Regulation of parathyroid hormone secretion.

0163-769X/91/1203-0291$03.00/0 Endocrine Reviews Copyright © 1991 by The Endocrine Society

Vol. 12, No. 3 Printed in U.S.A.

Regulation of Parathyroid Hormone Secretion * SUSAN L. POCOTTE, GERALD EHRENSTEIN, AND LORRAINE A. FITZPATRICK Laboratory of Biophysics (S.L.P., G.E.), National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892; and Endocrine Research Unit (L.A.F.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905

I. Introduction

II. The Role of Calcium in Regulating PTH Secretion

ARATHYROID hormone (PTH) maintains calcium homeostasis through its effects on target tissues, bone, and kidney. The parathyroid cell is unusual in that decreasing concentrations of calcium stimulate secretion of PTH, and increasing concentrations of calcium inhibit secretion of PTH. Thus, this cell, which is responsible for the critical maintenance of ionized blood calcium levels, is, in turn, regulated by calcium itself. The inverse relationship between extracellular calcium concentration and PTH secretion has been well established experimentally. However, the cellular mechanisms responsible for this relationship are poorly understood. This review will focus on several questions relating to the regulation of PTH secretion. Because of the physiological importance of the interaction between serum calcium and PTH, we will emphasize regulation of PTH by calcium. The role of other regulators will also be considered. The main questions we will address are: 1. Does intracellular or extracellular calcium regulate PTH secretion? 2. What are the mechanisms that allow extracellular calcium to influence the intracellular calcium concentration? 3. What is the mechanism for the inverse dependence of secretion on calcium concentration? 4. What is the role of cAMP and other second messengers in regulating PTH secretion? In order to understand the basic mechanisms involved in the exocytotic secretion of PTH, we will broaden the subject matter to include effects that occur at intracellular calcium concentrations that are below physiological concentrations, and we will compare PTH secretion with the secretion of other hormones.

Early whole animal studies in cow and goat (1, 2) and perfused isolated parathyroid glands (3) demonstrated that hypocalcemia increased PTH secretion and hypercalcemia decreased PTH secretion. Suspended bovine (4) and porcine (5) parathyroid cells were used to further study the effects of extracellular calcium on PTH secretion. These experiments demonstrated that PTH secretion is inversely proportional to extracellular calcium concentration in the physiological range. Since increased intracellular calcium concentrations stimulated secretion in other secretory cells (6, 7), the secretory phenomenon in parathyroid cells was described as paradoxical. Although the basic observation of an inverse dependence of PTH secretion on extracellular calcium concentration has been confirmed in many studies, it was observed that PTH secretion decreased when the extracellular calcium concentration was decreased to a very low level (8). Recent experiments have determined the doseresponse relationship for PTH secretion as a function of intracellular calcium concentration. As shown in Fig. 1, this dose-response relationship has been measured by two independent methods with the same qualitative result. One method involved the use of the calcium-sensitive fluorescent dye quin-2 to determine the intracellular calcium concentration (9), and the other method involved controlling the intracellular calcium concentration by permeabilizing the cells with electric pulses and adjusting the extracellular ionic concentrations (10). Both methods indicate that the dose-response relationship is biphasic with a peak at an intracellular calcium concentration of about 200 nM. Thus, the calcium concentrations corresponding to the ascending limb of the biphasic dose-response curve are below the physiological range. The importance of determining the complete doseresponse curve, however, is not in its direct correspondence to physiological conditions, but in the insight this curve may provide for understanding basic mechanisms and for resolving the parathyroid paradox.

P

Address requests for reprints to: Lorraine Fitzpatrick, M.D., 5-164 West Joseph Building, Endocrine Research Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905. * Supported in part by USPHS Grant DK-42572.

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several reasons. First of all, Fig. 1 applies to adult tissue, the tissue type that was used for ion channel measurements that we will compare with the secretion measurements, whereas the experiments of Oetting et al. (11) were performed on calf tissue. In addition, the results of Oetting et al. (11) imply that in the physiological region, PTH secretion increases with increasing intracellular calcium, whereas it has been demonstrated in intact cells that in the physiological region, PTH secretion decreases with increasing intracellular calcium (12, 13). Finally, the results of Fig. 1 provide two independent types of measurement that are consistent with each other. While extracellular calcium regulates the intracellular calcium concentration, it is the intracellular calcium pool that interacts to mediate the secretion of PTH. This conclusion follows from the consistency between the two measurements of the dose-response curve for PTH secretion in adult bovine tissue indicated in Fig. 1. In experiments corresponding to Fig. 1A, the intracellular calcium concentration at the peak of the dose-response curve is about 200 nM, and the corresponding extracellular calcium concentration is in the millimolar range. In experiments corresponding to Fig. IB, both the intracellular and extracellular calcium concentrations at the peak of the dose-response curve are about 200 nM. Thus, peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of whether the extracellular calcium concentration is in the submicromolar range, as in the permeabilized cell, or in the millimolar range, as in the intact cell.

