International review of cell and molecular biology

V O LU M E

T WO

E I G H T Y

INTERNATIONAL REVIEW OF

CELL AND MOLECULAR
BIOLOGY


INTERNATIONAL REVIEW
OF CELL AND MOLECULAR
BIOLOGY
Series Editors

GEOFFREY H. BOURNE
JAMES F. DANIELLI
KWANG W. JEON
MARTIN FRIEDLANDER
JONATHAN JARVIK

1949–1988
1949–1984
1967–
1984–1992
1993–1995

Editorial Advisory Board

ISAIAH ARKIN
PETER L. BEECH
ROBERT A. BLOODGOOD
DEAN BOK
KEITH BURRIDGE
HIROO FUKUDA
RAY H. GAVIN
MAY GRIFFITH
WILLIAM R. JEFFERY

KEITH LATHAM
WALLACE F. MARSHALL
BRUCE D. MCKEE
MICHAEL MELKONIAN
KEITH E. MOSTOV
ANDREAS OKSCHE
MANFRED SCHLIWA
TERUO SHIMMEN
ROBERT A. SMITH


V O LU M E

T WO

E I G H T Y

INTERNATIONAL REVIEW OF

CELL AND MOLECULAR
BIOLOGY

EDITED BY

KWANG W. JEON
Department of Biochemistry
University of Tennessee
Knoxville, Tennessee

AMSTERDAM • BOSTON • HEIDELBERG • LONDON
NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier


Front Cover Photography: Cover figure by Anders Lydik Garm
Academic Press is an imprint of Elsevier
525 B Street, Suite 1900, San Diego, CA 92101-4495, USA
30 Corporate Drive, Suite 400, Burlington, MA 01803, USA
32 Jamestown Road, London NW1 7BY, UK
Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands
First edition 2010
Copyright # 2010, Elsevier Inc. All Rights Reserved.
No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form
or by any means electronic, mechanical, photocopying, recording or otherwise without the prior
written permission of the publisher
Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in
Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier.
com. Alternatively you can submit your request online by visiting the Elsevier web site

at and selecting Obtaining permission to use Elsevier material.
Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a
matter of products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions or ideas contained in the material herein. Because of rapid advances in the
medical sciences, in particular, independent verification of diagnoses and drug dosages should be made.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
For information on all Academic Press publications
visit our website at elsevierdirect.com

ISBN: 978-0-12-381260-5

PRINTED AND BOUND IN USA
10 11 12
10 9 8 7 6 5 4 3 2 1


CONTENTS

Contributors

ix

1. Natriuretic Peptides in the Regulation of the
Hypothalamic–Pituitary–Adrenal Axis

1

Andrea Porzionato, Veronica Macchi, Marcin Rucinski,
Ludwik K. Malendowicz, and Raffaele De Caro
1. Introduction
2. Biology of Natriuretic Peptides and Their Receptors
3. Expression of Natriuretic Peptides and Their Receptors
in the HPA Axis
4. Effects of Natriuretic Peptides on the HPA Axis
5. Natriuretic Peptides and Pathophysiology of HPA Axis
6. Concluding Remarks
Acknowledgment
References

2. Evidence for Multiple Photosystems in Jellyfish

2
2
4
12
21
22
24
24

41

ăm
Anders Garm and Peter Ekstro
1. Multiple Photosystems
2. Photosensitivity in Cnidarians

3. Photosensory Organs in Hydromedusae
4. Photosensory Organs in Scyphomedusae
5. Photosensory Organs in Cubomedusae
6. Multiple Opsins in Cnidarians—Multiple Photosystems?
7. Conclusion
Acknowledgments
References

3. Membrane Trafficking in Protozoa: SNARE Proteins,
H+-ATPase, Actin, and Other Key Players in Ciliates

42
46
51
54
56
70
72
73
73

79

Helmut Plattner
1. Introduction
2. Factors Involved in the Regulation of Vesicle Trafficking
3. Features of SNAREs

80
88

108
v


vi

Contents

4. Exocytosis and Endocytosis
5. Possible SNARE Arrangement in Microdomains
and Membrane Fusion
6. Phagocytosis
7. Calcium-Binding Proteins and Calcium Sensors
8. Additional Aspects of Vesicle Trafficking
9. Emerging Aspects of Vesicle Trafficking in Ciliates
10. Concluding Remarks
Acknowledgments
References

4. New Insights into the Types and Function
of Proteases in Plastids

124
131
134
142
146
152
157
159

159

185

Yusuke Kato and Wataru Sakamoto
1. Introduction
2. Overview
3. Major Proteases
4. Processing Peptidases
5. Intramembrane Proteases
6. Other Proteases
7. Concluding Remarks
References

5. Impact of ATP-Binding Cassette Transporters
on Human Immunodeficiency Virus Therapy

186
188
190
201
204
206
207
209

219

Johanna Weiss and Walter Emil Haefeli
1. Introduction

2. Drug Therapy of HIV-1: Drug Classes and Site of Action
3. ABC-Transporters Influencing Drug Therapy
of HIV-1 Infections
4. Cell Models Investigating the Impact of ABC-Transporters
for HIV-1 Therapy
5. Anti-HIV-1 Drugs as Substrates, Inhibitors, and Inducers
of ABC-Transporters: In Vitro and In Vivo Findings
6. Clinically Relevant Drug Interactions with Anti-HIV-1 Drugs
Attributed to ABC-Transporters
7. ABC-Transporters, ‘‘Cellular’’ Resistance,
and Therapeutic Success
8. ABC-Transporter Polymorphisms and HIV-1
9. Concluding Remarks
References

