Effects of Lead, Mercury, and Methyl Mercury on Gap ... - CiteSeerX

1 downloads 0 Views 102KB Size Report
well as (B) intracellular applied Pb (5 μM in the patch electrode) did not change the coupling factor ..... Büsselberg D, Platt B, Micheal D, Carpenter DO, Haas HL.
Calcif Tissue Int (1998) 63:134–139

© 1998 Springer-Verlag New York Inc.

Laboratory Investigations Effects of Lead, Mercury, and Methyl Mercury on Gap Junctions and [Ca2+]i in Bone Cells K. Schirrmacher, M. Wiemann, D. Bingmann, D. Bu¨sselberg* Universita¨t-GH Essen, Institut fu¨r Physiologie, Hufelandstr. 55, 45122 Essen, Germany

Received: 6 May 1997 / Accepted: 15 October 1997

Abstract. Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca2+]i). Since heavy metals compete with Ca2+ at multiple sites and increased [Ca2+]i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca2+]i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb(e) (5 ␮mol/liter; n ⳱ 3) and Hg(e) (5 ␮mol/liter; n ⳱ 3) as well as Pb(i) (25 ␮mol/liter; n ⳱ 7) did not change the coupling (⌬MP2/⌬MP1). In contrast, MeHg(e) (1–10 ␮mol/liter; n ⳱ 6) and Hg(i) (艌5 ␮mol/liter; n ⳱ 8) reduced the coupling to 79.5 ± 19.3% and 62.4 ± 15.3%, respectively, within 15–20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb(e) (5 ␮mol/liter, n ⳱ 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca2+]i increase. Hg(e) (n ⳱ 48) slightly increased [Ca2+]i from 100 to 艋200 nmol/liter, whereas MeHg(e) (5 ␮mol/liter, n ⳱ 52) released Ca2+ from internal stores, thus increasing [Ca2+]i up to 2 ␮mol/liter. In conclusion, Pb(e), Pb(i) and Hg(e) do not affect gap junctional coupling per se. Since MeHg(e) and Hg(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca2+]i, which affects gap junction channels. Key words: Lead — Mercury — Methyl mercury — Gap junctions — Free intracellular Ca2+.

Heavy metals are ubiquitous and are taken up into the body by food, drinking water, and polluted air. Primary accumulation site for metals is the bone. It has been proven that the * Present address: Physiologie und Pathophysiologie, Universita¨t Go¨ttingen, Humboldtallee 23, 37073 Go¨ttingen, Germany Correspondence to: K. Schirrmacher

macro-distribution of lead is not uniform and clearly depends on the bone type, age, and gender [1]. However, this tissue is not a mere physiological sink, because metals can be mobilized in response to normal and pathological changes in bone metabolism, e.g., during pregnancy even many years after exposition [2]. As in other tissues the metabolic activities of bone cells may be directly impaired by metals. Beside these potential direct effects of heavy metals, indirect effects have to be taken into account: lead (Pb), mercury (Hg), or methyl mercury (MeHg) interact with Ca2+ and Ca2+-mediated processes. One major target site is the Ca2+ entry through Ca2+ selective membrane pores, out of which voltage-activated Ca2+ channel currents are most sensitive [3–5]. Voltage-activated Ca2+ channels are also present in bone cells [6–8]. It has been demonstrated that Pb competes directly with Ca2+ for a binding site at the channel [9]. Hg as well as MeHg also reduces ionic currents through voltage-operated Ca2+ channels [10]. In bone cells, at least Pb has complex effects [11], which may be partly attributed to perturbations of Ca2+-mediated and other sensitive signal transduction pathways in osteoblast-like cells [12–15]. Among other targets, gap junctions, which seem to be key elements in the signaling between bone cells [16, 17], may be affected directly and/or indirectly by heavy metals. This was tested in the present study on bone-derived cells, which, like bone cells in vivo [16] express gap junctions composed of connexin43 [18–20]. These channels for intercellular signaling of bone cells are controlled, e.g., by osteotropic hormones and second messengers [17, 20, 21], whose complex actions may be impaired by heavy metals as well. Moreover, Cx43 channels in osteoblast-like cells are sensitive to [Ca2+]i and close with rising [Ca2+]i [22]. Since heavy metals compete with Ca2+ for specific binding sites, the coupling strength might be sensitive to heavy metals. This was tested by analyzing actions of Pb, Hg, and MeHg on gap junctions and [Ca2+]i in rat osteoblast-like (ROB) cells using electrophysiological as well as Ca2+ imaging techniques. Materials and Methods Cell Cultures Calvarial fragments of newborn rats were explanted onto collagencoated coverslips and placed in plastic tubes (Nunclon, Germany)

