From the Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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The calcium-sensing receptor (CaR) is activated by small changes in extracellular calcium [Ca2+]o) in the physiological range, allowing the parathyroid gland to regulate serum [Ca2+]o; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and charged residues in the extracellular domain of the CaR appear critical for receptor activation through electrostatic interactions, suggesting that ionic strength could modulate CaR activation by polycationic agonists. Changes in the concentration of external NaCl potently altered the activation of the CaR by external Ca2+ and spermine. Ionic strength had an inverse effect on the sensitivity of CaR to its agonists, with lowering of ionic strength rendering the receptor more sensitive to activation by [Ca2+]o and raising of ionic strength producing the converse effect. Effects of osmolality could not account for the modulation seen with changes in NaCl. Other salts, which differed in the cationic or anionic species, showed shifts in the activation of the CaR by [Ca2+]o similar to that elicited by NaCl. Parathyroid cells were potently modulated by ionic strength, with addition of 40 mM NaCl shifting the EC50 for [Ca2+]o inhibition of parathyroid hormone by at least 0.5 mM. Several CaR-expressing tissues, including regions of the brain such as the subfornical organ and hypothalamus, could potentially use the CaR as a sensor for ionic strength and NaCl. The Journal guidelines state that the summary should be no longer than 200 words.
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INTRODUCTION |
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A calcium-sensing receptor (CaR)1 has been cloned that allows cells expressing this receptor to sense external Ca2+ within its physiological range of ~1.5 mM (1, 2). Initially cloned from bovine parathyroid cells, the CaR is highly expressed in the tissues involved in regulating [Ca2+]o including the parathyroid (PT), calcitonin-secreting cells of the thyroid (C cells), and several regions of the kidney (1, 3, 4). Interestingly, the CaR is also distributed in a number of other tissues which do not have well established roles in the control of [Ca2+]o. These include several regions of the brain (e.g. the subfornical organ and hypothalamus), the pituitary, collecting duct of the kidney, lung, and the intestines (1, 5-8). In many of these tissues the physiological role of the CaR is not understood. One possibility is that the CaR senses endogenous ligands other than [Ca2+]o, thus allowing the CaR to function in a number of specialized capacities in different CaR-expressing tissues.
The CaR is activated by both polyvalent cations and polycationic molecules that interact with the extracellular domain of the receptor (1, 9). This might take place through the screening of charged side chains of acidic or basic amino acids, rather than the more classical binding through hydrogen bonding and salt bridges. If its endogenous agonists act by screening charges on the CaR, then activation of the receptor by these ligands should be modulated by conditions such as changes in ionic strength (10). With the addition of salts, the ionic strength will increase and the ability of the polycationic ligand to activate the CaR should be diminished. Likewise, the removal of salts and the resultant decrease in ionic strength should have the opposite effect. These effects of ionic strength can be explained by changes in the Debye length of the electrical field surrounding the charged agonist. The Debye length is inversely proportional to the square root of the ionic strength of the extracellular solution. For example, addition of NaCl would increase the ionic strength of the solution and should increase the concentration of [Ca2+]o required for half-maximal activation of the CaR. Interestingly, the N-methyl-D-aspartate receptor shares some regions of homology with the CaR, and both receptors can be modulated by divalent cations, spermine and polycationic molecules such as neomycin (11-14). Furthermore, modulation of the N-methyl-D-aspartate receptor by spermine and pH are susceptible to ionic strength, suggesting that they may also act through charge screening (10).