220
221
224
230
236
257
259
262
264
265


Contents

6. New Insights into the Circadian Clock

in Chlamydomonas

vii

281

Takuya Matsuo and Masahiro Ishiura
1. Introduction
2. Behavioral and Physiological Circadian Rhythms
in Chlamydomonas
3. Circadian Oscillator in Chlamydomonas
4. Input Pathways to the Circadian Oscillator
in Chlamydomonas
5. Output Pathways from the Circadian Oscillator
in Chlamydomonas
6. Concluding Remarks
Acknowledgments
References
Index

282
285
288
302
303
307
308
308
315

This page intentionally left blank


CONTRIBUTORS

Raffaele De Caro
Department of Human Anatomy and Physiology, University of Padova, Padova,
Italy
ăm
Peter Ekstro
Department of Cell and Organism Biology, Lund University, Lund, Sweden
Anders Garm
Department of Comparative Zoology, University of Copenhagen, Copenhagen,
Denmark
Walter Emil Haefeli
Department of Clinical Pharmacology and Pharmacoepidemiology, University of
Heidelberg, Heidelberg, Germany
Masahiro Ishiura
Center for Gene Research and Division of Biological Science, Graduate School of
Science, Nagoya University, Nagoya, Japan
Yusuke Kato
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama,
Japan
Veronica Macchi
Department of Human Anatomy and Physiology, University of Padova, Padova,
Italy
Ludwik K. Malendowicz
Department of Histology and Embryology, Poznan University of Medical
Sciences, Poznan, Poland

Takuya Matsuo
Center for Gene Research and Division of Biological Science, Graduate School of
Science, Nagoya University, Nagoya, Japan
Helmut Plattner
Department of Biology, University of Konstanz, Konstanz, Germany
Andrea Porzionato
Department of Human Anatomy and Physiology, University of Padova, Padova,
Italy

ix


x

Contributors

Marcin Rucinski
Department of Histology and Embryology, Poznan University of Medical
Sciences, Poznan, Poland
Wataru Sakamoto
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama,
Japan
Johanna Weiss
Department of Clinical Pharmacology and Pharmacoepidemiology, University of
Heidelberg, Heidelberg, Germany


C H A P T E R

O N E

Natriuretic Peptides in the
Regulation of the Hypothalamic–
Pituitary–Adrenal Axis
Andrea Porzionato,* Veronica Macchi,* Marcin Rucinski,†
Ludwik K. Malendowicz,† and Raffaele De Caro*
Contents
1. Introduction
2. Biology of Natriuretic Peptides and Their Receptors
2.1. Natriuretic peptides
2.2. Natriuretic peptide receptors and their signaling mechanisms
3. Expression of Natriuretic Peptides and Their Receptors in the
HPA Axis
3.1. Hypothalamus
3.2. Pituitary gland
3.3. Adrenal cortex
3.4. Adrenal medulla
4. Effects of Natriuretic Peptides on the HPA Axis
4.1. Hypothalamus
4.2. Pituitary gland
4.3. Adrenal cortex
4.4. Adrenal medulla
5. Natriuretic Peptides and Pathophysiology of HPA Axis
5.1. Adrenocortical adenomas and carcinomas
5.2. Pheochromocytomas
6. Concluding Remarks
Acknowledgment
References

2

2
2
4
4
4
8
10
11
12
12
15
18
20
21
21
22
22
24
24

Abstract
Atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides act by binding to
three main subtypes of receptors, named NPR-A, -B, and -C. NPR-A and NPR-B are
coupled with guanylate cyclase. Not only NPR-C is involved in removing natriuretic
* Department of Human Anatomy and Physiology, University of Padova, Padova, Italy
Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland

{

International Review of Cell and Molecular Biology, Volume 280

ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80001-2

#

2010 Elsevier Inc.
All rights reserved.

1


2

Andrea Porzionato et al.

peptides from the circulation but it also acts through inhibition of adenylyl cyclase.
NPR-A binds ANP and BNP; NPR-B preferentially binds CNP; and NPR-C binds all
natriuretic peptides with similar affinities. All natriuretic peptides and their receptors are widely present in the hypothalamus, pituitary, adrenal cortex, and medulla.
In the hypothalamus, they reduce norepinephrine release, inhibit oxytocin, vasopressin, corticotropin-releasing factor, and luteinizing hormone-releasing
hormone release. In the hypophysis, natriuretic peptides inhibit basal and induced
ACTH release. Conversely, the effects of natriuretic peptides on secretion of growth,
luteinizing, and follicle-stimulating hormones are not clear. Natriuretic peptides are
known to inhibit basal and stimulated aldosterone secretion, through an increase
of intracellular cGMP, and to inhibit the growth of zona glomerulosa. Inhibition or
stimulation of glucocorticoid secretion by adrenocortical cells has been reported
on the basis of the species involved, and an indirect effect mediated by adrenalmedullary cells has been hypothesized. In the adrenal medulla, natriuretic peptides inhibit catecholamine release and increase catecholamine uptake. It appears
that natriuretic peptides may play a role in the pathophysiology of adrenocortical
neoplasias and pheochromocytomas.
Key Words: Natriuretic peptides, Hypothalamic–pituitary–adrenal axis, ACTH
secretion, Catecholamine secretion, Pheochromocytomas. ß 2010 Elsevier Inc.

1. Introduction
Numerous neuropeptides control the hypothalamic–pituitary–adrenal
(HPA) axis, acting on both its central and peripheral branch. Natriuretic
peptides are known to be included in this group of regulatory peptides, but
only a few review articles have been published regarding the role of natriuretic
peptides in the HPA axis, and mainly with reference to specific structures or
specific pathological conditions (Gutkowska et al., 1997; Wiedemann et al.,
2000). A comprehensive and updated review on the role of natriuretic peptides
in all the levels of the HPA axis is still lacking. Thus, after a synthetic account on
the biology of the natriuretic peptides system, we will herein review data
indicating how natriuretic peptides and their receptors are expressed in all
the anatomical components of the HPA axis, and are involved in the functional
regulation of HPA axis under both physiological and pathological conditions.

