help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ward, H. L.
Right arrow Articles by Bloom, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ward, H. L.
Right arrow Articles by Bloom, S. R.
Endocrinology Vol. 142, No. 8 3451-3456
Copyright © 2001 by The Endocrine Society

ARTICLES

The Actions of Tuberoinfundibular Peptide on the Hypothalamo-Pituitary Axes

H. L. Ward, C. J. Small, K. G. Murphy, A. R. Kennedy, M. A. Ghatei and S. R. Bloom

Metabolic Medicine, Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom W12 ONN

Address all correspondence and requests for reprints to: Professor S. R. Bloom, Metabolic Medicine, Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom. E-mail: s.bloom{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tuberoinfundibular peptide is a recently discovered agonist for the PTH receptor-2; the latter has a wide distribution including the external zone of the median eminence of the hypothalamus, suggesting a role in neuroendocrine function. We have investigated the effects of tuberoinfundibular peptide on the hypothalamo-pituitary axes in vitro and in vivo. Tuberoinfundibular peptide had effects on the hypothalamo-pituitary-adrenal axis with increased release of ACTH-releasing factor (tuberoinfundibular peptide 100 nM 4.4 ± 0.6 pmol/explant vs. control 2.9 ± 0.4 pmol/explant, P < 0.001) and increased release of arginine vasopressin (tuberoinfundibular peptide 100 nM 563.5 ± 55.5 fmol/explant vs. control 73.4 ± 9.6 fmol/explant, P < 0.01) from in vitro hypothalamic explants. Intracerebroventricular administration of tuberoinfundibular peptide and PTH(1–34) resulted in elevated plasma ACTH at 10 min post injection (saline 13.5 ± 2.1 pg/ml, tuberoinfundibular peptide 3 nmol 32.3 ± 4.0 pg/ml; P < 0.01 to saline: PTH(1–34) 10 nmol 28.9 ± 3.2 pg/ml: P < 0.05 to saline).

Tuberoinfundibular peptide also had both in vitro and in vivo effects on the hypothalamo-pituitary-gonadal axis with increased release of LH-releasing hormone (tuberoinfundibular peptide 100 nM 28.5 ± 5.1 fmol/explant vs. control 19.3 ± 2.5 fmol/explant, P < 0.05) from in vitro hypothalamic explants. Both intracerebroventricular and peripheral administration of tuberoinfundibular peptide had effects on the hypothalamo-pituitary-gonadal axis. Intracerebroventricular injection of tuberoinfundibular peptide increased plasma LH (tuberoinfundibular peptide 10 nmol 0.70 ± 0.09 ng/ml vs. saline 0.42 ± 0.04 ng/ml at 10 min, P < 0.05).

Intraperitoneal administration of tuberoinfundibular peptide also increased plasma LH (tuberoinfundibular peptide 30 nmol 0.53 ± 0.09 ng/ml vs. saline 0.21 ± 0.04 ng/ml at 10 min, P < 0.05). In addition to these actions on the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes, an increased release of GH-releasing factor (GRF) from hypothalamic explants (tuberoinfundibular peptide 100 nM 770.9 ± 90.7 pg/explant vs. control 657.8 ± 77.7 pg/explant, P < 0.01) was observed. Overall, these data show the actions of tuberoinfundibular peptide on the hypothalamo-pituitary axes and suggest that it may play a role in the control of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUBEROINFUNDIBULAR PEPTIDE (TIP39), a 39 amino acid peptide, was first identified in 1999 by Usdin et al. (1). It was isolated from bovine hypothalami and so far has not been identified in any other tissue or species. TIP39 has been demonstrated to bind with higher affinity than PTH to the rat parathyroid receptor 2 (PTH2-R), being twice as potent as PTH in stimulating cAMP accumulation in COS-7 cells transfected with the rat PTH2-R (1). Despite 50% homology (2) between the parathyroid receptor 1 (PTH1-R) and PTH2-R, the PTH1-R is not activated by TIP39 (1, 3). PTH is an 84 amino acid peptide produced from the parathyroid glands. TIP39 shares 9 of its amino acid residues with bovine PTH when they are aligned. To date there has been very little data published regarding the biological actions of TIP39. Studies have shown that the first 6 amino acids of TIP39 are important for receptor interaction; without them the fragment TIP (7–39) has a dramatically reduced affinity for the PTH2-R and acts as a PTH1-R antagonist (4, 5, 6, 7).

