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Treatment of frostbite with the calmodulin antagonists thioridazine and trifluoperazine
Gen. Pharraac. Vol. 20, No. 5, pp. 641-646, 1989 Printed in Great Britain. All rights reserved
0306-3623/89 $3.00+ 0.00 Copyright © 1989PergamonPress pie
TREATMENT OF FROSTBITE WITH THE CALMODULIN ANTAGONISTS THIORIDAZINE A N D TRIFLUOPERAZINE* RIVKA BEITNER,~"MALCA CHEN-ZION, YONIT SOFER-BA.~UKEVITZ, HAYA MORGENSTERN and HANNA BEN-PoP.AT Health Sciences Research Center, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel (Tel. 03-531-8224) (Received 1 December 1988)
Abstract--l. Thioridazine and trifluoperazine, which have been previously found in this laboratory to be the most effective calmodulin antagonists in treatment of burns, are shown here to be also effective in the treatment of frostbite. 2. Electron microscopic studies have revealed a complete reversal of both the vascular and skin tissue damage induced by frostbite. 3. The reversal of the vascular damage was also demonstrated by the ability of these compounds to abolish the increase in hemoglobin content in the skin. 4. The reversal of the skin tissue damage was also revealed by the ability of these compounds to raise the decreased ATP level and the reduced activities of 6-phosphogiuconate dehydrogenase and mitochondrial and soluble bexokinase in skin, induced by frostbite, to normal control levels. INTRODUCTION
METHODS
Previous experiments from our laboratory have revealed that several calmodulin antagonists are effective in treatment of skin burns having both therapeutic and prophylactic action (Beitner et al., 1989). They were shown to be effective when administered as an injectable solution, or when administered topically to the burned skin in the form of a cream. Of all the compounds investigated, the most effective in treatment of burns, were trifluoperazine and thioridazine, which are also known as the more potent calmodulin antagonists (Levin and Weiss, 1976, 1979; Weiss and Levin, 1978; Prozialeck and Weiss, 1982). These results prompted us to investigate whether these two calmodulin antagonists would also be effective in the treatment of frostbite injury. We have studied their effects on the same parameters, which characterize skin injury induced by burns (Beitner et al., 1989), namely, the content of hemoglobin, A T P and the activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase in skin. Here we show that the changes in these parameters induced by frostbite are similar to those induced by burns. The present results also reveal that the changes in all these parameters induced by frostbite can be abolished by topical treatment with thioridazine or trifluoperazine. Electron microscopy of blood capillaries and skin tissue from cold injured rats treated with thioridazine, clearly showed that this compound markedly abolished both the vascular and tissue damage.
Materials Trifluoperazine dihydrochloride, as well as enzymes and coenzymes, were either from Sigma Chemical Co., or from Boehringer Mannheim GmbH. Thioridazine hydrochloride was from Taro Pharmaceutical Co., Inc., Haifa, Israel. Other reagents were of the highest grade available from commercial sources. Induction and treatment of frostbite Charles River albino rats, aged 8-12 days (20-25 g body wt), were used. At this age, the animals are already well developed, but still hairless, thus no shaving is required. For each experiment, three groups of rats were used: (a) controls, which were neither frost-injured nor treated, (b) frost-injured rats, which received no treatment, and (c) frost-injured rats, which were treated topically with either thioridazine or trifluoperazine. Frostbite was induced in the rats under ether anesthesia by applying dry ice for 1 min to the skin in the abdomen area. Thioridazine or trifluoperazine were applied topically to the frost-injured area in the form of a cream (1 g of thiorida,fine mixed with 5 g of "Kamill Gtstichts-crenae-ffir normale Haut", or I g of trifluoperazine mixed with 5 g of "Oil of Olay" beauty fluid---Olay Company, Inc., Wilton), immediately following the induction of frostbite and reapplied 30 rain later and then every hour. After ~ hr from the induction of frostbite, the rats were killed by cervical dislocation and a section of the affected skin was surgically removed. Control rats, with and without frostbite, received the corresponding beauty cream alone. Preparation of the skins for transmission electron microscopy and extraction and assay of ATP, hemoglobin and enzymes, were as described previously (Beimer et al., 1989). R~UL~
*United States Patent No. 4,654,323 and No. 4,777,171. tCorrespondence should be addressed to: Professor Rivka Beitner, Ph.D., Department of Life-Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. 641
Figure 1 shows electron micrograph of blood capillaries in skin from a control not-injured rat (a), cold-injured rat which received no treatment (b) and cold-injured rat treated with thioridazine (c). It can
642
RIVKA BEITNERet al.
