Chapter 11 Regulation of cellular functions by extracellular calcium

Chapter 11

Regulation of Cellular Functions by Extracellular Calcium EDWARD F. NEMETH

Introduction Regulation of Systemic Ca Metabolism The Parathyroid Cell The C-Cell The Osteoclast Other Extracellular Ca^'*'-Sensing Cells Therapeutic Significance of Extracellular Ca "^ Receptors Summary

285 286 287 294 295 297 300 300

INTRODUCTION A biological phenomenon of increasing recognition is the peculiar ability of extracellular Ca^"^ to regulate the activity of certain specialized cells in the body. While most cells are insensitive to physiological changes in the level of Ca^"^ in the plasma or extracellular fluids, there are a variety of different cell types that can alter their behavior in response to changes in the extracellular Ca^^ concentration. Not

Principles of Medical Biology, Volume 4 Cell Chemistry and Physiology: Part II, pages 285-304 Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-806-4

285

286

EDWARD F. NEMETH

surprisingly, many of these cells are involved in the regulation of systemic Ca^"*" homeostasis. Notable among these is the parathyroid cell, whose secretory product, parathyroid hormone, plays a major role in regulating the level of plasma Ca^"^. Other cells sensitive to changes in the concentration of extracellular Ca^"^ are the parafollicular cells of the thyroid that secrete calcitonin and osteoclasts in the skeleton that resorb bone. Certain cells in the kidney, the gastrointestinal tract, the skin, and placental tissue also seem to be responsive to changes in the concentration of extracellular Ca^"^. In fact, for the parathyroid cell, extracellular Ca^"*" is the primary physiological stimulus regulating cellular function. The growing appreciation of the array of different cell types capable of sensing changes in the level of extracellular Ca^"^ has led to the concept that Ca^"^ can function as an extracellular signal, not unlike a hormone or neurotransmitter. This view complements the well-known messenger role of intracellular Ca^"^. Thus, just as intracellular Ca^"^ functions to control a variety of cellular functions as diverse as muscle contraction and cellular secretion, so too does extracellular Ca^^ function to regulate the activity of certain cells in the body. The action of extracellular Ca^"*" on some of these cells involves interaction with a cell surface Ca^"^ receptor protein which is coupled to effector mechanisms that regulate intracellular signals such as Ca^"^, diacylglycerol, and cyclic AMP. Extracellular Ca^"*" receptors are therefore functionally and mechanistically akin to more conventional membrane receptors that initially transduce changes in the concentration of an extracellular ligand into intracellular signals that regulate functional cellular responses. The difference is that the ligand for Ca^"*" receptors is an inorganic ion rather than an organic molecule or protein. This chapter will summarize the data suggesting a messenger role for extracellular Ca^"*" in regulating the activity of diverse cell types. Although all the cells discussed herein have been shown to respond to changes in the concentration of extracellular Ca^"^, the physiological significance of this response is not always obvious. By far the clearest understanding of the molecular events that enable a cell to detect and respond to extracellular Ca^"^, and its physiological significance, derives from studies of cells involved in the regulation of systemic Ca^"^ metabolism, especially parathyroid cells.

REGULATION OF SYSTEMIC Ca^^ METABOLISM Just as intracellular Ca^"^ functions to regulate a variety of cellular responses, so too does extracellular Ca^"*" function to control a variety of life-sustaining functions. Extracellular levels of Ca^"^ are important in maintaining the excitabili^ of nerve and muscle, in permitting thrombosis and cellular adhesion in general, and in proper bone formation. Because of this, the concentration of Ca^"*" in the plasma and extracellular fluids is under tight homeostatic control. In mammals, the level of Ca^"*" in the plasma and exfracellular fluids accounts for only a small percentage (about 0.1%) of the total body systemic calcium content, with

Regulation of Cellular Functions

287

the bulk (99%) stored in the teeth and bones. In humans, the concentration of total calcium in the plasma is 2.4 mM, but only about half of this (1.3 mM) is free, ionized calcium (Ca^"*"). Calcium binds to serum proteins (mostly albumin and globulins) and to various inorganic anions (mostly phosphate and citrate) and in this bound form, calcium is generally considered to be biologically inert. It is the concentration of ionized calcium in the plasma that regulates physiological responses and is the relevant variable sensed by the homeostatic control mechanism. The predominant control mechanism is endocrine and the principal factors regulating the level of plasma Ca^"^ are parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3. PTH, secreted by cells in the parathyroid gland, guards against hypocalcemia. PTH acts to increase the movement of Ca^"^ from bone to the circulation, and it additionally acts on the kidney to increase distal tubular calcium resorption and proximal tubular synthesis of 1,25-dihydroxyvitamin D3; the latter increases intestinal absorption of Ca^"^. All these actions tend to increase the level of Ca^"^ in the plasma. Increased circulating levels of Ca^"^, in turn, act in a negative feedback capacity to depress secretion of PTH. There is, therefore, a reciprocal relationship between the levels of plasma PTH and Ca^"^, and this simple yet elegant feedback loop is the principal mechanism regulating the level of plasma Ca^"^ (Mundy, 1989). In some species, an additional endocrine factor seems to play an important role in regulating plasma Ca^"^ homeostasis. This is the hormone calcitonin, secreted by parafollicular cells present throughout the thyroid gland. Like PTH, the secretion of calcitonin is regulated by changes in the level of plasma Ca^"^. The difference is that increasing the concentration of extracellular Ca^^ stimulates calcitonin secretion, whereas it inhibits PTH secretion. One site of action by calcitonin is in the kidneys where it stimulates excretion of Ca^"^. The predominant site of action of calcitonin, however, is in bone where it acts to inhibit ongoing osteoclastic bone resorption. This latter action causes a rapid inhibition of Ca^^ flux from bone into the circulation and this results in hypocalcemia. The physiological significance of this effect of calcitonin in adult humans is generally believed to be minor. Nonetheless, calcitonin can be used in pharmacological doses to inhibit bone resorption and is one treatment for bone diseases involving increased bone turnover, such as osteoporosis.

THE PARATHYROID CELL This is the classic cell type long known to be responsive to physiological changes in the concentration of plasma Ca^"^. Perhaps because PTH plays such a crucial role in regulating the level of plasma Ca^"^, its secretion is most responsive to the ambient Ca^^ concentration. In humans and some other species, PTH secretion can be increased by P-adrenergic receptor agonists, but the physiological significance of this is probably minor. The parathyroid glands do not receive significant neural input and, under physiological conditions, PTH secretion is not affected by a wide variety of neurotransmitters, hormones, or other extracellular signaling molecules

