Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha 1, alpha 2, alpha 1 beta and alpha 2 beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha 2 beta GlyR relative to the alpha 2 GlyR but not in the alpha 1 beta GlyR relative to the alpha 1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha 2 beta GlyR was transferred to the alpha 1 beta GlyR by the G2'A (alpha 1 to alpha 2 subunit) substitution. In addition, the alpha 1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha 1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Three-factor models versus time series models:quantifying time-dependencies of interactions between stimuli in cell biology and psychobiology for short longitudinal data

Signal integration determines cell fate on the cellular level, affects cognitive processes and affective responses on the behavioural level, and is likely to be involved in psychoneurobiological processes underlying mood disorders. Interactions between stimuli may subjected to time effects. Time-dependencies of interactions between stimuli typically lead to complex cell responses and complex responses on the behavioural level. We show that both three-factor models and time series models can be used to uncover such time-dependencies. However, we argue that for short longitudinal data the three factor modelling approach is more suitable. In order to illustrate both approaches, we re-analysed previously published short longitudinal data sets. We found that in human embryonic kidney 293 cells cells the interaction effect in the regulation of extracellular signal-regulated kinase (ERK) 1 signalling activation by insulin and epidermal growth factor is subjected to a time effect and dramatically decays at peak values of ERK activation. In contrast, we found that the interaction effect induced by hypoxia and tumour necrosis factor-alpha for the transcriptional activity of the human cyclo-oxygenase-2 promoter in HEK293 cells is time invariant at least in the first 12-h time window after stimulation. Furthermore, we applied the three-factor model to previously reported animal studies. In these studies, memory storage was found to be subjected to an interaction effect of the beta-adrenoceptor agonist clenbuterol and certain antagonists acting on the alpha-1-adrenoceptor / glucocorticoid-receptor system. Our model-based analysis suggests that only if the antagonist drug is administer in a critical time window, then the interaction effect is relevant.

Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity

The loss of dopamine in idiopathic or animal models of Parkinson's disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABAA receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity. © 2006 IBRO.

The synthesis and evaluation of small organic molecules as cholecystokinin antagonists

Cholecystokinin (CCK) is a peptide hormone, present in the alimentary and the CNS. It is the most abundant peptide in the brain. CCK has been implicated in a number of disorders. The link between CCK and anxiety was the basis for this research. A comprehensive discussion on the many types of CCK receptor antagonists is included. For the drug discovery process, a number of synthetic approaches have been investigated and alternative chemical approaches developed. 1,4-Benzodiazepine analogues were prepared, with substitutents In the 1,2 & 3- position of the benzodiazepine scaffold varied, and substituted 3-anilino benzodiazepines exhibited the greatest in vitro activity towards the CCKA receptor subtype. Through extensive screening, pyrazolinone-ureido derivatives were identified, optimised, SAR studied and re-screened. A comprehensive in vivo study on the most active analogue is included, which has a number of common structural features with L-36S, 260 including activity. Pyrazolinone-amide derivatives, bearing the tryptophan moiety were equally active. A number of existing and novel furan- 2(SH)-one building blocks were prepared, from which a selected mini-library of 4- amino-substituted furan-2(SH)-ones were prepared and evaluated. All synthesised compounds were evaluated in a CCK radiolabelled binding assay (CCKA & CCKB), with compounds demonstrating receptor selectivity and lead structures being discovered. The work in this thesis has identified a number of highly active prime structures, from which further investigations are essential in providing more in vitro & in vivo data and the need to prepare more analogues.