~k
Fig. 1. Electron micrograph of blood capillaries in skin from a control rat (a), frost-injured rat. which received no treatment (b) and frost-injured rat treated with thioridazine (c). Conditions for topical treatment of frostbite were as described in "Methods" ( x 8000).
be seen that frostbite induced a marked damage to the blood capillaries (b). We can see iysis of erythrocytes, which lost their colour (hemoglobin) to various degrees. We also see destruction of white blood cells, swollen mitochondria and damage of the endothelial cells. Treatment of the cold-injured rat with thioridazine (c), completely abolished the damage; the blood capillaries look normal like in the control not-injured rat (a). Figure 2 shows electron
micrograph of the skin tissue from a control (a), cold-injured (b) and cold-injured rat treated with thioridazine (c). In the control skin (a), we can see a normal fibroblast, with endoplasmic reticulum full with protein. In the cytoplasm we see many groups of polyribosomes. The nucleus structure is normal. Outside, we can see many groups of organized collagen fibers. Frostbite induced a severe damage of the fibroblast (b). The organization of the collagen fibers
Frostbite and calmodulin antagonists
643
Fig. 2. Electron micrograph of the skin tissue from a control rat (a), frost-injured rat, which received no treatment (b), and frost-injured rat treated with thioridazine (c). Conditions for topical treatment of frostbite were as described in "Methods" (x 6000).
is lost. We can see picnotic nucleus (the volume of the inactive heterochromatin is increased), indicating cell death. At the upper side, we can also see swollen mitochondria. All this damage disappears following treatment with thioridazine (c); we can see that the treated skin looks like the control normal skin. The release of hemoglobin from the cold-altered red cells [Fig. 1(b)], was estimated quantitatively by measuring hemoglobin content in the rat skin. As shown in Fig. 3, frostbite induced a marked increase
in hemoglobin content in the skin. Topical treatment with thioridazine or trifluoperazine (TFP), significantly reduced the elevated hemoglobin content in the frost-injured skin. The experiments demonstrated in Table 1 reveal that frostbite induced a marked decrease in ATP concentration in the rat skin. It can be seen that topical treatment with TFP or thioridazine reversed the reduction in ATP induced by frostbite to normal control levels.
6461
RIVKA BEITNER et al. Table 2. Effect of frostbite on the activity of 6-phosphogluconate dehydrogenase in rat skin and its reversal by topical treatment with thioridazine or trifluoperazine Conditions
~. o-~ @. Q
.i ~
•
az
1f m
.::'~
..: y?~ :'.'::.
6-Phosphogluconate dehydrogenase activity (munit/mg protein)
Control Frostbite TFP
17.73 ± 0.59 (6) 5.67 + 0.24 (6) 15.45 ± 0.41 (6)
Control Frostbite Thioridazine
16.85 ± 0.43 (7) 4.84 ± 0.29 (6) 13.69 ± 0.46 (5)
Conditions for topical treatment of frostbite were as described in "Methods." Values are means ± SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
.I •
0.1
Control Frostbite TFP
Control Frmltbit* Thlm'idozlne
Fig. 3. Increase in hemoglobin content in rat skin induced by frostbite and its reversal by topical treatment with trifluoperazine (TFP) or thioridazine. Conditions for topical treatment of frostbite were as described in "'Methods." Values are means + SE for 5-20 experiments. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005. The results presented in Table 2 show that frostbite induced a significant reduction in the activity of 6-phosphogluconate dehydrogenase in the rat skin to about one-third of the control levels. It can be seen that topical treatment with TFP or thioridazine abolished the decrease in this enzyme's activity, induced by frostbite. As shown in Table 3, frostbite also induced a marked reduction in the activity of soluble and mitochondrial hexokinase in the rat skin. Topical treatment of the frost-injured rats with TFP or thioridazine reversed these changes, also. DISCUSSION
The present results reveal that the changes in skin induced by frostbite are very similar to those induced by burns (Beitner et al., 1989), as judged by the Table I. Effect of frostbite on A T P concentration in rat and skin and its reversal by topical treatment with trifluoperazine or thioridazine Conditions