Ionic strength can have substantial effects on a number of different cell types, particularly those involved in the regulation of fluid volume, osmolality and extracellular sodium. The subfornical organ and hypothalamus regulate the secretion of vasopressin by sensing the systemic levels of various hormones, including angiotensin II, and the level of NaCl (15-17). Early studies using injections of NaCl into these regions of the brain suggested that there existed a sodium sensor, since NaCl injections were much more effective in the control of vasopressin and drinking behavior than a hyperosmotic but nonionic solution (18). Later research indicated that both ionic strength and osmolality may be sensed by the circumventricular organs and hypothalamic nuclei (e.g. paraventricular and supraoptic) (19-24). However, ionic strength and osmolality are intimately linked since NaCl is both the major salt and osmolyte in the extracellular fluid and blood. The mechanisms underlying osmotic and sodium sensing has not been fully delineated. Osmosensing is thought to involve changes in cell volume that result in the modulation of stretch-activated ion channels on the plasma membrane (23, 24). The existence of and mechanisms involved in sodium sensing remain poorly understood.
Some tissues normally experience large changes in ionic strength, such as the collecting duct of the kidney, where ionic strength and osmolality may act in a more independent manner since urea is a major contributor to the osmolality of the urine. Nevertheless, urinary salt composition can vary greatly for both sodium and potassium, whose concentrations can easily range from 50 to 200 mM. In addition, the area close to the plasma membrane of some nephron segments may experience large changes in local ionic strength when ion transport activity is high, particularly in the absence of water transport as in the renal thick ascending limb.
The following studies indicate that ionic strength is an important modulator of CaR activation. Changes in ionic strength can alter the electrical field surrounding polycationic CaR agonists, thus providing a potential mechanism for modulating their activation of the CaR. In addition, ionic strength might also modify the state of the receptor per se, leading to a change in the sensitivity of the CaR to its agonists. The ramifications of these actions of ionic strength are far reaching and must be considered wherever the CaR is expressed.
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EXPERIMENTAL PROCEDURES |
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Preparation of Dispersed Bovine Parathyroid Cells-- Dispersed parathyroid cells were prepared from parathyroid glands of 1-3 week-old calves using digestion with collagenase and DNase, and PTH secretion was assessed by radioimmunoassay as described previously (25).
Culturing and Maintenance of CaR-transfected and Untransfected HEK 293 Cells-- These cell lines were the generous gift of Dr. Kimberly Rogers, NPS Pharmaceuticals Inc., Salt Lake City, UT. The CaR-expressing HEK 293 cells were stably transfected with the human parathyroid CaR (26) and selected by hygromycin resistance. The transfected HEK 293 cells express the CaR on the membrane surface and are responsive to addition of CaR agonists to the external medium (26). Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 200 µg/ml hygromycin.
Measurement of [Ca2+]i Using Cell Population System-- Coverslips with near-confluent HEK cells were loaded with fura-2/AM and placed diagonally into thermostatted cuvettes equipped with a magnetic stirrer, using a modification of techniques we used previously (26). The bath solution was stirred at 37 °C, and CaR agonists were added to the desired final concentration. Excitation monochrometers were centered at 340 and 380 nm with emission light collected at 90° using a long-pass emission filter. The 340/380 excitation ratio of emitted light and in vitro calibrations were used to calculate [Ca2+]i as described previously (26).
Measurement of [3H]Inositol Phosphates-- The transfected cells were labeled with [3H]inositol (~10 µCi/106 cells) overnight in medium 199 (with 10 µl/ml penicillin-streptomycin, 10 mM Hepes, pH 7.5, and 15% bovine serum), washed with solution (10 mM LiCl, 0.5 mM MgSO4, 0.5 mM CaCl2, and 2 mg/ml bovine serum albumin in Eagle's MEM with Earle's salts), and then incubated with polyvalent cations for 30 min. The reactions were terminated with a final concentration of 10% trichloroacetic acid. After sedimentation of precipitated debris and removal of trichloroacetic acid by ether extraction, inositol phosphates in the aqueous phase were subsequently separated on Dowex anion exchange columns. The radioactive inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate were eluted stepwise with 0.2, 0.4, 0.8, and 1.2 M formate containing 0.1 M formic acid, respectively, and quantitated using a liquid scintillation counter (27).
