Human gastrointestinal sulfotransferases: identification and distribution

Sulfotransferases (STs) catalyze the sulfation of many structurally diverse molecules. Enzymatic assays and Western blots have been used to identify and characterize STs in the human gastrointestinal tract. Sulfation activities for 2-naphthol, dopamine, estradiol, and dehydroepiandrosterone (DHEA) from 23 donors were measured in cytosol prepared from stomach, duodenum, segments of small intestine, and colon and were compared to levels in human liver cytosol. Stomach and colon had low 2-naphthol and dopamine sulfation activities and almost no estradiol and DHEA sulfation activity. For all four substrates, small intestine has higher activities than both stomach and colon. Human small intestine 2-naphthol sulfation specific activity is approximately half that of human liver. Human small intestine dopamine sulfation activity is three times as high as that of human liver. While estrogen sulfation activity is about the same for both human intestine and human liver, human liver DHEA sulfation activity is about five times as high as that of human small intestine. The distribution of ST activities along the length of the small intestine was very different among different donors. Some donors had higher activity in the proximal segments of the small intestine, whereas other donors had higher activity in the distal segments of the small intestine. Our results also demonstrated high variation of small intestine sulfation activities compared with human liver activities among different donors. The Western blot results agreed with the enzymatic assay results. These results suggest that xenobiotics may regulate human small intestinal STs.     

Sulfation of dietary flavonoids by human sulfotransferases

1. Dietary flavonoids catechin, epicatechin, eriodictyol, and hesperetin were investigated as substrates and inhibitors of human sulfotransferases (hSULTs). Purified recombinant proteins and human intestine cytosol were used as enzyme sources.

2. hSULT1A1 and hSULT1A3 as well as human intestine cytosol can catalyse the sulfation of the investigated flavonoids.

3. Sulfation of catechin, epicatechin, eriodictyol, and hesperetin by recombinant hSULTs showed substrate inhibition at high flavonoid concentrations.

4. Hesperetin and eriodictyol are potent inhibitors of purified hSULT1A1, hSULT1A3, hSULT1E1, and hSULT2A1. Catechin and epicatechin inhibited hSULT1A1 and hSULT1A3, but not hSULT1E1 and hSULT2A1.

5. The sulfation efficacy and potency of inhibition is related to the C-ring structure of flavonoids. These results suggest that dietary flavonoids may regulate human SULT activity and, therefore, affect the regulation of hormones and neurotransmitters, detoxification of drugs, and the bioactivation of pro- carcinogens and pro-mutagens.

     

Pharmacogenetics of soluble sulfotransferases (SULTs)

Soluble sulfotransferases (SULTs) transfer the sulfo group from the cofactor 5-phosphoadenosine-3-phosphosulfate (PAPS) to nucleophilic sites of relatively small acceptor molecules including various hormones and numerous xenobiotics. Sulfo conjugation of xenobiotics can lead to the formation of polar, excretable products as well as reactive, potentially mutagenic and carcinogenic metabolites. Ten SULT genes encoding 11 proteins have been identified in the human. They differ in substrate specificity and tissue distribution. Genetic polymorphisms have been detected in all human SULT genes. The functional significance of any polymorphisms that do not affect the amino acid sequence has not yet been studied. Non-synonymous single-nucleotide exchanges have been observed in SULT1A1, 1A2, 1B1, 1C1, 1C2 and 2A1. Functional consequences have primarily been explored using cDNA-expressed alloenzymes. Furthermore, an Arg213His polymorphism in SULT1A1 has a strong influence on the level of enzyme protein and activity in platelets, which have been widely used for phenotyping. Compared to other xenobiotic-metabolizing enzymes, only few studies have been conducted on associations of SULT genotypes with diseases and other health-related parameters. Statistically significant associations were observed between the SULT1A1 genotype (Arg213His) and age, obesity and certain neoplasias (mammary, pulmonary, esophageal and urothelial cancer). However, these findings require corroboration and specification. The association with neoplasias appears to be complex and varies between subgroups. This is not surprising, as SULTs are involved in the activation of some carcinogens, in the inactivation of other carcinogens, and the regulation of many hormones. It is important to study these functions of SULTs in more detail and to take into account the corresponding environmental and endogenous exposures in epidemiological studies.     