288

EDWARD F. NEMETH

(in contrast to calcitonin secretion). It seems safe to say that extracellular Ca^"*" is the primary physiological stimulus regulating PTH secretion. The sensitivity of the parathyroid cell to the ambient Ca^^ concentration is remarkable: minimal and maximal rates of PTH secretion are obtained over a concentration range spanning only 1.5 mM. Significantly, the concentration of extracellular Ca^"^ causing halfmaximal inhibition of PTH secretion or the "set-point" for extracellular Ca^"^, is set precisely near normocalcemic levels (1.3 mM). Moreover, small changes in the level of extracellular Ca^"*" cause rapid (< 1 minute) changes in the rate of secretion of PTH (Brown et al., 1987). Thus, the parathyroid cell is exquisitely constructed to sense and rapidly respond to small, physiological changes in the concentration of extracellular Ca^"*". There have, therefore, been two distinct but related problems in understanding the cellular physiology of parathyroid cells: how do these cells detect such small changes in the concentration of extracellular Ca^"^ and how is this initial recognition event transduced into intracellular signals that regulate PTH secretion? Since the depressive effects of extracellular Ca^"*" on PTH secretion are observed in vitro using dissociated parathyroid cells, it is clear that extracellular Ca^"^ acts directly on parathyroid cells to regulate hormone secretion. While this has been known for many years, it is only quite recently that we have gained some insight into the molecular mechanisms used by parathyroid cells to sense extracellular Ca^"^ levels and thereby regulate PTH secretion. Studies undertaken during the 1970s and early 1980s using dissociated bovine and porcine parathyroid cells demonstrated that agents that cause increases in the levels of cyclic AMP stimulate PTH secretion (Brown et al., 1987). These agents included P-adrenergic agonists, dopamine, prostaglandin E2, and cholera toxin. In contrast, agents that decrease cellular cyclic AMP levels, such as a-adrenergic agonists and prostaglandin F2a, inhibit PTH secretion. Additional studies have suggested that cyclic AMP and extracellular Ca^"*" may regulate secretion of PTH from different intracellular pools: cyclic AMP regulates secretion from a storage pool, whereas extracellular Ca^"^ regulates secretion of PTH from a newly synthesized pool (Watson and Hanley, 1993). It is significant, however, that the magnitude of these responses (cyclic AMP levels and PTH secretion) are dependent on the concentration of extracellular Ca^"*" and increased levels of extracellular Ca^"*" block agonist-induced increases in cyclic AMP and PTH secretion. Moreover, extracellular Ca^"^ alone, while causing large changes in the secretion of PTH, causes relatively small changes in basal levels of cyclic AMP and does not alter the pattern of protein phosphorylation induced by cyclic AMP. Thus, there is an additional mechanism(s) used by extracellular Ca^"^ that can regulate PTH secretion independently of changes in cyclic AMP levels. There is considerable interest in the role cytosolic Ca^"^ may play in the regulation of PTH secretion. Increasing the concentration of extracellular Ca^^ evokes corresponding increases in the concentration of cytoplasmic Ca^"^ ([Ca^"*"]i)

Regulation of Cellular Functions

289

and these are associated with an inhibition of PTH secretion (Shoback et al., 1984). The inverse relationship between [Ca^"^]! and secretion is yet another peculiar aspect of parathyroid cell physiology. In most cells, increasing [Ca^"^]i evokes a stimulation of secretion. This general finding has led to the Ca^"^ hypothesis of stimulussecretion coupling which holds that cytosolic Ca^"^ activates or permits exocytotic secretion in diverse cell types (Douglas, 1974). In parathyroid cells, and in some other cells that appear to sense the ambient level of extracellular Ca^"^ (discussed below), cytoplasmic Ca^"^ appears to inhibit secretion. However, the exact role of cytoplasmic Ca^^ in controlling PTH secretion is far from clear. Studies in permeabilized parathyroid cells, in which Ca^"^ has direct access to the exocytotic machinery, have reported either no effect or a stimulation of PTH secretion when exposed to low levels of Ca^"^ that occur within the cell. Additionally, there is data from intact cells suggesting that cytosolic Ca^"^ can have both stimulatory (at low [Ca^"^]!) and inhibitory (at higher [Ca^^i) effects on PTH secretion (for review see Brown, 1991). Thus, there are numerous pieces of data suggesting some important signaling role for C3^oplasmic Ca^"^ in parathyroid cells, but the data are often discrepant and no explanatory model has yet emerged. Relatively more progress has been made in understanding the initial steps in stimulus-secretion coupling, namely, how parathyroid cells sense a change in the ambient Ca^"^ concentration and how this detection event is coupled to the regulation of intracellular signals. That extracellular Ca^^ might act through some receptor-like mechanism was initially suggested in 1983 based on electrophysiological measurements (LopezBameo and Armstrong, 1983). Measurements of [Ca^"^]i, however, provided more substantial evidence for an extracellular Ca^"^ receptor and led to a series of biochemical studies consistent with this notion. It was demonstrated that increases in [Ca^"^]i elicited by extracellular Ca^"^ arise from two mechanistically distinct events: the mobilization of intracellular Ca^"^ from a nonmitochondrial pool and the influx of extracellular Ca^"^ through voltage-insensitive channels (Nemeth and Scarpa, 1986,1987a). Moreover, a variety of extracellular di- and trivalent cations were all capable of causing the mobilization of intracellular Ca^"^ in parathyroid cells. Because trivalent cations are impermeant in parathyroid cells, and in cells generally, they must be acting at the cell surface to evoke the mobilization of intracellular Ca^^. Studies using monoclonal antibodies generated against parathyroid cells likewise suggested an action of extracellular Ca^"^ at the cell surface (Gylfe et al., 1990). Together, these results suggested the presence of a Ca^"^ receptor on the surface of parathyroid cells that is coupled to the mobilization of intracellular Ca^"^. Subsequent biochemical studies showed that increased levels of extracellular Ca^"^ evoked rapid increases in the formation of inositol 1,4,5-trisphosphate and diacylglycerol (Brown et al., 1990), two biochemical hallmarks of receptordependent mobilization of intracellular Ca^"^ in various other cells (Berridge, 1987). A significant piece of information was the finding that the inhibitory effects

290

EDWARD F. NEMETH

of extracellular Ca^"^ on cyclic AMP levels is blocked by pertussis toxin (Chen et al, 1989). This observation demonstrated that a Gj-like protein coupled the action of extracellular Ca^"^ at the cell surface to the regulation of adenylate cyclase activity. Such heterotrimeric G-proteins are used to link certain kinds of cell surface receptors to effector mechanisms in diverse cell types (Oilman, 1987). The data obtained with pertussis toxin suggest that the Ca^^ receptor is mechanistically akin to more conventional membrane receptors and is linked to adenylate cyclase by a Gj-like protein. In the aggregate, the results derived from physiological, biochemical, and immunological experiments are complementary and together provide strong evidence for the presence of a cell surface Ca^"^ receptor on parathyroid cells. These studies anticipated the recent functional expression, cloning, and sequencing of the parathyroid cell Ca^"*" receptor (Brown et al., 1993; Racke et al., 1993). Based on the accumulated evidence derived from these various studies, a model of how extracellular Ca^"*" acts on the parathyroid cell to regulate PTH secretion can be formulated (Figure 1). The model reflects to some degree the bias of the author but does incorporate and assemble in a testable manner nearly all the reproducible results obtained in parathyroid cells. On the surface of parathyroid cells is a Ca^"*" receptor protein that enables these cells to detect and respond to small, physiological changes in the concentration of extracellular Ca^"^. The cloning and sequencing of this receptor (Brown et al., 1993) shows that it is a member of the O-protein

Inhibition of PTH secretion Figure 1. Schematic representation of the receptor-dependent regulation of parathyroid cell function by extracellular Ca^"^. Increases in the concentration of extracellular Ca^"^ activate a cell surface Ca^"^ receptor which is linked, by G-proteins, to the inhibition of adenylate cyclase and stimulation of phospholipase C. The net result of Ca^"^ receptor activation is an increase in [Ca'^'*']^ which results from the mobilization of intracellular Ca^"^ and influx of extracellular Ca^"*^ through voltage-insensitive channels. Receptor activation is coupled to the inhibition of PTH secretion. AC, adenylate cyclase; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate.