2. Biology of Natriuretic Peptides and Their
Receptors
2.1. Natriuretic peptides
Natriuretic peptides represent a family of three hormones called atrial natriuretic peptides (ANP) (Kangawa and Matsuo, 1984), brain natriuretic peptides
(BNP) (Sudoh et al., 1988), and C-type natriuretic peptides (CNP) (Sudoh


3

Natriuretic Peptides and Adrenals

et al., 1990). ANP is a 28-amino acid peptide which has first been isolated from
human atrial extract (Kangawa and Matsuo, 1984). BNP and CNP have been
identified in the porcine brain (Sudoh et al., 1988, 1990). Figure 1.1 shows the
sequences of natriuretic peptides. All peptides contain the conserved sequence
FGXXXDRIGXXSGL. The flanking cysteines form a 17-amino acid disulfide-linked ring that is required for biological activity. In some tissues, CNP-53

is cleaved to CNP-22.

ANP
1

26

124

151

103

134

105

126

pro-ANP

ANP-28
URO
BNP
1

27
pro-BNP

g-BNP

(pro-BNP, in blood)
BNP-32
CNP
1

24
pro-CNP

CNP-53

CNP-22

Figure 1.1 Natriuretic peptide expression (prepro-ANP, -BNP, and -CNP). Each oval
represents 1-amino acid residue: yellow—the signal sequence; blue—part removed
during processing of propeptide to mature peptide; and red—mature peptide. Alternative processing of pro-ANP generates a 32-residue peptide called urodilatin (URO,
renal natriuretic peptide). Two variants of BNP are known: mature BNP-32 and in the
blood g-BNP (pro-BNP). CNP also is known in two variants: CNP-53 and CNP-22.


4

Andrea Porzionato et al.

2.2. Natriuretic peptide receptors and their signaling
mechanisms
The biological activity of the natriuretic peptides occurs via the activation of
three different receptors, which have been cloned and pharmacologically
characterized: NPR-A, NPR-B, and NPR-C. The first two receptors are
coupled with guanylate cyclase. They consist of an extracellular ligand-binding
domain, a short transmembrane region, a juxtamembranous protein kinasehomology domain, an alpha-helical or hinge region, and a C-terminal guanylyl

cyclase catalytic domain, receptor dimerization being essential for the activation
of the catalytic domain (reviewed in Anand-Srivastava and Trachte, 1993;
Kuhn, 2003; Maack, 1992; Potter et al., 2006, 2009). Alternative splicing of
NPR-A has recently been found to produce an isoform which does not bind
ANP and may inhibit ligand-inducible cGMP generation by forming heterodimers with the wild-type receptor (Hartmann et al., 2008). NPR-A is activated by ANP and BNP, ANP being more effective than BNP in stimulating
cGMP production. NPR-B binds with higher affinity CNP (Fig. 1.2). All
natriuretic peptide receptors are also known to be internalized and to some
extent recycled as a result of ligand binding (reviewed in Pandey, 2009).
NPR-C binds all three natriuretic peptides with relatively similar affinities (Maack, 1992). It is a disulfide-linked homodimer with a single
transmembrane domain which lacks the intracellular guanylate cyclase
domain but is able to internalize natriuretic peptides after binding. Thus,
it has first been considered to be involved in removing natriuretic peptides
from the circulation (Fig. 1.2). Nevertheless, following studies suggested
that NPR-C contains a 37-amino acid intracellular domain which is able to
inhibit the adenylyl cyclase and activate phospholipase C, through activation of Gi proteins. Moreover, NPR-C may also inhibit the mitogenactivated protein kinase pathway (signaling pathways of NPR-C reviewed
in Anand-Srivastava, 2005).

3. Expression of Natriuretic Peptides and Their
Receptors in the HPA Axis
3.1. Hypothalamus
ANP has first been identified in the rat hypothalamus by radioimmunoassay
(Glembotski et al., 1985; Tanaka et al., 1984) and its release in vitro from rat
hypothalamus has also been demonstrated (Shibasaki et al., 1986a; Tanaka and
Inagami, 1986). Although it must be considered that some authors reported
cross-reactions with neurophysins in immunohistochemistry of the rat hypothalamus, suggesting absence of ANP immunostaining in the hypothalamus
(Nilaver et al., 1989), ANP has been identified by immunohistochemistry in


ANP-28

BNP-32

COOH

H 2N

CNP-22

H2N

COOH

H2N

COOH

NPR-B
NPR-C

NPR-A

KHD
KHD
cGMP
GTP

cGMP

GTP
GC

GC
GTP
GTP

cGMP

cGMP

Figure 1.2 Interaction of ANP, BNP, and CNP natriuretic peptides with receptors NPA-R, NPB-R, and NPC-R. NPR-A and NPR-B are
membrane-bound guanylyl cyclases, NPR-C—not coupled to guanylyl cyclase—is involved in clearance and metabolism of natriuretic peptides.
KHD, kinase homology domain.


