the present invention involves a method for reducing the uptake of calcium by mammalian cells which are under the influence of a noxious stimuli . the type of noxious stimuli which create calcium uptake include , for example , metabolic inhibition , anoxia , ischemia and free radical attack . the following detailed description will be limited to reducing calcium uptake in cells subjected to metabolic inhibition or free radical attack with it being understood by those skilled in the art that the method has wide application to any situation where a cell is undergoing calcium uptake or influx in response to a noxious stimulus . the method in accordance with the present invention requires that the cell surface be treated with a sufficient amount of a cationic agent to alter the surface charge of the cell to reduce or prevent the uptake of calcium caused by the noxious stimuli . the amount of cationic agent which is used will vary depending upon the particular cationic agent , the particular cell type , the type of noxious stimulus to which the cell is being exposed , and the particular treatment process , ie . in vivo or in vitro treatment . the specific amount can be easily determined experimentally for each different situation . some general guidelines with respect to amounts of cationic agents will be given below in connection with the description of specific examples . the mammalian cell types which are amenable to treatment by the present invention include any mammalian cell which undergoes calcium uptake when exposed to noxious stimuli . the invention is especially well - suited for treating cells , such as muscle cells , which have a sarcolemma . exemplary cells include cardiac myocytes , in which the uptake has been directly demonstrated . other cells which have been demonstrated to have calcium leak channels would be expected to respond similarly , e . g . dystrophic skeletal muscle ( 12 ). the cationic agents which are suitable include organic cations such as protamine , dodecyltrimethylammonium bromide ( ddtma ), polymyxin b sulfate and polybrene . the greater the cationic charge , the more effective the agent . it is preferred that the cation carry as large a positive charge as possible without causing damage to the cell surface or otherwise disrupting the cell environment , especially when treatment is conducted in vivo . organic cations with relatively small positive charge are not preferred . gentamicin and other aminoglycoside antibiotics are not preferred because of their relatively low cationic charge . it was found that relatively large amounts ( 1 . 5 mm ) of these types of cationic agents were required in order to obtain any significant levels of calcium uptake inhibition . the procedure for exposing the cell surface to the cationic agent can vary widely provided that a sufficient amount of cationic agent is present to alter the cell surface sufficiently to reduce the uptake of calcium . for in vitro situations , the cationic agent is simply added to the cell culture . the concentration of cationic agent in the cell culture preferably will range from 5 μm to 50 μm for strong cationic agents . for example , cell culture concentrations of protamine at levels of 5 μm and above were found to completely prevent calcium overload . for in vivo situations , the cationic agent is preferably added to a pharmaceutically acceptable solution which is administered to the mammal . the cationic solution is preferably administered intravenously . the concentration of cationic agent in the pharmaceutically acceptable solution is chosen so that the ultimate concentration of cationic agent in the environment immediately surrounding the cell will be at least 5 μm . it is preferred that the minimum amount of cationic agent be utilized which is effective in eliminating calcium uptake so that any adverse effects on other cells in the body are minimized . suitable pharmaceutically acceptable solutions include saline or glucose solutions . the concentration of cationic agent in the pharmaceutically acceptable solution will range from about 5 μm to about 50 μm . the following example demonstrates the use of protamine to prevent the uptake of calcium by neonatal rat cardiomyocytes exposed to hydrogen peroxide . protamine is a strong polycationic polypeptide used extensively for heparin reversal after cardiopulmonary bypass . the drug is known to be negatively inotropic . the neonatal rat cardiomyocytes were cultured according to the procedure described by frank et al . ( 13 ). ca fluxes were measured by an on - line isotopic technique ( 13 ). the myocytes were cultured onto scintillant - containing discs which were mounted in a flow cell so that the cells were in direct contact with perfusate . the flow cell was placed between two opposing spectrophotometer tubes . cell - associated ca was then recorded in counts per minute . 