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Alterations in prostaglandin catabolism in rats chronically dosed with ethanol
BIOCHEMICAL
21, 246-252 (1979)
MEDICINE
Alterations
SAM
Department
in Prostaglandin Catabolism Chronically Dosed with Ethanol
N. PENNINGTON,
CARLTON P. SMITH, JOHN B. STRIDER, JR.
of Biochemistry, School of Medicine, Greenville, North Carolina
East 27834
in Rats
JR., AND
Carolina
University.
Received August 15, 1978
The prostaglandins (PGs) have been the subject of intensive investigation because of their wide distribution and marked biological activities. These cyclic, oxygenated, fatty acid derivatives have diverse effects at low concentrations in a multitude of biological systems and have had widespread clinical application in studies related to gastric secretion, hypertension, asthma, induction of labor, and contraception. At the molecular level, the PGs have been shown to influence, and to be influenced by, adenyl cyclase and appear to play a role in this control mechanism. Chemically, the prostaglandins and related compounds are synthesized from polyunsaturated fatty acids, e.g., arachidonic acid, via two common intermediate endoperoxides. Recent research has demonstrated that the intermediate endoperoxides (PGG, and PGH,) as well as thromboxane A, (TXA,) and prostacyclin (PGI,) are biologically active and suggests that these compounds may be of major importance in tissues such as blood vessels and platelets. The possibility that the physiological action of ethanol is linked in part to that of the prostaglandins is suggested by the fact that the sites of action of the two agents are similar: i.e., they both alter cardiovascular, hepatic, and gastrointestinal activities. Additionally, ethanol and prostaglandins have been shown to influence blood pressure. Since 1969 our laboratory has been investigating the effects of ethanol on arachidonic acid oxidative metabolism. Several laboratories, including our own, have demonstrated that chronic ethanol consumption, even in the presence of an adequate diet, reduces normal stores of arachidonic acid, the precursor of the prostaglandins, prostacyclin and thromboxanes. 246 oooG2944i79/03w&o7$o2.w0 Copyright @ 1979 by Academic Press, Inc. AU tights of reproduction in any form reserved.
CHANGES
IN
PROSTAGLANDIN
CATABOLISM
247
Accounting for the generally poor nutritional state of alcoholics, we suggest that the metabolism of prostaglandins, thromboxanes, and/or prostacyclin may be altered significantly in these individuals. This conclusion has been supported by results of recent studies in both man and experimental animals. Karppanen and Puurunen (1) observed that a 10% ethanol solution blocked gastric acid secretion in rats and that a prostaglandin synthesis inhibitor (indomethacin) blocked this effect. These data suggest that prostaglandin synthesis is stimulated by ethanol, an effect which may influence gastric ulcer formation. Similar effects of ethanol in man have been reported (2). Collier et al. (3) found that ethanol at high levels stimulates prostaglandin synthesis by bull seminal vesicles. Edgarian and Altura (4) reported a differential effect of ethanol on the response of arterial and venous smooth muscle to PGF%. These results all indicate that ethanol influences prostaglandin levels, generally by stimulation of prostaglandin synthesis. However, no studies of the effect of ethanol on prostaglandin degradation have been described. The biological degradation of prostaglandins, thromboxane AZ, and prostacyclin has been described. Thromboxane A, and prostacyclin are degraded rapidly in aqueous solutions. The prostaglandins, on the other hand, are more stable but can be degraded enzymatically. The results to be presented here suggest that the enzymatic degradation of certain prostaglandins is drastically decreased by chronic ethanol consumption. In these experiments, adult male animals (Holtzman rats) were chronically exposed to ethanol via a liquid diet. At the end of 5 weeks various tissues homogenates from these animals were assayed for levels of prostaglandin- 1Wzydroxy dehydrogenase and 15keto- 13,lQprostaglandin reductase. The results indicated that chronic ethanol consumption markedly inhibited prostaglandin catabolism even in the presence of appropriate amounts of oxidized cofactors. MATERIALS