electron microscopic appearance, as well as by the changes in hemoglobin, A T P and enzymes' activities. Thioridazine or TFP, which were previously found as the most effective calmodulin antagonists in treatment of skin burns (Beitner et al., 1988), were shown here to be also effective in treatment of frostbite. Topical application of these drugs to the frost-injured rats, abolished the decrease in ATP level (Table 1) and in the activities of 6-phosphogluconate dehydrogenase (Table 2) and mitochondrial and soluble hexokinase (Table 3), induced by frostbite in skin. The changes in these biochemical parameters in skin, correlated with the electron microscopic appearance of the skin tissue, which clearly showed the damage induced by frostbite and its reversal by treatment with thioridazine (Fig. 2). The calmodulin antagonists also had a protective action on the blood capillaries and erythrocyte membrane, as revealed by electron microscopy studies (Fig. 1), as well as the abolishment of hemoglobin increase in the skin induced by frostbite (Fig. 3). The present results, which reveal that the damage induced by frostbite is similar to that induced by bums, suggest that the same sequence of events occurs under both conditions. The proposed vicious cycle of events was described by us in detail, previously (Beimer et aL, 1989), and was based on our results obtained from several experimental models, which mimic tissue injury (Beitner et al., 1980, 1982, 1983, 1985; Lilling et al., 1983; Beery et al., 1980; Beitner and Frucht, 1983; Frucht et al., 1984b; Kaplansky and Beitner, 1984). Briefly, the proposed sequence of events in skin burns, frostbite or other skin injuries is as follows: In skin injuries, there is a Table 3. Effect of frostbite on the activity of soluble and mitochondrial hexokinase in rat skin and its reversal by topical treatment with thioridazine or trifluoperazine
ATP (pmol/kg wet wt)
Control Frostbite TFP
1087 --6 63 (8) 348 ± 48 (8) 979 4- 59 (14)
Control Frostbite Thioridaz/ne
1063 4- 27 (6) 376 ± 54 (6) 1003 _+ 49 (5)
Thioridazine or TFP were administered topically in the form of a cream, as described in "Methods.'" Values are means ± SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
Conditions
Mitochondrial hexokinase activity (munit/mg protein)
Soluble hexokinase activity (munit/mg protein)
Control Frostbite TFP
2.93 ± 0.I I (6) r.65 ~_ 0.07 (6) 3.22 ± 0.17 (6)
2.09 4- 0.07 (6) [.00 ± 0.05 (6) 1.99 ± 0.08 (6)
Control Frostbite Thioridazine
2.63 + 0.I I (7) 1.26 ± 0.07 (6) 1.84 ± 0.18 (5)
2.13 ± 0.02 (7) 0.85 ± 0.04 (6) 2.23 + 0.10 (5)
Conditions for topical treatment of frostbite were as described in "Methods." Values are means _+SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
Frostbite and calmodulin antagonists rapid release of bradykinin, serotonin and histamine, which are known to play a pathogenic role in tissue injury and inflammation. These hormones increase the intracellular concentration of Ca 2+, (for reviews see Berridge, 1984, 1985; Berridge and Irvine, 1984), which binds to calmodulin to form the active Ca 2+calmodulin complex. This complex activates the membrane-bound phospholipase A2 (Wong and Cheung, 1979; Moskowitz et al., 1983; Craven and De Rubertis, 1983). The activated phospholipase A 2 acts on the cell membrane phospholipids, causing cell membrane damage. This will lead to leakage of enzymes and may explain the marked decrease in the enzymes' activities in skin induced by frostbite (Tables 2 and 3). The Ca2+-calmodulin complex also activates glucose 1,6-bisphosphatase (Wakelam et al., 1982), which is the enzyme that degrades glucose 1,6-bisphosphate, the potent regulator of carbohydrate metabolism (for reviews see Bcitner, 1979, 1984, 1985). The decrease in the level of this regulator causes a decrease in glycolysis, with the resulting decrease in ATP concentration. The increase in intracellular Ca 2+ may also cause mitochondriai calcium overload, (see Figs 1 and 2), which depresses ATP production further. These mechanisms may account for the marked decrease in ATP level in skin induced by frostbite (Table I). The decrease in ATP also contributes to the destruction of the cell membrane (Weed et al., 1969; Wilkinson and Robinson, 1974; Thomson et al., 1975; R a j s e t al., 1980), which will cause an influx of external Ca 2+, raising further the intracellular Ca 2+ level that promotes tissue damage (Rasmussen and Palmieri, 1985). The calmodulin antagonists may act either by binding to the Ca 2+calmodulin complex and rending it substantially, biologically inert with regard to activation of phospholipase A 2 (Wong and Cheung, 1979; Walenga et al., 1981; Craven and DcRubertis, 1983) and glucose 1,6-bisphosphatase (Wakelam et al., 1982; Frucht et al., 1984a; Beitner et al., 1985), or by some additional or other actions not directly related to calmodulin antagonism. The present experiments suggest that the caimodulin antagonists thioridazine and trifluoperazine may be effective drugs in treatment of frostbite. Caimodulin antagonists have also recently shown promise in the treatment of psoriasis, several tumor types, and aging (for recent review, see Fairley, 1988). Acknowledgements--The authors thank Dr Z. Malik, Head of Electron Microscopy Unit and Mrs J. Hanania for carrying out the electron microscopy studies. We also thank Mrs Diane Radin for typing this manuscript. This work was supported in part by the Research and Development Committee, Bar-Ilan University, and the Health Sciences Research Center, Department of Life Sciences, Bar-Ban University, Ramat-Gan, Israel.
