Quercetagetin inhibits macrophage-derived chemokine in HaCaT human keratinocytes via the regulation of signal transducer and activator of transcription 1, suppressor of cytokine signalling 1 and transforming growth factor-ß1.

BACKGROUND: Inflammatory chemokines, such as macrophage-derived chemokine (MDC/CCL22), are elevated in the serum and lesioned skin of patients with atopic dermatitis (AD), and are ligands for C-C chemokine receptor 4, which is predominantly expressed on T helper 2 lymphocytes, basophils and natural killer cells. We have previously reported that quercetagetin has an inhibitory activity on inflammatory chemokines, which is induced by interferon (IFN)-γ and tumour necrosis factor (TNF)-α, occurring via inhibition of the signal transducer and activator of transcription 1 (STAT1) signal. OBJECTIVES: To investigate the specific mechanisms of quercetagetin on the STAT1 signal. METHODS: We confirmed the inhibitory activity of quercetagetin on MDC and STAT1 in HaCaT keratinocytes. The interaction between STAT1 and IFN-γR1 was investigated using immunoprecipitation. The small interfering RNA approach was used to investigate the role of suppressor of cytokine signalling 1 (SOCS1) and transforming growth factor (TGF)-ß1 induced by quercetagetin. RESULTS: Quercetagetin inhibited the expression of MDC at both the protein and mRNA levels in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. Moreover, quercetagetin inhibited the phosphorylation of STAT1 through upregulation of SOCS1. Increased expression of SOCS1 disrupted the binding of STAT1 to IFN-γR1. Furthermore, quercetagetin augmented the expression of TGF-ß1, which is known to modulate the immune response and inflammation. CONCLUSIONS: These results suggest that quercetagetin may be a potent inhibitor of the STAT1 signal, which could be a new molecular target for anti-inflammatory treatment, and may thus have therapeutic applications as an immune modulator in inflammatory diseases such as AD.

Postsynaptic action mechanism of somatostatin on the membrane excitability in spinal substantia gelatinosa neurons of juvenile rats.

We used tight-seal, whole-cell recording in juvenile rat spinal slices to investigate the action of somatostatin on substantia gelatinosa neurons. Bath application of somatostatin caused a robust and repeatable hyperpolarization or outward current in substantia gelatinosa neurons. Somatostatin inhibited spontaneous action potentials in subpopulation of substantia gelatinosa neurons. The amplitude of dorsal root-evoked excitatory postsynaptic currents and the frequency of spontaneous excitatory postsynaptic currents were not affected by somatostatin. The current induced by somatostatin developed almost instantaneously and did not show any time-dependent inactivation. The current-voltage relationship exhibited inward rectification. The conductance of somatostatin-sensitive current increased with the concentration of external K(+). The reversal potentials in different external K(+) concentrations were close to the K(+) equilibrium potentials. The effect of somatostatin was dose-dependent, with an EC(50) of 113 nM. The somatostatin-sensitive current was blocked by low concentration of extracellular Ba(2+) but not by glibenclamide, an inhibitor of ATP-sensitive K(+) channels. Hyperpolarization-activated cation current in a subpopulation of substantia gelatinosa neurons was not affected by somatostatin. In neurons recorded with an internal solution containing GTPgammaS, somatostatin induced outward current and hyperpolarization that did not reverse on washing. When the spontaneous induction of outward current with GTPgammaS was greatest, somatostatin did not induce any outward currents. Furthermore, intracellular dialysis of GDPbetaS, a G-protein antagonist, abolished the effect of somatostatin. In addition, SST-sensitive neurons were fewer in slices incubated with pertussis toxin than in adjacent control slices incubated without pertussis toxin. These results suggest that somatostatin decreases the postsynaptic membrane excitability of substantia gelatinosa neurons by a pertussis toxin-sensitive G-protein-mediated activation of an inwardly rectifying K(+) conductance.

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats.

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.

B cells of aged mice show decreased expansion in response to antigen, but are normal in effector function.

Increased dysfunction of the immune system with age can be attributed to developmental changes in cell types critical for proper immune responses. Previous studies have shown defects in humoral responses of aged individuals, but have not distinguished between aged T-cell/microenvironment and intrinsic B-cell defects. Here adoptive transfer of antigen-specific transgenic B cells compared early immunopoeisis from young and aged donors in a young recipient environment. B cells from aged donors demonstrated decreased antigen-induced expansion, particularly in the lymph nodes; however, they acquired a germinal center phenotype at frequencies similar to B cells from young donors. Additionally, aged B cells produced equivalent levels of antigen-specific antibody that underwent affinity maturation and isotype switching and demonstrated similar numbers of antibody-secreting cells of switched isotype. Thus, the ability of aged B cells to respond appropriately to T-dependent antigens and differentiate into high-affinity, isotype-switched, antibody-secreting cells appears to be intact.

Inhibition of apamin-sensitive K+ current by hypoxia in adult rat adrenal chromaffin cells.

The effect of hypoxia on small-conductance Ca(2+)-activated K+ current was investigated in a study of adult rat adrenomedullary chromaffin cells (AMCs), which were maintained in short-term culture. The nystatin-perforated, whole-cell patchclamp technique was used to study the effect of hypoxia with minimum perturbation of the intracellular milieu. Under voltage-clamp conditions, acute hypoxia (P(O2) approximately equal to 25 mmHg) suppressed the whole-cell outward currents of more than half the AMCs (24/46). This suppression was eliminated after application of apamin (400 nM), a selective inhibitor of small-conductance Ca(2+)-activated K+ current (I(SK)(Ca)) (n=5), suggesting that an apamin-sensitive component of whole-cell currents is suppressed during hypoxia. In contrast to I(SK)(Ca), Ca2+ current (I(Ca)) (n=10) was not affected by hypoxia. Finally, under current-clamp conditions, hypoxia reversibly depolarized the resting membrane potential of adult AMCs (34/40). Apamin, however, eliminated the hypoxia-induced depolarization (400 nM) (7/8), suggesting that hypoxic depolarization is related to the suppression of I(SK(Ca). From the above results, we conclude that adult AMCs are sensitive to hypoxia, and that I(SK)(Ca) contributes to the hypoxia-induced suppression of whole-cell outward current and depolarization of the resting membrane potential in adult AMCs.