DE10219545A1 - Regulation of apoptosis - Google Patents

Abstract

The present invention relates to the use of a substance for the detection of cation channels and the use of an active substance to influence cation channels for the prevention or treatment of diseases which are associated with a disturbed regulation of apoptosis.

Description

The present invention relates to the use of a substance for Detection of cation channels and the use of an active ingredient for influencing cation channels for prevention or Treatment of diseases with impaired regulation of apoptosis being related.

Apoptosis is the so-called programmed cell death. In a broader sense, this also includes cell death together, through genetic information of the affected cell itself is regulated. Apoptosis is the basis of a regulated one Embryogenesis (death of superfluous organ systems), tissue homeostasis (Protection against new growth) and function of the immune system (triggering of apoptosis in target cells by cytotoxic T lymphocytes and natural killer cells) and is affected by various factors (e.g. Receptor binding, protease effect) triggered. Apoptosis also plays a role also a role in cytostatic effects, radiation therapy and other therapeutic principles.

Scientific studies show that oxidative and osmotic stress, well-known triggers of apoptotic death nucleated cells [Gulbins et al. 2000, Michea et al. 2000, Rosette and Karin 1996] also cause erythrocytic apoptosis. Although erythrocytes contain neither nuclei nor mitochondria, they are able to undergo morphological changes such as those for apoptotic nucleated cells are typical [Daugas et al. 2001], although the erythrocytes are resistant to some apoptotic stimuli, e.g. B. against Serum withdrawal or the protein kinase C inhibitor staurosporin [Daugas et al. 2001].

Although apoptosis plays an important role in many disease courses plays are the molecular mechanisms that affect apoptosis mediate, and their importance for the course of the disease so far largely unclear.

Accordingly, the invention has the task of Clarification of molecular mechanisms new diagnostic and Find treatment approaches and provide active ingredients that are suitable for the therapeutic and diagnostic use for disorders of the Apoptosis regulation can be used.

Surprisingly, using the example of erythrocytes Mechanism through which these coreless tents in the programmed cell death or against well-known triggers of apoptotic death can be made resistant.

Accordingly, the object of the invention is through the subject of independent claims 1 to 4, 16, 22 and 26 solved. preferred Executions are in the dependent claims 5 to 15, 17 to 21, 23 to 25 and 27 called. The content of all these claims is hereby made by reference to the content of the description.

According to the invention, at least one substance can be used to detect the expression and / or function of cation channels in cells. This substance is used to diagnose diseases related to impaired regulation of apoptosis. This can be e.g. B. to the detection of genetic dispositions for certain diseases due to mutations, z. B. act on the cation channel. Under certain circumstances, a susceptibility to certain diseases can be diagnosed, so that, with the corresponding symptoms, it can quickly be concluded that the regulation of apoptosis, with the clinical symptoms associated therewith, is disturbed. The substance used for the detection of expression and / or function of cation channels can, for. B. is an antibody that is directed against the cation channel, and in a detection method known to those skilled in the art, such as. B. ELISA (enzyme linked immunosorbent assay) is used. Furthermore, e.g. B. with the help of the so-called PCR technique (polymerase chain reaction) this proof can be provided. In this molecular genetic method, certain DNA sections are selectively amplified. DNA sequences framed by two synthetic oligonucleotides are re-synthesized using DNA polymerases. Due to the exponential enrichment, starting from small amounts of DNA (10 ^-9 -10 ^-15 g), the DNA sections can be made detectable after repeating the process several times (20-40 cycles). To detect RNA sections, the RNA must be copied into DNA using an RNA-dependent DNA polymerase. The detection according to the invention can thus be carried out by selecting suitable DNA sections on the target protein, the cation channel. Further methods of how a known target protein or domains of a target protein composed of several subunits can be quantitatively and / or qualitatively detected are known to the person skilled in the art, and in this connection reference is made to the extensive corresponding specialist literature.

