Comp. Btochem. PhysioL Vol. 10315, No. 4, pp. 981-984, 1992 0305-0491/92 $$.00 + 0.00 Printed in Great Britain PerganumPremLid HISTIDINE DIPEPTIDE LEVELS IN AGEING AND HYPERTENSIVE RAT SKELETAL AND CARDIAC MUSCLES PETER JOHNSON* and JANET L. Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens, OH 45701, U.S.A. (Tel. 614 593-1744) (Received 6 May 1992) Almract--l. In rat skeletal muscles (Iongissimus dorsi and quadriceps femoris), carnosine and anserine levels decreased 35-50% during senescence, and were 35-45% lower in hypertem/ve rats compared to normoteusive levels. 2. In rat left ventricular cardiac muscle, although no free carnosine and anserine were detected, the total level of histidine dipeptides declined 22% during senescence and in hypertensive animals decreased 35% compared to normotensive levels. 3. The significance of these changes in relation to the possible antioxidant roles of histidine dipeptides in muscle is discussed. INTIIODUCTION The histidine dipeptide carnosine (~-alanyl-L-histidine) is found in a variety of animal tissues in different species, and is particularly abundant (at millimolar levels) in skeletal muscles. In addition to carnosine other histidine dipeptides such as auserine (/~-alanyl- L-l-methyl-histidine), homocamosine (y-amino- butyryl-L-histidine), ophidine (~8-alanyl-L-3-methyl- histidine) and the N-acetyl derivatives of free carnosine and anserine have been detected in muscle and other animal tissues (Boldyrev and Severin, 1990). Because of the relatively high concentrations of the histidine dipeptides in skeletal muscles and in other tissues such as the olfactory bulb (Margolis, 1974), considerable interest has centred on the putative roles of these compounds. Although the histidine dipeptides have previously been implicated in buffering properties, enzyme activity regulation, metal ion chelation and neuro- transmission (Boldyrev and Severin, 1990; Brown, 1981; Rochel and Margolis, 1982), more recent studies point to an/n v/vo antioxidant function for these compounds as scavengers of 02-derived free radicals. In particular, it has been shown that histidine dipeptides are effective hydroxyl and peroxyl radical scavengers (Aruoma et al., 1989; Boldyrev et aL, 1987, 1988; Kohen et al., 1988) and may have inhibitory effects on membrane lipid per- oxidation/n situ (Babizhayev, 1989; Yoshikawa et al., 1991). It has previously been suggested that similarities exist between the effects of ageing and hyper- tension on the myocardium (Lakatta, 1987), and it :[To whom correspondence should be addressed, at: Depart- ment of Chemistry, Ohio University, Athens, OH 45701, U.S.A. Abbreviation--OPA, o-phthalaldehyde. has recently been shown that increases in lipid per- oxidation may occur in skeletal and cardiac muscle as a result of the ageing process (Ji eta/., 1990) and in hypertension (Papies et al., 1989). We have there- fore investigated the concentrations of carnosine and other histidine dipeptides in skeletal and cardiac muscles of ageing and hypertensive rats in order to determine if there is a negative correlation between histidine dipeptide contents and the reported levels of lipid peroxidation. Our results indicate that decreases in the histidine dipeptide levels do occur in these muscles during agein 8 and as a result of hypertension. MATERIALSAND METHOI~ Animals For studies on ageing rats (Rattus rattus), male Fischer 344/NHsd BR rats of 3, 12 and 27 months of age were purchased from Harlan Sprague Dawley, Indianapolis. Normoteusive (WKY) and spontaneously hypertensive (SHR) male Wistar rats approximately 8 months of aBe were purchased from Taconic Farms, New York. Muscle extractpreparation Muscles were excised immediately after sacrifice of the animal and/mmediately cooled to and storedat -70 ° until furtheruse. Muscle samples of approximately 100rag were removed from the frozen sample and the sample was pulverized in a stainless steel macerator bathed in liquid N 2 (Pette and Reichnmun, 1982). The frozen muscle powder