AND METHODS
Animals and ethanol administration. Male Holtzman rats weighing 150-200 g at the start of the experiment were used. Ethanol in the form of a liquid diet (Lieber diet) was administered for 5 weeks and control animals received an isocaloric liquid diet with maltose-dextran substituted for the ethanol. Control animals were adjusted daily for the consumption of diet by matched experimental animal in the previous 24 hr. The diet mixtures were purchased from Bio-Serv Corporation. At the conclusion of the dosing period, the control animals weighed 330.7 + 16.2 g and the experimental animals weighed 234.8 2 22.4 g. Assay of prostaglandin degradation. Tissue assays were run in duplicate using total tissue homogenates. After depriving experimental animals of ethanol for 12 hr, all animals were sacrificed by decapitation and tissue
248
PENNINGTON,SMITH,
AND STRIDER
from lung and kidney collected as indicated. The tissue was rapidly excised and placed in ice-cold KC1 (1.15%) solution. After washing and mincing, the tissue was homogenized in ice-cold phosphate buffer, 0.05 M, pH 7.4. The homogenates were filtered through cheese cloth to remove debris and used immediately for the various assays. The catabolism of 14C-labeled prostaglandins (PGE, and PGF,,) was assayed according to the method of Pace-Asciak and Rangaraj (5,6) as modified in our lab. This modification combined both the NAD+- and NADP+-requiring enzymes and used diethyl ether to extract the products rather than an ethanol precipitation of protein. Following extraction of the reaction mixture, the ether extract was dried under N, and the residue redissolved in chloroform-methanol (1: 1). The samples were then spotted on silica gel H plates (0.25 mm) and developed in dioxane-benzeneacetic acid (50:50:2.5). The tic plates were dried in air and scanned by a thin-layer scanner (Berthold). Radioactive spots were identified by comparison with known standards run on the same plate and were scraped and quantitated by liquid scintillation counting (Beckman Model LS-233). This procedure gave good recoveries, e.g., several hundred assays for PGEz recovery gave a value of 80.0 +- 5.5%. This procedure worked well with minor modification for all tissue homogenates. The purity of the commercial 14C-labeled prostaglandins was checked by chromatography and found to be ~99% in all cases. Chemicals. NAD+ and NADP+ were purchased from Sigma Chemical Company. Radioactive prostaglandins were from New England Nuclear and Amersham Searle Corporation. All inorganic chemicals and organic solvents were purchased from Fisher Scientific. Solvents were redistilled and stored in dark bottles. RESULTS
AND DISCUSSION
Initial experiments were carried out to determine the appropriate parameters for the assay of each tissue with respect to time of incubation, amount of enzyme (protein), and cofactor concentration. As shown in Fig. 1, considerable variation exists with respect to reaction time for each tissue but in all cases assay conditions were obtained that yielded linear reaction rates relative to protein (enzyme) concentration. Overall, rat kidney contained the highest activity with respect to the catabolism of both PGE, and PGF,,. Chronic exposure to ethanol significantly lowered both the specific activity and activity per gram tissue in rat kidney for the formation of 15-keto-PGE, and 15keto-PGF, (Fig. 2). Under the assay conditions employed (excess NAD+ and NADP+ present, ethanol absent), rat kidney formed significant amounts of 15-ketoPGE, when PGFzu served as a substrate (Fig. 3). The formation of 15keto-PGE, was decreased in the same molar ratio in ethanol-treated
CHANGES
IN PROSTAGLANDIN
L
I
0.5
Relative
I
L
1
1.0
I.5
2.0
Enzyme
249
CATABOLISM
I
2.5
3.0
Concentration
FIG. 1. Relative enzyme (protein) curves used to determine assay conditions for various tissues. Lung catabolism of PGE, incubated 15 min. (0). lung catabolism of PGFp, incubated 1 hr (x), kidney catabolism of PGF, incubated 30 min. (IX), kidney catabolism of PGE, incubated 5 min. (A).
A
(6: (6) 00)
B
I.2 *
(9)
I
I
b
*
n. a.
n. s.