Furthermore, at least one active ingredient can be used according to the invention Prevention or treatment of diseases in cells can be used. After application, this active ingredient influences expression and / or Function of cation channels, with it becoming a particular Stimulation or inhibition of the cation channel comes. This results in an induction, alternatively an at least partial inhibition, the Apoptosis, i.e. H. generally in a regulation of apoptosis in the affected cells. On the one hand, this active ingredient can already be act substances described, on the other hand, the active ingredient z. B. a genetically modified mutation of the cation channel, or an up- / downstream member of the signal transduction cascade this cation channel. So it is z. B. possible with the help of Genetic engineering to produce active ingredients that are either a) a higher Have activity as their physiological equivalents, or b) none Can interact with their physiological inhibitors. A Another possibility is to provide the naturally occurring Signal elements. Inhibitors of the Signal transduction cascade are inhibited in turn, so as to activate the corresponding signal transduction cascade of the cation channel enable. This ingredient can then be used to make a drug or a pharmaceutical composition are provided, which is claimed in a further preferred embodiment.

In a further preferred embodiment, a method for Regulation of apoptosis claimed. In this procedure Cells brought into contact with an active ingredient, the active ingredient being the Influences expression and / or function of cation channels, in particular stimulates or inhibits, and thereby apoptosis in them Cells induced (when stimulated) or inhibited (when inhibited). Depending on the disease that is associated with apoptosis, you may do so by specifically influencing apoptosis, the course of the disease is positive to be influenced.

According to the invention, the influence, in particular the stimulation or inhibition, of cation channels has an influence and / or control of the intracellular Ca ²⁺ concentration, ie the influx of Ca ²⁺ ions into the cell. The cation channel is permeable to Ca ²⁺ , which allows Ca ^{2+ to} enter the cell. The channel itself is an unspecific cation channel. By increasing the intracellular Ca ²⁺ concentration, cellular apoptosis is stimulated under suitable conditions, and accordingly, by reducing the intracellular Ca ²⁺ concentration, cellular apoptosis is inhibited.

In general, according to the invention, all cells, i.e. H. both nucleated as also coreless cells, which can be influenced by influencing one Cation channel the apoptosis can be regulated. Under cells according to the invention, the cells are also included which are seedless, but have arisen from nucleated preliminary stages. In a In a particularly preferred embodiment, these cells are Blood cells, d. H. cells derived from bone marrow, in particular around erythrocytes. These nucleated cells are from the nucleated Precursors, the erythroblasts. During their development through maturation and division (erythropoiesis) the nucleus becomes the cell ejected, and in the cytoplasm finds the Hemoglobin formation takes place. The cell volume regulatory cation channels are but not only in erythrocytes, but also in several nucleated Cells expressed [Cabado et al. 1994, Chan & Nelson 1992, Gamper et al. 2000, Koch & Korbmacher 1999, Volk et al. 1995, Wehner et al. 1995, 2040]. After an increase in intracellular calcium too can induce cell death in nucleated cells [Green & Reed 1998] the activation of the cell volume-sensitive cation channels equally nucleated in triggering apoptotic death Cells play a role.

In a preferred embodiment, the active ingredient is or Substance directed against the cation channels themselves. For the active ingredient or the substance, in addition to those already described, can also be found under other about antisense sequences, genetically modified mutants the members of the signaling cascade of the cation channel, or are so-called "small molecular compounds". Further to are polynucleotides that code for a peptide that the Influences expression and / or function of cation channels, in particular stimulates or inhibits, or polypeptides, which preferably influence the expression and / or function of cation channels, in particular stimulate or inhibit. Furthermore, the active ingredient or the substance against activators, inhibitors, regulators and / or biological precursors of cation channels. This Activators, inhibitors, regulators and / or biological precursors can z. B. transcription factors relevant for the expression level of corresponding cation channel are responsible, and other known Activators, inhibitors, regulators and / or biological precursors to Be cation channels. Against these activators, inhibitors, Regulators and / or biological precursors can be used as an active ingredient or as Substance z. B. a polynucleotide encoding a peptide, or a Polypeptide and a "small molecular compound" can be used. Also the use of at least one oxidizing agent as an active ingredient or Substance is according to a further preferred embodiment includes.

