Departments of 1 Physiology and 2 Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+ insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.
mechanosensitive channels; mechanoelectrical feedback; lanthanides; chelation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() |
---|
THE IDENTIFICATION OF stretch-activated channels (SACs) in bacteria, plant, and animal cells has led to intense efforts to elucidate their physiological and pathophysiological roles (9, 27, 32). SACs are implicated in a wide range of responses to mechanical perturbations, including cell volume regulation, increased intracellular Ca2+, cell proliferation, gene expression, DNA synthesis, baroreceptor discharge, altered cardiac electrical activity, and release of atrial natriuretic factor (for review, see Ref. 9).
Gadolinium (Gd3+), a trivalent lanthanide, has emerged as the most commonly used tool to identify phenomena dependent on SACs (9). Millet and Pickard (25) originally postulated that Gd3+ blocks mechanosensitive ion channels on the basis of its ability to inhibit orientation of the roots of pea plants (Zea mays) in response to surface contact, thigmotropism, and gravity, geotropism. Direct evidence was provided by Yang and Sachs (41) who showed that Gd3+ blocks stretch-activated cation channels in Xenopus laevis oocytes. Often, 10 µM Gd3+ is sufficient to largely block cation SACs and thereby inhibit mechanosensitive processes (9, 27, 32). By extension, the inability of Gd3+ to modulate certain stretch-dependent events has been taken to imply that SACs are not involved.
The purpose of this communication is to highlight several methodological concerns. A review of recent literature indicates that Gd3+ sometimes is applied in the presence of anions that avidly bind free Gd3+ and effectively remove it from the experimental solution (4, 11, 13-16, 20-23, 29, 31, 33, 40). Notable among these anions are phosphate, carbonate, EGTA, sulfate, carboxylic acids, and albumin, which often are contained in physiological salt solutions and culture media. The use of Gd3+ with anions that avidly bind it can lead to false negative conclusions regarding the role of SACs in physiological processes. False negatives also can arise because several SACs are not blocked by Gd3+ (34, 41). On the other hand, false positives can arise because Gd3+ is not very specific (9).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Binding of
Gd3+.
Martell and Smith (24) and Evans (7) have provided critically reviewed
stability constants for the interaction of
Gd3+ with a number of inorganic
anions, carboxylic acids, and amine polycarboxylic acid chelators
(e.g., EGTA). Selected constants are listed in Table
1. For example, the equilibrium dissociation constants
for PO34 and
CO2
3 are given as
10
22.3 and
10
32.2, respectively. These
values indicate that physiological anions are high-affinity ligands for
Gd3+.
|
Other sources of error. False negative conclusions also can arise because some SACs have been shown to be insensitive to Gd3+ in studies conducted in the absence of anions with high affinity for this lanthanide. Among the Gd3+-insensitive SACs are K+-selective SACs in rat astrocytes (41) and snail (Lymnaea) neurons (34).
A final concern in using Gd3+ as a marker for SACs is that false positive findings may emerge because Gd3+ is not a specific antagonist. Besides SACs, Gd3+ can block L-type (1, 17, 18, 33), T-type (1, 26), and N-type Ca2+ (2, 3), Na+(6), K+ (6, 12), and Ca2+-activated Cl ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Joseph J. Feher for comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-46764 and HL-02798.
Address for reprint requests: C. M. Baumgarten, Dept. of Physiology, Medical College of Virginia, Richmond, VA 23298-0551.
Received 19 November 1997; accepted in final form 29 April 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() |
---|
1.
Biagi, B. A.,
and
J. J. Enyeart.
Gadolinium blocks low- and high-threshold calcium currents in pituitary cells.
Am. J. Physiol.
259 (Cell Physiol. 28):
C515-C520,
1990
2.
Bleakman, D.,
D. Bowman,
C. P. Bath,
P. F. Brust,
E. C. Johnson,
C. R. Deal,
R. J. Miller,
S. B. Ellis,
M. M. Harpold,
and
M. Hans.
Characteristics of a human N-type calcium channel expressed in HEK293 cells.
Neuropharmacology
34:
753-765,
1995[Medline].
3.
Boland, L. M.,
T. A. Brown,
and
R. Dingeldine.
Gadolinium block of calcium channels; influence of bicarbonate.
Brain Res.
563:
142-150,
1991[Medline].
4.
