The effects of remote ischaemic preconditioning on coronary artery function in patients with stable coronary artery disease.

BACKGROUND: Remote ischaemic preconditioning (RIPC) is a cardioprotective intervention invoking intermittent periods of ischaemia in a tissue or organ remote from the heart. The mechanisms of this effect are incompletely understood. We hypothesised that RIPC might enhance coronary vasodilatation by an endothelium-dependent mechanism. METHODS: We performed a prospective, randomised, sham-controlled, blinded clinical trial. Patients with stable coronary artery disease (CAD) undergoing elective invasive management were prospectively enrolled, and randomised to RIPC or sham (1:1) prior to angiography. Endothelial-dependent vasodilator function was assessed in a non-target coronary artery with intracoronary infusion of incremental acetylcholine doses (10-6, 10-5, 10-4mol/l). Venous blood was sampled pre- and post-RIPC or sham, and analysed for circulating markers of endothelial function. Coronary luminal diameter was assessed by quantitative coronary angiography. The primary outcome was the between-group difference in the mean percentage change in coronary luminal diameter following the maximal acetylcholine dose (Clinicaltrials.gov identifier: NCT02666235). RESULTS: 75 patients were enrolled. Following angiography, 60 patients (mean±SD age 57.5±8.5years; 80% male) were eligible and completed the protocol (n=30 RIPC, n=30 sham). The mean percentage change in coronary luminal diameter was -13.3±22.3% and -2.0±17.2% in the sham and RIPC groups respectively (difference 11.32%, 95%CI: 1.2- 21.4, p=0.032). This remained significant when age and sex were included as covariates (difference 11.01%, 95%CI: 1.01- 21.0, p=0.035). There were no between-group differences in endothelial-independent vasodilation, ECG parameters or circulating markers of endothelial function. CONCLUSIONS: RIPC attenuates the extent of vasoconstriction induced by intracoronary acetylcholine infusion. This endothelium-dependent mechanism may contribute to the cardioprotective effects of RIPC.

Cation-dependent leucine, alanine, and phenylalanine uptake at pH 10 in brush-border membrane vesicles from larval Manduca sexta midgut.

Using the rapid filtration technique, cation gradient driven leucine, alanine and phenylalanine uptake by brush-border membrane vesicles (BBMV) from the highly studied model insect, Manduca sexta, is characterized at the physiological pH of 10. The vesicles are sealed and nonspecific binding is small. Almost identical initial time courses of leucine uptake are obtained whether the vesicles are osmotically balanced initially or at equilibrium. The maximum accumulation values are also similar and the equilibrium values are identical with either treatment. Equilibrium is reached by 60 min. Amino acid accumulation is cation gradient dependent and is abolished by 18 microM valinomycin. Uptake of all three amino acids occurs over a broad pH range with maximum rates at approximately pH 10 and lower rates at pH 7.5. The cation selectivity of phenylalanine and alanine uptake changes with pH; the sequence is K+ > Na+ > Cs+ >> Rb+ = Li+ at pH 10.0, whereas K+ = Na+ at pH 8.0; the selectivity of leucine uptake is K+ = Na+ > Cs+ >> Rb+ = Li+ at pH 10. Maximum K+ driven accumulation of all three amino acids decreases with anions in the order: SCN- > NO3- > Cl- = CO(3)2- = So(4)2- = HPO(4)2- > gluconate-.Vmax values are similar for all three amino acids. There are large differences in initial uptake rates (leucine > phenylalanine = alanine), and maximum accumulation values (leucine > phenylalanine > alanine).

Neutral amino acid symport in larval Manduca sexta midgut brush-border membrane vesicles deduced from cation-dependent uptake of leucine, alanine, and phenylalanine.

Uptake of tritiated leucine, alanine, and phenylalanine was measured at the physiological pH of 10 by rapid filtration in brush-border membrane vesicles from the midgut of the larval tobacco hornworm, Manduca sexta. A 20-fold excess of unlabeled leucine, isoleucine, methionine, valine, alanine, lysine, histidine, phenylalanine, and glutamine inhibited uptake of leucine and phenylalanine, and six of these amino acids inhibited uptake of alanine, by more than 50% both in the presence and absence of a potassium ion gradient. These inhibitory amino acids also drove countertransport of leucine, alanine, and phenylalanine with accumulation ratios exceeding 2. These results are consistent with the hypothesis that leucine, alanine, and phenylalanine share a common uptake system - a broad scope B type symporter - which interacts strongly with half of the commonly occurring amino acids, interacts moderately with an additional quarter of them, but does not interact with cysteine, arginine, glutamate, aspartate, or proline.

Tyrosinase-catalyzed unusual oxidative dimerization of 1,2-dehydro-N-acetyldopamine.

Tyrosinase, which usually catalyzes the conversion of o-diphenols to o-benzoquinones, catalyzed an unusual oxidative dimerization of 1,2-dehydro-N-acetyl-dopamine to a benzodioxan derivative. The identity of the product was confirmed by UV, IR spectra, and NMR studies. During the oxidation, generation of a transient reactive intermediate could be witnessed by its characteristic visible absorption spectrum. Typical phenoloxidase inhibitors such as phenylthiourea, potassium cyanide, sodium azide, and sodium fluoride drastically inhibited the above reaction. Mimosine, a known competitive inhibitor of o-diphenoloxidase activity, also inhibited the new reaction competitively, suggesting that both the observed oxidative dimerization and the conventional quinone production are catalyzed by the same active site copper of tyrosinase. Based on our earlier findings (Sugumaran, M., and Lipke, H. (1983) FEBS Lett. 155, 65-68; Sugumaran, M. (1986) Biochemistry 25, 4489-4492) that phenoloxidases can produce quinone methides from certain 4-alkylcatechols, possible mechanisms for this new reaction are presented.

Annexins I, II and III are specific choline binding proteins.

We have isolated choline binding proteins from the plasma membrane fraction fraction of human lung epithelium-derived cell line (A549) by means of detergent solubilization, anion exchange and affinity chromatography. One of the affinity purified proteins had a specific choline binding activity of 44-57 pmol/mg, representing a two to three hundredfold enrichment relative to the specific activity of freshly prepared plasma membranes. The purified protein has a molecular mass of 38 kDa by SDS PAGE analysis and was identified as annexin II by N-terminal microsequencing. Annexin II, however, had not previously been known for choline binding activity. We therefore prepared a mixture of authentic annexins (I-V) from A549 cells. The mixture had a choline binding activity of 15 to 18 pmol/mg. The annexin mixture was subsequently affinity chromatographed on the choline-conjugated Sepharose 6B column. Analyses by SDS PAGE and immunoblot revealed that annexins I, II, and III are bound to the choline column while annexins IV and V did not. These results indicate that some of the annexins have specific choline binding activities.

Cloning and expression of rat lung acidic Ca(2+)-independent PLA2 and its organ distribution.

A clone for a rat acidic Ca(2+)-independent phospholipase A2 (aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T.S. Kim, C.S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2 activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was < 50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2 in the lung compared with the other organs and suggest translational control of aiPLA2 expression.