The classical actions of PTH on bone and calcium metabolism are mediated via the PTH1-R, at which TIP39 has little or no effect (1, 3). TIP39 alone among PTH and PTH-related protein (PTHrP) has a high binding affinity at the rat PTH2-R (1). The zebra fish is the only species in which a third PTH receptor has been identified (8). PTHrP acts via the PTH1-R (9) and can stimulate AVP release in vivo and in vitro from the supraoptic nucleus (SON), unlike PTH, and therefore maybe acting via an unidentified receptor (10, 11). The whole family of PTH-related peptides and receptors is expanding and to date evidence suggests that TIP39 or a TIP39-like peptide is the PTH2-R endogenous ligand.

Northern blotting shows that PTH2-R mRNA is highly expressed in the central nervous system (2). In situ hybridization studies have revealed PTH2-R mRNA expression in the periventricular area of the hypothalamus (12), where the majority of PTH2-R positive neurons can be dual stained with somatostatin (SST) (1). Other hypothalamic areas that express PTH2-R mRNA, shown by in situ hybridization, include the arcuate, ventromedial and the dorsal paraventricular nuclei plus the external zone of the median eminence. The external zone of the median eminence of the hypothalamus demonstrates positive staining with a PTH2-R selective antibody (13). A few cells of the anterior pituitary gland expressed PTH2-R mRNA, and these cells had a morphological appearance consistent with hormone-secreting cells (12). These studies demonstrate that the PTH2-R is located in hypothalamic areas known to be important in the control of anterior pituitary hormone secretion.

PTH-like immunoreactivity has been demonstrated in the central nervous system of mice, with nerve fibers extending to the external zone of the median eminence and terminating near the hypophyseal portal blood vessels (14). Circulating PTH is a sufficiently large molecule to require a transport system to cross the blood brain barrier. PTH mRNA has previously been detected in the hypothalamus (15, 16, 17); however Usdin et al. (3) were unable to repeat this during their investigations with the PTH2-R. Due to the low affinity of PTH for PTH2-R a search was commenced for a superior ligand for the central PTH2-receptors. This resulted in the discovery of TIP39, which is a high affinity agonist at the rat PTH2-R (1, 3, 18). At present, TIP39 is the best candidate for the endogenous ligand at the rat PTH2-R, the hypothalamic location of which suggests a neuroendocrine role. Therefore, the aim of this study was to determine the impact of TIP39 on the hypothalamo-pituitary axes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Wistar rats weighing 250–300 g (specific pathogen free, Imperial College School of Medicine, London, UK) were maintained under a controlled environment (temperature 21-23 C, 12-h light, 12-h dark cycle, lights on at 0700 h) with ad libitum access to food (RM1 diet SDS UK Ltd.) and water. All animal procedures were approved by the British Home Office Animals Scientific Procedures Act 1986 (Project License Nos. 90/1077 and 70/3888).

Materials
The reagents for the hypothalamic explant experiments were purchased from BDH Laboratory Supplies (Poole, Dorset, UK). TIP39, rat PTH (1–34), and GH-releasing peptide-6 (GHRP-6) were purchased from Peninsula (St. Helens, Merseyside, UK). Tissue culture materials were supplied by Life Sciences Technology (Paisley, Scotland, UK) and all other reagents by Merck Ltd. (Lutterworth, UK) or Sigma (Poole, UK).

Static incubation of medial basal hypothalamic explants
A static incubation system was used as previously described (19). Male Wistar rats were killed by decapitation and the whole brain removed immediately. The brain was mounted, ventral surface uppermost, and placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A 1.7-mm slice was taken from the basal hypothalamus and incubated in individual chambers containing 1 ml of artificial cerebrospinal fluid (aCSF) (126 mM NaCl, 0.09 mM Na2HPO4, 20 mM NaHCO3, 1.4 mM CaCl2, 0.09 mM MgSO4, 6 mM KCl, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 µg/ml aprotinin), which was equilibrated with 95% O2 and 5% CO2 (19). The tubes were placed on a platform in a water bath maintained at 37 C. After an initial 2-h equilibration period, each explant was incubated for 45 min in 600 µl aCSF (basal period) before being challenged with test peptide for 45 min. The viability of the tissue was confirmed by a final 45 min exposure to aCSF containing 56 mM KCl; isotonicity was maintained by substituting K+ for Na+. TIP39 was used in concentrations ranging from 1–100 nM. A standard concentration of ten times receptor EC50 is used to achieve tissue penetration. TIP39 100 nM is ten times the EC50 for the rat PTH2-R from published data (1). The active N-terminal of rat PTH (1–34) was used at a concentration of 100 nM (1). Explants were excluded from analysis if there was higher release during the basal period than the final, hyperkalaemic period.