K. Schirrmacher et al.: Effects of Heavy Metals on Gap Junctions

containing 2 ml of HEPES (N-[2-hydroxyethyl] piperazine-N⬘-2ethanesulfonic acid, 20 mmol/liter) buffered minimum essential medium (88 vol%). The media also contained fetal calf serum (10 vol%), glutamine (200 mmol/liter; 1 vol%), and penicillin (5000 IU/ml)/streptomycin (5000 ␮g/ml; 1 vol%). All media components were obtained from Flow Laboratories Meckenheim (Germany). The explants were maintained at 35°C, while nutrient medium was exchanged twice a week. Cells in vitro exhibit characteristics of the osteoblast-like phenotype, as they were able to form collagen and to mineralize [19].

Electrophysiological Recordings Two to six weeks after explantation of rat calvarial fragments osteoblast-like (ROB) cells were investigated in a saline that contained (concentrations in mmol/liter) NaCl 126, KCl 5, MgCl2 1.3, NaHCO3 6, CaCl2 2.4, and Tris-Cl 3. The solution was equilibrated with 5% CO2 in 95% O2, resulting in a pH of 6.9, to reduce formation of PbCO3 or HgCO3. A HEPES-buffered saline (HBS) containing (concentrations in mmol/liter) NaCl 140, KCl 3, CaCl2 1.8, MgSO4 1.3, KH2PO4 1.25, glucose 11, and HEPES 10 (pH 7.4) was used for the experiments with MeHg. As shown before, the pH values between 6.6 and 7.4 did not influence the electric and dye coupling of those cells [23]. For intracellular measurements, single-barreled micropipettes were prepared (ø 1 mm, inner filament, Hilgenberg, Germany) and filled with 2 mol/liter potassium methyl sulfate (110–140 M⍀). Patch electrodes were made from soft glass (Hilgenberg, Germany) in a two-step pulling procedure (P-87, Flaming/Brown, Sutter Instr., Germany). For whole cell recordings, glass pipettes were filled with a solution composed of (concentrations in mmol/liter) KCl 143, MgCl2 2, HEPES-KOH 10, adjusted to pH 7.2 with KOH (2–4 M⍀). The pipette was placed on the head-stage of an EPC 9 (HEKA, Germany) patch clamp probe. After formation of the seal (5–50 G⍀) [24], gentle suction was applied to gain access to the interior of the cell and to record the membrane potential in the current clamp mode. The bath reference electrode was a silver chloride wire in a glass tube filled with 2 mol/liter KCl. Cell cultures were placed into a recording chamber mounted on an inverted microscope (ID3, Zeiss, Germany). The chamber (1.5 × 5 cm2; volume 2 ml) was continuously perfused at a rate of 4–6 ml/minute with salt solution at room temperature. Under microscopic control, one cell was impaled with a conventional microelectrode advanced by a step motor-driven micromanipulator (Scientific Precision Instr., Germany), while the neighboring cell was patch clamped. By means of conventional Wheatstone bridges, the membrane potentials of the cells were shifted either by constant de- or hyperpolarizing current pulses (0.2 nA; A.M.P.I., Israel). The membrane potentials were monitored on a digital oscilloscope (Nicolet 410, USA), stored on discs, and plotted with a penrecorder (HP 7475A, Hewlett Packard, USA). The coupling factor of the cells was evaluated as ⌬MP2/⌬MP1 according to Loewenstein and Kanno [25].

