Molecular pharmacology
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Molecular pharmacology · Dec 2003
Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.
Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). ⋯ H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.
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Molecular pharmacology · Dec 2003
Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: implications for mitogen-activated protein kinase signaling.
Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. ⋯ However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.
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Molecular pharmacology · Aug 2003
Isoflurane and propofol inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve terminals.
Mounting electrophysiological evidence indicates that certain general anesthetics, volatile anesthetics in particular, depress excitatory synaptic transmission by presynaptic mechanisms. We studied the effects of representative general anesthetics on voltage-gated Na+ currents (INa) in nerve terminals isolated from rat neurohypophysis using patch-clamp electrophysiological analysis. Both isoflurane and propofol inhibited INa in a dose-dependent and reversible manner. ⋯ Marked effects on the voltage dependence and kinetics of inactivation and minimal effects on activation support preferential anesthetic interactions with the fast inactivated state of the Na+ channel. These results are consistent with direct inhibition of oxytocin and vasopressin release from the neurohypophysis by isoflurane and propofol. Inhibition of voltage-gated Na+ channels may contribute to the presynaptic effects of general anesthetics on nerve terminal excitability and neurotransmitter release.
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Molecular pharmacology · Aug 2003
Painful inflammation-induced increase in mu-opioid receptor binding and G-protein coupling in primary afferent neurons.
Opioids mediate their analgesic effects by activating mu-opioid receptors (MOR) not only within the central nervous system but also on peripheral sensory neurons. The peripheral analgesic effects of opioids are best described under inflammatory conditions (e.g., arthritis). The present study investigated the effects of inflammation on MOR binding and G-protein coupling of full versus partial MOR agonists in dorsal root ganglia (DRG) of primary afferent neurons. ⋯ In behavioral studies, administration of BUP produced significant antinociception only in inflamed but not in noninflamed paws. These findings show that inflammation causes changes in MOR binding and G-protein coupling in primary afferent neurons. They further underscore the important differences in clinical studies testing peripherally active opioids in inflammatory painful conditions.
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Molecular pharmacology · Jun 2003
Comparative StudyDiazonamide A and a synthetic structural analog: disruptive effects on mitosis and cellular microtubules and analysis of their interactions with tubulin.
The marine ascidian Diazona angulata was the source organism for the complex cytotoxic peptide diazonamide A. The molecular structure of this peptide was recently revised after synthesis of a biologically active analog of diazonamide A in which a single nitrogen atom was replaced by an oxygen atom. Diazonamide A causes cells to arrest in mitosis, and, after exposure to the drug, treated cells lose both interphase and spindle microtubules. ⋯ Nor were they able to stabilize the colchicine binding activity of tubulin. These observations indicate either that diazonamide A and the analog have a unique binding site on tubulin differing from the vinca alkaloid and dolastatin 10 binding sites, or that diazonamide A and the analog bind weakly to unpolymerized tubulin but strongly to microtubule ends. If the latter is correct, diazonamide A and its oxygen analog should have uniquely potent inhibitory effects on the dynamic properties of microtubules.