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Endocrinology Vol. 142, No. 1 290-298
Copyright © 2001 by The Endocrine Society

ARTICLES

Ca2+ Mobilization, Tyrosine Hydroxylase Activity, and Signaling Mechanisms in Cultured Porcine Adrenal Medullary Chromaffin Cells: Effects of Leptin1

Kazuhiro Takekoshi, Kiyoaki Ishii, Yasushi Kawakami, Kazumasa Isobe, Toru Nanmoku and Toshiaki Nakai

Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan

Address all correspondence and requests for reprints to: Kazuhiro Takekoshi, Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba 1–1-1 Tennoudai, Tsukuba, 305-8575, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin acts as a satiety factor, but there is also evidence that it affects energy expenditure. Leptin’s effects are mediated by its receptors, which function as activators of a Janus family of tyrosine kinases-signal transducer and activator of transcription (JAK-STAT) pathway. We have previously shown that murine recombinant leptin markedly induces both the release of catecholamine and tyrosine hydroxylase (TH) (rate-limiting enzyme in the biosynthesis of catecholamine)-messenger RNA (mRNA) levels, probably through Ob-Rb expressed in cultured porcine chromaffin cells. In the present study, we examined the effect of leptin on Ca2+ mobilization, TH enzyme activity, and signaling. Ca2+ channel blockers, nicardipine and {omega}-Conotoxin GVIA, each at 1 µM, were effective in inhibiting leptin-induced catecholamine secretion. When intracellular Ca2+ ([Ca2+]i) was measured in fura 2-loaded chromaffin cells, leptin was found to cause a sustained increase of Ca2+ by mobilizing Ca2+ from both extra- and intracellular pools. Additionally, leptin significantly stimulated inositol 1.4.5-triphosphate IP3 production in a dose-dependent manner. TH-activity is regulated by both TH enzyme activity and increased TH-mRNA levels accompanied by increased TH protein synthesis. Leptin (>=1 nM) significantly stimulated TH enzyme activity and increased the TH protein level, indicating that it stimulates catecholamine biosynthesis. In addition, removal of external Ca2+ completely inhibited leptin (100 nM)-induced TH enzyme activity. Leptin (>=1 nM) caused an increase in the activity of mitogen-activated protein kinases (MAPKs) that was accompanied by increased phosphorylation of STAT-3 and -5, but not STAT-1. Moreover, MAPK activity evoked by leptin(100 nM) and TH-mRNA caused by leptin (10 nM) were inhibited by 50 and 30 µM of PD-98059 (the MAP kinase kinase-1 inhibitor), respectively. These findings indicate that leptin activates voltage-dependent Ca2+ channels (VDCC), presumably L-type and N-type Ca2+ channels, as well as phospholipase C, and suggest that leptin-induced catecholamine secretion is mainly mediated by activation of VDCC. In addition, leptin stimulates the JAK-STAT pathway as well as increasing the levels of TH-mRNA levels through the MAPK pathway in porcine chromaffin cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE OB gene product, is mainly secreted by mature adipocytes (1). Leptin acts as a satiety factor, but there is also evidence that it affects energy expenditure (1, 2, 3, 4, 5). Indeed, it has been shown that leptin-induced increases in energy expenditure are mainly due to thermogenesis in brown adipose tissue BAT, which are regulated by sympathetic outflow through ß3-adrenoreceptors (3, 4, 5).

There are several alternatively spliced variants of the leptin receptor (6, 7). They are basically of two forms. The long form of the leptin receptor (Ob-Rb) is expressed at high levels in the hypothalamus, which is presumed to be the major site of action for leptin (6, 8, 9). They can associate with the Janus family of the tyrosine kinase-signal transducers and activators of transcription (JAK-STAT) (10, 11, 12, 13).

Leptin, however, is a member of the cytokine family and its signal transduction system may be similar to that of other cytokines, suggesting that pathways other than JAK-STAT may be involved. Indeed, it has been shown that mitogen-activated protein kinases (MAPKs) are involved in leptin stimulated signal transduction (14, 15, 16).

The leptin receptor is reported to be expressed in a number of peripheral organs such as the adrenal medullary chromaffin cells, indicating that leptin may directly affect catecholamine synthesis and release (17, 18, 19, 20, 21).

