Altered carbachol-induced contractile responses of rat jejunal smooth muscle following local myenteric plexus ablation

Alterations in smooth muscle responsiveness and neural pathways in adjacent tissue may occur after local myenteric denervation. The in vitro contractile responses of both longitudinal and circular muscle to the mixed muscarinic and nicotinic cholinergic agonist carbachol were determined 15, 30, and 45 days after localized myenteric plexus ablation. Denervated longitudinal muscle exhibited decreased responsiveness to carbachol at all times examined. Denervated circulated muscle was initially supersensitive, but with time became subsensitive. These changes probably reflect the loss of the nicotinic (neuronal) component of the action of carbachol. Muscle orad to the site of denervation appeared subsensitive, while muscle caudad to the lesion was supersensitive (circular) or unaffected (longitudinal). These results suggest that there are changes in ascending and descending neural pathways. Alterations in the cholinergic responsiveness of intestinal smooth muscle, both at and beyond the site of myenteric plexus ablation, may result in altered intestinal motility that could lead to functional obstruction.Supported by National Institutes of Health grant AM 32594.Contribution 209, Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin 53706.     

Aging alters mechanical and contractile properties of the Fisher 344/Nnia X Norway/Binia rat aorta

Vascular mechanical and contractile properties were compared in adult (6 months old) and very-aged (36 months old) Fischer 344/NNiaHSd X Brown Norway/BiNia (F344/NXBN) rats. Our previous work has indicated that aging is associated with aortic medial thickening. This morphological alteration was accompanied by a leftward shift in the aortic stress/strain curve indicating increased vessel stiffness in very-aged animals. Disruption of the endothelium as well as pretreatment of tissues with the nitric oxide (NO) donor sodium nitroprusside eliminated differences, suggesting a link between deficient endothelial NO release and reduced compliance in very-aged aortae. In addition, the Rho kinase inhibitor Y-27632 increased vessel compliance in both adult and very-aged tissues suggesting that the Rho cascade contributed to the stress/strain relationship. Maximal force developed in response to high potassium (K⁺) was reduced by ∼70% in intact and endothelium-denuded aortae from very-aged rats. In contrast to contractile force development, calcium-dependent stress relaxation was increased in very-aged aorta. Finally, gel electrophoresis indicated a significantly higher tissue content of myosin heavy chain and a higher ratio of SM1/SM2 isoforms with aging. The results suggest multiple molecular changes with aging, which may be expected to alter vascular tissue function.     

An improved procedure for the isolation of plasma membranes from rat mesenteric arteries.

A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.     

Expression levels of TRPC1 and TRPC6 ion channels are reciprocally altered in aging rat aorta: implications for age-related vasospastic disorders

We previously showed that the expression of transient receptor potential canonical (TRPC)6 ion channel elevated when TRPC1 was knocked down in A7r5 cultured vascular smooth muscle cells. Therefore, the purpose of this study was to explore whether TRPC6 is also upregulated in aging rat aorta comparable to that of TRPC1 in longitudinal in vivo aging model. We further investigated a possible causal relationship between altered phenylephrine-induced contractions and the expression levels of TRPC6, a purported essential component of alpha-adrenergic receptor signaling in aging aorta. Immunoblot analysis showed that TRPC1 protein levels significantly decreased whereas TRPC6 increased drastically in aorta from 16- to 20-month-old rats compared to that from 2 to 4 months. Immunohistochemical data demonstrated spatial changes in TRPC6 expression within the smooth muscle layers along with increased detection in the adventitia of the aged rat aorta. The phenylephrine-induced contractions were potentiated in aging aorta. In conclusion, based on this aging model, TRPC6 overexpression could be related with TRPC1 downregulation and might be responsible for the increased adrenoceptor sensitivity which contributes to the development of age-related vasospastic disorders.     

Age-related changes of P2X(1) receptor mRNA in the bladder detrusor from men with and without bladder outlet obstruction

The urinary bladder purinergic system is reported to change with age and with bladder dysfunction. Here, we examined the expression of purinergic P2X(1) receptors in detrusor and mucosa (urothelium+lamina propria) from male control bladder and investigated age-related P2X(1) receptor mRNA expression in control and obstructed detrusor. Biopsy specimens were obtained at cystoscopy from control patients (n=46, age range 30-86years) and patients diagnosed with outlet obstruction (n=29, 46-88years). Calponin expression (measured by RT-PCR) was similar in control and obstructed detrusor and did not change with age. Quantitative competitive RT-PCR was used to measure P2X(1) receptor and GAPDH mRNA in control and obstructed detrusor. P2X(1) receptor mRNA expression was 9-fold (p<0.0001) higher in the detrusor than in the mucosa. Expression of mRNA for the internal control GAPDH remained stable with age and across control and obstructed detrusor. No difference in P2X(1) receptor expression was observed between control and obstructed detrusor (p=0.35). However, an age-related decrease in P2X(1) mRNA expression was observed in control (n=27; p=0.0054; Spearman coefficient r=-0.520) but not obstructed detrusor (n=19; p=0.093; r=-0.396). Downregulation of P2X(1) mRNA expression might occur as a result of an increased component of neural ATP release in the aging bladder.     

