Angiotensin II subtype AT1 receptor blockade prevents hypertension and renal insufficiency induced by chronic NO-synthase inhibition in rats.

Aim of the present study was to investigate the influence of the angiotensin II (ANG II) subtype 1 (AT(1)) receptor blockers fonsartan and losartan on blood pressure, cardiac -dynamics and -metabolism as well as functional and morphological changes in the kidney of rats after long-term inhibition of the nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester (L-NAME). Oral chronic treatment with L-NAME in a dose of 25 mg/kg/d over 6 weeks caused a significant increase in systolic blood pressure (198+/-13 mmHg) when compared to untreated rats (144+/-4 mmHg). Animals receiving simultaneously L-NAME and fonsartan (10 mg/kg/d) or losartan (30 mg/kg/d) were protected against blood pressure increase. L-NAME treatment caused a significant decrease in glomerular filtration rate (GFR) from 4.52+/-0.81 to 1.34+/-0.26 ml/kg(-1)/min(-1) and renal plasma flow (RPF) from 10.52+/-1.29 ml/kg(-1)/min(-1) to 5.66+/-1.06 ml/kg(-1)/min(-1). Co-treatment with fonsartan and losartan prevented L-NAME-induced reduction in GFR and RPF. There was no difference in urine, sodium and potassium excretion in groups under investigation. Plasma renin activity (PRA) was further stimulated by fonsartan and losartan treatment. L-NAME produced a significant elevation in urinary protein excretion which was antagonised by both AT(1) blockers. Isolated hearts from animals treated with L-NAME showed a significant prolongation in the duration of ventricular fibrillation and a significant decrease in coronary flow as compared to control hearts. Treatment with fonsartan and losartan significantly decreased the duration of ventricular fibrillation as compared to L-NAME group. In addition, both AT(1) blockers given alone significantly reduced the duration of ventricular fibrillation as compared to hearts from untreated controls. During ischemia the cytosolic enzymes lactate dehydrogenase and creatine kinase as well as lactate in the coronary effluent were significantly increased in the L-NAME group. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased. Fonsartan and losartan treatment totally abolished these effects. Histological examination of kidneys revealed that simultaneous administration of fonsartan and losartan with L-NAME abolished L-NAME-induced arteriolar hyalinosis, segmental sclerosis of glomerular capillaries and focal tubular atrophies. In conclusion, long-term blockade of ANG II subtype AT(1) receptors by fonsartan and losartan prevented L-NAME-induced hypertension, renal insufficiency, as well as cardio-dynamic, cardio-metabolic, and morphological deteriorations.

S3226, a novel NHE3 inhibitor, attenuates ischemia-induced acute renal failure in rats.

BACKGROUND: Acute renal failure (ARF) remains a major problem in clinical nephrology characterized by sudden loss of the kidney function due to ischemia, trauma, and/or nephrotoxic drugs. The current therapy of ARF is symptomatic with mortality rates exceeding 50%. The aim of this study was to investigate the effects of an intravenous infusion of S3226 (3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride), a selective Na+/H+ exchange subtype 3 (NHE3) blocker, in ischemia-induced ARF in rats. In a second series of experiments cytosolic pH (pHi) changes in the kidney during ARF were continuously measured by means of nuclear magnetic resonance spectroscopy (MRS). METHODS: ARF was induced by bilateral occlusion of renal arteries for 40 minutes in three groups of anaesthetized Wistar rats. Control rats (N = 12) were infused with saline (6.25 mL/kg over 30 min) before occlusion and the compound groups (each N = 12) were infused with S3226 at a dose of 20 mg/kg over 30 minutes either before initiation of ischemia or immediately after release of clamps. Plasma creatinine (PCr), creatinine clearance (CCr), urine volume, sodium, and potassium excretion were determined up to seven days after release of clamps. In the second series of experiments in anaesthetized rats the left kidney was exposed by flank incision and fixed in a non-magnetic device. An inflatable cuff was positioned around the pedicle to induce ischemia without removing animals from the magnet. A double-tuned 1H-31P home-built surface coil was placed above the exposed kidney for the detection of pHi. RESULTS: At day 1 after ischemia CCr in the control group was significantly lower as compared to S3226-treated animals (control 0.30 +/- 0.05 vs. before 0.90 +/- 0.26 and reperfusion 0.83 +/- 0.15 mL/min/kg, respectively). PCr increased from 18 +/- 0.1 micromol/L before occlusion to 245 +/- 7 micromol/L in the control. The increase in PCr was significantly lower in the S3226 treated groups on days 1, 2, and 3 post-infusion. Fractional sodium excretion decreased significantly from 8.17% in the control to 1.42% and 1.88% in the treated groups. Renal pHi was significantly decreased by 0.15 units versus control during reperfusion. Histological examination of the kidneys on day 7 revealed pronounced reduction of tubular necrosis, dilatation, protein casts and cellular infiltration. CONCLUSIONS: These results demonstrate that an intravenous administration of S3226 acutely improves GFR and kidney function and structure in both treated groups. In addition, in a separate set of studies S3226 significantly decreased post-occlusion renal pHi values. Thus, the inhibition of NHE3 with S3226 may be beneficial in treatment of ischemic ARF.

Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron.

This study assessed the functional role of Na(+)/H(+) exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid (J(v)) and HCO(3)(-) (J(HCO3)) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J(v) and J(HCO3), but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J(HCO3) was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J(HCO3) was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

Role of Na(+)/H(+) exchanger NHE3 in nephron function: micropuncture studies with S3226, an inhibitor of NHE3.

Na(+)/H(+) exchanger NHE3 is expressed in the luminal membrane of proximal tubule and thin and thick ascending limb of Henle's loop. To further define its role, the novel NHE3 inhibitor S3226 was employed in micropuncture experiments in nephrons with superficial glomeruli of anesthetized rats. Microperfusion of proximal convoluted tubule with S3226 revealed a dose-dependent inhibition of reabsorption (IC(50) of 4-5 microM) with a maximum inhibition of 30% for fluid and Na(+). During microperfusion of Henle's loop (last superficial proximal to first superficial distal tubular loop), no effect of S3226 (10 or 30 microM) on the reabsorption of fluid or Na(+) was observed. Finally, S3226 (30 microM) left the tubuloglomerular feedback response unaltered as determined by the fall in proximal tubular stop-flow pressure in response to increasing loop of Henle perfusion rate. These studies indicate that NHE3 significantly contributes to fluid and Na(+) reabsorption in proximal convoluted tubule. NHE3 appears not to significantly contribute to fluid or Na(+) reabsorption in the loop of Henle (including the S3 segment of proximal tubule) or macula densa control of nephron filtration.

[Renal morphometric investigations in pre-eclampsia-like syndrome in a Wistar rat model]. / Nierenmorphometrische Untersuchungen an einem Präeklampsie-ähnlichen Modell bei der Wistarratte.

OBJECTIVE: Etiology and pathogenesis of renal changes in pregnancy-induced hypertension (PIH) remain somewhat controversial. Reducing the uterine perfusion leads to the development of systemic hypertension in animal models. Aim of this study was to investigate the effect of systemic hypertension on the kidney during pregnancy. MATERIAL AND METHODS: A rat model was used and the degree of the renal changes was quantified by morphometry. Normotensive pregnant and virgin animals served as a control. RESULTS: Hypertensive animals showed a decrease of the intravascular space in comparison to the cellular component. This difference was significant not only in pregnant (p = 0.0026) but also in virgin (p = 0.001) animals. CONCLUSIONS: These findings indicate that renal changes are usually found in normotensive pregnancies, while PIH strongly aggravates these changes.

[Animal experiment models of hypertension in pregnancy--an alternative to clinical studies?]. / Tierexperimentelle Modelle des Schwangerschaftshochdrucks--Eine Alternative zu klinischen Studien?