Fluorescence Optical Measurements of [Ca2+] Cells were loaded with 5 ␮mol/liter Fura-2-acetoxymethyl-ester (Fura-2/AM, Molecular Probes, Eugene, USA). Cultures were washed three times with HBS (control) containing (concentrations in mol/liter) NaCl 140, KCl 3, CaCl2 1.8, MgSO4 1.3, KH2PO4 1.25, glucose 11, and HEPES 10, pH 7.4. Stock solutions of heavy metals (see below) were added to this saline shortly before the experiments. HBS without CaCl2 was used as a nominally Ca2+free HBS. EGTA (ethyleneglycol-bis(-amino ethyl ether) N,N,N⬘,N⬘-tetraacetic acid) was not used to buffer external Ca2+ since it strongly binds heavy metals. Optical measurements of [Ca2+]i were performed at room temperature (20 ± 2°C). Cell cultures were mounted on an inverted microscope (Zeiss Axiovert 135TV, Germany). Solutions were changed by replacing the fluid within the experimental chamber (volume: 1 ml) by an equal volume of the desired one. To obtain nearly Ca2+-free conditions (in

135

the absence of Ca2+ chelatores) solutions were changed 10–20 times within 2–4 minutes. Intracellular free Ca2+ concentration ([Ca2+]i) was measured using ratio imaging (Attofluor RatioVision system, Atto Instruments, Rockville, MD, USA) with ratios being collected at 0.05 − 1 Hz. Relative changes of [Ca2+]i were calculated from the emission ratios (R; excitation 340 nm/380 nm; emission 500–530 nm, corrected for background fluorescence). Absolute [Ca2+]i in ROB cells was estimated according to the equation of Grynkiewicz et al. [26]: [Ca2+] ⳱ Kd * ␤ * [(R − Rmin) / (Rmax − R)] where Kd is the dissociation constant of fura-2 binding to Ca2+ (224 nmol/liter), ␤ is the maximal fluorescence at 380 nm divided by the minimal fluorescence at 380 nm, and R is the ratio of the two fluorescence intensities 340 nm/380 nm. Addition of 3 mol/liter KCl was used to obtain Rmax values. To obtain Rmin values in ROB cells pretreated with the Ca2+ ionophore 4-bromo-A23187 (2 ␮mol/liter) 11 mM of the Ca2+ chelator EGTA was used (Sigma, Germany). Ca2+ changes were measured in the soma, including the nuclear region. Total number of cells is given as n value in the text. Ca2+ transients of n cells from one experiment were averaged. Initial ratio values ranged from 0.3 to 0.6. Since it is known that Fura-2 can strongly bind heavy metals, the effect of Pb, Hg, and MeHg on Fura excitation/emission properties was tested in pilot experiments. Fura-2 (1 ␮mol/liter) was dissolved in a 100-fold diluted HBS, pH 7.4, containing either 0 or 15 ␮M Ca2+. Emission was collected at 530 nm. Excitation spectra were run from 300 to 400 nm using an Aminco-Bowman spectrophoto-fluorometer equipped with a 75W xenon lamp. Hg and MeHg (both 5 ␮mol/liter) did not (deviation 2 hours) at pH 6.9 reduced the dye coupling in an osteoblastic cell line [41], our previous study on ROB cells in primary culture showed that lowering extracellular or intracellular pH values from 7.4 to 6.6 did not acutely influence electric and dye coupling [23]. These results are confirmed by the present investigations, as extracellularly applied Pb or Hg at a pH of 6.9 did not diminish gap junction coupling. In contrast to the effects of Hg and MeHg on the electric activity of nerve cells [5, 10, 42], little is known about the influence of these metals on cell–to–cell communication between neighboring cells. Intracellular application of Hg (5 ␮mol/liter) via the patch pipette solution in one of the two coupled cells that induced a reduction of the initial coupling factor. However, extracellular application of 5 ␮mol/liter Hg, which increased [Ca2+]i to about 130 nmol/ liter (Fig. 3), had no effect on the electric coupling (Figs. 1, 2). One explanation could be that the rise of [Ca2+]i was subthreshold to cause gap junction channel closure in ROB cells [22]. Although intracellular Hg has the potency to affect the electric coupling in these cells, this metal failed to reduce the electric coupling when extracellularly applied because of insufficient permeation and/or too low increases of the [Ca2+]i. Our results are surprising as studies on he-

