to high K ÷ was slgmficantly reduced, despite a ~ 50 mln p e n o d o f washing between the cycles In the second cycle the K ÷ - c o n t r a c t e d VA was almost completely relaxed by the c a t e c h o l a m m e agonlst lsoprenallne In C M A A was the m o s t p o t e n t constrictor (Table 1) In n M concentrations N A partially relaxed the K + - e v o k e d c o n t r a c t i o n o f C M A (Table 2), while at higher c o n c e n t r a t i o n s N A was a constrictor, although w e a k e r than A (Table 1 a n d 2) The K ÷ -contractions in C M A were completely re-

obtained for each v a s o c o n s t r i c t o r and for the subsequent relaxation by e A N P o f the maximally evoked c o n t r a c t i o n I n d o m e t h a c l n and L - N M M A were a d d e d to u n p r o b e d vessels in separate series d u n n g the c o n t r a c t i o n with the v a s o c o n s t r i c t o r m question

Table 1 Relaxing effect of atrial natnuret~c peptlde in blood vessels of the Atlantic salmon

E

(mN)

°o Relaxation by eANP

ACh

A

ET- 1

ACh

A

ET- 1

142+01 112+03

(-) 17 1_+13~ _ 153+12b I

76_+01 (-)

3+1 93+5

(O) 21+8 ~ 32 _+7b

41_+6 (O)

+E VA CMA -E VA CMA

.....

*[-

L

13+10 5 1+ 1 5

(+)1 I-- 12 23 *L 94+10 b

,

* 61_+08 (-)

~] 8+2 ~ 87 + 16

(0~,,

35 +_ 47+7 b

57 +6 (O)

With propanolol (1 6/IM) added to the fish Ringer, b without propanolol Segments of the ventral aorta (VA) and dorsal aorta (DA) were examined before ( + E) and after (-E) probing of the endothehal hnmg The vessels were maximally contracted by agomsts and the response (Emax) lS given m mN Each vessel was relaxed by addzUon of atrial natrmretlc peptlde from eel (eANP) The relaxation obtained for 36 nM eANP and are shown and expressed m ° o decline of the maximal tension response of the respectwe agomst, of acetylchohne (ACh), 74 #M, of adrenahne (A), 33/~M and of endothehn (ET-1), 0 7 #M Values are means + S E M for vessels for (n = 6) fish significance for &fference (*P<0 05), no response (-), not tested (O)

4 ~terdrup It B Helle Regulatott Pepttde~ 53 11994) 89-101

94

laxed by lsoprenahne (Table 2) In the presence of the/~-agonlst (propanolol, 1 6/~M) the response to A (33 # M ) was insignificantly potentiated A potentiating effect of propanolol was more pronounced after mechanical probing of the endothehum (Table 1) The maximal contractions evoked by a high concentration of A ~ere partially relaxed by 36 nM eANP (Table 1) Although, mechanical probing of the endothehum resulted in considerable loss of contractile force evoked by the maximally effecUve concentration of A, there was no significant change in relative relaxation by eANP (Table 1) Indomethacln and L - N M M A did not alter the relaxing efficacy of 36 nM eANP on the A c o n t r a c t e d CMA The corresponding relaxations were 25 + 7 °o (n = 6) and 15 _+6°° (n = 6) with lndomethacln and L N M M A respectively, and were not significantly different from the control value (21 + 8°0, Table 1) O"

c 0

,o tO

<

30

E E

60"

AZ

"6 90" ID

n 27 <

120 -1 0

- '9

- '8

- '7

'6

Log [eANPI Fig 2 Relaxing effect of eel atr~onatrmreUc pepUde (eANP) on pre-contracted coehaco mesentenc arter'~ (CMA) from the 4tlantlc salmon ConcentraUon-response curves of e 4 N P were compared for unprobed ( + E) and probed ( - E) vessel segments eANP was added to the CMA precontracted v,~th acetylchollne (ACh) (E ..... XCh -- 74 /xM) The relaxaUon effects are expressed as "o relaxaUon of the maximal ACh-contracUon Values are means_+SEM (n-6)