Regulation of Cellular Functions

291

receptor superfamily, since the encoded polypeptide exhibits the classic seven transmembrane domain motif common to all such receptors (Strosberg, 1991). The bovine parathyroid cell Ca^"^ receptor is thus structurally homologous to other cell surface receptor proteins that initially transduce extracellular signals into cellular responses. The bovine and human (Garrett et al., 1995a) parathyroid cell Ca^"^ receptors are structurally homologous. Their molecular weight is about 120 kDa and they possess nine (eleven in human) potential glycosylation sites located on the putative extracellular domain. Although the parathyroid cell Ca^"^ receptor is rather large compared to many G-protein-coupled receptors, it is as large as one other subfamily. It turns out that the parathyroid cell Ca^"^ receptor exhibits a 25 to 35% sequence homology with metabotropic glutamate receptors. Glutamate is the principal excitatory neurotransmitter in the central nervous system and these metabotropic subtypes of glutamate receptors are coupled to the mobilization of intracellular Ca^"^ or inhibition of adenylyl cyclase (Schoepp et al., 1990), similarly to the parathyroid cell Ca^"^ receptor. Since the parathyroid cell Ca^"*" receptor responds to physiological changes in the levels of circulating Ca^^ (1-2 mM), it is not surprising that this receptor contains no EF hand domains characteristic of high-affmity Ca^'^-binding proteins like calmodulin (Heizmann and Hunziker, 1991). However, there are proteins that are known to bind Ca^"^ with low affinity. These proteins, such as calsequestrin and calreticulin, are present in the sarcoplasmic reticulum and endoplasmic reticulum, subcellular structures known to serve as intracellular reservoirs for Ca^"^ (Milner et al., 1992). These proteins contain highly acidic regions, especially runs of three or more acidic amino acid residues, which are thought to be responsible for low affinity Ca^"^ binding. The parathyroid cell Ca^"^ receptor contains three regions that are rich in acidic amino acids and these regions are on the putative extracellular portion of the receptor. Studies using chimeric receptor constructs have shown that the extracellular domain is necessary for activation of the receptor by extracellular Ca^-' (Hammerland et al., 1995). The Ca^"^ receptor is coupled to phospholipase C which breaks down inositol phospholipids to form inositol 1,4,5-trisphosphate and diacylglycerol. The former mobilizes intracellular Ca^"^ and the latter activates protein kinase C. It is believed that the Ca^"^ receptor is coupled to phospholipase C by a G-protein. Pertussis toxin does not affect the ability of extracellular Ca^"*" to increase inositol 1,4,5-trisphosphate levels, mobilize intracellular Ca^"*", or inhibit PTH secretion, so this putative G-protein is not Gj. The coupling G-protein might be related to Gq, as this G-protein couples a variety of receptors to the mobilization of intracellular Ca^'^'in other cells. Despite these uncertainties, there are already indications suggesting that the parathyroid cell Ca^"^ receptor uses conventional transmembrane signaling mechanisms to regulate intracellular messengers.

292

EDWARD F. NEMETH

Very little is known about how the Ca^"^ receptor couples to the influx of extracellular Ca^"^. Based on structural comparisons and functional expression studies, the parathyroid cell Ca^"*" receptor does not appear to function as a channel. The influx channel is apparently a distinct protein that is coupled to the Ca^^ receptor either through a G-protein, an intracellular signal(s), or some combination. Parathyroid cells are not electrically excitable and depolarization of the cells does not evoke an increase in [Ca^"^];, suggesting that parathyroid cells do not possess voltage-sensitive Ca^"*" channels. Electrophysiological studies likewise fail to reveal voltage-sensitive Ca^"*^ influx in parathyroid cells. The influx pathway in parathyroid cells is therefore akin to those voltage-insensitive, receptor-operated cation channels observed in various other cells (Nemeth, 1990). The two mechanisms for increasing [Ca^"^]i, mobilization of intracellular Ca^"^ and influx of extracellular Ca^"^, appear to have different functional roles in parathyroid cell physiology. Thus, various extracellular cations that do not promote Ca^"^ influx still inhibit PTH secretion, and secretion is not greatly affected when influx is blocked. It is primarily the mobilization of intracellular Ca^"^, rather than the influx of extracellular Ca^"*", that is associated with the regulation of PTH secretion. This does not necessarily negate a role for Ca^"^ influx in the more global process of stimulus-secretion coupling in parathyroid cells. Maintained hypercalcemic states lasting more than 30 minutes are associated with increased intracellular degradation of PTH, and the relative secretion of PTH fragments of intact hormone is increased at elevated levels of extracellular Ca^"^ (Cohn and MacGregor, 1981). It should be noted that [Ca^"^]i remains high as long as extracellular Ca^"^ remains elevated and it can be promptly decreased by blocking Ca^"^ influx, implying a constant rate of Ca^"*" cycling between cellular and extracellular compartments. The maintained elevation of [Ca^'^Jj under hypercalcemic conditions may be casually involved in regulating intracellular proteolysis of PTH. As noted above, the relationship between cytosolic Ca^"^ and PTH secretion is not clear-cut. There are experimental situations in which PTH secretion can be regulated by extracellular cations independently of changes in [Ca^"^]i. Such findings have led to the proposal that it is Ca^"*" receptor activation, rather than the associated increases in [Ca^"*"]}, that is the critical event regulating PTH secretion (Nemeth and Scarpa, 1987b). Activation of the Ca^"^ receptor presumably regulates the levels of additional or alternative intracellular signals that can influence PTH secretion. An attractive candidate in this regard is diacylglycerol and its target enzyme, protein kinase C. Activation of protein kinase C by diacylglycerol is believed to play a role in the regulation of exocytotic secretion in various secretory cells (Knight, 1986). Although the available data are not entirely consistent, much of it suggests that protein kinase C can modulate PTH secretion regulated by extracellular Ca^"^. It has been shown that activators of protein kinase C, like phorbol myristate acetate, decrease the ability of extracellular Ca^"^ to increase inositol

