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Endocrinology Vol. 143, No. 11 4210-4217
Copyright © 2002 by The Endocrine Society

ARTICLE

3',5'-Cyclic Adenosine Monophosphate Augments Intracellular Ca2+ Concentration and Gonadotropin-Releasing Hormone (GnRH) Release in Immortalized GnRH Neurons in an Na+-Dependent Manner

Keisuke Kaneishi, Yasuo Sakuma, Hisae Kobayashi and Masakatsu Kato

Department of Physiology, Nippon Medical School (K.K., Y.S., M.K.), Tokyo 113-8602, Japan; and Institute for Molecular and Cellular Regulation, Gunma University (H.K.), Maebashi 371-8511, Japan

Address all correspondence and requests for reprints to: Dr. Masakatsu Kato, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo, Tokyo 113-8602, Japan. E-mail: mkato{at}nms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In GT1-7 cells, cAMP increases the intracellular Ca2+ concentration ([Ca2+]i) through activation of the voltage-gated Ca2+ channels, thereby facilitating GnRH release. To activate these channels, the membrane potential must be depolarized. In the present study we hypothesize that cAMP depolarizes the cells by increasing the membrane Na+ permeability, as in the case of somatotrophs and pancreatic ß-cells. To examine this, we analyzed [Ca2+]i and [Na+]i in GT1-7 cells by an intracellular ion-imaging technique along with cAMP assay by RIA.

Forskolin, a direct activator of adenylyl cyclase, increased [Ca2+]i and [Na+]i via cAMP formation. The forskolin-induced increase in [Ca2+]i depended on the presence of Ca2+ and Na+ in the extracellular solution. This response was blocked by the voltage-gated Ca2+ channel blocker, nifedipine; the nonselective cation channel blocker, gadolinium (Gd3+); and the cyclic nucleotide-gated channel blocker, l-cis-diltiazem. In contrast, the forskolin-induced increase in [Na+]i depended only on extracellular Na+, not on Ca2+. Gd3+ and l-cis-diltiazem also blocked the increase in [Na+]i. Furthermore, the forskolin-induced increase in GnRH release was blunted in both low Ca2+ and low Na+ media. The results indicate that cAMP increases the membrane Na+ permeability, probably through nonselective cation channels on GT1-7 cells, thereby promoting GnRH release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH PLAYS A crucial role in reproductive physiology (1, 2), and its release is controlled by a number of neurotransmitters, including norepinephrine and dopamine. These monoamines stimulate GnRH release from GnRH neurons and its clonal cell line, GT1-7 cells (3), via formation of cAMP (4, 5, 6), so that cAMP is an important intracellular mediator in the process of GnRH secretion in these cells (7, 8). Forskolin, a direct activator of adenylyl cyclase, promotes cAMP formation and increases the intracellular Ca2+ concentration ([Ca2+]i) and GnRH release in GT1-7 cells (9, 10).

It is suggested that in GT1-7 cells cAMP activates the olfactory type of cyclic nucleotide-gated (CNG) channels that are permeable to cations, thereby controlling Ca2+ oscillation (11). Activation of this CNG channel may increase [Ca2+]i, because this channel is permeable to Ca2+ (12, 13, 14). On the other hand, cAMP depolarizes somatotrophs by increasing Na+ permeability in response to GHRH via protein kinase A (PKA) (15, 16, 17, 18), and in pancreatic ß-cells, glucagon-like peptide 1 causes depolarization by increasing Na+ permeability via cAMP (19, 20), so the Na+ permeability increase may play a pivotal role in the action of cAMP.

In the present study we investigated the involvement of Na+ permeability in the process of cAMP-mediated GnRH secretion from GT1-7 cells by the method of intracellular ion imaging and GnRH assay.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture of GT1-7 cells
GT1-7 cells (a gift from Dr. Richard I. Weiner, University of California, San Francisco, CA) were cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% fetal bovine serum (JRH, Lenexa, KS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) without phenol red. Cells were plated on poly-L-lysine-coated coverslips for intracellular Ca2+ imaging and Na+ imaging. The cultures were maintained at 37 C in 95% air and 5% CO2 until they reached confluence.

