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Endocrinology Vol. 143, No. 4 1521-1529
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

The Mutual Regulation of Arginine-Vasopressin and PTHrP Secretion in Dissociated Supraoptic Neurons

Shigeki Yamamoto, Isao Morimoto, Yoshiya Tanaka, Nobuyuki Yanagihara and Sumiya Eto

Departments of Internal Medicine of Mitsubishikagaku Hospital (S.Y.) and Inoue Hospital (I.M.), First Department of Internal Medicine (Y.T., S.E.), and Department of Pharmacology (N.Y.), School of Medicine, University of Occupational and Environmental Health (S.E.), Kitakyushu 806-0037, Japan

Address all correspondence and requests for reprints to: Shigeki Yamamoto, Department of Internal Medicine, Mitsubishikagaku Hospital, 13-1 Higashiouji, Yahatanishi-ku, Kitakyushu 806-0037, Japan. E-mail: . 5308940{at}cc.m-kagaku.co.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTHrP is detected in the supraoptic nucleus (SON) and paraventricular nucleus. We have recently demonstrated that PTHrP(1–34) is involved in AVP release and synthesis in the SON in vivo and in vitro. PTHrP and AVP, which act on blood vessels, may interact by autocrine and paracrine mechanisms in the central nervous system. The present study was undertaken to determine the mutual regulation of AVP and PTHrP secretion in dissociated magnocellular neurons of the SON. Both AVP and PTHrP existed in the dissociated SON neurons by immunohistochemistry. PTHrP(1–34) stimulated AVP secretion from the cells dose dependently, but PTHrP(7–34) and PTH(1–34) did not. PTHrP(1–34)-stimulated AVP secretion was associated with cAMP generation. PTHrP(1–34)-induced cAMP generation was inhibited by a 100-fold molar excess of PTHrP(7–34) but not by that of PTH(1–34). PTHrP(1–34) also stimulated AVP mRNA expression in the cells. These results are consistent with our previous observations that PTHrP(1–34) is involved in AVP secretion through a receptor distinct from type I PTH/PTHrP receptor. Next, AVP stimulated dose-dependent PTHrP release from the dissociated SON neurons. The AVP-induced PTHrP release was suppressed by both OPC-21268 (V1a receptor antagonist) and dP[Thy(Me)2]AVP (V1a/V1b receptor antagonist) but not by OPC-31260 (V2 receptor antagonist). AVP increased PKC activity dose dependently but not cAMP generation in the SON neurons. The AVP-stimulated PTHrP release was blocked by staurosporine (PKC inhibitor), nicardipine (L-type calcium channel blocker) or {omega}-agatoxin IVA (N type). Furthermore, AVP stimulated PTHrP mRNA expression for 12 h in the SON neurons. These results indicate that AVP caused increases in PTHrP secretion and its mRNA levels through V1a and/or V1b receptors in the SON neurons. Our observations, taken together, suggest that PTHrP stimulates AVP secretion into the extracellular space of the SON, which in turn leads to further secretion of AVP and PTHrP by an autocrine/paracrine mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTHrP PRODUCED FROM malignant tumors is a causal factor of hypercalcemia of malignancy (1). PTHrP and its transcripts are also expressed in various tissues, and PTHrP has been shown to play a role in a wide variety of normal developments and physiological processes (2). The gene of PTHrP is expressed in various brain regions, including the hypothalamus (3). Weaver et al. (4) have described that PTHrP transcripts were expressed in the paraventricular (PVN) and supraoptic nuclei (SON). We also observed that PTHrP-like immunoreactivity exists in the magnocellular parts of the PVN and SON in colchicine-treated rats (5). The magnocellular neurosecretory cells in the PVN and SON synthesize AVP and oxytocin and releases these peptides into the systemic circulation from their axon terminal in the neurohypophysis. Previously, we have demonstrated that PTHrP(1–34) induces AVP secretion from rat SON slices through a novel receptor distinct from the type I PTH/PTHrP receptor (6), and intracerebroventricular (icv) administration of PTHrP(1–34) causes an increase in plasma AVP levels and AVP mRNA expression in the SON and PVN through an A-kinase pathway but not oxytocin mRNA (7). These results suggest that PTHrP acts on the PVN and SON neurons to release AVP in a paracrine or autocrine mechanism. However, the physiological role of PTHrP-induced AVP secretion in vivo is unclear.

