James IV Article

Ion channels in osteoblasts:A story of two intracellular organelles

F. McDonald
GKT Dental Institute, Floor 22, Guys Tower, St Thomas Street, London, SE1 9RT
Correspondence to: F. McDonald, GKT Dental Institute, Floor 22, Guys Tower, St Thomas Street, London, SE1 9RT

Introduction Ions Signalling pathways

Apoptosis Summary References

Recent advances have highlighted a synchronous coordination of osteoblast and osteoclast activity, whereby, the osteoblast collates all signals applied to the bone and activates osteoclastic resorption.¹ The resorption of the bone and its matrix then releases growth factors held within the matrix (bone morphogenetic proteins, insulin-like growth factors and transforming growth factors) which then stimulate the osteoblast to lay down new osteoid.^2,3 In healthy adults there is a balance between bone deposition and bone loss and there is no net gain or loss, and the amount of calcium (Ca2+) ingested in the diet is equal to that which is excreted. In the early stages of life, the emphasis is on bone building and more Ca2+ is retained from the diet and more bone deposited as the skeleton matures. As we age, and, in particular in postmenopausal women, the osteoclastic activity outweighs the bone deposition and the patient loses bone becoming osteoporotic.⁴ The focus of the work reported here was to identify and dissect the various cytosolic intracellular signalling pathways within osteoblasts and establish the importance of each under different physiological conditions. In brief, two basic signalling pathways exist; one is linked with a seven transmembrane spanning protein and specific receptors for ligands (the G protein linked pathways) and one pathway is linked with protein phosphorylation especially of the tyrosine kinases. In general, G protein activity is associated with endocrine ligands such as parathyroid hormone (PTH) and (calcitonin has been linked with tyrosine kinase activity) tyrosine kinase activity is linked with adhesion of osteoblasts and recognition of the substrate to which they are attached. Our specific areas of interest are the ways in which Ca2+ activity within the cell is modified and used as a signal for further activation and possibly differential gene activation⁵

INTRODUCTION
Intracellular signalling pathways are the ways in which signals at the bone surface are transferred across to the nuclear material for subsequent cellular activation. One of the major signalling pathways at the cell surface is that involving ion channels. These are protein structures that cross the phospholipid cell layers allowing aqueous/water soluble materials to pass across. There are specific characteristics of these channels unique to the protein; there are many millions in any one cell.

They can be broadly characterised into ion channels in the plasma membrane or cell surface and the intracellular membrane of such intracellular organelles as mitochondria and endoplasmic/sarcoplasmic reticulum.

They are then characterised by the mechanism of opening; ligand gated (e.g. by a hormone such as parathyroid hormone (PTH) or voltage gated (i.e. controlled by the surrounding cell membrane which in turn is controlled by the distribution of ions). The specific ions include sodium (Na+), potassium (K+), chloride (Cl-), hydrogen (H+) and the major signalling intracellular ion calcium (Ca2+) 
(Figure 1).

Figure 1: A diagrammatic representation of ion channels associated with the plasma membrane. The channels crosses the bi-lipid layer and when closed (i) prevents passage of ion A along its concentration gradient. When open (ii) the ion is free to move along its concentration gradient until the ionic distribution is equal. This may be movement of ions into the cell such as Na+ or movement of ions out of the cell such as K+. The opening of the channels may be dependent upon other factors; distribution of ions either side of a membrane can produce a change in voltage at the membrane (iii) (calculated by the Nernst equation when considering single ions or the Goldman equation when examining multiple ions.). Equally the ion channel may be integrated with a receptor for a ligand (iv) such as PTH which then opens the channel.

Whilst many of these ion channel signalling processes may appear superficially irrelevant to orthodontics and our understanding of clinical practice, a direct parallel can be identified of voltage gated sodium channels in nervous tissue. Use of lignocaine and related pharmacological agents to obstruct these ion channels provides local anaesthesia in the day to day practice of dentistry.⁶

IONS
All ions have a role in non-excitable cell function and especially osteoblast cell function. These roles will be briefly considered. Each ion has a unique size, hence the specificity of the ion channels and, due to many proteins acting as ion transporters, a unique distribution of concentration across the cell membrane (Figure 2).⁷

Figure 2: The characteristics and concentrations of ions across the cell membrane. The numerical size of each ion is given together with the concentration of intra- and extra-cellular constituents across the cell membrane (mM-milliMolar).

