Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture

Yusaku Nakajima, Maki Uchiyama, Yasumasa Shirai, Yasuo Sakuma, and Masakatsu Kato

Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 µM) increased intracellular calcium concentration ([Ca2+]i) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+]i. These results suggest that ACh increases [Ca2+]i in FSCs by activating phospholipase C, presumably through activation of M1 receptors. The rise in [Ca2+]i could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.

cholinergic modulation; paracrine mechanism; calcium imaging; M1 receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FOLLICULOSTELLATE CELLS (FSCs) in the anterior pituitary do not secrete any known traditional pituitary hormones by themselves but exert inhibitory effects on the secretion of growth hormone (GH), prolactin (PRL), luteinizing hormone, or adrenocorticotropic hormone (ACTH) (1, 18). FSCs share similar characteristics with glial cells and, to some extent, with immune cells. For example, the glial protein S-100 is a specific marker for FSCs in the anterior pituitary (15). FSCs produce basic fibroblast growth factor (6) and possess nitric oxide synthase (4) as well as interleukin-6 (19). Several recent reports suggest that FSCs modulate hormone secretion through a paracrine mechanism within the anterior pituitary (17).

To date, it is poorly understood how the activity of FSCs is regulated. In the present study, we focused on the role played by ACh. ACh is synthesized by corticotrophs and by a subpopulation of lactotrophs in the anterior pituitary (3). Cholinergic modulation of GH and PRL release in the anterior pituitary has been demonstrated (2, 11, 21). Besides, muscarinic receptors have been found in rat anterior pituitary cells; however, their precise localization remains to be resolved (14, 16). From these lines of evidence, we hypothesized that ACh released from the endocrine pituitary cells could increase intracellular calcium concentration ([Ca2+]i) through muscarinic receptors on the FSCs and cause a variety of Ca2+-dependent cellular processes. A major question here is whether FSCs express functional cholinergic receptors and increase [Ca2+]i in response to ACh. To test this possibility, we examined the effect of ACh on primary-cultured FSCs by means of Ca2+ imaging.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary culture of rat anterior pituitary cells. The anterior pituitary was excised from male Wistar rats (250-300 g body wt) after decapitation by a guillotine. The pituitary was minced and incubated in 10 ml of Dulbecco's phosphate-buffered saline (-) (PBS, Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% trypsin (type III, Sigma, St. Louis, MO) and 0.25% collagenase (type I, Sigma) for 20 min at 37°C with a gentle stirring. After the incubation, pieces of the pituitary were transferred into 10 ml of PBS supplemented with 0.1 mg/ml trypsin inhibitor (type II, Sigma) and 4 U/ml deoxyribonuclease I (Sigma) and were dispersed by triturating with a 5-ml plastic pipette for 5 min. After washing with PBS, the cells were plated on poly-L-lysine-coated glass coverslips and incubated in MEM (Nissui Pharmaceutical) supplemented with 2 mM L-glutamine, 4% normal rat serum, and 0.2% BSA (fraction V, Sigma) for 3-5 days at 37°C in a humidified atmosphere of 5% CO2-95% air. Identification of FSCs is described in RESULTS.

Measurement of [Ca2+]i. Details of the imaging technique and superfusion system have been described previously (9). In brief, cultured cells were loaded by incubation with 1 µM Fura PE-3 AM (TefLabs, Austin, TX) for 60 min at 37°C. The coverslip was placed in a small superfusion chamber on the stage of a Nikon Diaphot microscope. [Ca2+]i was recorded by using the QuantiCell 700 system (Applied Imaging, Sunderland, UK). The cells were illuminated alternately at 340-nm 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 6 s. Ratios were converted to Ca2+ concentrations by the following equation (7): [Ca2+]i = kdbeta [(R - Rmin)/(Rmax - R)], where kd is the dissociation constant for Fura PE-3 Ca2+, R is the ratio, Rmin and Rmax are the ratio values of Fura PE-3 at zero and saturating [Ca2+]i, respectively, and beta  is the ratio of fluorescence at 380 nm for Fura PE-3 in saturating and zero [Ca2+]i.