Aspects of tic like behaviour and serotonergic control

Tic-like movements in rodents bear close similarities to those observed in humans both pharmacologically and morphologically. Pharmacologically, tics are modulated by serotonergic and dopaminergic systems and abnormalities of these systems have been reported in Tourette's Syndrome (TS). Therefore, serotonergic and dopaminergic modulation of tics induced by a thyrotrophin-releasing hormone (TRH) analogue were studied as possible models for TS. The TRH analogue MK771 induced a variety of tic like movements in mice; blinking fore-paw-licking and fore-paw-tremor were quantified and serotonergic and dopaminergic modulation was investigated. The selective dopamine D1 receptor antagonists SCH23390 and SCH39166 and dopamine D2 antagonists raclopride and sulpiride had no effect on MK771 induced blinking. The D1 antagonists attenuated fore-paw-tremor and -licking while the D2 antagonists were generally without effect on these behaviours. Ketanserin (5-HT2A/ alpha-1 antagonist) and ritanserin (5-HT2A/2C antagonist) were able to attenuate MK771-induced blinking and ketanserin, mianserin (5-HT2A/2C antagonist) and prazosin (alpha-1 adrenoceptor antagonist) were able to attenuate MK771-induced fore-paw-tremor and -licking. The 5-HT2C/2B antagonist SB200646A was without effect on blinking and fore-paw-licking but dose-dependently potentiated fore-paw-tremor. The 5-HT1A agonists 8-OH DPAT and buspirone attenuated blinking at the lower doses tested but were ineffective at the higher doses; the converse was found for fore-paw-licking and -tremor behaviours.The effects of these ligands appeared to be at a postsynaptic 5-HTlA site since para-chlorophenylalanine was without effect on the manipulation of these behaviours. (S)-W A Y100135 was without effect on MK771-induced behaviours, spontaneous and DOl-induced head shakes. Because kynurenine potentiates head shakes and plasma concentrations are raised in TS patients the effects of kynurenine on the 5-HT2A/2C agonist DOl mediated head shake were established. Kynurenine potentiated the DOl head shake. Attempts were made to correlate serotonergic unit activity with tic like behaviour in cats but this proved unsuccessful. However, the pharmacological understanding of 5-HTlA receptor function has been hampered because of the lack of selective antagonists for this site. For this reason the effects of the novel 5-HTlA antagonists (S)-WA Y- 100135 and WAY -100635 were tested on 5-HT single-unit activity recorded from the dorsal-raphe-nucleus in the behaving cat. Both drugs antagonised the suppression of unit activity caused by 8-0H DPAT. (S)-WA Y-100135 reduced unit activity whereas WAY-100635 increased it. This suggests that WAY-100635 is acting as an antagonist at the 5-HTlA somatodendritic autoreceptor and that (S)W A Y -100135 acts as a partial agonist at this site. Aspects of tic like behaviour and serotonergic control are discussed.

An investigation into the pharmacology of tics and tic-like movements

The study of tic-like movements in mice has demonstrated close parallels both in characteristics and in pharmacology with the tics which occur in TS. Head-shakes and/or other tic-like behaviours occurring spontaneously or induced by the selective 5-HT2/1C agonist DOI, alpha-melanocyte stimulating hormone, adrenocorticotrophic hormone (1-39), thyrotropin releasing hormone, or RX336-M were blocked when tested with neuroleptics such as haloperidol and/or the alpha-2 adrenoceptor agonist clonidine. The selective dopamine D1 antagonists SCH23390 and SCH39166 dose-dependently blocked spontaneous and DOI head-shakes but the selective dopamine D2 antagonists sulpiride and raclopride were ineffective. The 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone, gepirone, MDL 73005EF and buspirone (i.p) dose-dependently blocked DOI head-shakes, pindolol blocked the inhibitory effect of 8-OH-DPAT on DOI head-shakes. Parachlorophenylalanine blocked the inhibitory effect of 8-OH-DPAT and buspirone, suggesting that the 5-HT1A receptor involved is located presynaptically. The alpha-2 adrenoceptor antagonists yohimbine, idazoxan, 1-PP and RX811059 prevented the inhibitory effect of 8-OH-DPAT on DOI head-shakes suggesting that this 5-HT1A - 5-HT2 receptor interaction is under the modulatory control of adrenoceptors. Because kynurenine has previously been found to potentiate head-shaking, plasma kynurenine concentrations were measured in seven TS patients and were significantly higher than controls, but neopterin and biopterin were unchanged. The relationship between tic-like movements in rodents and their implications for understanding the aetiology and treatment of TS is discussed.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