Activity of Calcium Ions in Solutions of Varying Sodium Concentrations-- The CaCl2 activity was calculated using determinations of osmolality (Osmette A, Precision Scientific, Natick MA) of solutions containing differing concentrations of NaCl and CaCl2. In our standard solution containing 125 mM NaCl and 0.5 mM CaCl2, the osmolality was 272 mOsm. With addition of 5 and 10 mM CaCl2, the osmolality increased by 10 and 20 mOsm, respectively, yielding a mean activity coefficient of ~0.5 for CaCl2. Addition or removal of 80 mM NaCl had little effect on the changes in osmolality measured with addition of CaCl2.
The ionized Ca2+ concentration (the sum of electrostatically bound Ca2+ and active Ca2+) was measured using an ion-selective electrode electrolyte analyzer, AVL 987-S (AVL Scientific Corp., Roswell, GA). Solutions were made with Ca2+ concentration varying from 0.5 to 6 mM and NaCl concentrations varying from 45 to 205 mM. Hypo-osmotic reduction in NaCl concentration by 40 and 80 mM as well as hyperosmotic increase in NaCl concentration by 40 and 80 mM did not change the measurement of ionized Ca2+. ![]() |
RESULTS |
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Effect of NaCl on [Ca2+]o-evoked Activation of the Calcium-sensing Receptor-- The effect of ionic strength on activation of the calcium-sensing receptor by external Ca2+ was studied by adding or removing NaCl from the extracellular media followed by the elevation of [Ca2+]o. Changes in cytosolic calcium ([Ca2+]i) were used as an indicator of CaR activation in CaR-expressing HEK cells. Osmolality was not held constant in these experiments; rather both ionic strength and osmolality were allowed to change concomitantly, as would occur in vivo. Fig. 1A shows the effect of changing the concentration of NaCl on activation of the CaR by 2.5 mM [Ca2+]o in HEK 293 cells that have been stably transfected with the human CaR. When NaCl is elevated, the [Ca2+]i response to 2.5 mM [Ca2+]o is attenuated, while removal of NaCl produces an enhanced response. The dose-dependence of the effects of external NaCl on CaR activation by [Ca2+]o are shown in Fig. 1B. They indicate that peak [Ca2+]i transients, due to Ca2+ mobilization, as well as sustained [Ca2+]i increases, due to modification of Ca2+ movement across the plasma membrane, are modulated in a similar direction and magnitude by changes in NaCl concentration.
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Ionic Strength Modulation of CaR Activation by Spermine-- Two broad classes of CaR agonists are polyvalent cations (i.e. Ca2+ and Gd3+) and polycationic molecules (i.e. spermine and neomycin). To test the generality of the effects of ionic strength on CaR activation, the CaR-transfected HEK cells were stimulated by spermine in the presence of varying concentrations of NaCl. Similar to the effects on [Ca2+]o activation, ionic strength shifted the dose-response relationship for spermine so that the EC50 was inversely proportional to the ionic strength (Fig. 4, A and B). In the case of spermine, the relationship between ionic strength and EC50 was clearly nonlinear, a trend that may be magnified by the multiple positive charges spaced out along the spermine molecule.
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Effects of Changes in Ionic Strength versus Osmolality-- Experiments were also performed maintaining osmolality constant while NaCl was varied in the extracellular media. For removal of NaCl, the NaCl was substituted by sucrose (i.e. 40 mM NaCl was replaced by 80 mM sucrose), while addition of NaCl involved replacement of sucrose in a Hepes-buffered solution containing 80 mM sucrose with 40 mM NaCl. Fig. 5 shows that iososmotic changes in NaCl produced similar shifts in the dose-response relationship of [Ca2+]o activation of the CaR as hyperosmotic and hypo-osmotic changes in NaCl. The iso-osmotic comparisons were as follows: standard solution plus 80 mM sucrose (EC50 3.4 mM) versus standard solution plus 40 mM NaCl (EC50 4.6 mM) for hyperionic changes; and standard solution (EC50 3.6 mM) versus standard solution with the addition of 80 mM sucrose and the removal of 40 mM NaCl (EC50 2.4 mM) for hypoionic changes. Addition of 80 mM sucrose appeared to cause a slight (<10%) shift in the EC50 for CaR activation under all conditions suggesting a small osmotic or sucrose effect. Addition or removal of NaCl in solutions with or without sucrose had the same effect on EC50, indicating that the large shifts were due to ionic strength, not osmolality. Untransfected cells are not responsive to [Ca2+]o and other CaR agonist, regardless of the ionic strength.
