| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
First Department of Internal Medicine (S.Y., I.M., K.Z., S.E.) and Physiology (Y.U., H.Y.), School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan; and Department of Physiology (H.K.), Miyazaki Medical College, 5200 Kihara Kiyotake Miyazaki-gun, Miyazaki, 889–16, Japan
Address all correspondence and requests for reprints to: Isao Morimoto, First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807, Japan. E-mail: isaomo{at}med.uoeh-u.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Since the demonstration of PTHrP protein and messenger RNA (mRNA) and its receptor protein in a variety of tissues associated with the cardiovascular system, it has been suggested that PTHrP functions as an autocrine/paracrine modulator in the cardiovascular tissues (4, 5, 6, 7). PTHrP acts on vascular smooth muscle, where it is produced (8, 9), as a vasorelaxant (10, 11, 12). Iv administration of the amino-terminal PTHrP peptides induces hypotension in a specific manner (7, 13, 14). PTHrP also acts on heart muscle by increasing heart rate and contractility (4, 10, 15, 16). Furthermore, vasoconstrictor substances, such as angiotensin II, endotheline, and catecholamines stimulate PTHrP mRNA expression in vascular smooth muscle cells (7, 13). These results taken together suggest that PTHrP functions locally as an active autocrine or paracrine factor involving in the relaxation of vascular smooth muscle.
The type I PTH/PTHrP receptor is found in the brain, including the hypothalamus (17). Moreover, PTHrP is also abundant in the central nervous system (CNS) (18, 19). However, the physiological role of PTHrP in the CNS is not clear. Arginine-vasopressin (AVP), which has antidiuretic and pressor activities, is produced from hypothalamic magnocellular neurons in the supraoptic nuclei (SON) and paraventricular nuclei (PVN) (20). In our recent in vitro study, PTHrP(1–34), but not PTH(1–34), involved in the release of AVP from rat SON through a novel receptor distinct from a classical type I PTH/PTHrP receptor or a nonclassical type II PTH/PTHrP receptor that has not been cloned yet (21). In this study, we have examined the effect of intracerebroventricular (icv) administration of PTHrP(1–34) on AVP secretion and the expression of AVP mRNA in the SON of conscious rats. The results indicated that centrally administered PTHrP(1–34) causes the secretion of AVP from the hypothalamus through a novel receptor distinct from the PTH/PTHrP receptors reported previously.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animals
Male Wistar rats (150–200 g) were housed under alternate 12-h
periods of light and darkness at 23 C. Standard laboratory rat chow and
water were available ad libitum. One week before
experiments, the rats were anesthetized with pentobarbital sodium (50
mg/kg, ip) and positioned in a stereotaxic apparatus. A 21-gauge
stainless steel cannula was inserted into the right lateral ventricle
using the following stereotaxic coordinates: 0.8 mm posterior to
bregma, 1.4 mm lateral to midline, 4.0 mm below the surface of the
skull, according to the rat brain in sterotaxic coordinates (21a). The
cannula was fixed with dental cement and anchored to the skull with two
jeweler’s screws.
Experiments were done in the conscious, freely moving rat between 09.00 and 11.30 a.m. on the day, 1 week following recovery from surgery. PTHrP(1–34), PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), and rat PTH(1–34) were dissolved in 0.9% (wt/vol) NaCl and injected icv in a volume of 1 µl over a 1-min period with a Hamilton microsyringe. As a control, 1 µl of saline (vehicle) was injected. To verify the position of the icv injection site, Pontamine Sky Blue (Tokyo Kasei Industry, Tokyo, Japan) was administered through the cannula at the end of each experiment. Five rats were used for each group in all experiments.
All procedures were approved by the Animal Care Committee of the University of Occupational & Environmental of Health and complied with the guidelines of the Japan Physiological Council on Animal Care.
Effects of peptides on AVP release
Time-course study. PTHrP(1–34) (100 pmol/rat), rat
PTH(1–34) (100 pmol/rat), or saline (1 µl/rat) was injected icv 0,
15, 30, 60, or 180 min before decapitation.