For static incubation experiments, GT1-7 cells were cultured in 12-well plates as described above and were incubated for 15–30 min in normal perifusion medium before the experiments.

Measurement of [Ca2+]i and [Na+]i
Details of the imaging technique and the superfusion system were described previously (21). In brief, cells were loaded either with 2 µM fura-PE-3 acetoxymethyl ester (TefLabs, Austin, TX) for 1.5–2.0 h at 37 C for Ca2+ imaging or with 5 µM sodium-binding benzofuran isophthalate acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) for 2 h at room temperature for Na+ imaging. The coverslip was placed in a small superfusion chamber on the stage of an IX70 inverted microscope (Olympus Corp., Tokyo, Japan). [Ca2+]i and [Na+]i were recorded by using the QuantiCell 700 system (Applied Imaging, Sunderland, UK). The cells were illuminated alternately at 340- and 380-nm excitation wavelengths, and then 510-nm emission light images were captured by an image-intensifying, charge-coupled device camera (Photonics Science, Turnbridge Wells, UK). The time interval of each 340- to 380-nm ratio frame was 3 sec. Ratios were converted to Ca2+ concentrations as previously described (22) or to Na+ concentrations (23, 24).

Superfusion was performed with the normal perifusion medium containing 137.5 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.8 mM MgCl2, 0.6 mM NaHCO3, 10 mM glucose, 20 mM HEPES, and 0.1% BSA, and pH was adjusted to 7.4 with NaOH. Low Ca2+ medium was prepared by replacing Ca2+ with Mg2+ in normal perifusion medium. Low Na+ medium was prepared by substituting N-methyl-D-glucamine for Na+ in normal perifusion medium unless otherwise mentioned. The cells were continuously superfused at 37 C throughout the experiment, and the flow rate was approximately 1 ml/min. All drugs were applied through superfusion.

Assay of cAMP and GnRH
Cellular cAMP was extracted with 1.5 N perchloric acid, and pH was adjusted to 7 with 1.5 N KOH. The cAMP in the supernatant was first succinylated and then measured in duplicate by a double antibody RIA method with a cAMP assay kit (Yamasa Shoyu, Choshi, Japan). An ELISA kit (Peninsula Laboratories, Inc., San Carlos, CA) was used to determine the GnRH concentration in the incubation medium. All samples were assayed in duplicate or triplicate.

Reagents
Forskolin was purchased from Wako Chemicals (Osaka, Japan). Tetrodotoxin (TTX) was obtained from Sankyo Co., Ltd. (Tokyo, Japan); gadolinium chloride, cadmium chloride, norepinephrine, dopamine, H89, H7, adenosine 3',5'-cyclic monophosphothioate Rp-isomer (Rp-cAMPS), 8-bromo-cAMP, and l-cis-diltiazem (LCD) were obtained from Sigma (St. Louis, MO).

Statistical analysis
The results are expressed as the mean ± SEM unless otherwise mentioned. In the data from the imaging experiment, n indicates the number of cells. The differences between groups were analyzed by one-way ANOVA. In some experiments forskolin was applied twice with a 30-min wash interval, and the drugs were applied at the second application of forskolin. In this protocol a response to the first application was designated S1, and that to the second was designated S2. The effect of drugs was evaluated by the S2/S1 ratio. In these cases, paired t test was used. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Forskolin increased cellular cAMP level and [Ca2+]i
Forskolin increased the cAMP levels in GT1-7 cells in a concentration-dependent manner. Forskolin at 0.3 µM increased the cellular cAMP level to 10 ± 1.1 pmol/ml (n = 3) and at 20 µM to 93 ± 12 pmol/ml (n = 3) from the basal level of 2.1 ± 0.7 pmol/ml as shown in Fig. 1Go. Similarly, 1 µM norepinephrine and 1 µM dopamine increased the cAMP level to 27 ± 2.1 pmol/ml (n = 3) and 21 ± 3.1 pmol/ml (n = 3), respectively. Forskolin (0.1–30 µM) increased [Ca2+]i in GT1-7 cells. Concentration dependency was seen between 1–20 µM. The proportion of the responding cells was also concentration dependent (Fig. 2Go). Both 10 µM dopamine and 10 µM noradrenaline increased [Ca2+]i by 87 ± 11 (n = 112) and 105 ± 13 nM (n = 118), respectively. The responses were selectivity inhibited by SCH 23390 and propranolol.