Sodium is a ubiquitous ion in the extracelluar fluid. It is one of the ions most often associated with osmotic pressure of a fluid (the other being Cl-) and is the main initiator of an action potential in excitable tissue. In non-excitable tissue its action is less well defined. 

Potassium is the major intracellular ion and is very active electrochemically; small changes in intracellular concentrations of K+ can lead to an increase in activity of the cell typically to become hyperexcitable in the case of nerve or muscle. It is associated with ligand gated channels, the interaction of the ion channel and the receptor being mediated by a G protein. It has been demonstrated that K+ is an important ion as one of the first actions of cell stretching in both osteoblasts and fibroblasts.^8-10 We have also demonstrated the possibility that an excess load (in the form of strain energy density) exceeds the changes induced and, thus, poses the question of an upper limit of loading.¹¹ This ion has also been identified with apoptosis and mitochondrial membranes.¹²

Chloride ions, associated with Na+, are largely responsible for changing the volume of a cell. In an osteoblast, this change in volume can affect the ability of the cell to sense changes in its environment with either increased or decreased sensitivity. That is, if the osteocyte, which is an osteoblast but surrounded by bone in a lacuna (Figure 3a; the small canaliculi seen in the base of the lacuna will hold the processes through which the osteocytes communicate with one another) increases in volume in the confined space changes in skeletal shape can be easily sensed. If the cell decreases in size then the cell will become less sensitive. This manner of sensing the external deformation of the bone has led to the hypothesis of mechano-sensing by the osteocytes.¹³

Hydrogen ions relate to the intracellular pH although the changes in ion concentration can readily be modified by the changes in cell metabolism. Activation of the cells by various ligands can induce changes in H+ concentration.^14,15 Calcium²⁺ is the ubiquitous second messenger; hormones/ ligands being referred to as the primary messengers. It can be increased within the cytosolic material by passage from outside the cell or released from intracellular stores (Figure 3). The major intracellular store is the sarcoplasmic reticulum in excitable cells such as nerve and muscle and almost certainly the endoplasmic reticulum in non-excitable cells such as osteoblasts.^16,17 The function of Ca²⁺ as a second messenger has to be distinct from its role in calcification of the extracellular bone matrix. In the latter role, as a consequence of other intracellular signals, the ion is incorporated into the hydroxyapatite crystals laid down within the protein scaffold of bone. In the former role, the Ca²⁺ is instrumental in provoking a cellular response. As the main intracellular ion, Ca²⁺ has been identified as responsible for a variety of intracellular functions but more importantly, subtle differences in Ca²⁺ signal lead onto differing signals within cells. For example, a lymphocyte on first exposure to an antigen has one type of Ca²⁺ signal leading onto small amounts of antibody production; the second exposure to antigen leads onto a different Ca²⁺ signal and then a differing cell output of antibody.¹⁸ Any prolonged increase in intracellular Ca²⁺ leads to cell death or apoptosis.¹⁹

Figure 3: Representation of an osteoblast loaded with Fura-2 when unstimulated (a) and then when loaded with thrombin, a known G protein agonist (b). The change in Ca2+ concentration is significant and widespread throughout the cell.

SIGNALLING PATHWAYS
Signalling pathways can be divided into two distinct intracellular types; G protein and tyrosine kinase pathways. Both these basic mechanisms have an interaction with other signalling pathways and each is constructed of a unique protein that is reliant on the signal from the nucleus to allow the cell to manufacture the constituent parts. An example of this is seen with the interaction of oooestrogen and G protein pathways with a clear role of balanced maintenance ensuring an intact signalling mechanism. Changes in oooestrogen levels modify the levels of G protein which in turn modify the signal.²⁰ All the ionic signalling pathways may interact and co-exist in such a way that they share common proteins. A series of channels/transporters have been clearly identified (Figure 4).