macerate was then weighed, and suspended in 1.5 ml of 0.2 M lithium citrate pH 2.2 or pH 2.85 buffer and brietly homogenized in a 5 ml capacity Thomas glass-teflon hand homogenizer. Following centrifugation of the sample for 5 min at 500 I, the supernatant fluid was removed and mixed with approximately one half volume of 20% trichloroacetic acid. After incubation in ice for 10 rain, the sample was centrifuged to remove the pre~pitated protein, and the supernatant was extracted three times with an equal volume of diethyl ether in order to remove trichloroacetic acid. The solution was then adjusted to a final volume of 1.5 ml by the addition of lithium citrate buffer. 981
Transcript
Comp. Btochem. PhysioL Vol. 10315, No. 4, pp. 981-984, 1992 0305-0491/92 $$.00 + 0.00 Printed in Great Britain Perganum Prem Lid
HISTIDINE DIPEPTIDE LEVELS IN AGEING AND HYPERTENSIVE RAT SKELETAL
AND CARDIAC MUSCLES
PETER JOHNSON* and JANET L. Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens,
OH 45701, U.S.A. (Tel. 614 593-1744)
(Received 6 May 1992)
Almract--l. In rat skeletal muscles (Iongissimus dorsi and quadriceps femoris), carnosine and anserine levels decreased 35-50% during senescence, and were 35-45% lower in hypertem/ve rats compared to normoteusive levels.
2. In rat left ventricular cardiac muscle, although no free carnosine and anserine were detected, the total level of histidine dipeptides declined 22% during senescence and in hypertensive animals decreased 35% compared to normotensive levels.
3. The significance of these changes in relation to the possible antioxidant roles of histidine dipeptides in muscle is discussed.
INTIIODUCTION
The histidine dipeptide carnosine (~-alanyl-L-histidine) is found in a variety of animal tissues in different species, and is particularly abundant (at millimolar levels) in skeletal muscles. In addition to carnosine other histidine dipeptides such as auserine (/~-alanyl- L-l-methyl-histidine), homocamosine (y-amino- butyryl-L-histidine), ophidine (~8-alanyl-L-3-methyl- histidine) and the N-acetyl derivatives of free carnosine and anserine have been detected in muscle and other animal tissues (Boldyrev and Severin, 1990). Because of the relatively high concentrations of the histidine dipeptides in skeletal muscles and in other tissues such as the olfactory bulb (Margolis, 1974), considerable interest has centred on the putative roles of these compounds.
Although the histidine dipeptides have previously been implicated in buffering properties, enzyme activity regulation, metal ion chelation and neuro- transmission (Boldyrev and Severin, 1990; Brown, 1981; Rochel and Margolis, 1982), more recent studies point to a n / n v/vo antioxidant function for these compounds as scavengers of 02-derived free radicals. In particular, it has been shown that histidine dipeptides are effective hydroxyl and peroxyl radical scavengers (Aruoma et al., 1989; Boldyrev et aL, 1987, 1988; Kohen et al., 1988) and may have inhibitory effects on membrane lipid per- oxidation/n situ (Babizhayev, 1989; Yoshikawa et al., 1991).
It has previously been suggested that similarities exist between the effects of ageing and hyper- tension on the myocardium (Lakatta, 1987), and it
:[To whom correspondence should be addressed, at: Depart- ment of Chemistry, Ohio University, Athens, OH 45701, U.S.A.
Abbreviation--OPA, o-phthalaldehyde.
has recently been shown that increases in lipid per- oxidation may occur in skeletal and cardiac muscle as a result of the ageing process (Ji eta/., 1990) and in hypertension (Papies et al., 1989). We have there- fore investigated the concentrations of carnosine and other histidine dipeptides in skeletal and cardiac muscles of ageing and hypertensive rats in order to determine if there is a negative correlation between histidine dipeptide contents and the reported levels of lipid peroxidation. Our results indicate that decreases in the histidine dipeptide levels do occur in these muscles during agein 8 and as a result of hypertension.