Dose-response study. PTHrP(1–34), rat PTH(1–34), or PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (0, 1, 10, 50, 100, 200, or 400 pmol/rat) was injected icv 15 min before decapitation.
Effect of PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or PTH(1–34) pretreatment on PTHrP(1–34)-induced AVP secretion. To elucidate whether PTHrP(1–34)-induced AVP secretion is affected by PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or rat PTH(1–34), rats were pretreated with an icv injection of PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (200 pmol/rat) or PTH(1–34) (200 pmol/rat), and 10 min later PTHrP(1–34) (200 pmol/rat) was then injected icv. The blood samples were obtained at 15 min after the PTHrP(1–34) injection. The method of PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) pretreatment (dose and time) had been determined by preliminary experiments (data not shown).
Plasma AVP, sodium, and total protein measurement
After decapitation, the trunk blood was obtained for measurement
of various parameters. Blood samples were collected into chilled tubes
containing ethylenediaminetetraacetate (potassium salt). After
immediate separation at 4 C, plasma AVP was extracted through a Sep-pak
C18 cartridge (Waters Associates, Inc., Milford, MA) and measured using
an RIA kit (Mitsubishi Chemical Co., Ltd., Tokyo, Japan); the minimum
detectable dose was 0.063 pg/tube, and the 50% intercept was 0.728
pg/tube (22). Plasma sodium was measured using an autoanalyzer
(Hitachi, Tokyo, Japan) for estimation of the change in plasma
osmolality. Total protein was also measured by autoanalyzer for
estimation of the change in plasma volume (23).
Blood pressure measurement
Rats were reanesthetized on the day before the experiment, and a
polyethylene cannula was inserted into the right carotid artery for
blood pressure measurement. PTHrP(1–34), rat PTH(1–34) or
PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (0, 1, 10, 50, 100, 200, or 400 pmol/rat) was injected
icv, and arterial blood pressure was measured with a blood pressure
transducer (Gould Inc., Oxnard, CA) connected to cannula implanted into
carotid artery. Arterial blood pressure was recorded continuously for
180 min after the injection. Baseline values were recorded for 10 min
before the injection.
In situ hybridization histochemistry
Frozen coronal brain sections were cut at 12 µm in a cryostat
at -20 C and then mounted onto gelatin/chrome alum-coated slides that
were kept at -80 C until further processing. The position of the PVN
and SON were identified by reference to a stereotaxic atlas of the rat
brain (21a). The PVN and SON were chosen from four sections in a rat to
measure the density of autoradiography. The slides were warmed to room
temperature and allowed to dry for 10 min, then fixed in 4%
formaldehyde in PBS for 5 min, washed twice in PBS, and incubated in
0.9% NaCl containing 0.25% acetic anhydride (vol/vol) and 0.1
M triethanolamine (TEA) at room temperature for 10 min. The
sections were then dehydrated through 70% (1 min), 80% (1 min), 95%
(2 min) and 100% (1 min) ethanol and dilapidated in 100% chloroform
for 5 min. The slides were then partially rehydrated in 100% followed
by 95% ethanol and allowed to dry briefly in air. Hybridization with
complementary DNA (cDNA) probes of AVP (700 bp) (presented by Dr. D.
Richter, Hamburg, Germany) was carried out at 37 C overnight in 45
µl of buffer consisting of 50% formamide and 4 x SSC (1
x SSC = 150 mM NaCl and 15 mM sodium
citrate) containing 500 µg/ml sheared salmon sperm DNA (Sigma), 250
µl/ml baker’s yeast total RNA (Boehringer Manheim, GmbH, Mannheim,
Germany), 1 x Denhardt’s solution (0.02% Ficoll, 0.02%
polyvinylpyrrolidone, and 0.02% BSA) and 10% dextran sulfate (500,000
mol wt, Sigma), under a Nescofilm (Bando Chemical 1MD, Ltd., Osaka,
Japan) coverslip. A total of 5 x 105 cpm/slide were
used. After hybridization, the sections were washed for 1 h in
four changes of 1 x SSC at 55 C and for further 1 h in two
changes of 1 x SSC at room temperature. All sections were treated
simultaneously throughout to minimize the effects of variations in
hybridization and wash stringently. Hybridized sections of the PVN and
SON were opposed to autoradiography film (Hyperfilm, Amersham,
Buckinghamshire, UK) for 24 h. Quantitative image analysis was
undertaken with an MCID Image Analysis System (Imaging Research Inc.,
Ontario, Canada). The mean optical density of autradiographs was
measured by comparison with simultaneously exposed [14C]
microscale (Amersham).