6

Andrea Porzionato et al.

neurons of several mammal hypothalamic and nonhypothalamic brain structures, such as the septum, anteroventral region of the third ventricle (AV3V),
subfornical organum, paraventricular nucleus (PVN), preoptic, supraoptic
(SON), infundibular and ventromedial nuclei, lateral hypothalamus, organum
vasculosum lamina terminalis, median eminence, lamina terminalis, periaqueductal gray matter, parabrachial nucleus, solitary tract nucleus, tegmental lateral
dorsal nucleus, and periventricular regions (e.g., Chriguer et al., 2001;
Gutkowska et al., 1997; Jirikowski et al., 1986; Kawata et al., 1985; Raidoo
et al., 1998; Standaert et al., 1986a; Tanaka et al., 1984). Most ANP-immunoreactive neurons in the PVN belong to the parvocellular division ( Jirikowski
et al., 1986; Kawata et al., 1985), but colocalization of ANP and oxytocin (OT)
immunostaining has also been reported in some magnocellular neurons of the
magnocellular division of the PVN and SON (Chriguer et al., 2001;
Gutkowska et al., 1997; Jirikowski et al., 1986; Kawata et al., 1985). The
densest terminal fields of ANP-containing fibers have been reported in the

PVN of the hypothalamus, the bed nucleus of the stria terminalis, the interpeduncular nucleus, and the median eminence (Standaert et al., 1986a), where
ANP may modulate the release of anterior pituitary hormones (Franci et al.,
1990, 1992; Gutkowska et al., 1997). It has also been reported that ANPcontaining neurons in the PVN are the major source of ANP-containing nerve
terminals in the median eminence (Palkovits et al., 1987). ANP-immunoreactive fibers have also been observed in close proximity with oxytocinergic fibers
in the median eminence (Chriguer et al., 2001). In the hypophyseal portal
blood, ANP has been found in 3–4 times higher concentrations than in the
peripheral blood and the predominant species of IR-ANP in extracts of portal
blood from adult rats is ANP(5–28), whereas in peripheral blood is ANP(1–28)
(Lim et al., 1994). ANP mRNA has also been identified in the rat hypothalamus (Chen et al., 1992; Dagnino et al., 1991; Gardner et al., 1987; Komatsu
et al., 1992). The distribution of mRNA encoding prepro-ANP has also been
investigated in rat brain by in situ hybridization and the highest relative concentrations have been detected in the anteromedial preoptic nucleus of the
medial preoptic area (Gundlach and Knobe, 1992; Ryan et al., 1997).
Analysis through RT-PCR in the rat and monkey hypothalamus did not
identify BNP mRNA (Abdelalim et al., 2006; Langub et al., 1995). However,
radioimmunoassay studies have detected BNP in porcine (Ueda et al., 1988),
canine (Itoh et al., 1989), rat (Sone et al., 1991), human (Takahashi et al., 1992),
and ovine (Pemberton et al., 2002) hypothalamus. BNP-immunoreactive
fibers are also present in the PVN of the hypothalamus and many BNP-positive
neurons have been retrogradely labeled in the tuberomammillary nucleus of
the hypothalamus and in the pedunculopontine and laterodorsal tegmental
nuclei (Moga and Saper, 1994; Saper et al., 1989). An immunohistochemical
study on monkey hypothalamus revealed BNP-like immunoreactivity in the
form of clusters of granules in the PVN, SON, and periventricular area
(Abdelalim et al., 2006). These BNP-positive dots were located in neurons,


Natriuretic Peptides and Adrenals

7

oligodendrocytes, astrocytes, and microglial cells. It has been suggested that
BNP granules in the hypothalamus are originated from outside the hypothalamus and reach the hypothalamus through the subfornical organ (Abdelalim
et al., 2006) as high-density binding sites for BNP have been observed by
autoradiography in rat subfornical organ, SON, and paraventricular hypothalamic nucleus (Brown and Czarnecki, 1990) and NPR-A mRNA has been
found in the subfornical organ (Langub et al., 1995).
CNP has also been identified in the human hypothalamus in both high and
low molecular weight forms by using radioimmunoassay (Totsune et al.,
1994a). In the ovine hypothalamus, the concentration of CNP is much higher
than that of ANP, similar amounts of CNP-53- and CNP-22-like immunoreactive-CNP being present (Yandle et al., 1993). In the rat hypothalamus, the
highest CNP tissue concentrations have been found in the arcuate nucleus and
PVN (Herman et al., 1993; Minamino et al., 1993). Hybridization signals of
lower intensity were reported in the medial, median, and periventricular
preoptic area; the SON; dorsomedial, ventral premammillary, and lateral
mammillary nuclei; and in the posterior hypothalamic area (Herman et al.,
1993). Through in situ hybridization, prepro-CNP mRNA has also been
detected in the rat hypothalamus, particularly in the anteromedial preoptic
nucleus of the medial preoptic area (Ryan et al., 1997). CNP synthesis has also
been identified in immortalized luteinizing hormone-releasing hormone
(LHRH) neurons using RT-PCR, immunocytochemistry, and electron
microscopic immunohistochemistry and in these cells CNP also elevated
LHRH production in an autocrine manner (Middendorff et al., 1997). The
concentration of CNP in the cerebrospinal fluid has been reported to be one
order of magnitude greater than that of ANP (Kaneko et al., 1993).
Gibson et al. (1986) have found the highest levels of ANP binding in the
rat subfornical organ, area postrema and olfactory apparatus; moderate ANP
binding has been found throughout the brainstem and low levels in the
forebrain, diencephalon, basal ganglia, cortex, and cerebellum. ANP-binding sites have been identified in hypothalamic and nonhypothalamic structures in both rat and guinea pig (Mantyh et al., 1987). ANP-binding sites
have been identified in cerebral circumventricular organs, including the
subfornical organ and organum vasculosum of the lamina terminalis
(Mendelsohn et al., 1987). ANP-binding sites have also been reported in

the SON and in the magnocellular and parvocellular subdivisions of the
PVN in rat (Castre´n and Saavedra, 1989). In particular, high numbers of
ANP-binding sites have been reported in the circumventricular organs (the
organon vasculosum laminae terminalis, subfornical organum, and area
postrema) and selected hypothalamic (SON, median preoptic, and paraventricular) nuclei (Kurihara et al., 1987). ANP-binding sites have also been
reported in the median eminence, pineal gland, subfornical organ, choroid
plexus, but not in the magnocellular hypothalamic nuclei (Gerstberger
et al., 1992).