Figure 4: A broad spectrum of ion carriers/channels have been identified within cells both at the cell membrane and within the intracellular organelles

To date, we have identified specific signalling mechanisms within the human osteoblast. Firstly, mechanical deformation of cells leads to a rapid (within 500 ms) change in intracellular Ca²⁺ concentration but to enable this increase there has to be an intact G protein pathway.²¹ Blockage of the pathway by cholera (CTX) and pertussis toxin (PTX) clearly modifies the signal generated 
(Figure 5). Pertussis toxin prevents the two active components of the G protein, a and the ß. fragment, from separating and in so doing prevents a change in Ca2+. Whilst the CTX prevents the a and the ß. fragment from reconstituting and, therefore, exaggerates and prolongs the signal. The change in Ca²⁺ concentration is short-lived and the cell returns to the normal resting level.

Figure 5: The action of PTX prevents the disassociation of the G Protein aß. fragments and so prevents activation of pathways involving G proteins 

Figure 6: The two distinct signalling pathways within the cell both lead to release of Ca2+ from the endoplasmic reticulum. Whilst the common end-point is Ca2+, the consequences of such activation vary.

Secondly, we have identified the activation of a collagen peptide motif (DGEA) that appears to stimulate osteoblasts via a tyrosine kinase pathway.²² Herbimycin can block this signal whilst PTX and CTX have no effect on the signal (Figure 6). The precise nature of activation by this peptide has yet to be clarified although there are increases in the phosphorylation of focal adhesion kinase (FAK) (MW 125) which is typically associated with adhesion of a cell to a substrate.^23,24 This change in activity of the cell has been clearly illustrated when observing cells in vitro. A typical cell seeded onto a substrate (Figure 7a) will seek out a substrate until it is able to attach and change to a flatter more adapted cell (Figure 7b).²⁵ Upon attachment there appears to be an increase in Ca²⁺ (Figure 7c) which has been explained by activation of FAK after integrin clustering.²⁶ We were unable to identify a specific integrin antibody capable of obstructing the response from DGEA (the peptide sequence). More recently, there has been doubt cast on the role of this sequence on integrin activation especially the a₂ß₁. In platelets, the sequence for this type of activation has been determined as GFOGER.²⁷ However, DGEA is linked specifically with tyrosine kinase activity and could well act as a signal to maintain the phenotype of osteoblasts.

Figure 7: A cell when seeded in culture appears round (a) but once a substrate has been identified the cell attaches, assumes a flatter profile to the substrate (b) and there is a concomitant activation of intracellular Ca2+ (c)[Measured using Fura-2].²⁵

Subsequent to identifying the different stimuli that act upon the two types of pathways we have also identified a possible role of oooestrogen in maintaining the G protein based pathways. In a normal cell there are several classes of G protein; one of the classes is inhibitory (Gi) whilst one is stimulatory (Gs); other classes also exist including Gq and G12/¹³. Cells simulated by oestrogen have specific levels of the G proteins which will provoke a response when, for example, PTH targets the cell.²⁰ When oestrogen is withdrawn from these cells the levels change such that the response to PTH would change. This has subsequently been supported by data from patients when a combination of oestrogen and PTH therapy induced bone formation.²⁸