REFERENCES
Beery E., Klein S., Nordenberg J. and Beitncr R. (1980) The influence of Ca2+-ionophore A23187 on glucose-1,6diphosphate and ATP levels, and on the activities of phosphofructokinase and phosphoglucomutase, in isolated rat diaphragm muscle. Biochem. Int. 1, 526-521. Beitner R. (1979) The role of glucose-l,6-bisphosphatc in the regulation of carbohydrate metabolism in muscle. Trends Biochem. Sci. 4, 228-230.
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Beitner R. 0984) Control of levels of glucose 1,6-bisphosphate. Int. J. Biochem. 16, 579-585. Beitner R. 0985) Glucose 1,6-bisphosphate---the regulator of carbohydrate metabolism. In Regulation of Carbohydrate Metabolism, (Edited by Beitner R.) Vol. I, pp. 1-27. CRC Press, Florida. Beitner R. and Frucht H. (1983) Decrease in glucose 1,6-bisphosphate and activation of glucose 1,6-bisphosphatase in rat skin following the injection of phospholipase A2. IRCS Med. Sci. 11, 935-936. Beitner R,, Nordenberg J., Cohen T. J. and Beery E, (1980) The effects of phospholipase A and lysolecithin on glucose-l,6-diphosphate levels and on the activities of glucose-l,6-diphosphate phosphatase, phosphofructokinase and phosphoglucomutase in the isolated rat diaphragm muscle. Int. J. Biochem. !1, 467-472. Beitner R., Kaplansky M. and Frucht H. (1982) Effect of serotonin on glucose-l,6-diphoshate levels in rat muscle and skin. IRCS Med. Sci. 10, 300-301. Beitner R., Frucht H. and Kaplansky M. (1983) Changes in the levels of glucose 1,6-diphosphate and cyclic GMP, and in the activities of phosphofructokinase and phosphoglucomutas¢ induced by serotonin in muscle. Int. J. Biochem. 15, 935-940. Beitner R., Kaplansky M. and Frucht H. (1985) Trifluoperazine abolishes the actions of bradykinin on glucose 1,6-bisphosphate levels and on the activities of glucose 1,6-bisphosphatase, phosphofructokinase and phosphoglucomutase. Int. J. Biochem. 17, 545-550. Beitner R., Chen-Zion M., Sofer-Bassukevitz Y., Oster Y., Ben-Porat H. and Morgenstern H. (1989) Therapeutic and prophylactic treatment of skin burns with several calmodulin antagonists. Gen. Pharmac. 20, 165-173. Berridge M. J. (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345-360. Berridge M. J. (1985) Calcium-mobilizing receptors, membrane phosphoinositides and signal transduction. In Calcium in Biological Systems. (Edited by Rubin R. P., Weiss G. B. and Putney J. W. Jr) pp. 37--44. Plenum Press, New York. Berridge M. J. and h'vine R. F. (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312' 315-321. Craven P. A. and DeRubertis F. R. (1983) Ca 2+ Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. J. biol. Chem. 258, 4814-4823. Fairley J. A. (1988) Calcium and the skin. Arch. Dermatol. 124, 443-444. Frucht H., Kaplansky M. and Beitner R. (1984a) Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine. Biochem. Med. 31, 122-129. Frucht H, Lilling G. and Beimer R. (1984b) Influence of bradykinin on glucose 1,6-bisphosphate and cyclic GMP levels and on the activities of glucose 1,6-bisphosphatase, phosphofructokinase and phosphoglucomutase in muscle. Int. J. Biochem. 16, 397-402. Kaplansky M. and Beitner R. (1984) The antagonistic effect of trifluoperazine to the action of serotonin on glucose 1,6-bisphosphate and ATP levels in rat skin and muscle. IRCS Med. Sci. 12, 174-175. Lilling G., Frucht H., Ben-porat H. and Beitner R. (1983) Changes in glucose 1,6-bisphosphate levels and in the activity of glucose 1,6-bisphosphatase induced by bradykinin in rat skin. IRCS Med. Sci. 11, 130-131. Levin R. M. and Weiss B. (1976) Mechanism by which psychotropic drugs inhibit adenosine cyclic Y,5'monophosphate phosphodiesterase of brain. Molec. Pharmac. 12, 581-589.