In the present invention, it is possible to use known or as yet unknown active substances or substances. In a particularly preferred embodiment, the active substance or the substance is a so-called "small molecular compound", in particular one with a molecular weight (MW) <2,000. This "small molecular compound" is preferably amiloride (MW 229.65), ionomycin and / or their analogs. An example of an analog of amiloride is e.g. B. Ethylisopropylamiloride. Amiloride is the international short name for the potassium-retaining diuretic N-amidino-3,5-diamino-6-chloropyrazine-2-carbamide. Diuretics are generally drugs that, by inhibiting renal reabsorption, especially of Na ⁺ ions, increase the excretion of Na ⁺ , Cl ^- and HCO ₃ ^- ions and (indirectly) water, thereby reducing the plasma volume and improving congestion symptoms. Potassium-retaining or potassium-saving diuretics such as amiloride or triamterene inhibit sodium absorption in the distal tubule. They have a weak diuretic effect combined with potassium retention. Ionomycin is one of the so-called ionophores. This name, introduced by Pressmann in 1967 and composed of "ions" and "phor", summarizes the mostly macrocyclic compounds with a molecular weight (MW) generally <2,000, which reversibly form chelates or complexes with ions. Due to their relatively hydrophobic surface, they can transport these ions as carriers through biological membranes that are otherwise impermeable to ions. This carrier function enables the influx of Ca ²⁺ , which leads to an induction of apoptosis in the corresponding cells.

The invention can be used to treat all forms of diseases, those with a disturbed regulation, especially a disturbed one Inhibition or induction related to apoptosis recognize or treat. For diseases with a disturbed inhibition related, it is z. B. about neurodegenerative diseases, acute kidney failure, Thalassemia, sickle cell anemia, Glucose-6-phosphate dehydrogenase deficiency, iron deficiency anemia and / or immunodeficiency. A longer one Residence time of erythrocytes in the high osmolar renal medulla during acute kidney failure [Mason et al. 1986] can about activation of the channel trigger apoptosis, an effect which leads to the disruption of the Contributes to microcirculation. In addition, the cation channel at Disorders activated which lead to shrinkage of erythrocytes, such as sickle cell anemia [Joiner 1993, Lang et al. 1998], thalassemia [Mach-Pascual et al. 1996] or iron deficiency anemia [Jolobe 2000, Mach-Pascual et al. 1996]. The accelerated death of the Erythrocytes in these diseases can therefore be inhibited by Cation channel are at least partially weakened. Alternatively are the diseases with a disturbed Induction of apoptosis is related to tumor diseases and / or infections, well-known diseases in which one impaired inhibition of apoptosis is present. The invention also all other diseases caused by induction or inhibition of the Apoptosis can be treated via a cation channel.

According to the invention, the active ingredient or substance can be administered orally, administered intravenously, topically and / or by inhalation. The The form of administration and the dose can be chosen freely. there are known to play especially age, gender, as well as others Factors of the patient matter. The clinical picture, as well as Art and degree of disease must be selected when choosing the type of application and the dose of the active ingredient or substance are taken into account.

The invention further comprises a pharmaceutical composition, which contains at least one active ingredient which expression and / or Function of cation channels influenced, in particular stimulated or inhibited, and optionally a pharmaceutical carrier. there the active substance can be a polynucleotide which encodes a peptide, in particular a polypeptide, preferably this peptide has the expression and / or function of cation channels influenced, especially stimulated or inhibited. Alternatively, you can the active ingredient may be a peptide, preferably a polypeptide, and this Peptide or polypeptide preferably the expression and / or function influenced by cation channels, in particular stimulated or inhibited. The active ingredient may also be a "small molecular compound", preferably a "small molecular compound" with a molecular weight (MG) < 2,000. So it can be z. B. in the active ingredient by a so-called Act antisense sequence, d. H. a sequence that is able to the mRNA to form a double-strand duplex, and thereby the Prevent translation into a polypeptide. Also, e.g. B. the Gene sequence itself can be used to over- / under-express the To achieve cation channel, e.g. B. by incorporation into suitable vectors strong promoters or by constructing suitable mutants. Furthermore, the active ingredient can be the one already described Amiloride, ionomycin and / or their analogs, such as. B. Ethylisopropylamilorid act as well as an oxidizing agent. To further Characteristics of such a composition is based on the corresponding previous text of the description.