UNLABELLED: Because of ethical constraints upon research during human pregnancy and of the heterogeneity of preeclampsia a number of experimental animal models were used to study pathophysiology of preeclampsia. We reduced the uteroplacental flow by aortic clip technique in 72 SHR-rats in 4 groups (G 1 not gravid--no clip, G 2 not gravid--aortic clip, G 3 gravid--no clip, G 4 gravid--aortic clip). While gravidity normalised blood pressure in SHR (G 3), reduction of the uteroplacental flow significantly increased maternal blood pressure (G1 240/180 mmHG, G2 238/183 mmHg, G3 148/99 mmHG, G4 200/145 mmHg) and leads to significant higher hematocrit, significant lower plasma renin activity and aldosterone plasma concentration, as it is found in human superimposed preeclampsia. SDS-page electrophoresis proved a significant increase of urinary high density proteins. CONCLUSION: In comparison to chronical inhibition of NO-synthesis or chronical application of ultra-low-doses-endotoxin, reduction of uterine flow by aortic compression is a reliable animal model and leads in SHR to a superimposed preeclampsia-like syndrome with many similarities to human clinical findings. Animal models for preeclampsia are no alternative to clinical studies, but they are furthering our understanding of pathophysiology in many fields.

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

The bradykinin B2 receptor antagonist Icatibant (HOE 140) corrects avid Na+ retention in rats with CCl4-induced liver cirrhosis: possible role of enhanced microvascular leakage.

Avid Na+ retention is a hallmark of liver cirrhosis. The aim of this study was to investigate whether and how bradykinin is involved in Na+ retention in rats with CCl4-induced liver cirrhosis. To this end the bradykinin B2 receptor antagonist Icatibant (HOE 140) was used. On one hand, bradykinin has a renal natriuretic action. On the other hand, bradykinin is a potent mediator of both vasodilation and microvascular leakage. Both vascular mechanisms, which are reported for cirrhosis, could cause vascular underfilling and Na+ retention by activating the renin-angiotensin-aldosterone system. Icatibant normalised Na+ retention and reduced the hyperactivity of the renin-angiotensin-aldosterone system, suggesting a bradykinin-induced vascular disturbance. Icatibant had no significant effect on the mild hypotension which developed with CCl4 treatment. However, there was indirect evidence for enhanced microvascular leakage that was strongly inhibited by Icatibant. Our experimental results demonstrate that bradykinin is a key mediator of Na+ retention in liver cirrhosis and suggest that a bradykinin-induced increase in microvascular leakage is mainly responsible.

A new class of inhibitors of cAMP-mediated Cl- secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.

Previously we have shown that arylaminobenzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (Isc) induced by prostaglandin E2 (n = 7), vasoactive intestinal polypeptide (VIP, n = 5), adenosine (n = 3), cholera toxin (n = 4) and cAMP (n = 6), but not by ionomycin (n = 5) in distal rabbit colon half maximally (IC50) at 2 mumol/l from the mucosal and at 0.7 mumol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116 +/- 16 omega.cm2 to 136 +/- 21 omega.cm2 (n = 5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound within this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mumol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mumol/l 293 B had no effect on the membrane voltage across the basolateral membrane (Vbl) in non-stimulated crypt cells: -69 +/- 3 mV versus -67 +/- 3 mV (n = 10), whilst in the same cells 1 mmol/l Ba2+ depolarised Vbl significantly. However, 293 B depolarised Vbl significantly in the presence of 1 mumol/l forskolin: -45 +/- 4 mV versus -39 +/- 5 mV (n = 7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised Vbl from -40 +/- 5 mV to -30 +/- 4 mV (n = 19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mumol/l 293 B: -75 +/- 6 mV versus -75 +/- 6 mV (n = 6). Also 293 B had no effect on basal K+ conductance (n = 4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl- secretion and the basal K+ conductance is insufficient to support secretion.

Ramipril prevents the detrimental sequels of chronic NO synthase inhibition in rats: hypertension, cardiac hypertrophy and renal insufficiency.

