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J. Biol. Chem., Vol. 279, Issue 37, 38151-38159, September 10, 2004

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L-Amino Acids Regulate Parathyroid Hormone Secretion^*

Arthur D. Conigrave¶, Hee-Chang Mun, Leigh Delbridge||, Stephen J. Quinn**, Margaret Wilkinson||, and Edward M. Brown**

From the School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia, the ^||Departments of Surgery and Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia, and the ^**Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, June 8, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca²⁺-sensing receptor (CaR). Thus, Ca²⁺ ions, and to a lesser extent Mg²⁺ ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, L-amino acids acutely and reversibly activated the extracellular Ca²⁺-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual L-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca²⁺ ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca²⁺ (Ca²⁺_o), or spermine. The order of potency was L-Trp = L-Phe > L-His > L-Ala > L-Glu > L-Arg = L-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca²⁺ concentrations. At a Ca²⁺_o of 1.1 mM and an amino acid concentration of 1 mM, CaR-active amino acids (L-Phe = L-Trp > L-His = L-Ala), but not CaR-inactive amino acids (L-Leu and L-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca²⁺_o to 1.2 mM. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture (from 1x to 2x) also reversibly suppressed PTH secretion in the Ca²⁺_o concentration range 1.05–1.25 mM. In conclusion, L-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that L-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular Ca²⁺ ions are recognized as the principal physiological regulators of parathyroid hormone secretion acting to close a classical endocrine feedback loop, whereby PTH¹ elevates Ca²⁺_o, and elevated Ca²⁺_o, in turn, suppresses PTH secretion. A major insight into the mechanism by which Ca²⁺_o interacts with parathyroid cells was gained with the cloning of the extracellular Ca²⁺-sensing receptor (CaR) (1). This receptor, which belongs to subgroup C of the G-protein-coupled receptor superfamily, mediates the Ca²⁺_o-stimulated activation of intracellular signaling pathways in parathyroid cells leading to the activation of various enzymes including phosphatidyl inositol-specific phospholipase C and attendant intracellular Ca²⁺ mobilization (for reviews, see Refs. 2 and 3). Targeted deletion of the CaR in the CaR null mouse eliminates feedback control of PTH secretion and results in a severe form of neonatal hyperparathyroidism (4). A similar condition in humans, neonatal severe hyperparathyroidism, has been shown to arise in homozygotes or compound heterozygotes with inactivating mutations of the CaR (5) (for review see Ref. 3).

Molecular analysis of the CaR indicates that it is composed of several functional domains that are conserved across related members of subgroup C. The N-terminal Venus Fly Trap domains of many of these receptors recognize amino acids (especially glutamate) or the amino acid analog, -aminobutyric acid. More recent work indicates that several cloned members of the family are also broad-spectrum amino acid sensors. In mammals, these include the CaR, which is allosterically activated by aromatic, aliphatic, and polar amino acids as well as plasma-like amino acid mixtures (6) and a heterodimeric amino acid taste receptor, which has broad selectivity for aliphatic, polar, and charged amino acids but not aromatic amino acids (7). In human embryonic kidney (HEK)293 cells that stably express the human CaR, L-amino acids markedly enhanced the sensitivity of the receptor to Ca²⁺ and other cationic agonists including spermine and Gd³⁺ (6).

