瞬时受体电位香草酸亚型1在呼吸调节中的作用

瞬时受体电位香草酸亚型1是辣椒素的特异性受体,广泛分布于哺乳动物气道的感觉神经,被多种内外源性刺激(辣椒素、热、酸等)激活后,通过中枢及局部反射产生一系列生物学效应,如气道高反应性、神经源性炎症反应及咳嗽等.     

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.     

Impaired transient receptor potential vanilloid 1 in streptozotocin-induced diabetic hearts

Little is known about the effect of diabetes mellitus (DM) on transient receptor potential vanilloid 1 (TRPV1) located in cardiac sensory nerves. This study was performed to test the changes of TRPV1 and its main neuropeptides in diabetic hearts. DM was induced by intraperitoneal injection of streptozotocin (STZ) in C57BL/6J mice. The protein and mRNA expression of TRPV1, calcitonin gene-related peptide (CGRP) and substance P (SP) levels in hearts were measured, respectively. Compared with control mice, blood glucose was significantly increased in diabetic mice (P<0.05), while the protein and mRNA expression of TRPV1, CGRP and SP levels in hearts were essentially reduced in diabetic mice (P<0.05). TRPV1 and its main neuropeptides, CGRP and SP, in hearts were impaired during DM.     

Capsaicin-induced inhibition of platelet aggregation is not mediated by transient receptor potential vanilloid type 1

Capsaicin is an agonist of transient receptor potential vanilloid type 1 (TRPV1), in which it can act as a neuronal stimulant and result in nociception. Capsaicin also affects a variety of nonneuronal tissues, in which its mechanisms of action are less certain. The present study investigated whether the inhibitory effects of capsaicin on platelet aggregation are mediated via TRPV1. Venous whole blood obtained from beagle dogs (n = 6) was preincubated with capsaicin and/or the potent and selective competitive TRPV1 antagonist, A-993610 and then exposed to collagen (2 μg/ml). An aggregometer was used to quantify the platelet response. Capsaicin exposure inhibited collagen-induced platelet aggregation in a concentration-dependent manner, with significant effects at 10 and 30 μg capsaicin per millilitre. A-993610 alone (0.1-1.0 μg/ml) had no effects on collagen-induced platelet aggregation, nor did it have any effects on capsaicin's ability to inhibit platelet aggregation. The current results agree with previous findings that capsaicin can inhibit platelet aggregation. In addition, the present study demonstrates that capsaicin's inhibitory effect on collagen-induced canine platelet aggregation is not mediated by TRPV1.     

Deletion of transient receptor potential vanilloid type 1 receptors exaggerates renal damage in deoxycorticosterone acetate-salt hypertension

To determine whether the transient receptor potential vanilloid type 1 (TRPV1) channel provides protection against hypertension-induced renal damage, hypertension was induced by uninephrectomy and by giving deoxycorticosterone acetate (DOCA)-salt in wild-type (WT) and TRPV1-null mutant (TRPV1-/-) mice. Mean arterial pressure, as determined by radiotelemetry, increased significantly and reached the peak 7 days after DOCA-salt treatment in both WT and TRPV1-/- mice. There was no difference in mean arterial pressure between the 2 strains at the baseline or at the peak that lasted for 4 treatment weeks. DOCA-salt treatment in both WT and TRPV1-/- mice led to increased urinary excretion of albumin and 8-isoprostane, glomerulosclerosis, renal cortical tubulointerstitial injury, tubulointerstitial fibrosis, increased number of tubular proliferating cell nuclear antigen-positive cells, and renal monocyte/macrophage infiltration, all of which were much more severe in DOCA-salt-treated TRPV1-/- compared with DOCA-salt-treated WT mice. Renal TRPV1 protein expression, but not the renal anandamide content, was elevated in DOCA-salt-treated WT compared with vehicle-treated WT mice. Renal anandamide levels were markedly elevated in DOCA-salt-treated TRPV1-/- but not in vehicle-treated TRPV1-/- mice. Thus, our data show that ablation of the TRPV1 gene exacerbates renal damage induced by DOCA-salt hypertension, indicating that TRPV1 may constitute a protective mechanism against end-organ damage induced by hypertension.     