At the end of each experimental period, the aCSF was removed and stored at -20 C until measurement of hypothalamic releasing factors, TRH, VIP, ACTH-releasing factor (CRF), AVP, SST, GRF and LH-releasing hormone (LHRH), immunoreactivity by RIA. Each experiment was repeated such that for the hypothalami incubated with TIP39 100 nM n = 64 for CRF; n = 61 for AVP; n = 86 for VIP; n = 73 for LHRH; n = 74 for TRH; n = 66 for SST; and n = 44 for GRF. For the concentration dependence studies with TIP39 1 nM, n = 13 for CRF and n = 17 for LHRH and for TIP 10 nM n = 14 for CRF and n = 23 for LHRH. For the hypothalamic explants incubated with PTH (1–34), 100 nM n = 21 for CRF and n = 24 for LHRH.

Anterior pituitary dispersion
The pituitaries were dispersed using a previously described method (20). Monodispersed cells were plated at 2.0 x 105 cells per well in sterile poly-L-lysine coated 48-well plates. The cells were allowed to attach to the tissues culture wells for 4 h in serum-free DMEM then replaced with 1 ml of medium containing 10% heat-inactivated FCS. The cells were cultured at 37 C in air with 5% CO2 for 48 h before the secretion experiment when cells were washed twice in 1 ml of DMEM containing 0.1% BSA. After a 2 h preincubation, the medium was removed and replaced with 500 µl of medium containing the test substance and the cells incubated for 4 h with TIP39, concentrations ranging from 0.1–100 nM. CRF, GRF, TRH, and LHRH were used at 100 nM as positive controls; these concentrations have been previously shown to stimulate pituitary hormone release (21). Each experiment was repeated three times. The incubation and preincubation times were based on pilot studies and previously published work (20).

The effect of peripheral (ip) TIP39 on pituitary hormone secretion
Experiments were carried out during the early light phase (0900–1100 h). Male Wistar rats received an ip 0.5 ml injection of TIP39 30 nmol, GHRP-6 30 nmol (a synthetic GH secretagogue) or vehicle (0.9% saline). This dose of GHRP-6 is known to stimulate GH release (22). The rats were administered saline sham injections before the study to habituate them to the experimental procedure. Rats receiving TIP39, GHRP-6, or saline were decapitated and trunk blood collected at 10, 20, and 60 min following injection. Blood was collected into plastic tubes containing potassium EDTA (final concentration of 1.2–2 mg EDTA/ml blood) (Sarstedt, Leicester, UK). Those receiving GHRP-6 were killed at 20 min after injection when GHRP-6 has been shown to have a potent, stimulatory effect on GH levels (22). Each group had n = 8–10. Plasma was separated by centrifugation, immediately frozen and stored at -70 C until assay.

Intracerebroventricular (icv) cannulation
Stainless steel 22-gauge cannulae (Plastics One Inc., Roanoke VA) were inserted stereotactically into the third cerebral ventricle (0.8 mm caudal to the bregma in the midline, 6.5 mm below the surface of the skull) using a Kopf sterotactic frame. The coordinates were taken from the Paxinos and Watson atlas (23). Following a 7-day recovery period, an icv injection (2 µl) of angiotensin II (150 ng) was administered. Only those animals that demonstrated a prompt and sustained drinking response were included in the study. Cannulation and peptide administration were performed as described previously (24).

The effect of central (icv) TIP39 on hypothalamo-pituitary function
To show dose dependence groups of rat (n = 8–10 per group) were injected with either TIP39 (1 nmol, 3 nmol, or 10 nmol), PTH (1–34) 10 nmol or vehicle. To show the time course of TIP39 on hypothalamo-pituitary function groups of rats (n = 10–11 per group) were injected with either TIP39 10 nmol or vehicle control. TIP39 was dissolved in vehicle (0.9% saline) and administered in a total volume of 2 µl. Animals were habituated to the injection procedures by three icv injections before the study to minimize stress. Experiments were carried out during the early light phase (0900–1100 h). At 10 and 30 min following injection, rats were decapitated and trunk blood collected into plastic tubes containing potassium EDTA (final concentration of 1.2–2 mg EDTA/ml blood) (Sarstedt, Leicester, UK). Plasma was separated by centrifugation, immediately frozen and stored at -70 C until assay.

RIAs
TRH-IR and LHRH-IR levels in the aCSF were measured using reagents and methods kindly provided by H. M. Fraser, Medical Research Center Reproductive Biology Unit, Edinburgh (25). TSH, PRL, GH, and LH levels were assayed using reagents and methods provided by the NIDDK and the National Hormone and Pituitary Program (Dr. A. Parlow, Harbor University of California, Los Angeles Medical Center) as previously described (26). VIP, CRF, AVP, and SST were measured using established RIAs developed in this laboratory (27, 28, 29). ACTH was measured using reagents and methods from Euro-Diagnostica B. V. (Arnhem, The Netherlands). GRF was measured using reagents and methods from Phoenix Pharmaceuticals, Inc. (Belmont, CA). TIP39 did not cross-react with any of the assays used in this study.