8

Andrea Porzionato et al.

NPR-B mRNA has been observed to be expressed throughout the hypothalamus, in the magnocellular and parvocellular paraventricular, the arcuate,
and the SON, the median preoptic, anteroventral periventricular, tuberomammillary, ventromedial, and suprachiasmatic nuclei (Langub et al., 1995). The
three receptors have been identified in astrocyte glial and neuronal cultures
from the hypothalamus and brain stem of 1-day-old rats, with astrocytes
containing predominantly the ANP-A subtype and neurons predominantly
the ANP-B subtype (Sumners and Tang, 1992). NPR-A and -B mRNA have
also been identified in the GT1-7 cell line, an immortalized LHRH neuronal
cell line. All the natriuretic peptides elevated cGMP production in this cell line
with the following rank order of potency: CNP > ANP > BNP (Olcese
et al., 1994). NPR-C expression has also been found in mammalian hypothalamus (Peng et al., 1996; Sumners and Tang, 1992). In the human, ovine, and
rat hypothalamus, higher expression of CNP and NPR-B have been found
than of ANP, BNP, and NPR-A (Herman et al., 1993, 1996a; Komatsu et al.,
1991; Langub et al., 1995; Minamino et al., 1993; Pemberton et al., 2002).
Natriuretic peptide expression in the rat hypothalamus has also been
studied with reference to postnatal maturation. It has been found through
radioimmunoassay that ANP concentrations show a first increase in the

postnatal days 0–5 and a second one in the postnatal days 10–20, for a 16fold final increase ( Jankowski et al., 2004). Increments of ANP mRNA
have also been found by in situ hybridization in the septohypothalamic,
lateral, periventricular, and arcuate nuclei from postnatal day 4 until postnatal days 21–28 (Ryan and Gundlach, 1998). In rat SON and suprachiasmatic nuclei, ANP peptide and mRNA have been identified starting from
the 18th day of the fetal life (Lipari et al., 2005, 2007). CNP concentrations,
instead, increased steadily until postnatal day 60, when they were 3.7-fold
higher than at birth ( Jankowski et al., 2004). As regards concentrations of
the transcripts of the natriuretic peptides receptors in adult versus newborn
rats, higher NPR-A concentrations, lower NPR-C concentrations, and no
differences in NPR-B concentrations were found ( Jankowski et al., 2004).

3.2. Pituitary gland
ANP has been identified in the rat anterior pituitary by radioimmunoassay
(Gutkowska and Cantin, 1988) and ANP and BNP mRNA have been
identified in human pituitary by PCR (Gerbes et al., 1994). The presence
of all the three natriuretic peptides has been reported through radioimmunoassay in the ovine pituitary, CNP (15.84 pmol/g wet weight) showing
higher concentrations than ANP and BNP (0.25 and 0.26 pmol/g wet
weight) (Pemberton et al., 2002). In the ovine hypophysis, the CNP-53like IR-CNP was mainly present (Yandle et al., 1993). CNP has been
identified by radioimmunoassay in the anterior lobe and neurointermediate
lobe of the pituitary (Komatsu et al., 1991). ANP-like immunoreactivity has


Natriuretic Peptides and Adrenals

9

been detected in the rat posterior hypophysis (Gutkowska et al., 1987).
In particular, a low molecular weight peptide with a RP-HPLC pattern
similar to that of the synthetic rat 28-amino acid C-terminal (Ser 99-Tyr
126) ANP was found, together with an unidentified higher molecular
weight peptide (Gutkowska et al., 1987). An immunohistochemical study

on rat pituitary gland has found ANP-, BNP-, and CNP-immunoreactive
cells in the anterior lobe but not in the intermediate lobe of fetal and
maternal glands on day 21 of gestation, fetal samples showing fewer and
weakly stained cells (Chatelain et al., 2003). ANP has been localized by
immunohistochemistry (Gutkowska and Cantin, 1988; McKenzie et al.,
1985) and in situ hybridization (Morel et al., 1989a) in rat gonadotroph cells.
Its expression has also been reported through RT-PCR in LbT2 cells and
primary mouse pituitary tissue (Thompson et al., 2009). An in vivo ultrastructural autoradiographic approach through intravenous injection of
125
I-ANP has also demonstrated internalization of extracellular ANP by
gonadotroph cells (Morel et al., 1989a). BNP has not been found to be
expressed in gonadotroph aT3-1 and LbT2 cells and rat and mouse pituitaries (Thompson et al., 2009). Conversely, CNP has been localized in rat
and mouse LH-positive cells of the anterior pituitary and in aT3-1 and
LbT2 cells (McArdle et al., 1994; Thompson et al., 2009). Putative processing enzymes of CNP (Furin and peptidyl a-amidating monoxygenase
enzymes) have also been found to be expressed in aT3-1 cells and primary
mouse pituitaries. Transcriptional analyses revealed that CNP expression is
responsive to GNRH action in a protein kinase C and calcium-dependent
manner (Thompson et al., 2009). The CNP promoter has been reported to
work effectively also in somatomammotroph or somatotroph GH3 cells but
not in corticotroph AtT20 cells (Ohta et al., 1993).
ANP-binding sites have also been reported in the anterior pituitary in
rabbit (Gerstberger et al., 1992) and rat (Agui et al., 1989) and in the
posterior pituitary in guinea pig (Mantyh et al., 1986) and rabbit
(Gerstberger et al., 1992). NPR-A and -B have been isolated from a
human pituitary cDNA library (Chang et al., 1989; Wilcox et al., 1991).
In situ hybridization study in the anterior pituitary of rhesus monkey has
revealed NPR-A and NPR-B mRNA (Wilcox et al., 1991). NPR-B
mRNA has been identified in some cells of the anterior pituitary and in
pituicytes in the neural lobe (Herman et al., 1996a). Northern blot analysis
identified all three natriuretic peptide receptors in the mouse pituitary

