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Endocrinology Vol. 138, No. 5 2066-2072
Copyright © 1997 by The Endocrine Society

Articles

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

Shigeki Yamamoto, Isao Morimoto, Nobuyuki Yanagihara, Kazuya Zeki, Takashi Fujihira, Futoshi Izumi, Hiroshi Yamashita and Sumiya Eto

First Department of Internal Medicine, Department of Pharmacology (N.Y., F.I.), and First Department of Physiology (H.Y.), School of Medicine, University of Occupational and Environmental Health, Iseigaoka, Yahatanishi-ku, Kitakyushu, Japan

Address all correspondence and requests for reprints to: Dr. Isao Morimoto, First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH and PTH-related peptide (PTHrP) bind to a type I PTH/PTHrP receptor expressed in bone and kidney or a type II receptor in nonclassical target tissue with equal affinity and similar bioactivities. PTHrP is abundant in the central nervous system, but its physiological role remains unknown. Herein, we examined the role of PTHrP-(1–34) on arginine vasopressin (AVP) release from the rat supraoptic nucleus (SON). Application of PTHrP-(1–34) to SON slices caused an increase in AVP release in a concentration-dependent manner. Neither PTHrP-(7–34) nor PTH-(1–34) had any effect on AVP release from the SON. PTHrP-(1–34)-induced AVP release was antagonized by a large excess of PTHrP-(7–34) and by H89, an inhibitor of cAMP-dependent protein kinase (A kinase), but not by PTH-(1–34) or PTH-(13–34). PTHrP-(1–34), but not PTH-(1–34), also dose-dependently increased the levels of cAMP in the SON. 125I-Labeled PTHrP-(1–34) bound specifically to crude membranes isolated from the SON. Scatchard analysis showed a single class of binding sites for PTHrP-(1–34) with a Kd of 36.4 nM and a maximum binding capacity of 3.94 pmol/mg protein. No specific binding for 125I-labeled PTH-(1–34) was noted. The binding of 125I-labeled PTHrP-(1–34) was displaced by unlabeled PTHrP-(1–34) and unlabeled PTHrP-(7–34), but not by unlabeled PTH-(1–34). These findings suggest that PTHrP-(1–34), but not PTH-(1–34), causes the release of AVP from the SON through a novel receptor distinct from type I or II PTH/PTHrP receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH-RELATED protein (PTHrP) has been identified in tumors associated with humoral hypercalcemia of malignancy (1, 2, 3, 4, 5). PTHrP consists of 141 amino acid residues (1, 2, 6), and the first 13 amino acids exhibit 70% amino acid homology with PTH. Although PTHrP and PTH are expressed by different genes (1), the amino-terminal fragment of PTHrP and PTH interacts with a common PTH/PTHrP receptor (type I) in bone and kidney, which accounts for the clinical similarity of hypercalcemia of malignancy and hyperparathyroidism (7, 8).

PTHrP and its receptor protein have been demonstrated in a wide variety of normal tissues as well as in fetal organs (1, 2, 8, 9), suggesting that PTHrP functions as both a systemic and a local factor. Despite the similarities in action of PTH and PTHrP in bone and kidney, there are some differences in their biological activities (10). Recent studies (2, 11, 12, 13, 14) have indicated that a nonclassical PTH/PTHrP receptor (type II) is present in lymphocytes, insulinoma cells, keratinocytes, and squamous carcinoma cells. Stimulation of type II PTH/PTHrP receptors causes an increase in intracellular free calcium, but not cAMP. Moreover, PTHrP has a highly conserved sequence among species and an abundance of posttranslational processing sites (2, 15). The broad distribution of PTHrP and the PTH/PTHrP receptor in the brain suggests that PTHrP, unlike PTH, has specific functions in the brain (16). Thus, PTHrP may have different physiological roles via different PTH and/or PTHrP receptors.