Statistics
Data from the hypothalamic explant experiments was analyzed by paired t test between the basal, peptide, and high potassium periods. Data from the anterior pituitary dispersions was analyzed as percentage of the mean basal release and compared using ANOVA with post hoc Bonferroni Correction (ANOVA; Systat, Evanston, IL). Data from the ip study was analyzed by unpaired t test between vehicle and treatment group at each time point. Data from the icv study was analyzed using ANOVA with post hoc Bonferroni Correction. Results are shown as mean values ± SEM and in all cases P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of TIP39 on hormone secretion from medial basal hypothalamic explants
Incubation with TIP39 100 nM increased the secretion of several hypothalamic releasing factors compared with basal levels (Table 1Go). TIP39 100 nM increased CRF levels compared with the basal level unlike TIP39 1 nM, TIP39 10 nM, or PTH (1–34) 100 nM (TIP39 100 nM 314.8 ± 85.4% of basal release; P < 0.05) (Fig. 1Go). LHRH-IR release was also elevated after incubation with TIP39 100 nM unlike TIP39 1 nM, TIP39 10 nM or PTH (1–34) 100 nM (TIP39 100 nM 213.5 ± 53.1% of basal secretion, P < 0.05) (Fig. 2Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Hypothalamic releasing factors released from medial basal hypothalamic explants

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. The release of CRF from medial basal hypothalamic explants by TIP39 1 nM, 10 nM, and 100 nM; PTH 100 nM and 56 mM potassium as a percentage of basal release. Basal, peptide then potassium 56 mM incubation periods each of 45 min duration. Values are shown as mean ± SEM; *, P < 0.05 and ***, P < 0.01 by t test.

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. The release of LHRH-IR from medial basal hypothalamic explants by TIP39 1 nM, 10 nM, and 100 nM; PTH 100 nM and 56 mM potassium as a percentage of basal release. Basal, peptide then potassium 56 mM incubation periods each of 45 min duration. Values are shown as mean ± SEM; *, P < 0.05 and **, P < 0.01 by t test.

 
There was increased release of AVP, VIP, and GRF from in vitro hypothalamic explants following incubation with TIP39 100 nM (Table 1Go). Release of AVP compared with basal (TIP39 100 nM 563.5 ± 55.5 fmol/explant vs. control 454.3 ± 40.0 fmol/explant, P < 0.01), VIP release compared with basal (TIP39 100 nM 85.6 ± 8.4 fmol/explant vs. control 73.4 ± 9.6 fmol/explant, P < 0.05) and GRF release compared with basal (TIP39 100 nM 770.9 ± 90.7 pg/explant vs. control 657.8 ± 77.7 pg/explant, P < 0.01).

The effect of TIP39 on hormone secretion from dispersed anterior pituitary cells
TIP39 at concentrations from 0.1–100 nM did not alter the concentration of any pituitary hormones from dispersed anterior pituitary cells compared with the basal level; unlike the response to the known hypothalamic releasing factors used as positive controls. ACTH release from dispersed anterior pituitary cells (TIP39 100 nM 117.8 ± 9.1% of basal, n.s: CRF 100 nM 168.8 ± 7.5% of basal, P < 0.001). GH release from dispersed anterior pituitary cells (TIP39 100 nM 82.0 ± 7.3% of basal, n.s: GRF 100 nM 344.2 ± 27.2% of basal, P < 0.001). LH release from dispersed anterior pituitary cells (TIP39 100 nM 84.2 ± 8.0% of basal, n.s: LHRH 100 nM 201.2 ± 27.2% of basal, P < 0.001). PRL release from dispersed anterior pituitary cells (TIP39 100 nM 94.2 ± 3.5% of basal, n.s: TRH 100 nM 135.6 ± 6.4% of basal, P < 0.001). TSH release from dispersed anterior pituitary cells (TIP39 100 nM 92.0 ± 5.7% of basal, n.s: TRH 100 nM 135.9 ± 7.7% of basal, P < 0.05).

The effect of peripheral (ip) TIP39 on pituitary hormone secretion
The effect of TIP39 30 nmol on pituitary hormone secretion was assessed following ip administration. A significant increase was seen in the plasma levels of LH at 10 min post injection but not at the later time points (TIP39 vs. saline 0.53 ± 0.09 vs. 0.21 ± 0.04 ng/ml at 10 min, P < 0.05: 0.34 ± 0.09 vs. 0.26 ± 0.09 ng/ml at 20 min, n.s: 0.22 ± 0.06 vs. 0.11 ± 0.04 ng/ml at 60 min, n.s.).

