No abstract

Bradykinin (BK) is a peptide mediator released in inflammation that potently excites sympathetic neurons. We have studied the mechanism of this excitation in dissociated rat sympathetic neurons and found that at low nanomolar (EC50 = 0.9 nM) concentrations, BK inhibited the M-type K+ current IK(M). Studies with the selective antagonist Hoe140 revealed that this effect was mediated via the B2 receptor subtype, and mRNA encoding this receptor was identified in these neurons by RT-PCR. IK(M) inhibition was unaffected by Pertussis toxin or microinjection of antibodies to G alpha o but was selectively inhibited by microinjection of antibodies to G alpha q/11. Thus, BK is the most potent M current inhibitor yet described in mammalian neurons, and BK inhibition of M current is mediated by a G protein pathway similar to that activated by muscarinic acetylcholine receptors.

1. Microinjection of selective antibodies into superior cervical ganglion (SCG) neurones has identified the G‐protein alpha‐subunits mediating muscarinic receptor inhibition of M‐type K+ current (IK(M)) and alpha‐adrenoceptor inhibition of Ca2+ current (ICa). 2. Antibodies specific for G alpha q/11, but not those for G alpha o, reduced M‐current inhibition by the muscarinic agonist oxotremorine‐M, whereas anti‐G alpha o antibodies, but not anti‐G alpha q/11 or anti‐G alpha i1‐3 antibodies, reduced calcium current inhibition by noradrenaline. 3. Immunoblots with specific anti‐G‐protein antibodies demonstrated the presence of both G alpha q and G alpha 11, while G alpha o1 (but virtually no G alpha o2) was present. 4. We conclude that M1 muscarinic receptor inhibition of IK(M) is transduced by G alpha q and/or G alpha 11, and that G alpha o transduces alpha‐adrenoceptor inhibition of ICa.

Cannabinoid receptor ligands irreversibly inhibited peak voltage-activated Ca currents (44%) in NG108-15 cells; this inhibition was Pertussis toxin-sensitive. Inhibition was largely due to a reduction in the Co-conotoxin sensitive portion of high-voltage activated (HVA) current, although there was also a significant decrease in low-voltage activated current (56%) and in the nifedipine-sensitive portion of HVA current (41%).

Rat superior cervical ganglion (SCG) neurons express lowthreshold noninactivating M-type potassium channels (I K(M) ), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the G␣ q family (Caulfield et al., 1994). We have used DNA plasmids encoding antisense sequences against the 3Ј untranslated regions of G␣ subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of I K(M) . These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of G␣ q , muscarinic inhibition of I K(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate G␣ oA antisense plasmid. In contrast, depletion of G␣ 11 protein did not alter I K(M) inhibition. To determine whether the ␣ or ␤␥ subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of ␤ adrenergic receptor kinase 1 (␤ARK1), which binds free ␤␥ subunits. ␤ARK1 did not reduce muscarinic inhibition of I K(M) at a concentration of plasmid that can reduce ␤␥-mediated inhibition of calcium current (Delmas et al., 1998a). Also, expression of ␤ 1 ␥ 2 dimers did not alter the I K(M) density in SCG neurons. In contrast, I K(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of G␣ q and G␣ 11 . These data suggest that G␣ q is the principal mediator of muscarinic I K(M) inhibition in rat SCG neurons and that this more likely results from an effect of the ␣ subunit than the ␤␥ subunits of the G q heterotrimer.