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Specificity of Salts for Ionic Strength Effect-- The effects of NaCl may involve a sensor of sodium, chloride, or ionic strength. Different salts were used to determine the mechanism of the NaCl effect. Addition of 40 mM choline chloride or potassium chloride had the same effect on the EC50 for activation of the CaR-transfected HEK cells by [Ca2+]o as did addition of 40 mM NaCl (Fig. 6A). Likewise the addition of 40 mM sodium iodide or 40 mM sodium bicarbonate had similar effects as did 40 mM NaCl (Fig. 6b). These data indicate that neither sodium nor chloride are specific modulators of CaR activation, rather it is the change in ionic strength that modifies the activation of the CaR by [Ca2+]o.
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Effect of Ionic Strength on Other Receptors-- To determine if the effect of ionic strength was specific to the CaR, activation of endogenous G protein-coupled receptors on the HEK 293 cells were examined in solutions of varying concentrations of NaCl (Fig. 7, A and B). Purinergic and thrombin receptors were tested using ATP and thrombin receptor agonist peptide (SRLLRNP), respectively, as ligands. Determination of the EC50 for thrombin receptor activation indicates that optimal activation of the thrombin receptor was found at physiological NaCl concentration and ionic strength with little effect observed with subtraction or addition of 40 mM NaCl to the standard medium (Fig. 7c). With changes in NaCl of a 80 mM magnitude, the thrombin receptor became less sensitive to agonist stimulation. Activation of the purinergic receptor by ATP showed a slight trend toward reduced sensitivity with increasing ionic strength; however, removal or addition of 80 mM NaCl also resulted in a substantial decline in receptor sensitivity to agonist stimulation (Fig. 7D) as well as a blunted maximal [Ca2+]i response. Bradykinin produced only a small [Ca2+]i response in HEK 293 cells, which made it difficult to use when testing the effects of ionic strength on activation of this endogenous receptor. However, addition or removal of 40 mM NaCl also had little effect on the sensitivity toward bradykinin (data not shown).
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Ionic Strength and the Relative Degree of Activation for CaR as Well as ATP and Thrombin Receptors-- At a constant concentration of receptor agonist, changes in ionic strength will modulate the degree of CaR receptor activation. This is represented in Fig. 8 as the change in the magnitude of receptor activation at an agonist concentration equal to the EC50 concentration in our standard medium. With changes in ionic strength, the CaR shows a simple linear relationship between the change in receptor activation by either [Ca2+]o or spermine and ionic strength. These data suggest that ionic strength can both positively and negatively modulate CaR activation in the presence of a constant calcium concentration. In this way, the CaR could act as an ionic strength or NaCl sensor. Thrombin receptor agonist peptide and ATP stimulation shows a more complex relationship, indicating optimal or near-optimal receptor activation at physiological ionic strength and reduced activation with either increases or decreases in ionic strength.
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Ionic Strength and PTH Secretion-- To better understand the potential physiological impact of ionic strength on tissues expressing endogenous CaR, the effect of addition of NaCl on PTH secretion was assessed in bovine parathyroid cells. These experiments on the secretion of PTH showed a right shift in the [Ca2+]o-PTH concentration-response relationship, indicating a reduced sensitivity toward [Ca2+]o (Fig. 9) with increased ionic strength. Addition of 40 mM NaCl produced a [Ca2+]o set-point increase of 0.5 mM, when compared with the osmotic control of 80 mM sucrose addition.
