HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Short, A. D. Articles by Taylor, C. W. Search for Related Content PubMed PubMed Citation Articles by Short, A. D. Articles by Taylor, C. W. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 3, 1807-1813, January 21, 2000

Parathyroid Hormone Controls the Size of the Intracellular Ca²⁺ Stores Available to Receptors Linked to Inositol Trisphosphate Formation^*

Alison D. Short and Colin W. Taylor

From the Department of Pharmacology, Tennis Court Road, Cambridge, CB2 1QJ, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In HEK 293 cells stably expressing type 1 parathyroid (PTH) receptors, PTH stimulated release of intracellular Ca²⁺ stores in only 27% of cells, whereas 96% of cells responded to carbachol. However, in almost all cells PTH potentiated the response to carbachol by about 3-fold. Responses to carbachol did not desensitize, but only the first challenge in Ca²⁺-free medium caused an increase in [Ca²⁺]_i, indicating that the carbachol-sensitive Ca²⁺ stores had been emptied. Subsequent addition of PTH also failed to increase [Ca²⁺]_i, but when it was followed by carbachol there was a substantial increase in [Ca²⁺]_i. A similar potentiation was observed between ATP and PTH but not between carbachol and ATP. Intracellular heparin inhibited responses to carbachol and PTH, and pretreatment with ATP and carbachol abolished responses to PTH, suggesting that the effects of PTH involve inositol trisphosphate (IP₃) receptors. PTH neither stimulated detectable IP₃ formation nor affected the amount formed in response to ATP or carbachol. PTH stimulated cyclic AMP formation, but this was not the means whereby PTH potentiated Ca²⁺ signals. We suggest that PTH may regulate Ca²⁺ mobilization by facilitating translocation of Ca²⁺ between discrete intracellular stores and that it thereby regulates the size of the Ca²⁺ pool available to receptors linked to IP₃ formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Parathyroid hormone (PTH)¹ plays a central role in plasma Ca²⁺ homeostasis, and many tissues that are not involved in Ca²⁺ regulation also express PTH receptors (1). Two PTH receptor subtypes (types 1 and 2) have been identified; their cDNA sequences share 70% similarity (2-5) and identify them as members of a subfamily of G protein-coupled receptors to which the receptors for calcitonin, secretin, ACTH, and glucagon also belong.

It has long been accepted that PTH stimulates an increase in both intracellular cyclic AMP and [Ca²⁺]_i in many cell types (6, 7). The ability of PTH to activate two signaling pathways was generally assumed to result from its interaction with the two receptor subtypes, an assumption that gained support from the observation that the two pathways could be differentially stimulated by truncated forms of PTH (8). More recently, expression of recombinant receptors has established that type 1 (3, 9, 10) and type 2 (11) PTH receptors are each alone capable of stimulating increases in both [Ca²⁺]_i and intracellular cyclic AMP. Other members of the family of receptors to which the PTH receptor belongs share this ability to independently stimulate cyclic AMP formation and an increase in [Ca²⁺]_i (3, 12-14).

In some cells, the increase in [Ca²⁺]_i evoked by PTH is mediated largely by effects of cyclic AMP on Ca²⁺ entry (7), but in osteoblasts and kidney cells (6) and cells expressing recombinant type 1 (3, 9, 10) or type 2 (11) PTH receptors, PTH also stimulates release of intracellular Ca²⁺ stores. The means whereby PTH causes such Ca²⁺ mobilization is unclear. In some cells, PTH stimulates formation of inositol 1,4,5-trisphosphate (IP₃) (3, 6), which presumably then causes Ca²⁺ mobilization via IP₃ receptors. In other situations, PTH and agonists that stimulate IP₃ formation appear to release Ca²⁺ from different intracellular stores (9, 15), and PTH-stimulated Ca²⁺ mobilization occurs without detectable formation of IP₃ (10, 15) and in the presence of antagonists of IP₃ receptors (9, 10). Furthermore, PTH-evoked Ca²⁺ release appears not to be mediated by ryanodine receptors (9) or by either cyclic ADP-ribose or NAADP⁺ (9), both of which have been shown to stimulate Ca²⁺ release in other cells (16). In short, the ability of PTH to stimulate Ca²⁺ mobilization cannot easily be explained by the properties of known intracellular signaling pathways.