PTH has various effects on the cardiovascular system, such as systemic hypotension (10, 17, 18, 19) and direct cardiac stimulation (20). PTHrP and its messenger RNA (mRNA) are expressed in adult rat and human fetal cardiac myocytes and rat aortic smooth muscle (21, 22, 23, 24). PTHrP acts on vascular smooth muscle as a vasorelaxant (25, 26) and on heart muscle to increase the heart rate and contractility (21, 25, 26, 27, 28). Furthermore, vasoconstrictors, including angiotensin II, endothelin, and catecholamines, stimulate PTHrP mRNA expression in smooth muscle cells (25, 29). PTHrP increases endothelin production in the vascular smooth muscle (24). These findings indicate that PTHrP is a locally active autocrine or paracrine factor that is involved in the relaxation of vascular smooth muscle.

The PTH/PTHrP receptor is found in the brain (16, 29, 30), and PTHrP is also abundant in the central nervous system (CNS) (31, 32), but the physiological role of PTHrP in the CNS remains to be clarified. We speculated that PTHrP regulates the release of vasomodulators in the CNS. In a perfusion system of the isolated rat supraoptic nucleus (SON), we found an inhibitory effect of endothelin on the release of arginine vasopressin (AVP), which has antidiuretic and pressor activities. The perfusion system of rat SON slices is a useful tool with which to analyze the actions of several neuropeptides on the release of AVP in the hypothalamus (33). In this study, we examined the role of PTHrP-(1–34) on AVP release from rat SON-containing neurosecretory cells. We also characterized the specific binding of [125I]PTHrP to the SON, which is different from that of type I and II PTH/PTHrP receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic PTHrP-(1–34) and 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) were purchased from the Peptide Institute (Osaka, Japan). Rat PTH-(1–34), human PTH-(1–34), and human PTH-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) were purchased from the Peptide Institute and Sigma Chemical Co. (St. Louis, MO). Phenylmethylsulfonylfluoride, 3-isobutyl-1-methylxanthine (IBMX), and leupeptin were obtained from Sigma Chemical Co. H89, an inhibitor of A kinase, was from Seikagaku Co. (Tokyo, Japan). 125I-Labeled [Tyr34]PTHrP-(1–34) and 125I-labeled [Tyr34,Nle8,18]rat PTH-(1–34) were supplied by Dr. N. Tamura (Mitsubishi Chemical Co., Tokyo, Japan). The specific activity of both 125I-labeled [Tyr34]PTHrP-(1–34) and 125I-labeled [Tyr34,Nle8,18]rat PTH-(1–34) was 2162 Ci/mmol. Other chemicals used were of analytical grade from Nacalai Tesque (Kyoto, Japan).

Perfusion studies
Coronal hypothalamic slices containing the SON (400 µm in thickness) were cut by a vibrating slicer from the brain of adult male Wistar rats (100–150 g BW). Immediately after sectioning, the slices were carefully trimmed so that they contained only the SON and its perinuclear zone (34). We obtained five or six trimmed slices from each rat. The slices were placed in incubation medium (pH 7.3–7.5) containing 124 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 2.1 mM CaCl2, 20 mM NaHCO3, and 10 mM glucose (35) at room temperature and maintained for at least 30 min in an equilibrium state before being transferred to a perfusion chamber. The perfusion (incubation) medium was oxygenated with 95% O2 and 5% CO2, then perfused at the flow rate of 1.0 ml/min at room temperature. Perfusion medium containing chemicals at various concentrations was applied to the slices from separate storage bottles (34). The pH of the perfusing medium was not affected by the peptides at any concentration applied.

After a 30-min equilibration period, medium containing various concentrations (1 pM to 1 µM) of either 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), or PTH-(1–34) was perfused at 2-min intervals. The perfusates were collected every minute into polypropylene plastic tubes by a microfraction collector (Gilson Electronics, Oberlin, OH) and stored at -20 C until use. Thereafter, 60 mM KCl was perfused to induce maximum stimulation (33). In addition, we examined whether the PTHrP-(1–34)-stimulated AVP release from the SON is modulated 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), PTH-(1–34), PTH-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), or H89.

