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Methionine enkephalin accumulates in plasma but not in brain or cerebrospinal fluid of rats with acute toxic hepatitis
Neuroscience Letters. 141 (1992) 243 246 c~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
243
NSL 08771
Methionine enkephalin accumulates in plasma but not in brain or cerebrospinal fluid of rats with acute toxic hepatitis M a r k G. Swain", Melvyn R Heyes b, John Vergalla ~ and E. A n t h o n y Jones" "Liver Diseases Section, N1DDK and hSeetion on Anah'tieal Biochemistry, Laborato O, o[' Clh~ieal Seience, NIMH, National ln.vtitules o/Health, Bethesda, MD 20892 (USA) (Received 26 February 1992; Revised version received 21 April 1992: Accepted 22 April 1992) Key wor&v
Methionine enkephalin: Toxic hepatitis: Blood-to-brain transfer
In order to determine whether acute toxic hepatitis in the rat is associated with an accumulation of methionine enkephalin in plasma and increased blood-to-brain transfer of methionine enkephalin, immunoreactive methionine enkephalin levels were determined by radioimmunoassay in plasma, cerebrospinal fluid and whole brain samples from rats with thioacetamide induced acute toxic hepatitis. Thioacetamide treatment was associated with an 8.7-fold increase in plasma immunoreactive methionine enkephalin levels (P <- 0.005) 24 h alter treatment. However, this marked elevation in plasma immunoreactive methionine enkephalin levels was not associated with an increase in whole brain or cerebrospinal fluid immunoreactive methionine enkephalin levels. These data suggest that increased plasma-to-brain transfer of methionine enkephalin does not occur in this model of acute toxic hepatitis.
Methionine enkephalin is known to circulate in normal human and rat plasma at low concentration [4, 13]. Initial reports documented an impermeability of the blood-brain barrier to methionine enkephalin [12]. However, significant penetration of the blood-brain barrier has recently been described in rats exposed to a peripherally administered metabolically stable methionine enkephalin analogue, leading the authors to suggest that prolonged exposure of the blood brain barrier to elevated levels of methionine enkephalin may lead to its increased transport from plasma to brain [15]. This suggestion has been supported by the findings of other workers who have demonstrated significant transport of peripherally administered enkephalins from plasma to brain [21,22]. In addition, the permeability of the bloodbrain barrier has been shown to be non-specifically increased in a rabbit model of fulminant hepatic failure [2, 7]. Therefore, in acute hepatocellular disease plasma-tobrain transfer of endogenous opioids might be increased and this transfer may be facilitated by elevated plasma levels of methionine enkephalin that have been documented in humans with liver disease [19, 20]. Accordingly, increased centrally mediated opioid effects caused (brrespondenee." M.G. Swain, Liver Diseases Section, Bldg. 10, Rm. 4D-52, National Institutes of Health, Bethesda, MD 20892, USA. Fax: ( 1) (301) 402-0491
by plasma-derived endogenous opioids might contribute to the pathophysiology of acute hepatocellular disease. This study was designed to determine whether in a well characterized model of acute hepatocellular disease an increase in plasma levels of methionine enkephalin occurs and is associated with a concomitant increase in levels of methionine enkephalin in the brain. Acute toxic hepatitis was induced in male Sprague Dawley rats (200 250 g: Taconic Farms, Germantown, NY) by the intraperitoneal injection of the hepatotoxin thioacetamide (500 mg/kg in 1.0 ml of 0.9% NaCl: Sigma Chemicals, St. Louis, MO). Controls were treated with vehicle alone. This model of acute toxic hepatitis has been described in detail elsewhere [3]. Rats were anesthetized for sample collection prior to sacrifice (ketamine HC1 50 mg/ml, Parke-Davis, Morris Plains. N J: xylazine 100 mg/ml, Formenta Animal Health Co., Kansas City, MO: 8:1: v:v: dose 0.3 mg/rat). All samples were collected 24 h after injection when hepatocellular injury is maximal (M.G. Swain, personal observation). Blood was collected by inferior vena cava venipuncture, placed in prechilled EDTA containing glass tubes, and centrifuged at 760 x g for 15 min. The plasma was removed and then stored at -70°C until assayed for methionine enkephalin. The thoracic cavity was then opened and the inferior vena cava and thoracic aorta clamped. A 21 gauge needle was inserted into the left ventricle and a small hole
244