比较多巴胺和左旋多巴对大鼠肝脏芳基硫酸转移酶和雌激素硫酸转移酶的诱导作用（英文）

硫酸转移酶是主要的II相药物代谢酶之一。芳基硫酸转移酶包括rSULT1A1和rSULT1E1,能够催化酚类化合物发生硫酸化。多巴胺是中枢神经系统重要的神经递质,并且能调控各种外周功能。本研究旨在探究多巴胺和左旋多巴对大鼠肝脏硫酸转移酶（rSULT1A1,rSULT1E1）的作用。分别给予雄性大鼠、雌性大鼠不同浓度多巴胺（0,2,10,100 mg/kg/d）及左旋多巴（0,5,25,125 mg/kg/d）。采用real-time PCR以及western blot方法测定rSULT1A1和rSULT1E1的mRNA和蛋白表达;采用PNPS以及放射性的方法测定rSULT1A1和rSULT1E1活性。结果表明：多巴胺增加两种性别大鼠肝脏中rSULT1A1的表达和活性,而对rSULT1E1无显著作用;左旋多巴仅对雄性大鼠肝脏的rSULT1E1有诱导作用,但对两种大鼠肝脏中rSULT1A1以及雌性大鼠的rSULT1E1未产生显著作用。研究结果说明多巴胺在外周和中枢神经系统对rSULTA1和rSULTE1的诱导作用是不同的。     

Mechanisms of gender-specific regulation of mouse sulfotransferases (Sults)

1. Marked gender differences in the expression of sulfotransferases (Sults) are known to exist in several species including rats, mice and hamsters. However, the mechanism for this gender difference is not known. Therefore, in the present study, it was determined whether sex and/or growth hormone (GH) are responsible for the gender difference in the expression of Sults using gonadectomized (GNX), hypophysectomized (HX) and GH-releasing hormone receptor-deficient little (lit/lit) mouse models.

2. Sult1a1 and Papss2 in liver and kidney, and Sult1d1 in liver are female-predominant in mice because of suppressive effects of both androgens and male-pattern GH secretion. Sult2a1/a2 is the most markedly female-predominant Sult in mouse liver due to suppressive effects of androgens and male-pattern GH secretion, as well as stimulatory effects by estrogens and female-pattern GH secretion. Sult3a1 is female-predominant in mouse liver due to suppressive effects of androgens as well as stimulatory effects of estrogens and female-pattern GH secretion. Sult1c1 expression is male-predominant in mouse liver and kidney because of stimulatory effects of androgens in males. Sult4a1 expression is female-predominant in mouse brain due to stimulatory effects of estrogens.

3. In conclusion, gender-divergent Sults are mostly female-predominant and Sult1c1 is the only male-dominant Sult. The gender differences in expression of various mouse Sults are influenced by various mechanisms involving sex and/or GHs.

     

Sulphation of the heterocyclic amine 1,2,3,4-tetrahydroisoquinoline in the human liver and intestinal mucosa: interindividual variability

The sulphation rate of 1,2,3,4-tetrahydroisoquinoline (TIQ) was measured in the human liver and in the intestinal mucosa isolated from the transverse colon, ileum and duodenum. The rate (mean ± SD) of hepatic TIQ sulphation was 500 ± 174 pmol/min per mg in women (n=61) and 591 ± 201 in men (n=39; P=0.0087), varying over one order of magnitude in men and women. The sulphation rate of testosterone showed the same sex-dependent pattern and was correlated (r=0.6055; P<0.001) with that of TIQ. The frequency distribution of TIQ sulphation rate in human liver was bimodal: 70% of the population fell into the low-activity subgroup and the remaining 30% feel into the high-activity subgroup. In the colon (n=56), the rate of TIQ sulphation was 30.4 ± 15.6 pmol/min per mg and the values were similar in men and women (29.8 and 30.9 pmol/min per mg, respectively) but, varied over one order of magnitude and correlated (r=0.7231; P<0.001) with that of 4-nitrophenol. The rate of TIQ sulphation changed along the human bowel and mean (±SD) estimates for duodenum, ileum and transverse colon were 444 ± 25, 182 ± 87  and 30.4 ± 15.6 pmol/min per mg, respectively. The present results are consistent with the view that the heterocyclic amine TIQ is sulphated in the human liver and intestinal mucosa. TIQ-sulphotransferase activity varies among subjects and is mostly associated with the liver and duodenum. Received: 12 December 1996 / Accepted: 19 March 1997     

