Huoy-Rou Chang

Cutaneous blood flow and adrenoceptor response increase in segmental-type vitiligo lesions

It has been proposed that two types of vitiligo exist from the physiological and clinical points of view. Nonsegmental-type vitiligo is associated with autoimmune diseases while segmental-type vitiligo results from the dysfunction of sympathetic nerves in the affected area. Using laser Doppler flowmetry and iontophoresis for cutaneous microcirculatory assessments, we evaluated these two types of vitiligo in regard to their physiological changes. Ten patients with facial stable stage segmental-type vitiligo and ten stable nonsegmental-type vitiligo patients were selected for this study. Our results revealed that a nearly threefold increase in cutaneous blood flow was noticed in segmental-type vitiligo as compared to contralateral normal skin. In contrast, a 1.4-1.5 times difference was found among nonsegmental-type vitiligo, lesion side clinically normal skin and contralateral normal skin. There was a significant increase in cutaneous alpha- and beta-adrenoceptor response in segmental-type vitiligo lesions. However, no change in plasma catecholamines or adrenoceptor densities on blood cells was noticed. Our findings suggest that a dysfunction of the sympathetic nerves exists in the affected skin and plays a role in the pathogenesis of segmental-type vitiligo.

Expression of nitric oxide synthases in keratinocytes after UVB irradiation

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.

Reduced vascular β-adrenergic receptors and catecholamine response in rats with lead induced hypertension

Abstractβ-Adrenergic receptor-mediated relaxation of blood vessels declines in lead induced hypertension although the mechanism is unknown. We have utilized the aorta of lead hypertensive rats to investigate this problem. In an effort to elucidate the mechanism responsible for this alteration we examined plasma catecholamine levels, vascular β-adrenergic receptor density, and cyclic adenosine monophosphate (cAMP) production in lead hypertensive rats. The density of β-adrenergic receptors was 41% lower in the blood vessels of lead hypertensive rats compared with control rats. The corresponding apparent Kd values were not significantly different between two groups. The plasma catecholamine level was significantly higher in lead hypertensive rats compared with controls (P < 0.001). Stimulation of the vascular β-adrenoceptor resulted in significantly lower levels of cAMP in lead hypertensive rats compared with controls (P < 0.001). The present results suggest that there is reduced β-adrenoceptor density and diminished cAMP accumulation in blood vessels from lead hypertensive rats. Plasma catecholamine may play a role in the diminished β-adrenoceptor and responsiveness to cAMP-mediated vascular relaxation in lead exposure.

Change of cardiac β-adrenoceptors in lead-exposed rats

The effect of lead on cardiac beta-adrenoceptors was studied. Wistar rats used in these trials were divided into seven groups of ten animals each (A-G). Of these, groups B was given drinking water containing 0.01% lead acetate, group C 0.05%, group D 0.1%, group E 0.5%, group F 1% and group G 2% for a period of 60 days. Group A was given pure water. A radioligand-binding assay fulfilling strict criteria of receptor affinity and density was used to quantify cardiac beta-adrenoceptors. Application of a trend test indicated that both blood and heart lead levels increased significantly from group A to group G (A < G), but that beta-adrenoceptor density decreased (G < A), whereas Kd did not vary among the seven groups. Linear regression analysis showed that decrease of cardiac beta-adrenergic receptor density was closely related to elevation of blood and heart lead levels. The results show that lead exposure results in a reduction of cardiac beta-adrenoceptor density.

High concentration of magnolol induces hepatotoxicity under serum-reduced conditions