made in the right atrium. Ice-cold 0.9% NaCI was then perfused (2 ml/min) through the left ventricle. The perfusion was continued until the fluid returning to the right atrium was clear. The animal was then decapitated and the brain removed. Cerebrospinal fluid was collected as follows. Rats were anesthetized by an intraperitoneal injection of ketamine/xylazine (as above). The rats were then placed in a Kopf stereotaxic frame and the skin and muscles of the nape of the neck were dissected to expose the membrane over the cisterna magna. A cannula made from a 0.7 cm length of 25 gauge needle fixed to a 10 cm length of PE20 polypropylene tubing was attached to a 1 ml syringe. The needle was inserted into the cisternal space by hand and a 150-200 ¢tl sample of CSF was collected. Animals were then sacrificed by cervical dislocation. CSF samples from two animals were combined for the methionine enkephalin radioimmunoassay. Plasma and CSF samples were acidified with 1 N HC1 and passed over a C18 SepPak column (Waters, Milford, MA). The columns were washed twice with 5% acetic acid and eluted with 3 ml of methanol, The methanol eluates were dried under a stream of nitrogen. Dried samples were reconstituted for assay as previously described [17]. Whole brain methionine enkephalin was extracted using a modification of the method of Miller et al. [10]. Briefly, brains were placed in glass tubes containing ice cold 0.1 N HC1 (5:1 w:v) and were homogenized (Tissumizer, Tekmar, Cincinnati, OH; high setting for 15 s). The homogenates were then centrifuged at 50,000 × g at 4°C for 1 h. The pellet was resuspended in 0.1 N HC1 by homogenization and the samples subjected to a further centrifugation. The pooled supernatants were passed over C18 Sep-Pak columns, washed with 5% acetic acid, eluted with methanol, dried under a stream of nitrogen, and reconstituted for assay of methionine enkephalin. Dilutions were made as necessary. The radioimmunoassay of methionine enkephalin applied has been previously described [13, 17]. It employed a commercially available, C-terminus directed, highly specific antibody (INCSTAR, Stillwater, MN). Antibody cross-reactivities have been outlined previously [13]. Recovery was greater than 90% and inter- and intra-assay coefficients of variation 12% and 7% respectively. Data are expressed as means + S.E.M. and the significance of differences between means was determined using the unpaired Student's t-test. Treatment of rats with thioacetamide resulted in significant hepatocellular injury as documented by plasma biochemistry 24 h post-injection (aspartate aminotransferase: 1824 + 357 IU/liter; normal range 55-458 IU/ liter, MetPath Inc., Kensington, MD [3]). In the thioacetamide injected rats acute hepatocellular necrosis was associated with a striking elevation of plasma immunoreac-
1000
0) >
800
3
600
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400
E
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200
I t Saline TAA Injected Injected Fig. 1. Immunoreactive methionine enkephalin levels in plasma extracts from saline-injected and thioacetamide (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per mt plasma and represent means _+ S.E.M. o f at least 9 determinations ('P -< 0.005 vs. saline-injected controls).
tive methionine enkephalin levels with a mean concentration 8.7-fold higher than that in saline injected controls (Fig. 1; P - 0.005). The reason for the elevation of immunoreactive methionine enkephalin levels in plasma 24 h after thioacetamide injection is unknown but is consistent with findings in humans with hepatocellular injury [20]. These observations are of particular interest when the rapid metabolism of enkephalins in the plasma of normal rats is considered [6]. Increased release, decreased metabolism, or both might contribute to elevated plasma levels of immunoreactive methionine enkephalin. The source of circulating methionine enkephatin is poorly defined but the adrenals [16], the gut [14], sympathetic nerves [16], and the brain [1] have all been suggested as possibilities. Methionine enkephalin, like most hydrophilic peptides, is known to penetrate the normal blood-brain barrier poorly [12]. However, there is evidence suggesting that methionine enkephalin crosses the blood brain barrier by specific transport mechanisms [15, 21, 22], and that blood-to-brain transfer of methionine enkephalin is facilitated by prolonged exposure to elevated plasma lev-
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Fig. 2. Immunoreactive methionine enkephalin levels in whole brain homogenates from saline-injected and thioacetamidc (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per mg brain wet weight and represent m e a n s ± S.E.M. of at least 15 determinations ( P= n.s.).
