Hyperglycemia-induced inflammation and apoptosis have important roles in the pathogenesis of diabetic cardiomyopathy. We recently found that a novel curcumin derivative, C66, is able to reduce the high glucose (HG)-induced inflammatory response. This study was designed to investigate the protective effects on diabetic cardiomyopathy and its underlying mechanisms. Pretreatment with C66 significantly reduced HG-induced overexpression of inflammatory cytokines via inactivation of nuclear factor-κB in both H9c2 cells and neonatal cardiomyocytes. Furthermore, we showed that the inhibition of Jun NH2-terminal kinase (JNK) phosphorylation contributed to the protection of C66 from inflammation and cell apoptosis, which was validated by the use of SP600125 and dominant-negative JNK. The molecular docking and kinase activity assay confirmed direct binding of C66 to and inhibition of JNK. In mice with type 1 diabetes, the administration of C66 or SP600125 at 5 mg/kg significantly decreased the levels of plasma and cardiac tumor necrosis factor-α, accompanied by decreasing cardiac apoptosis, and, finally, improved histological abnormalities, fibrosis, and cardiac dysfunction without affecting hyperglycemia. Thus, this work demonstrated the therapeutic potential of the JNK-targeting compound C66 for the treatment of diabetic cardiomyopathy. Importantly, we indicated a critical role of JNK in diabetic heart injury, and suggested that JNK inhibition may be a feasible strategy for treating diabetic cardiomyopathy.

Several drugs have recently been reported to induce rapid antidepressant effects in clinical trials and rodent models. Although the cellular mechanisms involved remain unclear, reports suggest that increased glutamate transmission contributes to these effects. Here, we demonstrate that the antidepressant-like efficacy of three unique drugs, with reported rapid onset antidepressant properties, is coupled with a rapid transient rise in glutamate cycling in medial prefronal cortex (mPFC) of awake rats as measured by ex vivo 1H-[13C]-nuclear magnetic resonance spectroscopy. Rats were acutely pre-treated by intraperitoneal injection with a single dose of ketamine (1,3,10,30,80mg/kg), Ro 25-6981 (1,3,10mg/kg), scopolamine (5,25,100μg/kg) or vehicle (controls). At fixed times after drug injection animals received an intravenous infusion of [1,6-13C2]glucose for 8 min to enrich brain amino acid pools with 13C, followed by rapid euthanasia. The mPFC was dissected, extracted with ethanol and metabolite 13C enrichments measured. We found a clear dose dependent effect of ketamine and Ro 25-6981 on behavior and the percent of 13C-enrichment of glutamate, glutamine and GABA. Further, we also found an effect of scopolamine on both cycling and behavior. These studies demonstrate that three pharmacologically distinct classes of drugs, clinically related through their reported rapid antidepressant actions, share the common ability to rapidly stimulate glutamate cycling at doses pertinent for their antidepressant-like efficacy. We conclude that increased cycling precedes the antidepressant action at behaviorally effective doses and suggests the rapid change in cycling could be used to predict efficacy of novel agents or identify doses with antidepressant activity.

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphositespecific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.renin-angiotensin-aldosterone system | NCC | hypertension | renal electrolyte transport | distal convoluted tubule T he distal portion of the mammalian nephron plays a key role in water, electrolyte, and blood pressure homeostasis. Mutations that alter normal renal Na-Cl homeostasis in this nephron segment modulate blood pressure and result in diverse electrolyte abnormalities (1). One such rare Mendelian trait is Pseudohypoaldosteronism type II (PHAII, OMIM 145260), which is characterized by hypertension, hyperkalemia, and metabolic acidosis; these features can be corrected by low doses of thiazide diuretics, inhibitors of the Na-Cl cotransporter of the distal convoluted tubule (NCC).Genetic analysis of PHAII has revealed a previously unrecognized pathway that regulates blood pressure and electrolyte homeostasis in the distal nephron. Causative mutations have been found in four genes; two encode the serine-threonine kinases with-no-lysine 1 and 4 (WNK1 and WNK4) (2), and two encode Cullin 3 (CUL3) and Kelch-like 3 (KLHL3), components of an E3-RING ubiquitin ligase complex (3). At the time of the discovery of their causal relationship to this Mendelian disease, none of these proteins were known to play a role in electrolyte or blood pressure homeostasis. The biochemical mechanisms that link mutations to clinical phenoytpes are becoming understood. WNK4 is a Cl − -regulated kinase (4); when active, the kinase phosphorylates the kinases SPAK (Ste20-related proline alaninerich kinase) and OSR1 (oxidative stress responsive kinase), which in turn phosphorylate and activate the thiazide-sensitive Na-Cl cotransporter of the renal distal convoluted tubule (DCT). The phenotype of WNK4 knockout (WNK4-KO) mice recapitulates Gitelman syndrome (OMIM 263800), the mirror image of PHAII, sugg...