去甲肾上腺素与哌唑嗪对高血压大鼠动脉平滑肌细胞Na+-K+-ATPase活性及mRNA表达的影响

目的 观察去甲肾上腺素(NE)及其α1肾上腺素能受体(α1-AR)拮抗剂哌唑嗪(Prazosin)对自发性高血压大鼠(SHR)动脉血管平滑肌细胞Na -K -ATPase活性及mRNA表达的影响.方法 采用生化酶学方法和逆转录聚合酶链反应技术,观察不同浓度的NE和哌唑嗪对自发性高血压大鼠血管平滑肌细胞Na -K -ATPase活性和mRNA表达的影响.结果 与SHR组比较,NE(10-7 mol/L)能减弱Na -K -ATPase活性(3.98±0.14 vs 7.92±0.19,P＜0.01)及Na -K -ATPase α1-亚单位mRNA的表达(0.863±0.057 vs 1.307±0.051,P＜0.01),单用哌唑嗪对Na -K -ATPase活性及mRNA的表达均无影响(P＞0.05).哌唑嗪(10-7,10-6,10-5 mol/L)呈剂量依赖性减弱和阻断NE(10-7mol/L)对Na -K -ATPase活性(4.31±0.24,6.15±0.30,7.52±0.31)及Na -K -ATPase α1-亚单位mRNA表达(0.857±0.041,1.096±0.055,1.183±0.059)的影响(P＜0.01,P＜0.05).结论 NE抑制SHR Na -K -ATPase活性和下调Na -K -ATPase α1-亚单位mRNA表达,可能是通过α1-AR途径介导基因转录的下调.肾上腺素能1型受体拮抗剂哌唑嗪可通过阻断α1-AR途径抑制NE对自发性高血压大鼠血管平滑肌细胞Na -K -ATPase活性和Na -K -ATPase α1-亚单位mRNA表达的影响.     

五味子水提液对衰老模型小鼠肝细胞膜Na+-K+-ATPase、Mn-SOD和MDA的影响

目的探讨五味子水提液对衰老模型小鼠肝细胞膜Na^＋-K^＋-ATP酶（Na^＋-K^＋-ATPase）、锰超氧化物歧化酶（Mn-SOD）和丙二醛（MDA）的影响。方法以D-半乳糖（D-gal）建立人工致衰小鼠,观察五味子水提液对肝细胞膜Na^＋-K^＋-ATPase、Mn-SOD和MDA含量的影响。结果衰老小鼠肝细胞膜Na^＋-K^＋-ATPase和Mn-SOD活性均降低,MDA含量升高;五味子水提液可以提高肝细胞膜Na^＋-K^＋-ATPase和Mn-SOD活性（P〈0.01）,降低MDA含量（P〈0.01）。结论五味子水提液抗衰老机制可能与抑制脂质过氧化反应,降低MDA含量,提高Na^＋-K^＋-ATPase和Mn-SOD活性有关。     

替米沙坦对OK细胞Na+-K+ ATP酶活性的影响

目的探讨血管紧张素Ⅱ受体拮抗剂(ARB)替米沙坦和血管紧张素转换酶抑制剂(ACEI)苯那普利对负鼠近端小管上皮细胞(OK细胞)Na+-K+-ATP酶活性的影响.方法培养的OK细胞采用低渗方法制备细胞膜悬液,使用BCA-100蛋白质定量测定试剂盒测定膜蛋白;Na+-K+ ATP酶活性采用孔雀绿比色分析法测定释放的无机磷(Pi)含量,培养液中分别加入血管紧张素Ⅱ(Ang Ⅱ)、Ang Ⅱ+血管紧张素Ⅱ受体拮抗剂替米沙坦(Telmisartan)、Ang Ⅱ+血管紧张素转换酶抑制剂苯那普利(Benazepril),观察它们对OK细胞Na+-K+-ATP酶活性的影响.结果 (1)培养液中加入10-10 mol/L Ang Ⅱ组与对照组相比,OK细胞Na+-K+-ATP酶活性明显上升.(0.0972±0.0080 vs 0.0896±0.0065 μmol·L-1·mg pro-1·h-1, P<0.05)(2) 当培养液中同时加入10{10 mol/L Ang Ⅱ和10-9mol/L Telmisartan,与单加入10-10mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性明显降低.(0.0623±0.0053 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1,P<0.05)(3)当培养液中同时加入10-10 mol/L AngⅡ和10-9 mol/L Benazepril,与单加入10-10 mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性无明显变化.(0.1027±0.0166 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1, P>0.05).结论血管紧张素Ⅱ作为一种生长因子,不仅能刺激细胞增殖,又能调节近端小管的离子转运,增加Na+-K+-ATP酶活性;替米沙坦能抑制血管紧张素Ⅱ引起的OK细胞Na+-K+-ATP酶活性增加,而苯那普利则无此作用.     