The concentration of AVP in the perfusates was determined using a RIA kit (Mitsubishiyuka, Tokyo, Japan). The minimum detectable level was 0.063 pg/tube, and the 50% intercept was 0.728 pg/tube (36). The rates of AVP release were also assessed as changes in AVP release from the basal level (mean AVP release during 5 min) to the peak level (mean AVP release during 1 min) after the application of 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 or human PTH-(1–34). The inhibition rates were calculated as changes in the AVP release from the PTHrP-(1–34)-stimulated level (mean AVP release during 2 min) to that of the trough (mean AVP release during 2 min) after application of H89.

Generation of cyclic nucleotides in the rat SON
Rat SON slices and kidneys were placed in perfusion medium at room temperature for 1 h, then incubated with the same medium containing 0.3 mM IBMX for 10 min at 37 C. They were further incubated with or without 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/or rat PTH-(1–34) in medium containing 0.3 mM IBMX for 10 min. The slices were homogenized in the same medium containing 0.1% HCl (37). The samples were microcentrifuged for 15 min at 2500 x g, and the supernatants were removed and stored at -70 C until use. cAMP and cGMP contents in the supernatants were assayed using Yamasa cAMP and cGMP-kits (Choshi, Japan), respectively. Protein contents in the tissues were measured by the method of Lowry et al. (38). The cyclic nucleotide levels are expressed as picomoles per mg protein.

Membrane preparation
The brain SON, with its perinuclear zone, was rapidly dissected out from the rat and collected in ice-cold 0.25 M sucrose buffer containing 50 mM Tris-HCl (pH 7.4), 1.0 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, and 0.05 mM leupeptin (39). The tissues were homogenized with 8 vol of the same 0.25 M sucrose buffer and centrifuged at 3,000 x g for 15 min. The supernatants were then ultracentrifuged at 75,000 x g for 90 min. The membrane fraction was resuspended in the binding buffer and again centrifuged at 35,000 x g for 15 min, and the pellets were reconstituted in the binding buffer, containing 50 mM Tris-HCl buffer (pH 7.4), 4 mM MgCl2, 26 mM KCl, and 0.3% BSA (39) for binding studies. The protein contents were determined according to the method of Lowry et al. (38).

PTHrP-(1–34) binding studies in membrane fraction
The crude membranes obtained from the rat SON (~500–700 µg protein/tube) were incubated at 4 C for 60 min with various concentrations of either 125I-labeled [Tyr34]PTHrP-(1–34) or 125I-labeled [Tyr34,Nle8,18]rat PTH-(1–34) (250,000–300,000 cpm) in 0.2 ml of a buffer solution [50 mM Tris-HCl buffer (pH 7.4), 4 mM MgCl2, 26 mM KCl, and 0.3% BSA] with or without a 100-fold molar excess of unlabeled PTHrP-(1–34) or PTH-(1–34), respectively (40). After incubation, the mixture was laid on 300 µl ice-cold 2% BSA buffer [50 mM Tris-HCl (pH 7.4), 4 mM MgCl2, and 26 mM KCl] in a microcentrifuge tube. The bound and free fractions were separated by centrifugation (2,500 x g for 4 min) at 4 C. The supernatant was aspirated, and the tip of tube containing the bound fraction was cut. The radioactivity in the tip was counted using a Beckman 5,500 {gamma}-counter. Specific binding was determined by subtracting the averaged count of the nonspecific binding from that of the total binding. For competition analysis, the membrane fractions were incubated with radiolabeled PTHrP-(1–34) and various concentrations of unlabeled 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), or PTH-(1–34) at 4 C for 60 min.

Data analysis
The results are shown as the mean ± SEM of at least five experiments performed in duplicate. Significance was determined by ANOVA followed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PTHrP-(1–34) on AVP release from rat SON slices
The basal level of AVP release from the rat SON slices was 13.6 ± 3.6 pg/ml (Fig. 1Go). Incubating the slices with PTHrP-(1–34) caused an increase in AVP release in a concentration-dependent manner (0.1 nM to 1 µM Figs. 1Go and 2Go). The rate of AVP release induced by 1 µM PTHrP was similar to that caused by the addition of KCl (60 mM), suggesting that it was the maximal stimulation of AVP release from the SON. The minimal effective dose of PTHrP-(1–34) on AVP release was 0.1 nM, and the half-maximal effective dose (ED50) was 30 nM. 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), an antagonist of PTHrP-(1–34) (41), did not affect AVP release from the rat SON at concentrations of 0.1 nM to 1 µM (Fig. 2BGo). AVP release from the SON induced by 10 nM PTHrP-(1–34) was inhibited by superperfusion with 1 µM 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 the stimulatory effect of PTHrP-(1–34) recovered after 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) was removed from the perfusate (Fig. 3AGo). Even if different lots of rat and human PTH-(1–34) from two companies were used, they did not cause the release of AVP from the rat SON (Fig. 2CGo). Furthermore, neither 1 µM rat PTH-(1–34) nor human PTH-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) affected AVP release induced by 10 nM PTHrP-(1–34) (Fig. 3Go, B and C).



