An Experimental Circulatory Arrest Model in the Rat to Evaluate Calcium Antagonists in Cerebral Resuscitation

A circulatory arrest model in the rat was developed for use in cerebral and cardiac resuscitation studies. Whole-body ischemia was produced for 8 to 18 minutes by arresting the heart with a cold potassium chloride cardioplegic solution. Following cardiopulmonary resuscitation, minimal, standardized intensive care was provided. As the duration of ischemia was increased from 8 to 18 minutes, survival immediately following resuscitation decreased from 100% to 25%, and survival at 48 hours after ischemia decreased from 80% to 0%. Thirty per cent of the rats recovering from 11 minutes of ischemia suffered motor seizures. Survival and the incidence of motor seizures appear to be good measures of outcome following ischemic circulatory arrest. These measures can be used to test the possible anti-ischemic actions of calcium antagonists or other drugs

Influence of Adrenergic Drugs Upon Vital Organ Perfusion During CPR

To determine whether adrenergic drugs administered during cardiopulmonary resuscitation (CPR) alter the distribution of artificial card:l.ac output, we measu red regional blood flow and cardiac output using radioactive microspheres in 12 dogs. Ventricular fibrillation was induced electrically and CPR was immediately begun with a mechanical chest compressor and ventilator (Thurn per ( R) ) at 60 compressions/min, with a ventilation:compression ratio of 1:5, a compression duration of 0.5 sec, and a ventilation pressure of 20 em H 2 o. Compression force was sufficient to develop 40-50 mmHg peak intraesophageal pressure. After 30 sec of CPR, either 0.9% saline vehicle or 50 ug/kg of epinephrine, phenylephrine, or isoproterenol was administered through a central venous catheter. One minute later, microspheres were injected into the left ventricle. After 250 sec of CPR the ventricles were defibrillated electrically. Twenty minute recovery periods were interposed between each drug injection. accord:l.ng Each dog recei.ved to predetermlned all three drugs and saline sequence. Following saline, epinephrine, phenylephrine, and isoproterenol treatment respectively, cardiac output averaged 392, 319, 255, and 475 ml/min; bratn blood flow averaged 37, 54, 2 9 \u27 and 28 ml/min; heart blood flow averaged 25, 79, 26, and IS ml/min; and kidney blood flow averaged 44, 4, 16, and 29 ml/min. Epinephrine improved blood flow t6 the brain, probably because of its alpha adrenergic activity. Epinephrine improved blood flow to the heart during CPR much more than the other agents, probably because of its combined alpha and beta adrenergic activity. This effect may explain its superiority in restoring circulation after prolonged arrest and resuscitation. Isoproterenol should not be used in CPR because it shunts blood away from vital organs

Molecular Modeling Study for Inhibition Mechanism of Human Chymase and Its Application in Inhibitor Design

Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I) bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymasecomplexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most activecompound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitorymechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes

A Combination of Receptor-Based Pharmacophore Modeling & QM Techniques for Identification of Human Chymase Inhibitors

Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure–based pharmacophore models. One ligand–based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well

Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI) by lung epithelial cells: role of charge of the proteinase-inhibitor complex

<p>Abstract</p> <p>Background</p> <p>Secretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes <it>in vitro</it>.</p> <p>Methods</p> <p>Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.</p> <p>Results</p> <p>We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.</p> <p>Conclusion</p> <p>These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon <it>in vitro</it>, which may correlate with changes observed <it>in vivo</it>.</p

Thrombin Induces Macrophage Migration Inhibitory Factor Release and Upregulation in Urothelium: A Possible Contribution to Bladder Inflammation

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression

EFFECT OF DILTIAZEM ON BRAIN RESUSCITATION IN A RAT CIRCULATORY ARREST MODEL (CALCIUM BLOCKERS, CEREBRAL ISCHEMIA)

Intracellular calcium intoxication of brain and vascular smooth muscle cells has been hypothesized to be a major contributor to brain damage following cardiopulmonary arrest and resuscitation. I have measured the effects of diltiazem, a calcium entry blocker, on survival and neurological deficit (ND) following an average of 12 min of experimental cardio-respiratory arrest and resuscitation in rats. There was no statistically significant improvement in survival or ND, but there was significant amelioration of the incidence of motor seizures. There was a definite accumulation of calcium in the brain and heart following 12-15 min of ischemia and 60 min of reperfusion. Diltiazem significantly decreased brain calcium content after 12 min of ischemia but had no effect after 15 min. Brain blood flow was severely diminished to less than 20% of non-ischemic control flow following 12 min ischemia and 60 min reperfusion. Diltiazem did not improve brain blood flow in this setting. Thus, calcium does appear to be involved in post-ischemic encephalopathy perhaps directly on neurons and/or indirectly via a hypoperfusion effect. Diltiazem provided only a modest overall benefit, indicating it may not be the drug of choice in cerebral resuscitation of humans

Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases : Two Highly Specific Leu-Ases

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit beta-chymase and a guinea pig alpha-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species