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Endocrinology Vol. 140, No. 2 646-651
Copyright © 1999 by The Endocrine Society

ARTICLES

Parathyroid Hormone-Related Protein Markedly Potentiates Depolarization-Induced Catecholamine Release in PC12 Cells via L-Type Voltage-Sensitive Ca2+ Channels1

Michael L. Brines and Arthur E. Broadus

Section of Endocrinology (M.L.B., A.E.B.), Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520; and The Kenneth S. Warren Laboratories (M.L.B.), Tarrytown, New York 10591

Address all correspondence and requests for reprints to: Michael L. Brines, Ph.D., M.D., The Kenneth S. Warren Laboratories, 765 Old Saw Mill River Road, Tarrytown, New York 10591. E-mail: mbrines{at}kswl.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH-related protein (PTHrP) is a normal product of many excitable cells of the nervous and endocrine systems. Functions of PTHrP in these tissues are, however, currently unknown. Prior study has suggested that a relationship exists between PTHrP and the L-type voltage-sensitive Ca2+ channel (L-VSCC). For example, in cerebellar granule neurons PTHrP gene transcription is regulated by Ca2+ influx specifically through this channel. Amino-terminal PTHrP products signal via the widely expressed PTH/PTHrP receptor, which is linked to both protein kinase A and C. These second messengers are known modulators of L-VSCC conductance. To determine whether PTHrP can modulate L-VSCC function, we studied catecholamine secretion in a PC12 clone expressing the PTH/PTHrP receptor but not PTHrP. We found that PTHrP(1–36) (100 nM) to be an ineffective secretagogue for resting cells, but its presence markedly potentiates secretion to K+ depolarization. The PTHrP-augmented catecholamine secretion depends entirely upon L-VSCC Ca2+ influx and rapidly inactivates. Similar effects were produced by (Bu)2cAMP but not by carbachol. These observations support the hypothesis that PTHrP can regulate L-VSCC conductance. In the normal adrenal medulla that expresses both PTHrP and its receptor, PTHrP may act in an autocrine/paracrine fashion to modify catecholamine secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH-RELATED PROTEIN (PTHrP) was identified a decade ago as the tumor product that mediates humoral hypercalcemia of malignancy (reviewed in Refs. 1, 2). It is now known that PTHrP and PTH are two members of a small gene family and that one consequence of this heritage is a highly homologous sequence at the N terminus of each of these peptides. These N-terminal products appear to signal via a common G protein-coupled receptor known as the type I PTH/PTHrP receptor (reviewed in Refs. 3, 4). This receptor is the target of PTH’s classic regulatory effects on mineral metabolism in bone and kidney. While PTH is expressed in a very few sites, the type I receptor and PTHrP are widely expressed in adult and fetal tissues, the most common single pattern being PTHrP expression in epithelial surfaces and structures and receptor expression in immediately adjacent stromal cells (reviewed in Ref. 2). This pattern has been taken as prima facie evidence of paracrine PTHrP function. The best documented such function in the adult is as a compliance-regulatory factor in smooth muscle structures such as the stomach (5), uterus (6, 7), and urinary bladder (8); PTHrP is a stretch-induced product in these sites and acts as a potent smooth muscle relaxant that permits such structures to accommodate gradual filling. Recent gene manipulation experiments in mice have revealed that PTHrP also serves as a developmental regulatory molecule in the epidermis (9), the mammary epithelium (10), and chondrocytes/endochondral bone (11, 12). The specific function of PTHrP in a number of these sites is to control the rate at which cellular programs of differentiation proceed.

It is becoming clear that there are additional layers of complexity as regards both the ligand and receptor branches of this small signaling family. The PTHrP primary sequence is subject to posttranslational processing into midregion and C-terminal peptide species in a cell-specific fashion, and there is increasingly strong evidence for unique functions of each of these peptides, the former in the control of placental calcium transport and the latter in regulating osteoclast formation/activity (reviewed in Ref. 1 . Receptors for these products have yet to be identified. A second receptor has been cloned from a brain cDNA library and termed the PTH-2 receptor because it responds only to N-terminal PTH (13); a candidate ligand for this receptor that is not PTH has been partially purified from the hypothalamus (14). PTH itself is expressed in a few none-hypothalamic sites in the brain (15, 16, 17), and both the type I receptor and PTHrP are products of a number of neuronal populations (18, 19). Thus, in the central nervous system, there is evidence for at least two receptors that recognize N-terminal PTH-like proteins in a sequence-specific fashion and at least three PTH-like peptides that might signal via these receptors.