Superfusion was performed with a control solution containing (in mM): 137.5 NaCl, 5 KCl, 2.5 CaCl2, 0.8 MgCl2, 0.6 NaHCO3, 10 glucose, 20 HEPES, and 0.1% BSA, and the pH was adjusted to 7.4 with NaOH. Nominal Ca2+-free solution was prepared by replacing Ca2+ with Mg2+ in the control solution. This solution contained 10-20 µM Ca2+ (10). Excess K+ solution was prepared by replacing Na+ with K+ in the control solution. The cells were continuously superfused at 37°C throughout the experiment, and the flow rate was ~1 ml/min. All drugs were applied through superfusion.

Drugs. The following drugs were purchased from Wako Chemicals (Osaka, Japan): ACh, thapsigargin, U-73343, U-73122, and pirenzepine. Nifedipine, neomycin, and ryanodine were purchased from Sigma. SKF-96365 was from Biomol Research Laboratories (Plymouth Meeting, PA). Gadolinium chloride was from Nacalai Tesque (Kyoto, Japan).

Statistical analysis. ACh-induced maximum changes in [Ca2+]i (Delta [Ca2+]i) were considered significant when they exceeded 10 nM from basal [Ca2+]i. At least three independent experiments were made to draw conclusions. In each experiment, ACh was applied twice to the same cells with a 30-min wash period. The peak values of Delta [Ca2+]i in response to the first application and to the second challenge were designated as S1 and S2, respectively, and the S2-to-S1 ratio (S2/S1) was determined. In some experiments, the effects of drugs introduced between the initial and second applications of ACh were evaluated by frequency histograms constructed for S2/S1. The data were expressed as means ± SD. A paired t-test was used for statistical analysis. The significance level was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of FSCs in the cultured cells. It has been demonstrated that FSCs have distinctive morphological characteristics in the pituitary (15). In primary culture, FSCs extended thin cytoplasm (Fig. 1A, arrows). The thin cytoplasmic extension often formed processes in FSCs in culture. FSCs were confirmed by immunocytochemical staining of S-100 protein, a specific marker of FSCs in the anterior pituitary (Fig. 1B, arrows). Ca2+ images are shown in pseudo-color (Fig. 1, C-E). FSCs responded to 300 nM ACh (Fig. 1D) but not to 50 mM K+ (Fig. 1E). On the other hand, non-FSCs responded to 50 mM K+ but not to 300 nM ACh.


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Fig. 1.   Ca2+ imaging of folliculostellate cells (FSCs). Arrows indicate FSCs separated from endocrine cells in A-E. A: phase contrast microscopy of FSCs and other pituitary cells. B: same group of cells shown in immunofluorescent microscopy with Cy3-labeled anti-S-100 and FITC-labeled anti-growth hormone antibodies. C: Ca2+ images of the same cells under resting condition. D: Ca2+ images of the same cells observed with 300 nM ACh. FSCs increased [Ca2+]i. E: Ca2+ images of the cells observed with 50 mM K+. FSCs hardly responded in contrast to other pituitary cells. It should be noted that the FSCs located at upper right aggregated with endocrine cells. This disturbed the Ca2+ signal of FSCs in response to ACh and to excess K+. In this case, we excluded the response of FSCs from the data. Scale bars in A-E, 20 µm. Color bar at bottom indicates the intracellular calcium concentration ([Ca2+]i), 0-1,000 nM.

The time course of the responses is shown in Fig. 2, A and B. FSCs responded weakly to 50 mM K+ compared with non-FSCs. The average of peak Delta [Ca2+]i induced by 50 mM K+ was 42 ± 28 nM (n = 59 in 3 independent experiments) in FSCs and 646 ± 234 nM (n = 85 in 3 independent experiments) in non-FSCs (Fig. 2C). Additionally, non-FSCs did not respond to 300 nM ACh in these 85 cells. In the present experiment, therefore, cells were designated as FSCs if they showed distinctive morphology and relative unresponsiveness to excess K+ (peak Delta [Ca2+]i, <100 nM). We tested the validity of these criteria in 85 cells and found that 84 of them were stained by antisera to S-100 protein.


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Fig. 2.   Responses to excess K+ in FSCs and non-FSCs. A: FSCs responded to 300 nM ACh but only weakly to excess K+. B: non-FSCs responded to excess K+ but not to 300 nM ACh. C: excess K+-induced peak change in [Ca2+]i (Delta [Ca2+]i) in FSCs and non-FSCs. Each column indicates the mean ± SD of peak Delta [Ca2+]i (n = 59 in FSCs, n = 85 in non-FSCs).