重型肝炎患者血清对鼠Na+-K+ATP酶活性的影响

我们测定重型肝炎肝性脑病患者血清中中分子物质(middle molecularsubstances,MMS)的变化来探讨MMS在重型肝炎肝性脑病发病机制中的作用。     

内皮素B受体对肾近曲小管上皮细胞Na+-K+-ATP酶活性的影响

目的 探讨内皮素B(ETB)受体对肾脏近曲小管(RPT)上皮细胞Na -K -ATP酶活性的影响和机制.方法 以WKY(Wistar-Kyoto)大鼠RPT细胞为研究对象,Na -K -ATP酶活性采用哇巴因法进行测定.结果 ETB受体激动剂BQ3020能明显降低Na -K -ATP酶的活性,这一抑制作用呈浓度和时间依赖性,BQ3020 10-8 mol/L刺激15 min达最大效应,下降幅度达36.1%;用细胞膜钙通道阻断剂尼卡地平预先刺激细胞后,能够阻断ETB受体对Na -K -ATP酶的抑制效应;在无钙状态下,ETB受体激动剂BQ3020对Na -K -ATP酶的抑制效应丧失.结论 ETB受体在RPT处通过降低Na -K -ATP酶活性调节离子转运;细胞外钙内流参与了ETB受体对Na -K -ATP酶活性抑制作用的信号途径.     

幽门螺杆菌感染对肝硬化患者胃黏膜屏障功能的影响

胃黏膜的屏障功能，有赖于防御因素与损害因素的平衡，肝硬化患者亦如此。在防御众因素中，表皮生长因子（EGF）及前列腺素E2（PGE2）是重要的保护因子，其变化可反映黏膜防御能力的改变；而幽门螺杆菌（Hp）感染后常引起胃黏膜白细胞介素8（IL-8）及肿瘤坏死因子α（TNF-α）的增加，其增加幅度可反映黏膜损害的严重性。本研究通过观察肝硬化患者Hp感染后胃黏膜上述两组具有代表性的客观指标之变化，探讨Hp对肝硬化患者胃黏膜屏障功能的影响，从而较客观的阐明这类患者有无抗Hp的必要。     

SGB对糖尿病性面神经麻痹患者红细胞膜Na+-K+-ATP酶的影响

32例糖尿病合并面神经麻痹患者随机分为药物治疗组和星状神经节阻滞(SGB)组;观察两组不同时点的血清丙二醛(MDA)水平、超氧化物歧化酶(SOD)、红细胞膜Na -K -A1P酶活性及面部表情肌数量级量化评分.与治疗前比较,两组治疗后MDA水平明显降低,红细胞膜Na -K -ATP酶、SOD活性明显升高,面部表情肌数量级量化评分分值明显提高,以上变化SGB组均较药物治疗组明显.认为星状神经节阻滞能提高糖尿病性面神经麻痹患者红细胞膜Na -K -ATP酶活性,缩短痊愈时间.     