The invention further comprises a pharmaceutical composition, which has an effective amount of at least one active ingredient which Expression and / or function of activators, inhibitors, regulators and / or biological precursors influenced by cation channels, preferably stimulated or inhibited. This pharmaceutical The composition can optionally also be a pharmaceutical carrier contain. The activators, inhibitors, regulators and / or biological Precursors of cation channels can e.g. B. the above already mentioned transcription factors, as well as other known or so far unknown members of the signal transduction cascade of the Be cation channel. Also polynucleotides that encode a polypeptide that the Expression and / or function of activators, inhibitors, regulators and / or influence biological precursors of cation channels, preferably stimulate or inhibit in such Compositions may be included. Furthermore, so-called "small molecular compounds ", which preferably have a molecular weight (MG) of <2,000 and against activators, inhibitors, regulators and / or biological precursors of cation channels, and stimulate the expression or function of this channel or inhibit, can be used according to the invention. After all, it can the active ingredient is a peptide, preferably a polypeptide, preferably this peptide expresses and / or functions of Activators, inhibitors, regulators and / or biological precursors influenced by cation channels, in particular stimulated or inhibited. To The further features of such a composition are based on the previous corresponding text of the description referred.

Finally, the invention includes a diagnostic kit. In this kit is contain at least one substance for the detection of expression and / or function of cation channels is used. The diagnostic kit is used to identify vulnerabilities to Diseases associated with impaired regulation of apoptosis in Related. Such diagnostic kits can be targeted in one Diagnostic procedures are used, which for the detection of vulnerabilities against neurodegenerative diseases, acute kidney failure, Thalassemia, sickle cell anemia, Glucose-6-phosphate dehydrogenase deficiency, iron deficiency anemia, immunodeficiency, tumor disease and / or infections are used.

The features mentioned and other features of the invention result preferred from the description below Embodiments in connection with the subclaims and figures. The individual features can be used individually or in groups Combination with each other.

The pictures show:

Fig. 1 Ionomycin-induced erythrocyte apoptosis;

Fig. 2 Osmotically triggered erythrocytic apoptosis;

Fig. 3 Oxidatively induced erythrocytic apoptosis;

Fig. 4 Erythrocytic apoptosis caused by a lack of glucose.

methods

Erythrocytes were taken from healthy volunteers. The experiments were carried out at 37 ° C. in Ringer's solution of the following composition: 125 mM NaCl, 5 mM KCl, 1 mM MgSO ₄ , 32 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 mM glucose, 1 mM CaCl ₂ , pH = 7.4. For the nominal calcium-free solution, CaCl _{2 was} replaced by 1 mM ethylene glycol bis (β-aminoethyl ether) -N, N, N ', N'-tetra-acetic acid (EGTA). The osmolarity was increased to 850 mosmol / l by adding sucrose. Ionomycin was used in a concentration of 1 μM, amiloride in a concentration of 1 mM. The concentration of the solvent dimethyl sulfoxide DMSO was 0.1% in each case.

The FACS analysis (flow cytometric analysis) was carried out according to standard methods [Andree et al., 1990]. After incubation, the cells were washed in annexin binding buffer containing 125 mM NaCl, 10 mM HEPES; pH = 7.4) and 5 mM CaCl ₂ contained. The erythrocytes were diluted 1: 100 with Annexin-Flous (Böhringer Mannheim, Germany). After 15 min. the samples were diluted 1: 5 and analyzed by FACS (FACS-Calibur from Becton Dickinson). Forward scatter, sideward scatter, and annexin fluorescence (FL-1) were measured from the cells.

