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Endocrinology Vol. 143, No. 2 445-455
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Cell-Type Specific Messenger Functions of Extracellular Calcium in the Anterior Pituitary

Dragoslava Zivadinovic, Melanija Tomic, Davy Yuan and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Dr. Stanko Stojilkovic, Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca2+-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca2+ concentration ([Ca2+]e) induced transient elevations in the intracellular Ca2+ concentration ([Ca2+]i), and elevations in [Ca2+]e above physiological levels decreased [Ca2+]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca2+]i responses depended on the [Ca2+]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca2+ influx. Changes in [Ca2+]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca2+]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca2+]e independently of the status of voltage-gated Ca2+ influx. [Ca2+]e modulated GH secretion in a dose-dependent manner, with EC50 values of 0.75 and 2.25 mM and minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca2+]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca2+]e in the control of Ca2+ signaling and secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCIUM ACTS AS an extracellular and intracellular messenger to regulate a diverse array of cellular functions. The intracellular messenger functions of calcium, including control of exocytosis, are well defined (1). There has also been significant progress in understanding the extracellular messenger functions of Ca2+. Recently, cloned G protein-coupled calcium-sensing receptors (CaRs) (2) provide the molecular mechanism through which parathyroid cells and several other cell types detect small changes in the extracellular calcium concentration ([Ca2+]e) (3, 4, 5). These receptors are critical in maintaining [Ca2+]e relatively constant and may also play a role in the control of other functions (4). Calcium is not an exclusive agonist for these receptors; Mg2+ and other divalent cations can also activate CaRs and are frequently used to show the specificity of CaRs (6). Together with pheromone, taste, and putative odorant receptors, CaRs belong to group II of G protein-coupled receptors that probably operate through a pertussis toxin-sensitive signaling pathway. Activation of CaRs can also lead to stimulation of phospholipases C, A2, and D (5, 6).

Sensing [Ca2+]e is not limited to CaRs. It has been known for many years that changes in [Ca2+]e interfere with the gating properties of plasma membrane channels, which, in turn, affect spontaneous and receptor-controlled changes in the intracellular calcium concentration ([Ca2+]i). In general, the changes in [Ca2+]e could have nonspecific effects on cell excitability, e.g. by changing the ionic strength of the medium, the screening of the plasma membrane surface charge is affected. Also, the change in osmolality can induce mechanical stimulus on the membrane. Elevated [Ca2+]e usually decreases the excitability of cells by closing the gates of voltage-gated calcium channels (VGCCs), whereas lowered [Ca2+]e has the opposite effect. This leads to neuronal and muscle hyperexcitability, as was observed in patients with hypoparathyroidism (for review, see Ref. 7). More recent studies have identified several plasma membrane channels, including cyclic nucleotide-gated channels, human ether-a-go-go-like potassium channels, and sodium channels, as capable of detecting changes in [Ca2+]e and responding by changing the excitability of cells and the accompanying voltage-gated Ca2+ influx (VGCI) (8, 9, 10). Furthermore, VGCI influences the basal activity of several enzymes involved in intracellular signaling, including nitric oxide synthase, guanylyl cyclase, adenylyl cyclase, and phospholipase D (11, 12, 13). Thus, plasma membrane channels involved in the spontaneous and receptor-controlled electrical activity may also serve as Ca2+ sensors independently of CaRs and with a potential to affect [Ca2+]i and several other intracellular messengers.

Mouse and rat pituitary cells, AtT-20 immortalized cells, and human pituitary adenomas express CaRs (14, 15). A majority of secretory cell types also exhibit spontaneous firing of action potentials (APs), which promote Ca2+ influx through VGCCs and generate [Ca2+]i signals of sufficient amplitude to trigger hormone secretion (16, 17, 18, 19). Spontaneous excitability of pituitary cells is up- and down-regulated by several G protein-coupled receptors, including GHRH, CRF, pituitary adenylate cyclase-activating polypeptide, somatostatin, and dopamine (20, 21). However, potential extracellular messenger functions of calcium in the control of [Ca2+]i signaling and hormone secretion have been incompletely characterized. In this study we systematically examined the effects of lowering and elevating [Ca2+]e on [Ca2+]i and hormone secretion in three anterior pituitary cell types: somatotrophs, lactotrophs, and gonadotrophs. The results indicate that [Ca2+]e is not critical in controlling basal gonadotropin secretion, but plays an important role in spontaneous excitability and VGCI-dependent basal hormone secretion in somatotrophs and lactotrophs. In somatotrophs, but not lactotrophs, [Ca2+]e also stimulated exocytosis independently of [Ca2+]i, probably by activating CaRs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and treatments
Experiments were performed on anterior pituitary cells from normal postpubertal female Sprague Dawley rats obtained from Taconic Farms (Germantown, NY). Pituitary cells were dispersed as described previously (22) and cultured as mixed cells or enriched lactotrophs, somatotrophs, and gonadotrophs in medium 199 containing Earle’s salts, sodium bicarbonate, 10% heat-inactivated horse serum, and antibiotics. A two-stage Percoll discontinuous density gradient procedure (22) was used to obtain enriched lactotroph and somatotroph populations. In enriched lactotroph populations, lactotrophs were further identified by the addition of TRH. Gonadotrophs were initially identified by their cell type-specific morphology and subsequent to experimentation by the addition of GnRH, which stimulates baseline [Ca2+]i oscillations only in these cells (20).