!I
0.4
0.2
L
Kid
Ii
Lung-Fe,
E2
FIG. 2. (A) Catabolism of PGEI by kidney and lung tissue from control and ethanoltreated animals. In each pair, ethanol-treated animals are on the left and controls are on the right. Values are means with SEM shown. Numbers in parentheses are the number of animals in each group. See below for statistical significance. (B) Catabolism of PGF, by kidney and lung tissue. In each comparison, the ethanol-treated animals are on the left. Fti-E2 represents conversion of 15keto-PGF, to 15keto-PGE,. Values are mean with SEM shown. (a) Signilicant at the P < 0.01 level; (b) significant at the P < 0.10 level: (ns.) not significant.
250
PENNINGTON.
SMITH,
AND STRIDER
Ethanol
FIG. 3. Separation of PGF, catabolites from control and ethanol-treated animals. “PGEZ” represents IS-keto-PGE, formed via reversal of the 9-keto-reductase reaction in the presence of NAD+ and NADP+. Suppression of the 15-hydroxy-PG-dehydrogenase by ethanol is apparent as well as the relative ratios for the formation of 1%keto-PGF, and I5-keto-PGE,. No unmetabolized PGE, was observed.
I
B
FIG. 4. Specific activity of the 15-hydroxy-prostaglandin dehydrogenase in control and ethanol-treated rats. In each comparison, the ethanol-treated data is on the left. Values are means with SEM shown for the number of animals given in parentheses, (A) Kidney and hmg catabolism of F’GE,; (B) kidney and lung catabolism of PGF,. F&-E, represents the conversion of IS-keto-PCiF, to I5-keto-PGE,. (a) Significant at the P < 0.01 level; (c) significant at the P < 0.05 level.
CHANGES
IN PROSTAGLANDIN
CATABOLISM
251
animals as was the formation of lSketo-PGF,. Rat lung 154ydroxy PG dehydrogenase activity for PGEz was lower in ethanol-treated animals per milligram protein; however, the activity per gram tissue was not significantly altered. The marked decrease in PG catabolism in rat lung and kidney caused by chronic ethanol consumption, considered together with the reports of increased PG synthesis resulting from ethanol, may indicate a significant physiological imbalance in PG metabolism in the chronic alcoholic. It should be noted that the in vitro assays of prostaglandin degradation reported here were performed in the absence of ethanol but in the presence of adequate amounts of cofactors. In the individual chronically consuming ethanol, the redox ratio between oxidized and reduced cofactors (NAD+/NADH) appears to be lower resulting from increased formation of NADH in the liver and kidneys as a result of metabolism of ethanol by alcohol dehydrogenase. Thus, in viva, PG dehydrogenase activity may be further compromised. Kidney synthesis of PGE, from PGF,, via reversal of the 9-keto-PG reductase in the presence of oxidized cofactors has been reported previously (7). Under the assay conditions used in these experiments, no unmetabolized PGE, was observed. The synthesis of ISketo-PGE, from PGF, was influenced by ethanol but always in the same molar ratio as was 15-keto-PGF, synthesis. Thus, as previously reported (8), the synthesis of 15-keto-PGE, under these conditions appeared to be via 15keto-PGF,,. These results would also indicate that the 9-keto-reductase was not influenced by ethanol, but rather that the availability of appropriate substrate (15-keto-PGF,,) was influenced by ethanol. It is assumed that the differences observed in these experiments were the result of changes in the kinetic parameters of the ethanol-exposed enzymes. Further experiments are planned to test this hypothesis. Cause-and-effect relationships between the in V&-O changes observed in prostaglandin metabolism and any biochemical or clinical manifestation of chronic alcoholism remain to be determined. We would suggest, however, that the results obtained in this study may indicate a relationship between alcohol and prostaglandin metabolism that would be reflected in the hypertension associated with ethanol consumption. In addition, we believe that these results suggest that alterations in prostaglandin metabolism induced by ethanol may relate to several of the problems caused by ethanol during pregnancy and may underlie at least a portion of the condition known as fetal alcohol syndrome. SUMMARY
Previous research has demonstrated that prolonged exposure to ethanol results in alterations in the oxidative metabolism of arachidonic acid
252
PENNINGTON.
SMITH.
