Inhibition of Ca2+/calmodulin-dependent protein kinase II by arachidonic acid and its metabolites.

Abstract

A variety of evidence indicates that activation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in nerve terminals leads to enhanced neurotransmitter release. Arachidonic acid and its 12-lipoxygenase metabolite, 12-hydroperoxyicosatetraenoic acid (12-HPETE), have been suggested to act as second messengers mediating presynaptic inhibition of neurotransmitter release. In the present study it was found that CaM-kinase II, purified from rat brain cortex, was inhibited both by arachidonic acid (ICs5 = 24 FM) and by 12-HPETE (IC50 = 0.7 PAM). Neither substance inhibited CaM-kinase I or III, protein kinase C, or the catalytic subunit of cAMP-dependent protein kinase. Specific inhibition of Ca2+/calmodulin-dependent protein phosphorylation by arachidonic acid was also demonstrated in intact synaptic terminals (synaptosomes) isolated from rat forebrain. These results suggest that arachidonate and its metabolites may modulate synaptic function through the inhibition of CaM-kinase IIdependent protein phosphorylation.Within the presynaptic nerve terminal, stimulation of either of two Ca2"-dependent protein kinases, Ca2+/calmodulindependent kinase II (CaM-kinase II) or protein kinase C (PKC), leads to enhanced neurotransmitter release (for a review, see ref. 1). CaM-kinase II, a protein kinase with broad substrate specificity, is present throughout the mammalian nervous system (2). In nerve terminals, CaM-kinase II catalyzes the phosphorylation of several proteins, including synapsin I, a protein associated with synaptic vesicles (2). Phosphorylation of synapsin I by CaM-kinase II is thought to participate in presynaptic modulation of neurotransmitter release (3).In neurons, several neurotransmitters, including histamine, norepinephrine, glutamate, and bradykinin, stimulate the formation of free arachidonic acid (A4Ach) and of its lipoxygenase metabolites (4-7). Furthermore, A4Ach and its 12-lipoxygenase product, 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE), modulate ion conductances and produce presynaptic inhibition of neurotransmitter release in identified neurons of the mollusk Aplysia californica (8, 9). Moreover, modulation of gap junctions and ion channels by A4Ach and other fatty acids has been demonstrated in non-neuronal tissues (10-14).We have now investigated the actions of A4Ach and of its lipoxygenase-derived products on CaM-kinase II as one test of the possibility that these lipids achieve certain of their physiological effects through actions on this multifunctional protein kinase. (18), and CaM-kinase III and its 100-kDa substrate (elongation factor 2) from rat pancreas (19). Rat brain calcineurin (20), rat brain PKC (21), bovine heart catalytic subunit of cAMPdependent protein kinase (PKA) (22), and bovine caudate 32-kDa dopamine-and cAMP-regulated phosphoprotein (DARPP-32) (23) were purified and phosphorylated, when appropriate, as described, with minor modifications. MATERIALS AND METHODSProtein Kinase Assays. Standard CaM-kinase II assays were performed essentially as ...

Eicosanoid Activation of Protein Kinase C ϵ

“…We reported earlier that the 43-kDa polypeptide may be a substrate of Ca 2ϩ / calmodulin-dependent kinase (37). This would be consistent with these findings because lipoxygenase products are potent inhibitors of Ca 2ϩ /calmodulin-dependent kinase II (69). Thrombin treatment of GCPs for 30 s increased MARCKS phosphorylation, which could be inhibited by the PKC blocker Bis to below control levels.…”

Lipoxygenase metabolites of arachidonic acid in neuronal transmembrane signalling

“…Lipoxygenase-derived eicosanoids are potent and selective inhibitors of purified Ca2+-calmodulindependent protein kinase II (Ref. 37). 12-HPETE inhibited this enzyme with a half-maximal effect at a concentration of 0.7 ~t~a.…”

Essential Fatty Acids and the Brain

“…Further events in the process of neurotransmission, and eventual release of neurotransmitters from synaptic vesicles, are ushered in by activation of Ca 2+ -CM-PKs; here also, PUFA effects have been noted (46). The thousandfold concentration gradient between extra-and intracellular Ca 2+ is maintained by Ca-ATPase in neuronal membranes: Kearns and Haag have recently noted an inhibition of this enzyme by both DHA and EPA (47).…”