小剂量辣椒素对大鼠胃黏膜屏障的影响

目的:给予大鼠不同剂量、不同时期辣椒素(capsaicin,CAP),检测胃黏膜屏障、肝肾组织及血常规、血生化,探讨CAP对胃黏膜屏障的影响及肝肾脏器的安全性.方法:SD大鼠240只,随机分实验组与对照组,实验组分别给予0.1 mg/(kg·d)、1.0 mg/(kg·d)、5.0 mg/(kg·d)CAP饲料,分别于第1、7、14、28天检测血常规、血生化.处死大鼠检测胃黏膜屏障及肝肾组织.结果:各组大鼠状态均良好;大鼠体质量均增加,C组Ⅳ期体质量增加较缓慢,但与对照组D组相比无统计学意义.各组大鼠胃黏膜光滑,未见糜烂出血,Guth评分均为0分.HE染色光镜下观察实验组与对照组大鼠胃黏膜Masude评分无统计学差异(P0.05).血常规、血生化的检测:各实验组与对照组血常规、谷草转氨酶(aspartate transaminase,AST)、谷丙转氨酶(alanine transaminase,ALT)、血肌酐(crea,Cr)检测均无统计学差异.B组、C组血胆固醇(cholesterol,CHOL)及甘油三酯(triglyceride,TG)浓度在Ⅲ、Ⅳ期下降,与D组相比有统计学意义.各实验组大鼠肝肾组织HE染色均未见异常.结论:大鼠摄入CAP 0.1-5mg/(kg·d)饲料,1-28 d对胃黏膜屏障、肝肾组织无损伤,对大鼠血常规、AST、ALT、Cr均无影响.大鼠摄入CAP 1.0-5.0 mg/(kg·d)饲料14-28 d可以调节血脂代谢,降低血脂浓度.     

丹参注射液对老年冠心病患者肾上腺皮质功能的影响

本文通过对18例老年前期、老年期冠心病患者停用各种治广药物及低脂饮食2周后,采用丹参注射液24g/d静滴一个疗程(14次),于治疗前后采肘静脉血和留24h尿,行肾上腺皮质功能测定,并与同年龄正常人不服药组对照,发现血浆皮质醇及24h尿17-OHCS、17-KS均低于对照组,但无显著性差异,治疗后三项指标却有显著的增高。因此认为,丹参能增加冠状动脉血流量,改善冠心病人临床症状。其机理可能是通过升高肾上腺皮质激素水平来实现的。     

延胡索提取物对AMI大鼠模型心肌梗死面积及Na~+-K~+-ATPase、Ca~(2+)-ATPase活性的影响

目的观察延胡索提取物对急性心肌梗死(AMI)大鼠心肌梗死面积及心肌Na~+-K~+-ATPase、Ca~(2+)-ATPase活性的影响。方法体重200 g~220 g的健康Wistar大鼠,利用在体结扎冠状动脉前降支法建立急性心肌梗死模型,假手术组只穿线,不结扎。将造模成功的大鼠随机分为模型组(等量生理盐水)、美托洛尔组[2.25 mg/(kg·d)]、参松养心胶囊组[0.432 g/(kg·d)]、延胡索大剂量组[3.24 g/(kg·d)]、延胡索中剂量组[1.62 g/(kg·d)]、延胡索小剂量组[0.81 g/(kg·d)],加上正常组(等量生理盐水)与假手术组(等量生理盐水),共8组。连续灌胃1周后,末次给药或生理盐水后2 h取材。TTC法测量心肌梗死面积,ELISA法测量心肌Na~+-K~+-ATPase、Ca~(2+)-ATPase的活性。结果与模型组比较,除延胡索大剂量组外,其他各治疗组心肌梗死面积均缩小,差异有统计学意义(P0.05);其他各治疗组疗效相当,差异无统计学意义。与正常组比较,其余各组的Na~+-K~+-ATPase、Ca~(2+)-ATPase浓度值均减低(P0.01),与模型组比较,各治疗组均有所升高(P0.01)。结论延胡索提取物小、中剂量组均有明显缩小心肌梗死面积、提高Na~+-K~+-ATPase、Ca~(2+)-ATPase活性的作用,促进Na~+-Ca~(2+)交换,减轻细胞内钙超载,改善心肌缺血,促进心肌损伤修复,从而降低缺血性心律失常的发生。     

Effects of electroacupuncture on gastric mucosal blood flow and transmucosal potential difference in stress rats

ＥｆｅｃｔｓｏｆｅｌｅｃｔｒｏａｃｕｐｕｎｃｔｕｒｅｏｎｇａｓｔｒｉｃｍｕｃｏｓａｌｂｌｏｏｄｆｌｏｗａｎｄｔｒａｎｓｍｕｃｏｓａｌｐｏｔｅｎｔｉａｌｄｉｆｅｒｅｎｃｅｉｎｓｔｒｅｓｓｒａｔｓＸＵＧｕａｎＳｕｎ１，ＳＵＮＹｏｎｇ１，ＷＡＮＧＺｈｅ...     