Hormone secretion was monitored using cell column perfusion experiments, as previously described (23). Briefly, 1.5 x 107 cells were incubated with preswollen Cytodex-1 beads in 60-mm petri dishes for 2 d. The beads were then transferred to 0.5-ml chambers and perfused with Hanks’ medium 199 containing 20 mM HEPES, 0.1% BSA, and penicillin/streptomycin (1% of 100-fold concentrated) for 2 h at a flow rate of 0.8 ml/min and at 37 C to establish stable basal secretion. During the experiment, 1-min fractions were collected, stored at -20 C, and later assayed for GH, PRL, and LH contents using RIA. All reagents and standards were provided by the National Pituitary Agency and Dr. Parlow (Harbor-University of California-Los Angeles Medical Center, Torrance, CA).

Measurements of intracellular calcium ion concentration
For [Ca2+]i measurements, cells were incubated in Hanks’ medium 199 supplemented with 2 µM fura 2-AM (Molecular Probes, Inc., Eugene, OR) at 37 C for 60 min. Coverslips with cells were then washed with Krebs-Ringer buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. All recordings were made at room temperature in perfused pituitary cells at flow rates of 0.8 ml/min or higher. The ratio of light intensities, F340/F380, which reflects changes in Ca2+ concentration, was followed in several single cells simultaneously.

Inositol phosphate and cAMP measurements
Cells were plated at a density of 1 million/well in four-well plates. They were labeled with [3H]inositol (20 µCi/ml) for 24 h in inositol-free DMEM containing BSA (1 mg/ml), FCS (2%), and penicillin/streptomycin. The following day, cells were washed with inositol-free and Ca2+-containing medium 199 medium and preincubated for 30 min at 37 C in Ca2+-containing or Ca2+-deficient medium 199 supplemented with 10 mM LiCl to inhibit the phosphatases. This was followed by an additional 30-min incubation at 37 C in medium 199 adjusted with the desired Ca2+ concentration and in the presence of 10 mM LiCl. Incubation was stopped by the addition of ice-cold 10% perchloric acid. Extraction of InsP2 and InsP3 was performed by Dowex anion exchange chromatography, using Poly-Prep chromatography columns (Bio-Rad Laboratories, Inc., Richmond, CA). InsP2 and InsP3 were eluted with a mixture of 1 M ammonium formate and 0.1 M formic acid, and samples were counted on an LS 5600 scintillation counter (Beckman Coulter, Inc., Palo Alto, CA).

For cAMP measurements, cells (1 million/well) were plated in 24-well plates in serum-containing medium 199 and incubated overnight at 37 C under 5% CO2-air and saturated humidity. Before experiments, medium was removed, and cells were washed with serum-free medium 199 and stimulated at 37 C for 60 min. cAMP was measured in medium and dialyzed cells as previously described (11), using specific antisera provided by Albert Baukal (NICHD, Bethesda, MD). The antisera used in our RIAs are highly specific for cAMP, i.e. there was no cross-reactivity at 100 pmol or lower concentrations with cGMP. All samples were diluted two to five times, and cyclic nucleotide concentrations were estimated using standard curves ranging from 5 fmol to 1 pmol. The results shown are the sum of cAMP in medium and cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of lowered [Ca2+]e on basal [Ca2+]i and hormone secretion
In perfused pituitary cells, a decrease in [Ca2+]e from 2 to 0.05 mM induced a rapid and high amplitude spike [Ca2+]i response in somatotrophs and lactotrophs (on response), whereas gonadotrophs responded with either a small amplitude [Ca2+]i response or insensitivity. Figure 1Go shows the averaged [Ca2+]i response in the three cell types, each derived from at least 20 individual cells. The rise in [Ca2+]i was transient, followed by an exponential-like decrease to levels below the basal. The return of [Ca2+]e to initial (2 mM) levels initiated another spike [Ca2+]i response in somatotrophs and lactotrophs (off response), followed by a gradual decline to basal levels. The on and off [Ca2+]i responses were observed in spontaneously active and quiescent somatotrophs and lactotrophs. Figure 2Go illustrates the on response in single somatotrophs, two spontaneously active cells exhibiting high (A) and low (B) amplitude and frequency of [Ca2+]i spiking, and one quiescent cell (C).



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  		Figure 1. Effects of depletion and repletion of extracellular calcium on [Ca2+]i in somatotrophs, lactotrophs, and gonadotrophs. Tracings shown are means from at least 20 records. At the beginning and the end of experiments, recording was performed every second, and during the residual times recording was performed every 20 sec. Dotted lines illustrate the initial basal [Ca2+]i levels recorded at 2 mM [Ca2+]e. The first spike in [Ca2+]i is termed the on response, and the second the off response.

 


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  		Figure 2. Effects of depletion of extracellular calcium on [Ca2+]i in single somatotrophs; representative traces are shown. A and B, Spontaneously active somatotrophs. C, A quiescent somatotroph.

 
Two experiments were performed to exclude possible nonspecific effects of [Ca2+]e depletion. First, cells were perfused with Ca2+-deficient medium supplemented with 6 mM NaCl (in addition to already containing 120 mM), to maintain the same ionic strength (defined as 1/2{Sigma}cizi2) (7) as in 2 mM Ca2+-containing medium and therefore to maintain the same membrane surface charge screening throughout the experiment. The on and off [Ca2+]i responses recorded in these experimental conditions were comparable to those shown in Figs. 1Go and 2Go. Second, the addition of 6 mM glucose to Ca2+-deficient medium did not affect the pattern of [Ca2+]i responses, further indicating that the change in osmolality is not the cause for on and off [Ca2+]i responses (data not shown).

