G protein-coupled, extracellular Ca2+ (Ca o 2+ )-sensing receptor enables Ca o 2+ to function as a versatile extracellular first messenger

The cloning of a G protein-coupled, extracellular Ca²⁺ (Ca _o ²⁺ )-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca _o ²⁺ on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca _o ²⁺ metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca²⁺, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca²⁺ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.     

Complex formation with the Type B gamma-aminobutyric acid receptor affects the expression and signal transduction of the extracellular calcium-sensing receptor. Studies with HEK-293 cells and neurons

We co-immunoprecipitated the Ca(2+)-sensing receptor (CaR) and type B gamma-aminobutyric acid receptor (GABA-B-R) from human embryonic kidney (HEK)-293 cells expressing these receptors and from brain lysates where both receptors are present. CaRs extensively co-localized with the two subunits of the GABA-B-R (R1 and R2) in HEK-293 cell membranes and intracellular organelles. Coexpressing CaRs and GABA-B-R1s in HEK-293 cells suppressed the total cellular and cell surface expression of CaRs and inhibited phospholipase C activation in response to high extracellular [Ca(2+)] ([Ca(2+)](e)). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca(2+)](e). The latter effects of the GABA-B-R2 on the CaR were blunted by coexpressing the GABA-B-R1. Coexpressing the CaR with GABA-B-R1 or R2 enhanced the total cellular and cell surface expression of the GABA-B-R1 or R2, respectively. Studies with truncated CaRs indicated that the N-terminal extracellular domain of the CaR participated in the interaction of the CaR with the GABA-B-R1 and R2. In cultured mouse hippocampal neurons, CaRs co-localized with the GABA-B-R1 and R2. CaRs and GABA-B-R1s also co-immunoprecipitated from brain lysates. The expression of the CaR was increased in lysates from GABA-B-R1 knock-out mouse brains and in cultured hippocampal neurons with their GABA-B-R1 genes deleted in vitro. Thus, CaRs and GABA-B-R subunits can form heteromeric complexes in cells, and their interactions affect cell surface expression and signaling of CaR, which may contribute to extracellular Ca(2+)-dependent receptor activation in target tissues.     

Calcium sensing receptors as integrators of multiple metabolic signals

Calcium sensing receptors are critical to maintenance of organismal Ca2+ homeostasis, translating small changes in serum Ca2+ into changes in PTH secretion by the parathyroid glands and Ca2+ excretion by the kidneys. Calcium sensing receptors are also expressed in many cells and tissues not directly involved in Ca2+ homeostasis where their role(s) are less defined. Recent studies have demonstrated that calcium sensing receptors integrate a variety of metabolic signals, including polyvalent cations, pH, ionic strength, amino acids, and polypeptides, making CaR uniquely capable of generating cell- and tissue-specific responses, sensing not only Ca2+, but the local metabolic environment. The challenge for future studies is to define CaR responsiveness in each varied physiological context.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Ca2+ as an extracellular signal in bone

Bone is the major sink and store for calcium and it fulfils essential roles in the maintenance of extracellular free ionised calcium concentration ([Ca2+]e) within its homeostatic range (1.1-1.3 mM). In response to acute hypercalcaemia or hypocalcaemia, Ca2+ is rapidly transported into or out of bone. Bone turnover (and therefore bone Ca2+ turnover) achieves the long-term correction of the [Ca2+]e by the metabolic actions of osteoblasts and osteoclasts, as they respectively incorporate or release Ca2+ from bone. These processes are regulated by the actions of hormones, such as parathyroid hormone (PTH), the release of which is a function of the [Ca2+]e, and is regulated by the action of the Ca2+-sensing receptor (CaR) in the parathyroid gland. Tissue culture studies indicate that bone cells also directly respond to increasing and decreasing [Ca2+]e in their vicinity, independently of the systemic factors. Nevertheless, further studies are necessary to identify how the acute and long-term local changes in [Ca2+]e affect bone cells and the physiological processes they are involved in. Also, the molecular mechanisms which enable the bone cells to sense and respond to [Ca2+]e are not clear. Like the parathyroid cells, bone cells also express the CaR, and accumulating evidence indicates the involvement of this receptor in their responses to the changing extracellular ionic environment.     

