Different binding activities of A- and B-type natriuretic hormones in the heart of two Antarctic teleosts, the red-blooded Trematomus bernacchii and the hemoglobinless Chionodraco hamatus

ISSN 0300-9629/97/$17.00 PI1 SO300-9629(97)00007-8

Camp. Biochem. Physiol. Vol. 118A, No. 4. pp. 993-999, 1997 Copyright 0 1947 Elsevier Science Inc. All rights reserved.

ELSEVIER

Different Binding Activities of A-- and B-type Natriuretic Hormones in the Heart of Two Antarctic Teleosts, the Red/blooded Trematomus bernacchii and the Hemoglobinless Chionodruco hamatus Maria Carmeh

rota*j

Cerru,“s?_ Mm-cello Cunonuco, # Ruffuele Aciemo, § and Bruno

DEPARTMENTS OF *CELLULAR BIOLOGY, ~PHARMACO-BIOLOGY, AND $ECOLOCY, UNIVERSITY OF CALABRIA,87030 ARC.A~ACATA DJRENUE.COSENZA,ITALY;~INSTITIJTE OF PHYSIOLOGY. UNIVERSITY OF LECCE,71100 LECC.E, ITALY;ANJ) 80121 NAIWLI,ITAL\ “ZOOLOGICAL STATION“A. DOHRN,”VILLACOMUNAI.E,

ABSTRACT. tative and

Different

autor&ography hemoglobinless

were found bulhus

endocardium

and outer

binding

myncardium

sites

and

mltriuretic

hulbar

sites

were

natriuretic

indicated

peptide

tcr natriuretic ity with

prptides

respect

3.2, 9.7 layer

contained

detected

in all cardiac

In the presence different

of either

displacement

KEY

WORDS.

Antarctic

Chionodmco

notothenioid

hamatus,

heart,

like that of the other different

mones

that with their potent

vasorelaxant

peptide

molecular

properties

teleosts,

(2.1

diuretic,

natriuretic

wall (5). The

z

type NP [ANP] and C-type

NP [CNP])

similar to the NE’s identified

in mammals,

horanJ

ians, namely,

NP (BNP)

ANP,

B-type

NPs (i.e., A-

are structurally

very

birds and amphiband CNP

(37).

0.7 <

Porcine

brain

atrtum,

whereas

KL, <

262

of the

vcnrricular natriuretic

the

f

ventricular

90 pM).

peptlde

natriuretlc

pxwided

In both

or p’cxcinc brain

peptide,

a higher

of T. bernncchii.

able t
Jisplaccment

capx-

HIOC‘HEM I’HYSIOL

COMP

, auroradiogr,rphy,

peptidr

In

addition, mammalian BNP, whose release and gene expression is controlled in a specific manner by mechanical and stimuli, was shown to act as an important cardiac particularly in the ventricle (7,23). In fact, its was shown to increases in some cardiovascular ad-

A&frc\s rcprmt reywst.s to: R. Tom, Zool,@al Station “A. Dohrn,” Villa Comunalc. R@l?l Nap&, Italy. Tel. 081-5833204; 0984-401302; Fax 0817641 355; 09X4-493 160; E-mad: t(tfH@pr~h,~X.unlcal.ir. Rece~vrd 0 M;rrch 19%; revised 18 July 1996; accepted 1 August 1996.

brain

matriurcric

receptors

or pathologies,

or hypertension

role in osmoregu-

piscine

respectively),

rat atria1 natriuretic

atria1 natnuretic

synthesizes

of natriuretic

thr

peptide

layers

of Two classes of high and IGXWaffinity I n contrast, in C. hdrnLzt(s, both high and

in the atrium

bemacchli,

Trematomtds

vertebrates,

forms

play an important

and the vascular

endocrme hormone, secretion

numbers 66 FM).

unlabeled

that

natriuretic

and outer

by a single class of high afhmty

1.3 pM,

regions

showed

quantibrrnacchil

Inc.