Effects of [Ca2+]e on basal hormone secretion were also analyzed in perfused pituitary cells (Fig. 3Go). Depletion of [Ca2+]e from 2 to 0.05 mM and its repletion stimulated GH and PRL release, whereas LH release was not affected. The on PRL response was followed by a rapid inhibition of secretion below the basal levels (Fig. 3BGo), whereas the on GH response was accompanied by a sustained stimulation of secretion that lasted for 15–40 min (Fig. 3AGo). Thus, stimulation of GH secretion was also observed during the times when [Ca2+]i was reduced below the basal level (Fig. 1Go vs. Fig. 3Go).



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  		Figure 3. Effects of depletion and repletion of extracellular calcium on GH (A), PRL (B), and LH (C) secretion in perfused pituitary cells. Dotted lines illustrate the initial basal hormone secretion in cells perfused with 2 mM [Ca2+]e-containing medium.

 
In other cell types, CaRs are coupled to PLC, and their activation leads to an InsP3-induced rise in [Ca2+]i. As the patterns of [Ca2+]i responses observed during depletion and repletion of [Ca2+]e (Fig. 1Go) resemble those induced by calcium-mobilizing endothelin-1 (ET-1) in the same cells (23), we tested the hypothesis that spike [Ca2+]i responses in pituitary cells are generated by mobilization of intracellular Ca2+. Cells were treated with thapsigargin, a blocker of endoplasmic reticulum Ca2+-adenosine triphosphatase, for 30 min. This treatment effectively depleted the intracellular Ca2+ pool, as indicated by the inability of calcium ionophore ionomycin to elevate [Ca2+]i, but it did not abolish [Ca2+]i responses induced by the depletion of [Ca2+]e (Fig. 4Go). The amplitudes of [Ca2+]i responses were comparable in controls and thapsigargin-treated cells, further arguing against the hypothesis that activation of capacitative calcium entry accounts for [Ca2+]i responses.



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  		Figure 4. Independence of [Ca2+]e depletion-induced [Ca2+]i response on intracellular calcium stores in somatotrophs. [Ca2+]e depletion- and ionomycin-induced [Ca2+]i responses in control cells (A) and cells treated for 30 min with 10 µM thapsigargin, a blocker of endoplasmic reticulum Ca2+-adenosine triphosphatase (B). All traces shown are means derived from at least 10 records.

 
On the other hand, when somatotrophs were continuously perfused with nifedipine, a blocker of L-type Ca2+ channels, depletion and repletion of [Ca2+]e did not induce on and off [Ca2+]i responses (Fig. 5AGo). Lactotrophs were also insensitive to changes in [Ca2+]e when perfused with nifedipine-containing medium (not shown). In further experiments cells were perfused with Ca2+-containing medium in the presence or absence of nifedipine and subsequently with Ca2+-depleted medium with or without nifedipine. During perfusion with Ca2+-depleted medium, 1 µM ionomycin was added to estimate the intracellular Ca2+ content. As shown in Fig. 4BGo, no significant differences in the amplitude of ionomycin-induced [Ca2+]i responses were observed in cells bathed in nifedipine-containing and -free medium. These results suggest that a transient enhancement of VGCI, rather than the mobilization of intracellular Ca2+, accounts for the on and off [Ca2+]i responses.



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  		Figure 5. Dependence of the [Ca2+]e depletion-induced [Ca2+]i response on VGCI in somatotrophs. A, Lack of effect of [Ca2+]e depletion and repletion on [Ca2+]i in somatotrophs continuously perfused with 1 µM nifedipine. B, Effects of ionomycin on [Ca2+]i in cells bathed in Ca2+-depleted medium in the presence (upper trace) and absence (bottom trace) of nifedipine. All traces shown are means derived from at least 20 records.

 
The on and off [Ca2+]i responses were not observed when Ca2+ was substituted with equimolar concentrations of Mg2+. Figure 6AGo illustrates the absence of the on [Ca2+]i response in spontaneously active (upper trace) and quiescent (bottom trace) somatotrophs. As expected, spontaneous [Ca2+]i fluctuations in somatotrophs were abolished in Ca2+-deficient and Mg2+-containing medium, confirming their dependence on VGCI. These results indicate that there is a transient facilitation of VGCI and a sufficient Ca2+ gradient across the plasma membrane during [Ca2+]e depletion to generate the on [Ca2+]i response. In accordance, the amplitude and duration of [Ca2+]i response decreased progressively as the flow rate of [Ca2+]e increased from 0.8 to 2.5 ml/min (Fig. 6BGo). Furthermore, the on [Ca2+]i response was completely abolished when EGTA was added by pipette, rapidly reducing [Ca2+]e to about 100 nM, whereas the Ca2+-mobilizing ET-1 action was not affected (Fig. 6CGo).



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  		Figure 6. Characterization of the on spike [Ca2+]i response in somatotrophs. A, Effects of substitution of extracellular Ca2+ with equimolar Mg2+ on [Ca2+]i in single cells. B, Effect of flow rates on 50 µM extracellular Ca2+-induced on [Ca2+]i response. C, The lack of an on spike [Ca2+]i response upon rapid addition of EGTA. To demonstrate that the Ca2+-mobilizing pathway is still operative, cells were stimulated with ET-1. In B and C, traces shown are means from at least 20 records.

 
In parallel to the effects of EGTA on the [Ca2+]i response (Fig. 6CGo), GH secretion was inhibited in cells perfused with EGTA-containing medium (Fig. 7AGo). However, the level of inhibition was smaller than that observed during perfusion of cells with 2-mM Ca2+-containing medium in the presence of nifedipine. In a further parallel with [Ca2+]i measurements, depletion and repletion of [Ca2+]e in the presence of nifedipine did not induce on and off LH and PRL responses (Fig. 7BGo). However, depletion of [Ca2+]e led to a sustained increase in GH secretion, whereas PRL and LH secretion was not affected. As EGTA and nifedipine inhibited spontaneous VGCI and abolished on and off [Ca2+]i responses (Fig. 5Go), these results indicate that somatotrophs also respond to depletion of [Ca2+]e with stimulated GH secretion uncoupled to VGCI.



