title: Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole.
abstract: A two-micropipette current-clamp technique was used to record electrophysiological responses from the somatic muscle of Ascaris suum. Levamisole and acetylcholine were applied to the bag region of the muscle using a microperfusion system. Depolarizations produced by 10 s applications of 10 micro mol l(-1) levamisole or 20 s applications of 10 micro mol l(-1) acetylcholine were recorded. The effect on the peak membrane potential change of the kinase antagonists H-7, staurosporine, KN-93 and genistein was observed. H-7 (30 micro mol l(-1)), a non-selective antagonist of protein kinases A, C and G but which has little effect on Ca(2+)/calmodulin-dependent kinase II (CaM kinase II), did not produce a significant effect on the peak response to levamisole or acetylcholine. Staurosporine (1 micro mol l(-1)), a non-selective kinase antagonist that has effects on protein kinases A, C and G, CaM kinase and tyrosine kinase, reduced the mean peak membrane potential response to levamisole from 6.8 mV to 3.9 mV (P<0.0001) and the mean response to acetylcholine from 5.5 mV to 2.8 mV (P=0.0016). The difference between the effects of H-7 and staurosporine suggested the involvement of CaM kinase II and/or tyrosine kinase. KN-93, a selective CaM kinase II antagonist, reduced the mean peak response to levamisole from 6.2 mV to 2.7 mV (P=0.035) and the mean peak response of acetylcholine from 4.7 mV to 2.0 mV (P=0.0004). The effects indicated the involvement of CaM kinase II in the phosphorylation of levamisole and acetylcholine receptors. The effect of extracellular Ca(2+) on the response to levamisole was assessed by comparing responses to levamisole in normal and in low-Ca(2+) bathing solutions. The response to levamisole was greater in the presence of Ca(2+), an effect that may be explained by stimulation of CaM kinase II. Genistein (90 micro mol l(-1)), a selective tyrosine kinase antagonist, reduced peak membrane potential responses to levamisole from a mean of 6.4 mV to 3.3 mV (P=0.001). This effect indicated the involvement of tyrosine kinase in maintaining the receptor.
authors.affiliation: Department of Biomedical Sciences, Iowa State University, Ames 50011, USA.
authors.forename: Sasa M
authors.initials: SM
authors.lastname: Trailovic
authors.forename: Alan P
authors.initials: AP
authors.lastname: Robertson
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Clark
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Martin
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000378:metabolism; D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D019833:Genistein / Q000378:metabolism; D007978:Levamisole / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000378:metabolism; D014665:Vasodilator Agents / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011978:Receptors, Nicotinic; D014665:Vasodilator Agents; D007978:Levamisole; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D020794:Receptor Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; D000109:Acetylcholine; D002118:Calcium