lation and cardiovascular homeostasis (1,14,29,35,38). The cardiovascular role of NPs includes a direct action on the heart

data

justments

and releases (NPs)

and inner

of both notothenioA,

INTRODUCTION

The tish heart,

sites for rat atria1

capacities.

regions

of rat atria1 natriuretic

z

elevated

118,4;4:993-999, 1997. 0 1997 Elsevier Science

pcptide,

6.2

Trematomus

bmding

were characterized

-t 2.3 and

using in km

were evaluated

the red-blooded

endocardium

of the saturation

2 0.6 < k;l < 209 f

sites in all the heart

to that

and speck

analysis

peptide

teleosts,

ventricular

layer of T. bernacchii bulbar

experiments

Saturable

Scatchard

sites (1.8

fishes, competition

notothenioid

myocardium,

14 2

inner

binding

binding

hamatus.

t;shes.

(K\I =

the

pepride

low affinity

of two antarctic

ventricular

of both

of ‘2SI-rat atria1 natriuretic

activities

Chionodraco

in the atrium,

arteriosus

peptide

binding

in the heart

such as cardiac

As demonstrated

in other

views),

also in teleost

specific

selectivity

vertebrates

fish the various

for the different

tide receptors

(NPRs)

used for the mammalian

(37).

a

pep-

Following

NPRs

the

(34), the two

in fish have been called NPR-A

and

on the bases of their binding potencies toward the NPs and their linkage to the production of cGMP

(12,19,33).

NPR-B

affinity

demonstrated

for the other

NPs and was shown whereas

(see 3 I, 4 for reNPs have revealed

so far identitied

types of NPR described

higher

and/

types of natriuretic

nomenclature NPR-C various

hypertrophy

(21,25,28).

NPR-C,

a low affinity

homc&>gous

to be coupled

which

binds

for ANP,

a

and heterologuus

to a guanylate-cyclase,

with similar

affinity

all types

of NPs, is a guanylate-cyclase free receptor (11,19,33). The identification of these different types of NPR in organs such as the gills, kidney and intestine has provided a molecular target for the osmoregulatory functions of NPs in fish (12,19,33,X). These receptors have nlso been described in the eel heart (9,19,36). It remains to he analyzed by which mechanisms the functional effects reported in rhe fish car-

M. C. Cerra et al.

994

diovascular

system,

using both homologous

gous NPs (2,30,38), of the ligands

may be explained

with these

and/or

a cryostat (Cryocut E, Reichert-Jung, Nublock, Austria), mounted onto gelatin-chromium aluminum-coated slides

heterolo-

by the interactions

two types of cardiac

and dried

NP receptors.

under

vacuum

at 4°C. The

sections

were stored

Two unique functional traits of the antarctic Notothenioid fish are of interest in relation to the physiological sig-

in sealed boxes at -20°C before use or were immediately used. Heart sections were treated as previously described (8)

nificance

with

their

of the system

different

increase

(about

(about

atomus

activity

newnesi dealing

(13,32). to 9-fold

volume

C. hamatus,

higher

than

t~s, 4.02 ml/kg) hypervolemic distension control

( 16). A second

[C. aceratus, ml/kg

teleosts

acting

condition,

(13-71

(data not published),

64-l

ml/kg

presence

aorta pressure pump

with

of binding

endemic

presence tively.

and sluggish

In both

in plasma ported

(43).

a major

the

given

hormone

ability

in displacing

and bulbus

homogenates

In addition,

ventricular

compared

bottom

Louis, MO, USA),

1-28 (rANP)

rANP

binding

of both

BNP

(pBNP)

in the atrium,

binding

were then

were incubated

T. berecotype respecmaterial re-

heart,

we

to that

of

ventricle

Germany).

on other parallel

cassettes

together

temperature.

with

After

incubated All sec-

X 4) in 50 mM

a stream

were

of cold

transferred

slides containing

Amersham, sheet

the film was developed dipped

sections

([‘T] micro-scales,

to

plastic

Buckinghamshire,

was apposed

an exposure

to the sections

period

of 6 days,

for 5 min in Kodak D- 19 developer, acetic

acid, vol/

vol) for 20 set, fixed for 2 min in Kodak Rapid rinsed for 5 min in distilled water at 18°C.