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Levin R. M. and Weiss B. (1979) Selective binding of antipsychotics and other psychoactive agents to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. J. Pharmac. exp. Ther. 208, 454-459. Moskowitz N., Shapiro L., Shook W. and Puszkin S. (1983) Phospholipase A 2 modulation by calmodulin, prostaglandins and cyclic nucleotides. Biochem. hiophys. Res. Commun. 115, 94-99. Prozialeck W. C. and Weiss B. (1982) Inhibition of calmodulin by phenothiazines and related drugs: structure-activity relationships. J. Pharmac. exp. Ther. 222, 509 516. Rajs J., Sundberg M., H/irm T., Grandinsson M. and S6ndelund U. (1980) Interrelationships between ATP levels, enzyme leakage, gluconate uptake, trypan blue exclusion and length/width ratio of isolated rat cardiac myocytes subjected to anoxia and reoxygenation. J. Molec. cell. Cardiol. 12, 1227-1238. Rasmussen H. and Palmieri G. M. A. (1985) Altered cell calcium metabolism and human diseases. In Calcium in Biological Systems (Edited by Rubin R. P., Weiss G. B. and Putney R. W. Jr), pp. 551-560. Plenum Press, New York. Thomson W. H. S.., Sweetin J. C. and Hamilton I. J. D.
(1975) ATP and muscle enzyme efflux after physical exertion. Clin. Chim. Acta 59, 241245. Wakelam M. J. O., Emmerich M. and Pette D. (1982) The control of glucose-l,6-bisphosphatase by Ca 2÷ and calmodulin. Biochem. J. 208, 517-519. Walenga R. W., Opas E. E. and Feinstein M. B. (1981) Differential effects ofcalmodulin antagonists on phospholipases A 2 and C in thrombin-stimulated platelets. J. biol. Chem. 256, 12523-12528. Weed R. I., Lacelle P. L. and Merrill E. W. (1969) Metabolic dependence of red cell deformability. J. clin. Ira,est. 48, 795 809. Weiss B. and Levin R. M. (1978) Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsycotic agents. In Adt'ances in Cyclic Nucleotide Research (Edited by George W. J. and Ignarro L. J.), Vol. 9, pp. 285-303. Raven Press, New York. Wilkinson J. H. and Robinson J. M. (1974) Effect of ATP on release of intracellular enzymes from damages cells. Nature 249, 662~:~63. Wong, P. Y.-K. and Cheung W. Y. (1979) Calmodulin stimulates human platelet phospholipase A 2. Biochem. biophys. Res. Commun. 90, 473-480.