K. Schirrmacher et al.: Effects of Heavy Metals on Gap Junctions

patocytes have shown that extracellularly applied Hg easily enters these cells [43]. Therefore, we expected an effect of Hg independent of the mode of application. As the reduction of the electric coupling by intracellularly applied Hg2+ might be caused by the depletion of Ca2+ stores [43], it is most likely that this mechanism also plays a role in the reduction of electric coupling of ROB cells. Though extracellular Hg was ineffective, exposure of 1–10 ␮mol/liter MeHg reduced the electric coupling within 10–20 minutes (Figs. 1, 2). Simultaneously, the cell membrane depolarized and the cells uncoupled. This depolarization of the membrane induced by MeHg should not be responsible for the decreased electric coupling, as gap junctions between ROB cells were found to be insensitive to transjunctional voltage changes of up to ± 40 mV [23, 44]. Parallel experiments have shown that 5 ␮mol/liter MeHg induced an increase of [Ca2+]i to about 2 ␮mol/liter within 5 minutes (Fig. 3). The amplitude of this rise was similar to the release of Ca2+ from intracellular stores by A23187. Evidently, MeHg mobilizes Ca2+ from intracellular stores and, by this, affects the electric coupling via an increase of [Ca2+]i, as has been shown to occur after a single [Ca2+]i peak [45]. Since it has been shown that intracellular Hg or extracellular applied MeHg deplete Ca2+ stores [28, 43], the effect of these metals is most likely due to Ca2+ actions with gap junction channels. The present experiments suggest that the heavy metals used in the present study are not only disturbing Ca2+mediated processes in single bone cells, but also cell-to-cell communication via Ca2+ sensitive gap junctions. This is of particular importance as the cellular network of bone cells seems to be essential for signaling and nutrition purposes of this tissue [17]. As the bone is the major target organ for incorporation of heavy metals, some effects might be explained by disturbances of cell-to-cell communication between ROB cells observed in vitro. Besides Pb, other metals such as cadmium or aluminum interfere with bone (cf. [17]) and Ca2+ metabolism, and are classified as environmental risk factors for osteoporosis [15]. It is notable that also antiepileptic drugs such as carbamazepine or phenytoin weaken the electric coupling between cultured ROB cells by 40% [17, 23], and that this mechanism may result in osteomalacia which most often is attributed to disturbances of the vitamin D metabolism [46]. As the reduction of the electric coupling by intracellular Hg and extracellular MeHg was in the same order of magnitude as that of the antiepileptic drugs [17, 23], osteomalacia should also be expected after exposure to heavy metals. In conclusion, we found that intracellular Hg and extracellular MeHg, but not Pb, reduce functional electric coupling mediated by connexin43 gap junction channels in primary cultures of rat osteoblast-like cells. This effect was likely mediated by a short-term increase of [Ca2+]i released from internal stores. References 1. Aufderheide AC, Wittmers LE Jr (1992) Selected aspects of the spatial distribution of lead in bone. Neurotoxicology 13: 809–819 2. Klein M, Kaminsky P, Barbe F, Duc M (1994) Lead poisoning in pregnancy. Presse Med 23:576–580 3. Bu¨sselberg D, Platt B, Micheal D, Carpenter DO, Haas HL (1994) Mammalian voltage-activated calcium channel currents are blocked by Pb2+, Zn2+, and Al3+. J Neurophysiol 71:1491–1497