Epidermal growth factor increases intestinal calbindin-D9k and 1,25-dihydroxyvitamin D receptors in neonatal rats

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity

We tested the hypothesis that activation of transient receptor potential vanilloid type-1 (TRPV1) by capsaicin prevents adipogenesis. TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans were detected by immunoblotting and quantitative real-time RT-PCR. The effect of TRPV1 on cytosolic calcium was determined fluorometrically in 3T3-L1-preadipocytes and in human visceral fat tissue. Adipogenesis in stimulated 3T3-L1-preadipocytes was determined by oil red O-staining of intracellular lipid droplets, triglyceride levels, expression of peroxisome proliferator-activated receptor-gamma, and expression of fatty acid synthase. Long-term feeding experiments were undertaken in wild-type mice and TRPV1 knockout mice. We detected TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans. In vitro, the TRPV1 agonist capsaicin dose-dependently induced calcium influx and prevented the adipogenesis in stimulated 3T3-L1-preadipocytes. RNA interference knockdown of TRPV1 in 3T3-L1-preadipocytes attenuated capsaicin-induced calcium influx, and adipogenesis in stimulated 3T3-L1-preadipocytes was no longer prevented. During regular adipogenesis TRPV1 channels were downregulated which was accompanied by a significant and time-dependent reduction of calcium influx. Compared with lean counterparts in visceral adipose tissue from obese db/db and ob/ob mice, and from obese human male subjects we observed a reduced TRVP1 expression. The reduced TRPV1 expression in visceral adipose tissue from obese humans was accompanied by reduced capsaicin-induced calcium influx. The oral administration of capsaicin for 120 days prevented obesity in male wild type mice but not in TRPV1 knockout mice assigned to high fat diet. We conclude that the activation of TRPV1 channels by capsaicin prevented adipogenesis and obesity.     

Involvement of transient receptor potential vanilloid 1 in the vascular and hyperalgesic components of joint inflammation

OBJECTIVE: To investigate the endogenous involvement of transient receptor potential vanilloid 1 (TRPV1) in a model of knee joint inflammation in the mouse. METHODS: Following characterization of wild-type (WT) and TRPV1-knockout mice, inflammation was induced via intraarticular (IA) injection of Freund's complete adjuvant (CFA). Knee swelling was assessed as diameter, and inflammatory heat hyperalgesia was determined using the Hargreaves technique, for up to 3 weeks. At 18 hours, acute hyperpermeability was measured with 125I-albumin, and cytokines and myeloperoxidase activity, a marker of neutrophils, were assayed in synovial fluid extracts. The possibility that exogenous tumor necrosis factor alpha (TNFalpha) was involved in influencing TRPV1 activation was investigated in separate experiments. RESULTS: Increased levels of knee swelling, hyperpermeability, leukocyte accumulation, and TNFalpha were found in WT mice 18 hours after IA CFA treatment compared with saline treatment. Significantly less knee swelling and hyperpermeability were found in TRPV1-/- mice, but leukocyte accumulation and TNFalpha levels were similar in WT and TRPV1-/- mice. Knee swelling in response to CFA remained significantly higher for a longer period in WT mice compared with TRPV1-/- mice, with thermal hyperalgesic sensitivity observed at 24 hours and at 1 week in WT, but not TRPV1-/-, mice. Knee swelling was attenuated (P < 0.05) in TRPV1-/- compared with WT mice 4 hours after IA administration of TNFalpha. CONCLUSION: Our findings indicate that TRPV1 has a role in acute and chronic inflammation in the mouse knee joint. Thus, selective antagonism of TRPV1 should be considered as a potential target for treatment of acute and chronic joint inflammation.     

瞬时受体电位香草酸亚型1受体介导疼痛的机制和治疗应用研究进展

瞬时受体电位香草酸亚型1(TRPV1)自首次成功克隆以来作为与疼痛密切相关的伤害性感受器而备受关注,其分子结构与生理功能、分布特征、激活与抑制等得到广泛研究。TRPV1受体参与炎症、脂质氧化等生物过程也与疼痛息息相关。多种内、外源性增敏剂或抑制剂可调节TRPV1通道敏感性,从而介导疼痛信号传导。TRPV1还可能通过细胞内钙超载等机制导致伤害性感受器的消融,这可能是镇痛治疗的一种潜在有效途径。该文旨在对TRPV1受体介导疼痛的相关机制及其应用研究进展作一综述。     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

A novel mechanism contributing to development of Dahl salt-sensitive hypertension: role of the transient receptor potential vanilloid type 1