Because the CaR mediates the acute control of PTH secretion, the finding that it is allosterically activated by L-amino acids raises the possibility that PTH secretion is acutely regulated not only by adjustments in extracellular Ca²⁺_o but also by physiological changes in amino acid concentration. We have tested and confirmed this hypothesis in the current study on normal human parathyroid cells. The data indicate that L-amino acids and plasma-like mixtures of L-amino acids allosterically activate endogenous parathyroid CaRs contributing to the control of intracellular signaling pathways and PTH secretion. In the presence of physiological concentrations of extracellular Ca²⁺, L-amino acids (also at physiological concentrations) stereoselectively activated Ca²⁺ mobilization and inhibited PTH secretion. The data support the view that fluctuations in serum amino acid levels acting via the CaR acutely regulate PTH secretion and thus whole body calcium metabolism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Origin of Tissue and Preparation of Human Parathyroid Cells— Samples of normal human parathyroid transplants were obtained at neck surgery at the Royal North Shore Hospital, St. Leonards, New South Wales, Australia and the Mater Private Hospital, North Sydney, New South Wales, Australia under guidelines established by the relevant institutional ethics committees. The parathyroid tissue, typically taking the form of a single 1–1.5-mm diameter "chip", was transported to the laboratory in ice-cold Hanks' balanced salt solution (Invitrogen) containing CaCl₂ 1.25 mM. It was either used immediately or stored overnight at 4 °C in MEM (number 11380-037, Invitrogen). The tissue was then transferred into 10 ml of MEM that contained 1 mg/ml collagenase (type I, Worthington, Scimar, Victoria) and 0.2 mg/ml DNase I (type IV, Sigma). The composition of the MEM formulation was as follows: NaCl 137 mM, KCl 5.4 mM, NaHCO₃ 4.2 mM, CaCl₂ 1.2 mM; MgSO₄ 0.81 mM; Na₂HPO₄ 0.34 mM, KH₂PO₄ 0.44 mM, D-glucose 1 g/liter, Phenol Red 10 mg/liter, MEM amino acids, and vitamins. Collagenase was stored at –80 °C and brought out for weighing on ice or dry ice. The suspension was briefly oxygenated and then incubated at 37 °C. After 20 min, the enzyme solution was decanted and the parathyroid tissue was transferred into 5 ml of MEM that contained 1 mg/ml of bovine serum albumin (protease-free, Sigma A-3059). The tissue was then triturated by repeated passage (10–15 times) through the tip of a disposable 5-ml syringe (no needle attached). The cloudy suspension containing clumps of parathyroid cells was passed through a 200-µm pore-size nylon filter and then sedimented (Sorvall H-1000B rotor, 100 x g, 2 min). The cell pellet was gently resuspended and washed twice in 5 ml of bovine serum albumin-containing MEM (Invitrogen, 11575-032). It was finally resuspended in bovine serum albumin-containing MEM. The remaining pieces of undigested parathyroid tissue were returned to medium containing collagenase and DNase I and incubated for a further 20 min at 37 °C. The enzyme solution was then decanted, and the parathyroid tissue subjected to trituration and centrifugal isolation as above. In general, the second cell harvest was used for analyses of cell function because of greater cell yields; however, the first and second cell harvests were also frequently combined.

Amino Acid Solutions—Stock amino acid-containing solutions were routinely made up in physiological saline at 100 or 200 mM with the exception of tryptophan (50 mM because of poor solubility). The amino acid composition of the 1x basal amino acid solution that emulated fasting human amino acids (all L-isomers in physiological saline solution, pH 7.4) was as follows (in µM): 50 Phe, 50 Trp, 80 His, 60 Tyr, 300 Ala, 200 Thr, 30 Cys, 50 Asn, 600 Gln, 125 Ser, 30 Glu, 250 Gly, 180 Pro, 250 Val, 30 Met, 10 Asp, 200 Lys, 100 Arg, 75 Ile, and 150 Leu. Stocks of this basal amino acid solution (40-fold concentrated in amino acids) were made up in physiological saline solution and stored at –20 °C. They were thawed and diluted in amino acid-free physiological saline as required. The control physiological saline used in all microfluorimetry experiments had the following composition: 125 mM NaCl, 4.0 mM KCl, 0.2 or 0.5 mM CaCl₂, 0.5 mM MgCl₂, 20 mM HEPES (NaOH), 0.1% D-glucose, pH 7.4.