Although magnolol is cytoprotective against warm ischemia/reperfusion injury, its effect on cold preservation has not been fully investigated. This study aimed at examining whether magnolol maintains the liver graft integrity after cold preservation and elucidating the underlying mechanisms in terms of apoptotic signaling under both normothermic and hypothermic conditions. After being preserved in Ringers lactate (RL) at 4 degrees C for 6h ex vivo, the magnolol-treated grafts demonstrated significantly higher AST, ALT, and LDH levels in perfusates than those from negative controls. TUNEL staining showed no difference in the number of apoptotic nuclei in both groups, whereas a more intense apoptotic signal in magnolol-treated grafts was shown as compared with the controls. In vitro data showed no significant difference in viability of RL-preserved clone-9 hepatocytes between the magnolol-treated and control groups, while magnolol pretreatment at 30min before cold preservation prominently induced hepatocyte cell death. RT-PCR and Western blotting analyses revealed a suppression in Bcl-2, but an up-regulation in Bax expression in clone-9 cells after magnolol treatment. Magnolol suppressed the ratios of NF-kappaB to I-kappaBalpha protein contents and I-kappaBalpha phosphorylation induced by TNF-alpha, and potentiated mitochondrial cytochrome c release and subsequent caspase-3 cleavage. Conversely, caspase-3 inhibitor attenuated magnolol-induced hepatotoxicity. We concluded that magnolol could not protect liver grafts from cold ischemia/reperfusion injury. High concentration of magnolol under serum-reduced conditions attenuates NF-kappaB-mediated signaling and induces intrinsic apoptotic pathway, thereby inducing in vitro hepatotoxicity.

Alterations in β-adrenergic receptor density and adenylate cyclase activity in the rat brain treated chronically with lead

Behavioral and memory impairment resulting from lead exposure is well known but the mechanism is not. We utilized the brain of lead exposed rats to investigate this problem. In an effort to elucidate the mechanism responsible for this alteration we examined blood and brain lead levels, brain beta-adrenoceptor density and cyclic AMP production in lead exposed rats. Wistar rats used in these trials were divided into six groups of ten animals each. Five groups were given drinking water containing 0.05, 0.1, 0.5, 1 and 2% lead acetate for a period of 60 days. One group (control group, 0% lead acetate) was given pure water. Application of a trend test indicated that both blood and brain lead levels increased significantly from group 0% to group 2% (group 0% <group 0.05% <group 0.1% <group 0. 5%<group 1%<group 2%), but that brain beta-adrenoceptor density and cyclic AMP levels stimulated by isoproterenol decreased (group 2%<group 1%<group 0.5%<group 0.1%<group 0.05%<group 0%). Kd did not vary among the six groups and this indicated that the affinity of the remaining beta-adrenoceptors for [125I]iodocyanopindolol was not changed. Linear regression analysis showed that beta-adrenoceptor density and stimulated cyclic AMP level in brain was found to be negatively correlated with brain lead level (P<0.001). The results show lead exposure that may be the result of an alteration of beta-adrenergic receptor and adenylate cyclase activity in brain.

Nitric oxide enhances expression of raf kinase inhibitor protein in keratinocytes

Abstract:  Several reports have focused on the potential of nitric oxide (NO) to influence the proliferation and differentiation cascade in a number of mammalian cells. The purpose of this study was to determine the relationship between expression of raf kinase inhibitor protein (RKIP) and proliferation in keratinocyte with NO treatment. Normal human keratinocytes were treated with SNAP (NO donor) doses of 10−7, 10−6, 10−5, 10−4 and 0 m (control group) separately. Expression of protein and mRNA of RKIP, cell proliferation and apoptosis have been measured. These results showed that elevated expression of RKIP in keratinocyte with NO treatment may contribute to the pathological and physiological features of NO‐inhibited proliferation.

UVB irradiation induces decreased expression of β2-adrenergic receptors in cultured keratinocytes