Cell surface, Ca2+(cation)-sensing receptor(s): one or many?

In mammals Ca2+ concentration in the extracellular fluids ([Ca2+]o) is essential for a number of vital processes varying from bone mineralization to blood coagulation, regulation of enzymatic processes, modulation of permeability and excitability of plasma membranes. For this reason [Ca2+]o is under strict control of a complex homeostatic system that includes parathyroid glands, kidneys, bones and intestine. The extracellular Ca(2+)-sensing receptor (CaR) is an essential component of this system, regulating parathyroid hormone secretion, calcium (and magnesium) excretion by the kidney, bone remodeling and Ca2+ reabsorption by the gastrointestinal tract. Structurally, the CaR is a novel member of a growing G protein-coupled receptor superfamily, which includes metabotropic glutamate receptors (mGluRs) [1], [gamma]-aminoisobutyric acid (GABA-B) receptors [2] and vomeronasal organ receptors [3]. Initially identified from bovine parathyroid glands [4], within the 5 years following its identification CaR presence has rapidly been identified as extending to organs where the link with mineral ion metabolism has not been elucidated (i.e. brain, stomach, eye, skin and many other epithelial cells) (see [5] for review). The role of the receptor in these regions is largely unknown, but it appears to be somewhat related to phenomena such as chemotaxis, cell proliferation and programmed cell death. This review will describe the discovery of a novel class of ion-sensing receptor(s), receptor-effector coupling and the roles of the CaR inside and outside the Ca2+o homeostatic system.     

New insights into the pharmacology of the extracellular calcium sensing receptor

The extracellular calcium-sensing receptor (CaR) belongs to class III of G-protein coupled receptors. The CaR is expressed at the surface of the parathyroid cells and plays an essential role in the regulation of Ca2+ homeostasis through the control of parathyroid secretion. The CaR is activated by Ca2+ and Mg2+ present in the extracellular fluids, various di- and trivalent cations, L-aminoacids and charged molecules including several antibiotics. Calcimimetics potentiate the effect of Ca2+ and are proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. Calcilytics block the Ca2+-induced activation of the CaR. Three-dimensional models of the seven transmembrane domains of the human CaR have been used to identify specific residues implicated in the recognition of calcimimetics and calcilytics. These molecules should be useful for delineating the physiological roles played by the CaR in several tissues and for clarifying the direct effects attributed to extracellular Ca2+.     

Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca(2+)-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca(2+)] ([Ca(2+)](o))-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca(2+)](o) to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca(2+)](o) in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca(2+)](o) even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca(2+)](o)-induced PLC activation and or CaR expression.     

Cellular "sensing" of extracellular calcium (Ca(2+)(o)): emerging roles in regulating diverse physiological functions

The extracellular Ca(2+) (Ca(2+)(o))-sensing receptor (CaR) critically influences Ca(2+)(o) homeostasis by regulating parathyroid hormone (PTH) secretion and renal Ca(2+) handling. Moreover, its expression in intestinal and bone cells suggests roles in all of the organs involved in maintaining systemic Ca(2+)(o) homeostasis. This G-protein coupled receptor is also expressed in a wide variety of additional cells throughout the body. While our understanding of its role(s) outside of the system governing Ca(2+)(o) metabolism remains rudimentary, the CaR will probably emerge as a versatile regulator of diverse cellular functions, including proliferation, differentiation, apoptosis, gene expression and maintenance of membrane potential. Finally, the recently developed, "calcimimetic" CaR activators, exemplified by a NPS R-467 and NPS R-568, provide novel approaches to treating diseases that previously had no effective medical therapies: topic likewise covered in this review.     

The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that "senses" extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT-PCR using four CaR-specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR-specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti-CaR antiserum. In addition, raising Ca2+o from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a "calcimimetic" CaR activator (R-467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca(+)-activated K+ channel having a unitary conductance of 84+/-4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.     