Microfluorimetry for Determining Cytoplasmic Ca²⁺ Concentration— Parathyroid cells were loaded with fura-2/AM (1 µM, 20 min, 37 °C) in physiological saline solution containing bovine serum albumin 1 mg/ml. The cells were then sedimented (Sorvall H-1000B rotor, 100 x g, 2 min) and resuspended in albumin-free physiological saline solution. Fura-2/AM-loaded cells were transferred into a superfusion chamber and placed in the light path of a Nikon Diaphot microscope as described previously (8). Excitation was performed at alternating wavelengths (340 and 380 nm). The fluorescent light (F, peak 510 nm) was detected by a photomultiplier, and its digitized recording was achieved using Acknowledge software for Macintosh. The control superfusion solution had the following composition: 125 mM NaCl, 4.0 mM KCl, 0.2 or 0.5 mM CaCl₂, 0.5 mM MgCl₂, 20 mM HEPES (NaOH), 0.1% D-glucose, pH 7.4. Data for cytoplasmic free Ca²⁺ concentrations were expressed either as uncorrected excitation ratios (F340/F380) or converted to ionized Ca²⁺ concentration using a calibration procedure (8).

Determination of PTH Secretion—Perifusion of normal human parathyroid cells was undertaken in low molecular mass (4–5 kDa) cut-off gel filtration media so that intact PTH (9 kDa) would appear in the void volume. Gel filtration media were pre-equilibrated with physiological saline that contained 1x basal amino acid mixture and 1 mg/ml bovine albumin (protease-free, Sigma A-3905). 20,000–50,000 cells were loaded onto the surface of a 1-ml bed volume of Bio-Gel P-4 (nominal exclusion limit 4 kDa) and then gently covered with a 1-ml bed volume of Sephadex G-25 (medium, nominal exclusion limit 5 kDa) in a small perifusion column. After tubing connections were established downstream to a peristaltic pump and upstream to a reservoir, the column was suspended in a water bath (37 °C) and perifused at 1.5 ml/min with 37 °C equilibrated control physiological saline, 125 mM NaCl, 4.0 mM KCl, 1.25 mM CaCl₂, 1.0 mM MgCl₂, 0.8 mM NaH₂PO₄, 20 mM HEPES (NaOH, pH 7.4), 0.1% D-glucose, 1 mg/ml bovine albumin, 1x basal amino acid mixture. Prior to starting all experiments, the columns were perifused for a 20-min control period. Routinely, 2-min (i.e. 3-ml) samples were collected into tubes immersed in an ice bath, and the tubes were transferred to dry ice upon completion of each collection period. As required, solutions were changed to permit variations in extracellular calcium or amino acid concentration. All samples were stored at –80 °C until the analysis of intact human PTH using an Immulite 2000 autoanalyzer (Diagnostic Products Corporation, CA).

Statistical Analysis and Curve Fitting—The Ca²⁺ mobilization data were expressed as concentration-response curves and fitted to the following form of the Hill equation, R = b + (a – b)* Cⁿ/(eⁿ + Cⁿ), where R = fractional response, a = maximum fractional response, b = basal fractional response, C = extracellular Ca²⁺ concentration (in mM), e = EC₅₀ (the concentration of Ca²⁺ that induced a half-maximal response), and n = Hill coefficient.

The PTH secretion data were fitted to the following equation, S = a – (a – b)*Cⁿ/(iⁿ + Cⁿ) where S = secretory response, a = maximum response, b = basal response, C = extracellular Ca²⁺ concentration (in mM), i = IC₅₀ (the Ca²⁺_o concentration that yielded half-maximal inhibition of secretion), and n = Hill coefficient. The first of three or more data points, corresponding to a short equilibration period, was routinely ignored when calculating the mean secretion rate for any given treatment.