Further work has also identified the release of Ca²⁺ from two functionally distinct stores in the osteoblast. G protein linked stores have been linked with inositol trisphosphate stores whilst tyrosine kinase has been linked with ryanodine (Ry) activated stores.²⁹ Whilst the end point of cellular signalling appears to be Ca²⁺ increases in osteoblasts there appears to be preliminary data that the Ca²⁺ has a further change. Experiments with previously identified levels of DGEA (tyrosine kinase activity) and thrombin (G protein activity) lead onto differing profiles of early gene expression.³⁰ Cytosolic Ca²⁺ activates one pathway of gene expression mediated by the serum response element, whereas transcription mediated by the cyclic AMP response element-binding protein requires a nuclear Ca²⁺ signal, typically identified in osteoclasts by specific ligands.^31,32 A further function of Ca²⁺ entry is to charge the internal stores which in turn can then signal in the cytoplasm. Both InsP³ and Ry receptors (RyR) demonstrate a calcium-induced calcium release, which means that all Ca²⁺ signals should be ‘all or nothing’ based. However, this has been further clarified by the aspects of ‘elemental’ Ca²⁺ signalling organised by groups of channels signalling in a synchronised manner.³³ Thus, blips for InsP³ or quarks for RyR exist. The next stage of hierarchal signalling involves puffs and sparks found in both ocytes and cardiac cells. The grading can be completed by recruitment of many groups of intracellular receptors leading to a widespread Ca²⁺ signal.

Figure 8: Signaling through Fas requires the formation of a death-inducing signalling complex (DISC) that involves the cytoplasmic domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. In the induction phase (1) ligands acting at the cell membrane induce early signals, which differ depending on the ligand and cell type involved. During the commitment phase (2) the mechanisms for degradation are activated. Current evidence suggests that mitochondrial permeability transition pore opening is a major event. The final degradative stage of apoptosis (3) is characterised by fragmentation of DNA and cell shrinkage. [Bcl-2: The Bcl-2 family of proteins plays a central regulatory role in apoptosis. FADD: Fas-associated death domain. The cytoplasmic adaptor protein FADD is an essential component of the death-inducing signaling complexes (DISCs) that assemble when TNF receptor family members, such as Fas, are ligated. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. CD95: Apoptosis-inducing receptor CD95].

Localised productions of Ca²⁺ signals can lead to very high concentrations of Ca²⁺ in small areas of the cytoplasm which in turn activate processes that are not susceptible to this type of localised Ca²⁺ spike.³⁴ To further complicate Ca²⁺ signalling two other molecules, both of which are identifiable metabolites, have been identified which appear to activate distinct Ca²⁺ stores. These have been documented as cyclic adenosine diphosphate-ribose (cADPR), identified as the functional homologue to Ry and nicotinic acid dinucleotide phosphate (NAADP).^35,36 This complex nature of Ca²⁺ signalling, mainly identified in excitable tissue, also exists in non-excitable cells. For example, it is clearly established that differing Ca²⁺ signals promote different responses in gene regulation, a fact that has been confirmed in osteoblasts.^30,37 Differential gene transcription, for example, has been demonstrated in B lymphocytes by amplitude modulation of the Ca²⁺ pathway. Low concentrations of Ca²⁺ activate the nuclear factor of activated T cells and the Erk pathway. A large elevation stimulates a different set of transcriptional regulators including c-Jun kinase and NF-kB. In the case of lymphocytes, unexposed cells or naïve cells produce a large Ca²⁺ response, which induces proliferation and self-selection. The previously exposed cells produce a smaller signal due to their tolerance, which does not provoke proliferation. Instead there is blocking of plasma-cell differentiation and secretion of active antibodies.¹⁸

APOPTOSIS AND K+
Potassium ions, as briefly discussed are a major intracellular ion. There is confirmed evidence that PTH acts via K+ channels and is closely integrated with apoptosis. The stretching of cells and K+ activity appears closely related to PTH stimulation.^38,39 The activation, possibly by some metabolites of respiration such as cytochrome c, then activate K+ channels before inducing apopotosis (Figure 8).

Induction phase: Ligands acting at the cell membrane induce early signals, which differ depending on the ligand and cell type involved. Studies on Jurkat lymphoma cells show that n-type K channels, responsible for both membrane potential and volume regulation, are inhibited early in apoptosis.⁴⁰ If similar actions occur in osteoblasts then the stimulation of K+ channel opening by PTH could have a protective effect. The role of K+ channels early in apoptosis, however, remains controversial since other investigators have reported that K+ channel blockade prevents apoptosis in thymocytes.⁴¹