AVP can evoke in vitro its own release from rat hypothalamic tissue through a receptor-mediated mechanism (8, 9, 10) and stimulate PVN neuronal firing (11). We recently demonstrated that AVP stimulates CRH mRNA expression in the PVN by the local signal amplification mechanism through AVP receptors (12). Because PTHrP and AVP coexist with their receptors in the PVN and SON neurons, there may exist interrelations between PTHrP and AVP in the neurons.

This study was undertaken to evaluate the interaction of PTHrP and AVP using cultured dissociated SON neurons. The results indicated that in the dissociated SON neurons, PTHrP stimulates AVP secretion and its gene expression. Furthermore, AVP also acted on the cultured cells, stimulating PTHrP secretion and PTHrP mRNA expression through V1a or V1b receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic AVP, 1-desamino-8-D-arginine vasopressin (DDAVP), PTHrP(1–34), PTHrP(7–34), and rat PTH(1–34) were purchased from both the Peptide Institute (Osaka, Japan) and Sigma (St. Louis, MO). 3-Isobutyl-1-methylxanthine (IBMX), H89, staurosporine, nicardipine, {omega}-conotoxin GVIA (CgTx), {omega}-agatoxin IVA (AgTx), and [deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin (dP[Tyr(Me)2]AVP), a nonselective V1a/V1b receptor antagonist (8), were purchased from Sigma. OPC-21268 (1-{1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinolinone), a selective V1a receptor antagonist (9), and OPC-31260 {5-dimethylamino-1-[4-(2-methylbenzoyl-amino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine}, a selective V2 receptor antagonist (13), were presented by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Other chemicals used were from Nacalai Tesque (Kyoto, Japan).

Each calcium channel antagonist was added to the culture medium. Nicardipine, a dihydropyridine antagonist, was prepared as a concentrated stock solution in 95% ethanol. Final dilutions of nicardipine resulted in ethanol concentrations of less than 0.05%, which had no effect on calcium currents when delivered alone (14). The dihydropyridine solution was protected from ambient light. CgTx and AgTx were prepared as concentrated stock solutions in distilled water. Cytochrome C (0.01%) was added to the AgTx solution to saturate binding to sites on glass and plastic surfaces (15). At this concentration, cytochrome C had no effect on calcium currents (16).

Cell preparation
Dissociated cells in the region of newborn rat SON were obtained according to a similar procedure previously described (17). Newborn male Wistar rats (3–4 d old) were decapitated and each brain was removed. 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. Five or six trimmed slices were obtained from each rat brain. The slices were incubated for 20 min at 37 C in 10 ml of an oxygenated (100% O2) incubating medium containing both trypsin (0.1%) and DNase (0.1%). Following a double rinse with the incubation medium, individual tissue blocks were triturated with fire-polished pipette and the dissociated cell suspension was plated at a density of 2 x 106 cells for 1 ml of the medium in 35-mm dishes. The medium was replaced 48 h after the plating, and assay was performed 72 h after plating. The cells were adhered to the plastic plate, and the medium was replaced with fresh incubation medium with or without various doses (10-12 M to 10-6 M) of each peptide. The incubation medium was oxygenated with 95% O2 and 5% CO2 and kept at 37 C. The pH of the incubation media was not affected by the presence of peptides at the concentration used. The protein contents in the tissues were measured by the method of Lowry et al. (18).