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  		Figure 1. Effect of forskolin on cAMP accumulation. The cells were incubated with forskolin for 15 min at 37 C. Forskolin increased total cAMP levels in a concentration-dependent manner. Each column represents the mean ± SD for three independent experiments.

 


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  		Figure 2. Effect of forskolin on [Ca2+]i increase. A, Representative responses to forskolin. Forskolin was applied for 5 min, as indicated by the horizontal bar. B and C, Concentration-response relationship of forskolin to peak increase in {Delta}[Ca2+]i and proportion of responding cells. Each column in B represents the mean ± SEM.

 
To exclude possible side-effects of gadolinium and LCD on the cAMP level, we measured the forskolin-induced cAMP level in the presence of Gd3+ and LCD. The forskolin-induced cAMP level was not affected by either 100 µM Gd3+ or 100 µM LCD (data not shown).

Forskolin-induced increase in [Ca2+]i depended on the extracellular Ca2+ and Na+
Low Ca2+ medium decreased basal [Ca2+]i to 42 ± 1.2 nM from 56 ± 1.6 nM (P < 0.05; n = 52) and reversibly suppressed the forskolin-induced response (Fig. 3Go, A and F). The L-type Ca2+ channel blocker, nifedipine (10 µM), attenuated the response by 76 ± 4.5% (n = 56). Furthermore, the nonselective cation channel blocker, gadolinium (Gd3+; 10–100 µM) (25, 26), inhibited the response in a concentration-dependent manner (Fig. 3Go, C and F; n = 46–53). Low Na+ medium suppressed the forskolin-induced increase in [Ca2+]i by 86 ± 2.9% (10 mM [Na+]o; n = 80) and 66 ± 15% (30 mM [Na+]o; n = 107) as shown in Fig. 3Go, B and G. Blocker of the voltage-gated Na+ channel, TTX (1 µM), suppressed the response by 26 ± 7.0% (n = 124; Fig. 3Go, D and G). A blocker of the CNG channel, LCD (27), elicited a small rise in [Ca2+]i as previously reported (28) and suppressed the forskolin-induced response. Application of LCD at 100 and 300 µM reversibly suppressed the response by 86 ± 2.1% (n = 189) and 98 ± 1.0% (n = 117), respectively, but 40 µM LCD did not suppress the response in any of the 163 cells examined (Fig. 3Go, E and G). It should be noted that LCD blocks CNG channels when applied to the bath solution, although it has been shown that the blockade by LCD is more effective when intracellularly applied (27).



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  		Figure 3. The responses in Ca2+ imaging. A, The forskolin-induced [Ca2+]i increase disappeared in low Ca2+ medium. B, The forskolin-induced [Ca2+]i increase was reduced in low Na+ medium. C, The nonselective cation channel blocker, Gd3+ (100 µM), completely blocked the response. D, The voltage-gated Na+ channel blocker, TTX (1 µM), slightly suppressed the response. E, The CNG channel blocker, LCD (300 µM), completely blocked the response. F and G, Collective presentation of the effects of low Ca2+, nifedipine, Gd3+, low Na+, TTX, and LCD on the forskolin-induced [Ca2+]i increase. Each column indicates the mean of the S2/S1 with the SEM. *, P < 0.05, by paired t test.

 
To rule out the possible side-effect of N-methyl-D-glucamine in the experiment with low Na+ medium, we used mannitol instead of N-methyl-D-glucamine. Low Na+ medium (30 mM) with mannitol suppressed the response by 67 ± 3.0%. There was no difference in the inhibition caused by low Na+ medium prepared with either substitution.