The mammahan endothehn-1 ( E T 1 ) In VA ET-1 was a modest vasoconstrictor (Table 1), an effect which was not sustained and not significantly affected by endothehal probing S i m l larly, neither indomethacin nor L N M M A altered the rise in tension to E T 1 (not shown) In the E T l c o n t r a c t e d VA eANP was a partial relaxant This effect by eANP was not significantly reduced in segments pre incubated with lndomethacin (28 + 12"o, n = 6 ) or L - N M M A to the fish Ringer medmm (42 + 6°o, n = 6) 3 2 The cod VA and DA In these vessels ACh was without contractile e f fects up to 7 4 gM, independent of the state of the endothehum Isoprenahne (2 0 g M ) &d not give significant relaxation in VA or DA when preconstricted with high K ~ or ~ (not shown) Neither propanplol nor atropln was therefore added in the present e x perlments with the cod vessels Adrenahne (A) Adrenaline was a potent constrictor of VA (Fig 3A and Table 3) and this contraction was completely relaxed by eANP independent of endothelial probing, with a EDs0 of ~ 50 nM (Figs 3A and 4) Indomethacln and L-NMMA both enhanced the A e v o k e d constrictions m VA, but did not alter the relaxing effects of eANP (not shown) In DA the vasoconstriction evoked by A was weak (Fig 3B and Table 3) When eANP was added at 28 nM there ~ a s a dramatic decline in total tension which counteracted a considerable portion of the resting tension (Fig 3B and Fig 5) In the m e c h a n l call) probed DA the resting tension was completely abolished by eANP in the nM range Mammahan endothehn-i (ET-I ) In VA ET- 1 was a weak constrictor pepttde which was independent of the integrity of the endothehum (Table 3) These constrictions was partly relaxed by eANP (110 nM, Table 3)

A Sverdrup, K B Helle / Regulatory Pepndes 53 (1994) 89-101

95

Table 2 Relaxing effects of noradrenahne and lsoprenaline m blood vessels of the atlantic salmon Ventral aorta

Coehaco mesenterlc artery

unprobed mN

Probed mN

unprobed mN

probed mN

Klcyce 6_+031 36_+061 13+L1 noradrenahne 07gM 77/~M 32/~M h l g h K ÷ (2 cycle) Isoprenahne 04#M 16~ 5~M

57±20

°o relaxation

7+2 29+5 36__.6 mN 44+04

* * *

ns * *

3 1±7 -4±3 a -4±3" mN * 147±09 0o relaxatmn

*

35 ± 10 - 2 4 ± 10~ - 2 4 ± 10a mN 54±2 1

*

*

3±2 27+3 37±4 mN 22±04

21±6 48±7 77±7

* * *

25±6 62±8 88±8

* * *

* * *

74±6 84±7 96±4

* * *

67±3 85±3 93±2

a Contractions Segments of the ventral aorta and the colllace mesentenc artery were examined before ( + E) and after ( - E ) mechamcal probing of the lumen The vessels were maximally contracted by two cycles of high K + (80/IM) Each vessel was exposed to stepwlse increasing concentrations of noradrenaline of isoprenahne The responses to high K + are given in raN, and the catecholamlne agonists are expressed in ° o dechne of the mammal tension responce to K* Values are means _+S E M for vessels for 6 fish Slgmficance for difference P < 0 05 (*), not significant (ns)

ET-1 was, on the other hand, the most potent vasoconstrictor of DA, an effect that was sustmned and only weakly relaxed by eANP (110 nM, Table 3) Indomethacln significantly enhanced the ET- 1 evoked contractions of DA whereas L-NMMA was without effect (not shown) The relaxations by eANP was not modified by indomethacin (24 + 3 O/ ,o, n = 6)

4. Discussion In teleosts VA is the first possible target vessel for the myocardial release of ANP, analogous to the pulmonary artery of mr breathing vertebrates The present experiments clearly show that eANP is not a general relaxant of the precontracted VA in teleosts On the arterial side, in DA and major branches

such as CMA, eANP appears to be a potent relaxant in two distant related teleosts such as the salmon and the cod, consistent with previous findings for CMA in the trout [18] However, the conclusion that the vascular effects of A N P is similar in fish and mammals [18] is not substantiated by the present findings of diverse effects of eANP on these salmon and cod vessels It is noteworthy that eANP is without relaxing effect in the salmon VA which IS forcefully contracted by ACh, in accordance with previous reports for this [ 19,29] and other salmonldes [30-33] As presently shown both the high K + - and ACh-evoked contractions in the salmon VA were only moderately relaxed by the catecholamxne hormones ACh emerges as the most potent vasoconstrictor of the salmon VA for which neither eANP nor the catecholamines are potent direct relaxants This is in contrast to a recent