Among the many tissues coexpressing PTHrP and the type 1 receptor are excitable secretory cells. In these neurons and neuroendocrine cells the peptide is secreted in a regulated fashion upon depolarization (20, 21, 22) and is likely available to interact with nearby receptors. Results of our previous study (20) have demonstrated that cerebellar granule cells maintained in primary culture under depolarizing conditions express high levels of PTHrP messenger RNA (mRNA). PTHrP gene transcription critically depends upon Ca2+ influx mediated by high voltage-activated Ca2+ channels of the dihydropyridine-sensitive subtype (L-type voltage-sensitive Ca channels; L-VSCCs). Increases in PTHrP mRNA synthesis occur in a Ca2+/calmodulin kinase-dependent manner with a slow latency of onset of several hours. In the absence of L-VSCC Ca2+ influx, no transcription is observed, irrespective of the intracellular Ca2+ levels. These findings have recently been confirmed by Ono and co-workers (21).

Neuroendocrine cells share many characteristics with neurons, including electrical excitability and Ca2+-dependent secretion. A number of cell lines exist that are functionally intact and therefore of potential use for study of PTHrP action. One clonal cell type, which is derived from a mouse pheochromocytoma (PC12), is widely used to study stimulus-secretion coupling. PC12 cells not differentiated by nerve growth factor recapitulate much of the normal functional activity of the adrenal medulla, synthesizing high levels of catecholamines, principally dopamine and norepinephrine. These products are released in a regulated manner by depolarization, almost entirely by L-VSCC Ca2+ influx (23, 24). We have employed a clone that expresses PTH/PTHrP type 1 receptor mRNA but not PTHrP itself, which allows the effects of PTHrP to be observed without potential interference by release of endogenous peptide.

The PTHrP/PTH receptor has been shown by transfection studies to signal via both the protein kinase A and protein kinase C (PKC) second messenger cascades (25). Although pharmacological activation of either (or both) of these systems in undifferentiated PC12 cells is not secretagogic, protein kinase A or cAMP analogs markedly potentiate depolarization-induced secretion (26, 27). In contrast, the effects of PKC are controversial in undifferentiated PC12 cells, with both amplification and inhibition of secretion reported (28, 29). However, PKC is known to inhibit strongly L-VSSC Ca2+ influx (30, 31, 32), which is required for secretion of catechols in this cell line (23). Further, in PC12 cells receptor-mediated PKC stimulation markedly inhibits pharmacologically or physiologically induced cAMP generation (33). Thus, it was unclear a priori how PTH/PTHrP receptor occupancy might affect catecholamine secretion. We report here that PTHrP markedly but transiently increases catecholamine secretion via a L-VSSC-dependent mechanism. These observations are consistent with an autocrine/paracrine role for PTHrP to selectively amplify stimulus-secretion coupling within the adrenal medulla.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PC-12 cells (gift from L. Kaczmerek, Yale University) were maintained in growth medium consisting of RPMI 1640 (BRL Gibco, Gaithersburg, MD) supplemented by 10% (vol/vol) heat-inactivated horse serum and 5% FBS at 37 C in 95% O2 and 5% CO2 on standard polystyrene cultureware coated with poly-L-lysine (50 µg/ml). To prepare cells for perifusion study (see below), cells were lightly trypsinized, centrifuged, and added to 12-mm round glass coverslips coated with poly-L-lysine for use between 1 and 2 days after plating.

RNA was isolated and ribonuclease (RNase) protection analysis carried out on sister cultures as previously described (20) using RNA probes prepared from a 343-bp PvuII-Bgll rat PTHrP cDNA fragment, a 230-bp Sau3A-BamHI rat cyclophilin cDNA fragment, and a 259-bp fragment of the rat PTH/PTHrP receptor DNA. Cyclophilin was added as a loading control in each assay using a probe prepared with a reduced specific activity because of the high relative abundance of cyclophilin. Each sample was assessed using 20 µg total RNA.