P450酶和转运体的DNA甲基化调控：提示药物反应的个体差异

细胞色素P450酶和转运体的基因多态性已被公认是导致临床上药物反应个体差异的重要原因,但个体间某些代谢酶、转运体的基因型和表型不一致的现象不能完全用基因多态性来解释。表观遗传药理学从表观遗传学的角度来研究遗传因素与药物治疗的关系,为药物反应的个体差异提供了新的解释。P450酶和转运体都受表观遗传因素控制。最常见表观遗传调控机制是DNA甲基化,它不会改变基因的遗传代码,而影响基因的表达。由于它对基因组序列的维护,DNA甲基化可用来解释某些基因多态性与表型的不一致现象。本综述总结了DNA甲基化表观遗传调控机制对P450酶和转运体的基因表达影响的最新进展。     

Effect of scoparone on the hepatic sulfotransferase activity in mice

Effect of scoparone (6,7-dimethoxycoumarin) on the hepatic cytosolic sulfotransferase activity was investigated. After treatment with scoparone, hepatic cytosolic sulfotransferase activity was increased with dose and time-dependent manner as compared to control. The V _max value (control=1.33 n moles/mg protein/min, scoparone=2.39 n moles/mg protein/min) without affecting the K _m value for p-nitrophenol was increased by the scoparone treatment. Whereas, the hepatic cytosolic sulfotransferase was not changed by the addition of scoparonein vitro, and was strongly inhibited by the addition of metabolites of scoparone. The results obtained suggest that the characteristics of increase in the enzyme activity may include induction of enzyme proteins, and may be due to the metabolites of scoparone.     

Effects of delta 9-THC on human platelet phospholipids

The possible change in Platelet lipids after smoking delta 9-THC was studied in chronic hashish users. The fluctuations of total phospholipid content is related to alterations of individual phospholipids. Changes in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine are discussed in relation to membrane derangement leading to the increased rate of platelet lysis and aggregation under high doses of the drug.     

Rapid and reversible desensitisation of vascular and platelet alpha2 adrenoceptors

Summary 1. The effects of intravenous infusion with the alpha₂ adrenoceptor selective agonist alpha methylnoradrenaline on pressor responses to alpha adrenoceptor agonists, alpha₂ adrenoceptor mediated platelet aggregation and adenylate cyclase were examined in conscious rabbits. 2. Pressor responses to alpha methylnoradrenaline but not phenylephrine were decreased in a dose dependent manner during methylnoradrenaline infusion at all times examined. 3. Recovery of these responses after stopping infusion was dependent on both the dose infused and the duration of the infusion. 4. Alpha methylnoradrenaline infusion resulted in a dose and time dependent decrease in the pro-aggregatory response of platelet to adrenaline without any significant change in the response to ADP or in the number of [³H] yohimbine binding sites. 5. The ability of PGE₁ to stimulate adenylate cyclase was not influenced by alpha methylnoradrenaline infusions. However, reversal of this stimulation by adrenaline was decreased by relatively long (30 min) infusions of the highest dose of alpha methylnoradrenaline examined. 6. It is concluded that alpha methylnoradrenaline infusions resulted in desensitisation of all the alpha₂ adrenoceptor mediated responses examined. However the time course for the desensitisation apparently differed according to the response examined. Send offprint requests to C. A. Hamilton at the above address     