Mobilization of Ca2+ into adrenal medullary cells is important to induce catecholamine secretion. One important mechanism for regulating the intracellular concentration of calcium ([Ca2+]i) utilizes voltage-dependent Ca2+ channels (VDCC) that mobilize Ca2+ entirely from extracellular Ca2+ pools localized on the outer surface of chromaffin cell membranes (22). Another important mechanism for regulating the cytosolic Ca2+ level is the release of Ca2+ from intracellular storage sites by inositol 1.4.5-triphosphate (IP3) formation, following phospholipase C (PLC) activation (23).

Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamine (24). TH activity can be regulated by both short and long-term mechanisms. Short-term regulation of enzyme activity occurs at the posttranscriptional level. Central to this regulation is the phosphorylation of TH, which results in activation of the enzyme (25, 26). Indeed, TH is phosphorylated and activated by a variety of protein kinases (27, 28, 29). It has also been reported that mobilization of Ca2+ into adrenal medullary cells induces increased TH enzyme activity (30). A long-term regulation has been shown to be exerted at the TH protein synthesis level following TH-gene transcription (31). Similar to their effect on TH enzyme activity, several protein kinases, including PKA, also induce an increase in levels of TH-messenger RNA (mRNA) levels (32).

Using cultured porcine chromaffin cells (33), as indicated below, we have previously shown that: 1) Ob-Rb is expressed in these cells; (2) murine recombinant leptin (>=50 nM) markedly induces the release of catecholamine in a manner strongly dependent on extracellular Ca2+; 3) leptin (>=1 nM) stimulates TH-mRNA accumulation; and 4) leptin (>=1 nM) significantly stimulates cAMP production. These findings suggest that leptin directly stimulates catecholamine synthesis and release, which in turn may potentiate its anti-obesity effects.

The major aim of the present study was to delineate more precisely the mechanism of leptin-induced catecholamine synthesis and secretion in cultured porcine adrenal medullary cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless otherwise noted, all reagents were purchased from Wako Seiyaku (Tokyo, Japan). Recombinant murine leptin was purchased from Pepro Tech. (London, UK). The MEK-1 inhibitor, PD-098059, was purchased from New England Biolabs, Inc. (Beverly, MA). Xestospongin C was purchased from Calbiochem (CA).

Cell culture
Primary dissociated cells from porcine adrenal medulla were prepared and purified by the differential plating method as previously described (34). In brief, the cells (1 x 106) were plated into 35-mm polystyrene dishes and maintained as a monolayer culture in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS (Life Technologies, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1.3 µg/ml Fungizone in a humidified atmosphere of 5%CO2/95%O2 at 37C for 2–3 days, and then used for experiments.

Measurement of [Ca2+]i with fura 2
Cells were cultured on collagen-coated 96-well plates. The cells were incubated with 4 µM fura 2 acetoxymethylester at 37C for 30 min and washed twice with HEPES-buffered Krebs-buffer. The [Ca2+]i was measured in fura 2-loaded chromaffin cells with the Multi Cell-Based Assay System (FDSS2000, Hamamatsu Photonics, Hamamatsu, Japan) using excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm.

Determination of catecholamine content
Catecholamine concentrations in media were determined as previously described (34), using a catecholamine autoanalyzer (TOSOH, H8030, Japan) with a built-in high performance liquid chromatograph and a spectrofluorometer.

Measurement of the production of IP3
Measurement of the production of IP3 was carried out using the specific IP3 binding assay kit (Pharmacia & Upjohn, Piscataway, NJ). Briefly, the cells were washed twice with EM containing 0.5% BSA, then stimulated with various concentrations of leptin (1 nM~100 nM) for 10 min. The reaction was quenched by removing medium and rapidly mixing with an equal volume of ice-cold 15% trichloroacetic acid. After sedimentation of precipitates, supernatants were extracted three times with 10 volumes of H2O2-saturated diethyl ether, evaporated to dryness, and the pH adjusted to 7.5 with NaHCO3. The amount of IP3 in the sample was determined according to the manufacturer’s protocol for the assay kit.