The values are given as arithmetic mean ± SEM, the statistical analysis was performed using the paired or unpaired t Tests performed.

Studies have been conducted to investigate whether osmotic shock or oxidative stress are well-known triggers of apoptotic cell death in nucleated cells [Bortner & Cidlowski 1998, 1999, Lang et al. 1998, Maeno et al. 2000, Michea et al. 2000, Roger et al. 1999, Rosette &, Karin 1996] also drive erythrocytes into apoptosis. It could be shown that both measures and glucose deficiency trigger apoptosis. Surprisingly, apoptosis was prevented by removing the extracellular Ca ²⁺ . In further experiments it could be shown that the cation channel inhibitor amiloride can suppress the osmotic, oxidative or metabolically induced apoptosis. Amiloride inhibits a cation channel that is caused by osmotic shock [Huber et al. 2001] and oxidative stress [Duranton et al. 2001] is activated and Ca ^{2+ flows} into the cell [Duranton et al. 2001]. The inhibition of erythrocytic apoptosis by amiloride therefore shows that apoptosis is caused by activation of this channel. Findings in nucleated cells show that these cells can also be killed by activating the amiloride-sensitive cation channel.

Experiment 1 Increase in the cytosolic calcium concentration by the ionophore Ionomycin triggers erythrocytic apoptosis

Changes in cell volume (measured as forward scatter) after a 3-hour incubation of erythrocytes with 1 µM ionomycin in the absence of Ca ²⁺ (A) or in the presence of Ca ²⁺ (B). Phosphatidylserine asymmetry (measured as annexin binding) after 3 hours of incubation with ionomycin in the absence of Ca ²⁺ (C) or presence of Ca ²⁺ (D). Arithmetic mean (± SEM) of annexin-binding cells after 3 hours pretreatment with 1 µM ionomycin with or without Ca ²⁺ in the extracellular fluid (E).

The calcium ionophore ionomycin was used to check the importance of Ca ²⁺ for erythrocytic apoptosis. As shown in Fig. 1B, the administration of 1 µM ionomycin leads to massive, Ca ²⁺ -dependent cell shrinkage within 3 hours, as shown by the "forward scatter FC" of the fluorescence-activated cell sorting (FACS) can be. The FC decreases from a value of 428 ± 13 (n = 3) to a value of 121 ± 2 (n = 4). In addition, 1 µM ionomycin increases the binding of annexin from 4.1 ± 0.4% (n = 3) to 71.3 ± 3.2% (n = 4) ( Fig. 1 D). Ionomycin-induced cell shrinkage and annexin binding are both inhibited in the absence of extracellular Ca ²⁺ . The corresponding values are 337 ± 16 (n = 4) for the FC and 12.9 ± 3.1% (n = 4) for the annexin binding ( Fig. 1 E). Treatment of erythrocytes with ionomycin thus triggers two typical features of apoptosis, ie the breakdown of phosphatidylserine asymmetry (which leads to annexin binding) and cell shrinkage.

Experiment 2 Osmotic shock leads to erythrocyte annexin binding

Annexin binding of erythrocytes in isotonic medium (A) and in 850 mosmolar medium after addition of sucrose for 24 hours (B). The cells were further exposed to the hyperosmolar solution in the absence of Ca ²⁺ (C) or in the presence of 1 mM Ca ²⁺ but in the presence of 1 mM amiloride (D). Arithmetic mean (± SEM) of annexin-binding cells at various osmolarities after 24 and 48 hours (E). Arithmetic mean (± SEM) of annexin-binding cells that were incubated for 24 hours in 850 mosmol / l with or without Ca ²⁺ or with or without 1 mM amiloride (F).