Forskolin-induced increase in [Na+]i
Forskolin (20 µM) elicited a gradual increase in [Na+]i from a resting value of 13 ± 0.5 mM (n = 204). GT1-7 cells responded to forskolin with a peak increase in [Na+]i of 12 ± 0.7 mM (n = 204; Fig. 4Go, A and G). Cells were determined to be responsive when the difference in the ratio exceeded 0.15 in all Na+ imaging experiments presented here.



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  		Figure 4. The responses in Na+ imaging. A, Forskolin was applied for 5 min as indicated by the horizontal bar. Forskolin increased [Na+]i. B, Low Na+ medium reversibly suppressed the forskolin-induced [Na+]i increase in all cells examined. C, Low Ca2+ medium did not affect the forskolin-induced [Na+]i increase. D, TTX did not attenuate the forskolin-induced [Na+]i increase. E, LCD (40 µM) did not attenuate the forskolin-induced [Na+]i increase. F, LCD (300 µM) completely blocked the response. G, Collective presentation of the effects of Gd3+, low Na+, low Ca2+, TTX, and LCD on the forskolin-induced [Na+]i increase. Each column indicates the mean ± SEM. *, P < 0.05 vs. control.

 
As shown in Fig 4BGo, low (10 mM) Na+ medium did not affect the basal [Na+]i, but low Na+ medium almost completely suppressed the forskolin-induced response (Fig. 4Go, B and G). By contrast, low Ca2+ medium had no effect on the forskolin-induced increase in [Na+]i (n = 41; Fig. 4Go, C and G). Gd3+ (100 µM) reversibly suppressed the response (n = 85; Fig. 4GGo). TTX (1 µM) did not attenuate the peak increase in [Na+]i (Fig. 4Go, D and G). Application of 300 µM LCD decreased the basal level of [Na+]i to 8.0 ± 0.2 mM from the control level of 12 ± 0.6 mM (P < 0.05) and completely suppressed the forskolin-induced increase in [Na+]i (n = 71), but 40 µM LCD had no effect on either the basal or forskolin-induced increase in [Na+]i (n = 39; Fig. 4Go, E–G).

Effects of protein kinase inhibitors and cAMP analog
The PKA inhibitor, H89 (29), did not affect the forskolin-induced increase in either [Ca2+]i or [Na+]i (Fig. 5Go, A–D). The protein kinase A and C inhibitor, H7 (30), also did not inhibit the forskolin-induced [Ca2+]i increase (Fig. 5CGo). A membrane-permeable cAMP analog, 8-bromo-cAMP (2.5 mM), increased [Ca2+]i by 129 ± 24 nM (n = 52), but the specific cAMP antagonist, Rp-cAMPS, did not inhibit the forskolin-induced response as previously reported (28) (data not shown).



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  		Figure 5. Effect of PKA inhibitors in Ca2+ and Na+ imaging. A and B, PKA inhibitor, H89 (10 µM) did not affect the forskolin-induced [Ca2+]i and [Na+]i increases. C, Collective presentation of the effects of H89 and PKA and PKC inhibitor, H7 (10 µM), in Ca2+ imaging. Each column indicates the mean of the S2/S1 with the SEM. D, The effects of H89 on Na+ imaging are shown. Each column indicates the mean ± SEM.

 
Forskolin-induced GnRH release
Incubation of GT1-7 cells with 20 µM forskolin for 30 min increased the release of GnRH by 388 ± 136% (n = 4), but in low Ca2+ or low Na+ (10 mM) medium, forskolin did not increase GnRH release (n = 4; Fig. 6Go). The basal release of GnRH for 30 min was 0.33 ± 0.24 ng/ml (mean ± SD) in normal medium, 0.27 ± 0.15 ng/ml in low Ca2+ medium, and 0.71 ± 0.50 ng/ml in low Na+ medium (n = 6–7). There was no statistically significant difference among them.