Tyrosine hydroxylase enzyme activity
Tyrosine hydroxylase enzyme activity was measured using a method previously published by Kumai et al. (35). Experiments were initiated by replacing the medium with HEPES-buffered Krebs buffer containing various concentrations of leptin (1 nM~100 nM), and the cells were incubated at 37C for 10 min. The cells were then homogenized in 0.25 M sucrose (50 volumes) using a glass tissue grinder. The standard incubation medium consisted of the following components in a total volume of 250 µl: 100 µl tissue homogenate, 40 µl of 1 M sodium acetate buffer (pH 6.0), 40 µl of 1 mM L-tyrosine or D-tyrosine, 20 µl of 1 M 6-methyl-5,6,7,8-tetra-hydropterine in 1 M 2-mercaptoethanol, 20 µl of 20 mM catalase, and 30 µl water. The medium was incubated at 37 C for 30 min, and the reaction stopped with 1 M perchloric acid containing dihydroxy benzylamine as an internal standard and then 0.2 M EDTA in an ice bath. Finally, 1 M potassium carbonate and 0.2 M Tris-HCl (pH 8.5) containing 1% EDTA were added. The 3-(3,4-di-dihydroxyphenyl)-alanine (DOPA) was extracted using the aluminum oxide method. Forty microliters of extracted medium was mixed with 0.1 N NaOH and TSK-GEL ODS-120T (TOSOH, Tokyo, Japan) and analyzed by HPLC. The mobile phase consisted of the following components: 50 mM sodium acetate, 20 mM citric acid, 12.5 mM sodium octyl sulfate, 1 mM di-n-butylamine, and 0.134 mM EDTA. All separations were performed isocratically at a flow rate of 0.6 ml/min at 28 C. The detector potential was maintained at +0.65 V. The TH enzyme activity was calculated as the amount of DOPA formed from tyrosine per milligram of protein per minute.

Northern blot analysis
For Northern blot analysis, total RNA was extracted from the samples using an ISOGEN kit (Nippon Gene, Tokyo, Japan). The RNA concentration was determined spectrophotometrically (at 260 nm). RNA (10 µg sample) was fractionated by electrophoresis on 1% agarose/5% formaldehyde gels (80 V, 2 h). After staining with ethidium bromide and visual inspection of the UV fluorescence to confirm the presence of equal amounts of 18S and 28S ribosomal RNA in each lane, the RNA was transferred to a nitrocellulose membrane and hybridized to 32P-labeled probes. The probe used for TH was a 1.9 kb EcoRI fragment of pTHT1. Plasmid pTHT1 contained the full-length complementary DNA (cDNA) for human TH type 1 cDNA. This plasmid was developed by T. Nagatsu and was provided by the RIKEN Gene Bank (Ibaraki, Japan). The probe was labeled using a random primer extension labeling kit (NEN Life Science Products, Boston, MA). Rat glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA was used as an internal standard (CLONTECH Laboratories, Inc., Palo Alto, CA). Hybridization signals were scanned in an image analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Effect of removal of external Ca2+ on leptin-induced TH enzyme activty
Cells were treated with murine recombinant leptin (100 nM) in a Ca2+-free medium containing 0.1 mM EDTA for 10 min. TH enzyme activity was then measured as described above.

Western blot analysis for tyrosine hydroxylase
Western blot analyses were performed as previously described (36). In brief, the cells were solubilized with 0.1% SDS containing 1% Triton X-100, 1% sodium deoxycholate,and 20 mM Tris-HCl, pH 7.4. The supernatant containing 10 mg protein was separated on 10% SDS-polyacrylamide gels, then transferred to nitrocellulose using a Bio-Rad Laboratories, Inc. (Hercules, CA) Transblot apparatus. After transfer, the nitrocellulose sheets were incubated for 1 h with BLOTTO buffer (5% skimmed milk, 0.05% Triton X-100, 100 mM, NaCl 200 mM Tris-HCl, pH 7.4). The nitrocellulose membranes were then washed 3 times for 10 min with TBST solution (0.05% Triton X-100, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl), then for 1 h with 1 mg/ml of the monoclonal antibody to tyrosine hydroxylase (Roche Molecular Biochemicals, Mannheim, Germany). The nitrocellulose membranes were then washed 3 times for 10 min with TBST solution, then incubated for 1 h with HRP-labeled Protein A (Amersham Pharmacia Biotech, Buckinghamshire, UK). Finally, the blots were washed 3 times, incubated with ECL reagent (Amersham Pharmacia Biotech, UK) for 1 min and used to expose Polaroid films (ISO 3000).