0306-3623/89 $3.00+ 0.00 Copyright © 1989PergamonPress pie
TREATMENT OF FROSTBITE WITH THE CALMODULIN ANTAGONISTS THIORIDAZINE A N D TRIFLUOPERAZINE* RIVKA BEITNER,~"MALCA CHEN-ZION, YONIT SOFER-BA.~UKEVITZ, HAYA MORGENSTERN and HANNA BEN-PoP.AT Health Sciences Research Center, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel (Tel. 03-531-8224) (Received 1 December 1988)
Abstract--l. Thioridazine and trifluoperazine, which have been previously found in this laboratory to be the most effective calmodulin antagonists in treatment of burns, are shown here to be also effective in the treatment of frostbite. 2. Electron microscopic studies have revealed a complete reversal of both the vascular and skin tissue damage induced by frostbite. 3. The reversal of the vascular damage was also demonstrated by the ability of these compounds to abolish the increase in hemoglobin content in the skin. 4. The reversal of the skin tissue damage was also revealed by the ability of these compounds to raise the decreased ATP level and the reduced activities of 6-phosphogiuconate dehydrogenase and mitochondrial and soluble bexokinase in skin, induced by frostbite, to normal control levels. INTRODUCTION
METHODS
Previous experiments from our laboratory have revealed that several calmodulin antagonists are effective in treatment of skin burns having both therapeutic and prophylactic action (Beitner et al., 1989). They were shown to be effective when administered as an injectable solution, or when administered topically to the burned skin in the form of a cream. Of all the compounds investigated, the most effective in treatment of burns, were trifluoperazine and thioridazine, which are also known as the more potent calmodulin antagonists (Levin and Weiss, 1976, 1979; Weiss and Levin, 1978; Prozialeck and Weiss, 1982). These results prompted us to investigate whether these two calmodulin antagonists would also be effective in the treatment of frostbite injury. We have studied their effects on the same parameters, which characterize skin injury induced by burns (Beitner et al., 1989), namely, the content of hemoglobin, A T P and the activities of 6-phosphogluconate dehydrogenase and mitochondrial and soluble hexokinase in skin. Here we show that the changes in these parameters induced by frostbite are similar to those induced by burns. The present results also reveal that the changes in all these parameters induced by frostbite can be abolished by topical treatment with thioridazine or trifluoperazine. Electron microscopy of blood capillaries and skin tissue from cold injured rats treated with thioridazine, clearly showed that this compound markedly abolished both the vascular and tissue damage.
Materials Trifluoperazine dihydrochloride, as well as enzymes and coenzymes, were either from Sigma Chemical Co., or from Boehringer Mannheim GmbH. Thioridazine hydrochloride was from Taro Pharmaceutical Co., Inc., Haifa, Israel. Other reagents were of the highest grade available from commercial sources. Induction and treatment of frostbite Charles River albino rats, aged 8-12 days (20-25 g body wt), were used. At this age, the animals are already well developed, but still hairless, thus no shaving is required. For each experiment, three groups of rats were used: (a) controls, which were neither frost-injured nor treated, (b) frost-injured rats, which received no treatment, and (c) frost-injured rats, which were treated topically with either thioridazine or trifluoperazine. Frostbite was induced in the rats under ether anesthesia by applying dry ice for 1 min to the skin in the abdomen area. Thioridazine or trifluoperazine were applied topically to the frost-injured area in the form of a cream (1 g of thiorida,fine mixed with 5 g of "Kamill Gtstichts-crenae-ffir normale Haut", or I g of trifluoperazine mixed with 5 g of "Oil of Olay" beauty fluid---Olay Company, Inc., Wilton), immediately following the induction of frostbite and reapplied 30 rain later and then every hour. After ~ hr from the induction of frostbite, the rats were killed by cervical dislocation and a section of the affected skin was surgically removed. Control rats, with and without frostbite, received the corresponding beauty cream alone. Preparation of the skins for transmission electron microscopy and extraction and assay of ATP, hemoglobin and enzymes, were as described previously (Beimer et al., 1989). R~UL~
*United States Patent No. 4,654,323 and No. 4,777,171. tCorrespondence should be addressed to: Professor Rivka Beitner, Ph.D., Department of Life-Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. 641
Figure 1 shows electron micrograph of blood capillaries in skin from a control not-injured rat (a), cold-injured rat which received no treatment (b) and cold-injured rat treated with thioridazine (c). It can
642
RIVKA BEITNERet al.
~k
Fig. 1. Electron micrograph of blood capillaries in skin from a control rat (a), frost-injured rat. which received no treatment (b) and frost-injured rat treated with thioridazine (c). Conditions for topical treatment of frostbite were as described in "Methods" ( x 8000).
be seen that frostbite induced a marked damage to the blood capillaries (b). We can see iysis of erythrocytes, which lost their colour (hemoglobin) to various degrees. We also see destruction of white blood cells, swollen mitochondria and damage of the endothelial cells. Treatment of the cold-injured rat with thioridazine (c), completely abolished the damage; the blood capillaries look normal like in the control not-injured rat (a). Figure 2 shows electron
micrograph of the skin tissue from a control (a), cold-injured (b) and cold-injured rat treated with thioridazine (c). In the control skin (a), we can see a normal fibroblast, with endoplasmic reticulum full with protein. In the cytoplasm we see many groups of polyribosomes. The nucleus structure is normal. Outside, we can see many groups of organized collagen fibers. Frostbite induced a severe damage of the fibroblast (b). The organization of the collagen fibers
Frostbite and calmodulin antagonists
643
Fig. 2. Electron micrograph of the skin tissue from a control rat (a), frost-injured rat, which received no treatment (b), and frost-injured rat treated with thioridazine (c). Conditions for topical treatment of frostbite were as described in "Methods" (x 6000).