Regulation of Cellular Functions

293

1,4,5-trisphosphate and [Ca^"^], and decrease PTH secretion. This is reflected as a shift to the right in the concentration-response curve for extracellular Ca^"^ for each one of these parameters. Activation of protein kinase C thus decreases the sensitivity of parathyroid cells to regulation by extracellular Ca^"^ (Racke and Nemeth, 1993). In many other cell types, those receptors that are coupled to the mobilization of intracellular Ca^"^ are also sensitive to depressive effects of protein kinase C. In general, protein kinase C often acts in a negative feedback capacity to dampen signaling through the receptor-phospholipase C pathway. This seems to be one of its ftinctions in parathyroid cells, and it has been suggested that protein kinase C could directly phosphorylate the Ca^"^ receptor, thus decreasing its sensitivity to activation by extracellular Ca^"^ (Racke and Nemeth, 1993). In this regard, it is significant that the human parathyroid cell Ca^"^ receptor contains five potential protein kinase C phosphorylation sites on the putative cytoplesmic domain of the receptor (Garrett et al., 1995a). While the general mechanisms depicted in Figure 1 are supported by ample evidence, it should not be considered the penultimate model, and there are still many uncertainties. It is not clear, for instance, whether the same receptor protein couples to both adenylyl cyclase and phospholipase C. In fact, there is some data suggesting that the Ca^^ receptor on parathyroid cells is a much larger protein than the one described here (Juhlin et al., 1990). The mechanism(s) coupling the Ca^^ receptor to the influx of extracellular Ca^"^ is a topic that has been only tangentially studied. And despite much study, the role of cytosolic Ca^^ in the rapid regulation of PTH secretion is still uncertain. Nonetheless, some of the essential mechanisms comprising the initial events of stimulus-secretion coupling in parathyroid cells have been identified. These events, enabling the detection and membrane transduction of the extracellular Ca^"^ signal, are certainly involved in the acute secretory response of parathyroid cells. It seems reasonable to suppose that these same mechanisms are involved in longer term regulation of parathyroid cell functions such as synthesis of PTH and cellular proliferation. The synthesis of PTH (84 amino acids) follows the conventional pattern for proteins entering the regulated secretory pathway and is first transcribed as preproPTH (115 amino acids; Habener et al., 1984). Extracellular Ca^"^ regulates the synthesis of PTH by inhibiting transcription of preproPTH. There is evidence for a negative response element on the PTH gene sensitive to activation by Ca^"^ (Okazaki et al., 1991). Lowering the concentration of plasma Ca^"^ causes a threeto fourfold increase in message for PTH within two to three hours (Naveh-Many and Silver, 1990). Very small decreases from normocalcemia cause profound increases in the rate of synthesis of PTH. The parathyroid cell thus responds to a maintained hypocalcemic challenge by increasing both the secretion and synthesis of PTH. Hypocalcemic states lasting longer than several days are associated with hyperplasia and proliferation of parathyroid cells. It is uncertain if these latter events

294

EDWARD F. NEMETH

are regulated by the Ca^"*" receptor, but it seems possible they are. The new physiology to be learnedfromthe parathyroid cell is the significant role played by extracellular Ca^"*", which functions as an extracellular signaling ligand to control numerous mechanisms in parathyroid cells. All these mechanisms act in concert to protect the animal from hypocalcemia.

THE C-CELL Scattered throughout the thyroid gland are parafollicular or C-cells which secrete the hormone calcitonin. The C-cell, like the parathyroid cell, has long been known to respond to changes in the level of plasma Ca^"^ but the secretory products of these two cells affect plasma levels of Ca^"*" in an opposite manner: PTH causes hypercalcemia, whereas calcitonin causes hypocalcemia. The secretory responses of parathyroid cells and .C-cells are likewise regulated in opposite directions by extracellular Ca^"*". Increasing the concentration of extracellular Ca^"^ stimulates calcitonin secretion. Calcitonin then acts on target tissues to reduce the level of plasma Ca^"^ (Austin and Heath, 1981). C-cells have a different embryological origin than do parathyroid cells and derive from cells of the neural crest. Because of this, they seem to possess many properties of neuroendocrine cells. They synthesize various peptides and biogenic amines and they are electrically excitable. Most of our understanding of the cellular physiology of C-cells derives from studies using rat medullary thyroid carcinoma cells which have the advantage of being reasonably stable cell lines that express many of the characteristics believed to be representative of genuine C-cells. In these cells, increasing the concentration of extracellular Ca^"*" evokes corresponding increases in [Ca^"*']i as does depolarization of the cells by elevated levels of extracellular K^. The increases in [Ca^"^]i elicited by either of these stimuli is associated with a stimulation of calcitonin secretion (Fried and Tashjian, 1986; Muff et al., 1988). Thus, secretion in the C-cell seems to conform to the conventional Ca^"^ hypothesis of stimulus-secretion coupling, wherein cytosolic Ca^^ activates exocytotic secretion. The C-cell uses quite different mechanisms to respond to extracellular Ca^'^than does the parathyroid cell (Nemeth, 1990; Brown, 1991). In the C-cell, nearly all of the increase in [Ca^"^]} elicited by extracellular Ca^"^ resultsfrominflux; there is only a very minor contribution arising from the mobilization of intracellular Ca^"^. Moreover, in C-cells, the influx of extracellular Ca^^ is through voltage-sensitive Ca^"*^ channels. These channels have been characterized biophysically and pharmacologically and are very similar to the high-threshold, L-type Ca^"^ channels present throughout the body (Yamashita and Hagiwara, 1990). Currents through these channels can be affected by dihydropyridines. Dihydropyridines that block influx through these channels inhibit increases in [Ca^"*"]} evoked by extracellular Ca^"^, whereas those that potentiate iiiflux augment cytosolic Ca^"^ responses to extracel-

Regulation of Cellular Functions

295

lular Ca^"^. This contrasts with parathyroid cells, where dihydropyridines fail to influence cytosolic Ca^"^ responses evoked by extracellular Ca^^. How extracellular Ca^"^ regulates influx through the voltage-sensitive Ca^"^ channel(s) present on C-cells is far from clear. It apparently involves some novel mechanism because there are no known voltage-sensitive Ca^"^ channels on other cells that display this sensitivity to extracellular Ca^"^. Electrophysiological studies do not reveal any peculiar properties of the L-type channel in rat C-cell lines, so there is at present no reason to suppose that extracellular Ca^"^ affects the channel directly. It seems that there is some alternative mechanism that couples to the Ca^"^ channel and regulates its sensitivity to extracellular Ca^^. Medullary thyroid carcinoma cells and parafollicular cells express a Ca^"^ receptor which is probably identical to that expressed by parathyroid cells (Garrett et al., 1995b). Presumably it is linked to the voltage-sensitive Ca^"^ channel. At present, there is very little known about the longer term regulation of C-cell functions by extracellular Ca^"^. The available data is fragmentary and does not suggest profound regulatory influences of extracellular Ca^"^ on synthesis of calcitonin or cellular proliferation of C-cells.