K. Schirrmacher et al.: Effects of Heavy Metals on Gap Junctions

4. Bu¨sselberg D (1995) Calcium channels as target sites of heavy metals. Toxicol Lett 82/83:255–261 5. Leonhardt R, Pekel M, Platt B, Haas HL, Bu¨sselberg D (1996) Voltage-activated calcium channel currents of rat DRG neurons are reduced by mercuric chloride (HgCl2) and methyl mercury (CH3HgCl). Neurotoxicology 17:85–92 6. Bingmann D, Tetsch P, Massass R (1988) Membraneigenschaften von Zellen aus Knochen-explantaten (Membrane properties of cultured cells derived from calvarial explants). Z Zahna¨rztl Implantol IV:277–281 7. Chesnoy-Marchais D, Fritsch J (1988) Voltage-gated sodium and calcium currents in rat osteoblasts. J Physiol 398:291–311 8. Grygorczyk C, Grygorczyk R, Ferrier J (1989) Osteoblastic cells have L-type calcium channels. Bone Miner 7:137–148 9. Bu¨sselberg D (1991) Lead and zinc block a voltage-activated calcium channel of Aplysia neurons. J Neurophysiol 65:786– 795 10. Leonhardt R, Haas H, Bu¨sselberg D (1996) Methyl mercury reduces voltage activated currents of rat dorsal root ganglion neurons. Naunyn Schmiedebergs Arch Pharmacol 354:532– 538 11. Pounds JG, Long GJ, Rosen JF (1991) Cellular and molecular toxicity of lead in bone. Environ Health Perspect 9:17–32 12. Schanne FAX, Dowd TL, Gupta RK, Rosen JF (1989) Lead increases free Ca2+ concentration in cultured osteoblastic bone cells: simultaneous detection of intracellular free Pb2+ by 19 F-NMR. Proc Natl Acad Sci USA 86:5133–5135 13. Schanne FAX, Dowd TL, Gupta RK, Rosen JF (1990) Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR. Biochim Biophys Acta 1054:250–255 14. Long GF, Rosen JF (1992) Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism and collagen synthesis in clonal rat osteoblastic (ROS 17/2.8) cells. Toxicol Appl Pharmacol 114:63–70 15. Goyer RA, Epstein S, Bhattacharyya, Korach KS, Pounds J (1994) Environmental risk factors for osteoporosis. Environ Health Perspect 102:390–394 16. Doty SB (1981) Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509–512 17. Bingmann D, Schirrmacher K, Jones DB (1994) Signalling in bone: electrophysiological studies on cultured cells derived from calvarial fragments of rats. Cells Materials 4:275–286 18. Schiller PC, Mehta PP, Roos BA, Howard GA (1992) Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gapjunctional communication in osteoblastic cells. Mol Endocrinol 6:1433–1440 19. Schirrmacher K, Schmitz I, Winterhager E, Traub O, Bru¨mmer F, Jones D, Bingmann D (1992) Characterization of gap junctions between osteoblast-like cells in culture. Calcif Tissue Int 51:285–290 20. Donahue HJ, McLeod KJ, Rubin CT, Andersen J, Grine EA, Hertzberg EL, Brink PR (1995) Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function. J Bone Miner Res 10:881–889 21. Xia S-L, Ferrier J (1992) Propagation of a calcium pulse between osteoblastic cells. Biochem Biophys Res Comm 186: 1212–1219 22. Schirrmacher K, Nonhoff D, Wiemann M, Peterson-Grine E, Brink PR, Bingmann D (1996) Effects of calcium on gap junctions between osteoblast-like cells in culture. Calcif Tissue Int 59:259–264 23. Schirrmacher K, Bru¨mmer F, Du¨sing R, Bingmann D (1993) Dye and electric coupling between osteoblast-like cells in culture. Calcif Tissue Int 53:53–60 24. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches. Pflu¨gers Arch 391:85–100 25. Loewenstein WR, Kanno Y (1964) Studies on an epithelial (gland) cell junction: I. Modifications of surface membrane permeability. J Cell Biol 22:565–586