MATERIALS AND METHOI~
Animals
For studies on ageing rats (Rattus rattus), male Fischer 344/NHsd BR rats of 3, 12 and 27 months of age were purchased from Harlan Sprague Dawley, Indianapolis. Normoteusive (WKY) and spontaneously hypertensive (SHR) male Wistar rats approximately 8 months of aBe were purchased from Taconic Farms, New York.
Muscle extract preparation
Muscles were excised immediately after sacrifice of the animal and/mmediately cooled to and stored at -70 ° until further use. Muscle samples of approximately 100rag were removed from the frozen sample and the sample was pulverized in a stainless steel macerator bathed in liquid N 2 (Pette and Reichnmun, 1982). The frozen muscle powder macerate was then weighed, and suspended in 1.5 ml of 0.2 M lithium citrate pH 2.2 or pH 2.85 buffer and brietly homogenized in a 5 ml capacity Thomas glass-teflon hand homogenizer. Following centrifugation of the sample for 5 min at 500 I, the supernatant fluid was removed and mixed with approximately one half volume of 20% trichloroacetic acid. After incubation in ice for 10 rain, the sample was centrifuged to remove the pre~pitated protein, and the supernatant was extracted three times with an equal volume of diethyl ether in order to remove trichloroacetic acid. The solution was then adjusted to a final volume of 1.5 ml by the addition of lithium citrate buffer.
981
982 PETER JOHNSON and JANET L. HAm, mR
Dipeptide determinations
For analysis using the Physiological Fluids column on a Beckman 119CL amino acid analyser, 700/zl aliquots of deproteinized muscle extract were used. Standards of free (i.e. non-N-acetylated) carnosine and ansedne were used to calibrate the instrument, and results of the dipeptide con- tents of the samples were expressed as # M of dipeptide recovered per g wet weight of muscle.
Determination of the combined value of carnosine and anserine (including the N-acetylated derivatives of the dipeptides) in the muscle extract was performed by the OPA colorimetric procedure of Wolos et al. (1983). For these assays, 250-600/zi aliquots of the deproteinized muscle extracts were lyophilized, redissolved in 100/~1 of 5% tdchloroacetic acid solution, and then used directly in the assay procedure (2.5 ml final assay volume). The assay procedure was calibrated by a standard curve for carnosine, and results of determinations on muscle samples were expressed as/z M carnosine equivalents per g wet weight of muscle.
Statistical analysis
For both studies, six animals in each age group and six normotensive and hypertensive animals were used and mean values +_ SD were calculated from the data. Tests for signifi- cant differences between data sets from different muscle groups were made by the Student's t test and the confidence level (P value) for the difference was determined.
RESULTS
Histidine dipeptide levels in muscles of ageing rats
As determined on the amino acid analyser, age- related progressive decreases were observed in the free carnosine contents of the quadriceps femoris and longissimus dorsi muscles (Table 1), whereas anserine levels in these muscles increased during maturation (3-12 months) and then declined during senescence (12-27 months). Except for the difference beween the free anserine content in quadriceps femoris for 3 and 12 month-old rats, the changes observed between each age group were all statistically signifi- cant (P ~<0.05). On analysis of left ventricular cardiac muscle by the same procedure, no detectable amounts of free carnosine or anserine were detected in any of the muscle samples.
Determination of the levels of histidine dipeptides in skeletal and cardiac muscles was also performed by the OPA colorimetric method of Wolos et al. (1983). In these studies (Table 1), it was found that no significant changes in dipeptide levels occurred in the three muscles during maturation, but statisti- cally significant decreases (at or above 95% prob- ability) were observed in longissimus dorsi and left ventricular muscles during senescence.