Commitment phase: The mechanisms for degradation are activated. Current evidence suggests that mitochondria play a pivotal role at this stage, with opening of the mitochondrial permeability transition pore being a key event.^42-44 Interest in the role of mitochondria in apoptosis was stimulated with the realisation that members of the Bcl-2 family of proteins, whose action can be either pro- or antiapoptotic, localise mainly to the mitochondrial membrane.⁴⁵ Bcl-X_L, which is antiapoptotic, regulates mitochondrial membrane potential and volume homeostasis in mitochondria, implying that it also regulates ion transport across the membrane.⁴⁶ Bcl-2 also prevents opening of the permeability transition pore, although the mechanism by which this occurs is not known. A lowering of mitochondrial membrane potential appears to reduce the susceptibility to apoptosis in a number of systems.^46,47 It has been shown that both PTH and oestrogen stimulation lower mitochondrial membrane potential in chick osteoblasts, again providing a possible mechanism for their antiapoptotic effect.⁴⁸

Degradative stage: This is characterised by fragmentation of DNA and cell shrinkage, both of which have been correlated with a loss of K from the cell.^49,50 However, the vast body of evidence, to date, is indirect relying on the manipulation of the extracellular medium, in vitro translation of cell homogenates or measurement of [K]_I in already shrunken cells.^49,51

Our data, to date, have shown a change in K+ ions following stimulation of human osteoblast like cells; this was closely coordinated with G protein activity and is clearly identifiable with the specific opening of a K+ channel.^53,54

SUMMARY
Osteoblasts are pivotal to bone biology. Recent advances have highlighted a synchronous coordination of osteoblast and osteoclast activity whereby the osteoblast collates all signals 

applied to the bone and relays that information to the osteoclast via well characterised cytokines. The resorption of the bone and its matrix then releases growth factors held within the matrix which then stimulate the osteoblast to lay down new osteoid. The focus of this work reported here was to identify and dissect the various cytosolic intracellular signalling pathways within osteoblasts and establish the importance of each under different conditions comparable to the clinical setting.

Two basic pathways exist:

• Linked with a seven transmembrane spanning protein and specific receptors for ligands (the G protein linked pathways). One pathway is linked with protein phosphorylation, especially of the tyrosine kinases

• Our specific areas of interest are the ways in which calcium activity within the cell is modified and used as a signal for further activation, and possibly differential gene activation. To date, we have identified specific signalling mechanisms

• Firstly, mechanical deformation of cells leads to a rapid (within 500 ms) change in intracellular calcium concentration (Ca2+) but to enable this increase there has to be an intact G protein pathway

• Secondly, we have identified the activation of a collagen peptide motif that appears to stimulate osteoblasts via a tyrosine kinase pathway. The precise nature of activation by this peptide has yet to be clarified

• Subsequent to identifying the different stimuli that act upon the two types of pathways we have also identified a possible role of oestrogen in maintaining the G protein based pathways. Cells simulated by oestrogen have specific levels of the G proteins which will provoke a response when, for example, parathyroid hormone targets the cell. When oestrogen is withdrawn from these cells the levels change such that the response to PTH would change.

From our data it is clear that Ca²⁺ is essential to intracellular signalling pathways and the complexity of this signal may describe the variations of gene activity identified following variations of cell stimuli.