AND STRIDER
including lowered membrane levels of arachidonate, increased arachidonate peroxidation. and increased oxidation of arachidonate to prostaglandin E,. We now report that in the absence of ethanol, whole homogenates from both lung and kidney of adult Holtzman rats dosed chronically with ethanol (24 g/kg/24 hr) for 5 weeks differed significantly in their ability to catabolize prostaglandin E, (PGE,) and prostaglandin FBa (PGF,,) relative to lung and kidney homogenates from isocalorically treated controls. In the presence of NAD+, NADP+, and [l-‘4C]PGE, kidney homogenates from ethanol-treated animals formed significantly less 15-keto-PGE, than those of control animals. Both specific and total enzyme activities were suppressed in the ethanol-treated animals (3.35 + 0.67 nmol/min/mg protein versus 6.18 -t 0.96 nmollminimg protein for the controls, P < 0.05). Kidney metabolism of PGF,, to 15-keto-PGF,, was significantly depressed in terms of both total and specific activity and a significant amount of 15-keto-PGE, was formed from PGF2, by the kidney homogenates. Specific activity for the conversion of PGE, to 15-keto-PGE, in the lung was suppressed in ethanol-treated animals but the activity per gram tissue was not altered. Lung catabolism of PGFzu was not significantly altered by exposure to ethanol. ACKNOWLEDGMENTS This work was supported in part by a grant from the North Carolina Alcoholism Research Authority. Reference prostagiandins were kindly supplied by Dr. John Pike of Upjohn Company.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Karppanen, H., and Puurunen, J. Eur. J. Pharmacol, 35, 221 (1976). Beazell, J., and Ivy, A., Quart. J. Studies in Alcohol 1, 45 (1940). Collier, H. O., McDonald-Gibson, W. J., and Sneed, S. A., Lancer, 1, 1975, 702. Edgarian, H., and Altura, B. M., Experientia 32, 618 (1976). Pace-Asciak, C. R., and Rangaraj, G., J. Biol. Chem. 251, 3381 (1976). Pace-Asciak, C. R., and Rangaraj, G., Biochim. Biophys. Acta 529, 13 (1978). Pace-Asciak, C. R., J. Biol. Chem. 250, 2789 (1975). Hoult, J. R. S., and Moore, P. K., Brit. J. Pharmacot. 61, 615 (1977).
21, 246-252 (1979)
MEDICINE
Alterations
SAM
Department
in Prostaglandin Catabolism Chronically Dosed with Ethanol
N. PENNINGTON,
CARLTON P. SMITH, JOHN B. STRIDER, JR.
of Biochemistry, School of Medicine, Greenville, North Carolina
East 27834
in Rats
JR., AND
Carolina
University.
Received August 15, 1978
The prostaglandins (PGs) have been the subject of intensive investigation because of their wide distribution and marked biological activities. These cyclic, oxygenated, fatty acid derivatives have diverse effects at low concentrations in a multitude of biological systems and have had widespread clinical application in studies related to gastric secretion, hypertension, asthma, induction of labor, and contraception. At the molecular level, the PGs have been shown to influence, and to be influenced by, adenyl cyclase and appear to play a role in this control mechanism. Chemically, the prostaglandins and related compounds are synthesized from polyunsaturated fatty acids, e.g., arachidonic acid, via two common intermediate endoperoxides. Recent research has demonstrated that the intermediate endoperoxides (PGG, and PGH,) as well as thromboxane A, (TXA,) and prostacyclin (PGI,) are biologically active and suggests that these compounds may be of major importance in tissues such as blood vessels and platelets. The possibility that the physiological action of ethanol is linked in part to that of the prostaglandins is suggested by the fact that the sites of action of the two agents are similar: i.e., they both alter cardiovascular, hepatic, and gastrointestinal activities. Additionally, ethanol and prostaglandins have been shown to influence blood pressure. Since 1969 our laboratory has been investigating the effects of ethanol on arachidonic acid oxidative metabolism. Several laboratories, including our own, have demonstrated that chronic ethanol consumption, even in the presence of an adequate diet, reduces normal stores of arachidonic acid, the precursor of the prostaglandins, prostacyclin and thromboxanes. 246 oooG2944i79/03w&o7$o2.w0 Copyright @ 1979 by Academic Press, Inc. AU tights of reproduction in any form reserved.