III. Approach to the Parathyroid Paradox - log Calcium M

FIG. 1. Dose-response relationship for PTH secretion as a function of intracellular calcium concentration. A, For intact bovine parathyroid cells, with intracellular calcium concentrations as measured by quin-2. [Reprinted with permission from P. Nygren et al: FEBS Lett 213:195198,1987 (9).] B, For permeabilized bovine parathyroid cells, assuming intracellular and extracellular calcium concentrations are equal. O, No ATP; • , 2/mM ATP. [Reprinted with permission from S. L. Pocotte and G. Ehrenstein: Endocrinology 125:1587-1592, 1989 (10). © The Endocrine Society]

In contrast to Fig. IB, another study using electropermeabilized calf cells revealed a dose-response relationship for PTH secretion as a function of calcium that was also biphasic, but with a peak at a supraphysiological calcium concentration (11). The calcium concentrations at half-maximal and at peak secretion were 10~5 M and 4 x 10~4 M, respectively, and very little secretion occurred at 2 x 10~7 M calcium. The reason for the difference between the results of Oetting et al. (11) and measurements shown in Fig. 1 is not clear. In this review, we have based our discussion on the results of Fig. 1 for

Next, we will suggest how some of the results described above might lead to an explanation of the parathyroid paradox—the decrease in the secretion of PTH with increasing extracellular calcium concentration. The results that we will consider are the demonstration that intracellular calcium concentration is an important regulator of PTH secretion and the determination of the dose-response curve for secretion as a function of intracellular calcium concentration. A. Clues from the dose-response relationship for secretion as a function of intracellular calcium concentration

In Fig. 1, the rising phase at low calcium concentrations suggests an approach to the resolution of the parathyroid paradox. Since the dose-response curve at low calcium concentration in Fig. 1 is qualitatively similar to that for the secretion of other hormones, the mechanism for secretion of PTH in this subphysiological range of calcium concentration may be the same as the mechanism for the secretion of other hormones in the physiological range. According to this point of view, PTH


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secretion is unusual, in a quantitative rather than a qualitative way, in that the rising phase is shifted toward lower calcium concentration by about 1 order of magnitude. The concept of a quantitative difference between the secretion of PTH and the secretion of other hormones can be extended by noting that a falling phase at higher calcium concentrations has been found not only for PTH secretion, but also for chromaffin cell secretion, albeit at calcium concentrations several orders of magnitude higher than for secretion by parathyroid cells (14). The concept that the difference in the calcium dependence of PTH secretion and the secretion of most other hormones is a quantitative one leads to questioning the role of intracellular calcium for secretion in general. Before addressing this question directly, we will review experimental results on the electrical properties of parathyroid cells. The reason for this digression is that the electrical properties considered below lead to possible answers to this question. B. Electrical properties of parathyroid cells

Although increasing extracellular calcium concentration has little, if any, effect on the membrane potential of most cells, it causes a very large change in the membrane potential of mouse (15) and rat (16) parathyroid cells. The dose-response relationship for the membrane potential of rat parathyroid cells as a function of extracellular calcium concentration (16) is shown in Fig. 2. The relationship is biphasic with peak hyperpolarization at a calcium concentration of about 1 mM. The intracellular calcium concentration at the peak hyperpolarization cannot be estimated very accurately from available data on rat parathyroid cells, but a rough estimate made by comparing intracellular and extracellular calcium concentrations measured in bovine parathyroid cells (17) indicates that the peak hyperpolarization occurs at an intracellular calcium concentration of approximately 300 membrane potential TmVI 0-

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nM. In the range where increasing calcium concentrations cause a large membrane depolarization, the same calcium concentrations also cause a large increase in the electrical resistance of the membrane (16). In principle, this combination of effects could be explained by the closing of potassium channels, since open potassium channels tend to hyperpolarize cells and since the closing of any channel tends to increase membrane resistance. Because these effects are caused by calcium, the closing of calcium-activated potassium channels would explain the experimental results. This possible explanation was not seriously considered for some time, however, since extensive measurements on calcium-activated potassium channels in many cell types indicated that in the physiological range, increasing calcium concentration opens, rather than closes, these channels (18). C. Calcium-activated potassium channels in parathyroid cells The calcium-dependence of the open probability for calcium-activated potassium channels in parathyroid cell plasma membranes is unique. For other calcium-activated potassium channels, the open probability increases with increasing intracellular calcium concentration, at least up to the millimolar range of intracellular calcium (18). For calcium-activated potassium channels in parathyroid cells, however, the calcium-dependence of the open probability is biphasic, with a peak at an intracellular calcium concentration of about 200 nM (19). This result, which is shown in Fig. 3, can explain both the depolarization and the increased membrane resistance observed when the calcium concentration is increased, since for intracellular calcium concentrations above 200 nM, increasing calcium concentrations would tend to close the channels. It can also explain the biphasic nature of the relationship between membrane potential and calcium concentration (Fig. 2). Next, we will consider how the unusual properties of calcium-activated potassium channels in parathyroid cells might provide an explanation for the parathyroid paradox.

