These observations establish a signal function for Ca2+ downstream of the oxidative burst in the activation of a physiological cell death program in soybean cells that is similar to apoptosis in animals. That the characteristic cell corpse morphology is also induced in Arabidopsis and tobacco by different avirulence signals suggests that apoptosis may prove to be a common, but not necessarily ubiquitous, feature of incompatible plant-pathogen interactions. Emerging similarities between facets of hypersensitive disease resistance and the mammalian native immune system indicate that apoptosis is a widespread defence mechanism in eukaryotes.
The agonist selectivity for adenosine di-and triphosphates was determined for the human P2Y 1 receptor stably expressed in human 1321N1 astrocytoma cells and was studied under conditions in which nucleotide metabolism was both minimized and assessed. Cells were grown at low density on glass coverslips, encased in a flow-through chamber, and continuously superfused with medium, and Ca 2ϩ responses to nucleotides were quantified. Superfusion with high performance liquid chromatographically purified ADP, ATP, 2-methylthio-ADP, and 2-methylthio-ATP resulted in rapid Ca 2ϩ responses, with EC 50 values of 10 Ϯ 5, 304 Ϯ 51, 2 Ϯ 1, and 116 Ϯ 50 nM, respectively. Similar peak responses were observed with maximal concentrations of these four agonists and with the hydrolysisresistant adenine nucleoside triphosphate adenosine-5Ј-O-(3-thiotriphosphate (1998)] recently reported that, whereas ADP and 2-methylthio-ADP were agonists, ATP and 2-methylthio-ATP were weak antagonists in studies of the human P2Y 1 receptor expressed in human Jurkat cells. To assess whether differences in the degree of receptor reserve might explain this discrepancy of results, P2Y 1 receptor-expressing 1321N1 cells were incubated for 24 hr with adenosine-5Ј-O-(2-thiodiphosphate), with the goal of down-regulating the level of functional receptors. Pretreatment with adenosine-5Ј-O-(2-thiodiphosphate) resulted in a 10-fold rightward shift in the concentration-effect curve for ADP; in contrast, the agonist activity of ATP was completely abolished. Taken together, our results indicate that adenosine di-and triphosphates are agonists at the human P2Y 1 receptor. However, the intrinsic efficacy of ATP is less than that of ADP, and the capacity of ATP to activate second messenger responses through this receptor apparently depends on the degree of P2Y 1 receptor reserve.P2Y receptors are G protein-coupled receptors that are activated by extracellular nucleotides (Fredholm et al., 1994). Molecular cloning and heterologous protein expression have led to unambiguous identification of at least five mammalian P2Y receptor subtypes (Communi et al., 1997;Fredholm et al., 1997). Antagonists capable of discriminating among the subtypes of P2Y receptors are not generally available, and pharmacological characterization of these receptors has been limited to descriptions of the rank order of potencies for activation by nucleotide agonists.Metabolism of extracellular nucleotides complicates delineation of the agonist selectivity of P2Y receptors . For example, 1321N1 human astrocytoma cells (a cell line commonly used for heterologous expression of P2Y receptors) express both ectonucleotidase and extrafacial nucleoside diphosphokinase activities, which modify exogenously applied nucleotides (Lazarowski et al., 1997a(Lazarowski et al., , 1997b. Endogenous nucleotides also are released from these cells (Lazarowski et al., 1995(Lazarowski et al., , 1997a and other cells (Osipchuk and Cahalan, 1992;Grierson and Meldolesi, 1995;Schlosser et al., 1996;Grygorczyk and Han...
Over the last decade, many of the molecular components that mediate the transduction of taste signaling have been elucidated. The chemosensory receptors for taste have been identified as G protein-coupled receptors (GPCRs) and ion channels that are expressed on the surface of highly specialized taste sensory cells. Tastant molecules act as agonists, binding to and stabilizing active conformations of receptors, resulting in the initiation of signal transduction cascades. Taste signaling, therefore, should be amenable to the methods of pharmacology. This review focuses on the GPCR-mediated signaling of bitter, sweet, and umami tastes and emphasizes the opportunities for pharmacologic evaluation.
Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic β-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC₅₀ = 12 μM) and murine TRPM5 (IC₅₀ = 30 μM) heterologously expressed in HEK293 cells, but had no effect (up to 100 μM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)⁻ mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.
Rhesus monkeys were conditioned to press on levers and receive intravenous infusions of cocaine or ketamine. Experimental conditions provided several different doses of drug during each of two daily 130 min sessions; as a result, a dose-response curve relating rate of responding to dose/injection for self-administered drug was obtained within each session. Relative rate-maintaining effects of nomifensine and cocaine in monkeys on baseline conditions of cocaine self-administration, and rate-maintaining effects of ketamine, phencyclidine and MK-801 in monkeys on baseline conditions of ketamine self-administration, compared favorably with relative rate-maintaining effects of these substances obtained in more traditional paradigms.
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