ACh-induced rise in [Ca2+]i. Representative responses to various concentrations of ACh are shown (Fig. 3A). FSCs responded to ACh in a concentration-dependent manner at concentrations between 30 and 3,000 nM (Fig. 3B). No cells responded to 10 nM ACh. However, 30 nM and 100 nM ACh elicited responses in a substantial portion of the cells examined. Among 45 cells examined, 28 cells responded to 30 nM ACh with peak Delta [Ca2+]i of 53 ± 75 nM; 37 cells among 42 responded to 100 nM ACh with peak Delta [Ca2+]i of 120 ± 111 nM. All cells examined responded to ACh at a concentration of >= 300 nM. The averages of peak Delta [Ca2+]i induced by 300, 1,000, and 3,000 nM ACh were 229 ± 177, 301 ± 200, and 384 ± 235 nM, respectively.


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Fig. 3.   ACh-induced rise in [Ca2+]i. A: representative responses to different concentrations of ACh. ACh was applied for 3 min as indicated by horizontal bar. B: values are means ± SD of peak Delta [Ca2+]i in no. of cells indicated beside each symbol from 3-7 independent experiments.

ACh elicited a rapid initial increase of [Ca2+]i followed by a sustained phase (Fig. 4A). Similar responses were replicated by two sequential applications of ACh at an interval of 30 min. The peak Delta [Ca2+]i of the first application (211 ± 115 nM) was similar to that in the second application (194 ± 115 nM). The histogram for the frequency distribution of S2/S1 was ~1.0, with an average of 0.941 ± 0.327 (n = 90 in 4 independent experiments) as shown in Fig. 4B. This result was used as a control for the following experiments.


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Fig. 4.   Responses to two sequential applications of ACh. A: cells were stimulated twice with 300 nM ACh. A second stimulation, performed after a 30-min wash period, produced a similar response. ACh increased [Ca2+]i with an initial rapid rise followed by a relatively sustained increase. B: the population of the ratio of S2 to S1 (S2/S1) distributed ~1.0, and the average was 0.941 ± 0.327 (n = 90 in 4 independent experiments).

Muscarinic receptor antagonists. Two muscarinic antagonists, atropine (10 nM) and pirenzepine (10 nM), were applied 3 min before a second application of ACh. Typical examples are shown in Fig. 5. Both atropine (n = 54) and pirenzepine (n = 49) abolished the ACh-induced response in all cells examined. The responses recovered after a 30-min washout.


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Fig. 5.   Inhibition by muscarinic antagonists. A: atropine (10 nM) completely blocked the response (n = 54 in 4 independent experiments). B: pirenzepine (10 nM) also completely blocked the response (n = 49 in 4 independent experiments). These responses were reversed after a 30-min wash.

Ca2+-free medium and Ca2+ channel blockers. To determine whether an influx of extracellular Ca2+ or mobilization of intracellular Ca2+ contributed to changes in [Ca2+]i, Ca2+-free solution replaced the control solution 3 min before the second application of ACh. The Ca2+-free solution did not affect the initial rise in [Ca2+]i but reversibly suppressed the late sustained increase (Fig. 6A). The peak Delta [Ca2+]i was 152 ± 86 nM in the control and 134 ± 55 nM in the Ca2+-free solution. The frequency distribution of S2/S1 remained similar to that of control, and the average was 1.002 ± 0.436 (n = 53 in 5 independent experiments), as shown in Fig. 6B.


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Fig. 6.   Effect of Ca2+-free medium. A: removal of extracellular Ca2+ did not affect the initial rise of [Ca2+]i but reversibly suppressed the following sustained increase. B: distribution of S2/S1 of peak Delta [Ca2+]i was similar to control, and the average was 1.002 ± 0.436 (n = 53 in 5 independent experiments).