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  		Figure 1. A representative time course of the effects of various doses of PTHrP-(1–34) on AVP release from perfused rat SON slices. Coronal hypothalamic slices containing the SON were perfused with medium at a flow rate 1.0 ml/min at room temperature. After a 30-min equilibration period, media containing various concentrations (1 pM to 1 µM) of PTHrP-(1–34) were perfused at 2-min intervals. The perfusate was collected at 1-min intervals. The concentration of AVP in the perfusate was assayed by a RIA kit (see Materials and Methods). The experiments were repeated at least five times, and the results were similar to the data presented here.

 


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  		Figure 2. The dose-response relationship of AVP release from the rat SON by PTHrP-(1–34) (A), PTHrP-(7–34) (B), and rat PTH-(1–34) (C). The rates of AVP release were assessed as changes in AVP release from the basal (mean AVP release during 5 min) to the peak level (mean AVP release during 1 min) after the application of PTHrP-(1–34), PTHrP-(7–34), or PTH-(1–34). Each value is expressed as a percentage of the basal release of AVP. Data are the mean ± SEM of five independent experiments. *, P < 0.01.

 


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  		Figure 3. The effects of PTHrP-(7–34) (A), PTH-(1–34) (B), or PTH-(13–34) (C) on AVP release from rat SON by PTHrP-(1–34). The representative time course of effects was determined as discussed in Fig. 1Go. *, P < 0.01.

 
Effects of PTHrP-(1–34) on the levels of cyclic nucleotides in rat SON slices
PTHrP-(1–34) significantly increased the level of cAMP in rat SON slices in a concentration-dependent manner (1 nM to 1 µM), and the ED50 for cAMP accumulation was about 20 nM (Fig. 4AGo). Moreover, 1 µM of it caused a 2.1-fold increase in intracellular cAMP, whereas 10 µM forskolin induced a 3.0-fold increase in cAMP over the basal level. However, neither 1 µM 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 1 µM rat PTH-(1–34) increased cAMP accumulation in the slices. The stimulatory effect of 1 µM PTHrP-(1–34) on cAMP accumulation was attenuated 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) (100 µM), but not by rat or human PTH-(1–34) (100 µM). In the rat kidney slices, both PTHrP-(1–34) and rat PTH-(1–34) increased the accumulation of the intracellular cAMP, and 10 µM forskolin induced a 5.0-fold increase in cAMP over the basal level (Fig. 4BGo). Neither PTHrP-(1–34) nor PTH-(1–34) had any effect on the level of cGMP (data not shown).



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  		Figure 4. Effects of PTHrP-(1–34), PTH-(1–34), and forskolin on the level of cAMP in rat SON (A) and kidney (B). After a 10-min incubation at 37 C with 0.3 mM IBMX, the SON (A) and the kidney (B) were exposed to the various agents indicated for another 10 min in the presence of 0.3 mM IBMX. cAMP levels in the supernatants from the tissues were measured using a Yamasa cAMP kit. A, Rat SON: 1, control; 2, PTHrP-(1–34) (1 nM); 3, PTHrP-(1–34) (10 nM); 4, PTHrP-(1–34) (100 nM); 5, PTHrP-(1–34) (1 µM); 6, PTHrP-(7–34) (1 µM); 7, PTH-(1–34) (1 µM); 8, PTH-(13–34) (1 µM); 9, 1 µM PTHrP-(1–34) plus 100 µM PTHrP-(7–34); 10, 1 µM PTHrP-(1–34) plus 100 µM PTH-(1–34); 11, forskolin (10 µM). *, P < 0.01 vs. control. B, Rat kidney: 1, Control; 2, PTHrP-(1–34) (1 µM); 3, PTH-(1–34) (1 µM); 4, PTH-(1–34) (1 µM) incubated with the perfusion medium used in the perfusion experiments; 5 forskolin (10 µM). Data are the mean ± SEM of six experiments.