Christensen, O.,
and
E. K. Hoffmann.
Cell swelling activates K+ and Cl channels as well as nonselective, stretch-activated cation channels in Ehrlich ascites tumor cells.
J. Membr. Biol.
129:
13-36,
1992[Medline].
5.
Edsall, J. T.,
and
J. Wyman.
Biophysical Chemistry. Thermodynamics, Electrostatics, and the Biological Significance of the Properties of Matter. New York: Academic, 1958, vol. 1, p. 555-561.
6.
Elinder, F.,
and
P. Arhem.
Effects of gadolinium on ion channels in the myelinated axon of Xenopus laevis: four sites of action.
Biophys. J.
67:
71-83,
1994[Abstract].
7.
Evans, C. H.
Biochemistry of the Lanthanides. New York: Plenum, 1990, p. 18-22, 40-41, 85-210.
8.
Fatatis, A.,
A. Bassi,
M. R. Monsurro,
G. Sorrentino,
G. D. Mita,
G. F. Di Renzo,
and
L. Annunziato.
LAN-1: a human neuroblastoma cell line with M1 and M3 muscarinic receptor subtypes coupled to intracellular Ca2+ elevation and lacking Ca2+ channels activated by membrane depolarization.
J. Neurochem.
59:
1-9,
1992[Medline].
9.
Hamill, O. P.,
and
D. W. McBride, Jr.
The pharmacology of mechanogated membrane ion channels.
Pharmacol. Rev.
48:
231-252,
1996[Abstract].
10.
Hansen, D. E.,
M. Borganelli,
G. P. Stacy, Jr.,
and
L. K. Taylor.
Dose-dependent inhibition of stretch-induced arrhythmias by gadolinium in isolated canine ventricles. Evidence for a unique mode of antiarrhythmic action.
Circ. Res.
69:
820-831,
1991[Abstract].
11.
Hishikawa, K.,
T. Nakaki,
T. Marumo,
H. Suzuki,
R. Kato,
and
T. Saruta.
Pressure enhances endothelin-1 release from cultured human endothelial cells.
Hypertension
25:
449-452,
1995
12.
Hongo, K.,
C. Pascarel,
O. Cazorla,
F. Gannier,
J. Y. Le Guennec,
and
E. White.
Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes.
Exp. Physiol.
82:
647-656,
1997[Abstract].
13.
Jena, M.,
J. F. Minore,
and
W. C. O'Neill.
A volume-sensitive, IP3-insensitive Ca2+ store in vascular endothelial cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C316-C322,
1997
14.
Kim, D.
A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid.
J. Gen. Physiol.
100:
1021-1040,
1992[Abstract].
15.
Kim, D.
Novel cation-selective mechanosensitive ion channel in the atrial cell membrane.
Circ. Res.
72:
225-231,
1993[Abstract].
16.
Kim, D.,
C. D. Sladek,
C. Aguado-Velasco,
and
J. R. Mathiasen.
Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.
J. Physiol. (Lond.)
483:
643-660,
1995.
17.
Lacampagne, A.,
F. Gannier,
J. Argibay,
D. Garnier,
and
J. Y. Le Guennec.
The stretch-activated ion channel blocker gadolinium also blocks L-type calcium channels in isolated ventricular myocytes of the guinea-pig.
Biochim. Biophys. Acta
1191:
205-208,
1994[Medline].
18.
Lansman, J. B.
Blockade of current through single calcium channels by trivalent lanthanide cations. Effect of ionic radius on the rates of ion entry and exit.
J. Gen. Physiol.
95:
679-696,
1990[Abstract].
19.
Le Guennec, J. Y.,
A. Lacampagne,
and
D. Garnier.
Orthophosphate salts induce calcium current recovery from blockade by gadolinium in isolated guinea-pig ventricular myocytes.
Exp. Physiol.
81:
577-585,
1996[Abstract].
20.
Magishi, K.,
J. Kimura,
Y. Kubo,
and
Y. Abiko.
Exogenous lysophosphatidylcholine increases a non-selective cation current in guinea-pig ventricular myocytes.
Pflügers Arch.
432:
345-350,
1996[Medline].
21.
Malek, A. M.,
and
S. Izumo.
Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress.
J. Cell Sci.
109:
713-726,
1996
22.