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D. Possible resolution of the parathyroid paradox

•20 -30 •40 -50 -60 -70-BO0.5

1

1.5

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2.5ICa*+l mM

FIG. 2. Dose-response relationship for membrane potential as a function of extracellular calcium concentration of rat parathyroid cells. [Reproduced with permission from J. Lopez-Barneo and C. M. Armstrong: J Gen Physiol 82:269-294,1983 (16).]

The biphasic form of the dose-response curves for the calcium dependence of membrane potential, of secretion, and of the open probability of calcium-activated potassium channels in parathyroid cells and the similarity of the calcium concentration corresponding to the peak of each of these three curves suggest that either hyperpolarization or the opening of the channel is required for secretion. The hypothesis that hyperpolarization is required for secretion is not consistent with experiments in which parathyroid cells were depolarized by addition of extra-


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The above hypothesis predicts that for secretory cells in general, regardless of the shape of the secretion doseresponse curve, secretion and the probability that calcium-activated potassium channels are open should have a similar dependence on intracellular calcium concentration. As shown in Fig. 4, this has been found experimentally for acinar cells (25, 26) as well as for parathyroid cells (9, 19). Further experiments are required to determine whether this hypothesis is correct. If it is correct, it raises the further question of the mechanism by which opening calcium-activated channels leads to hormone secretion. A possible mechanism based on the opening of calcium-activated channels in secretory vesicles has recently been proposed (27). According to this hypothesis, the opening of calcium-activated channels in secreI.OT 100

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FIG. 3. Dose-response relationship for the probability that a calciumactivated postassium channel is open as a function of intracellular calcium concentration for bovine parathyroid cells. Measurements performed by patch-clamping inside-out patches. [Reproduced with permission from M. Jia et al.: Proc Natl Acad Sci USA 85:7236-7239, 1988 (19).]

cellular potassium. This hypothesis predicts that depolarization would reduce PTH secretion. The experimental results, however, indicate that depolarization decreases the intracellular calcium concentration and enhances secretion (20-22), thus ruling out the hypothesis. The other suggested possibility, that opening of calcium-activated potassium channels is required for secretion, actually involves two possible cases. Secretion could require the opening of calcium-activated potassium channels in the plasma membrane. Alternatively, secretion could require the opening of similar channels present in the secretory vesicles. Although such channels have not yet been reported, there is evidence that secretory vesicles in other cells contain calcium-activated channels (23, 24). If the secretory vesicles in parathyroid cells contain channels with properties similar to the ones reported for the plasma membrane, the opening of these channels would have the same calcium dependence shown in Fig. 3. Thus, it is interesting to consider the hypothesis that the opening of channels in either the plasma membrane or the secretory vesicles leads to secretion. Because of the similarity in the calcium dependence of the dose-response relations for secretion (Fig. 1) and for the probability that a calcium-activated potassium channel is open (Fig. 3), this hypothesis could provide a rationale for the shape of the dose-response curve for secretion, and hence a rationale for the parathyroid paradox.

0 -I

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1—I I I I I II

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1—I I I I III

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-5 Log Calcium Concentration (M)

FIG. 4. Comparison of calcium dependence of secretion (upper graph) and probability that a calcium-activated channel is open (lower graph) for parathyroid cells (circles) and acinar cells (squares). Parathyroid channel data are independent of membrane potential. Acinar channel data are for surface membrane potential of -40 mV, corresponding to a potential in the lumen of the vesicle that is +40 mV with respect to the cytoplasm. Acinar channel curve is based on probability data for 0 mV and shift along abscissa measured for -40 mV. Secretion data for parathyroid cells are taken from Ref. 9 and for acinar cells from Ref. 25. Channel data for parathyroid cells are from Ref. 19 and for acinar cells from Ref. 26.


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tory vesicles leads to a flow of potassium ions into the vesicle, and this, in turn, leads to a flow of anions and water into the vesicles. As a result, water is removed from the space between the vesicle membrane and the plasma membrane of the secretory cell. The removal of water from this space allows the two membranes to come in close contact and to fuse.