139

26. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450 27. Tomsig JL, Suszkiw JB (1990) Pb2+-induced secretion from bovine chromaffine cells: Fura-2 as a probe for Pb2+. Am J Physiol 259:C762–C768 28. Levesque PC, Hare MF, Atchison WD (1992) Inhibition of mitochondrial Ca2+ release diminishes the effectiveness of methyl mercury to release acetylcholine from synaptosomes. Toxicol Appl Pharmacol 115:11–20 29. Snitsarev VA, McNulty TJ, Taylor CW (1996) Endogenous heavy metal ions perturb Fura-2 measurements of basal and hormone-evoked Ca2+ signals. Biophys J 71:1048–1056 30. Hechtenberg S, Beyersmann D (1991) Inhibition of sarcoplasmic reticulum Ca2+-ATPase activity by cadmium, lead and mercury. Enzyme 45:109–115 31. Domann R, Wunder L, Bu¨sselberg D (1997) Lead reduces depolarization-induced calcium entry in cultured DRG neurons without crossing the cell membrane: Fura-2 measurements. Cell Mol Neurobiol 17:305–314 32. Pounds JG (1984) Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: a review. Neurotoxicology 5:295–332 33. Simons TJB (1986) Cellular interactions between lead and calcium. Br Med Bull 42:431–434 34. Habermann E, Crowell K, Janicki P (1983) Lead and other metals can substitute for Ca2+ in calmodulin. Arch Toxicol 54:61–70 35. Fullmer CS, Edelstein S, Wassermann RH (1985) Leadbinding properties of intestinal calcium-binding proteins. J Biol Chem 260:6816–6819 36. Markovac J, Goldstein GW (1988) Picomolar concentrations of lead stimulate protein kinase C. Nature (London) 343:71– 73 37. Markovac J, Goldstein GW (1988) Lead activates protein kinase C in immature rat brain microvessels. Toxicol Appl Pharmacol 96:14–23 38. Noma A, Tsuboi N (1987) Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea pig. J Physiol 382:193–211 39. Kolb H-A, Somogyi R (1991) Biochemical and biophysical analysis of cell-to-cell channels and regulation of gap junctional permeability. Rev Physiol Biochem Pharmacol 118:1– 47 40. Peracchia C, Shen L (1993) Gap junction channel reconstitution in artificial bilayers and evidence for calmodulin binding sites in MIP26 and connexins from rat heart, liver and Xenopus embryo. In: Hall J, Zampighi GA, Davis RM (eds) Progress in Cell Research. Elsevier, Amsterdam, vol 3, pp 163–170 41. Yamaguchi DT, Huang JT, Ma D (1995) Regulation of gap junction intercellular communication by pH in MC3T3-E1 osteoblastic cells. J Bone Miner Res 10:1891–1899 42. Yuan Y, Atchison WD (1994) Comparative effects of inorganic divalent mercury, methyl mercury and phenyl mercury on membrane excitability and synaptic transmission of CA1 neurons in hippocampal slices of the rat. Neurotoxicology 15:403–412 43. Nathanson MH, Mariwalla K, Ballatori N, Boyer JL (1995) Effects of Hg2+ on cytosolic Ca2+ in isolated skate hepatocytes. Cell Calcium 18:429–439 44. Schirrmacher K, Ramanan SV, Cronin K, Peterson E, Brink P (1997) Voltage sensitivity of gap junctions in rat osteoblastlike cells. Biochim Biophys Acta 1327:89–96 45. Schirrmacher K, Wiemann M, Rauen U, Nonhoff D, Bingmann D (1997) Impairment of dye coupling in osteoblast-like cells in vitro by elevated free intracellular calcium. Pflu¨gers Arch (Suppl)433:R109 46. Bell D, Pak ChYC, Zerwekh J, Barilla E, Vasko M (1979) Effect of phenytoin on bone and vitamin D metabolism. Ann Neurol 5:374–378