Inhibition of the angiotensin converting enzyme (ACE) with ramipril was studied in male Wistar rats during long-term inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME). Chronic treatment with L-NAME in a dose of 25 mg/kg per day over 6 weeks caused myocardial hypertrophy and a significant increase in systolic blood pressure (245 +/- 16 mmHg) as compared to controls (155 +/- 4 mmHg). Animals receiving simultaneously L-NAME and ramipril were protected against blood pressure increase and partially against myocardial hypertrophy. L-NAME caused a significant reduction in glomerular filtration rate (GFR: 2.56 +/- 0.73 ml.kg-1.min-1) and renal plasma flow (RPF: 6.93 +/- 1.70 ml.kg-1.min-1) as compared to control (GFR: 7.29 +/- 0.69, RPF: 21.36 +/- 2.33 ml.kg-1.min-1). Addition of ramipril prevented L-NAME-induced reduction in GFR and renal plasma flow. L-NAME produced an elevation in urinary protein excretion and serum creatinine and a decrease in potassium excretion which was antagonised by ramipril. L-NAME-induced increase in plasma renin activity (PRA) was further elevated with ramipril treatment. Isolated hearts from rats treated with L-NAME showed increased post-ischaemic reperfusion injuries. Compared to controls duration of ventricular fibrillation was increased and coronary flow reduced. During ischemia the cytosolic enzymes lactate dehydrogenase and creatine kinase, as well as lactate in the venous effluent were increased. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Furosemide enhances the release of endothelial kinins, nitric oxide and prostacyclin.

Despite a wealth of data, the mechanism of the direct dilator effect of furosemide on the systemic arterial and venous systems is far from being satisfactorily understood. Therefore, we investigated whether furosemide is capable of stimulating the production of the endogenous vasodilators nitric oxide and prostacyclin in primary cultured bovine aortic endothelial cells by an enhanced synthesis and release of endothelium-derived kinins. Nitric oxide production was assessed in terms of intracellular guanosine cyclic-3',5' monophosphate accumulation; kinin and prostacyclin release were determined by specific radioimmunoassays. Furosemide concentration- and time-dependently increased the formation of nitric oxide and prostacyclin. Maximal increases of both autacoids were already obtained after a 5-min incubation with 3 x 10(-7) to 10(-6) mol/l of furosemide. In the same concentration range, furosemide led to an enhanced release of kinins into the supernatant of the cells. This observation was supported by the inhibitory effect of the specific B2 kinin receptor antagonist icatibant (Hoe 140) on the furosemide-induced increase of nitric oxide and prostacyclin. Thus the hemodynamic effects, and in particular the direct early dilator effect, of furosemide may be explained in part by an enhanced endothelial synthesis and release of bradykinin and related kinins, which in turn stimulates endothelial autacoid formation via B2 kinin receptor activation.

Incidence of apparent restricted diffusion in three different models of cerebral infarction.

High speed magnetic resonance imaging (MRI) and short diffusion times are used to investigate the appearance of restricted diffusion in three different models of cerebral infarction. The models are: the middle cerebral artery occlusion (MCAO) model in the rat, the carotid occlusion model in the gerbil, and the Rose Bengal microvascular occlusion model in the rat. All three were investigated for 16 b-values equally spaced between 10 and 1510 s/mm2 using two distinct experiments. In the ct (constant time) experiment, the diffusion time was held constant at 11.7 ms while the b-value was varied with the gradient strength. In the cg (constant gradient) experiment, the gradient strength was held constant and the b-value increased by varying the diffusion time from 4.4 to 11.7 ms. A monoexponential decay of the signal intensity with b-value in the ct experiment accompanied by nonmonoexponential (NME) decay in the cg experiment is indicative of restricted diffusion. As this phenomenon is detectable only at short diffusion times, it cannot be due to restriction by impermeable membranes, and we have thus termed this apparent restriction. For the MCAO model and the carotid occlusion model, apparent restriction was found both inside the infarct territory and in some regions outside it. No definite evidence for restriction was found for the Rose Bengal model, which was, however, only studied from 24 h post-insult.

Diminished diuretic and natriuretic response to furosemide in potassium-depleted rats.