Western blot analysis for STAT proteins
Phosphorylation of STAT proteins from porcine adrenal medullary cells was measured using a Phospho Plus Stat Antibody Kit (New England Biolabs, Inc.) according to the manufacturer’s instruction manuals. Very briefly, cells were starved for 4 h and then the medium was replaced with HEPES-buffered Krebs buffer including leptin (5 nM). The incubation time was 5 min. The cells were washed with PBS, lysed in lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue). Lysates were immunoprecipitated with anti-STAT-1, 3 and-5 antibody. Immunoprecipitates were subjected to 7.5% SDS-PAGE and analyzed by immunoblotting using anti-phosphotyrosine antibody. The membrane was then stripped and reprobed using the respective anti-STAT antibodies.

MAPK kinase assays in vitro
MAPK kinase activity in porcine adrenal medullary cells was measured using a MAP Kinase Assay Kit as described previously (15) (New England Biolabs, Inc.). Very briefly, cells were starved for 4 h and then the medium was replaced with HEPES-buffered Krebs buffer including various concentrations of leptin (1 nM~100 nM). The incubation time was 15 min. Cell lysates were obtained as described above. Then, phospho-antibodies to p44/42 MAP kinase (Thr 202 and Tyr 204) were used to immunoprecipitate active MAP kinase from cell lysates. The resulting immunoprecipitate was then incubated with an Elk-1 fusion protein (GST fused to protein, derived from Escherichia coli expressing vector in which Elk-1 codons 307–428 were inserted). Phosphorylation of Elk-1 at Ser 383 was measured by Western blotting. The membrane was then stripped and reprobed using a control Elk-1 antibody (New England Biolabs, Inc.).

Statistical analysis
Data were analyzed between groups by one-way ANOVA by means of the Statview computer software program (Abacus Concepts, Inc., Berkeley, CA). When ANOVA showed significant differences, posthoc analysis was performed by Tukey’s test. P values less than 0.05 were considered significant. All data are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1) Effects of leptin on the [Ca2+]i concentration
We previously showed that leptin directly stimulates catecholamine release from cultured porcine adrenal chromaffin cells in a manner strongly dependent on extracellular Ca2+ (33). To confirm these findings, the mobilization of Ca2+ in response to leptin was examined in fura 2-loaded chromaffin cells. The concentrations of leptin used in this experiment were according to our previous study (33). As shown in Fig. 1aGo, leptin (100 nM) induced a sustained rise of [Ca2+]i in the presence of 2.2 mM Ca2+. When extracellular Ca2+ was removed by adding 5 mM EGTA at 8 min, the[Ca2+]i returned to baseline values. When extracellular Ca2+ was first chelated by the addition of 5 mM EGTA, subsequent addition of 100 nM leptin caused only a small sustained increase of [Ca2+]i (Fig. 1bGo). These findings indicate that leptin mobilizes Ca2+ from both extra- and intracellular pools in porcine adrenal chromaffin cells. The dose-relationship of leptin to [Ca2+]i was also confirmed. As shown in Fig. 1cGo, leptin (1 and 10 nM) did not affect the [Ca2+]i. However, leptin (50 and 100 nM) induced a significant rise of [Ca2+]i, which was similar to the stimulatory effects of catecholamine release induced by leptin (33).



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  		Figure 1. Effects of leptin on the[Ca2+]i concentration. a and b, Time-course changes in [Ca2+]i in fura 2-loaded chromaffin cells were measured using the system described in Materials and Methods. c, Dose-relationship of leptin on [Ca2+]i. The cells were stimulated by leptin (1, 10, 50, 100 nM). The values represent the means ± SD (n = 5~6). *, Significantly different (P < 0.05) from the basal level. [Ca2+]i was measured according to the method described in Materials and Methods.

 
2) Effects of Ca2+ channel blockers on leptin-induced [Ca2+]i
To establish whether L-type and/or N-type Ca2+ channels were functionally involved in (100 nM) leptin-induced [Ca2+]i, we examined the effects of nicardipine or {omega}-Conotoxin GVIA on leptin-induced [Ca2+]i (Fig. 2Go). As shown in Fig. 2Go, a and b, nicardipine and {omega}-Conotoxin GVIA significantly inhibited (100 nM) leptin-induced [Ca2+]i by 65% and 35%, respectively.



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  		Figure 2. Effects of Ca2+ channel blockers on leptin-induced[Ca2+]i. The cells were stimulated by leptin (100 nM) with or without nicardipine (L-type Ca2+ channel blocker: 1 µM) or {omega}-Conotoxin GVIA (N-type Ca2+ channel blocker: 1 µM). a, Representative data are shown. b, The values represent the means ± SD (n = 6). #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) as a control. [Ca2+]i was measured according to the method described in Materials and Methods.