In the present study, we examine the mechanisms underlying the effects of PTH on intracellular Ca²⁺ stores in human embryonic kidney 293 cells stably expressing type 1 PTH receptors (HEK/PTH-R1 cells).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- The full-length cDNA encoding the human type 1 PTH receptor in the vector, pcDNAI/neo, was a gift from Dr. K. Seuwen (Basel, Switzerland) (5). HEK 293 cells were from the European Collection of Animal Cell Cultures (Porton Down, UK). Human PTH (1-34), Rp-8-Br-cAMPS, ionomycin, Xestospongin A, wortmannin, and H89 were from Calbiochem (Nottingham, UK). Fura-2AM and Cascade Blue were from Molecular Probes (Leiden, The Netherlands). Forskolin, heparin, and poly-L-lysine were from Sigma. Tissue culture media, G-418, LipofectAMINE, and OptiMEM were from Life Technologies, Inc. The Biotrak cyclic AMP assay kit and D-myo-[2-³H] inositol were from Amersham Pharmacia Biotech.

Transfection and Cell Culture-- HEK 293 cells were transfected with the expression vector, pRezex-2, containing the complete coding sequence of the human type 1 PTH receptor (5). Cells expressing PTH receptors were selected by screening for those that responded to PTH with an increase in [Ca²⁺]_i (see below). Cells stably transfected with the PTH receptor (HEK/PTH-R1) were grown in a Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with fetal calf serum (10%), glutamine (2 mM), and G-418 (800 µg/ml). Responses to PTH were maintained during at least 40 passages.

Measurement of [Ca²⁺]_i-- HEK/PTH-R1 cells were plated (2.5 × 10⁶ cells/ml) onto glass coverslips coated with poly-L-lysine and grown for at least 2 days. Confluent cells were loaded with Fura-2 by incubation with Fura-2AM (1 µM, 60 min) dissolved in extracellular medium (ECM; 130 mM NaCl, 5.4 mM KCl, 0.8 mM NaH₂PO₄, 1.8 mM CaCl₂, 0.9 mM MgSO₄, 10 mM glucose, 20 mM Hepes, pH 7.4) at 20 °C for 45 min. The cells were then washed, and after 30 min in ECM, they were used for experiments. Previous experiments established that with this loading protocol, >95% of the Fura-2 fluorescence came from Fura-2 that was both cytosolic and hydrolyzed to its Ca²⁺-sensitive form (17). Single cell fluorescence imaging at 21 °C using IonVision software, corrections for background fluorescence, and calibration of fluorescence ratios (R_340/380) to [Ca²⁺]_i were performed as described previously (17). For microinjection with heparin, Fura-2-loaded cells bathed in nominally Ca²⁺-free ECM were injected with medium (27 mM K₂HPO₄, 8 mM NaH₂PO₄, 26 mM KH₂PO₄, pH 7.3) containing 10 mg/ml heparin and 1 mg/ml Cascade Blue as a marker for microinjected cells (18). After 30 min, cells were restored to Ca²⁺-containing ECM prior to measurement of [Ca²⁺]_i.

Measurement of Cyclic AMP Production-- Confluent cultures of HEK/PTH-R1 cells in 6-well plates were preincubated in ECM containing 3-isobutyl-1-methylxanthine (1 mM, 30 min). Agonists were then added, and the incubations were terminated after 10 min by the removal of the medium and the addition of cold 0.1 M HCl in 65% methanol (750 µl). After 30 min on ice, the contents of the wells were scraped, washed (750 µl acidified methanol) into tubes, and centrifuged (1000 × g, 30 min). The supernatants were dried under vacuum (60 °C, 4 h) and stored at 80 °C. A commercial enzyme-linked immunosorbent assay kit using the nonacetylation protocol specified by the manufacturer (Amersham Pharmacia Biotech) was used to determine the cyclic AMP contents of the samples.