The effects of Ca2+ channel blockers are shown in Fig. 7. All blockers were applied to the bath 3 min before the second application of ACh. Gadolinium (100 µM), a blocker of store-operated Ca2+ channels, did not affect the initial rise of [Ca2+]i but reversibly suppressed the following sustained increase (Fig. 7A). The values of peak Delta [Ca2+]i were 220 ± 105 nM in the control and 239 ± 143 nM with gadolinium. S2/S1 distributed similarly to the control, with an average of 1.068 ± 0.508 (n = 54 in 4 independent experiments) as shown in Fig. 7C. SKF-96365 (30 µM), a blocker of store-operated Ca2+ channels, also reversibly suppressed the sustained increase that followed (Fig. 7B). It was noted that the initial rise was partially inhibited (P < 0.01). The peak Delta [Ca2+]i was 244 ± 100 nM in the control and 181 ± 98 nM after SKF-96365. The distribution of S2/S1 shifted to the left, with an average of 0.813 ± 0.399 (n = 59 in 4 independent experiments) as shown in Fig. 7D. An L-type Ca2+ channel blocker, nifedipine (10 µM), had no effect. The peak Delta [Ca2+]i was 288 ± 206 nM in the control and 318 ± 214 nM with nifedipine. No difference was detected in the distribution of S2/S1 (n = 47 in 3 independent experiments).


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Fig. 7.   Effects of gadolinium and SKF-96365. A: gadolinium did not block the initial rise but reversibly suppressed the following sustained increase. B: SKF-96365 partially inhibited an initial rise and reversibly blocked the following sustained increase. C: distribution of S2/S1 of the peak Delta [Ca2+]i was similar to control, and the average was 1.068 ± 0.508 (n = 54 in 4 independent experiments). D: distribution of S2/S1 of the peak Delta [Ca2+]i was shifted to the left, and the average was 0.813 ± 0.399 (n = 59 in 4 independent experiments).

The contribution of intracellular Ca2+ stores was examined by experiments with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase (8). Thapsigargin (1 µM) was applied ~22 min before and throughout the second application of ACh. Thapsigargin alone caused a small and transient increase in [Ca2+]i (Fig. 8). Application of ACh, repeated after a 30-min interval, failed to induce any response in the presence of thapsigargin (n = 60 in 3 independent experiments).


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Fig. 8.   Effect of thapsigargin. After first application, 1 µM thapsigargin was introduced as indicated by horizontal solid bar, which caused a slow increase of [Ca2+]i in FSCs. The second application of ACh, performed 60 min from the first application, did not increase [Ca2+]i (n = 60 in 3 independent experiments).

Phospholipase C inhibitors and ryanodine. Inhibitors of phospholipase C, neomycin and U-73122, were used to examine whether ACh activates phospholipase C in the FSCs. Prior superfusion with neomycin (3 mM) for 1 h inhibited the initial rise in [Ca2+]i to 149 ± 115 nM from the control of 246 ± 135 nM (P < 0.01), which was partially reversed as shown in Fig. 9A. The distribution of S2/S1 was shifted to the left with an average of 0.617 ± 0.419 (n = 61 in 4 independent experiments) as shown in Fig. 9D. U-73343 (10 µM, 50-min prior superfusion), an inactive analog of U-73122, did not affect the response (Fig. 9B). The peak Delta [Ca2+]i was 179 ± 117 nM in the control and 180 ± 120 nM with U-73343. No difference was detected in the distribution of S2/S1 as shown in Fig. 9E (n = 63 in 4 independent experiments). U-73122 (10 µM, 50-min prior superfusion), an inhibitor of phospholipase C, suppressed the response to 96 ± 47 nM from a control of 177 ± 66 nM (P < 0.01), which was reversed as shown in Fig. 9C. The distribution of S2/S1 was shifted to the left, and the average was 0.570 ± 0.230 (n = 48 in 3 independent experiments) as shown in Fig. 9E.


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Fig. 9.   Effects of phospholipase C inhibitors. A: neomycin (3 mM), a blocker of phospholipase C, was introduced 1 h before ACh application and continuously superfused as indicated by horizontal solid bar. The exact time of application is indicated in parenthesis. Neomycin inhibited the rise of [Ca2+]i. This inhibition was partially reversed. B: U-73343 (10 µM), structural analog of U-73122 without inhibitory action, was applied for 50 min, as indicated in parenthesis, and washed with a control solution for 10 min before second ACh application. U-73343 did not affect the response. C: U-73122 (10 µM), an inhibitor of phospholipase C, was applied in the same time course as U-73343, and it reversibly suppressed the rise in [Ca2+]i. D: distribution of S2/S1 of the peak Delta [Ca2+]i was shifted to the left by neomycin, and the average was 0.617 ± 0.419 (n = 61 in 4 independent experiments). E: distribution of S2/S1 of the peak Delta [Ca2+]i was shifted to the left by U-73122 with an average of 0.570 ± 0.230 (n = 48 in 3 independent experiments). The distribution was not affected by U-73343, and the average was 1.066 ± 0.403 (n = 63 in 4 independent experiments).