els of enkephalin [15]. In galactosamine-induced acute hepatocellular injury in the rabbit the blood-to-brain transfer of ~-aminoisobutyric acid, a polar compound normally excluded from the central nervous system, is increased, a finding which can be explained by postulating a non-specific increase in blood brain barrier permeability in this model of acute toxic hepatitis [7]. If a non-specific increase in blood-to-brain transfer of methionine enkephalin occurs in acute severe hepatocellular disease an increase in CSF levels of methionine enkephalin would be expected. The finding of normal CSF levels of methionine enkephalin in the thioacetamide-induced model of acute toxic hepatitis may reflect a modulation of the transport system for this opioid. Support for increased central opioid effects in liver disease is available. Patients with cirrhosis are known to exhibit enhanced sensitivity to opiates such as morphine [8] and patients with pruritus complicating primary biliary cirrhosis exhibit a classical opiate withdrawal reaction when treated with the opiate receptor blocker nalmefene [18]. In addition, it has been proposed that endogenous opioids contribute to the pathogenesis of hepatic encephalopathy and this hypothesis is supported by reports of ameliorations of this syndrome in humans and rats by treatment with the opioid receptor antagonist naloxone [9, 11]. However, in thioacetamide-induced
Saline Injected
TAA Injected
Fig. 3. Immunoreactive methionine enkephalin levels in cerebrospinal fluid from saline-injected and thioacetamide (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per ml cerebrospinal fluid and represent means _+ S.E.M. of 5 determinalions (P-ms.).
acute hepatocellular injury in the rat, whole brain (Fig. 2) and cerebrospinal fluid (Fig. 3) immunoreactive methionine enkephalin levels were not elevated. These data are consistent with the findings of Ferenci et al. who found levels of immunoreactive methionine enkephalin in the frontal cortices of rabbits with galactosamineinduced fulminant hepatic t:ailure to be similar to those in controls [5]. The findings in this study do not exclude the possibility that central opioid effects in liver disease are mediated by endogenous opioids other than methionine enkephalin. Indeed in the rat model ofcholestasis due to bile duct resection methionine enkephalin accounted for only 5% of the increased total opioid activity in serum [171. Thus, we have shown that a striking accumulation of immunoreactive methionine enkephalin occurs in plasma in an animal model of acute toxic hepatitis. However, contrary to expectation, this finding is not associated with an increase in whole brain or cerebrospinal fluid immunoreactive methionine enkephalin levels. These results do not support the hypothesis that increased plasma-to-brain transport of methionine enkephalin occurs in and contributes to the pathophysiology of acute severe hepatocellular disease. 1 Banks. W.A., Kastin, A.J., Vischmam A.J., ('oy. D.H. and Straus,
246
2
3
4
5
6
7
8 9
10
11 12
S.L., Carrier-mediated transport of enkephalins and N-tyr-MlF-1 across blood-brain-barrier, Am. J. Physiol., 251 (1986) E477-E482. Bassett, M.L., Mullen, K.D., Scbolz, B., Fenstermacher, J.D. and Jones, E.A., Increased brain uptake of ?,-aminobutyric acid in a rabbit model of hepatic encephalopathy, Gastroenterology, 98 (1990) 747-757. Castro, J.A., D'Acosta, N., De Ferreyra, D., De Castro, E.C., Diaz, C.R., Comez, M.I. and De Fenos, O.M., Studies on thioacetamideinduced liver necrosis, Toxicol. Appl. Pharmacol., 30 (t974) 79 86. Clement-Jones, V., Lowry, RJ., Rees, L.H. and Besser, G.M., Metenkephalin circulates in human plasma, Nature, 283 (1980) 295 297. Ferenci, R, Pappas, S.C., Munson, RJ., Henson, K. and Jones, E.A., Changes in the status of neurotransmitter receptors in a rabbit model of hepatic encephatopathy, Hepatology, 4 (1984) 186 t 91. Hambrook, J.M., Morgan, B.A., Rance, M.J. and Smith, C.F.C., Mode of deactivation of the enkephalins by rat and human plasma and brain homogenates, Nature, 262 (1976) 782-783. Horowitz, M.E., Schafer, D.F., Molnar, P., Jones, E.A.. Blasberg, R.G., Patlak, C.S., Waggoner, J. and Fenstermacher, J.D., Increased blood-brain transfer in a rabbit model of acute liver failure, Gastroenterology, 84 (1983) 1003-1011. Laidlaw, J., Read, A.E. and Sherlock, S., Morphine tolerance in hepatic cirrhosis, Gastroenterology, 40 (1961 ) 389 396. Lisker-Melman, M., Moreno-Otero, R., Fromm, H.C., Gammal, S.H., Basile, A.S. and Jones, E.A., Behavioural amelioration of hepatic encephalopathy induced by an opiate receptor antagonist in a rat model, Hepatology, 8 (1988) A1247. Miller, R.J., Chang, K.J., Cooper, B. and Cuatrecasas, R, Radioimmunoassay and characterization of enkephalins in rat tissues. J. Biol. Chem., 253 (1978) 531.