Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the Dretro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4) In many nervous system disorders, including cerebral ischemia, traumatic brain injury and neurodegenerative diseases, overactivation of N-methyl-d-aspartate (NMDA) receptors leads to neuronal damage, resulting in neuronal loss and consequent severe neurological impairment. This cascade of neuronal injury, referred to as 'excitotoxicity', is still only partly understood. Dying neurons activate complex signal transduction events to trigger their death program, and the c-Jun N-terminal kinase (JNK) pathway plays an important role in this process. 1 D-retro-inverso form of JNK inhibitor (D-JNKI1) is an extremely potent neuroprotectant against excitotoxicity of cortical neurons and against different in vivo paradigms of neurodegeneration. [2][3][4] The active part of this peptide contains a retro-inverso form of a 20-amino-acid sequence (JBD 20 ) from the JNK-binding domain (JBD) of the scaffold protein JNK-interacting protein-1 (IB1/JIP-1), and it blocks the access of JNK to many of its targets. [5][6][7] Recently, Negri et al. 8,9 performed a detailed re-examination of the JBD-containing proteins and identified 19 different substrates of JNK. They then proved in a cell-free assay that the JBD 20 sequence prevented interactions and phosphorylations by JNK of nine of these targets. Among these nine substrates, we have studied the following four that might participate in regulating the death of cortical neurons: (1) MADD/DENN (MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells); (2-3) mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), the two direct upstream activators of JNK and (4) the scaffold protein IB1/JIP-1.,In particularly, MADD/DENN was identified as a substrate for JNK3, 10 the isoform most clearly involved in excitotoxicity 11,12 and mostly expressed in the brain. [10][11][12] Increasing evidence supports a strong correlation between low MADD/ DENN expression and neuronal loss. 13,14 MKK4 and MKK7 are the only known JNK activators. In some cell types, MKK4 activates JNK primarily by stress stimuli and MKK7 by inflammatory cytoki...

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These results show that the differences in angiotensin II receptor blockade observed with the various AT(1) antagonists are explained mainly by differences in dosing. When 160-mg or 320-mg doses were investigated, the effects of valsartan hardly differed from those obtained with recommended doses of irbesartan and candesartan.

In vitro studies have shown that telmisartan is an insurmountable angiotensin II subtype-1 (AT1) receptor antagonist. Herein, the molecular basis of this insurmountable antagonism has been investigated in vitro, and the effect of telmisartan has been compared in vivo with that of irbesartan and candesartan. Association and dissociation kinetics of telmisartan to AT1 receptors have been characterized in vitro on rat vascular smooth muscle cells (RVSMC) expressing solely the AT1 receptor subtype. In a second set of experiments, the antagonistic efficacy of single intravenous doses (0.1, 0.3, and 1 mg/kg) of telmisartan was compared with that of irbesartan (0.3, 1.0, 3.0, and 10.0 mg/kg) and candesartan (0.3 and 1 mg/kg) in conscious, normotensive, male Wistar rats. The results show that the specific binding of [ 3 H]telmisartan to the surface of living RVSMC is saturable and increases quickly to reach equilibrium within 1 h. Telmisartan dissociates very slowly from the receptor with a dissociation half-life (t 1/2 ) of 75 min, which is comparable with candesartan and almost 5 times slower than angiotensin II (AngII). In vivo, telmisartan blunts the blood pressure response to exogenous AngII dose dependently. The blockade is long lasting and remains significant at 24 h at doses Ͼ0.1 mg/kg. Ex vivo assessment of the AT1 receptor blockade using an in vitro AngII receptor binding assay shows similar results. When administered intravenously in rats, telmisartan is 10-fold more potent than irbesartan and comparable to candesartan. Taken together, our in vitro data show that the insurmountable antagonism of telmisartan is due at least in part to its very slow dissociation from AT1 receptors.

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An in vitro angiotensin II (AngII) receptor-binding assay was developed to monitor the degree of receptor blockade in standardized conditions. This in vitro method was validated by comparing its results with those obtained in vivo with the injection of exogenous AngII and the measurement of the AngII-induced changes in systolic blood pressure. For this purpose, 12 normotensive subjects were enrolled in a double-blind, four-way cross-over study comparing the AngII receptor blockade induced by a single oral dose of losartan (50 mg), valsartan (80 mg), irbesartan (150 mg), and placebo. A significant linear relationship between the two methods was found (r = 0.723, n = 191, P<.001). However, there exists a wide scatter of the in vivo data in the absence of active AngII receptor blockade. Thus, the relationship between the two methods is markedly improved (r = 0.87, n = 47, P<.001) when only measurements done 4 h after administration of the drugs are considered (maximal antagonist activity observed in vivo) suggesting that the two methods are equally effective in assessing the degree of AT-1 receptor blockade, but with a greatly reduced variability in the in vitro assay. In addition, the pharmacokinetic/pharmacodynamic analysis performed with the three antagonists suggest that the AT-1 receptor-binding assay works as a bioassay that integrates the antagonistic property of all active drug components of the plasma. This standardized in vitro-binding assay represents a simple, reproducible, and precise tool to characterize the pharmacodynamic profile of AngII receptor antagonists in humans.

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Background: The quantification of total (free + sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyra-mine, mainly in the form of sulfate conjugates (60-80%). Their determination requires the hydrolysis of the sul-fate ester moiety to allow electrochemical oxidation of the phenolic group. Commercially available urine calibrators and controls contain essentially free, unhydrolysable metanephrines which are not representative of native urines. The lack of appropriate calibrators may lead to uncertainty regarding the completion of the hy-drolysis of sulfated metanephrines, resulting in incorrect quantification. Methods: We used chemically synthesized sulfated metanephrines to establish whether the procedure most fre-quently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis. Results: We found that sulfated metanephrines differ in their optimum pH to obtain complete hydrolysis. Highest yields and minimal variance were established for incubation at pH 0.7-0.9 during 20 min. Conclusion: Urinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sul-fated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and pro-ficiency testing to improve inter-laboratory accuracy.