1 Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2 In the presence of indomethacin (3μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0 ± 0.1, n = 5). 3 Establishment of a rank order of potency using the EP4 > EP2 agonist 11-deoxy PGE1, and the EP2 > EP4 agonist PGE1-OH (mean pEC 50 of 7.6 ± 0.1 (n = 6) and 6.4 ± 0.1 (n = 4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations < 1 μM failed to relax the tissues. 4 Blockade of EP 4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration-response curves, exhibiting pA2 and pKB values of 5.7 ± 0.1, n = 3, and 8.4, n = 3, respectively. 5 The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3 ± 0.1 (n = 4) and 8.1 ± 0.1 (n = 9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGFα2 failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4 ± 0.3, n = 3). 6 These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

New drugs for type 2 diabetes mellitus:what is their place in therapy?

Oral therapy for type 2 diabetes mellitus, when used appropriately, can safely assist patients to achieve glycaemic targets in the short to medium term. However, the progressive nature of type 2 diabetes usually requires a combination of two or more oral agents in the longer term, often as a prelude to insulin therapy. Issues of safety and tolerability, notably weight gain, often limit the optimal application of anti-diabetic drugs such as sulforylureas and thiazolidinediones. Moreover, the impact of different drugs, even within a single class, on the risk of long-term vascular complications has come under scrutiny. For example, recent publication of evidence suggesting potential detrimental effects of rosiglitazone on myocardial events generated a heated debate and led to a reduction in use of this drug. In contrast, current evidence supports the view that pioglitazone has vasculoprotective properties. Both drugs are contraindicated in patients who are at risk of heart failure. An additional recently identified safety concern is an increased risk of fractures, especially in postmenopausal women. Several new drugs with glucose-lowering efficacy that may offer certain advantages have recently become available. These include (i) injectable glucagonlike peptide-1 (GLP-1) receptor agonists and oral dipeptidyl peptidase-4 (DPP-4) inhibitors; (ii) the amylin analogue pramlintide; and (iii) selective cannabinoid receptor-1 (CB1) antagonists. GLP-1 receptor agonists, such as exenatide, stimulate nutrient-induced insulin secretion and reduce inappropriate glucagon secretion while delaying gastric emptying and reducing appetite. These agents offer a low risk of hypoglycaemia combined with sustained weight loss. The DPP-4 inhibitors sitagliptin and vildagliptin are generally weight neutral, with less marked gastrointestinal adverse effects than the GLP-1 receptor agonists. Potential benefits of GLP-1 receptor stimulation on P cell neogenesis are under investigation. Pancreatitis has been reported in exenatide-treated patients. Pramlintide, an injected peptide used in combination with insulin, can reduce insulin dose and bodyweight. The CB1 receptor antagonist rimonabant promotes weight loss and has favourable effects on aspects of the metabolic syndrome, including the hyperglycaemia of type 2 diabetes. However, in 2007 the US FDA declined approval of rimonabant, requiring more data on adverse effects, notably depression. The future of dual peroxisome proliferator-activated receptor-alpha/gamma agonists, or glitazars, is presently uncertain following concerns about their safety. In conclusion, several new classes of drugs have recently become available in some countries that offer new options for treating type 2 diabetes. Beneficial or neutral effects on bodyweight are an attractive feature of the new drugs. However, the higher cost of these agents, coupled with an absence of long-term safety and clinical outcome data, need to be taken into consideration by clinicians and healthcare organizations.

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK 1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 CCK 1 for the CCK 1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg -1 was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine. © 2006 The Authors.