CHANGES
IN
PROSTAGLANDIN
CATABOLISM
247
Accounting for the generally poor nutritional state of alcoholics, we suggest that the metabolism of prostaglandins, thromboxanes, and/or prostacyclin may be altered significantly in these individuals. This conclusion has been supported by results of recent studies in both man and experimental animals. Karppanen and Puurunen (1) observed that a 10% ethanol solution blocked gastric acid secretion in rats and that a prostaglandin synthesis inhibitor (indomethacin) blocked this effect. These data suggest that prostaglandin synthesis is stimulated by ethanol, an effect which may influence gastric ulcer formation. Similar effects of ethanol in man have been reported (2). Collier et al. (3) found that ethanol at high levels stimulates prostaglandin synthesis by bull seminal vesicles. Edgarian and Altura (4) reported a differential effect of ethanol on the response of arterial and venous smooth muscle to PGF%. These results all indicate that ethanol influences prostaglandin levels, generally by stimulation of prostaglandin synthesis. However, no studies of the effect of ethanol on prostaglandin degradation have been described. The biological degradation of prostaglandins, thromboxane AZ, and prostacyclin has been described. Thromboxane A, and prostacyclin are degraded rapidly in aqueous solutions. The prostaglandins, on the other hand, are more stable but can be degraded enzymatically. The results to be presented here suggest that the enzymatic degradation of certain prostaglandins is drastically decreased by chronic ethanol consumption. In these experiments, adult male animals (Holtzman rats) were chronically exposed to ethanol via a liquid diet. At the end of 5 weeks various tissues homogenates from these animals were assayed for levels of prostaglandin- 1Wzydroxy dehydrogenase and 15keto- 13,lQprostaglandin reductase. The results indicated that chronic ethanol consumption markedly inhibited prostaglandin catabolism even in the presence of appropriate amounts of oxidized cofactors. MATERIALS
AND METHODS
Animals and ethanol administration. Male Holtzman rats weighing 150-200 g at the start of the experiment were used. Ethanol in the form of a liquid diet (Lieber diet) was administered for 5 weeks and control animals received an isocaloric liquid diet with maltose-dextran substituted for the ethanol. Control animals were adjusted daily for the consumption of diet by matched experimental animal in the previous 24 hr. The diet mixtures were purchased from Bio-Serv Corporation. At the conclusion of the dosing period, the control animals weighed 330.7 + 16.2 g and the experimental animals weighed 234.8 2 22.4 g. Assay of prostaglandin degradation. Tissue assays were run in duplicate using total tissue homogenates. After depriving experimental animals of ethanol for 12 hr, all animals were sacrificed by decapitation and tissue
248
PENNINGTON,SMITH,
AND STRIDER
from lung and kidney collected as indicated. The tissue was rapidly excised and placed in ice-cold KC1 (1.15%) solution. After washing and mincing, the tissue was homogenized in ice-cold phosphate buffer, 0.05 M, pH 7.4. The homogenates were filtered through cheese cloth to remove debris and used immediately for the various assays. The catabolism of 14C-labeled prostaglandins (PGE, and PGF,,) was assayed according to the method of Pace-Asciak and Rangaraj (5,6) as modified in our lab. This modification combined both the NAD+- and NADP+-requiring enzymes and used diethyl ether to extract the products rather than an ethanol precipitation of protein. Following extraction of the reaction mixture, the ether extract was dried under N, and the residue redissolved in chloroform-methanol (1: 1). The samples were then spotted on silica gel H plates (0.25 mm) and developed in dioxane-benzeneacetic acid (50:50:2.5). The tic