IV. Parathyroid Cell Plasma Membrane Calcium Sensors The relationship between intracellular and extracellular calcium concentrations has been measured by means of calcium-sensitive fluorescent dyes (9, 13, 17, 21). There are quantitative differences between results obtained with quin-2 and results obtained with fura-2, probably because of their differing buffering capacities. For quin-2, which has a higher buffering capacity, the steady state calcium concentration appears lower (180 nM vs. 323 nM), and the rapid transient changes of intracellular calcium are not detected (13). However, both dyes yield similar qualitative results regarding the proportional changes of extracellular and intracellular calcium concentration. A 1 mM change in extracellular calcium concentration corresponds to a change in the range of a few hundred nanomolar in intracellular calcium concentration. One of the most intriguing issues about parathyroid cell function is how the plasma membrane detects and responds to extracellular calcium to regulate intracellular calcium, resulting in the regulation of PTH secretion. There is now strong experimental evidence that the coupling between extracellular and intracellular calcium concentrations involves at least two distinct mechanisms. One mechanism is the direct movement of calcium ions across the plasma membrane through calcium-selective channels, a family of large glycoproteins that allow controlled entry of extracellular calcium into cells. The availability of agonists and antagonists for these channels has enhanced the study of their role in the regulation of secretory events. Calcium channel agonists open the channels, allowing entry of extracellular calcium into the cells, while calcium channel antagonists block the entry of calcium through the channels. Calcium channels have been classified into T, L, and N types, according to their electrophysiological properties and their response to different classes of pharmacological agents (28). For example, the dihydropyridine compounds predominantly affect the L-type calcium channel but also have effects on the T-type channel. Recently, a new class of calcium channels has been described, which responds to the dihydropyridine class of calcium channel agonists/antagonists but is not voltage-dependent (29).

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A second mechanism by which extracellular and intracellular calcium concentrations are coupled is through the release of calcium from intracellular stores in response to second messengers triggered by extracellular calcium. It is not clear whether these two entities are coupled to each other, such that the calcium receptor or sensor regulates the calcium channel, or whether they function separately. A wide variety of experiments provides evidence for the presence of calcium channels. As would be expected for channel-mediated calcium transport across the plasma membrane, calcium channel agonists (+)202791(30), a dihydropyridine, and maitotoxin (31), a nondihydropyridine potent activator of voltage-sensitive calcium channels, inhibit PTH secretion, and the calcium channel antagonist (—)202-791 (30) stimulates secretion. Pertussis toxin treatment prevented the agonist (+)202791 from inhibiting secretion, suggesting that a G protein is coupled to the calcium channel. Further evidence for calcium channels is the ability of mouse antibodies specific for purified a-subunits of the rat skeletal muscle dihydropyridine-sensitive calcium channel to block PTH secretion (32). Pertussis toxin treatment reduced the antisera effect, providing further evidence that a G protein is coupled to the calcium channel. Binding studies on parathyroid cell plasma membranes demonstrated specific binding of [125I]iodipine, a dihydropyridine-sensitive calcium channel ligand (33). Also, addition of the calcium-channel agonist maitotoxin (31) or increasing the extracellular calcium concentration (34) increased 45 Ca uptake into parathyroid glands. The calcium-channel agonists and antagonists (±) 202-791 considered above are pure enantiomers. Other studies have suggested that calcium-channel agonists stimulate, inhibit, or have no effect on PTH secretion (22, 31, 35-37), but many of the compounds used in these studies were racemic mixtures with mixed agonist and antagonist effects in diverse tissue types. Although it has been shown that the dihydropyridine class of drugs affect both L- and T-type calcium channels in other tissues, the nature of the calcium channel in parathyroid cells is unclear. Because of the wide variety of calcium channels that have recently been described in diverse tissues, there has been confusion in classification of calcium channels. Several investigators have described voltage-insensitive calcium channels (29, 38) that respond to dihydropyridine-like compounds. There are several reasons to infer that the calcium channel in the parathyroid cell may be of the voltage-insensitive type: depolarization of the cell by increasing the extracellular potassium concentration decreases the intracellular calcium concentration (21); depolarization does not affect either 45Ca uptake or efflux (34); and depolarization does not alter the effects of the calcium channel agents (±)