is lost. We can see picnotic nucleus (the volume of the inactive heterochromatin is increased), indicating cell death. At the upper side, we can also see swollen mitochondria. All this damage disappears following treatment with thioridazine (c); we can see that the treated skin looks like the control normal skin. The release of hemoglobin from the cold-altered red cells [Fig. 1(b)], was estimated quantitatively by measuring hemoglobin content in the rat skin. As shown in Fig. 3, frostbite induced a marked increase
in hemoglobin content in the skin. Topical treatment with thioridazine or trifluoperazine (TFP), significantly reduced the elevated hemoglobin content in the frost-injured skin. The experiments demonstrated in Table 1 reveal that frostbite induced a marked decrease in ATP concentration in the rat skin. It can be seen that topical treatment with TFP or thioridazine reversed the reduction in ATP induced by frostbite to normal control levels.
6461
RIVKA BEITNER et al. Table 2. Effect of frostbite on the activity of 6-phosphogluconate dehydrogenase in rat skin and its reversal by topical treatment with thioridazine or trifluoperazine Conditions
~. o-~ @. Q
.i ~
•
az
1f m
.::'~
..: y?~ :'.'::.
6-Phosphogluconate dehydrogenase activity (munit/mg protein)
Control Frostbite TFP
17.73 ± 0.59 (6) 5.67 + 0.24 (6) 15.45 ± 0.41 (6)
Control Frostbite Thioridazine
16.85 ± 0.43 (7) 4.84 ± 0.29 (6) 13.69 ± 0.46 (5)
Conditions for topical treatment of frostbite were as described in "Methods." Values are means ± SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
.I •
0.1
Control Frostbite TFP
Control Frmltbit* Thlm'idozlne
Fig. 3. Increase in hemoglobin content in rat skin induced by frostbite and its reversal by topical treatment with trifluoperazine (TFP) or thioridazine. Conditions for topical treatment of frostbite were as described in "'Methods." Values are means + SE for 5-20 experiments. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005. The results presented in Table 2 show that frostbite induced a significant reduction in the activity of 6-phosphogluconate dehydrogenase in the rat skin to about one-third of the control levels. It can be seen that topical treatment with TFP or thioridazine abolished the decrease in this enzyme's activity, induced by frostbite. As shown in Table 3, frostbite also induced a marked reduction in the activity of soluble and mitochondrial hexokinase in the rat skin. Topical treatment of the frost-injured rats with TFP or thioridazine reversed these changes, also. DISCUSSION
The present results reveal that the changes in skin induced by frostbite are very similar to those induced by burns (Beitner et al., 1989), as judged by the Table I. Effect of frostbite on A T P concentration in rat and skin and its reversal by topical treatment with trifluoperazine or thioridazine Conditions
electron microscopic appearance, as well as by the changes in hemoglobin, A T P and enzymes' activities. Thioridazine or TFP, which were previously found as the most effective calmodulin antagonists in treatment of skin burns (Beitner et al., 1988), were shown here to be also effective in treatment of frostbite. Topical application of these drugs to the frost-injured rats, abolished the decrease in ATP level (Table 1) and in the activities of 6-phosphogluconate dehydrogenase (Table 2) and mitochondrial and soluble hexokinase (Table 3), induced by frostbite in skin. The changes in these biochemical parameters in skin, correlated with the electron microscopic appearance of the skin tissue, which clearly showed the damage induced by frostbite and its reversal by treatment with thioridazine (Fig. 2). The calmodulin antagonists also had a protective action on the blood capillaries and erythrocyte membrane, as revealed by electron microscopy studies (Fig. 1), as well as the abolishment of hemoglobin increase in the skin induced by frostbite (Fig. 3). The present results, which reveal that the damage induced by frostbite is similar to that induced by bums, suggest that the same sequence of events occurs under both conditions. The proposed vicious cycle of events was described by us in detail, previously (Beimer et aL, 1989), and was based on our results obtained from several experimental models, which mimic tissue injury (Beitner et al., 1980, 1982, 1983, 1985; Lilling et al., 1983; Beery et al., 1980; Beitner and Frucht, 1983; Frucht et al., 1984b; Kaplansky and Beitner, 1984). Briefly, the proposed sequence of events in skin burns, frostbite or other skin injuries is as follows: In skin injuries, there is a Table 3. Effect of frostbite on the activity of soluble and mitochondrial hexokinase in rat skin and its reversal by topical treatment with thioridazine or trifluoperazine