There was no effect by TIP39 30 nmol on ACTH, GH, PRL, or TSH, compared with saline, at any time point investigated (data not shown). GHRP-6 was used as a positive control in this study and resulted in a significant rise in plasma GH (data not shown).

The effect of central (icv) TIP39 on pituitary hormone secretion
A significant increase in plasma ACTH was observed at 10 min post injection (Fig. 3aGo) for both TIP39 and PTH (1–34) but was not sustained at 30 min. Plasma ACTH (pg/ml) at 10 min post injection (saline 13.5 ± 2.1, TIP39 1 nmol 24.3 ± 2.8, TIP39 3 nmol 32.3 ± 4.0 (P < 0.01 to saline), TIP39 10 nmol 23.8 ± 3.6 and PTH (1–34) 10 nmol 28.9 ± 3.2 (P < 0.05 to saline). Plasma ACTH (pg/ml) at 30 min post injection (saline 12.5 ± 2.1 vs. TIP39 10 nmol 6.1 ± 0.8, n.s.).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. The release of plasma pituitary hormones 10 min after icv injection of saline (open bars) TIP39 (closed bars) 1, 3, or 10 nmol and PTH (1–34) 10 nmol (striped bars). A, ACTH values are shown as mean pg/ml ± SEM; B, LH values are shown as mean ng/ml ± SEM. *, P < 0.05 and **, P < 0.01 by ANOVA with post hoc Bonferroni correction.

 
A significant increase in plasma LH was observed at 10 min post injection of TIP39 10 nmol (Fig. 3BGo) but not at the lower doses studied or at 30 min post injection. Plasma LH (ng/ml) at 10 min post injection (saline 0.42 ± 0.04, TIP39 1 nmol 0.33 ± 0.04, TIP39 3 nmol 0.32 ± 0.06, TIP39 10 nmol 0.70 ± 0.09 (P < 0.05 to saline) and PTH (1–34) 10 nmol 0.49 ± 0.08). Plasma LH (ng/ml) at 30 min post injection (saline 0.24 ± 0.05 vs. TIP39 10 nmol 0.54 ± 0.11, n.s.).

There was no significant change in the levels of plasma GH, PRL, and TSH following icv injection of TIP39 or PTH (1–34). Plasma GH (ng/ml) at 10 min post injection (saline 44.5 ± 12.4, TIP39 1 nmol 44.5 ± 10.7, TIP39 3 nmol 26.3 ± 8.9, TIP39 10 nmol 20.8 ± 5.4 and PTH (1–34) 10 nmol 35.0 ± 7.8; all n.s. to saline). Plasma GH (ng/ml) at 30 min post injection (saline 32.2 ± 8.1 vs. TIP39 10 nmol 71.1 ± 19.0; P = 0.08). Plasma PRL (ng/ml) at 10 min post injection (saline 3.4 ± 0.7, TIP39 1 nmol 3.6 ± 1.0, TIP39 3 nmol 3.2 ± 0.4, TIP39 10 nmol 2.8 ± 0.7 and PTH (1–34) 10 nmol 3.4 ± 1.0; all n.s. to saline). Plasma PRL (ng/ml) at 30 min post injection (saline 2.3 ± 0.6 vs. TIP39 10 nmol 1.0 ± 0.2; n.s.). Plasma TSH (ng/ml) at 10 min post injection (saline 8.0 ± 0.7, TIP39 1nmol 11.3 ± 1.3, TIP39 3 nmol 5.8 ± 0.8, TIP39 10 nmol 7.1 ± 1.0 and PTH (1–34) 10 nmol 8.7 ± 0.9; all n.s. to saline). Plasma TSH (ng/ml) at 30 min post injection (saline 9.7 ± 0.8 vs. TIP39 10 nmol 5.6 ± 0.7; P = 0.06 to saline).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was undertaken to investigate the possible roles of TIP39 acting via the parathyroid receptor 2 (PTH2-R) on the hypothalamo-pituitary axes. PTH2-R has a wide distribution, and Northern blotting demonstrates the highest expression of PTH2-R mRNA to be in the central nervous system (2). Localization studies have shown PTH2-R mRNA in the hypothalamus including the periventricular area, the external zone of the median eminence, the dorsal paraventricular (PVN), arcuate and ventromedial nuclei (12). The anatomical position of this receptor would allow it to influence the hypothalamo-pituitary axes.