(Guild and Cramb, 1999). Analysis in alpha T3-1 and AtT-20 cell lines
did not confirm the presence of NPR-A mRNA, suggesting cGMP accumulation occurring via NPR-B (Gilkes et al., 1994; McArdle et al., 1994).
Ohta et al. (1993) have identified NPR-B in rat pituitary somatotroph and
somatolactotroph progenitor cells. In situ hybridization in rat anterior pituitary gland has revealed NPR-A, -B, and -C mRNA in lactotroph, corticotroph, and gonadotroph cells, but not in somatotroph or tyreotroph ones


10

Andrea Porzionato et al.

(Grandcle´ment et al., 1995; Thompson et al., 2009). NPR-C mRNA has
been identified by in situ hybridization not only in the rat anterior lobe but
also in the intermediate one (Herman et al., 1996b). Pituicytes cultured
from adult rat neurohypophyses have been found to possess high-affinity
binding sites for ANP, but ANP has been found not to modulate the basal or
electrically stimulated release of OT or vasopressin (VP) from the isolated
neurohypophysis in vitro (Luckman and Bicknell, 1991). NPR-B mRNA
has also been found in the pars intermedia and posterior of the pituitary
gland in the monkey (Wilcox et al., 1991) and rat (Konrad et al., 1992).
NPR-B mRNA was also observed in the neural lobe of the pituitary gland,
suggesting expression by pituicytes (Langub et al., 1995).

3.3. Adrenal cortex
Although Morel et al. (1988) did not report the presence of ANP mRNA in
the rat adrenal cortex and Lee et al. (1994) did not report BNP mRNA and
protein in the adrenal cortex by in situ hybridization and immunohistochemistry, ANP and BNP mRNA have been identified in human adrenal
gland (without distinction between cortex and medulla) by PCR (Gerbes
et al., 1994). Moreover, Lai et al. (2000) detected ANP mRNA and protein
by in situ hybridization and immunohistochemistry in the rat zona glomerulosa and outer region of the zona fasciculata, but not in the remaining part
of the zona fasciculata and in the zona reticularis. In bovine, CNP mRNA

has also been demonstrated by RT-PCR in the zona glomerulosa tissue and
cultured cells and CNP immunoreactivity has been localized in the outermost region of the adrenal cortex but not in the inner portion of the zona
fasciculata and zona reticularis (Kawai et al., 1996).
ANP-binding sites have been identified in the rat, guinea pig, rabbit,
bovine, and tree shrew adrenal zona glomerulosa (e.g., Chai et al., 1986; De
Le´an et al., 1984; Fuchs et al., 1986; Gerstberger et al., 1992; Lynch et al.,
1986; Mantyh et al., 1986; Mendelsohn et al., 1987; Morel et al., 1989b). In
particular, internalization of ANP in rat adrenal glomerulosa cells was also
demonstrated (Morel et al., 1989b). ANP-binding sites have also been
observed in the rat zona fasciculata (Chai et al., 1986) and in the tree
shrew and bovine zona fasciculata and reticularis (Fuchs et al., 1986;
Nunez et al., 1990). Lynch et al. (1986) also reported the presence of
ANP-binding sites in the rat zona fasciculata and reticularis, although at
lower levels. Developmental changes have also been reported in the expression of ANP receptors as in the 16-day-old rat ANP-binding sites are
present throughout the cortical area but at 20 days gestation and 1 day
postpartum ANP receptors are more numerous in the peripheral region
(Scott and Jennes, 1989). Conversely, rat adrenocortical autotransplants
regenerated from capsular-tissue fragments implanted in the musculus gracilis have been found not to significantly bind 125I-ANP (Belloni et al.,


Natriuretic Peptides and Adrenals

11

1993). BNP-binding sites have also been identified in bovine adrenocortical
membrane fractions (Higuchi et al., 1989).
In the rat zona glomerulosa cells, mRNA of the three natriuretic peptide
receptors have been identified (Grandcle´ment et al., 1997; Nagase et al., 1997;
Vaillancourt et al., 1997). The amount of NPR-A mRNA has been found to
be the highest (Grandcle´ment et al., 1997) and Western analysis using polyclonal anti-NPR-A and anti-NPR-B antibodies revealed the presence of

NPR-A but not of NPR-B proteins (Vaillancourt et al., 1997). Wilcox et al.
(1991) reported the presence of NPR-A but not NPR-B in the monkey zona
glomerulosa by in situ hybridization and observed clusters of NPR-C-positive
cells suggestive of endothelial, not necessarily secretory, cells. In the rat zona
fasciculata cells, NPR-A but not NPR-B and -C receptor’s mRNA has been
identified (Mulay et al., 1995; Vaillancourt et al., 1997). In the monkey zona
fasciculata and reticularis, mRNA of the three receptors was not identified in
secretory cells (Wilcox et al., 1991). NPR-A has also been identified in the
H295R human adrenocortical cell line (Bodart et al., 1996).
Plasma ANP concentrations are known to decrease after water deprivation
or hemorrhage and to increase after blood volume expansion. Conversely,
data concerning plasma ANP concentrations in response to salt-overloading
are contradictory. Water deprivation increases total number of ANP receptors in the adrenal gland of adult and maternal rats, but not of fetal ones
(Deloof et al., 1999; Lynch et al., 1986). In particular, the density of NPR-C
but not of NPR-B has been found to be increased (Deloof et al., 1999). Most
studies, with few exceptions (Deloof et al., 2000) reported downregulation of
the ANP receptors in the adrenal glands after salt-overloading (Lynch et al.,
1986; Sessions et al., 1992).