 
3) Effects of L-type or N-type Ca2+ channel blockers on leptin-induced catecholamine release
To confirm our findings as shown in Fig. 2Go, we examined the effect of nicardipine or {omega}-Conotoxin GVIA on leptin-induced catecholamine secretion. As shown Fig. 3Go, both nicardipine (L-type Ca2+ channel blocker: 1 µM) and {omega}-Conotoxin GVIA (N-type Ca2+ channel blocker: 1 µM) significantly inhibited (100 nM) leptin-induced catecholamine secretion by 66.8% and 35.3%, respectively, comparable with their inhibitory effects on [Ca2+]i. When both nicardipine and {omega}-Conotoxin GVIA were added, catecholamine secretion caused by (100 nM) leptin was inhibited by 97.5%.



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  		Figure 3. Effects of Ca2+ channel blockers on leptin-induced catecholamine release. The cells were stimulated by leptin (100 nM) with or without nicardipine (L-type Ca2+ channel blocker: 1 µM) or {omega}-Conotoxin GVIA (N-type Ca2+ channel blocker: 1 µM) or both for 10 min at 37 C and then the media were examined with a catecholamine analyzer according to the method described in Materials and Methods. Values represent the means ± SD (n = 6). Because the result of E was comparable to that of NE, only the findings on NE are presented. *, Significantly different (P < 0.05) from the basal level. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 
4 Effects of leptin on production of IP3
Because leptin was found to mobilize Ca2+ from an intracellular pool (Fig. 1bGo), we tested whether it could stimulate phospholipase C to produce IP3, a messenger for mobilization of Ca2+ from intracellular storage sites. As shown in Fig. 4Go, leptin (1, 10, and 100 nM) significantly induced increases of IP3 by approximately 1.6-, 2.7-, and 3.7-fold, respectively.



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  		Figure 4. Effects of leptin on IP3 production. Cells were incubated for 10 min with various concentrations (1 nM~100 nM) of leptin. The values represent the means ± SD (n = 4). *, Significantly different (P < 0.05) from the basal value. IP3 was measured according to the method described in Materials and Methods.

 
5) Effect of xestospongin C (a membrane-permeable blocker of IP3-mediated Ca2+release) on leptin-induced catecholamine release
Because leptin can induce Ca2+-mobilization from the intracellular pool following IP3 increases, we examined the effect of xestospongin C (XeC) (37) on leptin-induced catecholamine release. Pretreatment with 1 µM of XeC did not affect catecholamine release caused by leptin (data not shown). The concentration of XeC was set at 1 µM according to Narasimhan et al. (38) and Mattson et al. (39).

6) Effects of leptin on TH enzyme activity and its Ca2+-dependent stimulatory effects in porcine adrenal medullary cells
Because TH is the initial and the rate-limiting enzyme in catecholamine biosynthesis, we tested the effect of leptin on TH enzyme activity. As shown in Fig. 5Go, leptin (1, 10, and 100 nM) significantly increased TH activity by 7.8, 10.2, and 18.7%, respectively. Next, to clarify the role of external Ca2+ in leptin-induced TH enzyme activity, the effects of removal of external Ca2+ were tested. As shown in Fig. 5Go, removal of external Ca2+ completely inhibited leptin (100 nM)-induced TH enzyme activity. In addition, to determine the role of the intracellular Ca2+ in leptin-induced TH enzyme activity, the effect of XeC was also tested. Pretreatment with 1 µM of XeC did not affect TH enzyme activity evoked by leptin (100 nM) (data not shown).



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  		Figure 5. Effect of leptin on TH enzyme activity and its Ca2+-dependent stimulatory effects in porcine adrenal medullary cells. Cells were incubated for 10 min with various concentrations (1 nM~100 nM) of leptin or leptin (100 nM) under Ca2+-free conditions, as described in Materials and Methods. TH enzyme activity was then measured as described in Materials and Methods. The values represent the means ± SD (n = 6). *, Significantly different (P < 0.05) from the basal level. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 
7) Effects of leptin on TH protein level
To confirm increases of TH protein following TH-mRNA induction by leptin, as previously reported (33), the effects of leptin on TH protein level were examined. Cells were treated with leptin (1, 10, and 100 nM) for 24 h, and the TH protein level was examined by Western blotting. As shown in Fig. 6Go, a and b, leptin (1, 10, and 100 nM) induced significant increases of TH protein expression by approximately 1.2-, 1.7-, and 2.0-fold over the basal values, respectively.