Renal clearance and tubule microperfusion experiments were carried out to investigate the effects of chronic potassium depletion upon the renal response to furosemide. Rats kept on a potassium-deficient diet for 3 weeks developed hypokalemia, metabolic alkalosis, and decreased aldosterone levels. These rats responded to an oral administration of furosemide (32 mg/kg) with a blunted excretion rate of urine and sodium. Whereas furosemide increased fractional urine sodium excretion to 5.2% in control rats, the corresponding rate in potassium-depleted rats was 2.8%. The urinary excretion of furosemide was also significantly reduced during potassium depletion from 3.06 mg/kg in control rats to 0.97 mg/kg in potassium-depleted rats. In separate experiments, loops of Henle were pump-perfused with furosemide-containing solutions in control and potassium-depleted rats. No major modification of the inhibitory effects of furosemide on sodium transport was observed when the potassium concentration of the perfusion fluid was kept at the low levels expected in hypokalemic rats. Metabolic alkalosis unaccompanied by potassium deprivation did not decrease the diuretic response to furosemide. These experiments indicate that potassium deprivation reduces the diuretic effects of furosemide by mechanisms including diminished furosemide delivery to its tubule site of action.

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon.

Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (Vte) and resistance (Rte) were measured and the equivalent short-circuit current (Isc = Vte/Rte) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 mumol/l) Isc was -71 +/- 11 (n = 118) microA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to +32 +/- 4 (n = 118) microA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 mumol/l, s), known to induce Cl- secretion, generated an Isc of -143 +/- 8 (n = 92) microA/cm2. The arylaminobenzoate and Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced Isc reversibly with a half-maximal inhibition (IC50) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC50 to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-L-cysteine (m) preincubation. Pretreatment with N-acetyl-L-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and L-homocysteine (m) were more effective and reduced IC50 to approximately 50 mumol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/-1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl- conductance in the thick ascending limb of the loop of Henle (TAL).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of seasonal biorhythms on urinary excretion of enzymes and other parameters.

Urinary excretion of enzymes, electrolytes, creatinine, protein and the consumption of food and water in female and male Wistar rats was examined over two seasons of summer and wintertime, respectively. The evaluation of data revealed that gamma-glutamyltransferase, inorganic phosphate, and the lysosomal enzymes, N-acetyl-beta-D-glucosaminidase and acid phosphatase, were significantly different between the collection periods in both seasons. Other parameters showed significant differences only in male rats, whereas urinary electrolytes, with the exception of chloride, showed no significant differences.

Enzymuria in streptozotocin-diabetic rats.

Twenty four hour urine samples of male control and streptozotocin-diabetic Wistar rats were analysed for a series of commonly known kidney-specific enzymes, for electrolytes, creatinine, glucose, total protein and urine volume. The examination was done during two periods of 5 days between the 25th and 30th and the 32nd and 36th day after streptozotocin application. In the first period the animals had free access to food and water, whereas in the second period on days 32, 34 and 36 food was withdrawn. In the first observation period the diabetic rats showed increased excretion rates of 15 measured urinary parameters, while alanine aminopeptidase (EC 3.4.1.2) and gamma-glutamyltransferase (EC 2.3.2.2) activities were lowered and inorganic phosphate was unchanged. The removal of food resulted in decreased excretion values for alanine aminopeptidase, gamma-glutamyltransferase and total protein as compared with fasted nondiabetic animals. The activities of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), acid phosphatase (EC 3.1.3.2), lactate dehydrogenase (EC 1.1.1.27), pyruvate kinase (EC 2.7.1.40), C1-fructose 1.6-diphosphatase (EC 3.1.3.11) and the excretion values for sodium, calcium, magnesium, chloride and glucose were higher than in fasted nondiabetic rats. beta-Glucosidase (EC 3.2.1.21), potassium, inorganic phosphate, creatinine, and urine volume showed no differences between fasted diabetic and fasted control animals. The enzymes in the renal cortex at the end of the experiment showed only decreased activity of alanine aminopeptidase in diabetic rats. Lactate dehydrogenase, pyruvate kinase, beta-glucosidase, C1-fructose 1.6-diphosphatase and glucose 6-phosphatase (EC 3.1.3.9) were increased and gamma-glutamyltransferase, N-acetyl-beta-D-glucosaminidase, acid phosphatase and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) showed no change.(ABSTRACT TRUNCATED AT 250 WORDS)