 
Effect of H89 on PTHrP-(1–34)-induced AVP release from SON
We evaluated whether PTHrP-(1–34)-stimulated AVP release from rat SON is mediated through the A kinase system. H89, a selective inhibitor of A kinase (42), was superinfused with PTHrP-(1–34). H89 inhibited the AVP release stimulated by PTHrP-(1–34) in a concentration-dependent manner (Fig. 5Go).



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  		Figure 5. The effect of H89, an inhibitor of cAMP-dependent protein kinase, on PTHrP-(1–34)-stimulated AVP release from rat SON. A, A representative time course of the inhibitory effects on PTHrP-(1–34)-stimulated AVP release by superfusion with 160 nM H89. B, Dose-related inhibition by H89 on 10 nM PTHrP-(1–34)-stimulated AVP release from the rat SON.

 
Binding of 125I-labeled [Tyr34]PTHrP-(1–34) to crude membranes isolated from rat SON
The binding of 125I-labeled [Tyr34]PTHrP-(1–34) to a membrane fraction of the rat SON increased in a time-dependent manner, then reached a plateau at 20 min, which continued for up to 60 min (data not shown). When the membranes were incubated with various concentrations (15–150 nM) of 125I-labeled [Tyr34]PTHrP-(1–34), the specific binding of 125I-labeled [Tyr34]PTHrP-(1–34) was saturable (Fig. 6Go). Scatchard plot analysis revealed a single class of [125I]PTHrP-(1–34)-binding sites with an equilibrium dissociation constant (Kd) of 36.4 ± 6.4 nM and a maximum binding capacity (Bmax) of 3.94 ± 1.20 pmol/mg protein. However, there was no specific binding of 125I-labeled [Tyr34,Nle8,18]rat PTH-(1–34) to the membranes at any concentration (15–150 nM; data not shown).



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  		Figure 6. Specific binding of 125I-labeled [Tyr34]PTHrP-(1–34) to crude plasma membranes from the SON. The crude plasma membranes from the SON were incubated with various concentrations (15–150 nM) of 125I-labeled [Tyr34]PTHrP-(1–34) (250,000–300,000 cpm) at 4 C for 60 min in the presence (nonspecific binding) or absence (total binding) of a 100-fold molar excess of unlabeled PTHrP-(1–34). The bound and free fractions of 125I-labeled PTHrP-(1–34) were separated by centrifugation (see Materials and Methods). Specific binding ({blacksquare}) was defined as the difference between total ({circ}) and nonspecific ({triangleup}) binding. Data are the mean of five experiments in triplicate.

 
To study the structural specificity of [125I]PTHrP-(1–34) binding, the membranes were incubated with 30 nM [125I]PTHrP-(1–34) in the presence or absence of various concentrations of unlabeled 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). As shown in Fig. 7Go, unlabeled PTHrP-(1–34) and 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) competed for the specific binding of [125I]PTHrP-(1–34) in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) was 30 nM for PTHrP-(1–34) and 100 nM for 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). On the other hand, [125I]PTHrP-(1–34) binding to the membranes was not displaced by a 10-fold molar excess of rat PTH-(1–34), whereas 28% was displaced by a 100-fold molar excess.