-538. Ozsoylu, S. and Kocak, N., Naloxone in hepatic encephatopathy, Am. J. Dis. Child., 139 (1985) 749-750. Pardridge, W.M,, Frank, H.J.L., Cornford, E.M., Braun, L.D., Crane, P.D. and Oldendorf, W.H., Neuropeptides and the blood
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14
15
16
17
18 19
20
21
22
brain-barrier. In J.B. Martin, S. Reichlin and K.I.,. Bick (Ed~ i. Neurosecretion and Brain Peptides, Ra,,cn, New York. 1981, pp. 321 328. Pierzchala, K. and Van Loon, G.R.. Plasma native and peptidasederivable met-enkephalin responses to restraint stress, J. Clin. Invest., 85 (1990) 861 873. Polak, J.M., Bloom, S.R., Sullivan, S.N., Facer, R and Pearse, A.G.E., Enkephalin-like immunoreactivity in the human gastrointestinal tract, Lancet, i (1977) 972 974. Rapoport, S.l., Klee, W.A., Pettigrew, K.D. and Ohno, K., Entry of opioid peptides into the central nervous system, Science, 207 (1980) 84 86. Shultzberg, M., Lundberg, J.M., H6kfelt, T., Brandt, J., Elde, R.P and Goldstein, M., Enkephalinqike immunoreactivity in gland cells and nerve terminals of the adrenal medulla, Neuroscience, 2 (1978) 1169 1186. Swain, M.G., Rothman, R.B., Xu, H., Vergalla, J., Bergasa, N.V. and Jones, E.A., Endogenous opioids accumulate in plasma in a rat model of acute cholestasis, Gastroenterology, in press. Thornton, J.R. and Losowsky, M.S., Opioid peptides and primary biliary cirrhosis, Br. Med. J., 297 (1988) 1501- 1504. Thornton, J.R. and Losowsky, M.S., Plasma methionine enkephalin concentrations and prognosis in primary biliary cirrhosis, Br. Med. J.. 297 (1988) 1241- 1242. Thornton, J.R. and Losowsky, M.S., Methionine enkephalin is increased in plasma in acute liver disease and is present in bile and urine, J. Hepatol., 8 (1989) 53 59. Zlokovic, B.V., Begley, D.J. and Chain-Eliash, D.G., Blood--brain barrier permeability of leucine-enkephalin, D-aianine2-D-leucineL enkephalin and their N-terminal amino acid (tyrosine), Brain Res., 336 (1985) 125 132. Zlokovic, B.V., Lipovac, M.N., Begley, D.J., Davson, H. and Rakic, L., Transport of leucine-enkephalin across the blood-brain barrier in the perfused guinea pig brain, J. Neurochem., 49 (1987) 310 315.
243
NSL 08771
Methionine enkephalin accumulates in plasma but not in brain or cerebrospinal fluid of rats with acute toxic hepatitis M a r k G. Swain", Melvyn R Heyes b, John Vergalla ~ and E. A n t h o n y Jones" "Liver Diseases Section, N1DDK and hSeetion on Anah'tieal Biochemistry, Laborato O, o[' Clh~ieal Seience, NIMH, National ln.vtitules o/Health, Bethesda, MD 20892 (USA) (Received 26 February 1992; Revised version received 21 April 1992: Accepted 22 April 1992) Key wor&v
Methionine enkephalin: Toxic hepatitis: Blood-to-brain transfer
In order to determine whether acute toxic hepatitis in the rat is associated with an accumulation of methionine enkephalin in plasma and increased blood-to-brain transfer of methionine enkephalin, immunoreactive methionine enkephalin levels were determined by radioimmunoassay in plasma, cerebrospinal fluid and whole brain samples from rats with thioacetamide induced acute toxic hepatitis. Thioacetamide treatment was associated with an 8.7-fold increase in plasma immunoreactive methionine enkephalin levels (P <- 0.005) 24 h alter treatment. However, this marked elevation in plasma immunoreactive methionine enkephalin levels was not associated with an increase in whole brain or cerebrospinal fluid immunoreactive methionine enkephalin levels. These data suggest that increased plasma-to-brain transfer of methionine enkephalin does not occur in this model of acute toxic hepatitis.