Estimates of the curve-fitting parameters and their standard errors were obtained using MacCurveFit 1.5 for Macintosh. Differences between the parameter values obtained from concentration-response curves were tested for statistical significance using the F-test as described in Ref. 9. Differences in PTH secretion rates were tested for statistical significance using the paired or unpaired t tests. Other data are routinely expressed as means ± S.E. (number of experiments).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effects of Individual Amino Acids on Cytoplasmic Free Ca²⁺ Concentration and Sensitivity to Ca²⁺_o—Exposure of fura-2/AM-loaded normal human parathyroid cells to active amino acids including L-Phe (0.1–10 mM) at an extracellular Ca²⁺ concentration ([Ca²⁺]_o) below 0.2 mM had no effect on cytoplasmic free Ca²⁺ concentration [Ca²⁺]_i (not shown). However, at [Ca²⁺]_o 0.2 mM, L-Phe (0.1–10 mM) acutely elevated [Ca²⁺]_i (Fig. 1A), and similar effects were observed with L-Trp (Fig. 1B), L-His, and L-Ala (not shown) but not L-Ile, L-Leu (Fig. 1B), or L-Arg (not shown). CaR-active amino acids exhibited similar effects in the presence of the polycationic CaR agonist, spermine (not shown). The effects of amino acids on intracellular Ca²⁺ mobilization were stereoselective i.e. L-amino acids were much more effective than D-amino acids (Fig. 1). In addition, the effects of L-amino acids were concentration-dependent (Table I, Fig. 2). At a Ca²⁺_o concentration of 2.0 mM, the apparent EC₅₀s for L-Trp, L-Phe, L-His, and L-Ala were 0.04 ± 0.01 mM, 0.06 ± 0.01 mM, 0.1 ± 0.01 mM, and 0.41 ± 0.02 mM, respectively, and the order of potency was L-Trp = L-Phe > L-His > L-Ala > L-Glu > L-Leu = L-Arg (Table I). Individual L-amino acids were also effective at lower extracellular Ca²⁺ concentrations (1.5 and 1.0 mM); however, the EC₅₀ values increased progressively, and the amino acid-induced maximum responses fell progressively as the Ca²⁺ concentration fell (Fig. 2, C and D, Table I). An appreciation of the physiological significance of these effects can be gained by dividing the fasting plasma concentration of an amino acid by its EC₅₀ value (conc/EC₅₀). At 2.0 mM Ca²⁺_o, four of the seven amino acids tested, L-Trp, L-Phe, L-His, and L-Ala all exhibited conc/EC₅₀ ratios close to 1.0. At 1.5 and 1.0 mM Ca²⁺_o, the apparent effectiveness of these individual amino acids fell progressively (Table I). Between 1.0 and 1.5 mM Ca²⁺_o, encompassing the normal physiological range, the data indicate that physiologically significant modulation of the CaR could only arise if multiple CaR-active amino acids acted together in concert.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Acute reversible effects of L-amino acids on fura-2/AM-loaded normal human parathyroid cells. Parathyroid cells were prepared by collagenase digestion and loaded with fura-2/AM as described under "Experimental Procedures." The base-line extracellular Ca2+ concentration was 0.5 mM. A, effects of 10 mM D-Phe and L-Phe on intracellular Ca2+ mobilization. B, effects of 10 mM L-Ile, L-Leu, D-Trp, and L-Trp on intracellular Ca2+ mobilization. At 10 mM, both D-Trp and L-Trp also suppressed the sustained phase of the cytoplasmic free Ca2+ response as demonstrated in this record by the suppression in the base line upon the addition of D-Trp and transient overshoot upon the removal of L-Trp.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Concentration-dependent activation of intracellular Ca2+ mobilization in normal human parathyroid cells by L-Phe and L-Trp. A, the effect of stepwise increments in L-Phe concentration in the presence of 1.5 mM Ca2+. B, the effect of stepwise increments in L-Trp concentration in the presence of 1.5 mM Ca2+. C, concentration dependence of L-Phe-induced intracellular Ca2+ mobilization at various extracellular Ca2+ concentrations (1.0, 1.5, and 2.0 mM). D, concentration dependence of L-Trp-induced intracellular Ca2+ mobilization at various extracellular Ca2+ concentrations (1.0, 1.5, and 2.0 mM).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effect of L-Phe on extracellular Ca2+ sensitivity in normal human parathyroid cells. Parathyroid cells were prepared by collagenase digestion and loaded with fura-2/AM as described under "Experimental Procedures." A, the effect of stepwise increments in extracellular Ca2+ concentration (from 0.2 to 8.0 mM) in the absence or presence of 3 mM L-Phe; the base-line extracellular Ca2+ concentration was 0.2 mM. The arrowhead indicates the point of addition of 3 mM L-Phe in the presence of 0.2 mM Ca2+. B, concentration response curves generated from four experiments in which the data like those shown in (A) were obtained.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Impact of various -fold concentrations of a plasma-like amino acid mixture on intracellular Ca2+ mobilization and extracellular Ca2+ sensitivity. A, effect of increasing the -fold concentration of a plasma-like amino acid mixture on cytoplasmic free Ca2+ concentration in the presence of physiological saline containing 1.25 mM Ca2+; basal amino acid 1x (baa) corresponds to the amino acid composition of fasting plasma. B, effect of stepwise increments in [Ca2+]o (from 0.2 to 8.0 mM) in the absence or presence of various -fold concentrations of a fasting human plasma-like L-amino acid mixture including i, 0; ii, 0.2x; iii, 1x; iv, 2x; v, 5x. The arrowhead indicates the point of addition of the amino acid mixtures. C, impact of variations in -fold concentration of the plasma-like L-amino acid mixture on the extracellular Ca2+ concentration dependence of fura-2/AM-loaded cells. The -fold concentrations were, respectively, 0 (), 0.2x (), 0.5x (), 1x (), 2x (), and 5x (). The data were obtained from eight control experiments and four experiments for each of 0.2–5x. The error bars (all 20%) have been omitted for clarity. D, -fold concentration dependence of the receptor response to plasma-like amino acid mixtures at various Ca2+o concentrations between 0.5 and 2.0 mM.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Impact of elevated levels of L-Phe and L-Trp on PTH secretion from perifused parathyroid cells. Normal human parathyroid cells were perifused with physiological saline solution containing the 1x L-amino acid mixture and 1 mg/ml bovine serum albumin at 37 °C. The data in A and B are representative experiments showing acute responses to various concentrations of extracellular Ca2+ in the absence or presence of 3 mM L-Phe or 3 mM L-Trp, respectively. The data in C were derived from four L-Phe experiments and three L-Trp experiments. The experimental series in A and B were undertaken separately and exhibited an 2-fold difference in the maximum secretion rate when corrected for cell number. Accordingly, the PTH secretion data in C were normalized to the percent of maximum. The symbols are as follows, open circles, pre- and post-controls in cells exposed to L-Phe; open triangles, pre- and post-controls in cells exposed to L-Trp; closed circles, L-Phe; and closed triangles, L-Trp. The control physiological saline solution contained the 1x amino acid mixture as well as bovine serum albumin (1 mg/ml).