Colchicine treatment
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 the 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 fixed 1 mm above the left lateral ventricle, and colchicine dissolved in sterile distilled water (10 µg/µl) was administered icv (100 µg/rat). According to the method of previous reports (5), 2 d after central administration of colchicine, the brain was postfixed. After floating sections by a thickness of 40 µm, using microtome, they were incubated with the primary antibody of PTHrP at 4 C for 5 d.

Immunohistochemistry
Immunohistochemistry of the cultured cells obtained from newborn rat SON was performed according to procedures similar to those described previously (5). The cultures were fixed with buffered 4% paraformaldehyde and proceeded for immunocytochemistry. A rat anti-PTHrP(1–34)-NH2 antiserum and a rabbit anti-AVP antiserum (presented by Mitsubishi Chemical Co., Tokyo, Japan) were diluted 1:1000 in 0.1 M PBS containing 0.3% Triton X-100. The avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) in the cells was visualized with 0.02% 3–3'-diamino-benzidine and hydrogen peroxide for 20–30 min. All cultures for the independent experiment were processed simultaneously.

AVP and PTHrP measurement
The PTHrP levels of the supernatants were measured using an immunoradiometric assay kit (Mitsubishi Chemical Co.); the minimum detectable level was 1 pg/ml, and the 50% intercept was 10 pg/ml (19). Similarly, the AVP of the supernatants were extracted using a Sep-pak C18 cartridge (Waters Associates, Inc., Milford, MA), and then the levels were measured using a RIA kit (Mitsubishi Chemical Co.); the minimum detectable level was 0.2 pg/ml, and the 50% intercept was 2.33 pg/ml (20).

Generation of cyclic nucleotides in the dissociated SON neurons
Rat SON neurons were incubated with the same medium containing 0.3 mM IBMX for 10 min at 37 C. They were further incubated with or without either AVP or PTHrP in a medium containing 0.3 mM IBMX for 10 min. The cells were homogenized in the same medium containing 0.1% HCl (21). The samples were microcentrifuged for 15 min at 2,500 x g, and the supernatants were removed and stored at -70 C until use. The cAMP contents in the supernatants were assayed using [125I]Yamasa cAMP (Choshi, Japan). The protein contents in the tissues were measured by the method of Lowry et al. (18). The cyclic nucleotide levels are expressed as picomole per milligram protein.

Assay of PKC activity
After the dissociated SON neurons (2 x 106 cells/dish) were incubated with or without AVP, the cells were homogenized in buffer A, composed of 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 15 mM HEPES (pH 7.5), and then centrifuged at 105,000 x g for 30 min at 4 C (22). The supernatant was saved as a soluble enzyme, and the precipitate was homogenized in buffer A, containing 0.1% Triton X-100, and centrifuged. The resultant supernatant was used as a particulate enzyme. PKC activity was measured in a reaction mixture containing [{gamma}-32P]ATP (25 µM, 0.3 mCi), 200 µg/ml HI histone, enzyme, 20 µg/ml phosphatidylserine, 2 µg/ml 1,2-diolein, 5 mM MgCl2, and 50 mM Tris-HCl (pH 7.5) in the presence or absence of 0.4 mM CaCl2 and 5 mM EGTA. The reaction was carried out at 25 C for 5 min. The activity of PKC was defined as the kinase activity in the presence of CaCl2 minus the activity in the presence of EGTA without CaCl2.

Northern blot analyses
The total cellular RNA in the rat dissociated SON neurons 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 transferred to a nylon membrane filter (Amersham Pharmacia Biotech, Tokyo, Japan). The blots were hybridized with cDNA probes of AVP (kindly provided by Dr. D. Richter, Department of Neurobiology, UKE, Hamburg, Germany) and rat PTHrP (presented by Dr. A. Ootsuru, Nagasaki University, Japan). The 28S was visualized with 0.02% methylene blue or 0.5 M sodium acetate. To compare the density of each band, a Bioimage analyzer (BAS-2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) was used to accurately measure the density of AVP/PTHrP and 28S mRNA, respectively, and the results were evaluated as changes in AVP/28S or PTHrP/28S mRNA level after treatment.

