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Abstract The effect of extracellular calcium on humanosteoblast-like cells (HOS) has been demonstrated. An ex-perimental setup was used for applying defined rates ofchange in the extracellular calcium concentration. Theintracellular calcium concentration was monitored usingthe fluorescence dye fura-2. HOS cells showed qualita-tively different responses of the intracellular calcium con-centration to changes of the extracellular calcium concen-tration depending on its changing rate. A small rate causedonly a small and slow increase of the intracellular calciumconcentration, whereas faster changes are able to cause arapid transient increase followed by a sustained elevationof the internal calcium level. Surprisingly, both an increas-ing as well as a decreasing external calcium concentrationis able to cause cellular responses. These signals could bereduced by the IP3-inhibitor neomycin. We propose thatthe G-protein dependent signalling pathway of HOS cells

can not only sense the extracellular calcium concentrationbut also its time derivative.

Key words Extracellular calcium Calcium receptor Osteoblast-like cells Fura-2 Bone remodelling

Abbreviations IP3 inositol 1,4,5-trisphosphate [Ca2+]i

intracellular calcium concentration [Ca2+]e extracellularcalcium concentration cAMP adenosine 3,5-cyclicmonophosphate

Introduction

Recently a calcium-sensing receptor has been found in sev-eral cell types, such as parathyroid and kidney cells (Brownet al. 1993; Riccardi et al. 1995). This sensor is obviouslyinvolved in the regulation of the serum calcium level. The

direct response of this receptor to small variations of theextracellular calcium concentration leads to the activationof a cascade of cellular reactions. Spikes of the intracellu-lar calcium concentration as well as changes in cAMP leveland arachidonic acid release (Brown et al. 1993; Ruat et al.1995; Riccardi et al. 1995; Emanuel et al. 1996) werefound. These signals are supposed to influence the produc-tion and secretion of calciotropic hormones, maintainingcalcium homeostasis in the body (Brown 1991).

Similar receptors seem to exist even in bone cells (Zaidiet al. 1989; Honda et al. 1995; Leis et al. 1994; Kamiokaet al. 1995; Gao et al. 1996; Quarles et al. 1997). In osteo-blasts, for example, the rise of extracellular calcium pos-sibly stimulates processes of bone formation. In contrastto the calcium receptors of the above mentioned cells,which are monitoring the systemic calcium level in theblood, osteoblasts sense local concentrations, produced by

bone resorbing osteoclasts. These concentrations couldreach local values of up to 40 mM (Silver et al. 1988). Thisimplies local concentration gradients and probably varia-tions in a short time scale to which the cells could respond.

The process of bone remodelling includes not only theresponse of osteoblasts to the bone resorbing activity ofosteoclasts. One of the most investigated problems is boneadaptation according to mechanical loading. Thereforevarious hypotheses are proposed which include mechani-cal, electromechanical and direct electrical action. Osteo-blasts are known to be mechanosensitive, i.e. they respondto shear stress and vibrations (Reich et al. 1990; Lee andWong 1994; Klein-Nulend et al. 1995; Hung et al. 1996).Up to now, however, the molecular basis of these reactionsis not known. Is it possible that a connection exists betweenthe mechanoreceptors and the calcium-regulating systemand/or the location of the electrochemical processes in thecell membrane. On the other hand it is not known whetherreceptor and transporter are separate molecules, or whetherthe transporters themselves are stress-sensitive.

Some of our preliminary experiments indicated that hu-man osteoblast-like cells respond in a qualitatively differ-ent way to various kinds of change in the extracellular cal-cium concentration, especially to the time scale of a sud-

Eur Biophys J (1998) 27: 411416 Springer-Verlag 1998

Received: 9 October 1997 / Revised version: 19 February 1998 / Accepted: 22 February 1998

Beate Habel Roland Glaser

Human osteoblast-like cells respond not only to the extracellular calcium

concentration but also to its changing rate

BIOPHYSICS L E T T E R

B. Habel R. Glaser ()Institute of Biology/Experimental Biophysics,Humboldt-University Berlin, Invalidenstrasse 43,D-10115 Berlin, Germany

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den concentration rise. This effect corresponds to resultsof other authors (Fried and Tashijan 1986; Nemeth andScarpa 1987; Zaidi et al. 1989; Brown 1991). Unfortu-nately, these results could not be compared with each other,because in most cases there was no description of how theconcentration change had been done. Therefore we ex-tended the experimental setup to allow us to apply altera-tions of the calcium concentration near the cell on a welldefined time scale. We investigated the influence of vari-ous calcium concentrations and gradients on the inductionof intracellular calcium signals in single osteosarcoma

cells. In this paper we will show that human osteosarcomacells respond not only to the current concentration of theexternal calcium, but even to the time dependence of thisconcentration.