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202-791 on PTH secretion (Pocotte, S. L., G. Ehrenstein, and L. A. Fitzpatrick, submitted). These results suggest a calcium channel whose open probability does not increase with membrane depolarization. As measured by fura-2, the kinetics of intracellular calcium changes, in response to changes in extracellular divalent cations provide additional evidence for the existence of two types of calcium-sensing mechanisms. After an increase in the extracellular calcium concentration, both a transient increase and a steady state increase in intracellular calcium are observed (21). The transient increase can also be elicited by divalent cations other than calcium, suggesting that other divalent cations can also bind to the sensor that controls release of calcium from intracellular stores. The steady state increase is not elicited by divalent cations other than calcium, indicating either that these divalent cations cannot traverse the calcium channels or cannot activate calcium-dependent secretion. There is considerable evidence that the plasma membrane contains a sensor that couples extracellular calcium (or other divalent cations) to a second messenger system that stimulates the release of calcium from intracellular stores. This mechanism can be examined in the absence of extracellular calcium, since the sensor responds to a variety of divalent and trivalent cations. For example, in the absence of extracellular Ca, there is a transient increase in intracellular calcium in response to extracellular Mg, Sr, or Ba (21). The transient increase in intracellular calcium must come from intracellular stores, since there was no extracellular calcium present. Further evidence that the transient increase in calcium comes from intracellular stores is that it is accompanied by a transient increase in inositol trisphosphate, a second messenger that has been shown to be effective in causing the release of calcium from intracellular stores in parathyroid cells (39). The trivalent cation lanthanum might be expected to bind with higher affinity than calcium. This is clearly seen in the inhibition of PTH secretion by lanthanum, which has a set-point 1 order of magnitude lower than the set-point for calcium (40). There are a number of other differences between the effects of lanthanum and the effects of calcium. For example, pertussis toxin has little effect on the inhibition of PTH secretion by lanthanum, suggesting that extracellular lanthanum acts by means of only one of the mechanisms by which extracellular calcium regulates PTH secretion—the release of calcium from intracellular stores. The potent inhibition of PTH secretion by lanthanum is due to the ability of lanthanum to attenuate a second messenger within the parathyroid cell. One such possibility is inositol trisphosphate; however, experimental evidence with other trivalent cations of the lanthanide series suggests that

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calcium causes larger increases in inositol trisphosphate than do the lanthanides (41). A reduction in cAMP or other putative second messenger may be responsible for the profound inhibitory effect of lanthanum. Although it is clear that lanthanum decreases PTH secretion, there are differences in the reported effects of lanthanum on intracellular calcium concentration. One report indicates that the effect of lanthanum is to decrease the steady state component of intracellular calcium concentration (13), and another report indicates that lanthanum activates 45Ca influx and increases the intracellular calcium concentration (42). Results from experiments with different agents that affect plasma membrane molecules (43, 44) provide additional support for the existence of receptors that sense calcium. Treatment of parathyroid cells with Concanavalin-A, a lectin that inhibits binding of a variety of hormones to receptors, did the following: 1) attenuated the inhibitory effect of extracellular calcium on PTH secretion; 2) reduced by 25% the ability of increasing extracellular calcium concentrations to elevate intracellular calcium levels; 3) blocked the extracellular magnesium-evoked increase of intracellular calcium concentration; and 4) decreased the ability of extracellular calcium to increase intracellular inositol phosphates (45). Other agents that have been used to test for the presence of calcium receptors, monoclonal antiparathyroid antibodies to human parathyroid adenoma cells and trypsin, are not very specific and did not provide definitive results. For example, treatment of bovine parathyroid cell with the protease trypsin, in an attempt to inactivate the putative calcium receptor, prevented the effect of extracellular calcium on PTH secretion (44). However, trypsin did not alter the ability of extracellular calcium to elicit concentration changes of intracellular calcium concentration. PTH secretion is primarily regulated by calcium, as we have discussed above. It should be noted, however, that it has been shown that various other agents, e.g. dopamine, isoproterenol, and PRL, can modulate PTH secretion in the absence of extracellular calcium and without detectable changes in intracellular calcium (Table 1). For example, dopamine stimulation of parathyroid cells elevates intracellular cAMP and PTH secretion in the absence of extracellular calcium (46). Furthermore, dopamine does not elevate intracellular calcium, as measured by quin-2 (17). There is also a lack of correlation between PTH secretion and intracellular calcium concentration when the active form of vitamin D3 is added to parathyroid cells. In this case, the intracellular calcium concentration is significantly increased (47), but there is no change in PTH secretion (48). A possible explanation for the lack of correlation between PTH secretion and the measured intracellular calcium concentration in the



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TABLE I. Effects of various factors on PTH secretion, intracellular calcium concentration ([Ca]i), cAMP level, and IP3 level in parathyroid cells Factor

PTH

[Ca]i

cAMP

[Ca].lt Isoproteronol Osmolality PRL ATP Fluoride Lithium Potassium Dopamine

1

f

4

1

t —> ND

t T t

1 1 T T T

—» ND

t t ND 1

ND ND

t

IPs

T

ND ND ND ND

i

ND ND ND

[Ca]ext, Extracellular calcium concentration; ND, not determined.

several experiments described above is that secretion may be correlated with local changes in calcium concentration at discrete sites of fusion, and fluorescent dye measurements may not always detect these local changes. This explanation is consistent with the observation in chromaffin cells that, for equivalent elevations of cytosolic calcium as measured by fura-2, calcium from the external medium affects secretion, but calcium from intracellular stores does not (49).