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DISCUSSION |
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The CaR is activated by agonists possessing multiple positive charges, including polyvalent cations (i.e. Ca2+ and Gd3+) and polycationic molecules (i.e. spermine and neomycin) (1, 2). Given this shared characteristic of CaR ligands, it is quite likely that these agonists act on the extracellular domain through an electrochemical mechanism, rather than a biochemical one. One possibility is that these cationic agonists may screen negatively charged residues of the extracellular domain of CaR, thus altering the conformation of the receptor. Indeed, multiple negative residues are grouped within this domain, particularly in the amino acid sequence of 126-251 (1, 4, 5, 9).
If screening of charge is an important mechanism of agonist action, then activation of the CaR should be modulated by ionic strength. The Debye length of the electrical field around a soluble polycationic ligand is inversely proportional to the square root of the ionic strength. With a decreased ionic strength and a longer Debye length, the polycationic ligands should be more effective at screening negatively charged residues of the extracellular domain. Furthermore, the positively and negatively charged residues could have a greater impact on the conformation of the receptor, as their charge will not be screened as effectively by the reduced concentrations of monovalent ions present in the solution. Consistent with this model, decreased ionic strength allowed for more effective activation of the CaR by both Ca2+ and spermine. Likewise an increase in ionic strength and a decrease in the Debye length attenuates the actions of [Ca2+]o and spermine as if the affinity of the agonist for the receptor had been reduced.
The measurements of ionized Ca2+ by ion-sensitive
electrodes and unassociated CaCl2 by freezing-point
depression indicate that there are no large changes in the ionized
Ca2+ or active Ca2+ in the hypotonic or
hypertonic solutions used in our experiments, consistent with the
modest changes in the mean activity coefficient of CaCl2
previously reported for CaCl2 only solutions or
NaCl:CaCl2 mixtures (28-30). Our observed mean activity
coefficient for CaCl2 of ~0.5 is similar to that measured
by others and the subtle change in these parameter were beyond the
sensitivity of our equipment (28-30). The mean activity coefficient of
CaCl2, which represents the single ion activity
coefficients of Ca2+ and Cl, varies by less
than 10% in solutions of ionic strength between 0.1 and 0.2 M. Since the single ion activity coefficient of
Cl
changes with ionic strength and is the principle
counterion for Ca2+, the calculated single ion activity
coefficient of Ca2+ has a value ~0.3 in a NaCl solution
of 0.15 mol/kg ionic strength and can vary by 10-25% in solutions of
ionic strength between 0.1 and 0.2 M, depending on the
assumptions employed in the method of estimation (28-30). The
calculated single ion activity coefficient of Ca2+ also
varies slightly with concentration of added CaCl2. Using the mean activity coefficient values for CaCl2 reported in
Butler (28) and the Guggenheim assumption of the Debye-Huckel theory, a
~20% change in single ion activity coefficient of Ca2+
was calculated for solutions of ionic strength between 0.1 and 0.2 M. The EC50 for Ca2+ stimulation of
CaR-dependent cytosolic calcium responses was 1 mM in our standard HEPES-buffered solution when expressed
as Ca2+ activity and one-third of the shift in the
EC50, which is dependent on ionic strength, can be
accounted for by these changes in Ca2+ activity (28). Our
data and these estimations of Ca2+ activity suggest that
the effects of ionic strength on CaR activation may be due to changes
in the Ca2+ activity at a given ionized or added
Ca2+ concentration as well as changes in electrostatic
interactions within the receptor itself. We assume that the activity of
other polycationic agonists of the CaR will behave in a manner similar to that of Ca2+.