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  		Figure 7. VGCI independence of the effect of lowered [Ca2+]e on GH secretion. A, Comparison of the effects of 1 µM nifedipine and 2 mM EGTA on GH secretion. B, Effects of lowered [Ca2+]e on GH, PRL, and LH secretion in cells with inhibited VGCI.

 
Effects of elevated [Ca2+]e on basal [Ca2+]i and hormone secretion
Elevated [Ca2+]e was also an effective stimulus for changing the pattern of [Ca2+]i signaling and hormone secretion in pituitary cells. As shown in Fig. 8Go, elevation of [Ca2+]e in perfused pituitary cells from 2 to 5 mM was followed by a rapid decrease in [Ca2+]i in somatotrophs and lactotrophs and had practically no effect on gonadotrophs. The on [Ca2+]i response regularly seen during depletion of [Ca2+]e was never observed during elevation of [Ca2+]e. However, the secondary rise in [Ca2+]i was preserved in both somatotrophs and lactotrophs (indicated by dotted lines in Fig. 8Go).



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  		Figure 8. Effects of elevated [Ca2+]e on the [Ca2+]i response in somatotrophs, lactotrophs, and gonadotrophs. Traces shown are means from at least 20 records. Dotted lines illustrate initial [Ca2+]i levels.

 
Single cell calcium measurements revealed that none of the quiescent somatotrophs (n = 48) showed an increase in [Ca2+]i during the elevation of [Ca2+]e from 2 to 5 mM. In spontaneously active somatotrophs, 5 mM Ca2+ silenced both low frequency (Fig. 9Go, a trace) and high frequency (b and c traces) spiking cells. The partial recovery of spiking was observed in a fraction of cells (a and b traces). In quiescent cells, 5 mM [Ca2+]e did not change [Ca2+]i (d and e traces), whereas ET-1 induced a typical [Ca2+]i response in the same cells (e trace). Elevation in [Mg2+]e to 5 and 10 mM in the presence of 2 mM [Ca2+]e also silenced spontaneously active cells and was without effect in quiescent cells (not shown). Similar effects of elevated [Ca2+]e were observed in lactotrophs. As shown in Fig. 10Go, in a fraction of cells spontaneous [Ca2+]i transients were completely abolished (traces a and b), whereas in others baseline [Ca2+]i and the frequency of oscillations were reduced (traces c and d). Thus, raised [Ca2+]e inhibited VGCI in somatotrophs and lactotrophs, but was ineffective in activating the Ca2+-mobilizing pathway.



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  		Figure 9. Effects of elevated [Ca2+]e on [Ca2+]i in single somatotrophs. Traces shown are representative of high and low frequency spiking cells (a–c) and quiescent cells (d and e). To demonstrate that the Ca2+-mobilizing pathway is operative in these cells, somatotrophs were stimulated with ET-1 (e trace).

 


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  		Figure 10. Effects of elevated [Ca2+]e on [Ca2+]i in single lactotrophs. Traces shown are representative of spontaneously active cells. In quiescent lactotrophs, no changes in [Ca2+]i were observed during elevation of [Ca2+]e.

 
In parallel to [Ca2+]i profiles in lactotrophs, elevated [Ca2+]e transiently inhibited PRL secretion, and the return of [Ca2+]e to its initial concentration stimulated it (Fig. 11BGo). LH secretion also paralleled [Ca2+]i profiles in single gonadotrophs, i.e. both were ineffective in elevating [Ca2+]e (Fig. 11CGo). In contrast to [Ca2+]i profiles in somatotrophs, elevated [Ca2+]e stimulated GH secretion. This stimulation was transient, and the return to 2 mM [Ca2+]e was followed by an additional spike GH response (Fig. 11AGo). These results indicate that in somatotrophs, elevated [Ca2+]e stimulates GH secretion without activating the Ca2+ mobilization pathway and with inhibition of the VGCI pathway.



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  		Figure 11. Effects of transient elevation in [Ca2+]e on GH (A), PRL (B), and LH (C) secretion. Dotted lines illustrate initial basal hormone secretion at 2 mM [Ca2+]e.

 
Dose dependence of [Ca2+]e on intracellular signaling and GH secretion
In further experiments, the dose dependence of [Ca2+]e on GH secretion and intracellular signaling was examined. In secretory studies, columns were initially perfused with 1.8 mM [Ca2+]e-containing medium and subsequently with a lower or higher [Ca2+]e. Figure 12AGo illustrates the normalized peak secretory response from 12 columns. Both lowering and raising [Ca2+]e stimulated GH secretion in a dose-dependent manner, with EC50 values of 0.75 and 2.25 mM, respectively. A minimum GH secretion was observed around the physiological concentration of [Ca2+]e (Fig. 12AGo).



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  		Figure 12. Dose-dependent effects of [Ca2+]e on intracellular signaling and secretion in pituitary cells. A, Dose dependence of extracellular Ca2+ on GH secretion. Secretion in controls (1.8 mM [Ca2+]e) is expressed as 100%. B, Effects of extracellular Ca2+ and ET-1 on inositol phosphate accumulation. Main panel, Cells were preincubated in 1.8 mM Ca2+-containing medium, followed by 30-min incubation in medium containing variable Ca2+ concentrations. Inset, Cells were preincubated in 50 µM Ca2+-containing medium. ET, 100 nM. C, Dose dependence of extracellular calcium on cAMP accumulation in cells without and with inhibited phosphodiesterases by addition of 1 mM IBMX.