Materials and methods

Materials

The fluorescence dye fura-2 was obtained from MolecularProbes. DMEM: HAMs F12 (1 : 1), amphotericin, penicil-lin/streptomycin, fetal calf serum (FCS), trypsin/EDTAand Hanks salt solution were from Biochrom. Neomycinand Na2EDTA were from Sigma. CaCl2 2H2O were pur-chased from Fluka. Culture dishes were from Falcon.

Cell culture

Human osteogenic sarcoma cells (HOS, TE-85 from Amer-ican Type Culture Collection, Rockville, MD) were cul-tured routinely in DMEM: HAMs-F12 (1: 1) containing

10% FCS, 1% amphotericin and 1% penicillin/streptomy-cin in a humidified atmosphere of 5% CO2 . For every ex-periment, cells were subcultured and plated on glass cover-slips at 103 cells/ml and incubated overnight. The cover-slips formed the bottom of the experimental chamber formicroscopic observations. Its wall consisted of a glass ring(diameter 12 mm, 5 mm high). The use of these thin glasscoverslips instead of quartz coverslips did not effect the

fluorescence measurements.

Measurement of extracellular calciumby microelectrodes

Microeletrodes with a tip diameter of 20 30 m were pre-pared and filled with 10 mM CaCl2 and a calcium-iono-phore (Cocktail I from Fluka). To measure the real timecourse of the concentration rise in the funnel which cov-ers the cells (Fig. 1), the tips of the bent electrodes werepositioned horizontally under the opening of the funnel.

Measurement of intracellular calcium

The cells (HOS) were loaded with fura-2/AM (2 4 M,60 min, 37C) dissolved in Hanks salt solution containing1 mM CaCl2 (HSS). After washing, the cells were trans-ferred into HSS. The fluorescence was measured using aninverse fluorescence microscope (Attofluor, Carl Zeiss)equipped for alternate recording at two excitation wave-lengths (334/380 nm). In most of the experiments the flu-orescence ratio 334/380 nm was taken every 5 seconds.The intracellular calcium concentration was calculated us-ing calibrated concentrations of fura-2 salt. All experi-ments were performed at 37C.

The experimental setup to apply time dependent

external calcium concentrations

For our experiments we needed an experimental setupwhich allows one to measure the fura-2 fluorescence of thecells simultaneously during a controlled alteration of theexternal calcium concentration. The rate of concentrationchange should be adjustable independently of the hydro-dynamic conditions near the cells. These conditions, con-tradict each other. On the one hand, minimal shear stressshould be applied, protecting the cell from mechanical ex-citation. On the other hand, it should be possible to mod-ify the calcium concentration near the cell surface quicklyand without significant time delay. As a compromise, weused a setup, schematically demonstrated in Fig. 1. A fun-nel shaped opening was positioned over the microscopi-cally observed cells by a micromanipulator. The distancebetween the edge of this funnel and the coverslip was fixedby small pieces of a coverslip. An optimal flow rate of0.17 l/s was realised by pump I. Therefore the cells weresurrounded by a constant laminar flow of saline in all ex-

412

Fig. 1 Scheme of the experimental setup to apply definite rates ofchange of the extracellular calcium concentration. A funnel shapedopening was positioned over the observed cells and the distance wasfixed by small glass pieces. The time rise of calcium concentrationin the flow was achieved by a continuous concentration rise in themixing vessel. Pump I controlled the flow (0.17 l/s) from the mix-ing vessel to the coverslip. The rate of concentration rise in this ves-sel was determined by the concentration of calcium in the supplyvessel and/or by pump II

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periments, independent of the adjusted rate of concentra-tion change. The time rise in calcium concentration in thisflow was achieved by a continuous concentration rise inthe mixing vessel. The rate of concentration rise in this ves-sel was determined by the concentration of calcium in thesupply vessel and/or by pump II.