THE OSTEOCLAST The osteoclast is a relatively new addition to the list of extracellular Ca^'^-sensing cells. The osteoclast is primarily responsible for resorbing bone as part of the bone remodeling process and it accomplishes this task by secreting enzymes and protons. The former digests the organic components of bone (largely collagen), whereas the latter dissolves the inorganic matrix (hydroxyapatite: Caio(P04)6(OH)2). When activated, the osteoclast spreads and attaches tightly to the bone surface, effectively forming a sealed compartment beneath the cell. Actively resorbing osteoclasts are characterized morphologically by the presence of a ruffled border. This specialized part of the membrane is the site of secretion of enzymes, and additionally contains transport ATPases, some of which pump protons into the sealed compartment. The osteoclast, therefore, is a highly polarized cell, and the enzymes function together with the extremely low pH to dissolve the bone (Baron, 1989). There are many humoral and paracrine factors that turn osteoclasts on and off (Heersche, 1992; Mundy, 1992), but how these factors integrate the activity of osteoclasts into the more general scheme of bone remodeling is still far from understood. Certainly calcitonin is one of the more potent and effective hormonal factors that inhibit bone resorption. Osteoclasts possess calcitonin receptors that, when activated, inhibit secretion and cause the cells to round up. As discussed above, the rapid suppression of ongoing osteoclastic bone resorption by calcitonin can be readily monitored in vivo as a hypocalcemic response. PTH, on the other hand, activates osteoclasts. The conventional wisdom is that PTH acts indirectly, perhaps by affecting other cells in bone which then secrete some factor(s) that activates osteoclasts. Some more recent studies suggest that PTH may also have

296

EDWARD F. NEMETH

direct effects on osteoclasts. In addition, there are a wide array of cytokines and growth factors that can alter osteoclastic activity. During the process of osteoclastic bone resorption, the mineralized matrix is dissolved as the pH in the sealed compartment beneath the osteoclast falls to values of four to three. The dissolution of hydroxy appetite releases large amounts of Ca^"*" and its concentration is likely to build up in the forming lacunae in bone. Direct measurements of the concentration of extracellular Ca^"*" beneath osteoclasts in vitro reveal levels as high as 20 to 30 mM (Silver et al., 1988). Under physiological conditions, it is quite possible that the osteoclast is exposed to such high concentrations of extracellular Ca^"*". It was thus suggested that extracellular Ca^"^ controls osteoclastic activity (Teti and Zambonin-Zallone, 1987). It was subsequently shown that extracellular Ca^"^ caused increases in [Ca^'*"]i in isolated rat and avian osteoclasts in vitro (Malgaroli et al., 1989; Zaidi et al., 1989). It was additionally shown that extracellular Ca^"*" inhibited osteoclastic bone resorption in vitro. The concentrations of extracellular Ca^"^ producing these effects fall in the range of 5—20 mM. These concentrations are far in excess of the levels of Ca^"^ in plasma and most extracellular fluids but likely to be physiological for an actively resorbing osteoclast. There is thus mounting evidence suggesting that extracellular Ca^"^ released from the mineralized component of bone might function in a negative feedback capacity to depress osteoclastic activity. The physiological significance of this regulation by extracellular Ca^"^, particularly in the normal bone remodeling process, is yet to be determined. But the analogy to the parathyroid cell is obvious. In both cell types, extracellular Ca^"^ acts to increase [Ca^^]i and inhibit cellular function. We know comparatively little, however, about the mechanisms used by osteoclasts to detect and respond to changes in the concentration of extracellular Ca2^

Studies using isolated avian and rat osteoclasts suggest that increases in [Ca^"^]i evoked by extracellular Ca^"^ arise partly from the mobilization of intracellular Ca^"^ and also from influx of extracellular Ca^"^ (Zaidi et al., 1993). Like the parathyroid cell, the influx of extracellular Ca^"^ is through voltage-insensitive channels; osteoclasts appear to lack voltage-sensitive Ca^"^ channels, at least under the in vitro conditions necessary for their study. There is some evidence suggesting that voltage-sensitive Ca^"*" channels can be differentially expressed, depending on the composition of the substrate to which they are attached. The effects of extracellular Ca^"^ on [Ca^^^li and bone resorption can be mimicked by La^"*", suggesting that extracellular Ca^"*" acts at the osteoclast cell surface, perhaps by binding to an extracellular Ca^"*" receptor. However, because large populations of purified and viable mammalian osteoclasts are so difficult to obtain, there is scant biochemical data characterizing the transmembrane signaling mechanisms linked to the actions of extracellular Ca^"^ that affect [Ca^"*"]i and osteoclast function. The unavailability of tissue also limits efforts aimed at cloning the putative osteoclast Ca^"*" receptor.

Regalation of Cellular Functions

297

So the evidence supporting the presence of an extracellular Ca^"^ receptor on the osteoclast is fragmentary. There are two pieces of evidence suggesting that the extracellular Ca^"^ sensing mechanism(s) on the osteoclast is different from that on parathyroid cells and C-cells. In the first place, the concentration of extracellular Ca^"^ effective in altering osteoclastic activity is significantly higher than that which regulates the activity of parathyroid cells and C-cells (5 to 20 mM vs. 1 to 3 mM). Secondly, the extracellular -sensing mechanisms on osteoclasts and parathyroid cells are pharmacologically distinct. Organic compounds such as neomycin, which activate the parathyroid cell Ca^"^ receptor and increase [Ca^"^]i, are without effect on [Ca^"^]! in mammalian osteoclasts. The differential sensitivity to extracellular Ca^"^ and organic compounds suggests that the putative osteoclast Ca^"^ receptor is structurally distinct from that present on parathyroid cells and C-cells.

OTHER EXTRACELLULAR Ca^^-SENSING CELLS The cells discussed so far, particularly parathyroid cells and osteoclasts, play key roles in the regulation of body Ca^"*" homeostasis. The other main sites in the body that participate in body Ca^"^ metabolism are the kidney, the gastrointestinal tract, and, during pregnancy, the placenta. There is evidence that in each of these tissues there are cells that can sense and respond to changes in the concentration of extracellular Ca^"^. Proximal tubule cells of the kidney are the major site for the 1-hydroxylation of 25-hydroxyvitamin D3 to form 1,25-dihydroxyvitamin D3, the most biologically active form of vitamin D which affects a variety of cellular functions throughout the body, including regulation of PTH synthesis and intestinal uptake of dietary Ca^"*" (Kumar, 1986). 1,25-Dihydroxyvitamin D3 synthesis is increased by elevated plasma levels of PTH and decreased by hypercalcemia or hyperphosphatemia. In a series of elegant in vivo experiments, it was shown that the inhibitory effects of hypercalcemia occur independently of changes in plasma levels of PTH or phosphate (Matsumoto et al., 1987; Weisinger et al., 1989), suggesting that extracellular Ca^"^ might act directly on proximal tubule cells to regulate 1,25-dihydroxyvitamin D3 synthesis. Studies in vitro tend to support this. Thus, increased levels of extracellular Ca^"^ can block the stimulatory effects of PTH on cyclic AMP formation in isolated proximal tubule cells (Mathias and Brown, 1991) and this would be expected to lead to a decrease in the synthesis of 1,25-dihydroxyvitamin D3. Extracellular Ca^"^ might increase [Ca^"^]i in proximal tubule cells and this in itself would depress synthesis of 1,25-dihydroxyvitamin D3 since Ca^"*" can directly inhibit 1-hydroxylase activity. In the kidney, then, many of the ingredients necessary for creating a negative feedback loop akin to that seen in the parathyroid gland are present. A hypocalcemic state would stimulate 1,25-dihydroxyvitamin D3 synthesis, perhaps directly and also by increasing plasma levels of PTH. Elevated plasma levels of 1,25-dihydroxyvitamin D3 would increase the intestinal absorption