Role of neutrophils in gastric damage induced by platelet activating factor

Summary Platelet-activating factor (PAF) has recently been shown to be a potent ulcerogenic agent in the stomach and intestinal mucosa. Its exact mechanism of action is not yet known although histological studies suggest that vasocongestion is an important feature of PAF-induced damage. We have therefore studied the activity of various agents with different modes of action toward PAF-induced gastrointestinal lesions in the rat (PAF 2 g/kg i.v. ; macroscopic lesions of tissues scored 20 min later; arbitrary scale from 0 to 4). Drugs were administered either i. m., s. c. (5 min) or orally (30 min) before PAF injection. PAF-induced gastric lesions were strongly inhibited by the natural PAF-antagonist BN 52021 as well as by atropine sulphate and cimetidine which implicates cholinergic stimulation in the ulcerogenic activity of PAF. The somatostatin analog BIM 23014 was also very potent against PAF, perhaps by reducing the parasympathetic stimulation in the gastric wall as described for somatostatin. Allopurinol, which is a free radical scavenger also almost totally inhibited PAF-induced gastric damage, suggesting that neutrophils are involved in the mucosal lesions. The considerable inhibition of the gastric effects of PAF found in neutrophil-depleted animal supports this hypothesis. Theophylline and disodium cromoglycate, mast cell stabilizing drugs which were also active in our model, could act by protecting mast cell degranulation induced by free radicals released from activated neutrophils. A multifunctional process seems to determine the mucosal gastric damage induced by PAF, but parasympathetic stimulation and neutrophil activation play a major role in this pathology.Send offprint requests to A. Etienne at the above address     

Human platelet 5HT receptors: Characterisation and functional association

Normal human blood platelets in plasma were incubated at 2°C with tritiated 5-hydroxytryptamine ([³H]5HT), and the specific receptor binding was displaced by the addition of unlabelled 5HT. The kinetic parameters of this binding were established and a two-site model for the platelet 5HT receptor demonstrated. Site A has a K_D of 0.5–1 nM and capacity of 6–10 fmol/10^* platelets, and site B a K _D of 15–36 nM and capacity of 100–150 fmol/10⁸ platelets. Non-specific or non-receptor binding of [³H]5HT to platelets at 2°C was resolved into a passive linear component and an active saturable component sensitive to metabolic inhibition. Binding to the lower affinity 5HT receptor site was inhibited by drugs of the tricylic antidepressant type with IC₅₀ values similar to those against the active uptake component of non-specific binding as described. Isomers of the neuroleptic drug flupenthixol showed a differential and competitive antagonism of high affinity [³H]5HT binding. The 5HT antagonist methysergide, and pizotifen and mianserin also were competitive inhibitors at this site. The rank order of potency of these drugs correlated with thier action as inhibitors of 5HT induced platelet aggreation. It is conclude that bindin of [³H]5HT to intact human platelets satisfies all the critical for specific binding and that the two sites demonstrated, of high and lower affinity, are concerned with the functions of 5HT induced aggregation and 5HT uptake respectively.     

Size determination of binding polymers for [3H]imipramine and [3H]paroxetine in human platelet membranes

Imipramine and paroxetine both inhibit the transport of serotonin in serotonergic neurons and in platelets; furthermore specific high affinity binding sites for [3H]imipramine and [3H]paroxetine are located in these two cell types, probably on the serotonin transport mechanism. However, previous studies indicated that the binding site for [3H]imipramine was different from the binding site for [3H]paroxetine. We now report that the polymers on which the two binding sites are located have different molecular weights.     