Solutions employed were basal (nondepolarizing) in buffer containing (in millimolar concentration): HEPES, 10; CaCl2, 1.8; NaCl, 138; MgSO4, 0.8; KCl, 4.7; NaH2PO4, 1; glucose, 10; pargyline (to inhibit metabolism of dopamine), 0.05; pH 7.4. For stimulating (depolarizing) buffer, KCl was increased to 60 mM and NaCl was reciprocally reduced to maintain isotonicity. PC12 cells take up extracellular catecholamines in a Na+-dependent manner for storage within dense-core granules. To load PC12 cells, coverslips with attached PC12 cells were incubated with [3H]dopamine (1 µCi/ml; Amersham Pharmacia Biotech, Arlington Heights, IL.) in basal buffer with an additional 2 µM unlabeled dopamine [the Michaelis-Menton constant (Km) of uptake system has been reported to be 0.7–2 µM (34, 35)] for 90 min at 37 C.

Regulated secretion of PC12 cells was studied by monitoring the efflux of [3H]dopamine from labeled cells using a constant-temperature perifusion apparatus according to the protocol of O’Connor and Kimelberg (36) and Minnema and Michaelson (36, 37). Solutions could be instantaneously switched using a gang valve and were perifused at a constant rate of 1 ml/min through a coaxial heater (Warner Instrument Corp., Hamden, CT) to raise the buffer temperature to 37 C and delivered to [3H]dopamine-loaded cells maintained in a constant temperature chamber. Dead space within the perifusion device was ~0.1 ml and was accounted for in all experiments. Perifusate was collected each minute by a fraction collector, mixed with scintillation fluid (Optifluor, Packard Instruments, Meriden, CT), and radioactivity quantified using a scintillation counter (Packard). Each experiment was terminated by lysis and solubilization of the cells using 100 mM NaOH (0.5 ml) followed by neutralization with 100 mM HCl (0.5 ml), after which the remaining intracellular counts were determined. Radioactivity in perifusate was expressed as the percentage of total remaining intracellular radioactivity during each time interval.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RNase protection analyzes performed on this PC12 clone revealed abundant transcripts for PTH/PTHrP receptor but not for PTHrP (Fig. 1Go). This was true for cells maintained under nondepolarizing (basal) conditions as well as for cells depolarized for 3–5 h (25–60 mM K+), a condition that induces abundant PTHrP mRNA in cerebellar granule neurons (20, 21). Additionally, conditioned medium contained undetectable amounts of both PTHrP(1–36) and PTHrP(34–84) (immunoradiometric assay performed courtesy of A. Stewart; lower detection limit ~1 pmol; data not shown).



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  		Figure 1. Analysis of PTHrP, PTH/PTHrP receptor, and cyclophilin RNA of PC12 cells (lanes 1–4) and granule neurons (lanes 5–10) by protection assay. PC12 cell expressed appreciable levels of the PTH/PTHrP receptor, but no PTHrP mRNA, in control or prolonged depolarization. In contrast, granule neurons expressed abundant PTHrP RNA, which increased under depolarization. Granule neurons also express low levels of PTH/PTHrP receptor RNA, not seen under these exposure conditions. Conditions: Lane 1, PC 12 in 5 mM K buffer; lanes 2–4, PC 12 in 25 mM K for 60, 180, and 360 min, respectively; lane 5, granule neurons in 25 mM K buffer; lanes 6–10, granule cells in 50 mM K for 30, 60, 120, 180, and 240 min, respectively.

 
Cells subjected to perifusion adhered well to the poly-L-lysine/glass coverslips at the 1 ml/min flow rates used. After an initial 10-to 15-min washout period in nondepolarizing medium, [3H]dopamine release reached a steady state of ~1% of cellular stores per min (e.g. Fig. 2Go). An abrupt change to 60 mM K+ depolarizing medium elicited only a small, but reliable, increase in [3H]dopamine release, in the range of 0.3–0.5% of intracellular stores per min (Fig. 2Go, top; experiment repeated 16 times). Repetitive exposures to depolarizing buffer after a return to nondepolarizing medium from 5–45 min was ineffective in producing further increases in [3H]dopamine release.



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  		Figure 2. A, Sequental efflux of radiolabeled dopamine over 30 min with 5 mM K+ (5K) buffer followed by alternating 5-min periods of 60K and 5K shows a minimal but significant secretion upon depolarization. Generally, only the first stimulation period exhibited a clear response. B, In contrast, addition of PTHrP (100 nM) for at least 10 min produces a large and single burst of secretion.