View larger version (36K): [in this window] [in a new window]   FIG. 6. Impact of various amino acids and impact of an increase in the -fold concentration of the plasma-like amino acid mixture on PTH secretion. Normal human parathyroid cells were perifused with physiological saline solution containing the 1x L-amino acid mixture and 1 mg/ml bovine serum albumin at 37 °C. The data (means ± S.E.) in A and B were derived from three experiments in each case and show acute responses to various amino acids (all 1.0 mM). The cells exhibited marked sensitivity to a small change in extracellular Ca2+ concentration (from 1.2 to 1.1 mM) as well as sensitivity to CaR-active L-amino acids (see also Table III). In C the effect of an elevation in the -fold concentration of the amino acid mixture from 1 to 2x and the reversibility of this effect is shown from data obtained in five experiments in which cells were exposed to repetitive stepwise changes in ionized Ca2+o concentrations 1.25, 1.15, 1.05, and 1.25 mM. Basal amino acid 1x (baa) corresponds to the amino acid composition of fasting plasma.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The data described in this study support the conclusion that physiologically relevant increases in the concentrations of L-amino acids acutely activate the extracellular Ca²⁺-sensing receptor and reversibly inhibit PTH secretion from normal human parathyroid cells. These findings indicate that PTH secretion is under the acute physiological control of serum L-amino acid levels and provide a theoretical basis for a link between protein and calcium metabolism.