There was a robust release of CRF from medial basal hypothalamic explants following incubation with TIP39, which increased the level of CRF to over 300% of basal. Incubation with TIP39 also led to a significantly increased release of AVP from hypothalamic explants. PTH2-R mRNA expression has been clearly demonstrated in both the PVN and the external zone of the median eminence by in situ hybridization (12). There is an increase in plasma ACTH following icv administration of both TIP39 and PTH (1–34). The increased ACTH following icv injection of TIP39 could be mediated via an increase in hypothalamic CRF and AVP. PTH (1–34) did not increase of CRF from hypothalamic explants and others have shown that it does not increase AVP release (11). The hypothalamic mechanism of action by which PTH increases plasma ACTH is not clear from these investigations. It is possible that PTH, a weak agonist at the PTH2-R, increased CRF via PVN PTH2-receptors in the in the same manner as TIP39. However, a small change in CRF release in vitro was not observed. Further experiments using higher doses of PTH in vitro would be necessary to confirm this hypothesis. Considering the position of PTH2-R in the PVN and the external zone of the median eminence and the in vitro and in vivo effects of TIP39, these data suggest an activating role for TIP39 in the hypothalamo-pituitary-adrenal axis.

These results imply that TIP39 might act both directly and indirectly on the hypothalamo-pituitary-gonadal axis. Release of LHRH and VIP, from hypothalamic explants in vitro are increased following incubation with TIP39. VIP is known to release LHRH and LH in vitro from a combined hypothalamic-pituitary perifusion system (30). There is an increase in plasma LH after central administration of TIP39, and the in vitro data would suggest that this is mediated by the hypothalamic releasing factors LHRH and VIP. TIP39 30 nmol administered ip results in a significant release of LH 10 min post injection compared with saline. Although PTH2-R mRNA has been detected in anterior pituitary cells, which have the morphological appearance of hormone secreting cells (12), immunolocalization did not detect PTH2-R on the anterior pituitary, so the receptor number maybe low (13). TIP39 does not alter the release of any anterior pituitary hormone from monodispersed anterior pituitary cells. Disruption of the cell-to-cell communications of the pituitary gland may cause this disparity between the in vitro and in vivo effects of TIP39. Further investigation is required with these cell-to-cell communications preserved, possibly with perifused anterior pituitary fragments.

The combined results of the ip and icv studies may indicate distinct effects by TIP39 on the hypothalamus and pituitary. The blood brain barrier isolates the pituitary gland from the hypothalamus decreasing the likelihood of a direct action on the anterior pituitary following third ventricular administration of TIP39. In light of the rapid response of LH to peripheral TIP39, a direct pituitary action is possible. However, the PTH2-R is present on the external zone of the median eminence (13), which is accessible to circulating factors, where it could mediate the release of hypothalamic LHRH. Taken together our data suggest that TIP39 plays an important role in the regulation of the LH secretion but its site of action is unclear.

Immunolocalization studies show distinct labeling of PTH2-R in the periventricular area of the hypothalamus where there is costaining with SST (1). This anatomical distribution suggests a possible role for the PTH2-R in the control of GH. Our studies show a moderate increase in release of GRF but no effect on SST release from hypothalamic explants following treatment with TIP39. We detected no change in GH release from dispersed anterior pituitaries, and TIP39 did not alter GH release following ip administration of TIP39. Although, in our studies there was no statistically significant alteration in plasma GH following icv administration of TIP39, there is a recent report of GH suppression following icv administration of TIP39 with a decrease in pulsatility (31). Alterations in the release of hypothalamic GRF and changes in the pulsatility of GH release might possibly explain these findings, but further studies are clearly warranted.

The family of PTH peptides and receptors is increasing in size as new components are discovered. PTH2-R has a wide distribution and is found in the hypothalamus in areas that suggest a role in endocrine regulation. TIP39 is the most effective ligand at this receptor (1), and these investigations have revealed effects of TIP39 on the hypothalamo-pituitary control circuits (8, 32), particularly the gonadal axis. In the light of these preliminary findings, more studies are required to understand the physiological roles of TIP39.


   Acknowledgments
 
The authors wish to express their thanks to the hypothalamic group for their assistance with the in vivo experiments.


   Footnotes
 
This work was supported by the Medical Research Council (MRC). H.W. is a Wellcome Trust clinical training fellow. K.M. is funded by the MRC.

Abbreviations: aCSF, Artificial cerebrospinal fluid; CRF, ACTH-releasing factor; GRF, GH-releasing factor; GHRP, GH-releasing peptide; icv, intracerebroventricular; LH-releasing hormone; PTH1-R, parathyroid receptor 1; PTHrP, PTH-related protein; PVN, paraventricular nucleus; SON, supraoptic nucleus; SST, somatostatin; TIP39, tuberoinfundibular peptide.