Epidermal keratinocytes contain the β-adrenergic adenylate cyclase system, which when activated causes an accumulation of intracellular cyclic AMP through stimulatory guanosine 5-triphosphate (GTP) binding proteins [1]. Adrenergic receptors are responsible for selective recognition and binding of catecholamines, affecting epidermal cell proliferation and differentiation [2]. This β-adrenergic adenylate cyclase system has been found to be defective in several skin diseases including psoriasis [3, 4]. Ultraviolet radiation has been found to augment the epidermal beta adenylate cyclase response [5]. However, to the best of our knowledge, the effects of ultraviolet B (UVB) irradiation on the expression of β2-adrenergic receptors in cultured keratinocytes have not been reported. In the present study, we examined the effect of UVB irradiation on the expression of β2-adrenergic receptors in cultured keratinocytes using a 125I-iodocyanopindolol (ICYP) binding assay to assess receptor density. The keratinocytes were obtained from an adult foreskin from a routine circumcision. The keratinocytes were incubated in a serum-free medium according to the method of Boyce and Ham [6]. The harvested keratinocytes were divided into three groups which were irradiated with UVB at doses of 20, 50 and 75 mJ/cm2, respectively. Cell extracts were prepared 14 h after irradiation by ultrasonication followed by ultracentrifugation. A modification of the β-adrenergic receptor assay developed by Steinkraus et al. [7] was applied to bind ICYP to the keratinocyte membrane. The β-adrenergic receptor density (Bmax) and dissociation constant (Kd) for ICYP binding was determined from the saturation curves of specific binding analyzed by the Scatchard method [8]. Protein was measured according to the method of Lowry et al. [9] using bovine serum albumin as the standard. As shown in Table 1, the β2-adrenergic receptor density (Bmax) of normal control keratinocytes was 93.91 ± 11.02 fmol/mg membrane protein, and the value in UVBirradiated keratinocytes was 76.67 ± 8.81, 38.1 ± 7.13 and 35.47 ± 7.45 fmol/mg membrane protein after irradiation with 20, 50 and 75 mJ/cm2 UVB, respectively. This difference reached statistical significance (unpaired t-test, P < 0.05). The Kd values were not statistically significant (P > 0.05). On the other hand, a statistically significant negative correlation (r = 0.93, P < 0.05) was found beChee-Yin Chai · Hsin-Su Yu · Huoy-Rou Chang · Ying-Rong Lin · Chia-Li Yu

Hexavalent chromium inhibited the expression of RKIP of heart in vivo and in vitro

BACKGROUND Chromium (Cr) is considered to be a risk factor to the cardiovascular effects of fine particulate matter components to PM2.5 from traffic in highway patrol officers. RKIP (raf kinase inhibitor protein) is a physiological inhibitor of GRK-2 (G-protein-coupled receptor kinase 2) and affects β-adrenergic signaling and contractile activity in cardiomyocytes. OBJECTIVES In this study, we explored the change of RKIP in heart of chromium (VI)-exposed rats and cultured myocardial cells with chromium (VI) treatment. METHOD Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000, and 1250 ppm Na(2)Cr(2)O(7) and water for 60 days, respectively. Na(2)Cr(2)O(7) dose of 0.25, 0.5, 1.5, 3, 4.5, and 0 ppm (control group) was applied in cultured myocardial cells. The level of heart Cr (VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP was measured by Western blot method. The MTT assay was used to measure the toxicity of myocardial cells with Cr (VI) treatment. The apoptosis test of myocardial cells was determined by caspase-3 colorimetric assay kit. RESULT The result showed that the expression of RKIP in heart (in vivo) and myocardial cells (in vitro) was decreased following Cr (VI) dose-dependent treatment. CONCLUSION We suggested that the decrement of RKIP of heart and myocardial cells with Cr (VI) treatment resulted in the function of cardiovascular system decreased.

The expression of RKIP, RhoGDI, galectin, c‐Myc and p53 in gastrointestinal system of Cr(VI)‐exposed rats

Hexavalent chromium (CrVI) is considered to be a risk factor in the formation of human cancer. Raf kinase inhibitor protein (RKIP), Rho‐GDIα, galectin, c‐Myc and p53 play important roles in cancer formation. The purpose of this study was to determine if Cr(VI) induces the formation of gastrointestinal cancer. We explored the expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 in the colon and stomach in rats exposed to chromium (CrVI). Thirty Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000 and 1250 ppm Na2Cr2O7 and water for 60 days. The level of Cr(VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 of stomach and colon was measured by western blot. The gene expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 of the stomach and colon was determined by RT‐PCR. The results showed that the expression of p53 and Rho‐GDIα was decreased in the stomach and colon of rats with Cr(VI) treatment. The expression of RKIP was decreased in the stomach and colon of rats treated with high‐dose Cr(VI). The expression of c‐Myc and gelectin‐1 was increased in the stomach and colon of rats with Cr(VI) treatment. We concluded that the anomalous expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 might be a dangerous index of cancer formation in the stomach and colon of rats with Cr(VI) exposure. Copyright