Osmotic shock is known to trigger apoptosis in nucleated cells [Bortner & Cidlowski 1998, 1999, Lang et al. 1998, Maeno et al. 2000, Michea et al. 2000, Roger et al. 1999, Rosette &, Karin 1996]. As was surprisingly observed, osmotic shock also increases the annexin binding of coreless erythrocytes ( Fig. 2B, E). If erythrocytes are exposed to an osmolarity of 850 mosmol / l (addition of sucrose), the number of annexin-binding cells increases from 3.9 ± 0.3% (n = 4) to 51.4 ± 3.8% (n = 9) within 24 hours. A typical experiment is shown in Fig. 2B. Nominal absence of calcium significantly reduces the number of annexin-binding cells after osmotic shock to 21.1 ± 7.2% (n = 4, see Fig. 2C for a typical experiment and Fig. 2F for arithmetic mean ± SEM). In addition, the cation channel blocker amiloride (1 mM) significantly reduces the number of annexin-binding cells to 29.8 ± 4.0% (n = 5, see Fig. 2D for a typical experiment and Fig. 2F for arithmetic mean values ± SEM).

Experiment 3 Oxidative stress triggers erythrocytic apoptosis

Annexin binding before (A) and after (B, C, D) oxidation of erythrocytes with 0.66 tert-butyl hydroperoxide (tBOOH) for 15 minutes. After the oxidation, the cells were incubated for 24 hours in Ringer's solution with 1 mM Ca ²⁺ (B) or without Ca ²⁺ (C) or with 1 mM Ca ²⁺ and 1 mM amiloride (D). Arithmetic mean (± SEM) of annexin binding cells 24 hours after 15 minutes of treatment with 1mM tBOOH or 0.66mM tBOOH, in the presence or absence of 1mM Ca ²⁺ or in the presence of 1mM Ca ²⁺ and 1mM amiloride (E).

Oxidative stress caused by the administration of 0.66 mM or 1 mM tert-butyl hydroperoxide (tBOOH) led to massive shrinkage of erythrocytes, as by the change in the forward scatter in the FACS analysis of 370 ± 13 (n = 4) on 314 ± 51 (n = 4) or 167 ± 14 (n = 4). In a first series of experiments it was observed that treatment of the cells for 15 min with 0.66 mM tBOOH triggers significant annexin binding ( FIG. 3B). Annexin binding could be significantly reduced by omitting the extracellular calcium ( Fig. 3C) or by administering 1 mM amiloride ( Fig. 3D). As shown in Fig. 3E, 0.66 mM and 1 mM tBOOH increase the number of annexin-binding cells from 3.2 ± 0.5% (n = 4) to 26 ± 7.5% (n = 4) and 68.5 ± 5.3 (n = 4) , In the nominal absence of extracellular calcium, both oxidation-induced cell shrinkage and annexin binding are both reduced. The values for forward scatter in calcium-free incubation medium reached 339 ± 22 (n = 4) (0.66 mM tBOOH) and 307 ± 15 (n = 4) (1 mM tBOOH), compared to 314 ± 51 (n = 4) and 167 ± 14 (n = 4) in the presence of calcium. The numbers for annexin positive cells only reached 11.0 ± 1.4% (n = 4) (0.66 mM tBOOH) and 34.1 ± 2.6% (n = 4) (1 mM tBOOH) in the absence of calcium ( Fig. 3E). Accordingly, the omission of extracellular calcium inhibited the tBOOH-induced phosphatidylserine rearrangement by 66% and 53%, respectively. Likewise, the presence of 1mM amiloride diminished the effect of tBOOH even in the presence of 1mM Ca ²⁺ . The respective values for the forward scatter reached 356 ± 24.0 (n = 4) (0.66 mM tBOOH) and 291 ± 27 (n = 4) (1 mM tBOOH). In the presence of 1 mM amiloride, the number of annexin-positive cells was reduced to 7.9 ± 3.2% (0.66 mM tBOOH) and 37.7 ± 4.4% (1 mM tBOOH), respectively, a finding which showed the inhibition of the t-BOOH-induced annexin -Binding reflected by 80 or 48% ( Fig. 3E).