Statistics
The results were expressed as the meant ± SEM of at least five independent experiments. Statistical significance was determined by a one-way ANOVA and Fisher’s protected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemistry
The existence of PTHrP in the SON was examined by immunohistochemistry in colchicine-treated rats (Fig. 1AGo). Dissociated cells from rat brain SON exhibited good survival at least during the experiments, by the evaluation of cell viability (>95%), morphology, and contents of PTHrP and AVP. Immunohistochemistry of the dissociated SON neurons showed that PTHrP was expressed predominantly in the cytosol of the cultured SON neurons (Fig. 1BGo). Immunohistochemistry showed that AVP was also stained in the cytosol of the dissociated SON neurons (Fig. 1Go, B and C). Nonimmune serum was used for the negative control (Fig. 1DGo)



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  		Figure 1. Photomicrographs showing PTHrP-like immunoreactivity in the SON in a colchicine-treated rat. Scale bar, 100 µm. OC, Optic chiasma (A). Immunohistochemistry of PTHrP (B), AVP (C), and negative control (D) in the dissociated SON neurons.

 
PTHrP(1–34)-induced AVP secretion and AVP mRNA expression
The basal AVP levels in the media of the cultured rat SON neurons were 21.2 ± 3.2 pmol/mg protein. Application of PTHrP(1–34) caused an increase in AVP levels of the culture medium in a concentration-dependent manner [10-10 M to 10-6 M (Fig. 2AGo)]. The minimal effective dose of PTHrP(1–34) on the AVP secretion was 10-10 M, and the half-maximal effective dose (ED50) was 3 x 10-8 M. PTHrP(7–34), an antagonist of PTHrP(1–34) to the type I PTH/PTHrP receptor, did not affect AVP release from the rat dissociated SON neurons at concentrations of 10-10 M to 10-6 M (Fig. 2BGo). PTH(1–34) did not affect AVP levels in the culture medium at concentrations of 10-12 M to 10-8 M, but the levels were increased by PTH(1–34) of 10-7 M to 10-6 M (Fig. 2CGo).



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  		Figure 2. Dose response of AVP secretion from the dissociated SON neuron by PTHrP(1–34) (A), PTHrP(7–34) (B), and PTH(1–34) (C). Twenty minutes after the SON neurons were incubated with various doses of these peptides, the incubation media were collected and AVP levels in the incubation media were measured by an RIA. The values are expressed as mean ± SEM (n = 5). *, P < 0.01 vs. the values without the peptides.

 
We examined the expression of AVP mRNA in the cultured rat SON neurons by Northern blot analysis at 2, 6, and 12 h after the addition of 10-6 M PTHrP(1–34). PTHrP(1–34) stimulated AVP mRNA expression time dependently (Fig. 3AGo).



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  		Figure 3. AVP (A) and PTHrP (B) mRNA expression were shown in the dissociated SON neurons. Total RNA in the dissociated SON neurons incubated with 10-6 M AVP or PTHrP for 0, 2, 6, and 12 h was used for Northern gel analysis, as described in Materials and Methods. Left, A representative AVP (A) or PTHrP (B) mRNA expression in the SON neurons treated with PTHrP(1–34) and AVP, respectively. Right, The ratios of respective mRNA for 28S are expressed as the mean ± SEM obtained from three independent experiments. *, P < 0.01, vs. the value of 0 min.