4 5)erdrup k B Helle, Regulator~ Pepttdes 53 (1994) 89-101

96

eANP (nM)

A

3b

unprobed VA

10,

~1o

I'""'"'

~

H

1

HH

3

mHI

,,

I

,,

[mN

1

,~

, ,,

,<

2o

eANP (nM)

,

03

,

5 mln

3~

probed VA

,H H

, ,

3O

10

A (,uM)

0

"6

/

03

10

0

4~o

3

,,,, H

|

10

30

11o

46o ,

E

H,

i

,,,i

5 mln

40E ..c

A (gM)

"5 60g

unprobed DA 101 0 mN

30

eANP tnM) 110 ~0

-E so-

3

0'3

~o

3o

" ' 5 mln

A (~M)

10 rmN

probed DA

-10

30 I'"

0 3

"'

i

1

3

.,H

10

H

i

:30

110

400 i

z < 100 -10

oANP(nM)

O~

HH

5 n'lln

A ruM)

Fig 3 Cod (A) Ventral aorta (Va.) and (B) dorsal aorta (DA) Both vessels were contracted b~ increasing concentrations of adrenahne (A) and thereafter relaxed b) increasing concentrations of eel atrial namuretlc peptlde (eANP) Concentrationresponse c)cles were compared for unprobed and probed vessel segments

report [34] of relaxant effects of both mammalian A N P and the purified atrial and ventrlcular extracts from another teleost (Mvoaocephalus scorpms) when tested on its own aorta precontracted wtth either A or ACh Thus, species dependent differences with respect to A N P relaxation of the aortas are evident Mechanical or chemical impairment of the endothehum did not reveal any potent E D R F m VA A contrlbuUon of the endothehum towards reversing the weak eANP effect on the ACh-contracted VA was, however, m&cated The sustained contractaons to ACh which have not prewously been reported for other teleost or mammahan vessels with intact or probed endothehum, bears, on the other hand, similarities to the pattern of sustained ET- 1-evoked contracUons in mammahan blood vessels [35], but are clearly &fferent from the ET-1 evoked, reversible

+E

13.

-9

-'8 Log [eANP]

7

-6

Fig 4 Relaxmg effect of cel atrlonatrluretlc peptlde (eANP) on pre-contracted ventral aorta (VA) from the Cod Concentrationresponse cur, es ofeANP ,Aere compared for unprobed ( + E) and probed ( - E) vessel segments eA.NP ~as added to the VA precontracted with adrenaline (A) (Em~, x - 33 FM) The relaxation effects are expressed as"o relaxation of the maximal A,. contraction Values are means _+S E M (n = 6)

constriction of the salmon VA The mechamsm behind these sustained ACh-evoked contractions remmns a toptc for future studies In the cod, VA serves as the first effective target for the released ANP, and the A-evoked contractions in DA were completely relaxed in an endothehum-dependent manner The effect of eANP in DA was thus more dramatic, totally reversing not only the modest contractions evoked b~ A, but also reducing the resting tension Somewhat similar effect of A N P has been reported for the toadfish VA and the trout CMA, being relaxed below the resting tenslon without previous agonlst-lnduced constriction [ 11,18] Such dechnes in resting tension suggests an eANP-induced hyperpolarlzaUon of the smooth muscle cell membrane, quite unhke A N P effects so far reported for the mammahan aorta [36-39] Mammahan blood vessels also reveal heterogeneous responses to A N P [40,41], inaklng it difficult

97

A Sverdrup, K B Helle / Regulatory Pepndes 53 (1994) 89-I01 Table 3 Relaxing effect of atrml natnureUc pept~de in blood vessels of the cod Oo relaxation by eANP

E .... (mN)

