Inhibition of endoplasm reticulum stress by ghrelin protects against ischemia/reperfusion injury in rat heart.

Ghrelin is a multi-functional polypeptide with cardiovascular protective effects. We aimed to explore whether the cardioprotective effect of ghrelin is mediated by inhibiting myocardial endoplasmic reticulum stress (ERS). A Langendorff model of isolated rat heart was used with ischemia/reperfusion (I/R; 40/120 min). Cardiac function was monitored, and histomorphologic features, degree of myocardial injury, level of ERS markers, and number of apoptotic cardiomyocytes were determined. Compared with control group, the I/R group showed significantly decreased cardiac function, seriously damaged myocardial tissue, increased number of apoptotic cells, and overexpression of mRNA and protein of ERS markers. However, preadministration of ghrelin in vivo (10(-8)mol/kg, intraperitoneal injection, every 12h, twice in all) greatly ameliorated the damaged heart function, attenuated myocardial injury and apoptosis, and decreased the expression of ERS markers: it decreased the mRNA and protein levels of glucose-regulated protein78 (GRP78) and C/EBP homologous protein (CHOP), with reduced caspase-12 protein expression. Furthermore, in vitro, ghrelin directly inhibited the myocardial ERS response induced by tunicamycin or dithiothreitol in rat cardiac tissue. Ghrelin could protect the heart against I/R injury, at least in part, through inhibiting myocardial ERS.

Activation of glucuronidation through reduction of a disulfide bond in rat UDP-glucuronosyltransferase 1A6.

UDP-glucuronosyltransferase- (UGT-) dependent glucuronidation is an important detoxification process for many endogenous and exogenous compounds in mammals. Treatment of rat hepatic microsomes with the reducing reagent dithiothreitol (DTT) resulted in a significant increase in p-nitrophenol (p-NP) glucuronidation in a time- and concentration-dependent manner. The DTT-dependent activation of glucuronidation was specific for planar phenols but not for bilirubin or testosterone without membrane perturbation of the microsomes. p-NP glucuronidation in Gunn rat hepatic microsomes lacking UGT1 isozymes was not affected by DTT, indicating that UGT1A6 in the microsomes is mainly involved in the activation. The DTT-dependent activation was inhibited by 1,6-bis(maleimido)hexane (BMH) but not by N-ethylmaleimide, indicating that cross-linking between cysteine residues in UGT1A6 is responsible for the activation. Immunoblot analysis of rat hepatic microsomes on nonreducing SDS-PAGE gels revealed that most of the UGT1A6 migrated as a monomer, suggesting that DTT could affect an intramolecular disulfide bond in the UGT1A6 that may be responsible for the activation. To identify which of the ten cysteines in UGT1A6 are involved in the disulfide bond, rat UGT1A6 wild type and a set of mutants, each with a cysteine to serine substitution, were constructed and expressed in COS cells. Treatment of COS microsomes with DTT had no effect on the activity of the wild type but BMH showed significant inhibition, suggesting that UGT1A6 expressed in COS cells may be in the reduced and activated state. Replacement of either Cys 121 or Cys 125 with serine showed insensitivity to the BMH-dependent inhibition. These results demonstrate that both Cys 121 and Cys 125 are responsible for the activation of the activity through the disulfide bond in rat UGT1A6.

In vitro excystation of Paragonimus heterotremus metacercariae.

In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39-40 C, and osmolarity of 250-350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5-4 microg/ml), L-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1-8 microM), N-tosyl-L-phenylalanine chloromethyl ketone (0.1-0.4 mM), N alpha-p-tosyl-L-lysine chloromethyl ketone (25-200 microg/ml), iodoacetic acid (0.5-4 mM), and phenylmethylsulfonyl fluoride (1-4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.

The cell-specific anti-proliferative effect of reduced glutathione is mediated by gamma-glutamyl transpeptidase-dependent extracellular pro-oxidant reactions.