in a stop bath solution

Fixer and

The analysis relative

optical

the standard specific were

Jensities

binding

(SB) (TB)

the

quantity

equation

binding

(fmol/mg

on The

(NSB)]

p) on the

of 0.065 mg, which

content

found

in 1 mg of heart

(three

separate

determinations

data

were plotted

slope of the it was possible

converts based

standards.

from

nonspecific protein

protein

out by us-

that

wet tissue,

obtained -

to fmol/mg

saturation

system

from co-exposed

to the protein

The

was carried

analyzer

to fmol/mg

values

binding

converted

negative analyses,

image

curve derived

[(SB = total

(4% glacial

of the autoradiograms

ing a Zeiss “VIDAS”

for each animal) weighing

dried

New En-

Non-specitic

sections

for 8 min (2 min

and an “Ultrotilm”

at room

tissue.

Animals

St.

for 60 min

= 2200 Ci/mmol,

Dreieich,

the

bases of the calculated

(icetish)

(40 pus/ml), Company,

pH 7.4, at 4°C and dried under

standards

species.

and four C. hamatus

activity

Division,

cardboard

AND METHODS

Four T. bernacchii

bacitracin

Chemical

the sections

was determined

corresponds

MATERIALS

(Sigma

(specific

gland Nuclear

atria1

the

of C. 5 mM

(20°C) in the same buffer containing increasing concentrations (lo-” to lo-” M) of [‘L51]-rat atria1 natriuretic peptide

air. Subsequently,

of BNP as

in the mammalian

serum albumin,

(2 pg/ml)

Tris-HCl,

previously

the importance

of porcine

arteriosus

0.5% bovine

This

differ for the

has been

buffer

or 229 mM (for the buffer of T. bernacchii),

chymostatin

in

(for the buffer

(C. hama(41).

blood,

of ANP-like

aminomethane)-HCl

176 mM NaCl

of

(22-24°C)

washed

in the atrium,

dwellers, in their

the occurrence

and heart

MgCL,

a preincubation

tions

we investigated

of hemoglobin

fishes,

hamatus)

after

temperature

(C. hama-

remarkable

(rANI’)

(hydroxymethyl

7.4) containing

U.K.)

sites for rat ANP

or absence

(pH

Briefly,

X 2) at room

of 10-h M of cold rat ANP-(1-28).

seems to rely on a particular

considerations,

modifications.

(10 min

in the presence

ventricle and bulbus arteriosus of two notothenioids, nacchii and C. hamatus, that although show similar

rANP

[31]),

of NPs hormones.

On the basis of these

being

10

with that of small

as volume

associated

blood

(3)] is from 2-

large stroke volumes

low ventral

thus

(or icefish), in their

127-145

comparable

Na+/

of Trem-

in icefish

heart,

against

kPa),

teleosts

epithelia

Chaenichthydae

is able to displace

t~s, 3.0/2.0

seawater

some

20 min

50 mM Tris

(26), rela-

is likely due to a greater

in other

their enlarged

mammals,

temperate

to an

is represented

by the lack of hemoglobin

The blood

whereas

(lo),

in the gill and kidney

with the family

(18,42);

and

attributed

and Trematomus bernacchii

is characterized ml/kg

which

hormones

trait,

500 mOsm/kg)

than that of other

300 mOsm/kg),

K’ ATPase

One

concentration

osmolality

tively higher

by natriuretic

types.

in serum NaCl

by the plasma

trait,

formed

receptor

as SB/F vs SB, and from the using non-linear regression

curve, to determine

the mean dissociation

150-300g were caught by netting at the Italian base in Terranova Bay, (Ross Sea, Antarctica) during the 9th Italian expedition. After 1 week of stabulation in aerated water

constant (k’,,) value. The maximal binding capacity (B,,,,,,) was provided by the intercept of the curve at the abscissa. The saturation data were also evaluated using Ligand analy-

tanks at O”C, the animals were killed by decapitation. Their hearts were rapidly removed, freeze-mounted onto a cryostat

sis of “UltraFit” program (BIOSOFT, Cambridge, U.K.) fitted for Macintosh computer to check the presence of one

chuck using isopentane tor assay study.

or two classes

In Vitro Quantitative SATURATION

tions

and stored

before

recep-

Autoradiography STUDY. Transverse 16-pm secand C. hamatus hearts were cut with

BINDING

of T. hernacchii

at -80°C

of binding

sites.