 
Effects of PTHrP(1–34) on levels of cAMP in rat dissociated SON neurons
PTHrP(1–34) increased cAMP generation in the rat dissociated SON neurons in a concentration-dependent manner [10-9 M to 10-6 M (Fig. 4AGo)]. Intracellular cAMP levels were increased 2.3-fold by addition of 10-6 M PTHrP, compared with controls, and 10-5 M forskolin induced a 3.1-fold increase in cAMP over the basal levels. The addition of 10-6 M PTHrP(7–34) did not induce cAMP accumulation in the SON neurons. 10-8 M and 10-7 M PTH(1–34) did not affect cAMP accumulation in the SON neurons, but 10-6 M PTH(1–34) exhibited a slight increase in cAMP levels of the cells (P < 0.01). 10-6 M PTHrP(1–34)-induced cAMP accumulation was attenuated by pretreatment with 10-4 M PTHrP(7–34), but it was not modulated by 10-4 M PTH(1–34). We evaluated whether PTHrP(1–34)-stimulated AVP release from the rat dissociated SON neurons would be mediated through the PKA system. Pretreatment with 2 x 10-8 M H89, a selective inhibitor of PKA (23), blunted AVP release stimulated by 10-6 M PTHrP(1–34) (Fig. 4BGo).



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  		Figure 4. Effects of PTHrP(1–34), PTHrP(7–34), PTH(1–34), and forskolin on the levels of cAMP in rat dissociated SON neurons (A) and the effect of H89 on 10-6 M PTHrP(1–34)-induced AVP (B). After a 10-min incubation at 37 C with 0.3 mM IBMX, the dissociated SON neurons were exposed to the various agents indicated for another 10 min in the presence of 0.3 mM IBMX. The cAMP levels in the supernatants from the cultures were measured using a Yamasa cAMP kit. A, 1, Control; 2, PTHrP(1–34) 10-10 M; 3, PTHrP(1–34) 10-9 M; 4, PTHrP(1–34) 10-6 M; 5, PTHrP(7–34) 10-6 M; 6, PTH(1–34) 10-8 M; 7, PTH(1–34) 10-7 M; 8, PTH(1–34) 10-6 M; 9, 10-6 M PTHrP(1–34) + 10-4 M PTHrP(7–34); 10, 10-6 M PTHrP(1–34) + 10-4 M PTH(1–34); 11. forskolin 10-5 M. B, 1, control; 2, PTHrP(1–34) 10-6 M; 3, PTHrP(1–34) 10-6 M + H89 2 x 10-8 M; 4, H89 2 x 10-8 M. *, P < 0.01 vs. control. Data are means ± SEM of six experiments.

 
AVP-induced PTHrP secretion and PTHrP mRNA expression
The basal levels of PTHrP in the culture medium of rat dissociated SON neurons were 250 ± 5 fmol/mg protein. The addition of 10-6 M AVP stimulated PTHrP release into the culture medium from the neurons in a time-dependent manner and reached a plateau (223 ± 5 fmol/mg protein) at 20 min (Fig. 5AGo). Figure 5BGo shows the effect of various concentrations of AVP on PTHrP release from the dissociated SON neurons. AVP stimulated the release of PTHrP in a concentration-dependent manner (10-11 M to 10-6 M). The significant effect of AVP on PTHrP release from the cultured cells was observed at 10-11 M, and the ED50 was at approximately 10-9 M (Fig. 5BGo). We examined the expression of PTHrP mRNA in the dissociated SON neurons by Northern blot hybridization after the addition of AVP. As shown in Fig. 3BGo, AVP caused an increase in PTHrP mRNA expression time dependently.



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  		Figure 5. A, Time course of AVP-induced PTHrP secretion from the dissociated SON neurons. After incubation with AVP (10-6 M) for 5, 10, 20, and 30 min, PTHrP levels in the incubation media were measured by an IRMA. The values are expressed as mean ± SEM (n = 5). *, P < 0.01 vs. the values at 0 min. B, PTHrP levels in culture media of the dissociated SON neurons incubated with various doses of AVP for 20 min. The values are expressed as mean ± SEM (n = 5). *, P < 0.01 vs. the values without AVP.