Measurement of IP₃ Production-- HEK/PTH-R1 cells (2.5 × 10⁶ cells/ml) plated onto poly-L-lysine-coated glass coverslips were grown for 48 h in inositol-free Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum and D-myo-[2-³H] inositol (2 µCi/ml). Cells were washed twice in ECM and incubated for 15 s with the appropriate agents at 21 °C. Reactions were stopped by aspiration and addition of 0.5 ml of 1 mM ice-cold perchloric acid and kept on ice for 30 min. Acid extracts were neutralized and then loaded onto anion exchange columns from which the [³H]IP₃ fraction was eluted (18).

Analysis-- For most experiments, responses from 25-30 single cells on a coverslip were averaged. Statistical analyses were applied to the average results derived from independent measurements from several coverslips, where the sample size refers to the number of coverslips. Concentration-effect relationships were fitted to four-parameter logistic equations using least squares curve-fitting routines (Kaleidagraph, Synergy Software).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺ Mobilization Evoked by Type 1 PTH Receptors-- Stimulation of the endogenous muscarinic receptors of HEK 293 cells with carbachol (100 µM) evoked an increase in [Ca²⁺]_i irrespective of whether the cells had been transfected with PTH receptors (Fig. 1, A-C). PTH (1 µM), however, stimulated an increase in [Ca²⁺]_i only in HEK/PTH-R1 cells, and, in keeping with previous reports comparing PTH with agonists that stimulate IP₃ formation (9, 15, 19), the maximal response to PTH was only 21 ± 5% (n = 7) of that evoked by carbachol (Fig. 1B). In one previous study (19) but not in others (10, 11, 20, 21), it was claimed that HEK 293 cells express type 1 PTH receptors. Our results are consistent with most previous work in demonstrating that PTH evokes Ca²⁺ mobilization in HEK 293 cells only after transfection with type 1 PTH receptors (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   PTH stimulates release of intracellular Ca2+ stores in HEK/PTH-R1 cells. A-C, the effects of carbachol (100 µM, upper traces) and PTH (1 µM, lower traces) on [Ca2+]i were recorded in populations of mock-transfected cells (A) or HEK/PTH-R1 cells (B and C). Open bars denote the addition of PTH or carbachol. Extracellular Ca2+ was present for the recordings shown in A and B but absent from those shown in C. Each trace is representative of at least four similar recordings. D, a recording from a single cell is shown to illustrate typical increases in [Ca2+]i in a cell that responds both to PTH and carbachol (CCh).

Although the sustained phase of the Ca²⁺ signal evoked by carbachol and the peak amplitudes of the responses evoked by PTH or carbachol were reduced by removal of extracellular Ca²⁺ (Fig. 1, B and C), both agonists consistently evoked an increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺ (Fig. 1C), and responses to both were abolished after pretreatment with thapsigargin in Ca²⁺-free ECM (not shown). We conclude that both PTH and carbachol stimulate release of intracellular Ca²⁺ stores. Subsequent experiments addressed the mechanisms responsible for this Ca²⁺ mobilization.

Single cell analyses of HEK/PTH-R1 cells revealed that whereas 96 ± 5% (n = 12 coverslips) of cells responded to carbachol (100 µM) with an increase in [Ca²⁺]_i, only 27 ± 20% of cells responded to PTH (100 nM). In those cells that responded to PTH, the peak increase in [Ca²⁺]_i was 84 ± 16% (n = 11) of that evoked by carbachol in the same cells (Fig. 1D). Single cell analyses of HEK/PTH-R1 cells were used for all subsequent experiments (see "Experimental Procedures").

The magnitude of the responses to maximal concentrations of carbachol and PTH varied between experiments, although the relative amplitudes of the responses to the two stimuli were maintained. For each cell, we therefore expressed all responses as percentages of the response evoked by carbachol (100 µM) in normal ECM at the beginning of the experiment. After stimulation with carbachol in normal ECM, cells were allowed to recover for 5 min. Further stimulation then evoked indistinguishable responses; the peak increases in [Ca²⁺]_i were 93 ± 6% (n = 3) and 94 ± 8% (n = 3) of the initial response after the second and third stimulation with carbachol, indicating that responses to carbachol do not desensitize. In subsequent experiments, changes in [Ca²⁺]_i are expressed relative to the initial carbachol response recorded in normal ECM.

