Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Metabolic effects of sub-chronic ablation of the incretin receptors by daily administration of (Pro(3))GIP and exendin(9-39)amide in obese diabetic (ob/ob) mice

Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro(3))GIP and exendin(9-39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro(3))GIP, exendin(9-39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly (p <0.05) lower in (Pro(3))GIP-treated mice compared to control mice after just 9 days of treatment. (Pro(3))GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin (p <0.05 to p <0.001). The (Pro(3))GIP-treated group also exhibited significantly (p <0.05) reduced pancreatic insulin content. Acute administration of exendin(9-39) amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro(3))GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro(3)GIP) with exendin(9-39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9-39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.

Beneficial effects of sub-chronic activation of glucagon-like peptide-1 (GLP-1) receptors on deterioration of glucose homeostasis and insulin secretion in aging mice

Aging is associated with an increased incidence of glucose intolerance and type 2 diabetes. Glucagon-like peptide-1 (GLP-1) is an important insulinotropic peptide secreted from the gastrointestinal tract in response to nutrient absorption. The present study was designed to assess the sub-chronic glucose regulatory effects of the potent long-acting GLP-1 receptor agonist, (Val(8))GLP-1, in aging 45-49 week old mice. Daily injection of (Val$)GLP-1 (25 nmol/kg body weight) for 12 days had no significant effect on food intake, body weight, non-fasting plasma glucose and insulin concentrations. However, after 12 days, the glycaemic response to intraperitoneal glucose was improved (P <0.05) in (Val(8))GLP-1 treated mice. In keeping with this, glucose-mediated insulin secretion was enhanced (P <0.05) and insulin sensitivity improved (P <0.05) compared to controls. These data indicate that sub-chronic activation of the GLP-1 receptor by daily treatment with (Val(8))GLP-1 counters aspects of the age-related impairment of pancreatic beta-cell function and insulin sensitivity. 2006 Elsevier Inc. All rights reserved.

Significance of peroxisome proliferator-activated receptors in the cardiovascular system in health and disease.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that belong to the nuclear receptor superfamily. Three isoforms of PPAR have been identified, alpha, delta and gamma, which play distinct roles in the regulation of key metabolic processes, such as glucose and lipid redistribution. PPARalpha is expressed predominantly in the liver, kidney and heart, and is primarily involved in fatty acid oxidation. PPARgamma is mainly associated with adipose tissue, where it controls adipocyte differentiation and insulin sensitivity. PPARdelta is abundantly and ubiquitously expressed, but as yet its function has not been clearly defined. Activators of PPARalpha (fibrates) and gamma (thiazolidinediones) have been used clinically for a number of years in the treatment of hyperlipidaemia and to improve insulin sensitivity in diabetes. More recently, PPAR activation has been found to confer additional benefits on endothelial function, inflammation and thrombosis, suggesting that PPAR agonists may be good candidates for the treatment of cardiovascular disease. In this regard, it has been demonstrated that PPAR activators are capable of reducing blood pressure and attenuating the development of atherosclerosis and cardiac hypertrophy. This review will provide a detailed discussion of the current understanding of basic PPAR physiology, with particular reference to the cardiovascular system. It will also examine the evidence supporting the involvement of the different PPAR isoforms in cardiovascular disease and discuss the current and potential future clinical applications of PPAR activators.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors.

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission.

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M₄ allosteric potentiators. VU10010, the lead compound, potentiates the M₄ response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M₄ with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M₄ knockout mice. Selective regulation of excitatory transmission by M₄ suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.

Targeting death receptors in bladder, prostate and renal cancer

PURPOSE: We describe key components of normal and aberrant death receptor pathways, the association of these abnormalities with tumorigenesis in bladder, prostate and renal cancer, and their potential application in novel therapeutic strategies targeted toward patients with cancer.

MATERIALS AND METHODS: A MEDLINE literature search of the key words death receptors, TRAIL (tumor necrosis factor related apoptosis inducing ligand), FAS, bladder, prostate, renal and cancer was done to obtain information for review. A brief overview of the TRAIL and FAS death receptor pathways, and their relationship to apoptosis is described. Mechanisms that lead to nonfunction of these pathways and how they may contribute to tumorigenesis are linked. Current efforts to target death receptor pathways as a therapeutic strategy are highlighted.

RESULTS: Activation of tumor cell expressing death receptors by cytotoxic immune cells is the main mechanism by which the immune system eliminates malignant cells. Death receptor triggering induces a caspase cascade, leading to tumor cell apoptosis. Receptor gene mutation or hypermethylation, decoy receptor or splice variant over expression, and downstream inhibitor interference are examples of the ways that normal pathway functioning is lost in cancers of the bladder and prostate. Targeting death receptors directly through synthetic ligand administration and blocking downstream inhibitor molecules with siRNA or antisense oligonucleotides represent novel therapeutic strategies under development.

CONCLUSIONS: Research into the death receptor pathways has demonstrated the key role that pathway aberrations have in the initiation and progression of malignancies of the bladder, prostate and kidney. This new understanding has resulted in exciting approaches to restore the functionality of these pathways as a novel therapeutic strategy.