DISPLACEMENT STUDY. To compare the potency and specificity of unlabeled porcine BNP (pBNP, Sigma Chemical Company, St. Louis, MO, USA) in displacing [“‘II-rat ANP binding with respect to the unlabeled rANP in all cardiac chambers of both C. hamatus and T. bemacchii, a

Cardiac NP Binding in Antarctic

competition cedures that

binding

another

to those

series of adjacent

rat ANP

plus increasing

of either

unlabeled

heart

the Ligand computer.

analysis

binding

on the KLIvalues

(lo-”

of “UltraFit”

obtained

to lo-’

The relative

number

the competition

The concentration

was

100 pM of [“51]-

or of pBNP.

receptor

by elaborating

pro-

study except

(n = 8) sections

containing

concentrations

rat ANP

ity and the total

The binding

of the saturation

in the same buffer

obtained

995

study was performed.

were similar

incubated

Teleosts

for rANP

binding

program

of labeled

M) affinwas

data using

on a Macintosh

rat ANP

from the saturation

was based study.

RESULTS L.ax&ution

of

f’251J-rANP Binding Sites

Quantitative

autoradiography

the presence

of a heterogeneous

in the different

heart

binding

technique

pattern

chambers

showed

M) indicated

a highly

densities sulted

myocardial

in mtlrc labeled

the contrary, concerning

binding

value

Evaluation

ranging

from

of the silver

grain

the outer

media

and tunica revealed

and the endocardial

than

a difference

region whereas

Fig. 1.

See

labeling.

In both

layer (tunica level with

fish, the atria1 wall and the inner

intima) respect

demonstrated

the other

was observed

adventitia).

in C. hamatus,

presence

re-

cardiac

regions.

between

the two fish

layer of the bulbus

the

tissues

to that of the other

arteriosus

In T. brmacchii, of dense

it was characterized

On

(tunica

this hulbar

binding

levels,

by the lowest

Characterization

heart

bulhar binding

tissues.

BINDING

data showed

that

sites in a saturable ent heart

eters

STUDY

ANP.

SatUra-

was able to bind fashion

to NP

in the differ-

T. hrrnacchii and C. hamatus.

of both

saturation

curve of T. bernacchii

in Fig. 3. From the Scatchard

plots

atrium

is

of the ratio

of

to free ligand (SB/F) against the amount of bound (SB), it was possible to determine the binding paramin the cardiac

Table

regions

of both

t;sh as summarized

1. Figure 4 shows the Scatchard

In T. hernucchii, the various ized by different tricular

binding

endocardium

arteriosus,

plots obtained

in from

of T. bernacchii and C. humatus.

the atrium

(b)

OF [“‘II-RAT

[“‘I]-rANP

and heterogeneous

chambers

A representative shown

Receptors

of rANP

SATURATION

tion

hound lirand

(a)

an intermediate

that in both T. bernacchii and C. hamatus

revealed

the ventricular

specific

binding.