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  		Figure 6. Effect of leptin on the TH protein level in cultured porcine adrenal medullary cells. a, Cells were incubated for 24 h with leptin (1 nM~100 nM). TH protein levels were then measured by Western blot as described in Materials and Methods. Representative data from duplicate determinations are shown. b, The values represent the means ± SD (n = 6). *, Significantly different (P < 0.05) from basal level.

 
8) Effect of leptin on STAT activity in porcine chromaffin cells
To examine whether leptin activated STATs in chromaffin cells, the effects of leptin on STAT-1, -3, and -5 were tested. Tyrosine phosphorylation of STAT-1, -3, and -5 proteins, following leptin (5 nM) treatment, is shown in Fig. 7Go. The concentrations of leptin used in this experiment were equivalent to those observed in obese subjects (40). Tyrosine phosphorylation of STAT-3 and -5 was markedly elevated at 5 min, whereas phosphorylation of STAT-1 was not observed under the same experimental conditions.



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  		Figure 7. Effect of leptin on STAT activity in porcine chromaffin cells. Cells were incubated with leptin (5 nM) for 5 min. Phosphorylation of STAT-1, -3, and -5 proteins was measured as described in Materials and Methods. The lower panel shows the control STATs detected by the same blot after a stripping procedure. Representative data from duplicate determinations are shown.

 
9) Effect of leptin on MAPK activity
We examined the effect of leptin on MAPK activity (14, 15, 16). As shown in Fig 8Go, a and b, leptin (1, 10, and 100 nM) significantly induced increases of MAPK activity in a dose-dependent manner. In addition, the MAP kinase kinase (MEK)-1-inhibitor PD-098059 (50 µM) (41, 42), completely inhibited leptin-induced MAPK activity.



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  		Figure 8. Effect of leptin on MAPK activity in porcine chromaffin cells. a, Cells were incubated for 15 min with leptin alone (1 nM~100 nM) for the dose-response experiment. PD-098059 (50 µM) was added 1 h before leptin (100 nM) stimulation. MAPK activity was determined by the amount of phosphorylated Elk-1 (P-Elk-1: Ser383 Elk-1) substrate for MAPKs, as described in Materials and Methods. The lower panel shows the control Elk-1 detected by the same blot after a stripping procedure. Representative data from duplicate determinations are shown. b, The values represent the means ± SD (n = 4) of the densitometric measurement of the Phospho-Elk-1. *, Significantly different (P < 0.05) from basal value. #, Significantly different (P < 0.05) from value induced by leptin (100 nM).

 
10) Effects of PD-098059 on TH-mRNA levels induced by leptin
To clarify the involvement of MAPK activation in leptin-induced TH-mRNA accumulation, porcine chromaffin cells were treated with PD-098059 (10, 20, and 30 µM) for 1 h before 10 nM leptin stimulation. As shown in Fig. 9Go, a and b, PD-098059 inhibited TH mRNA induced by leptin in a dose-dependent manner. The IC 50 of PD-098059 was around 15 µM and 30 µM PD-098059 showed complete inhibition of leptin-induced TH mRNA.



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  		Figure 9. Effects of the MEK1 inhibitor on the TH-mRNA level induced by leptin. a, PD-098059 (10, 20, 30 µM) was added to the cells. One hour later, cells were stimulated by leptin (10 nM) and incubated for 8 h. RNA was then isolated and used for Northern blot analysis. The lower panel shows the control mRNA (glyceraldehyde phosphate dehydrogenase: GAPDH), which contained an amount of mRNA equivalent to that loaded in each lane. Representative data from duplicate determinations are shown. b, The values represent the means ± SD (n = 4) of the radioactivities (photostimulated luminescence minus background) of the TH-mRNA levels. *, Significantly different (P < 0.05) from basal value. #, Significantly different (P < 0.05) from value induced by leptin (10 nM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously reported that leptin directly stimulates catecholamine release from cultured porcine adrenal chromaffin cells in a manner highly dependent on extracellular Ca2+ (33). The present study further confirmed that leptin caused an increase of [Ca2+]i in an extracellular Ca2+-dependent manner (Fig. 1Go, a and c), confirming that leptin-induced catecholamine secretion is indeed dependent on external Ca2+ (Fig. 10Go). Moreover, leptin-induced [Ca2+]i and catecholamine secretion was markedly suppressed by nicardipine and {omega}-Conotoxin GVIA (Fig. 2Go, a and b, Fig. 3Go), indicating that VDCC, presumably both L-type and N-type Ca2+ channels, are involved in the mechanisms of leptin-induced catecholamine secretion.



