Pharmacology of stomoxytachykinin receptor depends on second messenger system

STKR is a neurokinin receptor derived from the stable fly, Stomoxys calcitrans. Insect tachykinin-related peptides, also referred to as "insectatachykinins", produce dose-dependent calcium and cyclic AMP responses in cultured Drosophila melanogaster Schneider 2 (S2) cells that were stably transfected with the cloned STKR cDNA. Pronounced differences in pharmacology were observed between agonist-induced calcium and cyclic AMP responses. The results indicate that the pharmacological properties of STKR depend on its coupling to a unique second messenger system. Therefore, a model postulating the existence of multiple active receptor conformations is proposed. This article presents the first evidence that an insect peptide receptor with dual coupling properties to second messenger systems can display agonist-dependent functional differences.     

Formation of angiotensin-(1-7) from angiotensin II by the venom of Conus geographus

The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.     

Insulin-related peptides and their conserved signal transduction pathway

The 'insulin superfamily' is an ancient category of small, structurally related proteins, such as insulin, insulin-like growth factors (IGF) and relaxin. Insulin-like signaling molecules have also been described in different invertebrates, including nematodes, mollusks, and insects. They initiate an evolutionary conserved signal transduction mechanism by binding to a heterotetrameric, membrane-spanning receptor tyrosine kinase. Recent physiological and genetic studies have revealed that, in different metazoans, the insulin signaling pathway plays a pivotal role in the regulation of a variety of interrelated, fundamental processes, such as metabolism, growth, reproduction and aging.     

Electrical stimulation reveals complex neuronal input and activation patterns in single myenteric guinea pig ganglia

The myenteric plexus plays a key role in the control of gastrointestinal motility. We used confocal calcium imaging to study responses to electrical train stimulation (ETS) of interganglionic fiber tracts in entire myenteric ganglia of the guinea pig small intestine. ETS induced calcium transients in a subset of neurons: 52.2% responded to oral ETS, 65.4% to aboral ETS, and 71.7% to simultaneous oral and aboral ETS. A total of 41.3% of the neurons displayed convergence of oral and aboral ETS-induced responses. Responses could be reversibly blocked with TTX (10(-)6 M), demonstrating involvement of neuronal conduction, and by removal of extracellular calcium. omega-Conotoxin (5 x 10(-7) M) blocked the majority of responses and reduced the amplitude of residual responses by 45%, indicating the involvement of N-type calcium channels. Staining for calbindin and calretinin did not reveal different response patterns in these immunohistochemically identified neurons. We conclude that, at least for ETS close to a ganglion, confocal calcium imaging reveals complex oral and aboral input to individual myenteric neurons rather than a polarization in spread of activity.     

Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation

Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (DeltaPsi(m)) was assessed with tetramethylrhodamine methyl ester. During ischemia, DeltaPsi(m) decreased to 24 +/- 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by Sytox Green was minimal during ischemia but averaged 66 +/- 7% after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological antioxidants attenuated the fall in DeltaPsi(m) during ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O(2) was scavenged from the perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in DeltaPsi(m) during ischemia. Finally, 143B-rho(0) osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of DeltaPsi(m) during ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during ischemia in the mitochondrial depolarization and subsequent cell death induced by ischemia and reperfusion in this model.     

ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (K(ATP)) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H(2)O(2), or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by K(ATP) channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the K(ATP) channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to K(ATP) channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N(omega)-nitro-l-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by K(ATP) channel activation. Coadministration of H(2)O(2) and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the K(ATP) channel. We conclude that ROS can trigger preconditioning by causing activation of the K(ATP) channel, which then induces generation of ROS and NO that are both required for preconditioning protection.     

The analysis of structure-anticancer and antiviral activity relationships for macrocyclic pyridinophanes and their analogues on the basis of 4D QSAR models (simplex representation of molecular structure)

A new 4D-QSAR approach has been considered. For all investigated molecules the 3D structural models have been created and the set of conformers (fourth dimension) have been used. Each conformer is represented as a system of different simplexes (tetratomic fragments of fixed structure, chirality and symmetry). The investigation of influence of molecular structure of macrocyclic pyridinophanes, their analogues and certain other compounds on anticancer and antiviral (anti-influenza, antiherpes and antiadenovirus) activity has been carried out by means of the 4D-QSAR. Statistic characteristics for QSAR of PLS (partial least squares) models are satisfactory (R = 0.92-0.97; CVR = 0.63-0.83). Molecular fragments increasing and decreasing biological activity were defined. This information may be useful for design, and direct synthesis of novel anticancer and antiviral agents.     

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.