 
Effect of AVP receptor antagonists on AVP-induced PTHrP secretion
AVP(10-6 M)-stimulated PTHrP release from the SON neurons was suppressed by pretreatment with 10-6 M OPC-21268, a V1a receptor antagonist (Fig. 6AGo). Pretreatment of dP[Tyr(Me)2]AVP (10-4 M), a V1a/V1b receptor antagonist, also inhibited a rise in PTHrP levels in the medium response to AVP (Fig. 6BGo). The suppressive effect was greater in dP[Tyr(Me)2] (2)AVP (10-4 M), compared with OPC-21268 (10-6 M). OPC-31260, a V2 receptor antagonist, did not modulate the AVP-stimulated PTHrP levels in the medium (Fig. 6CGo). The administration of OPC-21268 (10-6 M), OPC-31260 (10-6 M), or dP[Tyr(Me)2]AVP (10-4 M) alone had no significant effect on PTHrP levels (Fig. 6Go, A–C).



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  		Figure 6. The effects of OPC-21268, dP[Tyr(Me)2]AVP and OPC-31260 on AVP-induced PTHrP release from the dissociated SON neurons. A, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) + OPC-21268 (10-6 M); 4, OPC-21268 (10-6 M). B, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) + dP[Tyr(Me)2]AVP (10-4 M); 4, dP[Tyr(Me)2]AVP (10-4 M). C, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) + OPC-31268 (10-6 M); 4, OPC-31268 (10-6 M). The values are expressed as mean ± SEM (n = 5). *, P < 0.01 vs. the values of respective controls.

 
Effects of AVP on membrane PKC activity and of PKC inhibitor on AVP-induced PTHrP secretion
We examined the effects of AVP on the PKC translocation in the SON neurons. Incubation of cells with 10-6 M AVP for 5 min significantly increased the activity of PKC in the membrane fraction of the cultured SON neurons (Fig. 7AGo). The significant increase was evident at 3 min, and the maximum was at 5 min after incubation with AVP. The increase of PKC activity by AVP was dose dependent (Fig. 7BGo). The AVP (10-6 M)-induced PTHrP release from the SON neurons was suppressed by staurosporin, a selective inhibitor of PKC, dose dependently (24) (Fig. 7CGo).



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  		Figure 7. Involvement of PKC in AVP (10-6 M)-induced PTHrP secretion from the dissociated SON neurons. The cells were incubated for 5 min in the presence or absence of AVP. After the reaction, the cells were homogenized and centrifuged. PKC activity in soluble (cytosol) and (membrane) fractions was assayed, using H1 histone as a substrate. A, Time course of PKC activity (%) by AVP (10-6 M) administration. B, Dose response of AVP-induced PKC activity (%). C, Effect of staurosporine on AVP-induced PTHrP secretion from the dissociated SON neurons. 1, Control; 2, AVP (10-6 M); 3, AVP + staurosporine (10-8 M); 4, AVP + staurosporine (10-7 M); 5, AVP + staurosporine (10-6 M); 6, staurosporine (10-6 M). Respective values are expressed as mean ± SEM (n = 5). *, P < 0.01 vs. 0 min (A), without AVP (B), 10-6 M AVP (C).

 
Effects of calcium channel blockers on AVP-induced PTHrP secretion
At least three subtypes of voltage-dependent calcium channels have now been defined, based largely on the sensitivity of the channels to various ligands and toxins. High-voltage-activated Ca2+ channels can be divided into the dihydropyridine-sensitive L-type Ca2+ channel (25), CgTx-sensitive N-type Ca2+ channel (26), and AgTx-sensitive P-type Ca2+ channel (14).

We used selective organic Ca2+ channel antagonists to test for the presence of L-, N-, and P-type currents. We found that 10-5 M nicardipine, 10-5 M CgTx and 10-7 M AgTx caused about 40%, about 25% and about 59% inhibition of AVP (10-6 M)-induced PTHrP secretion from the dissociated SON neurons, respectively (Fig. 8Go, A–C). The suppressive effects of 10-5 M CgTx were less than those of 10-5 M nicardipine and 10-7 M AgTx and not significant.