45 ca 2 + is a low energy beta - emitter . the radioactivity contained within the perfusate is counted with very low efficiency (& lt ; 5 %) whereas that within the cells is counted with an efficiency of 39 %. in all of the examples the cells were initially perfused with non isotopic solution to remove any debris . perfusion with 45 ca 2 + containing solution was then commenced at 10 ml / minute and continued until the cells were labelled to asymptote . this normally took 25 - 30 minutes . the flow cell was then drained and refilled with a solution containing hydrogen peroxide . the cells were then incubated for 60 minutes with continuous recording of 45 ca labeling ( no flow ) prior to washout with non - isotopic perfusate . after asymptotic labeling with 45 ca , the cells were perfused with a solution containing protamine sulfate at varying concentrations . this did not significantly alter ca uptake . the flow cell was then drained and refilled with a solution containing 50 μm h 2 o 2 and protamine . the procedure was repeated with protamine in varying concentrations : 1 μm ( n = 4 ); 5 μm ( n = 6 ) and 50 μm ( n = 6 ). at the end of each experiment the discs were removed from the flow cell , allowed to dry and the cells were scraped onto a pre - weighed piece of filter paper . this was placed in an oven (& gt ; 100 ° c .) overnight and then weighed , permitting determination of the cellular dry weight . the specific activity of each isotopic solution was measured in a spectrophotometer ( beckman instruments ) and cell - associated ca was expressed in mmoles / kg dry cellular weight . the cell cultures were maintained at 37 ° c . at a concentration of 1 μm , protamine had no effect . at 5 μm , protamine completely prevented the ca overload . increasing the concentration above 5 μm had no additional effect . the results of the test for a protamine concentration of 5 μm is shown in fig1 . fig2 shows the test results for various concentrations of protamine . the effect of protamine , as described in this example was completely inhibited by neutralizing its positive charge with heparin . it is believed that the agent must have a positive charge which is sufficient to alter surface charge of the outer sarcolemmal leaflet . this penetration is necessary in order for the cation to exert its effect at the cell surface . the following example demonstrates the use of protamine to prevent the uptake of calcium by neonatal rat cardiomyocytes which are metabolically inhibited . exposure of neonatal rat cardiomycytes to 0 . 05 mm iodoacetic acid ( iaa ), as a glycolytic inhibitor , and 0 . 5 mm 2 - deoxyglucose ( 2 - dog ), as a non - metabolizable substrate , produces a graded depletion in intracellular atp and induces a significant ca leak . in the following example involving metabolic inhibition , these two compounds were included in the incubation medium . 2 - dog was added directly and iaa was added from a 1 m stock solution , dissolved in water . the concentration in the culture media was 0 . 5 mm for 2 - dog and 0 . 05 mm for iaa . the metabolically inhibited cells were treated with protamine in the same manner as example 1 . the results were similar to example 1 , i . e . protamine did not exert a significant effect at concentrations below 5 μm . at concentrations of 5 μm and higher , protamine provided a dramatic drop in calcium uptake . the results of this example are summarized and shown in fig3 . the following example demonstrates the use of polybrene ( hexamedronium bromide ) to prevent the uptake of calcium by neonatal rat cardiomyocytes which have been exposed to hydrogen peroxide or are metabolically inhibited . polybrene is a synthetic polycationic compound which is commercially available . the magnitude of the positive charge borne by the molecule is less than that of protamine . the same procedures were followed as in examples 1 and 2 , except that polybrene was substituted for protamine . polybrene also inhibited the calcium leak caused by either hydrogen peroxide or metabolic inhibition . the threshold concentration above which inhibition was significant was 25 μm . the results of the tests for the polybrene / hydrogen peroxide combination are summarized and displayed in fig4 . the result for the polybrene / metabolic inhibition combination are shown in fig5 . in this example , dodecyltrimethylammonium bromide ( ddtma ) was used as the cationic agent . the ddtma was tested in the same manner as the preceding examples wherein neonatal rat cardiomyocytes were subjected to metabolic inhibition using iodoacetic acid and 2 - deoxyglucose . the concentration of ddtma was 50 μm . the ddtma provided a significant inhibition of calcium uptake ( 14 ). in this example , polymyxin b sulfate ( pxb ) was used as the cationic agent . the pxb was tested in the same manner as example 4 . the concentration of pxb was 100 μm . in all cases , calcium uptake was substantially inhibited ( 14 ). the cationic agents of the present invention are particularly useful in