Northern blot analyses
Rat brain was removed after decapitation 60 min after icv
administration of PTHrP(1–34), PTH (1–34), PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (200
pmol/rat) or saline (1 µl/rat) as previously described (24). Then,
coronal hypothalamic slices containing the SON (400 µm in thickness)
were cut with a vibrating slicer. Immediately after sectioning, the
slices were carefully trimmed so that they contained only the SON and
its perinuclear zone. The total cellular RNA in the rat SON, obtained
as above, was isolated by an acid guanidium-phenol-chloroform
method using ISOGEN (Nippon Gene, Tokyo, Japan). The total RNA (10
µg) was electrophoresed in a 1% agarose gel and was transferred to a
nylon membrane filter (Amersham). The blots were hybridized with cDNA
probes of AVP (700 bp) (presented by Dr. D. Richter) and cyclophilin
labeled with 32P-deoxy-CTP (Amersham). Cyclophilin was used
as a housekeeping gene. To compare the density of each band, a Bioimage
Analyzer (BAS-2000; Fuji Film, Tokyo, Japan) was used to accurately
measure the density of AVP and cyclophilin mRNA, respectively, and the
results were evaluated as changes of AVP/cyclophilin mRNA level after
treatment.
Statistics
Results are expressed as mean ± SE. Comparison
between groups was performed by Student’s t test, except
where multiple comparison were made, when Dunnett’s test was
employed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). On the
other hand, AVP levels were not changed by the icv administration of
rat PTH(1–34) (100 pmol/rat) or saline. The increase in plasma levels
to PTHrP(1–34) was observed in a dose-dependent manner and ranged from
10–400 pmol/rat (Fig. 2, P < 0.01). The maximum effect (8.11 ± 0.41
vs. control 2.72 ± 0.52, P < 0.01)
was obtained at 200–400 pmol/rat of PTHrP(1–34), and the half-maximal
effective dose (ED50) was approximately 50 pmol/rat.
However, neither PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) nor PTH(1–34) had any effect on plasma
AVP levels within the same ranges (Fig. 2). Injection icv of rat or
human PTH(1–34) obtained from the two companies resulted in the same
effects.
|
|
).
|
),
suggesting that blood volume or plasma osmolality was not involved in
the AVP secretion in the experiments.
|
).
In situ hybridization histochemistry
A striking and distinct distribution of AVP mRNA was observed
within the hypothalamus in the studies of in situ
hybridization histochemistry. It was most abundant in the SON and PVN.
PTHrP(1–34) (200 pmol/rat) significantly stimulated AVP mRNA levels in
the SON and PVN at 30 min after icv injection. These levels reached a
maximum at approximately 180 min (Fig. 4). Icv injection of PTH(1–34) (200
pmol/rat) or saline did not induce an increase in AVP mRNA in the SON
and PVN (data not shown).