298

EDWARD F. NEMETH

of dietary calcium, resulting in increased circulating levels of Ca^"*". The rise of plasma Ca^"^ then acts directly on the proximal tubule cells of the kidney to depress synthesis of 1,25-dihydroxyvitamin D3. Extracellular Ca^"*" also blocks increases in the levels of cyclic AMP evoked by vasopressin in the medullary thick ascending limb of Henle's loop (Takaichi and Kurokawa, 1988). Significantly, this inhibitory effect is blocked by pretreatment with pertussis toxin, suggesting that extracellular Ca^"^ acts through a mechanism coupled to a Gj-like protein to depress adenylyl cyclase activity. The Ca^"^ receptor expressed in parathyroid cells and C-cells is also expressed in the kidney (Riccardi et al., 1995) and likely mediates the effects of extracellular Ca^"^ in the medullary thick ascending limb of Henle's loop. The effects of extracellular Ca^"^ observed in the proximal tubule could be mediated by this Ca^"*^ receptor or by an alternative receptor-like protein (Juhlin et al., 1987). In the gastrointestinal tract, there are only vague indications suggesting a physiologically important role for signaling by extracellular Ca^"^. Extracellular Ca^"*^ might participate in the regulation of gastrin secretion and may play a role in the proliferation of Goblet cells during embryonic development. Additional studies, with an eye towards the role of extracellular Ca^^ in regulating intestinal functions, are certainly warranted. During pregnancy, there are increased demands placed upon the maternal Ca^^ homeostatic system as the mother must now supply the Ca^"^ needed for skeletal development of the fetus (Chesney et al., 1992). One of the cells involved in the transport of Ca^^fromthe maternal to the fetal circulation is the cytotrophoblast of the placenta. There is convincing evidence showing that this cell type responds to increases in the concentration of extracellular Ca^"^ with corresponding increases in [Ca^^]i (Hellman et al., 1992). These evoked increases in [Ca^^]i are blocked by a monoclonal antibody which has been used to isolate a 500 kDa protein from placental cytotrophoblasts and it has been suggested that this protein fimctionsvas an extracellular Ca^"^ receptor (Juhlin et al., 1990). This protein clearly differs from the extracellular Ca^"^ receptor cloned from parathyroid cells, although this same monoclonal antibody blocks increases in [Ca^"*"]! evoked by extracellular Ca^"^ in parathyroid cells (Gylfe et al., 1990). Further studies are required to assess the possible role of this larger protein in regulating parathyroid cell function. The physiological significance of the extracellular Ca^"^ sensitivity of cytotrophoblasts is equally uncertain. Increasing the concentration of extracellular Ca^"^ has been shown to depress secretion of parathyroid hormone-related protein from cytotrophoblasts, and this protein has been implicated in the regulation of Ca^"^ transport in the placenta. Thus, there are various pieces of evidence suggesting that extracellular Ca^^, by actions on the cytotrophoblast, can regulate exchange of Ca^"*^ between the maternal and fetal circulation. Although not directly involved in the regulation of body Ca^^ metabolism, the juxtaglomerular cell of the kidney deserves mention because of the quite solid

299

Regulation of Cellular Functions Table 1. Extracellular Ca2+ -Sensing Cells in the Body Ceil Type Parathyroid cell Parafollicular cell Osteoclast Cytotrophoblast Kidney cells proximal tubule medullary thick ascending limb juxtaglomerular Gastrointestinal cells C-cell goblet Keratinocytes Mammary cells

Function Reguiated by Extraceiiular Ca PTH synthesis and secretion Calcitonin secretion Bone resorption Hormone secretion? Ca "^ transport? 1,25-diOH-vitamin D3 synthesis Urinary concentration Renin secretion Gastrin secretion Proliferation Proliferation Proliferation

evidence demonstrating the sensitivity of this cell to extracellular Ca^^. The juxtaglomerular cell secretes the enzyme renin which converts angiotensinogen to angiotensin I. Angiotensin I, in turn, is converted to angiotensin II by angiotensin converting enzyme. Angiotensin II acts directly and potently on vascular smooth muscle to constrict blood vessels, thus causing an increase in blood pressure. The juxtaglomerular cell therefore plays a key role in the regulation of blood pressure. Elevated levels of extracellular Ca^"^ cause increases in [Ca^"^]i and depress secretion of renin (Fray et al., 1987; Kurtz and Penner, 1989). The physiological importance of these effects of extracellular Ca^"^ on renin secretion are uncertain but an association between plasma Ca^"*" levels and hypertension has long been recognized (Bukoski and McCarron, 1988). From the above discussion, a general pattern emerges: extracellular Ca^"^ generally acts to depress cellular functions. The notable exception is the C-cell, where extracellular Ca^"^ acts to stimulate calcitonin secretion. The cell types known at present to respond to changes in the concentration of extracellular calcium are summarized in Table 1.

THERAPEUTIC SIGNIFICANCE OF EXTRACELLULAR CA^^ RECEPTORS Because extracellular Ca^"*" plays a key role in the regulation of certain cellular responses, it is possible that some disease states are intimately associated with cell surface Ca^"^ receptors. For example, in familial benign hypercalcemia, the set-point for extracellular Ca^"*" regulation of PTH secretion is increased (Khosla et al, 1993). Similar increases in the set-point for extracellular Ca^"^ are also observed in patients

300

EDWARD F. NEMETH

with primary hyperparathyroidism (Brown and Leboff, 1986). Curiously, parathyroid tissue from patients with hyperparathyroidism exhibit reduced staining with a monoclonal antibody that might interact with the parathyroid cell Ca^"^ receptor or some protein closely associated with it (Juhlin et al, 1988). There are thus some reasons for supposing that the expression of Ca^"^ receptors, or mechanisms regulating their activity may be altered in certain pathologic conditions. While it is not certain that all the cells reviewed here possess cell surface Ca^"*" receptors, certainly the parathyroid cell and the C-cell, and certain cells in the kidney do. These extracellular Ca^"^ receptors are structurally akin to many other G-protein-coupled receptors and function similarly to control the response of cells to changes in the concentration of extracellular Ca^"*". Such receptors have long been classic sites for pharmacological intervention in diverse disease states, so there is reason to suppose that extracellular Ca^"*" receptors will likewise be therapeutically relevant targets for new pharmaceuticals effective in the treatment of various disorders, especially those involving bone and mineral-related diseases. Drugs that mimic or potentiate the effects of extracellular Ca^"*" at Ca^"*" receptors are termed "calcimimetics," and act as receptor agonists. Conversely, drugs that block or depress the effects of extracellular Ca^"^ at Ca^"*" receptors are termed "calcilytics," and act as receptor antagonists. For example, calcimimetic drugs acting at the parathyroid cell Ca^"*" receptor would inhibit PTH secretion and be effective in the treatment of hyperparathyroidism. There are already compounds under development that act precisely in this manner. Cell surface Ca^"^ receptors thus provide novel and discrete molecular targets for new classes of drugs that mimic or antagonize the actions of extracellular Ca^"^ throughout the body.