2.

of T. bernacchii and C. ha-

mattts. Comparison of autoradiograms (Figs. 1 and 2) generated in the presence and absence of unlabeled rANP (lo-” 45 to 80% of total

FIG.

of [‘251]-rANP sites

the fitting

heart regions

patterns.

were character-

In the atrium,

in the vcn-

and in the outer

layer of the bulhus

of the Scatchard

plots

indicated

the

presence of only one class of rANP sites. In the atrium, an elevated number of high affinity rANP sites was found. B,,,,,, and I(,, values

Comparable ventricular spectively.

endocardium In contrast,

in the inner FIG. 1. Autoradiograms showing the distribution of NP binding sites in the atrium, ventricle and bulbus arteriosus of Trematomus bernacchii (la-d) and Chionodraco hamatus (Za-d). For total binding (a and c), the 16-pm sections were incubated in the presence of 100 pM [‘*‘I]~rat ANP-( l28). Non-specific binding (b and d) was determined in the presence of a 10e6 M of unlabeled rat ANP, as described in Materials and Methods. Bar, 1 mm. A, atrium; M, ventricular myocardium; E, ventricular endocardium; I, inner layer of the bulbus arteriosus; 0, outer layer of the bulbus arteriosus.

observed. ventricular served

bulbar

Site

layer, twc) classes

I demonstrated

myocardium,

in the inner

were also obtained

in the

and in the outer hulbar layer, rein the ventricular myocardium and

whereas

bulbar

very

of binding

sites were

low K,I values

higher

in the

K\I values were oh-

layer. For site 2, the highest

K~I

values were calculated in the inner bulbar layer. Contrary tO the results obtained in T. bernacchii, in all the cardiac regions of C. humatus, the presence of curvilinear plots, together with the subsequent titting of the curves, indicated that a two-site model is consistent with rANI’ receptors (Table 1). The atrium, the ventricular myocardium and the ventricular endocardium showed the presence

M. CL Cerra et al.

Atrium (rANI?)

Atrium (rANI?)

25

T. bernacchii

lk

T. bernacchii Bmax = 370 k 36 fmol/mg

12.5

SB

10

1

C. hamatus 0

Bmq CL

125I rANP (PM)

receptors

having

very similar

the high and the low affinities. ity was also shown the ventricular

The maximal

myocardium

In the case of the inner

bulbus

arteriosus,

number

site 2 demonstrated

the other

heart

of binding

of the other

and the outer slightly

chambers,

whereas

sites was shown

= 130 f 41 fmoYmg p

KdI = 8,9 f 3,0 pM

0

2

5-

capac-

in the atrium, endocar-

layers of the lower KLIval-

Bmax2 = 350 zk32 fmol/mg p

0

Kd, = 224 + 67 pM 0

2.5 -

0

q

m0

“a

100

300

200

400

SB (fmol/mg p)

for both

binding

and the ventricular

dium.

ues than

KC1values

to be in the same range

7.5

.

FIG. 3. Saturation curve of [‘Z51]-rat ANP-(1.28) specific binding (SB) in the atrium of Trematomus bernacchik Atria1 sections were incubated in the presence of increasing concentrations (lo-” to 10e9 M) of [‘251]-rat ANP-( l-28) and of 10m6M of cold rANP. The results were similar in three separate determinations.

of rANP

p

FIG. 4. Scatchard transformation of saturation curves of [‘z51]-rat ANP-( l-28) in the atrium of Trematomus bemacchiiand Chionodraco hamatus. The hound/free (SB/F) ratio plotted against bound (SB) produced similar results in three other experiments.

the maximum

to be similar

to those

regions.

DISPLACEMENT

STUDY

WITH

COLD rANI’ AND COLD pBNP.

specifity

all cardiac

re-

TABLE 1. Cardiac binding parameters obtained from saturation analysis in T. bemacchii Trematomus Cardiac Regions

Atrium Ventricle Myocardlum Endocardium B&us arterivsus Inner layer Outer layer

B maxi (fmollmg p)

Chionodraco

bemacchii (fmollmg

P)

370 r 36

14 2 3.2

177 !I 23 400 t 33

1.8 ? 0.6 9.7 k 2.3

271 ? 19

114 i 37 381 k 22

14 -c 6.1 6.2 ? 1.3

318 i 24

T. bemacchii, n = 4; C. hamutus, n = 4. The saturation

data were evaluatd

(2,

(fmollmg

P)

hamatus B maXI (fmollmg p)

B maxi

B mad (Z,

and C. hamatus

($

130 + 41

8.9 r

3.0

350 + 32

224 t 67

105 ? 37

159 t 29 118 t 36

8.5 z 2.0 6.7 ? 1.2

383 + 16 370 I 59

214 t 51 262 t 90

209 -t 66

148 2 37 107 ? 13

11 -t 3.2 2.1 2 0.7

348 I 80 304 ? 25

156 f 61 178 -c 41

‘15JrscriheJ

in MateriA

and Methods.