To determine the role of the transient receptor potential vanilloid type 1 (TRPV1) channels in development of hypertension in Dahl salt-sensitive (DS) rats fed a high-salt diet (HS), male DS and Dahl salt-resistant (DR) rats were maintained on a low-salt diet (LS) or HS for 3 weeks. HS significantly increased systolic blood pressure in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Intravenous bolus injection of capsazepine (3 mg/kg), a selective TRPV1 antagonist, significantly increased mean arterial pressure in conscious DR+HS rats compared with DR+LS, DS+/-HS, and DS+/-LS rats. In contrast, capsaicin (10 or 30 microg/kg), a selective TRPV1 agonist, dose-dependently decreased mean arterial pressure in all of the groups with the most profound magnitude in DR+HS rats compared with the other 3 groups. TRPV1 expression in mesenteric resistance arteries and the renal cortex and medulla, calcitonin gene-related peptide levels in dorsal root ganglia, and calcitonin gene-related peptide-positive sensory nerve density in mesenteric resistance arteries were significantly decreased in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Taken together, our data indicate that the TRPV1 receptor is activated and its expression upregulated during HS intake in DR rats, which acts to prevent salt-induced increases in blood pressure. In contrast, TRPV1 expression and function are impaired in DS rats, which renders DS rats sensitive to salt load in terms of blood pressure regulation.     

Gestational changes in calbindin-D9k in rat uterus, yolk sac, and placenta: implications for maternal-fetal calcium transport and uterine muscle function.

Calbindin-D9k was quantified and its cellular location was defined in uterus, yolk sac, and placenta. In late gestation (days 17 to term) coordinated induction of calbindin-D9k was seen in uterine epithelial lining cells and juxtaposed yolk sac visceral epithelium as well as the intraplacental yolk sac epithelium. The induction of calbindin-D9k in these cells coincided with the time of exponential fetal bone growth and maximal fetal accumulation of calcium, suggesting a role of the protein in these epithelial layers in maternal-fetal calcium transport. Dynamic changes also occurred in the calbindin-D9k contents of the two layers of uterine smooth muscle (outer longitudinal and inner circular) during mid- and late gestation. During early pregnancy (days 0-4), calbindin-D9k was present in the two smooth muscle layers. By midgestation (day 10), calbindin-D9k had decreased by a factor of 10 in these tissue layers. During late gestation calbindin-D9k rebounded in the inner circular smooth muscle layer. These uterine changes of early and midgestation were reproduced by the endocrine changes of pseudopregnancy. Progesterone appeared to be a good candidate for controlling the midgestational decrease of uterine muscle calbindin-D9k, as it blunted estrogen's induction of the protein in the muscle layers and stroma in a dose-dependent manner. Changes in myometrial calbindin-D9k may reflect variations in muscular calcium storage, thereby representing alterations in potential for contraction.     

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. CONCLUSION: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis.     

瞬时受体电位香草酸亚型1在小鼠心肌梗死后炎症中的保护作用

目的 研究瞬时受体电位香草酸亚型1(TRPV1)在心肌梗死(MI)后炎症中的保护作用.方法 在TRPV1基因敲除(TRPV1-/-)小鼠和野生型(WT)小鼠建立MI模型,监测梗死后第3天的梗死面积、炎症细胞渗透、炎症因子和趋化因子的表达水平、心功能和第7天的生存率.结果 TRPV1-/-小鼠MI后7 d内的生存率低于野生型小鼠(62.5%比82.1%,P<0.05).MI后第3天:TRPV1-/-小鼠的梗死面积(INF)大于野生型小鼠[INF/危险区面积(AAR):69.5%±3.1%比40.1%±2.6%,P<0.05];TBPV1-/-小鼠的血浆心肌钙蛋白Ⅰ水平[(0.98±0.16)ng/ml比(0.58±0.15)ng/ml,P<0.001]、中性粒细胞[(1142±112)/mm2比(779±50)/mm2,P<0.05]和巨噬细胞渗透[(1098±58)/mm2比(664±49)/mm2,P<0.01]均高于野生型小鼠.TRPV1-/-小鼠的肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6、巨噬细胞趋化蛋白(MCP)-1和巨噬细胞炎症蛋白(MIP)-2表达也明显高于野生型小鼠(均P<0.05).此外,与野生型小鼠比较,TRPV1-/-小鼠梗死后收缩末期和舒张末期内径进一步扩大,收缩功能恶化.结论 TRPV1基因缺失导致MI后生存率下降,炎症反应增强,心功能恶化,提示TRPV1可能通过抑制炎症和保存心功能而防止梗死扩散和心脏损伤.