those clinical situations where the heart has been deprived of its blood supply ( ischemia ) for variable periods of time and then resupply ( reperfusion ) is instituted . such conditions are typical of acute coronary occlusion treated by dissolution of the occluding clot by various enzymes , coronary occlusion treated by coronary angioplasty or reperfusion after interruption of coronary blood flow during cardiac surgical procedures . all such situations have a high risk of producing cellular calcium overload during the reperfusion period . the overload frequently produces cell death and , if sufficient numbers of cells are involved , death of the patient . in the above situations , the cationic agents are added directly to the reperfusion solution at a concentration found to be effective in preventing calcium overload under in vitro conditions , i . e . 5 μm for the most effective cations . at these concentrations the negative inotropic effect will be minimal and , when present , will be rapidly reversed when the cationic agent is discontinued . prevention of calcium overload in accordance with the present invention will increase the chance of cardiac cell survival in response to reperfusion . such survival is important since heart muscle is incapable of regeneration and non - reversible cell damage leads to heart failure and / or death . having thus described exemplary embodiments of the present invention , it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives , adaptations and modifications may be made within the scope of the present invention . accordingly , the present invention is not limited to the specific embodiments as illustrated herein , but is only limited by the following claims . 1 . farber j l : calcium dependence of toxic cell death : a final common pathway . science 1979 ; 206 : 700 - 702 . 2 . bourdillon , p d v ; poole - wilson , p a , ( 1981 ) effects of ischaemia and reperfusion on calcium exchange and mechanical function and mechanical function in isolated rabbit myocardium . cardiovasc res ; 15 : 121 - 130 . 3 . nayler , w g ; grau , a ; slade ( 1976 ) a protective effect of verapamil on hypoxic heart muscle . cardiovasc res ; 10 : 650 - 622 . 4 . allen , d g ; orchard , c h ( 1983 ) intracellular calcium concentration during hypoxia and metabolic inhibition in mammalian ventricular muscle . j . physiol ( london ); 339 : 107 - 122 . 4 . 5 . chien , k r ; engler , r ( 1990 ) superoxide dismutase enhances recovery following myocardial ischemia . am j physiol ; 248 : h637 - h643 . 6 . farber , j l ( 1979 ) calcium dependence of toxic cell death : a final common pathway . science ; 206 : 700 - 702 . 7 . tani , m ( 1990 ) mechanisms of ca 2 + overload in reperfused ischemic myocardium . annu rev physiol ; 52 : 543 - 559 . 8 . knopf , h ; theising , r ; moon , c h ; hirche , h j ( 1990 ) continuous determination of extracellular space and changes of k + , na + , ca 2 + , h + during global ischaemia in isolated rat hearts . j . mol cell cardiol ; 22 : 1259 - 1272 . 9 . ziegelstein , r c ; sweier , j l ; mellits , e d , younes , a ; lakatta , e g ; stern , m d ; silverman , h s ( 1992 ) dimethylthiourea , an oxygen radical scavenger protects isolated cardiac myocytes from hypoxic injury by inhibition of na - ca exchange and not by its antioxidant effects . circ res ; 70 : 804 - 811 . 10 . coulombe a ; lefevre i a ; baro i ; coraboeuf e : barium - and calcium permeable channels open at negative membrane potentials in rat ventricular myocytes . j membr biol 1989 ; 111 : 57 - 67 . 11 . benham c d , tsien r w : a novel receptor - operated ca 2 + - permeable channel activated by atp in smooth muscle . nature 1987 ; 328 : 275 - 278 . 12 . fong p , turner p r , denetclaw w f , steinhardt r a : increased activity of calcium leak channels in myotubes of duchenne human and mdx mouse origin . science 1990 : 250 : 673 - 676 . 13 . frank , j s ; langer , g a ; nudd , l m and seraydarian , k . the myocardial cell surface , its histochemistry and the effects of sialic acid and calcium removal on its structure and cellular ionic exchange . circ . res . 41 : 702 - 714 , 1977 . 14 . clague j . r . et al . : cationic amphiphiles prevent calcium leak induced by atp depletion in myocardial cells . circulation research , vol 72 , no . 1 , january 1993 .
a method for reducing calcium overload in cells to prevent the transition from reversible to irreversible injury . the cell surface is treated with an organic polycation selected from the group consisting of protamine , dodecyltrimethylammonium bromide , polymyxin b sulfate and polybrene which is capable of altering the cells surface charge sufficiently to eliminate or reduce the influx of calcium resulting from exposure to noxious stimuli . the method may be used both in vivo and in vitro . the method is effective in preventing calcium uptake in a variety of cell injury situations including metabolic inhibition and free radical attack .