VAw~ are meant k SE.

Cardiac NP Binding in Antarctic

gions

of T. bernacchii

examining

of unlabeled

was determined rANP

According to the subsequent data, the cardiac binding

shown to be in agreement saturation study. The comparison tion

997

and C. hamatus

the capability

radioligand. displacement

Teleosts

with

those

of the results

experiments

with

calculated

rANP

using unlabeled pBNP demonstrated differently able to displace [“‘I]-rANP ous cardiac shown

The application cold

pBNP

of the TB obtained

binding

of 100 pM of labeled revealed

rANP.

only in the atrium

rANP),

whereas

endocardium, outer

[“‘I]-rANP

with

binding.

layer of the bulbus

rANl?

as

of Fig. 5.

and 10-I’ M of with

that

25-

cold pBNP

potency

in competing

cold rANP

(pBNP

myocardium

lower concentrations

to displace

curves

In T. bernacchii,

in the ventricular

-M-

study in the presence

a higher

for the NPs sites as compared

inhibiobtained

comparable

in the saturation

the

and C. hamatus,

of lo-‘”

levels

of the were

from

to those

displacement

of concentrations

provided

T. bernacchii

this

that these NPs were binding in the vari-

of T. bernacchii

chambers

in the representative

elaboration parameters

of the competitive

unlabeled

by

to displace

arteriosus,

displacement

I

-13

-i2

-ii

-io

-b

-;1

-b

-‘6

-12

-11

-10

-9

-8

-7

-6

and in the

of cold rANP In both

”

>

were able

the inner

and the

the two cold peptides

demonstrated

a similar

displacement

curves being in part superimposed

capacity,

with the two in the inner

layer. A different

situation

was encountered

in C. hamatus,

in

which rANP displayed greater competing capabilities for cardiac NP sites than cold pBNP. Only in the inner layer of the bulbus arteriosus, the lower part of the displacement curve of cold pBNP showed that this peptide was more able to displace

[“‘I]-rANP

than

the unlabeled

rANP.

DISCUSSION The present gions

study demonstrates

of the

binless

C. hamatus

rANP

in

binding bulbus

are able to bind

a saturable

and

stable

levels were observed

and endocardium arteriosus

localization

that the different

T. bemacchii

red-blooded

of both

and

The

in the ventricular

NP labeling

that the

in the two nototheni-

is in contrast

with the most dense NP binding

found

in the

layer of the bulbus

teleosts

Log [peptide] M FIG. 5. Displacement curves of [‘“‘II-rat ANP-( le28) specific binding to the atrium of Trernatomus bemacchii and Chionodraco hamatus in the presence of increasing concentrations (lo-” to 10e6 M) of unlabeled rANP and pBNP, as described in Materials and Methods.

than the inner layer. This obser-

vation

inner

-13

layer of the

aids was shown to follow opposite patterns because, contrary to C. hamutus, the outer bulbar layer of T. bernacchii showed a more intense

01

[‘7]highest

myocardium

It is of relevance

of the bulbar NP binding

re-

hemoglo-

the radioligand manner.

fish and in the outer

of T. hernacchii.

heart

the

(8,Q). The signikance

arteriosus

of this inverted

usually of other

bulbar

NP

Antarctic rANP

tishes showed

binding

similar

a heterogeneity to that observed

of NP sites in [“‘I]in the heart

of two

binding could be clarified by the comparison of the putative levels of NI’s in the body fluids of these fishes, as for example in the pericardial fluid surrounding the outer bulbar layer.

other teleosts, the Conger eel (8) and the European eel Anguilla anffuilla (9). Interestingly, this zonation in cardiac NP binding occurs both in two anguilliform teleosts, and in