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  		Figure 6. Effect of forskolin on GnRH release. The cells were incubated with forskolin (20 µM) for 30 min at 37 C. Forskolin increased GnRH release in normal medium, but not in low Na+ medium and low Ca2+ medium. Each column represents the mean ± SEM of four independent experiments. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrated that forskolin increased cellular cAMP levels, [Ca2+]i, [Na+]i, and GnRH release in GT1-7 cells. Increases in [Ca2+]i and GnRH release depended on the presence of Ca2+ and Na+ in the extracellular solution. An increase in [Na+]i, however, depended on the presence of extracellular Na+, but not on Ca2+. The forskolin-induced increase in [Ca2+]i is due to an influx of Ca2+ through voltage-gated Ca2+ channels, because removal of extracellular Ca2+ or application of nifedipine suppressed or attenuated the rise in [Ca2+]i. Several reports also suggest an involvement of voltage-gated Ca2+ channels in the action of cAMP (31, 32).

The next question is how the voltage-gated Ca2+ channels are activated. One possibility is a phosphorylation of the voltage-gated Ca2+ channels, as shown in cardiac myocytes (33) and other types of cells (34, 35), but this may not be a major cause, because PKA inhibitors H89 and H7 did not suppress the forskolin-induced response. In addition, the response completely depended on the presence of extracellular Na+.

The Na+ dependency raises two possibilities. One is that the resting Na+ permeability plays a role as background current (36). In this case a decrease in K+ permeability effectively depolarizes the cells without a change in Na+ permeability, thereby activating the Ca2+ channels. If this is the case, we should not see an increase in [Na+]i induced by forskolin, because we would not expect a change in Na+ permeability. Our results, however, showed a clear rise in [Na+]i caused by forskolin, suggesting augmentation of Na+ permeability. Possible involvement of an increase in Na+ permeability is further supported by the results showing that a decrease in [Na+]o, Gd3+, and a blocker of the CNG channel (27), LCD, attenuated the response. It is therefore likely that cAMP augments membrane Na+ permeability, but the voltage-gated Na+ channel does not play a major role in the forskolin-induced response, because a blocker of the voltage-gated Na+ channel, TTX (1 µM), only slightly attenuated the response.

It has been suggested that the olfactory type of CNG channel mediates the cAMP action on the regulation of Ca2+ oscillation in GT1-7 cells (28). In cAMP-mediated release of GnRH, however, the olfactory types of CNG channels may not be involved, because this type of CNG channel is known to behave as an almost pure Ca2+-selective channel at a millimolar concentration of [Ca2+]o (37). If the CNG channel plays an important role in the forskolin-induced response, we should see a rise in [Ca2+]i even under 10 and 30 mM [Na+]o. In the present study we did not see a substantial rise in [Ca2+]i under such conditions. Therefore, it is unlikely that the olfactory-type CNG channel is a prerequisite for the forskolin-induced release of GnRH, although LCD at more than 100 µM blocked the response. For this blocking effect of LCD, we speculate that LCD would block channels other than the CNG channels. Alternatively, GT1-7 cells might express rod types of CNG channels, which are reported to have higher permeability to Na+ than the olfactory types (38). Therefore, the ion channels involved in the cAMP-mediated large and persistent rise in [Ca2+]i may differ from those involved in Ca2+ oscillation. Further studies are necessary to identify the ion channels involved and to determine through which mechanism cAMP activates this unidentified channel. It is unlikely, however, that cAMP action is mediated through PKA, because PKA inhibitors did not block the forskolin-induced response as previously shown by Weiner and colleagues (11, 28).

In conclusion, the results are in accordance with the idea that cAMP augments membrane Na+ permeability, probably through nonselective cation channels on GT1-7 cells, thereby promoting GnRH release.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research (10670071 and 13680883) from the Japan Society for the Promotion of Science.

Abbreviations: [Ca2+]i, Intracellular Ca2+ concentration; [Ca2+]o, extracellular Ca2+ concentration; CNG, cyclic nucleotide-gated; LCD, l-cis-diltiazem; [Na+]i, intracellular Na+ concentration; [Na+]o, extracellular Na+ concentration; PKA, protein kinase A; Rp-cAMPS, adenosine 3',5'-cyclic monophosphothioate Rp-isomer; TTX, tetrodotoxin.

Received May 13, 2002.

Accepted for publication July 11, 2002.


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 Introduction
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 Results
 Discussion
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