Cysteamine increases expression and activity of H^＋-K^＋-ATPase of gastric mucosal cells in weaning piglets

AIM: To determine the in vivo and in vivo effects of cysteamine (CS) on expression and activity of H(+)-K(+)-ATPase of gastric mucosal cells in weaning piglets. METHODS: Eighteen litters of newborn Xinhuai piglets were employed in the in vivo experiment and allocated to control and treatment groups. From 12 d of age (D12), piglets in control group were fed basal diet, while the treatment group received basal diet supplemented with 120 mg/kg CS. Piglets were weaned on D35 in both groups. Six piglets from each group (n = 6) were slaughtered on D28 (one week before weaning), D35 (weaning), D36.5, D38, D42, and D45 (36 h, 72 h, one week and 10 d after weaning), respectively. Semi-quantitative RT-PCR was performed to determine the levels of H(+)-K(+)-ATPase mRNA in gastric mucosa. H(+)-K(+)-ATPase activity in gastric mucosa homogenate was also determined. Gastric mucosal epithelial cells from piglets through primary cultures were used to further elucidate the effect of CS on expression and activity of H(+)-K(+)-ATPase in vivo. Cells were treated for 20 h with 0.001, 0.01, and 0.1 mg/mL of CS (n = 4), respectively. The mRNA expression of H(+)-K(+)-ATPase and somatostatin (SS) as well as the H(+)-K(+)-ATPase activity were determined. RESULTS: in vivo, both mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of control group exhibited a trend to increase from D28 to D45, reaching a peak on D45, but did not show significant age differences. Furthermore, neither the mRNA expression nor the activity of H(+)-K(+)-ATPase was affected significantly by weaning. CS increased the mRNA expression of H(+)-K(+)-ATPase by 73%, 53%, 30% and 39% on D28 (P = 0.014), D35 (P = 0.017), D42 (P = 0.013) and D45 (P = 0.046), respectively. In accordance with the mRNA expression, H(+)-K(+)-ATPase activities were significantly higher in treatment group than in control group on D35 (P = 0.043) and D45 (P = 0.040). In vivo, CS exhibited a dose-dependent effect on mRNA expression and activity of H+-K+-ATPase. Both H(+)-K(+)-ATPase mRNA expression and activity in gastric mucosal epithelial cells were significantly elevated after 20 h of exposure to the moderate (H(+)-K(+)-ATPase expression: P=0.03; H(+)-K(+)-ATPase activity: P = 0.014) and high concentrations (H(+)-K(+)-ATPase expression: P=0.017; H(+)-K(+)-ATPase activity: P = 0.022) of CS. Significant increases in SS mRNA expression were observed to accompany the elevation of H(+)-K(+)-ATPase expression and activity induced by the moderate (P = 0.024) and high concentrations (P = 0.022) of CS. Low concentration of CS exerted no effects either on expression and activity of H(+)-K(+)-ATPase or on SS mRNA expression in cultured gastric mucosal epithelial cells. CONCLUSION: No significant changes are observed in mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of piglets around weaning from D28 to D45. CS increases expression and activity of gastric H(+)-K(+)-ATPase in vivo and in vivo. SS is involved in mediating the effect of CS on gastric H(+)-K(+)-ATPase expression and activity in weaning piglets.     

Correlation between transmucosal potential difference and morphological damage during aspirin injury of gastric mucosa in rats

The potential difference (PD) that is maintained across healthy gastric mucosa is thought to be due to asymmetric ion pumping combined with resistance to back-diffusion of the separated charge. However, the structures that are responsible for this have not been clearly defined. This study examined the temporal changes in PD in rat stomach after injury by a single dose of aspirin. Multiple linear regression was used to compare this with the time course of several parameters of histological damage: (i) the per cent mucosal length showing superficial (confined to surface and gastric pits), deep (involving the isthmus or deeper in oxyntic glands) and total damage; (ii) the number of discrete erosions; and (iii) the total area of erosions per cm sectioned. Mucosal PD fell during the first 30–60 min after aspirin. Superficial damage appeared early and was already recovering by this time. The time course of deep damage more closely matched the alterations in PD and stepwise regression analysis showed that this could be predicted by the amount of deep damage alone (P < 0.001). Changes in transmucosal PD after acute aspirin injury probably reflect damage to structures in the oxyntic glands and not just the breaking of the surface and pit cell ‘barrier’.     