V. Guanine Nucleotide Regulatory Proteins Guanine nucleotide regulatory proteins (G proteins) are ubiquitous linkers between cell surface proteins and intracellular effector systems that transduce the hormonal signal generated by interaction between the hormone and receptor complex (50). A common feature of this large family of structurally and functionally related proteins is that they are all heterotrimers with subunits designated a, 0, and 7. Experiments on the inhibition of PTH release by prostaglandin F 2a and a-adrenergic agonists provided the first evidence that G proteins are involved in the regulation of PTH secretion (51, 52). Incubation of parathyroid cells with pertussis toxin prevented the inhibition normally caused by these agents. Since pertussis toxin is known to catalyze ADP ribosylation of the a-subunit of G proteins, thus inhibiting their normal activity, it was concluded that the G proteins play a role in regulating secretion. Of greater interest is the fact that inhibition of PTH release by calcium is prevented by incubation of parathyroid cells with pertussis toxin, implying that one or more G proteins are involved in coupling the extracellular calcium signal to suppression of PTH secretion (52). Several experiments have been performed to determine the mechanism for this involvement. Treatment of bovine parathyroid cells with pertussis toxin abolished the effects of both calcium channel agonist and antagonist (±)202-791 on PTH secretion (30), indicating that calcium channels may be linked with a G protein within the

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plasma membrane of the cell. A further test of the role of G proteins was provided by adding A23187, a lipidsoluble antibiotic known to transport calcium across cell membranes, to parathyroid cells with and without pertussis toxin treatment. It was found that A23187 caused suppression of PTH secretion regardless of pertussis toxin treatment. This result is consistent with the evidence for the role of G proteins in coupling extracellular calcium concentration to intracellular calcium concentration, since A23187 bypasses the physiological coupling mechanisms that are linked to G proteins (52). The possible role of G proteins in the cytosol or in intracellular organelles in regulating PTH secretion has also been examined directly, using electropermeabilized parathyroid cells (11, 53). In electropermeabilized adult parathyroid cells, GTP7S, a nonhydrolyzable guanine nucleotide that stimulates G proteins, did not alter the calcium concentration at which maximal PTH secretion was observed and had little, if any, effect on PTH secretion at low calcium concentrations. In electropermeabilized calf parathyroid cells, GppNHp and GTP7S stimulated PTH secretion at low calcium concentration. In general, GppNHp enhanced PTH secretion at all calcium concentrations tested, and the GppNHp effect was attenuated as the calcium concentration was increased.

VI. The Role of Second Messengers A. cAMP The observation that release of cAMP from bovine parathyroid explants and the accumulation of cAMP in parathyroid cells parallel the secretion of PTH in response to changes in the concentration of extracellular calcium gave rise to the hypothesis that cAMP mediates the regulation of PTH secretion by calcium. Furthermore, PTH secretion can be increased or decreased by a number of pharmacological agents that stimulate or inhibit parathyroid adenylate cyclase. Brown et al. (54) proposed that there is a close relationship between the inhibition of intracellular cAMP and PTH secretion regulated by divalent cations. These investigators suggested that the log-linear relationship between cAMP and PTH secretion makes the cell sensitive to the wide range of intracellular cAMP concentrations encountered with changes in ambient calcium concentrations. They further suggested that the inhibitory effects of divalent cations result from a direct effect on adenylate cyclase, but also indicated that the decrease in cAMP could equally reflect changes in phosphodiesterase activity. Several investigators have suggested that the modulation of intracellular cAMP levels that occurs in response to changes in extracellular calcium concentrations is small in amplitude compared to the cAMP response to



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/3-adrenergic or dopaminergic stimulation (55). Also, LeBoff et al. (56) indicate that the relatively large change in PTH secretion in response to a severalfold change in intracellular calcium concentration is accompanied by a relatively small change in cAMP. Thus, either there is a much larger amplification of the cAMP signal as a result of calcium stimulation than as a result of agonist stimulation or cAMP does not have a major role in calciumregulated PTH secretion. cAMP does seem to be an important second messenger in mediating PTH secretion in response to agonists. Isoproterenol, which acts through the /3-adrenergic receptor, is a classic example of mediation of PTH release by activation of adenylate cyclase. Many other examples, such as stimulation by epinephrine, prostaglandin F 2a ,

is a calcium-activated, phospholipid-dependent enzyme that plays an important role in intracellular events related to numerous cell functions. High extracellular calcium concentration increases the IP 3 concentration in the parathyroid cell. With the concomitant increase in DAG that occurs with increased levels of IP3, it might be expected that PKC activity would also be elevated by high extracellular calcium in the parathyroid cell. Although this makes it unlikely that PKC is the sole mediator of calcium-regulated PTH secretion, it is clear that activation of PKC may modulate PTH secretion at low levels of extracellular calcium. When PKC is activated by phorbol esters at extracellular calcium concentrations normally associated with inhibition of PTH secretion, PTH secretion is enhanced. On

and dopamine also suggest the importance of cAMP in

the other hand, at extracellular calcium concentrations

mediating PTH release in response to agonists.