ACh

A

ET- l

ACh

A

ET- 1

[--92+09 2 1+0 6

33_+07 I 11 9_+ 1 5

(O) (O)

65_+6 1 194_+56

49+17 15_+4

- 4 7_+0 9 1 0_+0 1

2 0+0 4 6 8~0 9

(©) (O)

59+ l0 462+ 127

+E VA DA

(-) (-)

-E

VA DA

*

(-) (-)

*

*

65_+21 17_+4

Segments of the ventral aorta (VA) and dorsal aorta (DA) were examined before ( + E) and after (-E) probing of the endothehal hnmg The vessels were maximally contracted by agonlsts and the response (Emax) is given In mN Each vessel was relaxed by addltmn of atnal natrluretlc pepude from eel (eANP) The relaxaUon obtained for 110 nM eANP are shown and expressed m °o dechne of the maximal tension response of the respective agonlst, of adrenahne (A), 33/aM and of endothehn (ET-1), 0 15/aM Values are means + S E M for vessels for (n = 6) fish Slgmficance for &fference (* P < 0 05), no response (-), not tested (O)

to demonstrate a role for this myocardial hormone in regulation of peripheral resistance (see review by Brenner [42]) For instance, the most potent relaxmg effect of A N P in the rat was found m the endothehum-denuded pulmonary artery pre-contractedwlththe ~l-analogue methoxamine [43] This vessel is the first vascular target for the release of A N P from the right atrium and is homologous to the teleost VA On the other hand, A N P does not relax the coronary arteries of guinea pigs [44] or the mesenterlc arteries of the rabbit [45] It is therefore hkely that A N P is a selective relaxant primarily of major vessels such as the pulmonary artery [43] and the aorta [43,45,46] and some veins [27,45] The low potency for eANP in the cod VA, reverslng the A-evoked contraction, suggest that the threshold for A N P may be higher in the cod VA than in the pulmonary artery of mammals, possibly reflecting a higher concentration of A N P in the blood leaving the heart of the teleost than the right atrium of mammals In the rat proximal aorta but not the thoracic and abdominal regions, A N P is of minor importance as a relaxant in segments already precontracted with NA or methoxamlne [43] This is in contrast to the potent, direct effect of eANP m the

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100"

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E ¢1 E

300"

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400"

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500"

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600 -

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-10

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Log [eANP] Fig 5 Relaxing effect of eel atrlonatrmretlc peptlde (eANP) on pre-contracted dorsal aorta (DA) from the Cod ConcentraUonresponse curves ofeANP were compared for unprobed ( + E) and probed ( - E) vessel segments eANP was added to the DA precontracted with adrenahne (A) ( E m a x a = 33/aM) The relaxation effects are expressed aS°o relaxation of the maximal AcontracUon Values are means _+S E M (n = 6)

98

4 S~erdrup A B Helle, Regulatorr PeptMes 53 (I994) 89-10l

cod DA and the salmon CMA On the other hand, pre-exposure of the proximal aorta to A N P resulted in a long term Inhibition of subsequent contractions evoked by NA or methoxamlne, but with no loss of resting tension [43] A similar pattern has not so far been obtained for contractions evoked b) A in the salmon C M A (n = 6, not shown) A u lde range of vascular effects may thus be elicited b) ANP, not only among mammals, but also among teleost species The reproduclblhty of the developed tension ~ as high In the series of blood vessels examined from the cod as well as from the salmon Morphologically the probing procedure, by gently t~lStlng a catheter in the lumen of the segments, had not lesioned the vascular smooth muscle la)er belo~ the internal membrane, consistent with our previous reports [19,29] The marked losses m maximal tension resulting from mechanical probing was not a general phenomenon in these teleost vessels It was not evident in the salmon VA when pre-contracted x~lth ACh or ET- 1, but seen in some series with high K + Loss of force ,s as conspicuous for the salmon CMA with all constrictors, including high K + In the cod, mechanical probing affected VA and DA to the same extent when exposed to high K + and the most potent constrictor for the respective vessels Taken together, these effects suggest that the vascular endothelium on both sides of the gills m the cod may produce endothelial-derived vasoconstrictor substances (EDCFs), so fai unidentified It is not unhkely to assume that also the gill endothehum ma) produce similar EDCFs, notabl) m the cod In the salmon, endothelial constriction of EDCFs was apparent mainl? on the arterial side of the gills and ma) thus reflect another significant distinction between the cod and the salmon vasculature In the salmon clear indications of endothelial derived relaxing factors (EDRFs) of prostanolc origin were obtained only for CMA, while ewdence for E D R F s of non-prostanolc origin ~as not detected, in accordance with previous findings in trout [33] In the cod, on the other hand, E D R F s of both pros-