3.4. Adrenal medulla
ANP, BNP, and CNP have been identified in rat, bovine, porcine, and human
adrenal medulla cells (e.g., Babinski et al., 1992; Dagnino et al., 1991; De Le´an
et al., 1985; Komatsu et al., 1991; Lai et al., 2000; Lee et al., 1993, 1994;
McKenzie et al., 1985; Minamino et al., 1993; Morel et al., 1988; Nawata et al.,
1991; Nguyen et al., 1990; Wolfensberger et al., 1995; reviewed in Kobayashi
et al., 1998). In situ hybridization identified ANP mRNA in noradrenergic cells
while immunohistochemistry identified ANP protein in both noradrenergic
and adrenergic cells, suggesting ANP synthesis in noradrenergic cells and
internalization in adrenergic ones (Morel et al., 1988). It has also been reported
that the majority ANP-immunoreactive chromaffin cells are the adrenergic

ones (Wolfensberger et al., 1995). Electrical stimulation of the splanchnic
nerves has been found to cause the release of ANP-like immunoreactive
material in isolated perfused calf adrenal glands (Duntas et al., 1993; Edwards
et al., 1990) and enhance the uptake of ANP by chromaffin cells (Edwards
et al., 1990).


12

Andrea Porzionato et al.

It has been hypothesized that ANP produced in the adrenal medulla may act
on the adrenal cortex (Lee et al., 1993, 1994; Nawata et al., 1991) and may be
involved in the regulation of blood flow and even in the zonation of the adrenal
cortex (Lee et al., 1994). 125I-ANP-binding sites have been identified by in vivo
autoradiography in rat adrenal medulla and by in vitro autoradiography in
bovine, guinea pig, tree shrew, rabbit, and rat adrenal medulla (Bormann
et al., 1989; Fuchs et al., 1986; Gerstberger et al., 1992; Konrad et al., 1992;
Maurer and Reubi, 1986; Morel et al., 1988; Niina et al., 1996). Specific
binding sites for ANP have been identified in the phaeochromocytoma cell line
PC12 (Boumezrag et al., 1988). 125I-ANP-binding sites, instead, have not
been identified in mouse, hamster, monkey, human, and in other studies in
bovine, guinea pig, and rat (Chai et al., 1986; Lynch et al., 1986; Mantyh et al.,
1986; Maurer and Reubi, 1986; Stewart et al., 1988). In rat, 125I-BNP
and125I-[Tyr0]-CNP-binding sites have also been identified (Konrad
et al., 1992). The number of ANP-binding sites has also been found to
increase regularly in fetal (day 17 of gestation and term) and neonatal (weeks
1 and 4) rats (Deloof et al., 1994).
NPR-A and NPR-B mRNA, but not NPR-C mRNA, have been identified by in situ hybridization in adrenal chromaffin cells of monkey (Wilcox
et al., 1991). This finding is in keeping with displacing of 125I-ANP

and125I-BNP bindings by ANP and BNP but not by selective analogues
for NPR-C in rat and bovine (Konrad et al., 1992; Niina et al., 1996). In
rat adrenal medulla, the mRNA of the three subtypes has been found by in
situ hybridization, the amount of NPR-A mRNA being the highest
(Grandcle´ment et al., 1997). The above receptors were selectively present
in adrenaline-containing chromaffin cells and not in the noradrenalinecontaining ones (Grandcle´ment et al., 1997).
NPR-A mRNA expression has also been reported to be significantly
increased in the adrenal medulla of adult pro-ANP gene-disrupted mice
(O’Tierney et al., 2007).

4. Effects of Natriuretic Peptides on the
HPA Axis
4.1. Hypothalamus
ANP has been found to modulate the membrane excitability of neurons of the
lateral septal nucleus, lateral paraolfactory area, bed nucleus of the anterior
commissure, and medial preoptic area (Wong et al., 1986). ANP has been
found to produce significant increases in blood pressure and heart rate when
injected into the preoptic suprachiasmatic nucleus, suggesting it may play an
important role in central cardiovascular regulatory mechanisms (reviewed in
Oparil et al., 1996). Moreover, intracerebroventricular injection of ANP has