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  		Figure 8. Effects of pretreatment with organic Ca2+ channel antagonists (L-, N-, P-type) on AVP-induced PTHrP secretion from the dissociated SON neurons. A, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) + nicardipine (10-5 M); 4, nicardipine (10-5 M). B, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) + CgTx (10-5 M); 4, CgTx (10-5 M). C, 1, Control; 2, AVP (10-6 M); 3, AVP (10-6 M) +AgTx (10-7 M); 4, AgTx (10-7 M). The values are expressed as mean ± SEM (n = 5). *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have already reported that the application of PTHrP(1–34) to rat SON slices caused an increase in AVP release through the A kinase pathway (6). The icv administration of PTHrP(1–34) also induced AVP release in blood and AVP mRNA expression in the SON and PVN (7). However, PTH(1–34) did not have these effects in vitro or in vivo (6, 7). These results, including a binding study using crude membranes of the rat SON (6), suggest that PTHrP(1–34), but not PTH(1–34), causes the release of AVP from the SON involving cAMP through a novel receptor distinct from the type I PTH/PTHrP receptor (6), causing an increase in intracellular cAMP. In this study, we got the similar evidence in the culture system of dissociated SON neurons to those that had already been established by us (17). That is, PTHrP(1–34) stimulated AVP release and cAMP generation from the cultured SON neurons. The PTHrP(1–34)-induced AVP release was blocked by H89. These observations indicate that PTHrP(1–34) stimulates AVP release from the cultured SON neurons through PKA. PTH(1–34) and PTHrP(1–34) bind to the type I PTH/PTHrP receptor with equal affinity to activate A kinase and have the same degree of bioactivity in bone and kidney (27, 28). However, 10-12 M to 10-8 M PTH(1–34) did not stimulate AVP release from the dissociated SON neurons, and 10-6 M PTH(1–34) induced AVP release and activated A kinase in the neurons. The results may be associated with a weak binding of PTH(1–34) to the crude membranes of the SON because 28% of PTHrP(1–34) binding to the membranes was displaced by a 100-fold molar excess of PTH(1–34) (6). In this study, PTHrP(7–34) was not involved in AVP release from the cultured cells and cAMP generation in the cells. Therefore, the results observed in the dissociated SON neurons are consistent with our previous in vitro and in vivo studies (6, 7), that PTHrP(1–34) stimulates AVP release through a novel receptor distinct from the classical PTH/PTHrP receptor.

AVP receptors and their transcripts exist on magnocellular vasopressinergic neurons (29). AVP is released locally into the extracellular space of the SON and PVN (30) and can evoke its own release through a receptor-mediated mechanism (31, 32, 33), which leads to the most efficient mechanism for systemic AVP release. The dissociated SON neurons contained AVP as well as PTHrP in the immunohistochemical study. It is suggested that coregulation and/or mutual regulation exists between AVP and PTHrP. Surprisingly, an application of AVP stimulates the release of PTHrP from the dissociated SON neurons in a concentration-dependent manner (10-12 M to 10-6 M). PTHrP mRNA expression in the SON neurons was also stimulated by AVP. A significant stimulation of PTHrP release induced by AVP was observed at 10-10 M, and the ED50 by AVP was at approximately 10-8 M (Fig. 5BGo). AVP increased PKC activity in the neurons, and AVP-induced PTHrP release was inhibited by staurosporine, a PKC inhibitor. These results, taken together, indicate that AVP stimulates AVP release from the SON cells linked to PKC.

