Osmotic activation of a Ca2+-dependent phospholipase C pathway that regulates ∆N TRPV1-mediated currents in rat supraoptic neurons.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus regulate body fluid balance by releasing the hormones vasopressin (VP) and oxytocin (OT) in an osmolality-dependent manner. Elevations of external osmolality increase MNC firing and hormone release. MNC osmosensitivity is largely due to activation of a mechanosensitive non-selective cation current that responds to osmotically-evoked changes in MNC volume and is mediated by an N-terminal variant of the TRPV1 channel (∆N TRPV1). We report a novel mechanism by which increases in osmolality may modulate ∆N TRPV1-mediated currents and thus influence MNC electrical behaviour. We showed previously that acute elevations of external osmolality activate the enzyme phospholipase C (PLC) in isolated MNCs. We now show that the osmotic activation of PLC has a time course and dose-dependence that is consistent with a role in MNC osmosensitivity and that it contributes to the osmotically-evoked increase in non-selective cation current in MNCs through a protein kinase C-dependent pathway. We furthermore show that the mechanism of osmotic activation of PLC requires an increase in internal Ca2+ that depends on influx through L-type Ca2+ channels. Our data therefore suggest that MNCs possess an osmotically-activated Ca2+-dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation.

Osmotic activation of phospholipase C triggers structural adaptation in osmosensitive rat supraoptic neurons.

The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC.

The Ca2+ channel ß2 subunit is selectively targeted to the axon terminals of supraoptic neurons.

The assembly of high voltage-activated Ca(2+) channels with different ß subunits influences channel properties and possibly subcellular targeting. We studied ß subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca(V)ß subunits (Ca(V)ß(1)-Ca(V)ß(4)) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 ß subunits are expressed in both locations, but that Ca(V)ß(2) had the highest relative expression in the neurohypophysis. These data suggest that the Ca(V)ß(2) subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca(2+) channels.

Pharmacological profile of AW-814141, a novel, potent, selective and orally active inhibitor of p38 MAP kinase.

The p38 mitogen activated protein kinase (MAPK) is a key signaling molecule that plays a crucial role in the progression of various inflammatory diseases such as rheumatoid arthritis (RA), asthma and chronic obstructive pulmonary disease. The objective of the present study was to evaluate the anti-inflammatory activity of a p38 MAPK inhibitor, AW-814141. AW-814141 inhibited enzymatic activity of recombinant p38-alpha and beta isoforms with IC(50) value of 100nM and 158nM, respectively. AW-814141 also inhibited the release of tumor necrosis factor (TNF)-alpha by lipopolysaccharide (LPS) treated human peripheral blood mononuclear cells with an IC(50) value of 212nM and demonstrated selectivity against a panel of few kinases. Oral administration of AW-814141 (10mpk) in LPS-injected mice resulted in a significant reduction in TNF-alpha production in the circulation. In a carrageenan-induced rat paw edema model and collagen-induced arthritis model (CIA), AW-814141 dose dependently inhibited paw swelling. In different in vivo efficacy models, efficacy of AW-814141 was found to be better as compared to the reference compounds (Vx-745 and BIRB-796). This study demonstrated that AW-814141 is a novel p38 MAPK inhibitor and it displays promising in vitro and in vivo anti-inflammatory activities and can be used for the treatment of rheumatoid arthritis.

Ranbezolid, a novel oxazolidinone antibacterial: in vivo characterisation of monoamine oxidase inhibitory potential in conscious rats.

Ranbezolid, a novel oxazolidinone antibacterial, competitively inhibits monoamine oxidase-A (MAO-A), in vitro. The consequences of MAO-A inhibition was evaluated in vivo, by testing interaction of Ranbezolid with tyramine (in solution or mixed with feed), and amine containing cold remedies on pressor response in conscious rats. Single and repeat doses of Ranbezolid (50 mg/kg, p.o.) did not affect pressor response to tyramine (5 or 15 mg/kg), but potentiated the same after a single dose of 100 mg/kg. Co-administration of Ranbezolid with tyramine in feed or with cold remedies also did not potentiate the respective pressor responses. These results suggest that Ranbezolid exhibits minimal cardiovascular liability associated with MAO-A inhibition.