Effect of ethanol on canine gastric epithelial ultrastructure and transmucosal potential difference

We correlated changes in the gastric transmucosal potential difference (PD), as an indicator of the integrity of the gastric mucosal barrier, with morphological evidence of injury in dogs which had received either intragastric saline or 5, 10, 15, or 30% ethanol. Increasing degrees of morphological damage were accompanied by greater, more rapid changes in PD. Furthermore, ultrastructural changes occurred within surface epithelial cells, not in the deeper parietal or zymogen cells, and initially did not involve disruption of the apical cell membrane. Typically, the tight junctions also were not affected, although in a minority of dogs small bleblike separations of the tight junctions were seen. We consider the gastric mucosal barrier to be represented morphologically by the interconnecting sheet of gastric epithelial cells and that ethanol breaks the barrier by first causing intracellular injury.This study was supported by Veterans Administration funds, MRIS #690-2037.     

Studies on the Na+-K+-ATPase in myocardial infarction

Changes in the cardiac sarcolemma in myocardial infarction were studied by both determination of Na+-K+-ATPase activity and SDS gel electrophoretic analysis of sarcolemmal proteins in the canine heart. Ninety minutes after coronary ligation, Na+-K+-ATPase activity in ischemic myocardium was decreased significantly to approximately 36% of that of non-ischemic myocardium, and it remained at the lower level for 28 days. By SDS gel electrophoresis, reduction of the protein band with molecular weight of 111,000, which is suggestive of the main component of ATPase, was observed simultaneously with the reduction of Na+-K+-ATPase activity. These results indicate that ischemia for 90 minutes produces substructural changes in the sarcolemma indicating irreversible myocardial changes.     

The change and significance of the Na+-K+-ATPase alpha-subunit in ouabain-hypertensive rats.

Ouabain has recently been identified as an endogenous Na+-K+ pump inhibitor having a close association with hypertension. However, some patients with hypertention do not show high levels of endogenous ouabain (EO), and patients with high EO levels do not necessarily suffer from hypertention. It is believed that the Na+-K+-ATPase activity in essential hypertension does not undergo homogenous change. The present study was designed, therefore, to investigate the expression and the significance of the Na+-K+-ATPase alpha-subunit isoforms in kidney tissue in ouabain-hypertensive rats. Ouabain was administered chronically to establish a model of ouabain-hypertensive rats. Biochemical analysis, cytobiology and sABC immunohistochemistry were they used to assay for expression of Na+-K+-ATPase alpha-subunit isoforms in kidney tissue. After the first week of receiving ouabain, 65% (n=13) of rats had hypertension. After the second week, the blood pressure of these 13 hypertensive rats was increased significantly compared to the baseline and control levels (p<0.05). The plasma renin activity was normal, and angiotensin II and aldosterone levels were increased significantly in these rats (p<0.05). But in the other 35% (n=7) of rats of the experimental group, there was no apparent increase in blood pressure after receiving ouabain. The plasma ouabain level in the non-hypertensive subgroup was significantly higher than that in the hypertensive subgroup, but the 86Rb intake and the number of 3H-ouabain binding sites did not decrease. The Na+-K+-ATPase activity showed non-homogeneous changes. In hypertensive rats, the expression levels of ouabain paralleled the degree of hypertension (r=0.88, p<0.05). The positive granules were mainly scattered in the cytoblastoma of the reticular zone of adrenal cortex. There were thus different levels of expression of Na+-K+-ATPase alpha-subunit isoforms in this model. In the hypertension subgroup the alpha1 was most strongly expressed, followed by the alpha2 and alpha3 isforms. But in the non-hypertensive subgroup the order was alpha3 > alpha2 > alpha1. The positive granular was mainly scattered in the convoluted tubules of the kidney. These results suggest that the high level of ouabain and the change of the Na+-K+-ATPase alpha-subunit isoforms may play a critical role in hypertension.     