Malysz, J.,
D. Richardson,
L. Farraway,
M.-O. Christen,
and
J. D. Huizinga.
Generation of slow wave type action potentials in the mouse small intestine involves a non-L-type calcium channel.
Can. J. Physiol. Pharmacol.
73:
1502-1511,
1995[Medline].
23.
Marchenko, S. M.,
and
S. O. Sage.
A novel mechanosensitive cationic channel from the endothelium of rat aorta.
J. Physiol. (Lond.)
498:
419-425,
1997[Abstract].
24.
Martell, A. E.,
and
R. E. Smith.
Critical Stability Constants. Inorganic Complexes. New York: Plenum, 1974, vol. 4, p. 37, 56-57, 269.
25.
Millet, B.,
and
B. G. Pickard.
Gadolinium ion is an inhibitor suitable for testing the putative role of stretch-activated ion channels in geotropism and thigmotropism (Abstract).
Biophys. J.
53:
155a,
1988.
26.
Mlinar, B.,
and
J. J. Enyeart.
Block of current through T-type calcium channels by trivalent metal cations and nickel in neural rat and human cells.
J. Physiol. (Lond.)
469:
639-652,
1993[Abstract].
27.
Morris, C. E.
Mechanosensitive ion channels.
J. Membr. Biol.
113:
93-107,
1990[Medline].
28.
Nakazawa, K.,
M. Liu,
K. Inoue,
and
Y. Ohno.
Potent inhibition by trivalent cations of ATP-gated channels.
Eur. J. Pharmacol.
325:
237-243,
1997[Medline].
29.
Olson, J. E.,
C. Alexander,
D. A. Feller,
M. L. Clayman,
and
E. M. Ramnath.
Hypoosmotic volume regulation of astrocytes in elevated extracellular potassium.
J. Neurosci. Res.
40:
333-342,
1995[Medline].
30.
Perrin, D. D.,
and
B. Dempsey.
Buffers for pH and Metal Ion Control. London: Chapman and Hall, 1974, p. 6, 18-20, 62-64, 104, 157-163.
31.
Quasthoff, S.
A mechanosensitive K+ channel with fast-gating kinetics on human axons blocked by gadolinium ions.
Neurosci. Lett.
169:
39-42,
1994[Medline].
32.
Sachs, F.
Mechanical transduction in biological systems.
Crit. Rev. Biomed. Eng.
16:
141-169,
1988[Medline].
33.
Sadoshima, J.,
T. Takahashi,
L. Jahn,
and
S. Izumo.
Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes.
Proc. Natl. Acad. Sci. USA
89:
9905-9909,
1992[Abstract].
34.
Small, D. L.,
and
C. E. Morris.
Pharmacology of stretch-activated K channels in Lymnaea neurons.
Br. J. Pharmacol.
114:
180-186,
1995[Abstract].
35.
Stacy, G. P., Jr.,
R. L. Jobe,
L. K. Taylor,
and
D. E. Hansen.
Stretch-induced depolarizations as a trigger of arrhythmias in isolated canine left ventricles.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H613-H621,
1992
36.
Takano, H.,
and
S. A. Glantz.
Gadolinium attenuates the upward shift of the left ventricular diastolic pressure-volume relation during pacing-induced ischemia in dogs.
Circulation
91:
1575-1587,
1995
37.
Tokimasa, T.,
and
R. A. North.
Effects of barium, lanthanum and gadolinium on endogenous chloride and potassium currents in Xenopus oocytes.
J. Physiol. (Lond.)
496:
677-686,
1996[Abstract].
38.
Ward, H.,
and
E. White.
Reduction in the contraction and intracellular calcium transient of single rat ventricular myocytes by gadolinium and the attenuation of these effects by extracellular NaH2PO4.
Exp. Physiol.
79:
107-110,
1994[Abstract].
39.
Weast, R. C.
Handbook of Chemistry and Physics (67th ed.). Boca Raton, FL: CRC, 1987, p. D-190.
40.
Yamazaki, T.,
I. Komuro,
S. Kudoh,
Y. Zou,
R. Nagai,
R. Aikawa,
H. Uozumi,
and
Y. Yazaki.
Role of ionic channels and exchangers in mechanical stretch-induced cardiomyocyte hypertrophy.
Circ. Res.
82:
430-437,
1998
41.
Yang, X.-C.,
and
F. Sachs.
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
Science
243:
1068-1071,
1989[Medline].