ATP (pmol/kg wet wt)
Control Frostbite TFP
1087 --6 63 (8) 348 ± 48 (8) 979 4- 59 (14)
Control Frostbite Thioridaz/ne
1063 4- 27 (6) 376 ± 54 (6) 1003 _+ 49 (5)
Thioridazine or TFP were administered topically in the form of a cream, as described in "Methods.'" Values are means ± SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
Conditions
Mitochondrial hexokinase activity (munit/mg protein)
Soluble hexokinase activity (munit/mg protein)
Control Frostbite TFP
2.93 ± 0.I I (6) r.65 ~_ 0.07 (6) 3.22 ± 0.17 (6)
2.09 4- 0.07 (6) [.00 ± 0.05 (6) 1.99 ± 0.08 (6)
Control Frostbite Thioridazine
2.63 + 0.I I (7) 1.26 ± 0.07 (6) 1.84 ± 0.18 (5)
2.13 ± 0.02 (7) 0.85 ± 0.04 (6) 2.23 + 0.10 (5)
Conditions for topical treatment of frostbite were as described in "Methods." Values are means _+SE, with the number of experiments in parentheses. P-values (frostbite vs control or calmodulin-antagonist vs frostbite) <0.005.
Frostbite and calmodulin antagonists rapid release of bradykinin, serotonin and histamine, which are known to play a pathogenic role in tissue injury and inflammation. These hormones increase the intracellular concentration of Ca 2+, (for reviews see Berridge, 1984, 1985; Berridge and Irvine, 1984), which binds to calmodulin to form the active Ca 2+calmodulin complex. This complex activates the membrane-bound phospholipase A2 (Wong and Cheung, 1979; Moskowitz et al., 1983; Craven and De Rubertis, 1983). The activated phospholipase A 2 acts on the cell membrane phospholipids, causing cell membrane damage. This will lead to leakage of enzymes and may explain the marked decrease in the enzymes' activities in skin induced by frostbite (Tables 2 and 3). The Ca2+-calmodulin complex also activates glucose 1,6-bisphosphatase (Wakelam et al., 1982), which is the enzyme that degrades glucose 1,6-bisphosphate, the potent regulator of carbohydrate metabolism (for reviews see Bcitner, 1979, 1984, 1985). The decrease in the level of this regulator causes a decrease in glycolysis, with the resulting decrease in ATP concentration. The increase in intracellular Ca 2+ may also cause mitochondriai calcium overload, (see Figs 1 and 2), which depresses ATP production further. These mechanisms may account for the marked decrease in ATP level in skin induced by frostbite (Table I). The decrease in ATP also contributes to the destruction of the cell membrane (Weed et al., 1969; Wilkinson and Robinson, 1974; Thomson et al., 1975; R a j s e t al., 1980), which will cause an influx of external Ca 2+, raising further the intracellular Ca 2+ level that promotes tissue damage (Rasmussen and Palmieri, 1985). The calmodulin antagonists may act either by binding to the Ca 2+calmodulin complex and rending it substantially, biologically inert with regard to activation of phospholipase A 2 (Wong and Cheung, 1979; Walenga et al., 1981; Craven and DcRubertis, 1983) and glucose 1,6-bisphosphatase (Wakelam et al., 1982; Frucht et al., 1984a; Beitner et al., 1985), or by some additional or other actions not directly related to calmodulin antagonism. The present experiments suggest that the caimodulin antagonists thioridazine and trifluoperazine may be effective drugs in treatment of frostbite. Caimodulin antagonists have also recently shown promise in the treatment of psoriasis, several tumor types, and aging (for recent review, see Fairley, 1988). Acknowledgements--The authors thank Dr Z. Malik, Head of Electron Microscopy Unit and Mrs J. Hanania for carrying out the electron microscopy studies. We also thank Mrs Diane Radin for typing this manuscript. This work was supported in part by the Research and Development Committee, Bar-Ilan University, and the Health Sciences Research Center, Department of Life Sciences, Bar-Ban University, Ramat-Gan, Israel.
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