 
In accordance with the lack of effect of lowering or raising [Ca2+]e on Ca2+ mobilization, inositol phosphate levels were not affected by changes in [Ca2+]e. Figure 12BGo (main panel) illustrates an experiment in which cells were preincubated in 1.8 mM [Ca2+]e-containing medium, followed by 30-min incubation in medium with variable [Ca2+]e. The inset illustrates an experiment with cells preincubated for 30 min in 50 µM [Ca2+]e-containing medium, followed by 30-min incubation in medium containing 5 mM [Ca2+]e. In both experimental conditions, 100 nM ET-1, a calcium-mobilizing agonist for somatotrophs and lactotrophs, induced significant increases in inositol phosphate accumulation. These results further strengthen the conclusion that [Ca2+]e stimulates GH secretion in a PLC-independent manner. It is also unlikely that cAMP is a messenger mediating [Ca2+]e-induced GH secretion. First, cAMP levels were not significantly affected by changing [Ca2+]e from 50 µM to 5 mM when phosphodiesterases were not inhibited (Fig. 12CGo). Second, a progressive dose-dependent effect of [Ca2+]e on cAMP levels was observed when cells were bathed in the presence of a nonselective phosphodiesterase inhibitor, in contrast to the bidirectional changes in GH secretion (Fig. 12AGo).

Finally, we examined the dependence of VGCI on the decrease in [Ca2+]e near to physiological concentrations in somatotrophs, lactotrophs, and gonadotrophs. A decrease in [Ca2+]e concentration from 2 to 0.6 mM induced an increase in the mean values of [Ca2+]i in mixed pituitary cells (Fig. 13AGo). Single cell analysis revealed that a decrease in [Ca2+]e from 1.3 to 0.7 mM initiated [Ca2+]i transients in quiescent somatotrophs (Fig. 13BGo, b trace) and lactotrophs (d trace) and increased the amplitude and/or frequency of [Ca2+]i transients in spontaneously active somatotrophs (a trace) and lactotrophs (c trace), whereas gonadotrophs were not affected (e trace).



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  		Figure 13. Dependence of excitability of pituitary cells on [Ca2+]e. A, Mean values of [Ca2+]i in cells perfused in 2 and 0.6 mM [Ca2+]e. B, Pattern of [Ca2+]i signaling in single somatotrophs (a and b traces), lactotrophs (c and d traces), and gonadotrophs (e trace) during a transient depletion of [Ca2+]e from 1.3 to 0.7 mM. Representative traces are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Extracellular Ca2+ is critical to the secretory activity of many neuroendocrine and endocrine cells. In pituitary cells, secretion is controlled by VGCI, which occurs spontaneously and in a receptor-regulated manner, and elevates [Ca2+]i to the levels needed to activate exocytosis. Spontaneous firing of APs was observed in cultured and immortalized pituitary cells and is accompanied by fluctuations in [Ca2+]i (20). In lactotrophs and immortalized GH cells, spontaneous excitability and basal PRL release are inhibited by nifedipine, an L-type Ca2+ channel blocker, whereas Bay K 8644, an L-type Ca2+ channel agonist, increased the frequency of APs and [Ca2+]i transients (16, 24, 25, 26) as well as hormone secretion (27). Spontaneous firing of APs and VGCI-dependent hormone release are up- and down-regulated by several Gs/Gi/o protein-coupled receptors, including GHRH, CRF, somatostatin, and dopamine (20). Extracellular Ca2+ is also important for sustained [Ca2+]i signaling during activation of Ca2+-mobilizing Gq/11-coupled receptors expressed in pituitary cells, including GnRH, TRH, and ETA receptors (20, 28). Activation of these receptors leads to an early spike in [Ca2+]i due to InsP3-induced mobilization of Ca2+ from endoplasmic reticulum. In GnRH- and TRH-stimulated cells, this was accompanied by a sustained Ca2+ influx through VGCCs. Capacitative Ca2+ entry through voltage-insensitive calcium channels may also participate in sustained Ca2+ influx in lactotrophs (29). Finally, pituitary cells express several ATP-gated receptor channels that conduct Ca2+ and facilitate VGCI (22).

In addition to serving as a reservoir for spontaneous and receptor-controlled [Ca2+]i signaling, extracellular Ca2+ may act as an extracellular messenger in pituitary cells to modulate VGCI. In this study we examined the mechanisms underlying the [Ca2+]e dependence of basal hormone secretion from anterior pituitary somatotrophs, lactotrophs, and gonadotrophs and the potential role of this ion in the control of hormone secretion. In general, unstimulated cells secrete in a constitutive and regulated manner, the regulation occurring via AP-driven Ca2+ influx and Ca2+-dependent exocytosis (30, 31, 32, 33). Our results in perfused anterior pituitary cells indicated that basal GH and PRL secretion was much higher than basal LH secretion and occurred in a regulated manner. As shown in other studies (26, 34, 35), the majority of basal GH and PRL secretion was extracellular Ca2+-dependent and sensitive to the blockade of VGCI through L-type channels. In contrast, basal LH secretion was low, at the level of detection by RIA, and was not affected by the addition of nifedipine.