Extracellular calcium-sensing receptors in fishes.

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

Extracellular calcium-sensing receptors in fishes

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

pH Sensing by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is activated by small changes in the ionic extracellular calcium concentration (Ca(o)) within the physiological range, allowing the parathyroid gland to regulate serum Ca(o); however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and our previous studies suggest that charged residues in the extracellular domain of the CaR are critical for receptor activation through electrostatic interactions. Therefore, pH could also potentially modulate CaR activation by its polycationic agonists. Changes in the concentration of extracellular H(+) substantially altered the activation of the CaR by Ca(o) and other CaR agonists. The effects of external pH on the CaR's sensitivity to its agonists were observed for both acidic and basic deviations from physiological pH of 7.4, with increases in pH rendering the receptor more sensitive to activation by Ca(o) and decreases in pH producing the converse effect. At pH values more acidic than 5.5, CaR sensitivity to its agonists showed some recovery. Changes in the intracellular pH could not account for the effects of external pH on CaR sensitivity to its agonists. Other G-protein-coupled receptors, which are endogenously expressed in human embryonic kidney 293 cells, showed little change in activity with alterations in external pH or effects opposite those found for the CaR. Extracellular pH directly alters the CaR in the case of Ca(o) and Mg(o) activation; however, the charges on many organic and inorganic agonists are pH-dependent. Activating CaR mutations show reduced pH(o) modulation, suggesting a molecular mechanism for increased CaR activity at physiological pH(o). Several CaR-expressing tissues, including regions of the stomach, the kidney, bone, and the brain, could potentially use the CaR as a sensor for pH and acid-base status.     

Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is a key player in Ca(2+)(o) homeostasis. The activity of CaR can be potentiated by various l-amino acids. In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by l-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC(50) for stimulation by Ca(2+)(o) and a modest increase in its maximal activity. In addition, mutating Ser-169 and Ser-171 together with Ser-170 yielded a more complete block of the phenylalanine modulation than did the single mutation. The presence of the triple mutation, S169A/S170A/S171A, also eliminated phenylalanine potentiation of the activities of heterodimeric receptors in which one of the monomeric receptors had intact triple serines (A877Stop). The putative amino acid binding site of the CaR is probably close to or structurally dependent on the Ca(2+)(o) binding sites of the receptor, because mutant CaRs with mutations in the putative amino acid binding site exhibited severely reduced responses to Ca(2+)(o).     

Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line.

We examined recycling of heparan sulfate (HS) proteoglycans and transferrin receptor (Tf-R) in a rat parathyroid cell line. While extracellular Ca2+ concentration ([Ca2+]e) regulates the recycling of HS proteoglycans in parathyroid cells, such that HS proteoglycans only recycle when [Ca2+]e is lowered below physiological levels, recycling of Tf-R occurs equally well both in 0.05 mM (low) and 2 mM (high) [Ca2+]e. Inhibiting endocytosis chemically with phenylarsine oxide or at low temperature (4 degrees C) did not abolish the effects of changing [Ca2+]e on HS proteoglycans in the recycling compartment even though transport of HS proteoglycans from the Golgi complex to the cell surface was inhibited in low [Ca2+]e. Microtubules are not involved in the recycling of HS proteoglycans or of Tf-R since nocodazole did not affect these processes. Inhibiting the increase of intracellular Ca2+ by an intracellular Ca2+ chelator sustained recycling of HS proteoglycans even in the presence of high [Ca2+]e. These observations show that the exocytosis pathway of HS proteoglycans in the recycling compartment is specifically regulated by [Ca2+]e, whereas that for constitutive secretion is not. Therefore, the recycling of HS proteoglycans may be directly related to some functions of parathyroid cells regulated by [Ca2+]e. Although the mechanism by which [Ca2+]e regulates the exocytosis and recycling of HS proteoglycans is uncertain, it is suggested that an increase of intracellular Ca2+ is necessary, but not necessarily sufficient, for inhibiting their exocytosis.