First this setup was used just to increase the calcium

concentration continuously near the cells. Later we usedthe same setup to investigate the diminution of the extra-cellular calcium concentration. This is possible in the sameway be continuously adding EDTA into the mixing vessel.

The calcium concentration in the mixing vessel can bedescribed by the following equations:

(1)

(2)

where c2 (t) is the current calcium concentration in the

mixing vessel,V

(t) is the volume of the mixing vessel,

c1

is the calcium concentration in the supply vessel, V20 isthe volume of the mixing vessel at t=0, V12 is the vol-ume flow rate from supply to mixing vessel and V23 fromthe mixing vessel to the coverslip.

For the condition at t= 0, c2 (t) = c20 , this equation canbe solved:

(3)

The slope in the interesting time interval is assumed to beconstant.

These calculations were checked experimentally usingion-selective microelectrodes. Figure 2 demonstrates themeasured values and the curve theoretically predicted. Itshows that the above description is sufficiently correct forsmall rates of calcium increase. At higher rates, especiallyat the beginning, an increased rise of the concentration wasmeasured, which later became flatter and closer to the theo-retical curves. Furthermore, we observed concentrationchanges at the electrodes even before they were theoreti-cally expected. This expectation is based on the dead timeof the system, which was difficult to estimate exactly. This

c t c c c t V V

V

V

V V

2 1 20 112 23

20

1

12

12 23

( ) ( )

= + +

dc t

dtc t

V

V t

V

V tc

V t V V V t

22

12 121

20 12 23

( )( )

( )

( )

( ) ( )

+ =

= +

dead time was determined experimentally by observing thepassage of small air bubbles in the tube. However, in thereal case a fast central tongue of the streaming fluid cancause locally increased concentration gradients. Further-more, the influx of the fluid into the funnel can effect thestreaming profile. Thus, the time difference increases withincreasing slope of the concentration curves.

Results

Experiments with increasing concentrationsof external calcium

Two typical experiments, demonstrating the response ofcells to different rates of increasing the external calciumconcentration are shown in Fig. 3. Only the fast change ofthe extracellular calcium concentration was able to causea rapid overshooting increase of the calcium concentration(spike) in the cell. The increase of intracellular calciumcontinued even when the external concentration had al-ready reached the plateau. In the case of slower externalconcentration rises, no intracellular calcium spike could beobserved. The internal calcium concentration was simplyadjusted to correspond to the new extracellular conditions.Thus, this process took place as long as the external con-centration was rising. In all experiments the external cal-cium concentration rose from 1 mM to a final concentra-tion of 10 mM more or less rapidly. Consequently, the levelof the finally reached internal calcium concentration wasalways the same.

We classified the time course of the intracellular cal-cium concentration of every single cell either as slow in-crease, as fast overshooting reaction (spike) or as no

reaction. In the latter class we counted all cells whoseintracellular calcium concentration rose by less then 25%with respect to the initial value. The dependence of therelative occurrence of intracellular calcium spikes on therate of concentration changes (dc/dt) is a sigmoid function(Fig. 4). For these experiments with different changingrates of the extracellular concentration from 1 mM to10 mM, the critical rate is (0.0460.015) mM/s. The varia-tions in the experimental results increase with the concen-tration rate. This can be explained by the deviation betweenthe calculated and the actually measured values of the ex-ternal concentration rise, as demonstrated in Fig. 2.

Pre-treatment with neomycin

To clarify whether the phosphatidyl inositol turnovercontributes to the generation of the intracellular calciumsignal, the HOS cells were pre-treated with neomycin(1 M, 1 h) which inhibits the activity of phospholipase C(Lipsky and Leitman 1982). Neomycin was also present inthe measuring buffer. In these experiments the effect ofhigh extracellular calcium concentrations was strongly re-duced (Fig. 3 D). Some cells showed a biphasic behaviour

413

Fig. 2A C Measured versus predicted extracellular calcium con-centrations near the cells for three different rates of concentra-tion rises (dc/dt). The extracellular calcium concentration was meas-ured using ion-selective microelectrodes. The theoretical concen-tration rate is dc/dt= 0.008 mM/s (A), dc/dt= 0.070 mM/s (B), dc/dt=0.700 mM/s (C)

A B C

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in the following sense: they responded to increased extra-cellular calcium with a slight increase of intracellular cal-cium (see Fig. 3 D), but after ten minutes a calcium spikeappeared suddenly.