Cross-potentiation of [Ca²⁺]_i Signals by Carbachol and PTH-- PTH (100 nM) alone stimulated Ca²⁺ mobilization from only a fraction (27 ± 20%) of HEK/PTH-R1 cells, but it caused an increase in [Ca²⁺]_i in most cells (86 ± 12%, n = 12 coverslips) when added immediately after carbachol (100 µM) (Fig. 2B). From the average responses of each cell in the field, the peak rise in [Ca²⁺]_i evoked by PTH after carbachol was more than 10-fold greater than that evoked by PTH alone (160 ± 10%, n = 13; and 12 ± 7%, n = 11, respectively) (Fig. 2C). The interaction between PTH and carbachol was also evident when cells were first stimulated with PTH and then with carbachol; the peak rise in [Ca²⁺]_i evoked by carbachol alone was 71 ± 8% (n = 13) of the initial response, and after pretreatment with PTH it increased to 245 ± 10% (n = 8) (Fig. 2, A and C). Similar results were obtained in Ca²⁺-containing ECM (Fig. 2C).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Cross-potentiation of the Ca2+ mobilization evoked by PTH and carbachol in HEK/PTH-R1 cells. A and B, cells were first stimulated with carbachol (CCh) (100 µM) in Ca2+-containing ECM (reference response) before removal of extracellular Ca2+ and successive stimulation with PTH (100 nM) and then carbachol (100 µM) (A) or carbachol and then PTH (B). Traces show average responses of about 40 cells on a single coverslip and are typical of recordings from at least eight coverslips. C, results from experiments similar to those in A and B are summarized with the peak rise in [Ca2+]i (percentage of that initially evoked by carbachol in Ca2+-containing ECM) shown after simulation with carbachol or PTH alone or in rapid succession. Open bars denote results obtained in Ca2+-free ECM, and filled bars show responses in Ca2+-containing ECM. Results are means ± S.E. of 8-13 independent experiments.

PTH increased both the maximal amplitude and the sensitivity of the response to carbachol (Fig. 3A). The half-maximal effect (EC₅₀) of carbachol was reduced from 71 ± 6 µM (n = 3) in control cells to 25 ± 3 µM (n = 3) in the presence of PTH. The EC₅₀ for the effect of PTH in potentiating responses to carbachol (100 µM) was 7.8 ± 0.5 nM (Fig. 3B).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effects of PTH on carbachol-evoked Ca2+ mobilization in HEK/PTH-R1 cells. A, cells in Ca2+-free-ECM were stimulated with the indicated concentrations of carbachol alone () or after pretreatment with PTH (100 nM, 2 min) (). Results (means ± S.E., n = 3) show the peak rise in [Ca2+]i expressed relative to the reference response to carbachol (CCh). B, cells in Ca2+-free ECM were stimulated with carbachol (100 µM, 1 min) before addition of the indicated concentrations of PTH. The peak rises in [Ca2+]i detected after addition of PTH are shown (means ± S.E., n = 3) relative to the reference response to carbachol.

Because most HEK/PTH-R1 cells respond to PTH after carbachol pretreatment, whereas untransfected cells are unresponsive, we conclude that even though few HEK/PTH-R1 cells respond directly to PTH alone, most express functional PTH receptors. Carbachol unmasks a latent ability of PTH to stimulate release of intracellular Ca²⁺ stores, and PTH increases by 2-3 fold the Ca²⁺ mobilization evoked by carbachol, which alone stimulates Ca²⁺ mobilization in all cells. Potentiation of the responses to carbachol by PTH occurred despite [Ca²⁺]_i having returned to its basal level (Fig. 2A), indicating that an increase in [Ca²⁺]_i is not directly responsible for the effect.