Receptor for activated C Kinase 1 protein binds to and activates the human estrogen receptor α

The classical concept of estrogen receptor (ER) activation is that steroid passes the cell membrane, binds to its specific protein receptor in the cell's cytoplasm and the steroid-receptor complex travels to the nucleus where it activates responsive genes. This basic idea has been challenged by results of experiments demonstrating insulin-like growth factor 1 (IGF-1) activation of the ER in the complete absence of estrogen suggesting at least one other mechanism of ER activation not involving steroid. One explanation is that activation of the cell surface IGF-1 receptor leads to synthesis of an intracellular protein(s) able to bind to and stimulate the ER. Based on results using the two-hybrid system, coimmunoprecipitation and transfection-luciferase assays, we herein show that one of these proteins could well be receptor for activated C kinase 1 (RACK-1). Using the human ER type α (ER-α) as bait, a cloned complementary deoxyribonucleic acid (cDNA) library from IGF-1 treated human breast cancer MCF-7 cells was screened for ER-α - protein interactions. Many positive clones were obtained which contained the RACK-1 cDNA sequence. Coimmunoprecipitation of in-vitro translation products of the ER-α and RACK-1 confirmed the interaction between the two proteins. Transfection studies using the estrogen response element spliced to a luciferase reporter gene revealed that constitutive RACK-1 expression was able to powerfully stimulate ER-α activity under estrogen-free conditions. This effect could be enhanced by 17β-estradiol (E2) and blocked by tamoxifen, an E2 antagonist. These results show that RACK-1 is able to activate the ER-α in the absence of E2, although together with the latter, enhanced effects occur. Since RACK-1 gene expression is stimulated by IGF-1, it is distinctly possible that RACK-1 is the mediator of the stimulatory effects of IGF-1 on ER-α. © 2014 JMS.

STOPPFrail (Screening Tool of Older Persons Prescriptions in Frail Adults with Limited Life Expectancy): Consensus Validation

Background: To validate STOPPFrail, a list of explicit criteria for potentially inappropriate medications (PIMs) in frailer older adults with limited life expectancy. A Delphi consensus survey of an expert panel (n = 17) comprising specialists in geriatric medicine, clinical pharmacology, palliative care, psychiatry of old age, clinical pharmacy and general practice.
Methods: STOPPFrail criteria was initially created by the authors based on clinical
experience and appraisal of the available literature. Criteria were organised according to physiological system. Each criterion was accompanied by an explanation. Panellists ranked their agreement with each criterion on a 5-point Likert scale and invited to provide written feedback. Criteria with a median Likert response of 4/5 (agree/strongly agree) and a 25th centile of ≥4 were included in the final criteria.
Results: Three Delphi rounds were required. All panellists completed all rounds. Thirty criteria were proposed for inclusion; 26 were accepted. No new criteria were added. The first two criteria suggest deprescribing medications with no indication or where compliance is poor. The remaining 24 criteria include lipid-lowering therapies, alpha-blockers for hypertension, anti-platelets, neuroleptics, proton pump inhibitors, H-2 receptor antagonists, anti-spasmodics, theophylline, leukotriene antagonists, calcium supplements, bone anti-resorptive therapy, selective oestrogen receptor modulators, non-steroidal antiinflammatories, corticosteroids, 5-alpha reductase inhibitors, alpha-1 selective blockers, muscarinic antagonists, oral diabetic agents, ACE-inhibitors, angiotensin receptor blockers, systemic oestrogens, multivitamins, nutritional supplements and prophylactic antibiotics. Anticoagulants and anti-depressants were excluded. Despite incorporation of panellists’ suggestions, memantine and acetyl-cholinesterase inhibitors remained inconclusive.
Conclusion: STOPPFrail comprises 26 criteria, which have been judged by broad consensus, to be potentially inappropriate in frailer older patients with limited life expectancy. STOPPFrail may assist in deprescribing medications in these patients.

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure

Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol 109: 702-709, 2010. First published July 1, 2010; doi: 10.1152/japplphysiol.00281.2010.-The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca(2+) abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha(2A)/alpha(2C) adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca(2+) handling-related protein expression by Western blot. alpha(2A)/alpha(2C)ARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca(2+) release-related proteins [alpha(1)-, alpha(2)-, and beta(1)-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca(2+) reuptake-related proteins [sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA) 1/2 and Na(+)/Ca(2+) exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca(2+) handling toward WT levels. We provide evidence that Ca(2+) handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca(2+) handling proteins.