Furthermore it is known, that neomycin is able to act asa stimulus itself in causing an intracellular calcium in-crease. This, however, did not influence our measurementsbecause neomycin had already been added 1 h before thecalcium measurement started and it was present during thewhole experiment. On the other hand we could show thatafter a sufficient relaxation period cells could respond sev-eral times to a stimulus with a calcium spike. Furthermore,the cells did not show an anomalous behaviour in these ex-periments. So, the cells should have been able to respond

to external calcium concentrations, even if neomycin hascaused a calcium signal.

Experiments with decreasing concentrationsof external calcium

If the observed cellular reactions on increasing extracellu-lar calcium are based on a receptormediated process, thensimilar behaviour is also expected in the case of decreas-ing concentrations. Sufficiently fast changes of the con-centration of free calcium were obtained by continuouslyadding EDTA to the external solution.

We found that even a decreasing extracellular calcium

concentration affects the intracellular calcium concentra-tion (Fig. 5). Again there were two qualitatively distinctreactions depending on the rate of concentration change:A fast change caused a transient increase of the intracel-lular calcium concentration, which is sometimes followedby a slower, sustained increase. Slower changes of the ex-ternal concentration again caused a simple adjustment ofthe intracellular calcium concentration to the extracellularconcentration.

Discussion

An increase of the intracellular calcium concentration as aresponse to high extracellular calcium concentrations hasalready been found for cells of parathyreoidea (Brown1991), kidney (Riccardi et al. 1995), brain (Ruat et al.1995; Emanuel et al. 1996), placenta (Juhlin et al. 1990),and for keratinocytes (Reiss et al. 1991), osteoclasts (Zaidiet al. 1989) and osteocytes (Kamioka et al. 1994). Brownet al. (1993) isolated and cloned a calcium sensitive recep-tor of parathyroid cells and identified its primary structure.Thus, at least in these cells the response to extracellular

414

Fig. 3A D The intracellular calcium concentration in response todifferent rates of increasing the extracellular calcium concentration.A The time course of the extracellular calcium concentration (dc/dt):1.73 mM/s (solid line); 0.03 mM/s (dotted line). B D Intracellularcalcium concentration in the case of dc/dt= 0.03 mM/s (B),dc/dt =1.73 mM/s (C). In D the cells were pre-treated with 1 M neo-mycin. Neomycin was also present in the measuring buffer;dc/dt =0.87 mM/s. The curves B D represents mean values of 34cells

Fig. 4 Percentage of cells with an intracellular calcium spike in re-lation to varying changing rates of the extracellular calcium concen-tration. Every point represents results of one independent experiment,including 30 50 cells. The data were fitted (r2= 0.82) by the sigmoid

function: y = yy y

x

x

minmax min

+

+

150

n

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calcium was found not simply to be caused by a calciumchannel, but rather by a special receptor molecule.

Leis et al. (1994) found a corresponding behaviour inmurine osteoblast-like cells. Our own experiments confirmthese results. We found that human osteoblast-like cells in-crease their internal calcium concentration as a result of

rapidly increased as well as decreased external concentra-tion. There, even very small concentration changes are ef-fective. This indicates that it is not simply a process of asmall change of the 104 : 1 relation of external to internalconcentration, but rather a result of a signal transforma-tion. There are two hypothetical possibilities describingthe behaviour of the cells. Both have to include dynamicproperties. First an ion channel could act, which is coupledin some way to a calcium sensor. This protein could con-trol the calcium influx according to the existing concen-tration gradient. In the following a calcium induced cal-cium release can lead to intracellular calcium spikes. Aslow concentration change gives the cells more time to han-dle this, so no spike is observed. However, decreasing theexternal concentration nearly down to zero should preventan internal calcium increase. Contradicting this expecta-tion we again found a calcium spike (Fig. 5 C), forcing usto reject the idea of a receptor coupled ion channel. Thesesignals particularly suggest the intracellular origin of theinvolved calcium.