PTH Allows Carbachol to Recruit Additional Ca²⁺ Stores-- In Ca²⁺-free ECM, carbachol caused a transient increase in [Ca²⁺]_i in HEK/PTH-R1 cells, but subsequent challenges with carbachol failed to increase [Ca²⁺]_i, consistent with the first stimulation having fully emptied the carbachol-sensitive Ca²⁺ stores (Fig. 4A). PTH alone also failed to increase [Ca²⁺]_i, when added 5 min after carbachol had been removed (Fig. 4A). However, a further challenge with carbachol after addition of PTH evoked a large transient increase in [Ca²⁺]_i, equivalent to 135 ± 15% (n = 3) of a standard response to carbachol (Fig. 4A). We conclude that PTH allows carbachol to release intracellular Ca²⁺ stores that would otherwise be insensitive to carbachol. Similar results were obtained when the order of addition of carbachol and PTH was reversed. PTH caused a substantial mobilization of intracellular Ca²⁺ stores when added immediately after carbachol had been removed, even though the carbachol itself evoked no increase in [Ca²⁺]_i because the carbachol-sensitive Ca²⁺ stores had already been emptied by prior stimulation (Fig. 4B).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Intracellular Ca2+ stores that are otherwise insensitive to carbachol can be released by a combination of carbachol and PTH. A, HEK/PTH-R1 cells were repeatedly stimulated with carbachol (100 µM) in Ca2+-free ECM and then with PTH (100 nM) before a further stimulation with carbachol (CCh). B, PTH added immediately after the second addition of carbachol evoked a substantial increase in [Ca2+]i, despite the lack of response to carbachol alone. Traces show the average changes in [Ca2+]i in at least 15 cells from a single experiment; similar results were obtained in three experiments.

Cross-potentiation of [Ca²⁺]_i Signals by ATP and PTH-- ATP also stimulates IP₃ formation and Ca²⁺ mobilization in HEK/PTH-R1 cells; the Ca²⁺ mobilization evoked by 10 µM ATP was 74 ± 5% (n = 7) of the standard carbachol response (Fig. 5A). Although only 71 ± 7% (n = 6) of cells responded to ATP, in the responsive cells the synergistic interaction with PTH was again evident. PTH added immediately after removal of ATP caused a substantial increase in [Ca²⁺]_i; the response to PTH was 160 ± 30% (n = 4) of the standard carbachol response (Fig. 5A). When PTH was added first, the increase in [Ca²⁺]_i evoked by subsequent addition of ATP was increased to 159 ± 28% (n = 3) of the standard carbachol response (Fig. 5B). There was no synergistic interaction between ATP and carbachol (not shown). Carbachol did not increase the fraction of cells that responded to ATP (73 ± 11% before carbachol, and 74 ± 4% after carbachol, n = 4), and in cells that responded to both stimuli, the increase in [Ca²⁺]_i evoked by combined application of carbachol and ATP (198 ± 5%) was similar to the sum of the responses evoked by either agonist alone (71 ± 5 and 97 ± 35% for carbachol and ATP, respectively).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Synergistic effects of ATP and PTH on Ca2+ mobilization in HEK/PTH-R1 cells. A, ATP (10 µM) evoked Ca2+ release in HEK/PTH-R1 cells and PTH (100 nM) added immediately after completion of the response to ATP evoked a substantial increase in [Ca2+]i. B, PTH itself caused a minimal increase in [Ca2+]i but massively potentiated that evoked by ATP. Similar results were obtained in three experiments. CCh, carbachol.

Mechanism of Action of PTH: the Role of IP₃ Receptors-- Responses to IP₃ are positively cooperative (22). PTH might therefore have potentiated responses to carbachol and ATP by either directly stimulating formation of a subthreshold level of IP₃ or by increasing the amount of IP₃ made in response to ATP or carbachol. Neither explanation seems likely because PTH (10 µM) neither directly stimulated detectable IP₃ formation (100 ± 23% of control, n = 6) nor affected the amount made in response to ATP (174 ± 23 and 166 ± 14% in the absence and presence of PTH) or carbachol (246 ± 31 and 266 ± 34%). Ca²⁺ stimulates IP₃ receptors (22), but this cannot explain the ability of PTH to potentiate responses to carbachol and ATP because the potentiation occurred without an increase in [Ca²⁺]_i (Figs. 2, 4, and 5).