SUMMARY It is now recognized that extracellular Ca^"^ can regulate the functional activity of particular types of cells in the body. Many of these cells are involved in maintaining body Ca^^ homeostasis and are present in certain endocrine glands and in bone, kidney, and the intestine. Notable among these cells are parathyroid cells which secrete parathyroid hormone (PTH). PTH acts on bone and kidney to increase the level of Ca^"*" in blood and extracellular fluids and plays a major role in maintaining body homeostasis. Parafollicular cells in the thyroid, or C-cells, secrete the hormone calcitonin which acts to decrease plasma levels of Ca^"^. The secretion of both PTH and calcitonin is regulated by changes in the concentration of extracellular Ca^"^: increased levels of extracellular Ca^"*^ inhibit PTH secretion and stimulate calcitonin secretion. The effects of extracellular Ca^"^ are mediated by a cell surface Ca^"^ receptor protein. The parathyroid cell Ca^"*" receptor has been cloned and is a member of the G protein-coupled receptor superfamily. In parathyroid cells, the Ca^"^ receptor is coupled to phospholipase C and its activation by extracellular Ca^"*^ results in the inositol 1,4,5-trisphosphate-induced release of intracellular Ca^"^, which is associated with an inhibition of PTH secretion. Other cells, such as

Regulation of Cellular Functions

301

osteoclasts in bone are also responsive to changes in the concentration of extracellular Ca^"^, although the structure of the putative Ca^^ receptor on these cells is still unknown. The recognition of a wide array of cells scattered throughout the body that can detect and respond to changes in the concentration of extracellular Ca^"*" provides evidence for a signaling role of extracellular Ca^"*" that is functionally akin to molecular ligands such as hormones and neurotransmitters. The cell surface Ca^"^ receptors expressed on these cells provide novel molecular targets for new drugs to treat a variety of disease states. REFERENCES Austin, L.A. & Heath, H.I. (1981). Calcitonin. Physiology and pathophysiology. N. Engl. J. Med. 304, 269-278. Baron, R. (1989). Molecular mechanisms of bone resorption by the osteoclast. Anat. Record 224, 317-324. Berridge, M.J. (1987). Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Ann. Rev. Biochem. 56, 159-193. Brown, E.M. (1991). Extracellular Ca^"^ sensing, regulation of parathyroid cell function, and role of Ca^"*" and other ions as extracellular (first) messengers. Physiol. Rev. 71, 371-411. Brown, E.M. & Leboff, M.S. (1986). Pathophysiology of hyperparathyroidism. Prog. Surg. 18, 13-22. Brown, E.M., LeBoff, M.S., Getting, M., Posillico, J.T., & Chen, C. (1987). Secretory control in normal and abnormal parathyroid tissue. Rec. Prog. Horm. Res. 43, 337-382. Brown, E.M., Chen, C.J., Kifor, O., LeBoff, M.S., El-Hajj, G., Fajtova, V., & Rubin, L.T. (1990). Ca2- -sensing, second messengers, and the control of parathyroid hormone secretion. Cell Calcium 11,333-337. Brown, E.M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., liediger, M., & Lytton, J. (1993). Cloning and characterization of an extracellular Ca^"^-sensing receptor from bovine parathyroid. Nature 366, 575-580. Bukoski, R.D. & McCarron, D.A. (1988). Calcium and hypertension. In: Calcium in Drug Actions (Baker, P.P., ed.), pp. 467-487, Springer-Verlag, New York. Chen, C.J., Bamett, J.V., Congo, D.A., & Brown, E.M. (1989). Divalent cations suppress 3',5'-adenosine monophosphate accumulation by stimulating a pertussis toxin-sensitive guanine nucleotide-binding protein in cultured bovine parathyroid cells. Endocrinol. 124, 233-239. Chesney, R.W., Specker, B.L., Mimouni, P., & McKay, C.P. (1992). Mineral metabolism during pregnancy and lactation. In: Disorders of Bone and Mineral Metabolism (Coe, F.L. & Favus, M.J., eds.), pp. 383-393, Raven Press, New York. Cohn, D.V. & MacGregor, R.R. (1981). The biosynthesis, intracellular processing, and secretion of parathormone. Endocrine Rev. 2, 1-26. Douglas, W. W. (1974). Involvement of calcium in exocytosis and the exocytosis-vesiculation sequence. Biochem. Soc. Symp. 39, 1-28. Fried, R.M. & Tashjian, A.H., Jr. (1986). Unusual sensitivity of cytosolic free Ca^"*" to changes in extracellular Ca^"*" in rat C-cells. J. Biol. Chem. 261, 7669-7674. Fray, J.C.S., Park, C.S., & Valentine, A.N.D. (1987). Calcium and the control of renin secretion. Endocrine Rev. 8, 53-93. Garrett, J.E., Capuano, I.V., Hammerland, L.G., Hung, B.C.P., Brown, E.M., Hebert, S.C, Nemeth, E.F., & Fuller, F. (1995a). Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J. Biol. Chem. 270, 12919-12925.