Methionine enkephalin is known to circulate in normal human and rat plasma at low concentration [4, 13]. Initial reports documented an impermeability of the blood-brain barrier to methionine enkephalin [12]. However, significant penetration of the blood-brain barrier has recently been described in rats exposed to a peripherally administered metabolically stable methionine enkephalin analogue, leading the authors to suggest that prolonged exposure of the blood brain barrier to elevated levels of methionine enkephalin may lead to its increased transport from plasma to brain [15]. This suggestion has been supported by the findings of other workers who have demonstrated significant transport of peripherally administered enkephalins from plasma to brain [21,22]. In addition, the permeability of the bloodbrain barrier has been shown to be non-specifically increased in a rabbit model of fulminant hepatic failure [2, 7]. Therefore, in acute hepatocellular disease plasma-tobrain transfer of endogenous opioids might be increased and this transfer may be facilitated by elevated plasma levels of methionine enkephalin that have been documented in humans with liver disease [19, 20]. Accordingly, increased centrally mediated opioid effects caused (brrespondenee." M.G. Swain, Liver Diseases Section, Bldg. 10, Rm. 4D-52, National Institutes of Health, Bethesda, MD 20892, USA. Fax: ( 1) (301) 402-0491
by plasma-derived endogenous opioids might contribute to the pathophysiology of acute hepatocellular disease. This study was designed to determine whether in a well characterized model of acute hepatocellular disease an increase in plasma levels of methionine enkephalin occurs and is associated with a concomitant increase in levels of methionine enkephalin in the brain. Acute toxic hepatitis was induced in male Sprague Dawley rats (200 250 g: Taconic Farms, Germantown, NY) by the intraperitoneal injection of the hepatotoxin thioacetamide (500 mg/kg in 1.0 ml of 0.9% NaCl: Sigma Chemicals, St. Louis, MO). Controls were treated with vehicle alone. This model of acute toxic hepatitis has been described in detail elsewhere [3]. Rats were anesthetized for sample collection prior to sacrifice (ketamine HC1 50 mg/ml, Parke-Davis, Morris Plains. N J: xylazine 100 mg/ml, Formenta Animal Health Co., Kansas City, MO: 8:1: v:v: dose 0.3 mg/rat). All samples were collected 24 h after injection when hepatocellular injury is maximal (M.G. Swain, personal observation). Blood was collected by inferior vena cava venipuncture, placed in prechilled EDTA containing glass tubes, and centrifuged at 760 x g for 15 min. The plasma was removed and then stored at -70°C until assayed for methionine enkephalin. The thoracic cavity was then opened and the inferior vena cava and thoracic aorta clamped. A 21 gauge needle was inserted into the left ventricle and a small hole
244
made in the right atrium. Ice-cold 0.9% NaCI was then perfused (2 ml/min) through the left ventricle. The perfusion was continued until the fluid returning to the right atrium was clear. The animal was then decapitated and the brain removed. Cerebrospinal fluid was collected as follows. Rats were anesthetized by an intraperitoneal injection of ketamine/xylazine (as above). The rats were then placed in a Kopf stereotaxic frame and the skin and muscles of the nape of the neck were dissected to expose the membrane over the cisterna magna. A cannula made from a 0.7 cm length of 25 gauge needle fixed to a 10 cm length of PE20 polypropylene tubing was attached to a 1 ml syringe. The needle was inserted into the cisternal space by hand and a 150-200 ¢tl sample of CSF was collected. Animals were then sacrificed by cervical dislocation. CSF samples from two animals were combined for the methionine enkephalin radioimmunoassay. Plasma and CSF samples were acidified with 1 N HC1 and passed over a C18 SepPak column (Waters, Milford, MA). The columns were washed twice with 5% acetic acid and eluted with 3 ml of methanol, The methanol eluates were dried under a stream of nitrogen. Dried samples were reconstituted for assay as previously described [17]. Whole brain methionine enkephalin was extracted using a modification of the method of Miller et al. [10]. Briefly, brains were placed in glass tubes containing ice cold 0.1 N HC1 (5:1 w:v) and were homogenized (Tissumizer, Tekmar, Cincinnati, OH; high setting for 15 s). The homogenates were then centrifuged at 50,000 × g at 4°C for 1 h. The pellet was resuspended in 0.1 N HC1 by homogenization and the samples subjected to a further centrifugation. The pooled supernatants were passed over C18 Sep-Pak columns, washed with 5% acetic