Xestospongin, which has been shown to block cerebellar IP₃ receptors (23), proved not to be useful in resolving the involvement of IP₃ receptors in the response to PTH. A range of concentrations (10-50 µM) and preincubation periods (10-40 min) failed to separate direct effects of Xestospongin on the emptying of intracellular Ca²⁺ stores from inhibition of IP₃ receptors (not shown) (18). Two lines of evidence are, however, consistent with IP₃ receptors mediating the enhanced Ca²⁺ release evoked by ATP or carbachol with PTH. First, and in contrast with a previous report (10), microinjected heparin (10 mg/ml in the injection pipette) was similarly effective in blocking both the PTH- and carbachol-evoked increase in [Ca²⁺]_i (Fig. 6). Second, after combined application of ATP and carbachol to release the IP₃-sensitive Ca²⁺ stores, the subsequent response to PTH was almost abolished (down from 160 ± 10 to 25 ± 22%, n = 7).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Intracellular heparin abolishes responses to both PTH and carbachol. A, cells were microinjected with either heparin (10 mg/ml in the pipette) or control medium and allowed to equilibrate for at least 30 min. The traces show the average changes in [Ca2+]i evoked by carbachol (100 µM) or PTH (100 nM) in 19 control cells (dashed line) and 16 heparin-injected cells (solid line). B, The graph shows results from many experiments in which responses to carbachol (CCh, 100 µM) and PTH (100 nM) were compared in control and heparin-injected cells. Each point (the average of at least 10 cells) shows the response to each stimulus in heparin-injected cells expressed as a percentage of the response from control cells. The results indicate that heparin inhibits responses to both agonists to a similar degree.

A plausible explanation for these observations is that PTH generates a signal that causes IP₃ receptors to become more sensitive to IP₃. The increase in [Ca²⁺]_i evoked by PTH alone in some cells might then result from a sufficient basal level of IP₃ to cause Ca²⁺ mobilization from sensitized IP₃ receptors. The effects of PTH were persistent; even 10 min after removal of PTH, enhanced increases in [Ca²⁺]_i were observed after carbachol addition (not shown); and this presumably reflects the slow dissociation of PTH from its high affinity receptor (3). However, the ability of carbachol to enhance the rise in [Ca²⁺]_i evoked by PTH was short-lived and reversed within the ~10 s taken for the three washes required to completely remove the carbachol-containing medium, in keeping with the relatively low affinity of muscarinic receptors for carbachol and the rapid metabolism of IP₃.

Cyclic AMP Does Not Mediate the Effects of PTH on Ca²⁺ Mobilization-- PTH stimulates adenylyl cyclase activity in HEK 293 cells with an EC₅₀ of 20 ± 7 nM (n = 3; not shown); cyclic AMP might therefore have mediated the increases in [Ca²⁺]_i evoked by PTH. Cyclic AMP-dependent protein kinase (PKA) phosphorylates IP₃ receptors, and, although the functional consequences differ between cell types, in hepatocytes, which like HEK 293 cells express largely type 2 IP₃ receptors (24), phosphorylation increases the sensitivity of IP₃ receptors to IP₃ (25). Forskolin was previously shown not to mimic the effects of activating type 1 PTH receptors on [Ca²⁺]_i (10), but forskolin (5 µM) alone only very modestly increased intracellular cyclic AMP (from 0.53 ± 0.05 to 0.91 ± 0.13 pmol/well, n = 3) relative to the increase evoked by PTH (to 3.04 ± 0.05 pmol/well). Similarly modest effects of forskolin on the endogenous adenylyl cyclase activity of HEK 293 cells has been reported previously (26). However, several additional lines of evidence demonstrate that neither cyclic AMP nor PKA mediate the effects of PTH on intracellular Ca²⁺ stores. First, pretreatment with 8-bromo cyclic AMP (50 µM, 5 min) reduced the Ca²⁺ mobilization evoked by carbachol (Fig. 7A). Second, pretreatment of HEK/PTH-R1 cells with either of two inhibitors of PKA, Rp-8-Br-cAMPS (20 µM, 15 min) (27) or H89 (5 µM, 10 min) (28), affected neither the response to PTH alone nor its ability to potentiate the response to carbachol (Fig. 7B). That these inhibitors had effectively blocked activation of PKA is confirmed by their ability to reverse the inhibition of carbachol-evoked Ca²⁺ mobilization caused by 8-bromo-cAMP (Fig. 7A). Third, 3-isobutyl-1-methylxanthine, which by inhibiting cyclic AMP degradation would be expected to potentiate responses to PTH if they were mediated by cyclic AMP, had the opposite effect. Pretreatment with 3-isobutyl-1-methylxanthine (1 mM, 20 min) abolished the potentiation of carbachol responses by either submaximal (6 nM) or maximal (100 nM) concentrations of PTH (not shown). We conclude that the ability of PTH to potentiate carbachol-evoked Ca²⁺ mobilization is not mediated by cAMP. Indeed PKA, by a mechanism that we have not addressed further, appears to cause desensitization of either muscarinic or PTH receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   The effects of PTH on Ca2+ mobilization are not mediated by PKA. A, the increase in [Ca2+]i evoked by carbachol (CCh; 100 µM; percentage of relative response) is shown after pretreatment with the indicated combinations of 8-Br-cAMP (50 µM, 5 min), Rp-8-Br-cAMPS (20 µM, 5 min), and H89 (5 µM, 10 min). The results (expressed relative to the reference response to carbachol, means ± S.E., n = 3) demonstrate that the inhibition of carbachol-evoked Ca2+ mobilization caused by 8-Br-cAMP is reversed by inhibitors of PKA (Rp-8-Br-cAMPS and H89). B, experiments similar to those shown in Fig. 2 were used to determine the effects of preincubation with Rp-8-Br-cAMPS (20 µM, 5 min) or H89 (5 µM, 10 min) on the peak rise in [Ca2+]i evoked by carbachol (100 µM) and PTH (100 nM) alone or in rapid succession. The results (means ± S.E., n = 3) demonstrate that inhibition of PKA does not prevent PTH from potentiating responses to carbachol.

