Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

BACKGROUND: Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. METHODS AND RESULTS: SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCß knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCß inhibitor LY333531. NADH (30-300 µmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCß small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCß expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCß inhibitor LY333531. CONCLUSIONS: NADH-induced inhibition of endothelial SK channel function is mediated via PKCß. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Nalbuphine attenuates morphine-induced scratching by inhibiting PKCß-dependent microglial activation and p38 phosphorylation in male mice.

Morphine-induced scratching (MIS) is a common adverse effect associated with the use of morphine as analgesia after surgery. However, the treatment of MIS is less than satisfactory due to its unclear mechanism, which needs to be enunciated. We found that intrathecal (i.t.) injections of morphine significantly enhanced scratching behavior in C57BL/6J male mice as well as increased the expressions of protein kinase C ß (PKCß), phosphorylated p38 mitogen-activated protein kinases (MAPK), and ionized calcium-binding adapter molecule 1 (Iba1) within spinal cord dorsal horn. Conversely, using the kappa opioid receptor antagonist nalbuphine significantly attenuated scratching behavior, reduced PKCß expression and p38 phosphorylation, and decreased spinal dorsal horn microglial activation, while PKCδ and KOR expression elevated. Spinal PKCß silencing mitigated MIS and microglial activation. Still, knockdown of PKCδ reversed the inhibitory effect of nalbuphine on MIS and microglial activation, indicating that PKCδ is indispensable for the antipruritic effects of nalbuphine. In contrast, PKCß is crucial for inducing microglial activation in MIS in male mice. Our findings show a distinct itch cascade of morphine, PKCß/p38MAPK, and microglial activation, but an anti-MIS pathway of nalbuphine, PKCδ/KOR, and neuron activation.

Protein kinase C-ß distinctly regulates blood-brain barrier-forming capacity of Brain Microvascular endothelial cells and outgrowth endothelial cells.

Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-ß (PKC-ß) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-ß inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-ß protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-ß signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.

Therapeutic hypothermia augments the restorative effects of PKC-ß and Nox2 inhibition on an in vitro model of human blood-brain barrier.

To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-ß (PKC-ß) and Nox2 inhibition on an in vitro model of human blood-brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-ß inhibitor (LY333531, 0.05 µM) or a Nox2 inhibitor (gp91ds-tat, 50 µM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-ß or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-ß or Nox2 pathways.

Hepatocyte-specific PKCß deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis.

OBJECTIVE: Nonalcoholic hepatic steatosis, also known as fatty liver, is a uniform response of the liver to hyperlipidic-hypercaloric diet intake. However, the post-ingestive signals and mechanistic processes driving hepatic steatosis are not well understood. Emerging data demonstrate that protein kinase C beta (PKCß), a lipid-sensitive kinase, plays a critical role in energy metabolism and adaptation to environmental and nutritional stimuli. Despite its powerful effect on glucose and lipid metabolism, knowledge of the physiological roles of hepatic PKCß in energy homeostasis is limited. METHODS: The floxed-PKCß and hepatocyte-specific PKCß-deficient mouse models were generated to study the in vivo role of hepatocyte PKCß on diet-induced hepatic steatosis, lipid metabolism, and mitochondrial function. RESULTS: We report that hepatocyte-specific PKCß deficiency protects mice from development of hepatic steatosis induced by high-fat diet, without affecting body weight gain. This protection is associated with attenuation of SREBP-1c transactivation and improved hepatic mitochondrial respiratory chain. Lipidomic analysis identified significant increases in the critical mitochondrial inner membrane lipid, cardiolipin, in PKCß-deficient livers compared to control. Moreover, hepatocyte PKCß deficiency had no significant effect on either hepatic or whole-body insulin sensitivity supporting dissociation between hepatic steatosis and insulin resistance. CONCLUSIONS: The above data indicate that hepatocyte PKCß is a key focus of dietary lipid perception and is essential for efficient storage of dietary lipids in liver largely through coordinating energy utilization and lipogenesis during post-prandial period. These results highlight the importance of hepatic PKCß as a drug target for obesity-associated nonalcoholic hepatic steatosis.

Immunostimulatory effects of RACK1 pseudosubstrate in human leukocytes obtained from young and old donors.

Aims of this study were to investigate the ability of RACK1 pseudosubstrate alone or in combination with classical immune stimuli to activate human leukocytes, and to restore age-associated immune defects.A total of 25 donors (17 old donors, 77-79 yrs; 8 young donors, 25-34 yrs) were enrolled. To evaluate the effect of RACK1 pseudosubstrate on cytokine production and CD86 expression the whole blood assay was used. Cultures were treated with RACK1 pseudosubstrate in the presence or absence of lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) and incubated for 24 h or 48 h for LPS-induced CD86 expression, TNF-α, IL-6, IL-8, IL-10 production, and PHA-induced IL-4, IL-10, IFN-γ, respectively. RACK1 pseudosubstrate alone induced IL-6, IL-8, and CD86 expression in both young and old donors, and IFN-γ in old donors. In combination with LPS an increase in IL-8, IL-10 and TNF-α was observed, also resulting in restoration of age-associated defective production, while no changes in the other parameters investigated were found.Even if based on a small sample size, these results suggest the possibility to by-pass some of age-associated immune alterations, which may be beneficial in situations were natural immune stimulation is required, and highlight a different role of PKCß in immune cells activation.