The effect of ionic strength on the activation of the CaR depends, in part, on the agonist under investigation (Fig. 8). The shift in the EC50 for activation by [Ca2+]o appeared linear and was ~0.25 (7%) mM, not adjusted for Ca2+ activity, per 10 mM (7-8%) change in ionic strength (i.e. equivalent percent change in EC50 for a given change in ionic strength). The effects on CaR activation by spermine were less linear with a 20 mM (~15%) change in ionic strength from basal solutions producing at least a 35% shift in EC50 for spermine (i.e. twice the percent change in EC50 for a given percent change in ionic strength). While other ligands were not tested, it is likely that the greater the number of positive charges on the given agonist, the greater the effect of ionic strength will be on the activation of this agonist of the CaR. This conclusion may be important in the brain. Extracellular polyamine levels are high and polyamines may be released in synaptic regions during neuronal stimulation (31). Furthermore, the ionic composition of the extracellular solution around the neuron and, in particular, the synapses can change dramatically during neuronal activity.
Our initial studies varied NaCl in order to change the ionic strength of the media. However, other salts were tested that differed in the cationic or anionic species, including ions with different predicted membrane permeabilities. In all cases, there were shifts in the activation of the CaR by [Ca2+]o similar in magnitude to that elicited by NaCl. There was little difference in the effects of salts where the cation was varied but the anion remains chloride. Replacing the chloride with other anion species of similar molar solubilities for their calcium salts yielded comparable results. Only NaCl was tested for effects of decreases in ionic strength, as these were the principal ions in the standard Hepes-buffered solution; however there are no data suggesting that a separate mechanism exists to explain the effects of decreased versus increased ionic strength. Effects of osmolality cannot account for the modulation seen with ionic strength, since experiments using isosmotic solutions with different ionic strengths gave results similar to solutions where NaCl was simply added or removed without replacement by an uncharged osmolyte. This ionic strength effect on receptor activation appears specific to the CaR since the sensitivity of the endogenous purinergic and thrombin receptors to activation by external ATP and thrombin receptor peptide agonist, respectively, showed only modest effect with changes in the ionic strength of the external media, exhibiting bell-shaped relationships with the optimal activation at physiological levels of ionic strength.
The importance of modulation of the CaR by ionic strength on tissue and whole body physiology remains to be established. Several tissues offer interesting possibilities for interactions between NaCl, the chief contributor to ionic strength, and Ca2+. In the kidney, the CaR is strongly expressed on the basolateral membrane of the cortical thick ascending limb (4, 32). Na+ reabsorption is regulated by the CaR and other factors, and Ca2+ handling is linked to Na+ movement in this segment of the nephron, which is nearly impermeant to water. Activation of the CaR appears to be linked to inhibition of Na+ and Ca2+ reabsorption (32, 33). Enhanced Na+ and an equivalent amount of Ca2+ reabsorption at low [Ca2+]o and high levels of PTH without accompanying water movement, for example, will lead to accumulation of these ions at the extracellular side of the basolateral membrane. In the case of Ca2+ accumulation, this should lead to the activation of the CaR and inhibition of reabsorption. Without the modulation of the CaR by ionic strength, Na+ reabsorption could be predominately regulated by Ca2+. However, concomitant Na+ reabsorption and accumulation at the basolateral membrane will lead to an attenuation of CaR activation and less inhibition of ion reabsorption. Thus both Na+ and Ca2+ may interact to modulate CaR activation and the control of Na+ and Ca2+ reabsorption, allowing for continued reabsorption of both ions.
In the collecting duct, the CaR is located on the apical membrane and may be involved in the regulation of the availability and activity of water channels and bulk water reabsorption from the urine at this point in the nephron (8). Concentrated urine will typically have a high concentration of both Na+ and Ca2+. If the concentrations of these ions change in concert, the increased ionic strength with high Na+ concentrations will allow the CaR on the apical membrane of collecting duct to adjust its sensitivity to [Ca2+]o thereby coordinating [Ca2+]o with water and salt handling. Indeed, this ability of increased ionic strength to lower the apparent affinity of the CaR for Ca2+ may be critical to the functioning of the CaR in some saltwater animals, where both ionic strength and Ca2+ levels are often much higher in the ingested fluid as well as in the serum and urine than that found in freshwater and land animals.