Furthermore, somatotrophs and lactotrophs, but not gonadotrophs, responded to a decrease in [Ca2+]e and its return to physiological concentrations by elevating [Ca2+]i transiently. The amplitudes of [Ca2+]i responses depended on [Ca2+]e and its changing rate and were cell type specific. Although the nonspecific effects of [Ca2+]e are unlikely to account for these effects (e.g. 2 mM CaCl2 accounts only for ~2% of the ionic strength of medium used for perfusion and ~3% of its osmotic strength), we examined possible effects on the membrane surface charge screening and osmolality on on and off responses in [Ca2+]i and excluded the participation of these phenomena in the formation of response. Depletion and repletion of [Ca2+]e may also affect cellular Ca2+ homeostasis, which in other cell types is sufficient to trigger capacitative calcium entry (36). In pituitary cells this is probably not the case. First, ionomycin experiments revealed that the intracellular Ca2+ pool is not significantly affected by depletion of [Ca2+]e. Second, we did not observe amplification of [Ca2+]i responses in thapsigargin-treated cells, a treatment commonly used to activate capacitative calcium influx (37).

The presence of spontaneous firing of APs in all three cell types (16) and the lack of effect of [Ca2+]e on VGCI and LH secretion in gonadotrophs further indicate the specificity of [Ca2+]e action on cell excitability, i.e. the selective expression of a Ca2+-sensing plasma membrane channel in somatotrophs and lactotrophs. Our results indicate that the on and off [Ca2+]i responses were driven by an increase in VGCI through L-type Ca2+ channels and were sufficient to transiently stimulate GH and PRL secretion. In contrast, elevation of [Ca2+]e above physiological levels led to a transient decrease in the spontaneous excitability of cells, [Ca2+]i, and PRL secretion, whereas LH secretion was not affected. However, the cell type-specific amplitudes of on and off [Ca2+]i responses do not correlate with the density of L-type Ca2+ channels in three cell types (38), suggesting that the frequency of spiking and/or duration of APs were affected by depletion and repletion of [Ca2+]e.

Anterior pituitary cells express two pacemaker currents, tetrodotoxin-sensitive Na+ and Ca2+-conducting T-type currents (39, 40). Lactotrophs and somatotrophs exhibited low expression levels of Na+ channels in contrast to gonadotrophs (38), arguing against the role of these channels in on and off [Ca2+]i responses. On the other hand, the rank order of the on response parallels the expression level of T-type Ca2+ channels as well as Ca2+-activated large conductance potassium channels in these cells (38). Furthermore, pituitary cells express cyclic nucleotide-gated channels (41) and ether-a-go-go-like-type voltage-gated K+ channels (42, 43), which are members of a channel family known to respond to change in [Ca2+]e by modulating their gating properties, and consequently VGCI (8, 10). The divalent ion sensitivity of VGCI was consistent with cyclic nucleotide-gated channels acting as a [Ca2+]e sensor. Specifically, Mg2+ represents an effective cation in inhibiting cyclic nucleotide-gated channels (10), but it cannot substitute Ca2+ for the control of human ether-a-go-go-like potassium channel gating (8). Thus, the change in conductivity of several channels could account for the observed effects, indicating that further studies are needed to clarify the mechanism of transient activation of VGCI by depletion and repletion of [Ca2+]e and channels involved in this process.

Pituitary cells have an additional mechanism to detect [Ca2+]e. G Protein-coupled CaRs (120 kDa), originally identified and cloned from bovine parathyroid and rat kidney (2, 44), are also expressed in mouse and rat pituitary cells, AtT-20 immortalized cells, and human pituitary adenomas (14, 15). Furthermore, elevated [Ca2+]e affects [Ca2+]i and ACTH secretion in AtT-20 cells and GHRH-stimulated GH secretion in human pituitary adenomas (14, 15). Here we show that in rat somatotrophs changes in [Ca2+]e stimulate GH secretion not only by increasing the excitability of cells, but also independently of the status of VGCI. The CaR-like effect is cell type specific, i.e. it is observed only in somatotrophs.

If operative in somatotrophs, however, it is not clear how CaRs stimulate GH secretion. In a search for the intracellular messenger mediating the action of [Ca2+]e, we looked at the Ca2+-mobilizing action of lowered and raised [Ca2+]e (5). When VGCI was blocked, none of the cell types responded to changes in [Ca2+]e by elevations in [Ca2+]i. Furthermore, no changes in inositol phosphate accumulation were observed in pituitary cells in response to an increase in [Ca2+]e from 0.05 to 5 mM, in contrast to AtT-20 immortalized cells (14). In parallel to results in AtT-20 cells (14) we observed a concentration-dependent rise in cAMP accumulation when cells were bathed in medium containing phosphodiesterase inhibitors. This could indicate the coupling of CaR to adenylyl cyclase signaling pathway in pituitary cells, but also that adenylyl cyclases and/or phosphodiesterases are sensitive to VGCI (11, 12, 13). In accordance with the second hypothesis, no changes in cAMP levels were observed when cells were bathed in medium without phosphodiesterase inhibitors.

The ability of [Ca2+]e to stimulate GH secretion in a [Ca2+]i-independent manner is not an artifact generated by our GH measurements, as indicated in experiments featuring stimulation of secretion by GHRH and ET-1 and its inhibition by somatostatin and nifedipine (16, 23). Furthermore, although unconventional, [Ca2+]i-independent stimulation of GH secretion by [Ca2+]e is not unique for somatotrophs. CaRs in parathyroid cells activate PLC and Ca2+ mobilization from intracellular stores, but the Ca2+-mobilizing action of CaRs in these cells is accompanied by an inhibition of PTH secretion (6), suggesting the paradoxical role of [Ca2+]i in the control of exocytosis or that a messenger(s) other than Ca2+ mediates the action of CaRs on secretion. In accord with the second hypothesis, it has been shown recently that G protein-coupled receptors can modulate exocytotic fusion downstream of Ca2+ entry, and that this inhibition is mediated by ß/{gamma}-subunits (45).