The second hypothesis postulates a special calcium re-ceptor, which can release calcium from intracellular storesvia the formation of second messengers. That model per-mits an internal calcium spike for both cases, increasing as

well as decreasing external concentration. It is supportedby the experiments with neomycin (Fig. 3 D), indicatingthat IP3 is involved in the generation of the observed cal-cium signals. Furthermore, we found that human osteosar-coma cells respond not only to the external calcium con-centration (ce), but, additionally, to its time derivative(dce/dt), i. e. to the rate of external concentration change.To investigate this cellular response, we used an experi-

mental setup which allowed us to change this time deriv-ative, without changing the hydrodynamic conditions nearthe cells (Fig. 1). However, this setup fulfils these condi-tions only approximately. The comparison of the measuredrates of calcium concentration changes near the measuredcells with the calculated curves, shows some deviations(Fig. 2) for fast concentration changes.

The main questions are: how do these deviations mod-ify our results, and: how one could avoid these difficul-ties? In general, the flow in the tube and in the funnel ofthis system can be characterised by very small Reynoldsnumbers. Therefore it is supposed to be laminar. A para-bolic velocity profile in the tube, however, causes (in thecase of increasing concentrations in the mixing vessel) theconcentration near the centre of the tube to be higher thanin the periphery. Furthermore, reflections at the coverslipmay produce additional heterogeneities. These deviationsincrease with increasing rates of concentration changes.This weakness of the experimental setup could have beenavoided only by slowing down the rate of concentrationchange. This, however, would not bring us to the region ofinterest. Therefore our results can only qualitatively indi-cate the response of the cells to the rate of calcium con-centration changes.

The experiments which were done with neomycin pre-treatment indicate that the IP3 signalling pathway is at leastpartly involved. The above mentioned biphasic behaviour

of some cells suggests that a further mechanism could ex-ist for increasing the intracellular calcium concentration.For instance a slow calcium influx could lead to a calciuminduced calcium release (CICR).

Reiss et al. (1991) found that in the case of high extra-cellular calcium concentrations the calcium efflux as wellas the influx of keratinocytes is increased. We suppose thatthese transports are very important to establish an intracel-lular steady state concentration of calcium. In our experi-ments the adjustment of the intracellular calcium concen-tration to a slowly changing extracellular concentrationdemonstrates this fact. Furthermore, the finally reachedintracellular concentration is nearly the same in all our ex-periments done with different concentration rates but withequal final concentrations.

Consequently, at least two different processes of cal-cium control are going on in the cell, which overlap eachother in their action. First, there is a slow adjustment of thecellular calcium level, which is realised by transport mech-anisms localised in the cell membrane. Second, a rapid IP

3

mediated process exists, which leads to calcium releasefrom intracellular stores. In contrast to the first process,the second one is not only influenced by concentrations butalso by the rate of change of the extracellular calcium con-

415

Fig. 5A C The intracellular calcium concentration in response todifferent rates of decreasing the extracellular calcium concentration.A The time course of the extracellular concentrations (dc/dt): 0.035 mM/s (smooth line); 0.002 mM/s (dotted line). B, C Intra-cellular calcium concentration in the case of dc/dt = 0.002 (B), anddc/dt= 0.035 mM/s (C). The curves B, C represents mean values of32 cells

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centration. It is stimulated only in the case of sufficientlyfast changing rates, independent of their direction.

We suggest that there could be a G-protein coupled cal-cium sensing receptor in the osteoblast-like cell line HOS,similar to that found in other cells. It could be responsiblefor this IP3 mediated process. This assumption is supportedby Gao et al. (1996) who has detected mRNA of the para-thyroid calcium receptor in some human osteoblastic cell

lines. It should not be identical to the already cloned andcharacterised calcium receptor of parathyroid and kidneycells. The biophysical mechanism, explaining the percep-tion of the time derivative is still not known. One can im-agine a twofold calcium dependent response to the extra-cellular stimulus where one way leads to some kind of ac-tivation and the other, with a time delay, to an inactivationof processes which cause an increase of intracellular cal-cium. Molecular investigations are needed to clarify thisquestion.

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