Sodium and Ionic Strength Sensing by the Calcium Receptor
1998; Elsevier BV; Volume: 273; Issue: 31 Linguagem: Inglês
10.1074/jbc.273.31.19579
ISSN1083-351X
AutoresStephen J. Quinn, Olga Kifor, Sunita Trivedi, Rubén Díaz, Peter Vassilev, Edward M. Brown,
Tópico(s)Ion Transport and Channel Regulation
ResumoThe 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+]osimilar 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. 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+]osimilar 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. A calcium-sensing receptor (CaR) 1The abbreviations used are: CaR, calcium-sensing receptor; PTH, parathyroid; HEK, human embryo kidney. 1The abbreviations used are: CaR, calcium-sensing receptor; PTH, parathyroid; HEK, human embryo kidney. has been cloned that allows cells expressing this receptor to sense external Ca2+ within its physiological range of ∼1.5 mm (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 2Brown E.M. Physiol. Rev. 1991; 71: 371-411Crossref PubMed Scopus (622) Google Scholar). 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 (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 3Garrett J.E. Tamir H. Kifor O. Simin R.T. Rogers K.V. Mithal A. Gagel R.F. Brown E.M. Endocrinology. 1995; 136: 5202-5211Crossref PubMed Google Scholar, 4Riccardi D. Park J. Lee W.-S. Gamba G. Brown E.M. Hebert S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (434) Google Scholar). 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 (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 5Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (342) Google Scholar, 6Rogers K.V. Dunn C.E. Brown E.M. Hebert S.C. Brain Res. 1997; 744: 47-56Crossref PubMed Scopus (98) Google Scholar, 7Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (97) Google Scholar, 8Sands J.M. Naruse M. Baum M. Jo I. Hebert S.C. Brown E.M. Harris H.W. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (77) Google Scholar). 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 (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 9Quinn S.J. Ye C.-P. Diaz R. Kifor O. Bai M. Vassilev P. Brown E. Am. J. Physiol. 1997; 273: C1315-C1323Crossref PubMed Google Scholar). 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 (10Traynelis S.F. Hartley M. Heinemann S.F. Science. 1993; 268: 873-876Crossref Scopus (344) Google Scholar). 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, theN-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 (11Pullan L.M. Stumpo R. Powel R.J. Paschetto K.A. Britt M. J. Neurochem. 1992; 59: 2087-2093Crossref PubMed Scopus (38) Google Scholar, 12Reynolds I.J. Miller R.J. Eur. J. Pharmacol. 1988; 151: 103-112Crossref PubMed Scopus (103) Google Scholar, 13Rock D.M. MacDonald R.L. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 463-482Crossref PubMed Scopus (150) Google Scholar, 14Romano C. Williams K. Carter C. Neuropharmacology of Polyamines. Academic Press, London1994: 81-106Google Scholar). Furthermore, modulation of theN-methyl-d-aspartate receptor by spermine and pH are susceptible to ionic strength, suggesting that they may also act through charge screening (10Traynelis S.F. Hartley M. Heinemann S.F. Science. 1993; 268: 873-876Crossref Scopus (344) Google Scholar). 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 (15Denton D.A. McKinley M.J. Weisinger R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7397-7404Crossref PubMed Scopus (133) Google Scholar, 16Johnson A.K. Cunningham J.T. Thunhorst R.L. Clin. Exp. Pharm. Physiol. 1996; 23: 183-191Crossref PubMed Scopus (143) Google Scholar, 17McKinley M.J. Pennington G.L. Oldfield B.J. Clin. Exp. Pharm. Physiol. 1996; 23: 271-281Crossref PubMed Scopus (89) Google Scholar). 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 (18Fitzsimons J.T. Acta Physiol. Scand. 1989; 583: 15-25Google Scholar). Later research indicated that both ionic strength and osmolality may be sensed by the circumventricular organs and hypothalamic nuclei (e.g. paraventricular and supraoptic) (19Thornton S.N. deCaro G. Epstein A.N. Massi M. The Physiology of Thirst and Sodium Appetite. Plenum Press, New York1984: 103-108Google Scholar, 20Thrasher T.N. Brown C.J. Keil L.C. Ramsay D.J. Am. J. Physiol. 1980; 238: R333-339PubMed Google Scholar, 21Weisinger R.S. Denton D.A. McKinley M.J. Simpson J.B. Tarjan E. deCaro G. Epstein A.N. Massi M. The Physiology of Thirst and Sodium Appetite. Plenum Press, New York1984: 485-490Google Scholar, 22Tarjan E. Cox P. Denton D.A. McKinley M.J. Weisinger R.S. deCaro G. Epstein A.N. Massi M. The Physiology of Thirst and Sodium Appetite. Plenum Press, New York1984: 473-478Google Scholar, 23Leng G. Mason W.T. Dyer R.G. Prog. Neuroendocrinol. 1982; 34: 75-82Crossref PubMed Scopus (109) Google Scholar, 24Oliet S.H.R. Bourque C.W. Trends Neucrosci. 1994; 17: 340-344Abstract Full Text PDF PubMed Scopus (35) Google Scholar). 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 (23Leng G. Mason W.T. Dyer R.G. Prog. Neuroendocrinol. 1982; 34: 75-82Crossref PubMed Scopus (109) Google Scholar, 24Oliet S.H.R. Bourque C.W. Trends Neucrosci. 1994; 17: 340-344Abstract Full Text PDF PubMed Scopus (35) Google Scholar). 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 receptorper 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. 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 (25LeBoff M.S. Shoback D. Brown E.M. Thatcher J. Lembruno R. Beudoin D. Henry M. Wilson R. Pallotta J. Marynick S. Stock J. Leight G. J. Clin. Invest. 