Experiment 4

A lack of glucose induces erythrocyte apoptosis

Annexin binding of erythrocytes after 48 hours incubation in the presence (A) or in the absence of glucose (B). The cells were further exposed to a glucose-free solution without Ca ²⁺ (C) or with 1 mM Ca ²⁺ and 1 mM amiloride (D). Arithmetic mean (± SEM) of annexin-binding cells that were exposed to glucose deprivation for 24 or 48 hours with or without the presence of Ca ²⁺ or 1 mM amiloride (E).

Glucose deficiency has previously been shown to weaken the antioxidant mechanisms of erythrocytes. The effect of glucose deficiency was therefore investigated ( Fig. 4A-E). In the presence of glucose, 3.1 ± 0.6% (n = 4) of the erythrocytes bound annexin. Removal of glucose from the extracellular medium increased the number of annexin-binding cells to 11.2 ± 2.2% (n = 4) after 24 hours and to 49.4 ± 5.7% (n = 4) after 48 hours. The increase in annexin-binding cells was significantly reduced in the nominal absence of calcium. The corresponding values were 10.4 ± 2.5% (n = 4) after 24 hours and 10.0 ± 1.9% (n = 4) after 48 hours. In the same way, the effect of glucose withdrawal was weakened by amiloride (1 mM). The respective values were 5.7 ± 1.8% (n = 4) after 24 hours and 11.7 ± 1.9% (n = 4) after 48 hours ( Fig. 4E).

Recent findings have shown that the Ca ²⁺ ionophore ionomycin triggers erythrocytic cell shrinkage and annexin binding of erythrocytes, both typical indications of apoptosis in nucleated cells [Bratosin et al. 2001, Berg et al. 2001, Daugas et al. 2001]. How and under what conditions the Ca ²⁺ concentration is increased in erythrocytes has so far remained unclear. With the present results, the importance of an amiloride-sensitive, cell volume-regulated cation channel for the triggering of apoptotic cell death was shown for the first time. The channels were characterized earlier, their inhibitory bark by amiloride [Huber et al. 2001, Duranton et al. 2002] and their sensitivity to osmotic [Huber et al. 2001] and oxidative [Duranton et al. 2002] shown stress.

The erythrocytic cell shrinkage induced by oxidative stress results from an activation of Ca ²⁺ sensitive K ⁺ channels in the erythrocytic cell membrane, which leads to erythrocyte loss of KCl via hyperpolarization of the cell membrane [Bookchin et al. 1987, Brugnara et al. 1993].

The mechanisms shown play a role in limiting the Erythrocyte life. The presentation of phosphatidylserine on the cell surface stimulates the uptake of erythrocytes Macrophages [Boas et al. 1998, Romero & Romero 1999]. Therefore encourages an increase in intracellular calcium concentration as seen in aging erythrocytes occurs [Kiefer & Snyder 2000, Romero & Romero 1999], the removal of the affected erythrocytes from the bloodstream.

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Claims (27)