The most striking finding in this study is the cell type-specific action of [Ca2+]e. In gonadotrophs, there is a clear dissociation between spontaneous firing of APs and basal secretion. Although about 50% of gonadotrophs were found to fire APs, basal secretion was low and was not affected by nifedipine (16). Consistent with this observation, changes in [Ca2+]e were ineffective in modulating [Ca2+]i. LH secretion was also not affected, indicating that [Ca2+]e is an unlikely extracellular messenger for the control of gonadotropin release. In lactotrophs, the exclusive dependence of basal PRL secretion on spontaneous VGCI and the [Ca2+]e sensitivity of VGCI indicate a potential role of [Ca2+]e as a carrier through VGCCs as well as a regulator of spontaneous electrical activity. Such in vitro effects of [Ca2+]e are consistent with the hypothesis that in vivo hypercalcemia should lead to a decrease in basal PRL secretion, whereas hypocalcemia should stimulate PRL secretion. In somatotrophs, [Ca2+]e has the most complex role, as a carrier through VGCCs and as an extracellular messenger acting on two classes of sensors. This, in turn, leads to the bidirectional dose dependence of [Ca2+]e on GH release, with a minimum response near the physiological concentration and facilitation of secretion in response to lowered and raised [Ca2+]e. As GH is intimately involved in the control of bone matrix (46), it is also reasonable to speculate that these [Ca2+]e sensors serve as feedback detectors that modulate GH secretion.

In conclusion, our results indicate a dual role of [Ca2+]e in intracellular signaling and basal hormone secretion, as a carrier through VGCCs and as an extracellular messenger. The carrier function of [Ca2+]e is critical for basal GH and PRL, but not LH, release. The messenger functions of [Ca2+]e are mediated by two sensors, a plasma membrane channel participating in spontaneous firing of APs and a CaR-like sensor. The first mechanism is operative in somatotrophs and lactotrophs, whereas the CaR-like sensor is operative only in somatotrophs. The range of [Ca2+]e required to change the excitability of somatotrophs and lactotrophs and to stimulate GH secretion independently of VGCI supports the hypothesis that [Ca2+]e has the potential to act as an extracellular messenger in physiological and pathophysiological conditions.


   Footnotes
 
Abbreviations: AP, Action potential; [Ca2+]e, extracellular Ca2+ concentration; [Ca2+]i, intracellular Ca2+ concentration; CaR, calcium-sensing receptor; ET-1, endothelin-1; VGCC, voltage-gated calcium channels; VGCI, voltage-gated Ca2+ influx.

Received July 27, 2001.