|
, total RNA from the SON treated with
PTHrP(1–34) showed a stronger hybridization-positive (0.7 kb) band
(2-fold as an arbitrary unit of AVP mRNA/cyclophilin mRNA) against cDNA
of AVP compared with that without PTHrP treatment. However, icv
injection of PTH(1–34) (200 pmol/rat) had no effects on AVP mRNA
expression in the SON. The PTHrP(1–34) (200 pmol/rat)-stimulated AVP
mRNA expression was suppressed by pretreatment with PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (200
pmol/rat), but not by that with PTH(1–34) (200 pmol/rat).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PTHrP mRNA has been detected by in situ hybridization in the hypothalamus, especially in the SON and PVN (26), where PTH mRNA transcripts are also expressed (26, 27). PTHrP(1–34) is also identified in the posterior pituitary (19), although it is not clear whether PTHrP(1–34) acts directly on the posterior pituitary to secrete AVP. We are convinced that PTHrP(1–34) acts on hypothalamic nuclei, including the anterventral third ventricle (AV3V), the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), PVN, and SON, to secrete AVP. Accumulating evidence reveals that the AV3V region and SFO are an important site for regulating the body water and electrolyte homeostasis in the CNS (28). Therefore, icv administration of PTHrP(1–34) stimulates to secrete plasma AVP into blood from the SON and magnocellular neurons of the PVN through the AV3V region and SFO.
This effect of PTHrP(1–34) was dose dependent (10–400 pmol/rat) with an ED50 of approximately 50 pmol/rat. The threshold concentration of PTHrP to evoke AVP release was approximately 10 pmol/rat. A previous report (16) indicated that the systemic and regional hemodynamics were affected by the iv administration of 1 nmol/100 g BW of PTHrP in conscious, unrestrained rats. Although PTHrP(1–34) in the cerebrospinal fluid (CSF) of rats has not been demonstrated, a question arises whether PTHrP(1–34) concentrations used in these experiments are physiological or pharmacological effect on AVP secretion. PTHrP induces AVP release through an autocrine or paracrine mechanism as a neuromodulator. The concentration of PTHrP near the cell body and dendrites may be much higher than that of the plasma and CSF. The content of PTHrP in the rat SON measured by RIA was 0.42 ± 0.1 pmol/mg protein (n = 12). We don’t know whether the levels in the SON are comparable with the dose of PTHrP required to stimulate AVP release from the SON through a paracrine or autocrine mechanism.
The plasma AVP levels induced by PTHrP infusion in this study were similar to the levels observed after the restriction of water and food intake for 24 h in our experiments (9.50 ± 0.51 pg/ml, n = 4). In previous studies (29, 30), plasma AVP levels in response to hyperosmolality induced by ip injection of hyperosmotic saline, hypovolemia by ip injection of polyethylene glycol or icv administration of pituitary adenylate cyclase-activating polypeptide were equal to the levels observed by icv administration of PTHrP(1–34). In our in vitro studies, a desensitization of PTHrP-induced AVP release from the SON was not observed by consecutive stimulation of PTHrP. Though we do not examine the effect of chronic icv infusion of PTHrP on AVP secretion, chronic PTHrP stimulation in the SON may cause the excess of blood AVP, which leads to syndrome of inappropriate secretion of ADH (SIADH).
Both PTH(1–34) and PTHrP(1–34) bind to a G protein-coupled PTH
receptor (type I PTH/PTHrP receptor) with equal affinity to stimulate
the generation of cAMP and also have the same degree of bioactivities
in bone and kidney (1, 31, 32, 33). The first 13 amino acids of PTH and
PTHrP exhibit amino acid homology and have biological activity upon
bone and kidney (1). The C-terminal amino acid sequences (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) of
both peptides, despite having no primary sequence homology, are
functionally important in binding to a type I PTH/PTHrP receptor
expressed on bone and kidney (2, 34). PTH(1–34) and amino-terminal
PTHrP also exerts biochemical effects through nonclassical PTH/PTHrP
receptor (type II) linked with intracellular Ca2+ in
lymphocytes, insulinoma cells, keratinocytes, and squamous carcinoma
cell lines (35, 36, 37). In this study, icv administration of PTH(1–34)
did not affect plasma AVP levels and AVP mRNA expression in the
hypothalamus determined by Northern blot analyses and in
situ hybridization histochemistry. These in vivo
results are consistent with our previous in vitro
observations that PTHrP(1–34), but not PTH(1–34), stimulate AVP
release from the SON slices through a receptor distinct from the type I
or type II PTH/PTHrP receptors (21). The in vivo effects
of PTHrP(1–34) were antagonized by preadministration of
PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), a competitive antagonist, but not by that of
PTH(1–34) (Fig. 3), indicating that the bioactivity of PTHrP(1–34) on
AVP secretion from the rat SON is located in the N-terminus and it
binds to the receptors at the C-terminus as same as the results of our
previous in vitro studies (21).