tanolc and non-prostanolc origin were indicated in the VA, while lndomethacln-sensltlVe E D R F s were indicated in the DA Thus, the cod endothehum could be distinguished from that in the salmon by its apparent production of not only E D C F s but also of E D R F s on both sides of the gills The effects of eANP being largely independent of the endothehum in the cod VA, was in contrast dramatically dependent on the endothelial lntegrat) in DA In this \ essel the intact endothellum apparently counteracted the pronounced loss in resting tension m response to eANP in the mechanxcall) probed DA, possibly b) release of EDCFs The results of these in vitro experiments on the cod vessels make it evident that eANP ma) act on both sides of the gills, although b) different mechanisms Our results are thus consistent with the report on marked h)potenslon Induced b) A N F when injected into the cod circulation causing declines in both VA and DA blood pressures [20] The lack of eANP relaxation of the salmon VA precontracted by ACh, and weak relaxation b) lSOprenallne, strongly suggest that, unhke in the cod, the salmon VA is not a target for either the myocardial release of A N P or the head kldne) release of catecholamlnes The blood pressures in salmon VA is not )et known, but ma) differ from that in the cod in which the mean pressure over the gills drops from 4 6 m m H g i n VA to 29 mmHg in DA [12,47] This in contrast to ralnbo~ trout from which mean pressure drops from 39 mmHg in VA to 31 mmHg in D A, have been reported [13,14] The salmonldes ma) thus have developed another strateg) for regulating blood flou through VA b) sustained contractions evoked b) the paras)mpathetlc neurotransmltter A s)nergism between/3-adrenerglc relaxation and ANP ~vas observed in the pulmonar) arter) of the rat [43] Whether a similar s)nerglsm exist fol relaxation of the salmon VA remains to be elucidated The ~ascular A N P receptor in teleosts is presumabl) coupled to the mcinbrane bound guanylatc c)clase, analogous to the nlammallan receptor [48,49] The functions of c G M P m cells are xersatlle de-

A Sverdrup, K B Helle / Regulatory PeptMe~ 53 (1994) 89-101

pending on the second messenger systems expressed and acttvated in each type of cells [42,50-52] A detailed study of the cellular mechanisms behmd the apparent hyperpolartzatlon presently observed with eANP on the A-exposed cod DA should be further elucidated, but lies outside of the scope of the present study It has been postulated that ANP may play a central role also m fish osmoregulatton [34,53,54] Receptors for ANP have so far been shown only in the kidney of the hagfish [55] The presence of ANPreceptors in the fish kidney and the role of ANP in osmoregulatlon thus remains to be elucidated In conclusion, the present findmgs show that the myocardial release of ANP m teleosts may have diverse and species-specific effects, as Illustrated m the major vessels taken from both sides of the gills In two species as distantly related as the cod and the Atlantic salmon These and other regions of the teleost vasculature emerge as fruitful models for comparative studtes, not only of the unidentified vasoconstrtctors and vasodtlators of endothelial origin [ 19,29], but also of the role of ANP and the mechanisms behind the diverse effects of this myocardial hormone

[ 2]

[ 3]

[ 4]

[ 5]

[ 6]

[ 7]

[ 8]

Acknowledgement [ 9]

This study was supported by grants from the Norweglan Fishery Research Council, from the Bergen Umverslty Research Foundation and from the Faculty of Science and Mathemetlcs at the Umverslty of Bergen Technical assistance from Reldar Handeg~rd and Jan Ove Wedaa at Bergen High Technology Centre and from Berlt Karln Hausvlk, Department of Anatomy and Cell biology, the University of Bergen is gratefully acknowledged

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99

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