Egbert Scholtens

Dose-dependent shifts in the sulfation and glucuronidation of phenolic compounds in the rat in vivo and in isolated hepatocytes: The role of saturation of phenolsulfotransferase

Abstract The role of enzyme-kinetic parameters of sulfotransferase and UDP-glucuronyltransferase in the balance between sulfation and glucuronidation of various phenolic substrates was studied in the rat in vivo after i.v. administration and in isolated hepatocytes. A pronounced shift from sulfation to glucuronidation was observed in vivo upon increasing the dose of two phenols, harmol and phenol. Similar shifts were found when these compounds were incubated with isolated hepatocytes. However, the shift from sulfation to glucuronidation was small when 4-chlorophenol, or absent when 4- t -butylphenol were given in vivo . Such shifts were also absent when 4-chlorophenol and 4- t -butylphenol were incubated at increasing concentrations with isolated hepatocytes. The in vivo results with the various phenols were very similar to the conjugation patterns found in isolated hepatocytes. This suggests that these conjugations in hepatocytes are regulated by similar factors as in the intact animal. In isolated hepatocytes at most 16 per cent of the available pool of inorganic sulfate was consumed during the incubation. Since Cheng and Levy have shown [ J. biol. Chem. 255 , 2637 (1980)] that uptake of inorganic sulfate by hepatocytes is very rapid, the present results suggest that the limitation of sulfation of harmol and phenol at increasing dose was caused by saturation of the overall sulfation process by the acceptor substrate, rather than by depletion of inorganic sulfate.

Absorption, serum levels and urinary excretion of inorganic sulfate after oral administration of sodium sulfate in the conscious rat

The absorption of inorganic sulfate after ingestion was investigated in rats. After oral administration of Na235SO4, 35S radioactivity was measurable in plasma already after 15 min and its plasma concentration reached a peak after about 1.5--2 h. The 35S-radioactivity excreted in urine during 24 h after ingestion of Na235SO4 together with varying amounts of unlabelled Na2SO4 (0.25--5.0 mmol Na2SO4 per rat) indicated an almost complete absorption of inorganic sulfate from the gastrointestinal tract. Determination of the inorganic sulfate concentration in rat serum 2 h after oral administration of 5.0 mmol Na2SO4 revealed a three-fold increase in serum sulfate concentration. The data suggest a rapid and almost complete absorption of inorganic sulfate after oral administration in the rat. Its importance in relation to the sulfate availability for sulfate conjugation of drugs is discussed.

Increased expression of cardiac angiotensin II type 1 (AT1) receptors decreases myocardial microvessel density after experimental myocardial infarction

OBJECTIVE To study the effects of increased levels of myocardial angiotensin II type 1 (AT(1)) receptor on microvascular growth following myocardial infarction (MI). METHODS MI was created in transgenic rats (TGR) with a cardioselective overexpression of the AT(1) receptor. We used Sprague-Dawley (SD) rats as controls. Some of the rats were treated with the selective AT(1) receptor blocker losartan (Los). Rats were sacrificed after 3 weeks. RESULTS MI caused left ventricular (LV) hypertrophy and LV dysfunction in both SD and TGR, which was prevented by AT(1) receptor blockade. Furthermore, MI decreased microvessel density in the non-infarcted myocardium (SD MI: 1653+/-37/mm(2), P<0.01 vs. sham-operated controls), however, microvessel density decreased significantly more in TGR with MI (1298+/-33/mm(2), P<0.01 vs. SD MI). AT(1) receptor blockade restored microvessel density (SD MI Los: 2046+/-195/mm(2); TGR MI Los: 1742+/-47/mm(2); P<0.01 vs. untreated). The differences in microvessel density were still present after correction for LV hypertrophy. The increase in microvessel density after AT(1) receptor blockade was not accompanied by increased myocardial vascular endothelial growth factor (VEGF) levels. Microvessel density correlated with parameters of myocardial stretch, such as LV end-diastolic pressure (-0.681, P<0.001) and N-ANP (-0.424, P=0.01). CONCLUSIONS Microvessel density after MI is decreased when the AT(1) receptor is overexpressed, and this is amenable to AT(1) receptor blockade. This suggests that efficacy of AT(1) receptor blockers post-MI may not only be due to attenuation of LV remodeling, but also to a stimulatory effect on angiogenesis.