AVP exerts its regulatory effects through interaction with specific plasma membrane receptors (34). Three types of AVP receptors (V1a, V1b, and V2) are defined on the basis of the second messenger system to which they are coupled and their affinity profile for various AVP agonists and antagonists. The respective receptors are also produced from different genes and are expressed in different organs (34). The V1a receptor is detected in the brain, including PVN and SON of the hypothalamus, the liver, and blood vessels (35, 36). The V1b receptor, detected in the anterior pituitary, is involved in ACTH release (37). The V2 receptor is localized in the kidney and is coupled to adenylyl cyclase (38). In the present study, a V1a receptor antagonist (OPC-21268) or a nonselective V1a/V1b antagonist (dP[Tyr(Me)2]AVP) attenuated the AVP- induced PTHrP release from the dissociated SON neurons, whereas no suppressive effects were seen by a V2 receptor antagonist (OPC-31260). Recent studies indicated that V1a and V1b receptor transcripts were detected in the magnocellular neurons of SON and PVN (30, 39). V1a and V1b receptors are coupled to PLC and phosphoinositol hydrolysis (40, 41). In this study, AVP-induced PTHrP secretion from the dissociated SON neurons was associated with PKC activity and Ca2+ mobilization. Furthermore, in the study of the blocking of voltage-dependent calcium channels, L- and P-type channels were linked to AVP-induced PTHrP release in the SON neurons. The estimate obtained by blocking of the L- and P-type calcium channels in this study was very similar to those recently reported by Fisher and Bourque (42) in the dissociated SON neurons. However, N-type calcium channel may not be involved in the AVP-induced PTHrP release because the suppressive effect by blocking of N type was less than 25% and not statistically significant. DDAVP is considered a standard vasopressin V2 receptor selective agonist with a potent antidiuretic effect through the V2 receptor (without the induction of vasoconstriction through the V1a receptor). A recent report (43), however, demonstrated that DDAVP, which is a synthetic agonist, acts as an agonist on the V1b receptor, as it does on the V2 receptor, causing accumulation of intracellular Ca2+ in pancreatic islet cells. Although DDAVP is not normally present in the brain and DDAVP does not enter the brain, when given peripherally, we observed that DDAVP stimulated the release of PTHrP from the dissociated SON neurons in a dose-dependent manner (10-11 M to 10-6 M) (data not shown). These observations, taken together, suggest that AVP acts on the dissociated SON neurons, releasing PTHrP not only through the V1a receptor but also through the V1b receptor.

PTHrP has been shown to play a role in a wide variety of normal development and physiological processes as a local hormone. PTHrP acts on vascular smooth muscle, where it is produced, as a vasorelaxant (44, 45, 46). In contrast, AVP acts on vascular smooth muscle as a vasoconstrictor. The magnocellular AVP neurons of the SON are controlled by cholinergic and noradrenergic afferent fibers, neuropeptides, and neurotransmitters (47), and the regulation of AVP secretion is well defined. PTHrP exists in the central nervous system. However, the central role of PTHrP is not clarified. From our observations, it is suggested that PTHrP stimulates AVP secretion into systemic circulation and the extracellular space of the SON, which, in turn, leads to further secretion of AVP and PTHrP by an autocrine/paracrine mechanism. We cannot explain the meaning of the mutual regulations of AVP and PTHrP observed in the dissociated SON neurons. Because the release of PTHrP from SON neurons was lower than that of AVP when calculated by a molar basis in this study, it is speculated that PTHrP acts on the SON as a local factor. We need further studies to clarify the central role of PTHrP, the mutual regulation of AVP and PTHrP in the hypothalamus, and the physiological importance for their relationship in vivo.


   Footnotes
 
Abbreviations: AgTx, {omega}-Agatoxin IVA; CgTx, {omega}-conotoxin GVIA; DDAVP, 1-desamino-8-D-arginine vasopressin; dP[Tyr(Me)2]AVP, [deamino-Pen1,O-Me-Tyr2, Arg8]-vasopressin; ED50, half-maximal effective dose; IBMX, 3-isobutyl-1-methylxanthine; icv, intracerebroventricular; PVN, paraventricular; SON, supraoptic nuclei.

Received August 13, 2001.

Accepted for publication November 30, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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