1. Use at least one substance for the detection of Expression and / or function of cation channels in cells, for Diagnosis of diseases with a disturbed regulation related to apoptosis. 2. Use of at least one active ingredient for influencing, in particular to stimulate or inhibit expression and / or function of cation channels in cells, for prevention or treatment of disorders associated with a disorder Regulation of apoptosis related. 3. Use of at least one active ingredient for influencing, in particular to stimulate or inhibit expression and / or function of cation channels in cells, for the production a drug or a pharmaceutical Composition for the prevention or treatment of diseases associated with an impaired regulation of apoptosis stand. 4. A method for regulating apoptosis, characterized in that that cells are brought into contact with an active ingredient which influences the expression and / or function of cation channels, in particular stimulates or inhibits, and thereby apoptosis induced or inhibited in these cells. 5. Use or method according to one of claims 2 to 4, characterized in that the influence, in particular stimulation or inhibition, of cation channels has an influence and / or control of the Ca ²⁺ influx into the cells. 6. Use or method according to one of the preceding Claims, characterized in that the cells are Blood cells, especially erythrocytes. 7. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the Substance is directed against cation channels. 8. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the Substance against activators, inhibitors, regulators and / or biological precursor is directed by cation channels. 9. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the Substance is a polynucleotide, which is a peptide, in particular a Polypeptide, encoded, preferably this peptide Influences expression and / or function of cation channels, especially stimulated or inhibited. 10. Use or method according to one of claims 1 to 8, characterized in that the active ingredient or substance Is peptide, preferably a polypeptide, preferably this peptide has the expression and / or function of cation channels influenced, especially stimulated or inhibited. 11. Use or method according to one of the preceding Claims, characterized in that the active ingredient or the Substance a "small molecular compound", preferably a "small molecular compound "with a molecular weight (MG) <2,000 is. 12. Use or method according to claim 11, characterized characterized in that the active ingredient or the substance amiloride or a Is analog. 13. Use or method according to one of claims 1 to 11, characterized in that the active substance or the substance is an oxidizing agent. 14. Use or method according to one of the preceding Claims, characterized in that it is in the Diseases with a disturbed regulation, especially one impaired inhibition related to apoptosis about neurodegenerative diseases, acute kidney failure, Thalassemia, sickle cell anemia, Glucose-6-phosphate dehydrogenase deficiency, iron deficiency anemia and / or immunodeficiency is. 15. Use or method according to one of claims 1 to 13, characterized in that the diseases that with a disturbed regulation, especially a disturbed one Inducing apoptosis related to tumors and / or infections. 16. Pharmaceutical composition comprising an effective Amount of at least one active ingredient, the expression and / or Function of cation channels influenced, in particular stimulated or inhibited, and optionally a pharmaceutical Carrier. 17. Pharmaceutical composition according to claim 16, characterized characterized in that the active ingredient is a polynucleotide which encodes a peptide, in particular a polypeptide, where preferably this peptide expresses and / or functions of Cation channels influenced, in particular stimulated or inhibited. 18. Pharmaceutical composition according to claim 16, characterized characterized in that the active ingredient is a peptide, preferably a Polypeptide, preferably this peptide is the expression and / or function of cation channels influenced, in particular stimulates or inhibits. 19. Pharmaceutical composition according to one of the claims 16 to 18, characterized in that the active ingredient is a "small molecular compound ", preferably a" small molecular compound "with a molecular weight (MW) <2,000. 20. Pharmaceutical composition according to claim 19, characterized characterized in that the active ingredient is amiloride or an analog. 21. Pharmaceutical composition according to one of the claims 16 to 19, characterized in that the active ingredient is a is oxidizing agent. 22. Pharmaceutical composition comprising an effective Amount of at least one active ingredient, the expression and / or Function of activators, inhibitors, regulators and / or biological precursors influenced by cation channels, in particular stimulates or inhibits, and optionally one pharmaceutical carrier. 23. Pharmaceutical composition according to claim 22, characterized characterized in that the active ingredient is a polynucleotide which encodes a peptide, in particular a polypeptide, where preferably this peptide expresses and / or functions of Activators, inhibitors, regulators and / or biological precursors influenced by cation channels, in particular stimulated or inhibited. 24. Pharmaceutical composition according to claim 22, characterized characterized in that the active ingredient is a peptide, preferably a Polypeptide, preferably this peptide is the expression and / or function of activators, inhibitors, regulators and / or biological precursors of cation channels, especially stimulates or inhibits. 25. Pharmaceutical composition according to one of the claims 22 to 24, characterized in that the active ingredient is a "small molecular compound ", preferably a" small molecular compound "with a molecular weight (MW) <2,000. 26. Diagnostic kit comprising at least one substance for detection of expression and / or function of cation channels for Detection of susceptibility to diseases associated with a disturbed regulation of apoptosis. 27. Diagnostic kit according to claim 26 for the detection of vulnerabilities against neurodegenerative diseases, acute Kidney failure, thalassemia, sickle cell anemia, glucose-6-phosphate Dehydrogenase deficiency, iron deficiency anemia, immunodeficiency, Tumor diseases and / or infections.