that stimulate PTH secretion, phorbol esters have no effect on secretion (60). Since PTH secretion is influenced by both calcium concentration and PKC activity, it is of interest to consider their possible interaction. However, the effect of PKC activation on intracellular calcium mobilization in parathyroid cells is controversial. One report using the fluorescent calcium indicator quin-2 suggests that during enhancement of PTH secretion by phorbol esters, the cytosolic calcium concentration decreased (61). Other studies report no change in intracellular calcium (62).

B. Inositol phosphates As we have previously indicated, there is considerable experimental evidence that the plasma membrane contains a sensor that couples extracellular calcium to a second messenger system that stimulates the release of calcium from intracellular stores. The second messenger system is likely to include the turnover of phosphoinositides, since in many cells these compounds are implicated in the control of the intracellular calcium concentration and in cellular secretion. The turnover of phosphoinositides is part of a signal transduction pathway that generates inositol trisphosphate (IP3) and diacylglycerol (DAG) by means of phosphatidylinositol-4.5bisphosphate (PIP2) hydrolysis within plasma membranes. In the parathyroid cell, the concentrations of both IP 3 and DAG are increased in response to an increase in the concentration of extracellular calcium, suggesting that PIP 2 hydrolysis may be involved in the inhibition of PTH secretion (57). In saponin-permeabilized parathyroid cells, IP 3 causes release of intracellular calcium (58). Further work demonstrated a variety of secretory responses to other products of phospholipid hydrolysis, such as dilauroyl, dioleoyl, and dipalmitoyl phosphatidic acids (59). The diversity of response suggests that these compounds may also influence other second messengers.

f

C. Protein kinase C (PKC) In many cell types, the turnover of phosphoinositides occurs after the interaction of biologically active substances with specific cell surface receptors. Polyphosphoinositide breakdown is an important mechanism for signal transduction that generates two intracellular second messengers, IP 3 and DAG. IP 3 mobilizes calcium from intracellular stores, and DAG activates PKC. PKC

VII. Other Factors that Affect PTH Secretion A. ATP The addition of ATP or ATP7S to parathyroid cells evokes a rapid and transient increase in the intracellular calcium concentration followed by a small steady state increase (63). The calcium transient is elicited even in the absence of extracellular calcium and is inhibited by extracellular Ca, Mg, or Sr. These results suggest that extracellular ATP acts as an agonist for a purinoreceptor coupled to a second messenger system that stimulates the release of calcium from the same intracellular pool that is activated by extracellular divalent cations. Extracellular ATP may also alter the flux of calcium through calcium channels. B. Vitamin D The two major hormones that regulate calcium homeostasis are PTH and the active form of Vitamin D3 [1,25(OH)2D3]. Of interest is the relationship between PTH and 1,25(OH)2D3. Parathyroid glands possess specific receptors for 1,25(OH)2D3 (64) and a calcium binding protein, suggesting that the parathyroid gland may be a target tissue for vitamin D action. The administration of 1,25(OH)2D3 to patients with chronic renal failure



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or primary hyperparathyroidism (65) can reduce serum levels of PTH. Several studies suggest that in vivo and in vitro, 1,25(OH)2D3 inhibits PTH secretion. Some investigators suggest that 1,25(OH)2D3 does not have a direct effect on PTH secretion (66). Binding of 1,25(OH)2D3 by parathyroid tissue is reduced in chronic renal failure and may contribute to the pathogenesis of secondary hyperparathyroidism (67). In bovine parathyroid cells, 1,25(OH)2D3 (10~8 M) rapidly increased the intracellular calcium concentration as determined by Indo-1 fluorescence (47). Because of the rapid action of 1,25(OH)2D3 on intracellular calcium, these authors propose that this effect is independent of genomic activation. Recent studies have focused on the steroid-like hormone effects of 1,25(OH)2D3 in decreasing or increasing gene transcription in biological systems. Silver, et al. (68) demonstrated suppression of mRNA for pre-proPTH by 1,25(OH)2D3 in a specific, dose-responsive, and reversible manner. The effects occurred in physiological doses (10~u M) and were larger with 1,25(OH)2D3 than with 24,25(OH)2D3. Using the nuclear runoff technique, the synthesis of pre-proPTH mRNA was altered as early as 1-2 h after exposure to 1,25(OH)2D3 (69). Kremer et al. (70) evaluated protooncogene expression in cultured bovine parathyroid cells. 1,25(OH)2D3 may modulate parathyroid cell proliferation, since addition of this metabolite to quiescent cells abolishes the expression of the cmyc gene without change in expression of the c-fos gene. Circulating levels of calcium and 1,25(OH)2D3 are important in controlling PTH secretion in chronic renal failure. The relative contribution of each regulator in the pathogenesis of parathyroid hyperplasia remains unknown. Further studies of normal and histologically abnormal parathyroid tissue will be necessary to determine the physiological role of 1,25(OH)2D3] in the regulation of PTH synthesis and secretion.