L-Amino acids, including L-Phe, L-Trp, and L-Ala but not L-Arg, L-Leu, and L-Ile stereoselectively activated intracellular Ca²⁺ mobilization from fura-2/AM-loaded normal human parathyroid cells in the presence of the CaR agonist Ca²⁺_o (Figs. 1 and 2, Table I). In addition, CaR-active amino acids markedly enhanced the sensitivity of the receptor to Ca²⁺_o (Figs. 3 and 4, Table II). The amino acid selectivity of these responses, L-Trp = L-Phe > L-His > L-Ala = L-Glu > L-Leu = L-Arg, closely resembles that described previously for the cloned human CaR stably expressed in HEK293 cells (6, 10). In addition, an L-amino acid mixture that emulated the composition of fasting human plasma also activated intracellular Ca²⁺ mobilization and enhanced the sensitivity of the receptor to extracellular Ca²⁺ (Fig. 4). A threshold extracellular Ca²⁺ concentration for the effects of plasma-like L-amino acid mixtures was detected around 0.5 mM (Fig. 4). The Ca²⁺ mobilization response to a change in -fold concentration of the plasma-like L-amino acid mixture from 0.5 to 2x (the range normally described between protein restriction and high protein intake) took the form of a small decrease in EC₅₀ (from 1.7 to 1.6 mM) and an increase in maximal response. A decrease in the EC₅₀ for Ca²⁺_o of 0.1 mM would be expected to induce a physiologically significant reduction in the Ca²⁺_o set-point (i.e. IC₅₀) for PTH secretion, because the normal range for ionized Ca²⁺_o is extremely tight (1.1–1.3 mM). Furthermore, an increase in the maximum response of the receptor would be expected to lower the minimum level of PTH secretion observed at high Ca²⁺_o. The observed effects of CaR-active amino acids on the IC₅₀ for Ca²⁺_o and minimum level of PTH secretion (Figs. 5 and 6) are consistent with both predictions.

CaR-active amino acids including L-Phe, L-Trp, L-His, and L-Ala stereoselectively suppressed PTH secretion from normal human parathyroid cells that were perifused with physiological saline solution that contained a 1-fold mixture of L-amino acids that emulated normal fasting human plasma (11–13). CaR-inactive amino acids including L-Arg and L-Leu had no inhibitory effect (Fig. 6, A and B, Table III). Consistent with the concept that aromatic and aliphatic L-amino acids are allosteric activators of the CaR, L-Phe enhanced the Ca²⁺ concentration dependence of intact PTH secretion, lowering the IC₅₀ for Ca²⁺_o from 1.15 to 1.05 mM. The inhibitory effects of L-Phe and L-Trp (Fig. 5) as well as the inhibitory effect of raising the -fold concentration of the plasma-like amino acid mixture from 1 to 2x (Fig. 6) were observed in the physiological Ca²⁺_o concentration range i.e. from 1.1 to 1.3 mM. The data indicate that amino acids are also effective in the wider pathophysiological range from 0.8 to 1.5 mM and above.

In the current work the observed effects of amino acids are not simply pharmacological, because plasma-like amino acid mixtures, as well as individual amino acids, are effective CaR activators (Figs. 4 and 6, Table II). The effects are also not simply "permissive," because the response of the receptor was clearly submaximal in the presence of the 1x mixture (Figs. 4 and 6, Table II). The conclusion that physiologically relevant fluctuations in the plasma levels of amino acids modulate the Ca²⁺_o sensitivity of CaR and parathyroid hormone secretion relies on the observed impact of variations in amino acid concentration in the context of a normal fasting amino acid mixture. Most important was the observed impact of an increase in the -fold concentration of the plasma-like amino acid mixture from 1 to 2x, which reversibly suppressed PTH secretion by 25–40% at all ionized Ca²⁺_o concentrations tested in the range 1.05–1.25 mM (Fig. 6C). A -fold concentration change of this magnitude approximates what is observed in response to a protein-rich meal (11, 13–15) or between the 24-h trough and peak levels of the normal circadian rhythm (16, 17). In addition, the observed inhibitory effects of individual L-amino acids on PTH secretion in the presence of the 1x mixture (Figs. 5 and 6, Table III) indicate that there is a "reserve" amino acid-inducible sensitivity even under normal physiological conditions.