The circumventricular organs and hypothalamus are thought to control in large measure the secretion of vasopressin and other hormones of the pituitary (15-17). The circumventricular organs such as the subfornical act as windows to the systemic circulation for the brain, since the vasculature is highly permeable due to the presence of a fenestrated endothelium allowing the neurons in these organs to lie outside of the blood-brain barrier. For example, systemic angiotensin II increases thirst and drinking behavior through activation of receptors located on neurons of the subfornical organ. These neurons are also sensitive to changes in osmolality and ionic strength through alterations in NaCl (34-36). Similar receptors and sensitivities are found in neurons of the hypothalamus, suggesting that the same systems and pathways found in the circumventricular organs are often present in the hypothalamus where additional information processing occurs (23, 24, 37).
Interestingly, CaR density is very high in the subfornical organ and in some nuclei of the hypothalamus (6). The purpose for such high levels of CaR expression is not understood. One possibility is the regulation of a "calcium appetite" with calcium-deficient animals being driven to consume calcium-rich food and drink (38). Such a calcium appetite has been documented for the rat and the chicken when hypocalcemia is induced either through surgical removal of the parathyroids and/or maintenance on a calcium-deficient diet (38-40). These data suggest that there is a central control of calcium intake, similar to that for salt and water. The CaR may also participate in the regulation of water and salt handling through the modulation of CaR activation by ionic strength. Thus small changes in serum ionic strength could modify the secretion of vasopressin and other pituitary hormones, in part through modulation of CaR activity even at a constant level of [Ca2+]o. Our in vitro experiments with bovine parathyroid cells indicate that control of PTH secretion can also be highly sensitive to changes in ionic strength or serum sodium, potentially with a 10 mM (7-8%) change in NaCl yielding a 0.1 mM (7-8%) shift in the calcium set-point. It is curious that existing studies have not observed a clear relationship between serum sodium or osmolality and PTH/[Ca2+]o levels. One confounding factor could be the balancing of activation and attenuation which would occur with parallel serum changes in [Ca2+]o and NaCl. Studies which focus on this relationship between ionic strength and PTH/[Ca2+]o homeostasis may provide better understanding of this potential modulating factor.
In many parts of the body, sodium, calcium and water regulation appear to be linked. In the kidney, sodium reabsorption helps to set the transepithelial potential and the passive paracellular movement of calcium in the cortical thick ascending limb. The collecting duct is presented with special homeostatic complexities by virtue of mediating urinary concentration while still not allowing conditions for calcium stone formation. The intestines are sites for regulated absorption of both sodium and calcium and represent a critical site for ion and water homeostatis. The CaR appears to combine the sensing of both calcium and sodium in one receptor. By having elevation of [Ca2+]o activate the receptor and elevation of sodium attenuate receptor stimulation, a feedback control exists within the receptor itself. Sodium and calcium transport often occur together and, in general, changes in water handling (i.e. in the collecting duct) will change the molality of both ions in the same direction. Thus the antagonist effects of calcium and sodium (ionic strength) offers a built-in negative feedback mechanism. This arrangement could, however, be problematic when it is prudent for sodium and calcium handling to be dissociated.
In summary, the CaR can sense changes in ionic strength independently of alterations in osmolality and the ionic species used to alter ionic strength. Ionic strength is related inversely to the potency of polycationic agonist activation of the receptor. Changes in ionic strength appear to modulate the different classes of polycationic CaR agonists in a similar manner. The ability of ionic strength to modify CaR function appears to be related to the mode of agonist activation, which likely involves screening of charged residues on the extracellular domain of the receptor.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK41415, DK44588, and DK48330 (to E. B.), HL 42120 (to S. Q.), and the St. Giles Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Brigham and Womens'
Hospital, Endocrine-Hypertension Division, 221 Longwood Ave., Boston,
MA 02115. Tel.: 617-732-5630; Fax: 617-732-5764.
1 The abbreviations used are: CaR, calcium-sensing receptor; PTH, parathyroid; HEK, human embryo kidney.
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REFERENCES |
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