In mammals, this fluid was recently shown to contain both ANP and BNP, probably involved in the modulation of neurnnal and/or heart muscle function acting from the peri-

these endemic natothenioids that in the last 20 millions of years have phyletically diversified in the isolation of the Antarctic habitat (13). The characterization of the cardiac NP receptors revealed

cardial

side (20). The remaining

cardiac

regions

of the two

998

M. C. Cerra

et al.

numbers of both high and low affinity NP

disc regions, seems to better bind rANP than pBNP. On the

binding sites were present in all regions of C. hamums and in the ventricular myocardium and inner bulbar layer of T.

contrary, in all heart chambers of C. hamatus, two classes of high and low affinity NP sites with the ranking order of

bernacchii. Contrarily,

potency,

that elevated

the atrium, ventricular

endocardium

and outer layer of the bulbus arteriosus of T. bemacchii were

rANP

> pBNP,

were demonstrated.

Although

by one class of high affinity NP binding sites.

caution must be taken when analyzing data provided by application of the only available heterologous ligands, such

These binding paratneters are in good agreement with those

results obtained in both species are potentially useful toward

calculated for the NP receptors from other piscine tissues, as in the case of the single class of the gill NPR from the

in fish. Moreover, the putative occurrence

characterized

freshwater Japanese eel that demonstrated

a I& value of 250

the understanding

of the physiological

significance

of NPs

of a receptor type

able to mediate the functions of BNP or a BNP-like peptide,

pM (33) and of the high and low affinity NPR found in the

especially in the atrium of T. bernacchii,

gills from the toadfish ( 11) and the seawater European eel

ther research.

may

challenge

fur-

(6) that showed Kd values ranging from 37 pM to 11 nM. The heterogeneous

cardiac binding pattern of T. bernacchii

resembles that recently found in A. nnguilla in which one population

of NPR was identified

two classes of receptors

in the inner

of A. anguilla (9) and of T. bemacchii, it is noteworthy

that

in C. hanatus, all cardiac regions possess two classes of NP sites of high and low affinities. The evidence

provided by

is that among the various teleost species

so far analyzed, only the inner bulbar layer demonstrates constant

NP binding activity represented

binding sites. Although dress the functional

a

by two classes of

this work does not specifically ad-

significance

of such constant

receptor

pattern of the inner bulbar layer, it suggests, however, that this pattern is independent influences

and/or

from

from either the environmental cardiac

adjustments

because

it

equally occurs both in the enlarged heart of C. hamatus and in the heart of the other fish species. On the basis of the competitive

studies, it is interesting

to note that pBNP is able to bind to NP sites in the heart of both notothenioids,

but it shows a higher displacement

capacity with respect to that of rANP in the atrium of T. bernacchii.

There

is evidence

that in mammals,

the heart

contains binding sites common for both ANP and BNP (27) that interact with BNP at different affinities (22,44). although

the bulk of information

obtained

using the

(12,19,36),

recently

In fish,

dealing with NPR was

available

homologous

in the rectal gland of the elasmobranch

NPs

Squalus

acanthias, the heterologous

pBNP was recently shown to specifically bind two types of NPRs (17). BNP has not yet been identified in fish; however, the elevated sequence homology shown by this type of NP with respect to the other piscine NPs (39) strengthens the results of the displacement study performed in the various cardiac regions of T. bemmand suggests that NP binding sites able to be bound by a peptide similar to BNP exist. Moreover, it is of relevance that in the atrium of T. bewmcchii, BNP binds to the NP sites better than rANP.

chii and C. hamatus

In conclusion,

in T. bernacchii

Nazion&

di Ricerche

in the atrium, whereas

were demonstrated

layer of the bulbus arteriosus (9). With respect to the hearts

these observations

This research was supported by the Programma in Ant&de (PNRA) (B. Tom).

at least two types of NP

binding sites may be hypothesized. A first site, which is characterized by a high affinity for pBNP, is present in the atrium, whereas the other(s), common to the remaining car-

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