acid, eluted with methanol, dried under a stream of nitrogen, and reconstituted for assay of methionine enkephalin. Dilutions were made as necessary. The radioimmunoassay of methionine enkephalin applied has been previously described [13, 17]. It employed a commercially available, C-terminus directed, highly specific antibody (INCSTAR, Stillwater, MN). Antibody cross-reactivities have been outlined previously [13]. Recovery was greater than 90% and inter- and intra-assay coefficients of variation 12% and 7% respectively. Data are expressed as means + S.E.M. and the significance of differences between means was determined using the unpaired Student's t-test. Treatment of rats with thioacetamide resulted in significant hepatocellular injury as documented by plasma biochemistry 24 h post-injection (aspartate aminotransferase: 1824 + 357 IU/liter; normal range 55-458 IU/ liter, MetPath Inc., Kensington, MD [3]). In the thioacetamide injected rats acute hepatocellular necrosis was associated with a striking elevation of plasma immunoreac-
1000
0) >
800
3
600
|_I. 0 C
400
E
_E E i G.
200
I t Saline TAA Injected Injected Fig. 1. Immunoreactive methionine enkephalin levels in plasma extracts from saline-injected and thioacetamide (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per mt plasma and represent means _+ S.E.M. o f at least 9 determinations ('P -< 0.005 vs. saline-injected controls).
tive methionine enkephalin levels with a mean concentration 8.7-fold higher than that in saline injected controls (Fig. 1; P - 0.005). The reason for the elevation of immunoreactive methionine enkephalin levels in plasma 24 h after thioacetamide injection is unknown but is consistent with findings in humans with hepatocellular injury [20]. These observations are of particular interest when the rapid metabolism of enkephalins in the plasma of normal rats is considered [6]. Increased release, decreased metabolism, or both might contribute to elevated plasma levels of immunoreactive methionine enkephalin. The source of circulating methionine enkephatin is poorly defined but the adrenals [16], the gut [14], sympathetic nerves [16], and the brain [1] have all been suggested as possibilities. Methionine enkephalin, like most hydrophilic peptides, is known to penetrate the normal blood-brain barrier poorly [12]. However, there is evidence suggesting that methionine enkephalin crosses the blood brain barrier by specific transport mechanisms [15, 21, 22], and that blood-to-brain transfer of methionine enkephalin is facilitated by prolonged exposure to elevated plasma lev-
245 J
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Fig. 2. Immunoreactive methionine enkephalin levels in whole brain homogenates from saline-injected and thioacetamidc (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per mg brain wet weight and represent m e a n s ± S.E.M. of at least 15 determinations ( P= n.s.).
els of enkephalin [15]. In galactosamine-induced acute hepatocellular injury in the rabbit the blood-to-brain transfer of ~-aminoisobutyric acid, a polar compound normally excluded from the central nervous system, is increased, a finding which can be explained by postulating a non-specific increase in blood brain barrier permeability in this model of acute toxic hepatitis [7]. If a non-specific increase in blood-to-brain transfer of methionine enkephalin occurs in acute severe hepatocellular disease an increase in CSF levels of methionine enkephalin would be expected. The finding of normal CSF levels of methionine enkephalin in the thioacetamide-induced model of acute toxic hepatitis may reflect a modulation of the transport system for this opioid. Support for increased central opioid effects in liver disease is available. Patients with cirrhosis are known to exhibit enhanced sensitivity to opiates such as morphine [8] and patients with pruritus complicating primary biliary cirrhosis exhibit a classical opiate withdrawal reaction when treated with the opiate receptor blocker nalmefene [18]. In addition, it has been proposed that endogenous opioids contribute to the pathogenesis of hepatic encephalopathy and this hypothesis is supported by reports of ameliorations of this syndrome in humans and rats by treatment with the opioid receptor antagonist naloxone [9, 11]. However, in thioacetamide-induced
Saline Injected
TAA Injected
Fig. 3. Immunoreactive methionine enkephalin levels in cerebrospinal fluid from saline-injected and thioacetamide (TAA)-injected rats 24 h after injection. Results are expressed as pg methionine enkephalin per ml cerebrospinal fluid and represent means _+ S.E.M. of 5 determinalions (P-ms.).