Decrease in myocardial Na+-K+-ATPase activity and ouabain binding sites in hypercholesterolemic rabbits

Objectives The purpose of this study was to explore the effect of high dietary cholesterol on the lipid composition, Na⁺–K⁺-ATPase activity and ouabain receptor property of the myocardial sarcolemma.Methods Male New Zealand white rabbits were fed with standard chow or standard chow supplemented with 0.5% (w/w) cholesterol and 10% (w/w) coconut oil to induce hypercholesterolemia. After 8 weeks, the rabbits were sacrificed; a myocardial sarcolemma fraction was then prepared from the left ventricular myocardium and analyzed for lipid composition. Assay of Na⁺–K⁺-ATPase activity and³H-ouabain binding studies were performed in the myocardial sarcolemma from the control and cholesterol-fed rabbits.Results The cholesterol content, but not the phospholipid content, of the sarcolemma was significantly greater in the cholesterol-fed group, thus, resulting in an increased cholesterol/phospholipid molar ratio in the cholesterol-fed group. In addition, a decrease in Na⁺–K⁺-ATPase activity was also found in this group. The decrease in Na⁺–K⁺-ATPase activity was selective, since the Mg⁺⁺-ATPase and 5-nucleotidase activities remained unchanged. In the³H-ouabain binding study, a decrease in the number of maximum binding sites, but not the binding affinity, for³H-ouabain was foundie the cholesterol-fed group.Conclusions High dietary cholesterol induces higher levels of cholesterol not only in the plasma, but also in the myocardial sarcolemma. These changes result in decreased myocardial Na⁺–K⁺-ATPase activity mediated by a reduction in the maximum number of binding sites for ouabain but not a change in binding affinity.     

Aldosterone increases Na+ -K+ -ATPase activity in skeletal muscle of patients with Conn's syndrome

Objective In Conn’s syndrome, hypokalaemia normally results from renal potassium loss because of the effect of excess aldosterone on Na⁺‐K⁺‐ATPase in principal cells. Little is known about the effect of aldosterone on cellular potassium redistribution in skeletal muscle. Our study determined the effect of aldosterone on muscle Na⁺‐K⁺‐ATPase. Design Muscle biopsies were taken from six patients immediately before and 1 month after adrenalectomy. Ten age‐matched subjects with normal levels of circulating aldosterone served as controls. Results Average plasma aldosterone was significantly higher in presurgery (235·0 ± 51·1 pg/ml) than postsurgery (64·5 ± 25·1 pg/ml) patients. Similarly, Na⁺‐K⁺‐ATPase activity, relative mRNA expression of α₂ (not α₁ or α₃) and β₁ (not β₂ or β₃), and protein abundance of α₂ and β₁ subunits were greater in pre‐ than postsurgery samples (128·7 ± 12·3 vs 79·4 ± 13·3 nmol·mg/protein/h, 2·45 ± 0·31 vs 1·04 ± 0·17, 1·92 ± 0·22 vs1·02 ± 0·14, 2·17 ± 0·33 vs 0·98 ± 0·09 and 1·70 ± 0·17 vs 0·90 ± 0·17, respectively, all P < 0·05). The activity and mRNA expression of the α₂ and β₁ subunits correlated well with plasma aldosterone levels (r = 0·71, r = 0·75 and r = 0·78, respectively, all P < 0·01). Conclusions Our study provides the first evidence in human skeletal muscle that increased plasma aldosterone leads to increased Na⁺‐K⁺‐ATPase activity via increases in α₂ and β₁ subunit mRNAs and their protein expressions. The increased activity may contribute in part to the induction of hypokalaemia in patients with Conn’s syndrome.     

Effect of lysolecithin on gastric mucosal structure and potential difference.

The effect of lysolecithin (100 mg%) on the guinea-pig gastric mucosa was studied by instilling a solution for 30 minutes, preceded and followed by 100 MN HCl into 10 total gastric pouches. Ten control animals had HCl throughout. Lysolecithin produced a significant change in transmucosal potential difference, macroscopic erosions, and mucosal damage on histology and electron scanning microscopy. None of these changes was seen in the control animals. This is further evidence that the reflux of lysolecithin from the duodenum is an important factor in causing active gastritis and gastric erosions.