Received December 19, 2000.

Accepted for publication April 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Usdin TB, Hoare SR, Wang T, Mezey E, Kowalak JA 1999 TIP39: a new neuropeptide and PTH2-R agonist from hypothalamus. Nat Neurosci 2:941–943[CrossRef][Medline]
  2. Usdin TB, Gruber C, Bonner TI 1995 Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor. J Biol Chem 270:15455–15458[Abstract/Free Full Text]
  3. Hoare SR, Bonner TI, Usdin TB 1999 Comparison of rat and human parathyroid hormone 2 (PTH2) receptor activation: PTH is a low potency partial agonist at the rat PTH2 receptor. Endocrinology 140:4419–4425[Abstract/Free Full Text]
  4. Piserchio A, Usdin T, Mierke DF 2000 Structure of tuberoinfundibular peptide of 39 residues. J Biol Chem 275:27284–27290[Abstract/Free Full Text]
  5. Hoare SR, Clark JA, Usdin TB 2000 Molecular determinants of tuberoinfundibular peptide of 39 residues (TIP39) selectivity for the parathyroid hormone-2 (PTH2) receptor. N-terminal truncation of TIP39 reverses PTH2 receptor/PTH1 receptor binding selectivity. J Biol Chem 275:27274–27283[Abstract/Free Full Text]
  6. Hoare SR, Usdin TB 2000 Tuberoinfundibular peptide (7–39) [TIP(7–39)], a novel, selective, high- affinity antagonist for the parathyroid hormone-1 receptor with no detectable agonist activity. J Pharmacol Exp Ther 295:761–770[Abstract/Free Full Text]
  7. Jonsson KB, John MR, Gensure RC, Gardella TJ, Juppner H 2001 Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist. Endocrinology 142:704–709[Abstract/Free Full Text]
  8. Rubin DA, Juppner H 1999 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 274:28185–28190[Abstract/Free Full Text]
  9. Behar V, Pines M, Nakamoto C, et al. 1996 The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor. Endocrinology 137:2748–2757[Abstract]
  10. Yamamoto S, Morimoto I, Zeki K, et al. 1998 Centrally administered parathyroid hormone (PTH)-related protein(1–34) but not PTH(1–34) stimulates arginine-vasopressin secretion and its messenger ribonucleic acid expression in supraoptic nucleus of the conscious rats. Endocrinology 139:383–388[Abstract/Free Full Text]
  11. Yamamoto S, Morimoto I, Yanagihara N, Zeki K, Fujihira T, Izumi F, Yamashita H, Eto S 1997 Parathyroid hormone-related peptide-(1–34) [PTHrP-(1–34)] induces vasopressin release from the rat supraoptic nucleus in vitro through a novel receptor distinct from a type I or type II PTH/PTHrP receptor. Endocrinology 138:2066–2072[Abstract/Free Full Text]
  12. Usdin TB, Bonner TI, Harta G, Mezey E 1996 Distribution of parathyroid hormone-2 receptor messenger ribonucleic acid in rat. Endocrinology 137:4285–4297[Abstract]
  13. Usdin TB, Hilton J, Vertesi T, Harta G, Segre G, Mezey E 1999 Distribution of the parathyroid hormone 2 receptor in rat: immunolocalization reveals expression by several endocrine cells. Endocrinology 140:3363–3371[Abstract/Free Full Text]
  14. Pang PK, Kaneko T, Harvey S 1988 Immunocytochemical distribution of PTH immunoreactivity in vertebrate brains. Am J Physiol 255:R643–R647
  15. Nutley MT, Parimi SA, Harvey S 1995 Sequence analysis of hypothalamic parathyroid hormone messenger ribonucleic acid. Endocrinology 136:5600–5607[Abstract]
  16. Fraser RA, Kronenberg HM, Pang PK, Harvey S 1990 Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus. Endocrinology 127:2517–2522[Abstract]
  17. Gunther T, Chen ZF, Kim J, et al. 2000 Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 406:199–203[CrossRef][Medline]
  18. Usdin TB 1997 Evidence for a parathyroid hormone-2 receptor selective ligand in the hypothalamus. Endocrinology 138:831–834[Abstract/Free Full Text]
  19. Stanley SA, Small CJ, Kim MS, et al. 1999 Agouti-related peptide (Agrp) stimulates the hypothalamo pituitary gonadal axis in vivo and in vitro in male rats. Endocrinology 140:5459–5462[Abstract/Free Full Text]
  20. Todd JF, Small CJ, Akinsanya KO, Stanley SA, Smith DM, Bloom SR 1998 Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 139:4222–4229[Abstract/Free Full Text]
  21. Wynick D, Venetikou MS, Critchley R, Burrin JM, Bloom SR 1990 Flow cytometric analysis of functional anterior pituitary cells from female rats. J Endocrinol 126:261–268[Abstract]