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  		Figure 7. Effects of unlabeled PTHrP-(1–34), PTHrP-(7–34), and PTH-(1–34) on specific 125I-labeled PTHrP-(1–34) binding. The crude membranes were incubated with 30 nM 125I-labeled PTHrP-(1–34) at 4 C for 60 min in the presence or absence of various concentrations of unlabeled PTHrP-(1–34) (•), PTHrP-(7–34) ({blacktriangleup}), or PTH-(1–34) ({blacksquare}). Each point is a percentage of the specific 125I-labeled PTHrP-(1–34) binding. Data are the mean of five experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The wide distribution of PTHrP and of PTH/PTHrP receptors in the brain (29, 30, 31, 32) indicates that PTHrP has autocrine or paracrine effects as a neuromodulator. In this study, we demonstrated that PTHrP-(1–34) stimulates the release of AVP from the isolated rat SON. This effect of PTHrP-(1–34) was dose dependent (0.1 nM to 1 µM), with an ED50 of 30 nM, and a dose of 0.1 nM significantly increased AVP release (P < 0.01). The effects of PTHrP-(1–34) on AVP release were antagonized by a 100-fold molar excess 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, 35, 36, 37), a competitive antagonist in classical target tissues, indicating that the bioactivity of PTHrP-(1–34) on AVP release from the rat SON is located in the N-terminus, and it binds to the receptors at the C-terminus. Other studies of PTHrP biochemical responses have shown that the ED50 in PTHrP-(1–34)-stimulated adenylate cyclase activity was 0.1–30 nM in bone cells and 2.5–100 nM in renal cells (10, 43, 44, 45), and that the value for relaxation of smooth muscle by PTHrP was 0.25–10 nM (26, 46). These ED50 values are comparable to that of PTHrP-(1–34) in the present study. Our in vitro results using a perfusion system suggest that PTHrP-(1–34) acts as a neuroendocrine modulator that causes the release of AVP from the SON. AVP exhibits antidiuretic and pressor activities, the latter resulting from a constricting effect on vascular smooth muscle. Studies in vivo and in vitro (1, 2, 17, 18, 19, 20, 25, 47) have indicated that PTHrP is a hypotensive peptide and functions in the relaxation of vascular smooth muscle as a local paracrine or autocrine factor. Therefore, PTHrP not only acts directly on smooth muscle cells as a vasorelaxant, but it also stimulates AVP release from the SON indirectly, resulting in the constriction of vascular smooth muscle. PTHrP may have a homeostatic role in regulating the smooth muscle tone of blood vessels in the CNS and peripheral system.

Both PTH-(1–34) and PTHrP-(1–34) bind to a G protein-coupled PTH receptor with equal affinity to stimulate the generation of cAMP and have the same degree of bioactivity in bone and kidney (1, 6, 47, 48, 49, 50). The N-terminal amino acid sequences of PTH and PTHrP have biological activity on bone and kidney (1, 5, 6). The amino acid sequences, 14–34, of both peptides, despite having no primary sequence homology, are functionally important in binding to a type I PTH/PTHrP receptor expressed in bone and kidney (2, 4, 8, 10). PTH-(1–34) and amino-terminal PTHrP also exert biochemical effects in a number of nonclassical target tissues with an equivalent pathway (2, 10). Both peptides appear to stimulate cAMP production in smooth muscle through the type I receptor (2, 10, 23, 43). On the other hand, in lymphocytes (14), insulinoma cells (13), keratinocytes, and squamous carcinoma cell lines (12), PTH-(1–34) and amino-terminal PTHrP increase intracellular Ca2+, but not cAMP, suggesting the presence of alternative type II PTH/PTHrP receptors on these cells (12) that share some regions of homology with the type I receptor (11).

In this study, PTHrP-(1–34)-stimulated AVP release was inhibited by H89, an inhibitor of A kinase, and PTHrP-(1–34) also stimulated cAMP accumulation in the SON, which was antagonized by a large excess 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). These findings suggest that the receptor on the SON for PTHrP-(1–34) is related, at least in part, to a A kinase signaling system. PTH-(1–34) did not induce AVP release or cAMP accumulation in the SON. Furthermore, a 100-fold molar excess of rat PTH-(1–34) or human PTH-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) did not modulate the AVP release and cAMP generation in the SON induced by PTHrP-(1–34). These findings suggest that the receptor for PTHrP-(1–34) on the SON neuron involved in AVP release and cAMP generation is distinct from the type I or type II PTH/PTHrP receptor.