REFERENCES
1. Lacey DL, Timms E, Tan H.-L, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165-76.
2. Haydn JM, Mohan S, Baylink DJ. The insulin-like growth factor system and the coupling of formation to resorption. Bone 1995; 17: 93S-98S.
3. Mundy GR. Local control of bone formation by osteoblasts. Clin Orthopaed Rel Res 1995; 313: 19-26.
4. McDonald F. Bone homeostasis: extracellular calcium levels and their control MacMillan Life Science Encyclopaedia, 2000: Article 2004.
5. Thomas A, Hall SL, Nicolas V, Lau KH, Farley JR. Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells. Calcif Tiss Int 1995; 56:268-273.
6. Robinson P, Pitt TR, McDonald F. (2000) Local Anaesthesia in Dentistry, Butterworth Heineman 2000.
7. Sperelakis N. Cell Physiology Source Book 3 rd Edition 2001 Academic Press, SanDiego.
8. McDonald F, Houston WJB. The affect of mechanical deformation on the distribution of potassium ions across the cell membrane of sutural cells. Calcif Tiss Int 1992; 50: 547-552.
9. Leeves MA, McDonald F. The effect of mechanical deformation on the distribution of ions in fibroblasts. Amer J Orthodont Dento-fac Orthoped 1995; 107: 625-632.
10. Davidson RM. Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells. J Membr Biol. 1993; 131:81-92.
11. McDonald F, Yettram AL. The loading of cells and a possible upper limit of load response with respect to strain energy density. J Biomed Mat Res 1995; 29: 1577-1585.
12. Hughes FM, Cidlowski JA. Potassium is a critical regulator of apoptotic enzymes in vitro and in vivo. Adv Enzyme Regul 1999; 39:157-171.
13. Mullender MG, Huiskes R. Osteocytes and bone lining cells: which are the best candidates for mechano-sensors in cancellous bone? Bone 1997; 20: 527-32.
14. Green J, Yamaguchi DT, Kleeman CR, Muallem S. Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca²⁺ ions. J Gen Physiol 1990; 95:121-45.
15. Hoyland J, McDonald F, Pourghadiri M. External ligands effecting intracellular hydrogen ion concentration in human osteoblasts. XXXIV Int Cong Physiol Sci 2001; P117 (Abstract).
16. Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium 2002;32:235-49.
17. Berridge MJ. The AM and FM of calcium signalling. Nature 1997;386:759-60.
18. Bachmann MF, Mariathasan S, Bouchard D, Speiser DE, Ohashi PS. Four types of Ca2+ signals in naive CD8+ cytotoxic T cells after stimulation with T cell agonists, partial agonists and antagonists. Eur J Immunol 1997;27: 3414-9.
19. Berridge MJ, Bootman MD, Lipp P. Calcium-a life and death signal. Nature 1998;395:645-8.
20. Papaioannou S, Tumber AM, Meikle MC, McDonald F. G-protein signalling pathways and ooestrogen: A role of balanced maintenance in osteoblasts. Biochim Biophys Acta (mol Cell Res) 1999; 1449: 284-292.
21. McDonald F, Somasundaram B, McCann TJ, Mason WT, Meikle MC. Calcium waves in fluid flow stimulated osteoblasts are G protein mediated. Arch Biochem Biophys 1996; 326:31-38.
22. McCann TJ, Mason WT, Meikle MC, McDonald F. A collagen peptide motif activates tyrosine kinase-dependent calcium signalling pathways in human osteoblast-like cells. Matrix Biol 1997; 16: 271-280.
23. Richardson A, Parsons JT. Signal transduction through integrins: a central role for focal adhesion kinase? Bioessays 1995; 17: 229-36.
24. Bister D, Meikle MC, McDonald F. Adhesion of human osteoblast-like cells: tyrosine kinase dependent phosphorylation of focal adhesion kinase. J Bone Min Res 2000; 15:1235.
25. McCann TJ, Meikle MC, Northrop AJ, Mason WT, McDonald F. An integrin binding collagen peptide motif activates calcium signalling in SaOS-2. Calcif Tiss Int 1995; 57: 322.
26. Bister D, Meikle MC, McDonald F. Adhesion of human osteoblastlike cells to a collagen peptide motif: tyrosine kinase dependent phosphorylation of focal adhesion kinase. Mol Biol Cell 1998; 9: 358.
27. Knight CG, Morton LF, Peachey AR, Tuckwell DS, Farndale RW, Barnes MJ. The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens. J Biol Chem 2000; 275: 35-40.