Conclusions-- We conclude, in keeping with previous work (29), that PTH-mediated Ca²⁺ mobilization does not result from production of IP₃; PTH does not stimulate detectable formation of IP₃, it has no detectable effect on the IP₃ formation evoked by ATP or carbachol, and the lack of synergy between ATP and carbachol indicates that the IP₃ formed in response to these receptors is incapable of mimicking PTH. Nor are the potentiated Ca²⁺ signals evoked by PTH mediated by cyclic AMP (Fig. 7), by Ca²⁺-induced sensitization of IP₃ receptors (Figs. 2, 4, and 5), or by phosphatidylinositol-3-kinase, because wortmannin (1 µM) did not prevent PTH from potentiating responses to carbachol (not shown). Our data are nevertheless consistent with IP₃ receptors being involved in responses to PTH (Fig. 6). We suggest that activation of type 1 PTH receptors stimulates formation of an intracellular messenger that allows more complete emptying of intracellular Ca²⁺ stores than is possible with maximal concentrations of either ATP or carbachol alone. To account for the larger maximal response to carbachol in the presence of PTH (Fig. 3A), we suggest that PTH may facilitate translocation of Ca²⁺ between discrete intracellular Ca²⁺ stores (Fig. 8), such that stores lacking IP₃ receptors are brought into continuity with those expressing them. Such a model would account for the small amount of Ca²⁺ release evoked by PTH alone (triggered by basal levels of IP₃) (Fig. 1C), for the increase in the size of the pool released by maximal concentrations of carbachol or ATP (Figs. 4 and 5B), and for the increased sensitivity of the stores to carbachol (Fig. 3A).

View larger version (9K): [in this window] [in a new window]   Fig. 8.   A possible mechanism for the effects of PTH on intracellular Ca2+ stores. The type 1 PTH receptor, via a mechanism that does not require cyclic AMP or PI-3-kinase, is proposed to allow IP3-sensitive Ca2+ stores to become linked to those lacking IP3 receptors. PTH thereby increases the size of the intracellular Ca2+ stores available to receptors (for ATP and carbachol (CCh)) that stimulate IP3 formation.