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  		Figure 10. Outline of proposed model for the effects of leptin on catecholamine release and TH transcription in chromaffin cells. Results are suggested by the current experiments, together with data that were the basis of our previous reports (33 ). Binding of leptin to its receptor (Ob-Rb) causes stimulation of the JAK-STAT pathway as well as increasing the levels of TH-mRNA through the MAPK pathway. In addition, cAMP production induced by leptin may converge to the MAP kinase pathway via the small G protein Rap1-dependent pathway, instead of Ras. Leptin-induced catecholamine secretion is indeed dependent on external Ca2+ via VDCC, whereas the release of Ca2+ from intracellular storage sites by IP3 formation, following PLC activation plays only a small part in catecholamine secretion. PKA, cAMP-dependent protein kinase A; ER, endoplasmic reticulum. Broad arrows, signaling pathways suggested or confirmed by the current experiments.

 
Although we have no evidence that the increases of IP3 are associated with increased cytosolic Ca2+ in our experimental system, we showed that leptin induced the mobilization of Ca2+ from intracellular storage sites, accompanied by increases of inositol IP3, a messenger for mobilization of Ca2+ from intracellular storage sites (Fig. 1bGo, Fig. 4Go). These findings indicate that leptin causes a sustained increase of [Ca2+]i by mobilizing Ca2+ from both extra- and intracellular pools. However, leptin (100 nM) caused little catecholamine secretion in the absence of extracellular Ca2+ as previously reported (33). Consistent with this, our previous finding, we showed that xestospongin C (Xe C: a membrane-permeable blocker of IP3-mediated Ca2+ release) (37, 39) did not affect catecholamine release caused by leptin (data not shown). It was demonstrated by Westhead et al. (43) that Ca2+ entering across the plasma membrane was much more effective at triggering exocytosis than the Ca2+ released from internal stores. This led them to claim that Ca2+ released from intracellular storage sites does not induce exocytosis. Thus, it is possible that mobilization of Ca2+ from extracellular but not intracellular pools may contribute to catecholamine secretion induced by leptin (Fig. 10Go).

In the present study, we have demonstrated that leptin-induced significant increases of TH enzyme activity (EC50: around 10 nM) (Fig. 5Go). In addition, leptin significantly induced increases of TH protein (Fig. 6Go, a and b), confirming that it stimulates increases in the level of TH protein as a result of TH-mRNA increases, as previously reported (33). Thus, these findings indicate that leptin directly stimulates catecholamine synthesis.

The present study showed that removal of external Ca2+ completely suppressed leptin (100 nM)-induced TH enzyme activity, indicating that external Ca2+ plays a critical role in regulating TH enzyme activity as well as catecholamine release. It has been shown that phosphorylation sites on TH can be identified as Ser8, Ser19, Ser31, and Ser40 in bovine chromaffin cells (44, 45). Our findings also support the suggestion that leptin-induced phosphorylation sites on TH may be highly dependent on extracellular Ca2+. However, precise mechanisms by which external Ca2+ exerts its stimulatory effect on TH enzyme activity are unclear. Further studies (i.e. to determine which kinases mediate these phosphorylations and which phosphorylations cause TH-enzyme activation following leptin stimulation) will be required to clarify these points. In contrast, Xe C did not alter leptin (100 nM)-induced TH enzyme activity (data not shown), suggesting that internal Ca2+ may have a minimal role in regulating TH enzyme activity.

Mounting evidence suggests that STAT proteins including STAT-1, -3, and -5 are involved in the mechanisms of leptin signal transduction (10, 11, 12, 13, 46). In the present study, leptin caused tyrosine phosphorylation of STAT-3 and -5 in porcine chromaffin cells (Figs. 7Go and 10Go). However, phosphorylation of STAT-1 was not observed under the present experimental conditions. These findings strongly suggest that STATs are involved in the signal transduction mediated by leptin in chromaffin cells. Because Ob-Rb contains a box-2 motif that is a prerequisite for activation of the JAK-STAT pathway (7, 9), it is also possible that leptin exerts its function through Ob-Rb in chromaffin cells (Fig. 10Go). However, Vaisse et al. (12) reported that leptin failed to stimulate STAT-3 activation in rat adrenal cells, which is discordant with the findings presented here. The reasons for this difference are unclear. Those investigators measured STAT activity using electromobility shift assays (EMSA) rather than Western blot analysis as used in the present study. Also, species differences (porcine vs. rat), differences in experimental conditions (in vitro vs. in vivo) should be considered.