plates were dried in air and scanned by a thin-layer scanner (Berthold). Radioactive spots were identified by comparison with known standards run on the same plate and were scraped and quantitated by liquid scintillation counting (Beckman Model LS-233). This procedure gave good recoveries, e.g., several hundred assays for PGEz recovery gave a value of 80.0 +- 5.5%. This procedure worked well with minor modification for all tissue homogenates. The purity of the commercial 14C-labeled prostaglandins was checked by chromatography and found to be ~99% in all cases. Chemicals. NAD+ and NADP+ were purchased from Sigma Chemical Company. Radioactive prostaglandins were from New England Nuclear and Amersham Searle Corporation. All inorganic chemicals and organic solvents were purchased from Fisher Scientific. Solvents were redistilled and stored in dark bottles. RESULTS
AND DISCUSSION
Initial experiments were carried out to determine the appropriate parameters for the assay of each tissue with respect to time of incubation, amount of enzyme (protein), and cofactor concentration. As shown in Fig. 1, considerable variation exists with respect to reaction time for each tissue but in all cases assay conditions were obtained that yielded linear reaction rates relative to protein (enzyme) concentration. Overall, rat kidney contained the highest activity with respect to the catabolism of both PGE, and PGF,,. Chronic exposure to ethanol significantly lowered both the specific activity and activity per gram tissue in rat kidney for the formation of 15-keto-PGE, and 15keto-PGF, (Fig. 2). Under the assay conditions employed (excess NAD+ and NADP+ present, ethanol absent), rat kidney formed significant amounts of 15-ketoPGE, when PGFzu served as a substrate (Fig. 3). The formation of 15keto-PGE, was decreased in the same molar ratio in ethanol-treated
CHANGES
IN PROSTAGLANDIN
L
I
0.5
Relative
I
L
1
1.0
I.5
2.0
Enzyme
249
CATABOLISM
I
2.5
3.0
Concentration
FIG. 1. Relative enzyme (protein) curves used to determine assay conditions for various tissues. Lung catabolism of PGE, incubated 15 min. (0). lung catabolism of PGFp, incubated 1 hr (x), kidney catabolism of PGF, incubated 30 min. (IX), kidney catabolism of PGE, incubated 5 min. (A).
A
(6: (6) 00)
B
I.2 *
(9)
I
I
b
*
n. a.
n. s.
!I
0.4
0.2
L
Kid
Ii
Lung-Fe,
E2
FIG. 2. (A) Catabolism of PGEI by kidney and lung tissue from control and ethanoltreated animals. In each pair, ethanol-treated animals are on the left and controls are on the right. Values are means with SEM shown. Numbers in parentheses are the number of animals in each group. See below for statistical significance. (B) Catabolism of PGF, by kidney and lung tissue. In each comparison, the ethanol-treated animals are on the left. Fti-E2 represents conversion of 15keto-PGF, to 15keto-PGE,. Values are mean with SEM shown. (a) Signilicant at the P < 0.01 level; (b) significant at the P < 0.10 level: (ns.) not significant.
250
PENNINGTON.
SMITH,
AND STRIDER
Ethanol
FIG. 3. Separation of PGF, catabolites from control and ethanol-treated animals. “PGEZ” represents IS-keto-PGE, formed via reversal of the 9-keto-reductase reaction in the presence of NAD+ and NADP+. Suppression of the 15-hydroxy-PG-dehydrogenase by ethanol is apparent as well as the relative ratios for the formation of 1%keto-PGF, and I5-keto-PGE,. No unmetabolized PGE, was observed.
I
B
FIG. 4. Specific activity of the 15-hydroxy-prostaglandin dehydrogenase in control and ethanol-treated rats. In each comparison, the ethanol-treated data is on the left. Values are means with SEM shown for the number of animals given in parentheses, (A) Kidney and hmg catabolism of F’GE,; (B) kidney and lung catabolism of PGF,. F&-E, represents the conversion of IS-keto-PCiF, to I5-keto-PGE,. (a) Significant at the P < 0.01 level; (c) significant at the P < 0.05 level.