 
Addition of PTHrP (100 nM) to the nondepolarizing basal buffer did not change [3H]dopamine release (Figs. 2BGo and 3AGo; experiment repeated six times). In contrast, cells that had been previously exposed to PTHrP responded with a large release of [3H]dopamine, reaching about 4–5% of total content over the first few minutes (Fig. 2Go, bottom; range 3.5–10%; experiment repeated eight times), followed by a smaller second phase (1.5–2%) maintained for an additional 3–5 min (e.g. Fig. 3AGo). Repeat exposure to PTHrP did not further affect [3H]dopamine release (Fig. 2BGo). The potentiation of [3H]dopamine release by PTHrP was slow in onset, requiring a minimum of ~10 min. Thus, simultaneous exposure of previously untreated cells to depolarizing buffer containing PTHrP did not affect [3H]dopamine release (Fig. 4BGo; experiment repeated five times). Although of slow latency of onset, the amplifying effect of PTHrP was characterized by rapid offset, as a change to depolarizing buffer without PTHrP in a previously primed culture did not affect catecholamine release (Fig. 4Go, top; experiment repeated six times), in contrast to when PTHrP was present during depolarization (Fig. 3AGo; experiment repeated seven times). The PTHrP-enhanced release of [3H]dopamine was completely blocked by addition of the dihydropyridine nitredepine [100 nM (Fig. 3BGo; experiment repeated three times)]. Nitredipine alone did not effect the spontaneous (basal) [3H]dopamine release.



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  		Figure 3. The burst of [3H]dopamine secretion facilitated by PTHrP (A) is dependent upon L channel Ca2+ influx as nitrendipine (100 nM) completely abolishes secretion (B). Note that the basal secretion rate (~1% of [3H]dopamine content/min) is not nitrendipine sensitive.

 


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  		Figure 4. PTHrP is required during depolarization to augment [3H]dopamine secretion, even after prolonged preexposure (A). Exposure to PTHrP is not associated with stimulated secretion in the absence of priming (B).

 
To compare the magnitude and temporal pattern of PTHrP-induced release to that of cAMP alone, the freely diffusable cAMP analog (Bu)2cAMP (1 mM) was substituted for PTHrP in the perfusion buffers. Cells exposed to (Bu)2cAMP responded to depolarization in a similar manner to those exposed to PTHrP, except that the second, smaller release phase was distinctly prolonged (Fig. 5AGo; experiment repeated five times). Unlike PTHrP, cells primed with (Bu)2cAMP responded to depolarizing buffer without a continued presence of (Bu)2cAMP (data not shown). The presence of (Bu)2cAMP did not increase the effectiveness of the cholinergic secretagogue carbachol (1 mM) (Fig. 5BGo, experiment repeated three times). Finally, prior exposure of cells to depolarizing buffer alone for 5 min before the addition of PTHrP effectively prevented subsequent potentiation of either PTHrP (Fig. 6Go, top) or (Bu)2cAMP (Fig. 6Go, bottom).



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  		Figure 5. A, The potentiated secretion of [3H]dopamine associated with PTHrP exposure is duplicated by use of the cAMP analog (Bu)2cAMP (500 µM), but with a much longer decay. B, Potentiation of secretion by (Bu)2cAMP is not observed with the secretagogue carbachol, which acts through acetylcholinergic receptors.

 


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  		Figure 6. Brief periods of depolarization (60K) block subsequent stimulated secretion to either PTHrP or (Bu)2cAMP (bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The observed catecholamine secretory response can be fully accounted for by a cAMP effect mediated by the type 1 receptor. This pattern is duplicated in magnitude by the cAMP analog, although the time course is more prolonged, probably a consequence of its long biological half-life when compared with native cAMP. The temporal relationships of the interplay of PTHrP, cAMP, depolarization, and regulated secretion suggest that PTHrP may act to enable brief, precise release of stored catecholamines. Under nondepolarizing conditions the addition of either PTHrP or cAMP analogs does not increase dopamine secretion. This behavior would be predicted for a process that depends upon L-VSCC Ca2+ for regulated secretion, as the probability of finding a high conductance L-VSCC [i.e. phosphorylated (38)] is very low under nondepolarizing conditions. When depolarized, however, the L-VSCCs are in a conducting favored state, and under this condition either PTHrP or (Bu)2cAMP initiate Ca2+ influx and secretion. The secretory response rapidly inactivates within 5 min and, at least during the duration of the experimental observations (up to 45 min), remains off. This rapid termination can be partially accounted for by voltage-dependent effects, as a 5 min predepolarization effectively eliminated a secretory response for at least 15 min after exposure. Such voltage inactivation has been described previously in PC12 cells (39). However, the much slower termination of secretion seen with (Bu)2cAMP suggests that rapid termination of the effects of cAMP is primarily biochemical and could swamp the rapid effects of phosphatases that dephosphorylate and inactivate conductance of L-VSCCs (40, 41).