C. Glucocorticoids

To test the effects of glucocorticoids on PTH secretion, Sugimoto et al. (71) incubated dexamethasone with primary parathyroid cell cultures for 48 h. While 1,25(OH)2D3 inhibited PTH release, glucocorticoids significantly stimulated PTH release in a dose-dependent manner. Addition of 1,25(OH)2D3 in the presence of dexamethasone decreased PTH secretion, although not to the control level. The parathyroid gland as a site of action for glucocorticoids is an attractive hypothesis since glucocorticoid-associated osteopenia is marked by aggressive bone resorption.

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D. Dopamine Dopamine transiently increases bovine PTH in vitro and in vivo. Dopamine stimulates adenylate cyclase in the bovine parathyroid gland via a G protein. Identification of a dopamine receptor on parathyroid glands remained elusive until the discovery of the Dl receptor in calf parathyroid gland (72). Interactions of dopamine with its receptor are modulated by Gpp(NH)p, presumably through a stimulatory G protein. In humans, infusions of dopamine (4 jig/kg-min) raised PTH levels in patients undergoing open heart surgery but is without effect under normal physiological conditions (73). E. Estrogen Estrogen has the recognized action in mineral homeostasis of inhibiting bone resorption. Estrogen has also been used to decrease serum calcium concentrations in primary hyperparathyroidism, although PTH levels in these studies remained unchanged or increased (74). Studies on isolated bovine parathyroid tissue suggest that estrogen and progesterone stimulate PTH secretion (75), and estradiol and progesterone stimulate PTH secretion from abnormal human parathyroid tissue (76). The physiological relevance of these findings and the mechanism by which estrogen and progesterone stimulate PTH release remain to be elucidated. VIII. Summary Calcium is the most important physiological regulator of PTH secretion. Peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of the extracellular calcium concentration. We suggest, therefore, that intracellular calcium concentration is a regulator of PTH secretion that maintains calcium homeostasis. Other factors may be responsible for modulation of the intracellular calcium concentration, ultimately modulating PTH secretion. The "paradoxical" nature of the dependence of PTH secretion on the calcium concentration may be explained by considering PTH secretion to be unusual in a quantitative, rather than a qualitative, fashion. A possible mechanism for the control of PTH secretion by intracellular calcium, which involves calcium-activated potassium channels, is proposed. The parathyroid cell plasma membrane contains several sensors or channels by means of which the cell senses extracellular calcium. It is not clear whether these entities are coupled to each other or whether they function independently. Guanine nucleotide regulatory proteins are transducers of extracellular signals, including calcium. Several other second messengers that influence PTH secretion


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have also been described, but possible interactions between these messengers have not yet been determined.

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Parathyroid hormone secretion in magnesium deficiency.

Parathyroid hormone secretion during exchange transfusion.

Parathyroid hormone secretion in primary hyperparathyroidism.

Does cimetidine inhibit parathyroid hormone secretion?

Stimulation of the secretion of parathyroid hormone during hypoglycemic stress.

Effects of transdermal estrogen replacement on parathyroid hormone secretion.

Non-autonomy of parathyroid hormone secretion in acute primary hyperparathyroidism.

Effect of somatostatin on parathyroid hormone and calcitonin secretion.

Role of adrenergic stimuli in parathyroid hormone secretion in man.

Circadian rhythm and pulsatility of parathyroid hormone secretion in man.

Fluoride Modulates Parathyroid Hormone Secretion in vivo and in vitro.

Angiotensin-Converting Enzyme Inhibition and Parathyroid Hormone Secretion.

Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells.

Avian parathyroid glands in organ culture: secretion of parathyroid hormone and calcitonin.

[Regulation of growth hormone-releasing hormone gene expression and secretion].

Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent nonsuppressible component of secretion.

The by-pass of tissue hormone stores during the secretion of newly synthesized parathyroid hormone.

The effect of 1,25-dihydroxycholecalciferol on the parathyroid hormone secretion of porcine parathyroid glands and human parathyroid adenomas in vitro.

Parathyroid hormone and the regulation of acid-base balance.

Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol.

The carboxy-terminus of parathyroid hormone is essential for hormone processing and secretion.

Calcium-dependent intracellular degradation of parathyroid hormone: a possible mechanism for the regulation of hormone stores.

[Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)].

Neuroendocrine regulation of pulsatile luteinizing hormone secretion in elderly men.