The oxidation of L- and D-cysteine to inorganic sulfate and taurine in the rat

Oral administration of L-cysteine to rats (8 mmol/kg body wt.) caused a rapid increase of the concentration of cystine in serum, from less than 5 micro M in controls to about 200 micro M. Concomitantly, the serum concentration of inorganic sulfate increased, reaching a peak 2 h after L-cysteine administration; this level, twice the control level, was maintained for 4 h. Serum sulfate returned to control concentration 23 h after L-cysteine administration. The urinary excretion of inorganic sulfate during the 24 h after administration increased considerably, and 33% of the dose of L-cysteine was recovered as inorganic sulfate in urine. Consumption of comparable amounts of L-cysteine via the food caused the same increase in urinary excretion of sulfate, but did not increase the concentration of sulfate in serum. After oral administration of D-cysteine (8 mmol/kg body wt.), very high cystine levels were reached in serum (mean concentration about 1500 microM); the sulfate concentration was already maximal 30 min after administration. The increase in urinary excretion of sulfate after D-cysteine was also higher than after L-cysteine administration: 55% of the dose. Possible routes for the rapid degradation of D-cysteine to inorganic sulfate are discussed. The administration of L-cysteine also caused an increase the serum concentration of taurine, while methionine was not influenced. D-Cysteine did not increase the serum concentration of taurine, indicating that it is probably not or only slowly converted to taurine.

An evaluation of methods to decrease the availability of inorganic sulphate for sulphate conjugation in the rat in vivo

Abstract The concentration of inorganic sulphate in serum of the rat (about 0.9 mM) could be lowered in the following three different ways. (1) Oral administration of sodium chloride (8 mmol/kg) decreased serum sulphate within 2 hr to 0.5 mM. Eight hours after administration serum sulphate had returned to the control level. (2) Feeding of a low-protein diet (8 per cent casein, without supplements of sulphur-containing amino acids or inorganic sulphate salts) reduced urinary sulphate excretion in 2 days to 10 per cent of control. Concomitantly, serum sulphate was decreased to half the control level. (3) Paracetamol (1.0 or 1.5 mmol/kg, orally), a substrate of sulphation, reduced serum sulphate within 3 hr to 30 per cent of control. Eight hours after administration the sulphate concentration tended to rise again. Fasting initially increased serum sulphate; after 3 days of fasting still considerable amounts of inorganic sulphate were excreted in urine (50–70 per cent of control). Even after 3 days serum sulphate was not yet significantly decreased below control. Lowering of the serum sulphate concentration results in a decreased availability of inorganic sulphate. Sulphation of a high dose of phenol (266 μmol/kg) was decreased at a serum concentration of sulphate of 0.3 mM, presumably because sulphate was depleted by the high dose of phenol. Feeding the low-protein diet, however, caused no decrease in sulphation at a tracer dose of [ 14 C]-phenol (1.25 μmol/kg), while paracetamol pretreatment did cause a decrease in the fraction of the dose that became sulphated, probably because remaining unconjugated paracetamol competed with phenol for sulphation; the tracer dose of [ 14 C]-phenol did not further deplete sulphate. The findings are discussed in relation to implications for the toxicity of many xenobiotics that are eliminated as sulphate conjugates.

[4] Collection of metabolites in bile and urine from the rat

Publisher Summary The procedure for the collection of metabolites from the rat primarily includes the administration of drugs. Further, for the cannulation of the bile duct, a 2-cm incision is made across the abdomen just below the liver. The overlying liver lobes and gut are gently moved out of the way, and the duodenum is gently pulled. The bile duct is noted as a thin vessel that lies between the hilus, where it leaves the liver, and the point at which it joins the duodenum. The upper part of the bile duct is free and should be used for catheterization at about 1 cm from the liver hilus. In this case, the pancreatic ducts are left intact and mixing of bile with pancreatic secretions is avoided. To collect bile for a prolonged period, the bile is collected external to the animal and to the cage by way of a swivel joint that is connected to polyethylene tubing. An alternative method is to implant a reservoir into the peritoneal cavity of the rat for the collection of bile. To study the role of the liver in the conjugation of xenobiotics, two other operative procedures may be useful. A bypass may be used to temporarily exclude the liver from the circulation. Another useful method is the cannulation of a side branch of the portal vein to both administer compounds and to sample directly at the portal circulation. Additionally, a simple way to collect the metabolites of a compound excreted in urine (and feces) is to place the rats in metabolism cages and collect urine and feces separately.