1985; 75: 49-57Crossref PubMed Scopus (121) Google Scholar). 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 (26Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) 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 (26Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 200 μg/ml hygromycin. 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 (26Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). 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 (26Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). 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 mformic acid, respectively, and quantitated using a liquid scintillation counter (27Brown E.M. El-Hajj Fuleihan G. Chen C.J. Kifor O. Endocrinology. 1990; 127: 1064-1071Crossref PubMed Scopus (93) Google Scholar). 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 mmCaCl2, 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 mmdid not change the measurement of ionized Ca2+. 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 occurin vivo. Fig. 1 Ashows 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.1 B. 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. The concentration-response relationships for activation of the CaR by [Ca2+]o were compared in the presence of varying concentrations of NaCl (Fig.2 A). With no alteration in NaCl, the threshold for cell activation is ∼1.5 mmcalcium with an EC50 of ∼3.5 mm calcium. With addition of NaCl in 20 mm increments, the threshold and EC50 both shift to the right suggesting either a change in the CaR or the ability of [Ca2+]oto activate the receptor. The change in EC50 was linear with changes in NaCl both above and below that in our standard solution (Fig. 2 B). Agonist activation of the CaR leads to stimulation of phospholipase C and increased production of inositol phosphates in CaR-transfected HEK cells as well as cells expressing the receptor endogenously. To ensure that the ionic strength effect described above involved activation of the CaR, inositol phosphate production was also examined during stimulation in the presence of different NaCl concentrations. As with the [Ca2+]i responses, inositol phosphate accumulation was likewise shifted in a similar manner, with enhanced accumulation observed with decreased ionic strength and the converse with increased ionic strength (Fig.3). 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. 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 mmNaCl. 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. 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. 6 A). Likewise the addition of 40 mm sodium iodide or 40 mm sodium bicarbonate had similar effects as did 40 mm NaCl (Fig. 6 b). 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. 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. 7 c). 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. 7 D) as well as a blunted maximal [Ca2+]i response. Bradykinin produced only a small [Ca2+]iresponse 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). 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. 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. 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) (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 2Brown E.M. Physiol. Rev. 1991; 71: 371-411Crossref PubMed Scopus (622) Google Scholar). 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 (1Brown E.M. Gamba G. Riccardi D. Lombardi D. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2327) Google Scholar, 4Riccardi D. Park J. Lee W.-S. Gamba G. Brown E.M. Hebert S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (434) Google Scholar, 5Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (342) Google Scholar, 9Quinn S.J. Ye C.-P. Diaz R. Kifor O. Bai M. Vassilev P. Brown E. Am. J. Physiol. 1997; 273: C1315-C1323Crossref PubMed Google Scholar). 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 CaCl2previously reported for CaCl2 only solutions or NaCl:CaCl2 mixtures (28Butler J.N. Biophys. J. 1968; 8: 1426-1433Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 29Bates R.G. Staples B.R. Robinson R.A. Anal. Chem. 1970; 42: 867-871Crossref Scopus (195) Google Scholar, 30Siggaard-Andersen O. Thode J. Fogh-Andersen N. Scand. J. clin. Lab. Invest. 1983; 43: 11-16Crossref Scopus (28) Google Scholar). 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 (28Butler J.N. Biophys. J. 1968; 8: 1426-1433Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 29Bates R.G. Staples B.R. Robinson R.A. Anal. Chem. 1970; 42: 867-871Crossref Scopus (195) Google Scholar, 30Siggaard-Andersen O. Thode J. Fogh-Andersen N. Scand. J. clin. Lab. Invest. 1983; 43: 11-16Crossref Scopus (28) Google Scholar). 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 0.2m. 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 (28Butler J.N. Biophys. J. 1968; 8: 1426-1433Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 29Bates R.G. Staples B.R. Robinson R.A. Anal. Chem. 1970; 42: 867-871Crossref Scopus (195) Google Scholar, 30Siggaard-Andersen O. Thode J. Fogh-Andersen N. Scand. J. clin. Lab. Invest. 1983; 43: 11-16Crossref Scopus (28) Google Scholar). 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 (28Butler J.N. Biophys. J. 1968; 8: 1426-1433Abstract Full Text PDF PubMed Scopus (68) Google Scholar) 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 0.2m. The EC50 for Ca2+
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