Accepted for publication October 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berridge MJ, Irvine RF 1989 Inositol phosphates and cell signalling. Nature 341:197–205[CrossRef][Medline]
  2. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
  3. Brown EM, Vassilev PM, Hebert SC 1996 Calcium ions as extracellular messengers. Cell 83:679–682
  4. Brown EM 1991 Extracellular Ca2+ sensing, regulation of parathyroid cell function, and role of Ca2+ and other ions as extracellular (first) messengers. Physiol Rev 71:371–411[Free Full Text]
  5. Brown EM 1999 Physiology and Pathophysiology of the extracellular calcium-sensing receptor. Am J Med 106:238–253[CrossRef][Medline]
  6. Chattopadhyay N 2000 Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor. Int J Biochem Cell Biol 32:789–804[CrossRef][Medline]
  7. Hille B 1991 Ionic channels of excitable membranes. Sunderland: Sinauer Associates
  8. Johnson Jr JP, Balser JR, Bennett PB 2001 A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels. J Neurosci 21:4143–4153[Abstract/Free Full Text]
  9. Su H, Kirson ED, Yaari Y 2001 Extracellular calcium modulates persistent sodium current-dependent burst-firing in hippocampal pyramidal neurons. J Neurosci 21:4173–4182[Abstract/Free Full Text]
  10. Zagotta WN, Siegelbaum SA 1996 Structure and function of cyclic nucleotide-gated channels. Annu Rev Neurosci 19:235–263[CrossRef][Medline]
  11. Andric SA, Kostic TS, Tomic M, Koshimizu T, Stojilkovic SS 2001 Dependence of soluble guanylyl cyclase activity on calcium signaling in pituitary cells. J Biol Chem 276:844–849[Abstract/Free Full Text]
  12. Beavo JA 1995 Cyclic-nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75:725–748[Abstract/Free Full Text]
  13. Decorte MN, Jaffard R, Cooper DM 1998 Ca2+-sensitive adenylyl cyclases, key integrators of cellular signalling. Life Sci 62:1647–1652[CrossRef][Medline]
  14. Emanuel RL, Adler GK, Kifor O, Quinn SJ, Fuller F, Krapcho K, Brown EM 1996 Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line. Mol Endocrinol 10:555–565[Abstract/Free Full Text]
  15. Romoli R, Lania A, Mantovani G, Corbetta S, Persani L, Spada A 1999 Expression of calcium-sensing receptor and characterization of intracellular signaling in human pituitary adenomas. J Clin Endocrinol Metab 84:2848–2853[Abstract/Free Full Text]
  16. Van Goor F, Zivadinovic D, Matinez-Fuentes AJ, Stojilkovic SS 2001 Dependence of pituitary hormone secretion on the pattern of spontaneous voltage-gated calcium influx: cell-type specific action potential-secretion coupling. J Biol Chem 276:33840–33846[Abstract/Free Full Text]
  17. Valentijn JA, Louiset E, Vaudry H, Cazin L 1991 Dopamine-induced inhibition of action potentials in cultured frog pituitary melanotrophs is mediated through activation of potassium channels and inhibition of calcium and sodium channels. Neuroscience 42:29–39[CrossRef][Medline]
  18. Sankaranarayanan S, Simasko SM 1996 A role for a background sodium current in spontaneous action potentials and secretion from rat lactotrophs. Am J Physiol 271:C1927–C1934
  19. Ingram CD, Bicknell RJ, Mason WT 1986 Intracellular recordings from bovine anterior pituitary cells: modulation of spontaneous activity by regulators of prolactin secretion. Endocrinology 119:2508–2515[Abstract/Free Full Text]
  20. Stojilkovic SS, Catt KJ 1992 Calcium oscillations in anterior pituitary cells. Endocr Rev 13:256–280[Abstract/Free Full Text]
  21. Kwiecien R, Hammond C 1998 Differential management of Ca2+ oscillations by anterior pituitary cells: a comparative overview. Neuroendocrinology 68:135–151[CrossRef][Medline]
  22. Koshimizu T, Tomic M, Wong AOL, Zivadinovic D, Stojilkovic SS 2000 Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells. Endocrinology 141:4091–4099[Abstract/Free Full Text]
  23. Tomic M, Zivadinovic D, Van Goor F, Yuan D, Koshimizu T, Stojilkovic SS 1999 Expression of Ca2+-mobilizing endothelinA receptors and their role in the control of Ca2+ influx and growth hormone secretion in pituitary somatotrophs. J Neurosci 19:7721–7731[Abstract/Free Full Text]
  24. Kwiecien R, Robert C, Cannon R, Vigues S, Arnoux A, Kordon C, Hammond C 1998 Endogenous pacemaker activity of rat tumour somatotrophs. J Physiol 508:883–905[Abstract/Free Full Text]
  25. Charles AC, Piros ET, Evans CJ, Hales TG 1999 L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells. J Biol Chem 274:7508–7515[Abstract/Free Full Text]
  26. Holl RW, Thorner MO, Mandell GL, Sullivan JA, Sinha YN, Leong DA 1988 Spontaneous oscillations of intracellular calcium and growth hormone secretion. J Biol Chem 263:9682–9685[Abstract/Free Full Text]
  27. Stojilkovic SS, Izumi S-I, Catt KJ 1988 Participation of voltage-sensitive calcium channels in pituitary hormone secretion. J Biol Chem 263:13054–13061[Abstract/Free Full Text]
  28. Yu R, Ashworth R, Hinkle PM 1998 Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes. Thyroid 8:887–894[Medline]
  29. Hinkle PM, Nelson EJ, Ashworth R 1996 Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells. Trends Endocrinol Metab 7:370–374[CrossRef][Medline]
  30. von Ruden L, Neher E 1993 A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. Science 262:1061–1064[Abstract/Free Full Text]
  31. Mulkey RM, Zucker RS 1991 Action potentials must admit calcium to evoke transmitter release. Nature 350:153–155[CrossRef][Medline]
  32. Jackson MB, Konnerth A, Augustine GJ 1991 Action potnetial broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals. Proc Natl Acad Sci USA 88:380–384[Abstract/Free Full Text]
  33. Lim NF, Nowycky MC, Bookman RJ 1990 Direct measurement of exocytosis and calcium currents in single vertebrate nerve terminals. Nature 344:449–451[CrossRef][Medline]
  34. Cota G, Hiriart M, Horta J, Torres-Escalante JL 1990 Calcium channels and basal prolactin secretion in single male rat lactotropes. Am J Physiol 259:C949–C959
  35. Enyeart JJ, Sheu SS, Hinkle PM 1987 Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells. J Biol Chem 262:3154–3159[Abstract/Free Full Text]
  36. Yue L, Peng J-B, Hediger MA, Clapham DE 2001 CaT1 manifests the pore properties of the calcium-release-activated calcium channels. Nature 410:705–708[CrossRef][Medline]
  37. Bird GSJ, Putney JW 1993 Inhibition of thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. J Biol Chem 268:21486–21488[Abstract/Free Full Text]
  38. Van Goor F, Zivadinovic D, Stojilkovic SS 2001 Differential expression of ionic channels in anterior pituitary cells. Mol Endocrinol 15:1222–1236[Abstract/Free Full Text]
  39. Tse A, Hille B 1993 Role of voltage-gated Na+ and Ca2+ channels in gonadotropin-releasing hormone-induced membrane potential changes in identified rat gonadotropes. Endocrinology 132:1475–1481[Abstract/Free Full Text]
  40. Marchetti C, Childs GV, Brown AM 1987 Membrane currents of identified isolated rat corticotropes and gonadotropes. Am J Physiol 252:E340–E346
  41. Tomic M, Koshimizu T, Yuan D, Andric SA, Zivadinovic D, Stojilkovic SS 1999 Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs. J Biol Chem 274:35693–35702[Abstract/Free Full Text]
  42. Schafer R, Wulfsen I, Behrens S, Weinsberg F, Bauer CK, Schwartz JR 1999 The erg-like potassium current in rat lactotrophs. J Physiol 518:401–416[Abstract/Free Full Text]
  43. Schledermann W, Wulfsen I, Schwartz JR, Bauer CK 2001 Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via native signal cascade. J Physiol 357:593–615
  44. Riccardi D, Park J, Lee W-S, Gamba G, Brown EM, Hebert SC 1995 Cloning and functional expression of a rat kidney extracellular Ca2+-sensing receptor. Proc Natl Acad Sci USA 92:131–135[Abstract/Free Full Text]
  45. Blackmer T, Larsen EC, Takahashi M, Martin TFJ, Alford S, Hamm HE 2001 G protein bg subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry. Science 292:293–297[Abstract/Free Full Text]
  46. Ohlsson C, Bangtsson B-A, Isaksson OGP, Andreassen TT, Slootweg MC 1998 Growth hormone and bone. Endocr Rev 19:55–79[Abstract/Free Full Text]



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