Table 1. Histidine dipeptide levels in skeletal and cardiac muscles of rats of various ages
Total Muscle Age Camosine Anserine dipeptides
L. dorsi 3 1.08 ± 0.20 1.41 ± 0.25 6.68 ± 0.81 P < 0.05 P < 0.001 NSD
12 0.79 ± 0.22 2.20 ± 0.26 3.88 ± 0.96 P < 0.05 P < 0.01 P < 0.01
Values are reported as/~mol dipeptide/g wet weight of muscle ± SD. Carnosine and anserine values were determined using the amino acid analyser, and total dipeptide levels were measured as carnosine equivalents by the OPA procedure. The P value for significant difference between dipeptide levels in the same muscle type at different ages (in months) is shown between the values compared. NSD indicaU:s there was no significant different between the values (P < 0.2 or larger).
Histidine dipeptide levels in muscles of normotensive and spontaneously hypertensive rats
Analysis of free carnosine and anserine levels in WKY and SHR (spontaneously hypertensive) rat skeletal muscles (Table 2) revealed that the levels of free carnosine and anserine in the two skeletal muscles were significantly lower (P values ~<0.05) in the SHR than in the same muscles of age- and sex-matched normotensive WKY rats. Cardiac muscle from the left ventricle of both types of rat did not contain detectable amounts of either free dipeptide as determined by the amino acid analyser procedure.
Determination of histidine dipeptide levels in the three muscles by the OPA procedure (Table 2) indi- cated that in each muscle, the measured levels in the SHR rats were significantly lower (P ~< 0.05) than in the WKY rats.
DISCUSSION
The results which show a decline in free carnosine content in skeletal muscles during maturation are similar to those of an earlier study (Marlin et al., 1989) in which it was shown that carnosine levels decreased in equine middle gluteal muscle between one and four years of age (i.e. into adulthood). Our
Table 2. Histidine dipeptide levels in skeletal and cardiac muscles of normotensive (WKY) and spontaneously hypertensive (SHR) rats
Muscle WKY/SHR Carnosine Anserine Total dipeptides
L. dorsi WKY 1.53 -6 0.33 5.85 -6 1.29 16.28 -6 2.68 P < 0.05 P < 0.05 P < 0.02
SHR 1.00 + 0.34 3.71 + 0.95 10.88 + 3.64
Q. femoris WKY 1.17 + 0.28 4.42 ± 0.62 14.18 ± 1.96 P < 0.01 P < 0.001 P < 0.001
SHR 0.76 ± 0.18 2.42 ± 0.43 8.38 ± t.60
Cardiac WKY 0 0 14.31 -6 1.96 left ventricle P < 0.05
SHR 0 0 9.25 ± 1.86
See Table I footnote for definitions of values reported.
Histidine dipeptides in rat muscles 983
studies also show that the decline in carnosine con- tent in skeletal muscles continues during senetcence. Free anserine levels also decreased in the skeletal muscles during senescence, but during maturation (i.e. from 3 to 12 months of age in the rat) the levels were found either to increase or remain unchanged. The lack of a decrease in anserine contents of the skeletal muscles during maturation may be explained in part by the reported ontogenic increase in the rate of synthesis of anserine from carnosine (Boldyrev and Severin, 1990). Although the cause of the decreases in histidine dipeptide levels remains unidentified, the changes may be related in part to the observation (Lenney et al., 1982) that the levels of carnosinase increase during ageing, implying that there might be an elevation in the rate of degradation of the dipeptides during ageing. Further evidence of an age-related decline in tissue contents of histidine dipeptides has also been reported by Takahashi (1981), who determined that the levels of homo- carnosine in human cerebrospinal fluid decreased during puberty.
Because of reports of the presence of significant levels of N-acetylated carnosine and anserine in rat cardiac muscle (O'Dowd et al., 1988), we also estimated total histidine dipeptide levels by the OPA procedure of Wolos et al. (1983) which, unlike the amino acid analyser procedure, can detect the N-acetylated dipeptides. The results from these studies showed that total hisfidine dipeptide levels in the two skeletal muscles and in the left ventricu- lar muscle were unaltered during maturation, but decreased significantly in longissimus dorsi and left ventricular muscles during senescence. Although the left ventricular muscle contained no detectable amounts of free carnosine and anserine, the total amounts of histidine dipeptides in the cardiac muscle estimated by the OPA procedure were found to be comparable with the levels found in skeletal muscles.