acute hepatocellular injury in the rat, whole brain (Fig. 2) and cerebrospinal fluid (Fig. 3) immunoreactive methionine enkephalin levels were not elevated. These data are consistent with the findings of Ferenci et al. who found levels of immunoreactive methionine enkephalin in the frontal cortices of rabbits with galactosamineinduced fulminant hepatic t:ailure to be similar to those in controls [5]. The findings in this study do not exclude the possibility that central opioid effects in liver disease are mediated by endogenous opioids other than methionine enkephalin. Indeed in the rat model ofcholestasis due to bile duct resection methionine enkephalin accounted for only 5% of the increased total opioid activity in serum [171. Thus, we have shown that a striking accumulation of immunoreactive methionine enkephalin occurs in plasma in an animal model of acute toxic hepatitis. However, contrary to expectation, this finding is not associated with an increase in whole brain or cerebrospinal fluid immunoreactive methionine enkephalin levels. These results do not support the hypothesis that increased plasma-to-brain transport of methionine enkephalin occurs in and contributes to the pathophysiology of acute severe hepatocellular disease. 1 Banks. W.A., Kastin, A.J., Vischmam A.J., ('oy. D.H. and Straus,
246
2
3
4
5
6
7
8 9
10
11 12
S.L., Carrier-mediated transport of enkephalins and N-tyr-MlF-1 across blood-brain-barrier, Am. J. Physiol., 251 (1986) E477-E482. Bassett, M.L., Mullen, K.D., Scbolz, B., Fenstermacher, J.D. and Jones, E.A., Increased brain uptake of ?,-aminobutyric acid in a rabbit model of hepatic encephalopathy, Gastroenterology, 98 (1990) 747-757. Castro, J.A., D'Acosta, N., De Ferreyra, D., De Castro, E.C., Diaz, C.R., Comez, M.I. and De Fenos, O.M., Studies on thioacetamideinduced liver necrosis, Toxicol. Appl. Pharmacol., 30 (t974) 79 86. Clement-Jones, V., Lowry, RJ., Rees, L.H. and Besser, G.M., Metenkephalin circulates in human plasma, Nature, 283 (1980) 295 297. Ferenci, R, Pappas, S.C., Munson, RJ., Henson, K. and Jones, E.A., Changes in the status of neurotransmitter receptors in a rabbit model of hepatic encephatopathy, Hepatology, 4 (1984) 186 t 91. Hambrook, J.M., Morgan, B.A., Rance, M.J. and Smith, C.F.C., Mode of deactivation of the enkephalins by rat and human plasma and brain homogenates, Nature, 262 (1976) 782-783. Horowitz, M.E., Schafer, D.F., Molnar, P., Jones, E.A.. Blasberg, R.G., Patlak, C.S., Waggoner, J. and Fenstermacher, J.D., Increased blood-brain transfer in a rabbit model of acute liver failure, Gastroenterology, 84 (1983) 1003-1011. Laidlaw, J., Read, A.E. and Sherlock, S., Morphine tolerance in hepatic cirrhosis, Gastroenterology, 40 (1961 ) 389 396. Lisker-Melman, M., Moreno-Otero, R., Fromm, H.C., Gammal, S.H., Basile, A.S. and Jones, E.A., Behavioural amelioration of hepatic encephalopathy induced by an opiate receptor antagonist in a rat model, Hepatology, 8 (1988) A1247. Miller, R.J., Chang, K.J., Cooper, B. and Cuatrecasas, R, Radioimmunoassay and characterization of enkephalins in rat tissues. J. Biol. Chem., 253 (1978) 531.-538. Ozsoylu, S. and Kocak, N., Naloxone in hepatic encephatopathy, Am. J. Dis. Child., 139 (1985) 749-750. Pardridge, W.M,, Frank, H.J.L., Cornford, E.M., Braun, L.D., Crane, P.D. and Oldendorf, W.H., Neuropeptides and the blood
13
14
15
16
17
18 19
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