Characterization of soluble platelet guanylyl cyclase with peptide antibodies

Summary Soluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the ₁- and ₁-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the ₁-subunit which did not react with the human platelet but with the bovine platelet ₁-subunit. Furthermore the human platelet ₁-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.Abbreviations GC_pep2 YGPEVWEDIKKEA (one letter code) - GC_pep3 SRKNTGTEETEQDEN - GC_pep5 VYKVETVGDKYMTVSGLP - GC_pep8 KKDVEEANANFLGKASGID - TBS-T Tris-buffered saline, containing 0.0501o Tween 20 Correspondence to E. Böhme at the above address     

Reduced basal nitric oxide bioavailability and platelet activation in young spontaneously hypertensive rats

OBJECTIVE: To investigate the role of basal nitric oxide (NO) bioavailability for platelet activation in young spontaneously hypertensive rats before onset of hypertension. Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) in platelets was used as a sensitive monitor of in vivo NO bioavailability. METHODS AND RESULTS: Whole blood samples were taken from 10-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In vivo surface-expression of P-selectin and platelet-binding of fibrinogen were assessed by flow cytometry. Platelet VASP-phosphorylation at its serine 239 (Ser239) and serine 157 (Ser157) residues was assessed using specific antibodies to determine NO bioavailability in vivo, and compared with endothelial vasomotor function. The increment in vascular tone following inhibition of NO-synthase in slightly preconstricted aortic rings was reduced indicating less NO formation under physiological stimulation (WKY 71.1+/-4.1%; SHR 57.8+/-2.4%, P<0.05). In vivo platelet VASP-phosphorylation was significantly reduced at both phosphorylation sites in SHR (mean fluorescence for Ser239: WKY: 15.2+/-0.6; SHR: 11.7+/-0.5, P<0.01; Ser157: WKY: 53.0+/-3.0; SHR: 35.0+/-3.5, P<0.05). Surface-expression of P-selectin and membrane-bound fibrinogen were significantly enhanced in SHR compared with WKY (P-selectin: WKY: 23.2+/-3.4; SHR 58.3+/-7.9, P<0.001; platelet-bound fibrinogen: WKY: 8.6+/-0.5; SHR: 13.5+/-1.1, P<0.001). In vitro preincubation of platelets with the NO donor sodium nitroprusside normalized platelet surface-expression of P-selectin in SHR. CONCLUSION: Using VASP-phosphorylation as a sensitive monitor of in vivo NO bioavailability, these data provide evidence that reduced vascular NO formation in vivo contributes to increased platelet activation in young SHR.     

Human platelet imipramine recognition sites: Biochemical and pharmacological characterization

The influence of the membrane environment on the integrity of the human platelet [³H]-imipramine recognition site was examined. When platelet membranes were isolated in a buffer containing enzyme inhibitors (EDTA, EGTA and antiproteases) a significantly greater number of high affinity [³H]-imipramine binding sites was observed. A calcium-stimulated degradation of imipramine sites was also demonstrated. This degradation occurred in vitro over physiologically relevant time periods. Furthermore, inactivation of imipramine binding was achieved by very low concentrations (1C₅₀=5 μg/ml) of phospholipase A₂. Specific serotonin reuptake inhibitors were potent displacers of [³H]-imipraminebinding; histamine (H₁), alpha-adrenergic (α₁),, and muscarinic agents were much less active. The receptor was shown to be proteinaceous in nature due to its sensitivity to proteases, heat denaturation and chemical modification with N-ethylmaleimide. From these results it is proposed that membrane lipid perturbations, catalyzed by calcium, may control expression of platelet [³H]-imipramine sites. The relation of this recognition site to aminergic systems and the possible relevancy to the action of antidepressants are addressed.     

Inhibition of adrenaline-induced platelet aggregation by the orally administered α-adrenergic receptor blocker phentolamine (regitine®)

Summary In eight healthy volunteers pretreatment with phentolamine 40 mg p o inhibited platelet aggregation (1st and 2nd phases) induced by low concentrations of adrenaline (2, 1 and 0.5 µM) in plasma from blood sampled 30 min after administration of the compound. The lower the concentration of adrenaline used, the greater was the degree of inhibition elicited. These results are indicative of competitive inhibition of the action of adrenaline on the platelet membrane by phentolamine. Four to six hours after administration of the compound, the aggregation characteristics had reverted to normal. It is concluded that the increased tendency toward platelet aggregation associated with elevated blood levels of catecholamines can be prevented by therapeutic doses of phentolamine.