We have shown earlier that extracellular GSH can exert a cell-specific growth-inhibitory effect on human tumor cells. In the present study, 2 human ovarian carcinoma cell lines (A2780 and IGROV-1) were used to investigate the biochemical basis of the GSH growth-inhibitory effect. Whereas cells were resistant, A2780 cells were sensitive to a 1 hr exposure to GSH, as assessed by the growth inhibition assay. Analysis of relevant GSH-dependent enzymes indicated that A2780 cells had low level of GSH S-transferase, glutathione reductase and gamma-glutamyl transpeptidase (gamma-GT) activities in comparison with those of IGROV-1 cells, and GSH peroxidase activity was undetectable in A2780 cells. The GSH effect was reversed by catalase and by dithiothreitol, indicating the occurrence of oxidative phenomena resulting in the impairment of critical cellular thiols. Indeed treatment of cells with H(2)O(2) also resulted in growth inhibition, which was more marked in A2780 cells. The gamma-glutamyl acceptor glycylglycine, a co-substrate for gamma-GT, potentiated the growth-inhibitory effect of GSH, which in contrast was decreased by the gamma-GT inhibitors, serine-borate complex and acivicin, suggesting that the production of reactive forms of oxygen (probably H(2)O(2)) was mediated by cysteinyl-glycine after GSH hydrolysis. The results support that the growth-inhibitory effect of low GSH concentration is the result of oxidative damage related to extracellular GSH metabolism.

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.

Inactivation of angiotensin II receptors in bovine adrenal cortex by dithiothreitol: further evidence for the essential nature of disulfide bonds.

Dithiothreitol (DTT), a disulfide reducing agent, significantly decreased specific (( 3H]angiotensin II binding to membrane-bound and solubilized bovine adrenal cortical receptors. Scatchard analysis indicated a reduction in the maximum number of membrane-bound binding sites without change in affinity. The effect of DTT on membrane-bound receptors was readily reversed by the sulfhydryl oxidizing agent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), indicating that its action upon specific[3H]angiotensin II binding is mediated via reduction of disulfide bonds rather than other properties of this agent. Preincubation of the membranes with unlabeled angiotensin II, but not its biologically inactive precursor angiotensin I, protected the receptor from deactivation by DTT. The data provide evidence that disulfide bonds essential for[3H]angiotensin II receptor binding in bovine adrenal cortex are located at, or near, the active sites of the receptor.

Acetylcholine receptor: modification of synaptic gating mechanism after treatment with a disulfide bond reducing agent.

Reduction of a 'reactive' disulfide bond in the postsyraptic membrane of the frog neuromuscular junction by dithiothreitol (DTT) decreases both the sensitivity of the membrane to applied acetylcholine (ACh) and the amplitude of the single 'shot effect'. Analysis of ACh induced conductance fluctuations in voltage clamped frog endplates indicates that DTT reduces both the amplitude gamma and duration tau of the elementary conductance events. The mean control value of gamma was 18.5-10(-12) omega-1 with no significant dependence on temperature. The mean control values pi were 2.3 msec at 7-9 degrees C and 0.94 msec at 20-22 degrees C. At 7-9 degrees C 1m7 DTT (20-50 min after application) reduced gamma to 61% of the control value and at 20-22 degrees C to 39%, while tau was reduced to 70% at both temperature ranges. The dose-response curve for iontophoretically applied ACh indicates that neither the total number of ionic channels nor the cooperativity within the receptors are changed. However, the affinity of ACh for the receptor sites was reduced. All effects of DTT were fully reversed by the oxidizing agent 5,5'-dithie-bis-(2-nitro-benzoic acid) (DTNB).

Acetylcholine noise: analysis after chemical modification of receptor.

The elementary voltage pulses ("shot effects") produced by the action of acetylcholine molecules on the receptor were studied by analyzing the membrane voltage fluctuations ("noise") after acetylcholine application at the frog neuromuscular junction. The amplitude of these pulses was decreased after treatment with a disulfide-bond reducing agent. The shot effect may thus depend on the structure or conformation of the receptor molecule.