Links between protein and calcium metabolism have been previously identified including evidence that elevated dietary protein intake promotes urinary calcium excretion (18, 19) and reduced protein intake induces secondary hyperparathyroidism in the absence of changes in plasma-ionized Ca²⁺ concentration (20). The finding that the CaR in normal human parathyroid cells is modulated by physiologically relevant variations in amino acid levels provides a possible explanation for secondary hyperparathyroidism in human subjects on moderately low protein diets (20). In this respect, the impact of fluctuations in the concentrations of CaR-active amino acids on PTH secretion in vivo requires analysis. Similar modulation of CaRs by amino acids in renal thick ascending limb cells, which are a major site of CaR-regulated calcium transport (for review see Ref. 2), may contribute to the mechanism by which elevated protein intake provokes hypercalciuria.

Although the impact of -fold concentrations of plasma-like amino acid mixtures on PTH secretion has obvious physiological implications, the selectivity of the CaR for aromatic and aliphatic amino acids seems puzzling. It might be wondered whether situations arise physiologically whereby selective elevations or reductions in the serum levels of these amino acids are associated with, respectively, suppressed or elevated serum levels of PTH. The established patterns of the circadian rhythms for extracellular Ca²⁺, PTH, and L-amino acids may provide an answer. Normal human subjects exhibit a highly reproducible nocturnal peak in the serum PTH level that occurs independent of changes in plasma ionized Ca²⁺ concentration (21, 22). The origin of this peak, which is reported to be defective in patients with osteoporosis (23) and is not driven by changes in plasma-ionized Ca²⁺ concentration (24, 25), is currently unclear. However, it coincides with a 30% drop in the serum levels of aromatic amino acids (16, 26). The data described in the current study indicate that this association may be causative i.e. the nocturnal drop in the serum levels of aromatic and other amino acids may drive the nocturnal peak in PTH. In turn, then octurnal PTH peak may drive the osteoblast-dependent synthesis of bone in a manner comparable with that described for daily subcutaneous injections of PTH in patients with osteoporosis (27).

The finding that normal parathyroid cells are regulated by physiologically relevant variations in amino acid levels indicates that fluctuations in amino acid levels may modulate CaR-dependent cell function in other settings. With respect to whole body calcium metabolism, this may include CaR-expressing cells of the renal tubules as discussed above (28). Amino acids may also regulate CaR-expressing cells in the gastroin-testinal tract such as gastrin-secreting G cells (29) and acid-secreting gastric parietal cells (30). Exposure of these cells to elevations in amino acid levels following the ingestion, digestion, and absorption of a protein-rich meal results in enhanced gastrin release and gastric acid production, respectively (see Ref. 31).

In conclusion, the evaluation of the Ca²⁺_o-sensing receptor in normal human parathyroid cells, confirms its sensitivity to L-amino acids (6, 10). CaR-active L-amino acids acutely and reversibly inhibit PTH secretion in physiologically relevant concentration ranges for ionized Ca²⁺_o and amino acids. Amino acids, like Ca²⁺ ions, should be viewed as physiological regulators of parathyroid hormone secretion.

	FOOTNOTES

 
^* This work was supported by the NHMRC of Australia and Grants DK41415, DK48330, and DK52005 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ Received early mentoring in endocrine research from Prof. Niels A. Thorn of the University of Copenhagen, Denmark. To whom correspondence should be addressed: School of Molecular and Microbial Biosciences (G08), University of Sydney, New South Wales 2006, Australia. Fax: 612-9351-4726; E-mail: a.conigrave{at}mmb.usyd.edu.au.

¹ The abbreviations used are: PTH, parathyroid hormone; CaR, Ca²⁺-sensing receptor; MEM, minimal Eagle's medium.

	ACKNOWLEDGMENTS

 
We thank Linda Critchley and Cameron Wood for the excellent technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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