302

EDWARD F. NEMETH

Garrett, J.E., Tamir, H., Kifor, O., Simin, R.T., Rogers, K.V., Mithal, A., Gagel, R.F., & Brown, E.M. (1995b). Calcitonin-secreting cells of the thyroid express an extracellular calcium receptor gene. Endocrinology 136, 5202-5211. Oilman, A.G. (1987). G proteins: Transducers of receptor-generated signals. Ann. Rev. Biochem. 56, 615-649. Gylfe, E., Johlin, C, Akerstrom, G., Klareskog, L., Rask, L., & Rastad, J. (1990). Monoclonal antiparathyroid antibodies—tools for studies of the regulation ofcytoplasmic calcium and function of parathyroid and other antibody-reactive cells. Cell Calcium 11, 329-332. Habener, J.F., Rosenblatt, M., & Potts, J.T. Jr. (1984). Parathyroid hormone: Biochemical aspects of biosynthesis, secretion, action, and metabolism. Physiol. Rev. 64,985-1053. Hammerland, L.G., Krapcho, K.J., Alasti, N., Simin, R., Garrett, J.E., Capuano, I.V., Hung, B.C.P., & Fuller, F.H. (1995). Cation binding determinants of the calcium receptor revealed by functional analysis of chimeric receptors and a deletion mutant. J. Bone Min. Res. 10, SI 56. Heersche, J.N.M. (1992). Systemic factors regulating osteoclast function. In: Biology and Physiology of the Osteoclast (Rifkin, B.R. & Gay, C.V., eds.), pp. 151-169, CRC Press, Boca Raton, FL. Hellman, P., Ridefelt, P., Juhlin, C, Akerstrom, 0., Rastad, J., & Gylfe, E. (1992). Parathyroid-like regulation of parathyroid-hormone-related protein release and cytoplasmic calcium in cytotrophoblast cells of human placenta. Arch. Biochem. Biophys. 293,174-180. Heizmann, C.W. & Hunziker, W. (1991). Intracellular calcium-binding proteins: More sites than insights. TIBS 16,98-103. Juhlin, C, Holmdahl, R., Johansson, H., Rastad, J., Akerstrom, 0., & Klareskog, L. (1987). Monoclonal antibodies with exclusive reactivity against parathyroid cells and tubule cells of the kidney. Proc. Natl. Acad. Sci. USA 84, 2990-2994. Juhlin, C, Klareskog, L., Nygren, P., Ljunghall, S., Gylfe, E., Rastad, J., & Akerstrom, 0 . (1988). Hyperparathyroidism is associated with reduced expression of a parathyroid calcium receptor mechanism defined by monoclonal antiparathyroid antibodies. Endo 122, 2999-3001. Juhlin, C, Lundgren, S., Johnsson, H., Lorentzen, J., Rask, L., Larsson, E., Rastad, J., Akerstrom, 0., & Klareskog, L. (1990). 500-kilodalton calcium sensor regulating cytoplasmic Ca^"^ in cytotrophoblast cells of human placenta. J. Biol. Chem. 265, 8275-8279. Khosla, S., Ebeling, P.R., Firek, A.F., Burritt, M.M., Kao, P.C., & Heath, H. (1993). Calcium infusion suggests a "set-point" abnormality of parathyroid gland function in familial benign hypercalcemia and more complex disturbances in primary hyperparathyroidism. J. Clin. Endo. Metab. 76, 715-720. Knight, D.E. (1986). Calcium and exocytosis. In: Calcium and the Cell Ciba Foundation Symposium, Vol. 122, pp. 250-265, John Wiley & Sons, New York. Kumar, R. (1986). The metabolism and mechanism of action of 1,25-dihydroxyvitamin D3. Kidney Intl. 30, 79S-803. Kurtz, A. & Penner, R. (1989). Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells. Proc. Natl. Acad. Sci. USA 86, 3423-3427. Lopez-Bameo, J. & Armstrong, CM. (1983). Depolarizing response of rat parathyroid cells to divalent cations. J. Gen. Physiol. 82, Malgaroli, A., Meldolesi, J., Zambonin-Zallone, A., & Teti, A. (1989). Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin. J. Biol. Chem. 264, 14342-14347. Mathias, R.S. & Brown, E.M. (1991). Divalent cations modulate PTH-dependent 3',5'-cyclic adenosine monophosphate production in renal proximal tubular cells. Endocrinol. 128, 3005-3012. Matsumoto, T., Ideda, K., Morita, K., Fukumoto, S., Takahashi, H., & Ogata, E. (1987). Blood Ca^"^ modulates responsiveness of renal 25(OH)D3-la-hydroxylase to PTH in rats. Am. J. Physiol. 253, E503-E507.

Regulation of Cellular Functions

303

Milner, R.E., Famulski, K.S., & Michalak, M. (1992). Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells. Molec. Cell Biochem. 112, 1-13. Muff, R., Nemeth, E.F., Haller-Brem, S., & Fischer, J.A. (1988). Regulation of hormone secretion and cytosolic Ca^"^ by extracellular Ca^^ in parathyroid cells and C-cells: Role of voltage-sensitive Ca^"^ channels. Arch. Biochem. Biophys. 265, 128-135. Mundy, G.R. (1989). Calcium Homeostasis: Hypercalcemia and Hypocalcemia. Martin Dunitz Ltd., London. Mundy, G.R. (1992). Local factors regulating osteoclast function. In: Biology and Physiology of the Osteoclast (Rifkin, B.R. & Gay, C.V., eds.), pp. 171-185, CRC Press, Boca Raton, FL. Naveh-Many, T. & Silver, J. (1990). Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J. Clin. Invest. 86, 1313-1319. Nemeth, E.F. (1990). Regulation of cytosolic calcium by extracellular divalent cations in C-cells and parathyroid cells. Cell Calcium 11, 323-327. Nemeth, E.F. & Scarpa, A. (1986). Cytosolic Ca^"*" and the regulation of secretion in parathyroid cells. FEBS Lett. 203, 15-19. Nemeth, E.F. & Scarpa, A. (1987a). Rapid mobilization of cellular Ca^"*" in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor. J. Biol. Chem. 262, 5188-5196. Nemeth, E.F. & Scarpa, A. (1987b). Are changes in intracellular free calcium necessary for regulating secretion in parathyroid cells? Ann. New York Acad. Sci. 493, Okazaki, T., Zajac, J.D., Igarashi, T., Ogata, E., & Kronenberg, H.M. (1991). Negative regulatory elements in the human parathyroid hormone gene. J. Biol. Chem. 266, 21903—21910. Racke, F.K., Hammerland, L.G., Dubyak, G.R., & Nemeth, E.F. (1993). Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes. FEBS Lett. 333, 132-136. Racke, F.K. & Nemeth, E.F. (1993). Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C. J. Physiol. 468, 141-162. Riccardi, D., Park, J., Lee, W.-S., Gamba, G., Brown, E.M., & Hebert, S.C. (1995). Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc. Natl. Acad. Sci. USA 92, 131-135. Schoepp, D., Bockaert, J., & Sladeczek, F. (1990). Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors. TIPS 11, 508-515. Shoback, D.M., Thatcher, J., Leombruno, R., & Brown, E.M. (1984). Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells. Proc. Natl. Acad. Sci. USA 81,3113-3117. Silver, I.A., Murrills, R.J., & Etherington, D.J. (1988). Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell. Res. 175,266-276. Strosberg, A.D. (1991). Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins. Eur. J. Biochem. 196, 1-10. Takaichi, K. & Kurokawa, K. (1988). Inhibitory guanosine triphosphate-binding protein-mediated regulation of vasopressin action in isolated single medullary tubules of mouse kidney. J. Clin. Invest. 82, 1437-1444. Teti, A. & Zambonin-Zallone, A. (1987). A working hypothesis: Calcium concentration controls directly osteoclast activity. In: Calcium Regulation and Bone Metabolism. Basic and Clinical Aspects (Cohn, D.V., Martin, T.J., & Meunier, P.J., eds.), Vol. 9, pp. 358-362, Excerpta Medica, New York. Watson, P.H. & Hanley, D.A. (1993). Parathyroid hormone: Regulation of synthesis and secretion. Clin. Invest. Med. 16,58-77. Weisinger, J.R., Favus, M.J., Langman, C.B., & Bushinsky, D.A. (1989). Regulation of 1,25-dihydroxyvitamin D3 by calcium in the parathyroid^ctomized, parathyroid hormone-replete rat. J. Bone Min. Res. 4, 929-935.

304

EDWARD F. NEMETH

Yamashita, N. & Hagiwara, S. (1990). Membrane depolarization and intracellular Ca^^ increase caused by high external Ca^"^ in a rat calcitonin-secreting cell line. J. Physiol. 431, 243-267. Zaidi, M., Datta, H.K., Patchell, A., Moonga, B., & Maclntyre, I. (1989). Calcium-activated intracellular calcium elevation: A novel mechanism of osteoclast regulation. Biochem. Biophys. Res. Comm. 163, 1461-1465. Zaidi, M., Alam, A.S.M.T., Shankar, V.S., Bax, B.E., Bax, CM., Moonga, B.S., Bevis, P.J.R., Stevens, C, Blake, D.R., Pazianas, M., & Huang, C.L.H. (1993). Cellular biology of bone resorption. Biol. Rev. 68, 197-264.