Undifferentiated PC12 cells have been reported to express predominantly voltage-activated Ca2+ channels of the L-VSCC type, in contrast to PC12 cells that have been differentiated by exposure to nerve growth factor, which express L-VSCCs and N-VSCCs about equally (23). The finding that nitredipine completely blocks release in our system is consistent with involvement of L-VSCCs alone in secretion. However, the ~1% basal secretory rate clearly does not depend upon L-VSCC flux, as it is unaffected by dihydropyridine blockers. Other investigators have demonstrated that removal of extracellular calcium abolishes this basal efflux (42).

Phosphorylation of L-VSCCs is a common mechanism of regulation of Ca2+ conductance in excitable cells via a variety of protein kinase systems [reviewed by Xiong and Sperelakis(43)]. However, the direction of modulation, and therefore the biological effect, varies for each kinase in different tissues. For neurons and excitable endocrine cells, cAMP-dependent phosphorylation generally amplifies conductance (38, 44), similar to cardiac muscle (41). In vascular smooth muscle, on the other hand, cAMP-dependent phosphorylation inhibits L-VSCC conductance, producing relaxation. PKC also regulates L-VSCC conductance but appears to do so in many tissues indirectly through phosphorylation effects on a regulatory protein. Further, the response of ionic currents to PKC activation in the same cell type appears to vary between tissues and experimental conditions and may arise from the presence of multiple PKC isoforms or from the physiological state of each individual cell.

The effectiveness of PTHrP [and (Bu)2cAMP] in stimulating secretion likely arises from the known positive effects on L-VSCC channel conductance that arises from phosphorylation of the {alpha}-subunit of the L-VSCC by translocation of the catalytic subunit of protein kinase A to the cell membrane (41). This same electrophysiological study showed that intracellular phosphatases reverse this augmented conductance within a few seconds, and this probably accounts for the observation that stimulated secretion is not maintained, in spite of large remaining intracellular dopamine stores. Additionally, actions of these phosphatases could also explain why withdrawal of PTHrP from the medium is accompanied by an rapid loss of augmented secretion. Thus, the characteristics we observed would predict that PTHrP receptor occupancy would give a temporally narrow, large pulse of catecholamines released only from cells experiencing appreciable ambient PTHrP concentrations.

The adrenal medulla is a complex tissue with multiple cell types, including dense nerve terminals from extrinsic and intrinsic innervation (45). The detailed cellular distribution of PTHrP and the type 1 receptor have not yet been reported for the adrenal medulla. However, the presence of both peptide and receptor in clonal human pheochromocytomas (46) shows that a single chromaffin cell type can make both protein products. As PTHrP itself is released by a L-VSCC-dependent mechanism, it could feedback in an autocrine/paracrine manner to modulate catecholamine secretion. The presence of a potentiating receptor on a secretory cell predicts that its physiological role is one of allowing a large but focused burst of secretion only under conditions in which adequate priming has occurred and in this manner, would ensure a precise release of secretory materials only under appropriate conditions. This, together with the rapid inactivation we observed, would serve to limit the secretion of catecholamines, which are highly toxic if not regulated to within a narrow physiological range (e.g. Refs. 47, 48). Whether this theme of autoregulation of L-VSCC-dependent secretion is also true for other secretory cells and neurons is a question currently under investigation.


   Acknowledgments
 
We thank M. Pouresmail for technical assistance in performing the efflux studies and E. Holt and B. Dryer for performing the RNase protection analyzes.


   Footnotes
 
1 Supported by NIH Grants AR-30102 and DK-45735. Back

Received May 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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