At present we can only speculate on the mechanisms linking PTH receptors to the transfer of Ca²⁺ between intracellular stores, although it is tempting to suggest that they may be related to the many reports linking small G proteins with Ca²⁺ translocation between intracellular stores. For example, previous studies had demonstrated that GTP facilitated transfer of Ca²⁺ between IP₃-sensitive and -insensitive stores by a process that required GTP hydrolysis and probably involved membrane fusion, the cytoskeleton, and a monomeric G protein (30-32). Inositol 1,3,4,5-tetrakisphosphate has also been reported to allow IP₃ to more completely empty intracellular Ca²⁺ stores and to perhaps thereby also promote increased capacitative Ca²⁺ entry (33, 34). Again monomeric G proteins are implicated, because the effects of inositol 1,3,4,5-tetrakisphosphate on IP₃-evoked Ca²⁺ mobilization appear to be mediated by a member of the GAP1 family of GTPase-activating proteins (33, 35). Indeed monomeric G proteins are involved in every aspect of intracellular membrane trafficking (36), including that between the organelles, endoplasmic reticulum and the Golgi, known to respond to IP₃ (37).

Most extracellular stimuli cause Ca²⁺ mobilization by stimulating formation of the IP₃ that gates intracellular Ca²⁺ channels. We suggest that PTH may be the first example of a hormone shown to regulate Ca²⁺ mobilization by regulating the size of the Ca²⁺ pool to which IP₃ has access.

	ACKNOWLEDGEMENT

We thank Gavin Winston, who was supported by a grant from the Physiological Society, for help with experiments.

	FOOTNOTES

^* This work was supported by Wellcome Trust Grant 039662.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.

To whom correspondence should be addressed. Tel./Fax: 44-1223-334058; E-mail: cwt1000@cam.ac.uk.

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; ECM, extracellular medium; H89, N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; HEK/PTH-R1 cells, human embryonic kidney 293 cells stably transfected with human type 1 PTH receptor; IP₃, inositol 1,4,5-trisphosphate; PKA, cyclic AMP-dependent protein kinase; Rp-8-Br-cAMPS, Rp-isomer of 8-bromo-adenosine 3',5'-cyclic monophosphorothioate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. C. Tovey, S. G. Dedos, E. J.A. Taylor, J. E. Church, and C. W. Taylor Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP J. Cell Biol., October 20, 2008; 183(2): 297 - 311. [Abstract] [Full Text] [PDF]

				A. Skupin, H. Kettenmann, U. Winkler, M. Wartenberg, H. Sauer, S. C. Tovey, C. W. Taylor, and M. Falcke How Does Intracellular Ca2+ Oscillate: By Chance or by the Clock? Biophys. J., March 15, 2008; 94(6): 2404 - 2411. [Abstract] [Full Text] [PDF]

				T. D. Werry, G. F. Wilkinson, and G. B. Willars Cross Talk between P2Y2 Nucleotide Receptors and CXC Chemokine Receptor 2 Resulting in Enhanced Ca2+ Signaling Involves Enhancement of Phospholipase C Activity and Is Enabled by Incremental Ca2+ Release in Human Embryonic Kidney Cells J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 661 - 669. [Abstract] [Full Text] [PDF]

				R. P. Guttmann, S. Sokol, D. L. Baker, K. L. Simpkins, Y. Dong, and D. R. Lynch Proteolysis of the N-Methyl-D-Aspartate Receptor by Calpain in Situ J. Pharmacol. Exp. Ther., September 1, 2002; 302(3): 1023 - 1030. [Abstract] [Full Text] [PDF]

				V. Correa, A. M. Riley, S. Shuto, G. Horne, E. P. Nerou, R. D. Marwood, B. V. L. Potter, and C. W. Taylor Structural Determinants of Adenophostin A Activity at Inositol Trisphosphate Receptors Mol. Pharmacol., April 16, 2001; 59(5): 1206 - 1215. [Abstract] [Full Text]

				K. A. Buckley, S. C. Wagstaff, G. McKay, A. Gaw, R. A. Hipskind, G. Bilbe, J. A. Gallagher, and W. B. Bowler Parathyroid Hormone Potentiates Nucleotide-induced [Ca2+]i Release in Rat Osteoblasts Independently of Gq Activation or Cyclic Monophosphate Accumulation. A MECHANISM FOR LOCALIZING SYSTEMIC RESPONSES IN BONE J. Biol. Chem., March 16, 2001; 276(12): 9565 - 9571. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Short, A. D. Articles by Taylor, C. W. Search for Related Content PubMed PubMed Citation Articles by Short, A. D. Articles by Taylor, C. W. Social Bookmarking                      What's this?