  22. Bowers CY, Momany FA, Reynolds GA, Hong A 1984 On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114:1537–1545[Abstract]
  23. Paxinos G, Watson C The rat brain in stereotactic coordinates. 1998 ed. 4. San Diego: Academic Press, Inc.
  24. Turton MD, O’Shea D, Gunn I, et al. 1996 A role for glucagon-like peptide-1 in the central regulation of feeding [see comments]. Nature 379:69–72[CrossRef][Medline]
  25. Beak SA, Heath MM, Small CJ, et al. 1998 Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line. J Clin Invest 101:1334–1341
  26. Beak SA, Small CJ, Ilovaiskaia I, et al. 1996 Glucagon-like peptide-1 (GLP-1) releases thyrotropin (TSH): characterization of binding sites for GLP-1 on {alpha}-TSH cells. Endocrinology 137:4130–4138[Abstract]
  27. Bloom SR, Long RG Radioimmunoassays of gut regulatory peptides. 1982 Philadelphia: Saunders
  28. Jones PM, Ghatei MA, Steel J, et al. 1989 Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125:334–341[Abstract]
  29. Satoh F, Beak SA, Small CJ, et al. 2000 Characterization of human and rat glucagon-like peptide-1 receptors in the neurointermediate lobe: lack of coupling to either stimulation or inhibition of adenylyl cyclase. Endocrinology 141:1301–1309[Abstract/Free Full Text]
  30. Ohtsuka S, Miyake A, Nishizaki T, Tasaka K, Tanizawa O 1988 Vasoactive intestinal peptide stimulates gonadotropin-releasing hormone release from rat hypothalamus in vitro. Acta Endocrinol (Copenh) 117:399–402[Medline]
  31. Wang T, Edwards GL, Lange GD, Parlow AF, Usdin T 2000 Brain administration of tuberoinfundibular peptide of 39 residues inhibits growth hormone secretion
  32. Hoare SR, Rubin DA, Juppner H, Usdin TB 2000 Evaluating the ligand specificity of zebrafish parathyroid hormone (PTH) receptors: comparison of PTH, PTH-related protein, and tuberoinfundibular peptide of 39 residues. Endocrinology 141:3080–3086[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
T. B. Usdin, M. Paciga, T. Riordan, J. Kuo, A. Parmelee, G. Petukova, R. D. Camerini-Otero, and E. Mezey
Tuberoinfundibular Peptide of 39 Residues Is Required for Germ Cell Development
Endocrinology, September 1, 2008; 149(9): 4292 - 4300.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. Ross, P. Engel, Y. Abdallah, W. Kummer, and K. D. Schluter
Tuberoinfundibular Peptide of 39 Residues: A New Mediator of Cardiac Function via Nitric Oxide Production in the Rat Heart
Endocrinology, May 1, 2005; 146(5): 2221 - 2228.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. R. Papasani, R. C. Gensure, Y.-L. Yan, Y. Gunes, J. H. Postlethwait, B. Ponugoti, M. R. John, H. Juppner, and D. A. Rubin
Identification and Characterization of the Zebrafish and Fugu Genes Encoding Tuberoinfundibular Peptide 39
Endocrinology, November 1, 2004; 145(11): 5294 - 5304.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
A. Bisello, D. Manen, D. D. Pierroz, T. B. Usdin, R. Rizzoli, and S. L. Ferrari
Agonist-Specific Regulation of Parathyroid Hormone (PTH) Receptor Type 2 Activity: Structural and Functional Analysis of PTH- and Tuberoinfundibular Peptide (TIP) 39-Stimulated Desensitization and Internalization
Mol. Endocrinol., June 1, 2004; 18(6): 1486 - 1498.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
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]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Dobolyi, H. Ueda, H. Uchida, M. Palkovits, and T. B. Usdin
Anatomical and physiological evidence for involvement of tuberoinfundibular peptide of 39 residues in nociception
PNAS, January 24, 2002; (2002) 42416199.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Dobolyi, H. Ueda, H. Uchida, M. Palkovits, and T. B. Usdin
Anatomical and physiological evidence for involvement of tuberoinfundibular peptide of 39 residues in nociception
PNAS, February 5, 2002; 99(3): 1651 - 1656.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ward, H. L.
Right arrow Articles by Bloom, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ward, H. L.
Right arrow Articles by Bloom, S. R.

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