Natriuretic Peptides and Adrenals

13

been found to inhibit dehydration- and angiotensin II-induced water intake in
conscious, unrestrained rats (Antunes-Rodrigues et al., 1985).
ANP, BNP, and CNP have been found to reduce both spontaneous and
acetylcholine, Kỵ and angiotensin II-evoked norepinephrine release in slices of

rat hypothalamus (Giridhar et al., 1992; Vatta et al., 1996). ANP has been
found to increase neuronal norepinephrine uptake in hypothalamus
(Fernandez et al., 1993) and in organum vasculosum lamina terminalis and
organum subfornical (Vatta et al., 1995) of rat. BNP and CNP have also been
found to increase neuronal norepinephrine uptake in slices of rat hypothalamus
and, particularly, independently of the hypothalamic nucleus involved (preoptic, periventricular, paraventricular, SON, and arcuate nuclei; median eminence) (Rodriguez Fermepin et al., 2000; Vatta et al., 1996). ANP has been
found to diminish monoamine oxidase activity, but not catechol-O-methyl
transferase activity and the formation of deaminates metabolites, in rat hypothalamus slices (Vatta et al., 1998). Moreover, centrally applied ANP has been
reported to increase the hypothalamic content of NE, diminish its utilization
and turnover, inhibit basal and KCl-evoked tyrosine hydroxylase activity, and
increase cyclic GMP levels (Vatta et al., 1999).
Experimental studies on rats have shown that ANP microinjections into the
third ventricle do not change basal levels of OT but attenuate the increase in
OT secretion induced by hyperosmolarity (Chriguer et al., 2001; Gutkowska
et al., 1997; Lewandowska et al., 1992; McCann et al., 1996; Poole et al.,
1987). ANP has also been found to markedly inhibit OT release in vitro from
the isolated neurointermediate lobe both under basal condition as well as
during stimulation (Lewandowska et al., 1992; Poole et al., 1987).
ANP has been proven to be a potent inhibitor of VP neurons of the PVN in
anesthetized rats (Okuya and Yamashita, 1987; Standaert et al., 1987). Intravenous infusion of ANP has been found to reduce dehydration and hemorrhageinduced VP release in the rat (Samson, 1985). ANP has been reported to inhibit
the basal and stimulated release of VP in hypothalamo-neurohypophyseal slice
preparations and in superfused rat posterior pituitary gland ( Januszewicz et al.,
1986; Obana et al., 1985). ANP has also been found to inhibit VP release in vitro
from the neurointermediate lobes both under basal condition as well as during
stimulation (Lewandowska et al., 1992; Poole et al., 1987). Intracerebroventricular injections of ANP, BNP, or CNP have been found to show inhibitory
effects on the VP secretion (e.g., Iitake et al., 1986; Lewandowska et al., 1992;
Makino et al., 1992; Poole et al., 1987; Samson et al., 1991; Shirakami et al.,
1993). The three natriuretic peptides have also been reported to inhibit the
basal secretion of VP from rat SON neurons in dissociated cell preparations,
CNP being the most potent inhibitory factor (Yamamoto et al., 1997).

Reduction of VP plasma levels due to central ANP stimulus has been observed
in both euhydrated and dehydrated sheeps (Lee et al., 1987) and rats
(Manzanares et al., 1990). In rats, inhibition of VP secretion was not accompanied by modifications in the concentrations of 3,4-dihydroxyphenylacetic


14

Andrea Porzionato et al.

acid and dopamine, indicating that ANP-induced suppression of VP secretion
is not mediated by tuberohypophysial or tuberoinfundibular dopaminergic
neurons (Manzanares et al., 1990). Conversely, in dehydrated but not in
euhydrated rabbits, infusion of ANP has also been found to inhibit secretion
of VP (Gerstberger et al., 1992). ANP and BNP have also been found to
decrease the firing rate and hyperpolarize the membrane potential in phasically
firing (putative VP) but not in nonphasically firing (putative OT) neurons of
SON; inhibition of cGMP synthesis was also reported in neurons of SON
(Akamatsu et al., 1993). ANP and BNP have been found to inhibit AV3V
neurons, suggesting direct actions of the peptides on drinking, and in the SON,
these peptides inhibited selectively putative VP neurons but not putative OT
neurons, suggesting direct actions of the peptides on VP secretion (Yamamoto
et al., 1995). The central inhibition of OT and VP release from the magnocellular neurosecretory cells by ANP has been suggested to be mediated by
presynaptic inhibition of glutamate release from osmoreceptor afferents
derived from the organum vasculosum lamina terminalis (Richard and
Bourque, 1996). Experiments through injection of highly specific antiserum
against ANP into the third cerebral ventricle of rats also showed that the
inhibitory role in suppressing ACTH release during stress is in part mediated
by inhibition of VP release (Franci et al., 1992). Conversely, it has also been
reported an increase of the plasma VP response to acute moderate hemorrhage
after intracerebroventricular injection of CNP (Charles et al., 1995).

It has also been demonstrated that CNP has a potent and selective
inhibitory effect on magnocellular cells of SON and PVN, which is
mediated by NPR-C (Rose et al., 2005). Moreover, since NPR-C binds
all natriuretic peptides with equal affinity (Levin et al., 1998), it has been
suggested that this receptor could mediate the hypothalamic effects by the
other natriuretic peptides (Rose et al., 2005).
It has been reported that intracerebroventricular injection of ANP in rats
does not modify tuberoinfundibular dopaminergic neuronal activity and serum
prolactin levels, but it attenuates the stimulatory effects of angiotensin II on
tuberoinfundibular dopaminergic neuronal activity, negatively modulating
also the inhibitory effect on serum prolactin level (Yen and Pan, 1997). ANP
and BNP have been reported to cause a dose-dependent increase in dopamine
accumulation in cultured rat hypothalamic cells through an increase in intracellular cGMP concentration (Kadowaki et al., 1992). Franci et al. (1992) also
reported a role by ANP in augmenting the prolactin release in stress through a
hypothalamic action. On the other hand, CNP has been found to stimulate
prolactin secretion in rats by a hypothalamic site of action (Huang et al., 1992a;
Samson et al., 1995).
In rat, ANP has been found to inhibit acetylcholine- and KCl-induced
release of corticotrophin-releasing factor in vitro (Grossman et al., 1993;
Ibanez-Santos et al., 1990; Takao et al., 1988) and to increase its immunoreactivity in the hypothalamus in vivo (Biro´ et al., 1996). In humans,