A single class of 125I-labeled [Tyr34]PTHrP-(1–34)-binding site was identified by a binding study using crude membranes of the rat SON. The PTHrP-(1–34) binding affinity (Kd, 36.4 nM) was lower than that for the type I PTH/PTHrP receptor in bone (Kd, 1–3 nM) (10, 45), kidney (Kd, 0.3–13.8 nM) (10, 47, 49), or vascular smooth muscle cells (Kd, 1.8 nM) (28). However, it was higher than that for the type II PTH/PTHrP receptor in rat insulinoma cells (Kd, 240 nM) (13). The Kd value of 36.4 nM for receptor binding seems to be comparable to those of AVP release (30 nM) and the ED50 (20 nM) on cAMP accumulation. Our study also demonstrated that there was no specific binding of 125I-labeled [Tyr34,Nle8,18]rat PTH-(1–34) at any concentration (15–150 nM) in the same SON membranes. The specific binding of 125I-labeled [Tyr34]PTHrP-(1–34) was reversed by unlabeled PTHrP-(1–34) and 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), whereas the displacement by PTHrP-(1–34) was 3-fold greater than that 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) (IC50, 30 vs. 100 nM). In contrast, unlabeled rat PTH-(1–34) had a slight effect on [125I]PTHrP-(1–34) binding only at high concentrations. These findings support the idea that the PTHrP-(1–34) receptor on the SON is a novel receptor differing from the type I or type II PTH/PTHrP receptor. There are interesting observations that PTHrP-(1–36) has biological properties that are qualitatively distinct from those of PTH-(1–34), as PTHrP-(1–36), but not PTH-(1–34), promoted epidermal growth factor-dependent transformation of NRK 49F cells in soft agar and biosynthesis of fibronectin by human dermal fibroblasts (51). These findings suggest the existence of another receptor distinct from the type I or II PTH/PTHrP receptor associated with PTHrP-(1–34), but not PTH-(1–34).

In previous studies of the localization of type I PTH/PTHrP receptor mRNA, no specific hybridization or a faint hybridization signal for type I PTHrP receptor was detected in rat SON (16). We could not detect type I PTH/PTHrP receptor mRNA in rat SON by Northern blot analysis using a complementary DNA probe encoding the full-length rat type I receptor (data not shown). SON may express the type I PTH/PTHrP receptor at low level, as the transcript generally is not observed in a Northern blot in nonclassical target tissues, in contrast with bone and kidney. Multiple transcripts of type I receptor mRNA were observed in various tissues (2, 9). The type I PTH/PTHrP receptor gene contains 15 exons with multiple intervening sequence, suggesting that there is ample opportunity for alternative splicing of transcripts (2). The posttranscriptional modification of type II PTH/PTHrP receptor does not appear to be involved in this receptor, as the novel receptor transduced signals in a manner distinct from type II PTH/PTHrP receptor. Taken together, the receptor observed in this study could rise from a different gene or through alternative processing of a type I PTH/PTHrP receptor gene or protein, which can bind PTHrP-(1–34), but not PTH-(1–34).

A PTHrP mRNA has been detected by in situ hybridization in the hypothalamus, especially in the SON (52), where PTH mRNA transcripts are also expressed (52, 53). Immunohistochemical studies indicated that PTH immunoreactivity is limited to the perikarya in the hypothalamus (54). Usdin et al. (55) reported that a PTH2 receptor is abundant in the brain and pancreas. Despite the high degree of similarity between the PTH2 receptor and type I PTH/PTHrP receptors, the former is activated by PTH, but not PTHrP (55, 56). The PTH2 receptor does not mediate the major effect of PTH on calcium and phosphonate metabolism, suggesting that the receptor is involved in CNS function. PTH and PTHrP may act on the hypothalamus through different receptors, namely PTH functions through the PTH2 receptor and PTHrP interacts with the novel receptor observed in this study.

In conclusion, our data suggest that PTHrP-(1–34) stimulates the release of AVP linked with cAMP through a novel PTHrP receptor in rat SON neurons. This receptor could rise from a different gene or through alternative processing of type I PTH/PTHrP receptor gene or protein.

Received October 18, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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