CHANGES
IN PROSTAGLANDIN
CATABOLISM
251
animals as was the formation of lSketo-PGF,. Rat lung 154ydroxy PG dehydrogenase activity for PGEz was lower in ethanol-treated animals per milligram protein; however, the activity per gram tissue was not significantly altered. The marked decrease in PG catabolism in rat lung and kidney caused by chronic ethanol consumption, considered together with the reports of increased PG synthesis resulting from ethanol, may indicate a significant physiological imbalance in PG metabolism in the chronic alcoholic. It should be noted that the in vitro assays of prostaglandin degradation reported here were performed in the absence of ethanol but in the presence of adequate amounts of cofactors. In the individual chronically consuming ethanol, the redox ratio between oxidized and reduced cofactors (NAD+/NADH) appears to be lower resulting from increased formation of NADH in the liver and kidneys as a result of metabolism of ethanol by alcohol dehydrogenase. Thus, in viva, PG dehydrogenase activity may be further compromised. Kidney synthesis of PGE, from PGF,, via reversal of the 9-keto-PG reductase in the presence of oxidized cofactors has been reported previously (7). Under the assay conditions used in these experiments, no unmetabolized PGE, was observed. The synthesis of ISketo-PGE, from PGF, was influenced by ethanol but always in the same molar ratio as was 15-keto-PGF, synthesis. Thus, as previously reported (8), the synthesis of 15-keto-PGE, under these conditions appeared to be via 15keto-PGF,,. These results would also indicate that the 9-keto-reductase was not influenced by ethanol, but rather that the availability of appropriate substrate (15-keto-PGF,,) was influenced by ethanol. It is assumed that the differences observed in these experiments were the result of changes in the kinetic parameters of the ethanol-exposed enzymes. Further experiments are planned to test this hypothesis. Cause-and-effect relationships between the in V&-O changes observed in prostaglandin metabolism and any biochemical or clinical manifestation of chronic alcoholism remain to be determined. We would suggest, however, that the results obtained in this study may indicate a relationship between alcohol and prostaglandin metabolism that would be reflected in the hypertension associated with ethanol consumption. In addition, we believe that these results suggest that alterations in prostaglandin metabolism induced by ethanol may relate to several of the problems caused by ethanol during pregnancy and may underlie at least a portion of the condition known as fetal alcohol syndrome. SUMMARY
Previous research has demonstrated that prolonged exposure to ethanol results in alterations in the oxidative metabolism of arachidonic acid
252
PENNINGTON.
SMITH.
AND STRIDER
including lowered membrane levels of arachidonate, increased arachidonate peroxidation. and increased oxidation of arachidonate to prostaglandin E,. We now report that in the absence of ethanol, whole homogenates from both lung and kidney of adult Holtzman rats dosed chronically with ethanol (24 g/kg/24 hr) for 5 weeks differed significantly in their ability to catabolize prostaglandin E, (PGE,) and prostaglandin FBa (PGF,,) relative to lung and kidney homogenates from isocalorically treated controls. In the presence of NAD+, NADP+, and [l-‘4C]PGE, kidney homogenates from ethanol-treated animals formed significantly less 15-keto-PGE, than those of control animals. Both specific and total enzyme activities were suppressed in the ethanol-treated animals (3.35 + 0.67 nmol/min/mg protein versus 6.18 -t 0.96 nmollminimg protein for the controls, P < 0.05). Kidney metabolism of PGF,, to 15-keto-PGF,, was significantly depressed in terms of both total and specific activity and a significant amount of 15-keto-PGE, was formed from PGF2, by the kidney homogenates. Specific activity for the conversion of PGE, to 15-keto-PGE, in the lung was suppressed in ethanol-treated animals but the activity per gram tissue was not altered. Lung catabolism of PGFzu was not significantly altered by exposure to ethanol. ACKNOWLEDGMENTS This work was supported in part by a grant from the North Carolina Alcoholism Research Authority. Reference prostagiandins were kindly supplied by Dr. John Pike of Upjohn Company.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Karppanen, H., and Puurunen, J. Eur. J. Pharmacol, 35, 221 (1976). Beazell, J., and Ivy, A., Quart. J. Studies in Alcohol 1, 45 (1940). Collier, H. O., McDonald-Gibson, W. J., and Sneed, S. A., Lancer, 1, 1975, 702. Edgarian, H., and Altura, B. M., Experientia 32, 618 (1976). Pace-Asciak, C. R., and Rangaraj, G., J. Biol. Chem. 251, 3381 (1976). Pace-Asciak, C. R., and Rangaraj, G., Biochim. Biophys. Acta 529, 13 (1978). Pace-Asciak, C. R., J. Biol. Chem. 250, 2789 (1975). Hoult, J. R. S., and Moore, P. K., Brit. J. Pharmacot. 61, 615 (1977).