PTHrP and type I PTH/PTHrP receptors are widely distributed in the brain (17, 18, 19, 38). Recent studies (39, 40) have demonstrated that a PTH2 receptor, which is activated by PTH, but not PTHrP, is abundant in the CNS (19). Usdin also suggests the presence of new peptide linked with PTH2 receptor in extracts prepared from bovine hypothalamus (41). The PTH2 receptor does not mediate the major effect of PTH on calcium and phosphate metabolism, suggesting that PTH2 receptor may be a neurotransmitter receptor. PTH and PTHrP may act on the hypothalamus through different receptors: PTH2 receptor and the novel receptor observed in our studies, respectively. Further studies are needed for understanding a physiological role of PTHrP in the CNS.
In conclusion, our in vivo data suggest that PTHrP(1–34) stimulates AVP secretion from the rat SON neurons through a novel PTHrP receptor distinct from the type I or type II PTH/PTHrP receptors.
Received June 16, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Sugimura, T. Murase, S. Ishizaki, K. Tachikawa, H. Arima, Y. Miura, T. B. Usdin, and Y. Oiso Centrally Administered Tuberoinfundibular Peptide of 39 Residues Inhibits Arginine Vasopressin Release in Conscious Rats Endocrinology, July 1, 2003; 144(7): 2791 - 2796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yamamoto, I. Morimoto, Y. Tanaka, N. Yanagihara, and S. Eto The Mutual Regulation of Arginine-Vasopressin and PTHrP Secretion in Dissociated Supraoptic Neurons Endocrinology, April 1, 2002; 143(4): 1521 - 1529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Guerreiro, J. Fuentes, D. M. Power, P. M. Ingleton, G. Flik, and A. V. M. Canario Parathyroid hormone-related protein: a calcium regulatory factor in sea bream (Sparus aurata L.) larvae Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2001; 281(3): R855 - R860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Ward, C. J. Small, K. G. Murphy, A. R. Kennedy, M. A. Ghatei, and S. R. Bloom The Actions of Tuberoinfundibular Peptide on the Hypothalamo-Pituitary Axes Endocrinology, August 1, 2001; 142(8): 3451 - 3456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. J. Hoare, D. A. Rubin, H. Juppner, and T. B. Usdin Evaluating the Ligand Specificity of Zebrafish Parathyroid Hormone (PTH) Receptors: Comparison of PTH, PTH-Related Protein, and Tuberoinfundibular Peptide of 39 Residues Endocrinology, September 1, 2000; 141(9): 3080 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Rubin and H. Juppner Zebrafish Express the Common Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor (PTH1R) and a Novel Receptor (PTH3R) That Is Preferentially Activated by Mammalian and Fugufish Parathyroid Hormone-related Peptide J. Biol. Chem., October 1, 1999; 274(40): 28185 - 28190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Abe, H. Suzuki, M. Notoya, T. Yamamoto, and S. Hirose Ig-Hepta, a Novel Member of the G Protein-coupled Hepta-helical Receptor (GPCR) Family That Has Immunoglobulin-like Repeats in a Long N-terminal Extracellular Domain and Defines a New Subfamily of GPCRs J. Biol. Chem., July 9, 1999; 274(28): 19957 - 19964. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), rat PTH(1–34)
(
) injected icv at a dose of 100 pmol/rat, or vehicle (
). After
the indicated periods, the animals were decapitated, and plasma AVP
levels were measured as described in Materials and
Methods. The values are expressed as mean ±
SE (n = 5). *, P < 0.01
vs. control.
