In brains of the rabbit, pig, and human, expression of the high‐affinity Na+‐d‐glucose cotransporter SGLT1 and of the protein RS1, which alters the activity of SGLT1, was demonstrated. In situ hybridization showed that SGLT1 and RS1 are transcribed in pyramidal cells of brain cortex and hippocampus and in Purkinje cells of cerebellum. In neurons of pig brain SGLT1 protein was demonstrated by western blotting with synaptosomal membranes and by immunohistochemistry, which showed SGLT1 in pyramidal and Purkinje cells. To test whether SGLT1 in neurons may be activated during increased d‐glucose consumption, an epileptic seizure was induced in rat brain, and the uptake of specific nonmetabolized substrates of SGLT1 {[14C]methyl‐α‐d‐glucopyranoside ([14C]AMG)} and of Na+‐independent transporters {2‐deoxy‐d‐[14C]glucose([14C]2‐DG)} was analyzed by autoradiography. During the seizure the uptake of AMG and 2‐DG was increased in the focus. Within two hours after the seizure 2‐DG uptake in the focus returned to normal. In contrast, the AMG uptake in the focus area was still increased 1 day later. The data show that the high‐affinity Na+‐d‐glucose cotransporter SGLT1 is expressed in neurons and can be up‐regulated.

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the ␣ isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.