Arachidonic acid induces phosphorylation of an 18 kDa protein in electrically permeabilised rat islets of Langerhans.

Arachidonic acid (AA) was shown to induce concentration-dependent, calcium-independent, in situ phosphorylation of a protein of approximate molecular weight 18 kDa in electrically permeabilised rat islets of Langerhans. This protein did not appear to be a substrate for protein kinase C (PKC) since stimulation of PKC by 4 beta phorbol myristate acetate (4 beta PMA) did not result in 32P incorporation into an 18 kDa protein, and since AA-induced phosphorylation was observed in islets in which PKC had been down-regulated by prolonged exposure of islets to 4 beta PMA. These results suggest that AA stimulates protein phosphorylation by a mechanism other than PKC activation.

Arachidonic acid-induced insulin secretion from rat islets of Langerhans is not mediated by protein phosphorylation.

Arachidonic acid (AA) stimulated protein phosphorylation in electrically permeabilised islets, most notably of an islet protein of approximate molecular weight 18 kDa. This protein did not appear to be a substrate for cAMP-dependent protein kinase. The AA-induced protein phosphorylation was mediated by unmetabolised AA since the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or the cyclooxygenase inhibitor, indomethacin, did not significantly reduce AA-induced phosphorylation. Although saturated fatty acids did not stimulate phosphorylation of islet proteins, a number of cis-unsaturated fatty acids, other than AA, induced 32P incorporation into an 18 kDa protein. However, some fatty acids which stimulated protein phosphorylation had no effect on insulin secretion in experiments where AA clearly stimulated insulin secretion. AA stimulated protein kinase C (PKC) activity extracted from islets but several fatty acids which induced protein phosphorylation had no significant effect on PKC activity in vitro. 50 nM staurosporine had no effect on AA-induced protein phosphorylation but this concentration of staurosporine markedly inhibited PKC activity. 200 nM staurosporine caused complete inhibition of the AA-induced phosphorylation without having any effect on AA-induced insulin secretion. These results suggest that AA and some other fatty acids can promote 32P incorporation into islet proteins, independently of PKC activation, and that AA-induced phosphorylation is not required for insulin secretory responses to AA.

Effects of sodium on PKC translocation; relationship to neurotransmitter release.

Our previous work using Na+ channel activators such tityustoxin (TsTX), indicated that local increases in Na+ modulate glutamate release from synaptosomes. We have now investigated the role of the Ca2+/phospholipid-dependent protein kinase (PKC) in mediating this effect. TsTX and KCl stimulate 'fast' glutamate release to the same extent but TsTX is more effective than KCl in enhancing the 'slow' phase of release. KCl greatly stimulates PKC translocation. However, TsTX inhibits basal and phorbol ester-induced translocation while the Na(+)-ionophore, gramicidin D, has no effect. Taken together, these data suggest TsTX mediated localized Na+ entry inhibits PKC translocation and that this effect may be associated with recruitment of vesicles to the readily releasable pool.

Protein phosphorylation in the regulation of insulin secretion: the use of site-directed inhibitory peptides in electrically permeabilised islets of Langerhans.

We have used electrically permeabilised rat islets of Langerhans to investigate the role of protein phosphorylation in the regulation of insulin secretion using pseudosubstrate inhibitory peptides for cyclic AMP-dependent protein kinase (PKA) and for protein kinase C (PKC). The protein kinase inhibitor (PKI) peptide, PKI(6-22), completely inhibited the effects of cyclic AMP on islet PKA activity in vitro, on endogenous protein phosphorylation and on insulin secretion. This peptide had no significant effect on islet PKC activity in vitro, on Ca(2+)-induced protein phosphorylation and on secretory responses to Ca2+ or to the PKC activator, 4 beta-phorbol myristate acetate (PMA). The PKC pseudosubstrate inhibitory peptide, PKC(19-36), caused a marked inhibition of islet PKC activity in vitro and inhibite PMA-induced insulin secretion without affecting secretory responses to cyclic AMP and Ca2+. These results demonstrate that PKA- and PKC-induced protein phosphorylation is obligatory for cyclic AMP- and PMA-stimulated insulin secretion, respectively, and suggest that there is little "crosstalk" between the response elements of the secretory pathways to the different second messengers, at least after the generation of the messengers within the beta-cells.

Interleukin-10 modulates neuronal threshold of vulnerability to ischaemic damage.

Interleukin-10 (IL-10) is a powerful suppressor of cellular immune responses, with a postulated role in brain inflammation. First, we have evaluated the role of this cytokine in ischaemic brain damage using IL-10 knockout (IL-10-/-) mice. The middle cerebral artery (MCA) was occluded in either IL-10-/- or wild-type animals of corresponding strain (C57Bl/6) and age. Infarct volume was assessed 24 h later in serial brain sections. Brain infarct produced by MCA occlusion was 30% larger in the IL-10-/- than in wild-type mice (21. 8 +/- 1.2 vs. 16.9 +/- 1.0 mm3, respectively; P < 0.01; Student's t-test). To further characterize these findings, studies were extended to in vitro models. Primary neuronal cortical cultures derived from IL-10-/- animals were more susceptible to both excitotoxicity and combined oxygen-glucose deprivation compared with cell cultures from wild-type mice. Moreover, when added to the culture medium, recombinant murine IL-10 (0.1-100 ng/mL) exerted a concentration-dependent prevention of neuronal damage induced by excitotoxicity in both cortical and cerebellar granule cell cultures taken from either strain. The accordance of in vivo and in vitro data allows us to suggest a potential neuroprotective role of IL-10 against cerebral ischaemia when administered exogenously or made available from endogenous sources.

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures.

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.