28. Cosman F, Nieves J, Woelfert L, Formica C, Gordon S, Shen V, Lindsay R. Parathyroid hormone added to established hormone therapy; effects on vertebral fracture and maintenance of bone mass after parathyroid hormone withdrawal. J Bone Min Res 2001; 16: 925-931.
29. McCann TJ, Keyte JW, Terranova G, Papaioannou S, Mason WT, Meikle MC et al. An analysis of calcium release by DGEA: Mobilisation of two functionally distinct internal stores in Saos-2 cells. Amer J Physiol: Cell Physiol 1998; 275: C33-C41.
30. Varley H, Papaioannou S, Meikle MC, McDonald F. Expression of early onset genes following differential ligand stimulation. J Bone Min Res 2000; 15: S382, SU209 (Abstract).
31. Hardingham GE, Chawla S, Johnson CM, Balding H. Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression Nature 1997; 385: 260-265.
32. Shankar G, Davison I, Helfrich MH, Mason WT, Horton MA. Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts. J Cell Sci 1993; 105: 61-68.
33. Bootman MD, Niggli E, Berridge MJ and Lipp P. Imaging the hierarchial Ca²⁺ signalling system in HeLa cells. J Physiol 1997; 499: 307-314.
34. Hajnoczky G, Robb-Gaspers LD, Seitz MB and Thomas AP. Decoding of the cytosolic calcium oscillations in the mitochondria. Cell 1995; 82: 415-442.
35. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 1997; 386: 855-858.
36. Aarhus R, Graeff RM, Dickey DM, Walseth TF, Lee HC. ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium mobilising metabolite from NADP J Biol Chem 1995; 270: 30327-30333.
37. Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kurosaki T, Iino M Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J 1999; 18:1303-8.
38. Duncan R, Misler S Voltage-activated and stretch-activated Ba2+ conducting channels in an osteoblast-like cell line (UMR 106). FEBS Lett 1989; 251: 17-21.
39. Platoshyn O, Zhang S, McDaniel SS, Yuan JX. Cytochrome c activates K+ channels before inducing apoptosis. Am J Physiol Cell Physiol 2002; 283: C1298-305.
40. Szabo I, Gulbins E, Apfel H, Zhang X, Barth P, Busch AE et al. Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T-lymphocytes upon Fas stimulation. J Biol Chem 1996; 271: 20465-20469.
41. Dallaporta B, Marchetti P, de Pablo MA, Maisse C, Duc H-T, Metivier D et al. Plasma membrane potential in thymocyte apoptosis. J Immunol 1999;162: 6534-6542
42. Green D, Kroemer G. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol 1998; 8: 267-271.
43. Kroemer G, Dallaporta B, Resche-Rigon M.The mitochondrial death/life regulator in apoptosis and necrosis. Ann Rev Physiol 1998; 60: 619-642
44. Gulbins E, Jekle A, Ferlinz K, Grassme H, Lang F. Physiology of apoptosis. Am J Physiol 2000; 279: F605-F615.
45. Krajewskji S, Tanaka S, Takayama S, Schiber MJ, Fenton W, Reed JC. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane. Cancer Res 1993; 53: 4701-4714.
46. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell 1997; 91: 627-637.
47. Dey R, Moraes CT. Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-XL in osteosarcoma cells. J Biol Chem 2000; 275: 7087-7094.
48. Troyan MB, Gilman VR, Gay CV. Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol. Exp Cell Res 1997; 233: 274-280.
49. Hughes FM, Cidlowski JA. Potassium is a critical regulator of apoptotic enzymes in vitro and in vivo. Adv.Enzyme Regul 1999; 39:157-171.
50. Bortner CD, Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 1998; 56: 1549-1559.
51. Bilney AJ, Murray AW. Pro- and anti-apoptotic effects of K+ in HeLa Cells. Febs Lett 1998; 424: 221-224.
52. Walev I, Resko K, Palmer M, Valera A, Bhakdi S. Potassium inhibited processing of IL-1ß in human monocytes. EMBO J 1995;14:1607-1614.
53. Chatoo AH, Arrebola F, McDonald F, Warley A. An analysis of ionic and volumetric changes in osteoblasts following G protein activation. J Physiol 2001; 535P.
54. Goodyear F, McDonald F, Warley A. PTH stimulation of Saos-2 osteoblastlike cells; evidence for potassium channel involvement. Biochem Soc Trans 2002; 3X: 132.

Copyright: 19 November 2003