Ryanodine (20 µM) did not increase [Ca2+]i of FSCs and did not affect the peak Delta [Ca2+]i induced by ACh. The peak Delta [Ca2+]i was 233 ± 130 nM in the control and 227 ± 127 nM with ryanodine. No difference was detected in the distribution of S2/S1 (n = 47 in 4 independent experiments).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments demonstrated that ACh increased [Ca2+]i of FSCs in a concentration-dependent manner. Because this response was blocked completely by atropine and pirenzepine, the response is likely to be mediated by a muscarinic receptor. Pirenzepine is relatively specific to the M1 receptor subtype but also blocks M4 receptors with a similar potency (5). As we will discuss, the response of FSCs to ACh is likely to involve an activation of phospholipase C via Gq/11. Because M1 but not M4 receptors are coupled to Gq/11 (20), the present action of ACh is most likely mediated by the M1 receptor.

The initial rise in [Ca2+]i was not affected by the removal of extracellular Ca2+ but was completely blocked by an inhibitor of the endoplasmic reticulum Ca2+-ATPase, thapsigargin (8). These results indicate that the initial rise in [Ca2+]i was caused by mobilization of Ca2+ from internal stores. However, the late sustained increase in [Ca2+]i depended completely on the presence of extracellular Ca2+. This phase was almost completely blocked by gadolinium, which is known to block store-operated Ca2+ influx (12). Another inhibitor of store-operated Ca2+ entry, SKF-96365 (13), also caused a partial suppression. Thus the late sustained phase is probably due to an influx of extracellular Ca2+. Involvement of voltage-gated Ca2+ channels, however, is unlikely, because FSCs are not excitable cells and express few of these channels. Indeed, FSCs weakly increased the [Ca2+]i in response to 50 mM K+. In the present study, nifedipine, a blocker of the L-type Ca2+ channel, did not affect the response at all. Based on the observation that two different inhibitors of phospholipase C, neomycin and U-73122, both suppressed the ACh-induced response, we propose that ACh activates phospholipase C and promotes production of D-myo-inositol 1,4,5-trisphosphate in the FSCs.

It should be noted, however, that we found that U-73343, an inactive analog of U-73122, inhibited the response to ACh when U-73343 was present simultaneously with ACh (data not shown). This nonspecific action of U-73343 may be due to competitive inhibition on ACh action, because the inhibition was reversed by increasing the concentration of ACh (our preliminary results). We did not further investigate this point but simply washed out U-73343 before ACh application and found that the 10-min wash completely removed the nonspecific action of U-73343. Therefore, we applied U-73122 in the same time course as U-73343, which must reveal a specific action of U-73122 to inhibit phospholipase C.

An involvement of ryanodine receptors is unlikely, because ryanodine did not increase [Ca2+]i of FSCs and did not affect the peak Delta [Ca2+]i induced by ACh.

In the anterior pituitary, ACh is synthesized by corticotrophs and by a small proportion of lactotrophs (3). ACh may be secreted alone or with ACTH and PRL. To date, ACh has been considered to act on somatotrophs and lactotrophs through their M2 receptors to decrease the secretion of GH and PRL (2). The present findings indicate that FSCs are another target of ACh action in the anterior pituitary. The rise in [Ca2+]i in FSCs may stimulate a variety of Ca2+-dependent cellular processes, including synthesis and release of bioactive substances, which in turn act on the endocrine cells.

In conclusion, ACh increased [Ca2+]i of FSCs by activating phospholipase C through M1 receptor activation. The present results, taken together with previous findings, suggest that ACh could function as a paracrine factor acting on FSCs in the anterior pituitary.


    ACKNOWLEDGEMENTS

We are grateful to Professor V. N. Luine for critical reading of the manuscript.


    FOOTNOTES

This work was supported in part by Grants-in-Aid 08680872, 10670071, and 10480227 from the Ministry of Education, Science, Sports and Culture of Japan.

Address for reprint requests and other correspondence: M. Kato, Dept. of Physiology, Nippon Medical School, Tokyo 113-8602, Japan (E-mail: mkato{at}nms.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 July 2000; accepted in final form 15 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 280(4):E608-E615
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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