Protective Effects of Captopril against Ischemia/Reperfusion-Induced Ventricular Arrhythmias in Vitro and in Vivo

The effects of the converting enzyme inhibitor captopril on the susceptibility of the heart to ventricular arrhythmias following ischemia, both in vitro and in vivo, were studied. In isolated rat hearts, captopril, administered either before or at the end of ischemia, reduced ventricular fibrillation upon reperfusion after 15 minutes of local ischemia. Reduction of purine overflow, improvement in contractility, and increase in coronary blood flow occurred concomitantly. In vivo, a closed-chest pig model was used to determine the effects of captopril, administered at the end of ischemia and continued orally, on the susceptibility to ventricular arrhythmias during the chronic phase of myocardial infarction. Myocardial ischemia was induced by 60-minute inflation of a balloon catheter in the left anterior descending coronary artery. Upon reperfusion, an accelerated idioventricular rhythm occurred, both in 10 untreated and in 10 captopril-treated animals. Creatine kinase levels during the reperfusion period were significantly lower after captopril treatment. Two weeks after the short-term experiments, monomorphic ventricular tachycardia could be induced with programmed electrical stimulation in six of eight surviving untreated pigs. In contrast, in none of the six surviving captopril-treated animals was ventricular tachycardia inducible. Thus, early intervention with captopril during the development phase of myocardial infarction may have beneficial effects on the subsequent development of ventricular arrhythmias. Salvage of ischemic myocardium, improvement in ventricular function, beneficial effects on coronary flow, and decreased activity of the sympathetic nervous system may all contribute.

Glucuronidation and sulfation in the rat in vivo: The role of the liver and the intestine in the in vivo clearance of 4-methylumbelliferone

The role of the liver in the conjugation of 4-methylumbelliferone (4MU), mainly glucuronidation, was investigated in the rat in vivo. The liver extracted 4MU almost completely (97%) during steady-state infusion, as measured by the difference between 4MU concentration in portal and hepatic venous blood. Previously, it was shown that the intestinal region extracts 40% of the 4MU of the incoming arterial blood. The liver and the gastrointestinal region are so efficient that their conjugation activity can account for total body clearance of 4MU (50-60 ml/min per kg). These results and other evidence on extrahepatic conjugation of phenolic substrates suggest that glucuronidation may be limited to the liver, (the kidney) and the gastrointestinal region, while sulfation may occur more widespread throughout the body. Protein binding studies showed the sulfate conjugate to be even more protein-bound than unconjugated 4MU, while 4MU glucuronide was much less bound to rat plasma proteins.

β-Adrenoceptor density in chronic infarcted myocardium: a subtype specific decrease of β1-adrenoceptor density

Abstract β-adrenoceptor density is altered in different cardiac diseases. In heart failure β-adrenoceptor density is down regulated but in acute myocardial ischemia β-adrenoceptor density is up regulated. In hearts with myocardial infarction total β-adrenoceptor density is decreased shortly after myocardial infarction. Aims and methods: To investigate whether total β-adrenoceptor number is altered in the chronic phase after myocardial infarction, and to identify the specificity of alteration, we studied male Wistar rats ( n =18) which underwent a ligation of the left coronary artery or a sham operation. Twelve weeks after coronary ligation, rats were sacrificed and hearts were excised, perfused to obtain blood-free myocardium and frozen in liquid nitrogen. Infarcted myocardium was identified visually and separated from non-infarcted myocardium. Total β-adrenoceptor number was calculated in fmol (−)-[ 125 I]iodocyanopindolol specifically bound/mg protein and the relative amount of β 1 - and β 2 -adrenoceptor density was measured by inhibition of (−)-[ 125 I] iodocyanopindolol binding with CGP 20712 A. Results: Total β-adrenoceptor number in infarcted myocardium was significantly decreased (25.7±1.4 vs. 24.9±2.2 vs. 20.1±3.2 fmol/mg protein ( P =0.03) resp. Sham vs. Non-infarcted vs. Infarcted myocardium), due to a decrease of only β 1 -adrenoceptor density (14.7±0.61 vs. 12.7±1.09 vs. 4.84±0.96 fmol/mg protein ( P =0.004) resp.), whereas the β 2 -adrenoceptor density and the dissociation constant ( K d ) were not significantly decreased. Conclusion: In the infarcted myocardium total β-adrenoceptor density is decreased due to a decreased β 1 -adrenoceptor density at 12 weeks after myocardial infarction.

The dependence of the rate of sulphate conjugation on the plasma concentration of inorganic sulphate in the rat in vivo

The dependence of the rate of sulphation of harmol [7-hydroxy-1-methyl-9H-pyrido(3,4-b)indole] on the plasma concn of inorganic sulphate was estimated in the rat in vivo. This was done in rats that were fed a low protein diet for 4 days to deplete the pool of inorganic sulphate, which decreased the plasma concn of sulphate from 0.9 mM normally to about 0.15 mM. Infusion of sodium sulphate at a rate that was stepwise increased, yielded a range of constant plasma sulphate levels below and above the physiological concn in plasma. By a constant infusion of harmol (6 mumoles/min/kg body wt), a steady state in the excretion of harmol sulphate could be reached at various plasma concns of sulphate. The apparent Km for inorganic sulphate was approx 0.3 mM. The relevance of these data in the evaluation of the effects of changes in the availability of inorganic sulphate on sulphate conjugation of xenobiotics is discussed.