As shown in Table 3, both the amino acid analyser and OPA methods gave similar percentage decreases in histidine dipeptide levels in skeletal muscles during senescence and in comparisons of normotensive and hypertensive animals, although the absolute levels of dipeptides were different for the Fischer and Wistar
~'ains of rat. Table 3 also shows that the results obtained from the coiorimetric assays were in general a factor of between two and three times higher than the figures for the sum of free carnosine and anserine obtained from the amino acid analyser. Such increases are consi~ent with data on the ratios of N-acetylated to free dipeptides in muscle (O'Dowd et al., 1988), although part of these increases may also be the result of overestimation of total histidine dipeptide levels caused by reactions of other com- pounds in muscle with OPA (Wolos et al., 1983).
It has previously been reported that whereas the rate of fipid peroxidation in skeletal muscles of ageing rats is significantly increased, the activities of the antioxidant enzymes catalase, superoxide dismutase, glutathione-S-transferase and glutathione reductase are either unchanged or elevated during ageing (Ji et al., 1990, 1991). These same studies also showed that the activity level of another antioxidant enzyme, glutathione peroxidase, either increased or decreased during ageing depending on the particular muscle studied and on the physical activity patterns of the animals. As these results suggest that changes in the activities of the antioxidant enzymes may not protect muscle against increased fipid peroxidation, it is tempting to speculate that the diminished levels of histidine dipeptides in senescent muscles may in part be a factor in the increased levels of lipid per- oxidation, on the basis of the antioxidant properties of the histidine dipeptides.
Increased levels of lipid peroxidation have also been observed in the myocardium of hypertensive animals (Papies et al., 1989), and it has been suggested that this may be caused by the presence of elevated levels of O2-derived free radicals result- ing from decreased levels of antioxidants and antioxidant.related enzymes (Krzanowski, 1991; Salonen, 1991). Our results which show a decrease in histidine dipeptides in the left ventricular muscle of the myocardium therefore are consistent with this proposal on the basis of the antioxidant properties of the histidine dipeptides.
Previous studies have also established that in spon- taneous or induced hypertension, skeletal muscles as well as the myocardium undergo biochemical
Table 3. Comparimn of histidine dipeptide levels in rat mascla determined on the amino acid analyser and by the OPA procedure
Animal status A B C D Column B
Muscle and Mature Senescent Normotensive Hypertensive or D as % rat strain Analysis (12 too) (27 too) (WKY) (SHR) of A or C L. dorsi AAA
The levels of dipeptides are in ttM/s wet weillht of muscle, and the fillures are the tuna of free carnosine and anaerine levels determined by the amino acid analysis (AAA) procedure, or the total histidine dipeptide levels estimated by the OPA procedure.
984 l~a~R JOHNSON and J^NFr L. HAr, O~R
changes, including an increase in the ratio of fast to slow fibers and changes in the levels of glycolytic and citric acid cycle-associated enzymes (Ben Bachir- Lamrini et al., 1990; Favier et al., 1989). Our studies now show that the levels of histidine dipeptides in two different skeletal muscles of spontaneously hypertensive rats are decreased in comparison to the normotensive levels, and it will be interesting to determine if lipid peroxidation in these skeletal muscles is increased in hypertensive animals.
As shown in Table 3, the decreases in histidine dipeptide contents in hypertensive muscles were simi- lar to the decreases observed in the ageing studies on the same muscles, and these similarities suggest that, in addition to hypertension and ageing causing simi- lar changes in the myocardium, there may be compar- able changes that also occur in skeletal muscles as a result of hypertension and ageing. Further studies on the levels of antioxidant enzymes in muscles may therefore provide additional evidence about the possibility that O2-derived free radicals can cause abnormafities in hypertensive skeletal muscle as well as in the hypertensive myocardium and in ageing skeletal muscle.
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