In the present study, we demonstrated that leptin significantly induced activation of MAPK in a dose-dependent manner in chromaffin cells (Fig. 8Go, a and b). Moreover, MAPK activity (Fig. 8Go, a and b) and TH-mRNA evoked by leptin (Fig. 9Go, a and b) were inhibited by 50 and 30 µM of PD-98059, respectively, confirming that leptin induces TH-mRNA via the MAPK cascade in chromaffin cells. From these observations, together with those of previous studies (14, 15, 16, 18), it appears that MAPKs are essential for induction of TH-mRNA caused by leptin in chromaffin cells (Fig. 10Go).

Recent studies have shown that STATs are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for MAPK, suggesting that the MAPK and JAK/STAT pathways are intimately linked at various steps (47, 48). Indeed, a close link between the JAK/STAT and MAPK pathways was reported to be involved in the mechanisms of action of ß-interferon and oncostatin M in Hela and U-4A cells (49). Thus, it is possible that the TH-mRNA induced by leptin may be mediated by the MAPK pathway through activation of JAK/STAT-mediated pathways (Fig. 10Go).

Although the precise signal transduction pathways responsible for leptin-induced increases of TH-mRNA levels are unknown, it was suggested that MAP kinase stimulates activation of Fos and Jun, resulting in activation of AP-1 activity (50, 51). Transcription of the TH gene is regulated by several transcriptional factors through binding to the 5'-flanking sequence of the TH gene (52, 53). In particular, the binding of the AP-1 (Fos/Jun) heterodimer to the TH-TPA-response element (TRE) site is a prerequisite for TH gene activation mechanisms in chromaffin cells (54). Indeed, Raizada et al. recently demonstrated that Ang induced TH-mRNA through activation of AP-1-binding activity in a MAPK-dependent manner (55). Thus, it is tempting to speculate that increases of TH gene expression induced by leptin are mediated by increases of some transcriptional factors at the specific TRE site of the TH 5'-flanking region (Fig. 10Go).

We previously reported that leptin induced increases of cAMP production (33). This finding suggests that the effects of leptin on TH-mRNA levels are mediated, at least in part, by the cAMP/PKA/CRE pathway. Indeed, another important site of TH gene regulation is CRE in the promoter region that is regulated by the cAMP/PKA pathway (32). However, Vossler et al. recently reported that PKA directly activates the small G protein Rap1 instead of Ras, which in turn, results in the activation of MAP kinase in rat pheochromocytoma PC12 cells (56). Consistent with this finding, the present study showed that PD-098059 completely abolished the leptin-stimulated TH-mRNA induction (Fig. 9Go). Thus, it is still possible that the cAMP production induced by leptin may converge to the MAP kinase pathway via the Rap1-dependent pathway (Fig. 10Go). Further studies will be needed to clarify the interaction and/or relationship between the PKA and MAPK pathways induced by leptin in chromaffin cells.

We showed that high concentrations of leptin (50 nM), probably supraphysiological, were associated with increased Ca2+ and release of catecholamines, whereas other effects such as TH enzyme activity and changes in TH-mRNA levels were observed at lower concentrations of leptin. More recently, Yanagihara et al. (57) reported that human recombinant leptin (3–30 nM) stimulated catecholamine synthesis without affecting catecholamine secretion in cultured bovine adrenal medullary cells, which is in agreement with our study. The physiological relevance of this difference remains to be clarified. It should be noted, however, that the adrenal gland is surrounded by periadrenal fat tissue, shown to contain abundant leptin (21, 58). Subsequently, the local concentration of leptin may reach a level much higher than that in the plasma, as suggested by Bornstein et al. (21).


   Acknowledgments
 
The authors thank Kohei Sawada, Ph.D. (Senior Scientist, Department of Drug Discovery, Tsukuba Research Laboratories, Eizai Co., Ltd.) for measurement of [Ca2+]i mobilization.


   Footnotes
 
1 This work was supported in part by a grant (No. 11770624) from the Ministry of Education and by the University of Tsukuba Research Project. Back

Received May 4, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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