Weakness of whole muscles in mice deficient in Cu, Zn superoxide dismutase is not explained by defects at the level of the contractile apparatus

Mice deficient in Cu,Zn superoxide dismutase (Sod1^−/− mice) demonstrate elevated oxidative stress associated with rapid age-related declines in muscle mass and force. The decline in mass for muscles of Sod1^−/− mice is explained by a loss of muscle fibers, but the mechanism underlying the weakness is not clear. We hypothesized that the reduced maximum isometric force (F_o) normalized by cross-sectional area (specific F_o) for whole muscles of Sod1^−/− compared with wild-type (WT) mice is due to decreased specific F_o of individual fibers. Force generation was measured for permeabilized fibers from muscles of Sod1^−/− and WT mice at 8 and 20 months of age. WT mice were also studied at 28 months to determine whether any deficits observed for fibers from Sod1^−/− mice were similar to those observed in old WT mice. No effects of genotype were observed for F_o or specific F_o at either 8 or 20 months, and no age-associated decrease in specific F_o was observed for fibers from Sod1^−/− mice, whereas specific F_o for fibers of WT mice decreased by 20 % by 28 months. Oxidative stress has also been associated with decreased maximum velocity of shortening (V_max), and we found a 10 % lower V_max for fibers from Sod1^−/−compared with WT mice at 20 months. We conclude that the low specific F_o of muscles of Sod1^−/− mice is not explained by damage to contractile proteins. Moreover, the properties of fibers of Sod1^−/− mice do not recapitulate those observed with aging in WT animals.     

Aging changes in the periodontal bone of F344/N rat

The aim of this research was to determine whether a rat was an adequate laboratory animal model for periodontal research on elderly humans. Thirty-two F344/NSlc female rats ranged between 30 and 1000 days of age were used. The alveolar bone loss around the molars was assessed by a morphometric method. A significant correlation was found between age and the amount of alveolar bone loss. For further analysis, the rats were grouped into four by age; 30–60 days, 220–430 days, 640–850 days, and more than 850 days. The means of alveolar bone loss were compared between age groups. It was found that the resorption of the alveolar bone around the molars of the rats continued until they were 1000-days-old, and this trend was stronger in the mandible than the maxilla. It was suggested that rats could be used as adequate laboratory animals for periodontal research.     

缝隙连接蛋白-43在膀胱出口部分梗阻的大鼠逼尿肌细胞膜中的表达

目的探讨缝隙连接蛋白-43(Cx-43)在膀胱出口部分梗阻大鼠逼尿肌细胞膜中表达的变化。方法 33只Wistar大鼠分为模型组和假手术组,两组大鼠分别于术后2、4和8 w处死,同时进行膀胱内测压、缝隙连接数量、形态变化及Cx-43的检测。结果在模型组中缝隙连接的数量显著降低,并且术后4 w Cx-43在模型组大鼠逼尿肌细胞膜上的表达显著降低。模型组大鼠逼尿肌细胞膜上的缝隙连接数量也明显减少。结论正常的排尿过程是通过逼尿肌细胞间的缝隙连接直接作用的结果。缝隙连接介导的细胞间通讯功能障碍可能是导致排尿功能障碍的原因。     

Effects of contractile protein phosphorylation on force development in permeabilized rat cardiac myocytes

The phosphorylation status of myofibrillar proteins influences the Ca²⁺ responsiveness of the myofilaments,but the contribution of and the interaction between the individual components is poorly characterized. Therefore, in Langendorff perfused rat hearts (n=30), the phosphorylation levels of cardiac myosin binding protein-C (cMyBP-C), troponin I and T (cTnI, cTnT) and myosin light chain 1 and 2 (MLC-1, MLC-2) were determined by 1- and 2-dimensional gel electrophoresis. Isometric force development, its Ca²⁺-sensitivity, the rate of tension redevelopment (k_tr) and passive force (F_pas) were studied at optimal sarcomere length (2.2 μm) in mechanically isolated,permeabilized cardiomyocytes at 15 °C. Protein phosphorylation was varied by: 1) blocking spontaneous cardiac activity by lidocaine (0.35 mM; Quiescence); 2) electrical stimulation of the hearts at 5 Hz (Contraction) and 3. treatment of contracting hearts with Isoprenaline (1 μM). MLC-2 phosphorylation was increased in the Contraction group almost 2-fold, relative to the Quiescence group, whereas cMyBP-C and cTnI phosphorylation remained the same. Isoprenaline resulted in 3.7-fold increases in both cMyBP-C and cTnI phosphorylation, but did not result in a further increase in MLC-2 phosphorylation.No significant differences were found in maximum force and k_tr between groups, both before and after protein kinase A (PKA) treatment. Ca²⁺-sensitivity in the Contraction and Isoprenaline groups was significantly reduced in comparison to the Quiescence group. These differences were largely abolished by PKA and F_pas was reduced. These results highlight the impact of PKA-dependent phosphorylation on Ca²⁺-sensitivity and provide evidence for an interaction between the effects of TnI and MLC-2 phosphorylation.     

β-磷酸甘油诱导体外培养大鼠主动脉平滑肌细胞的钙化

目的:研究β磷酸甘油对体外培养的大鼠主动脉平滑肌细胞的钙化作用。方法:采用组织块培养法体外培养大鼠主动脉平滑肌细胞。细胞分为钙化组和正常组。钙化组加入10mmol/Lβ磷酸甘油,0.1μmol/L胰岛素及50μg/L维生素C（钙化培养基）诱导细胞钙化,继续培养14d。茜素红S染色观察钙结节计数并测定细胞钙沉积含量。采用四唑盐比色法（MTT）测量细胞增殖。结果:钙化组钙结节计数为（7.4±3.8）%,较正常组（4.3±1.9）%显著增加（P<0.05）;细胞钙沉积含量为（5.4±0.4）mmol/g蛋白,较正常组（1.2±0.5）mmol/g蛋白显著增加（P<0.05）。MTT检测细胞增殖,钙化组细胞增殖为0.071±0.011,较正常组0.040±0.009明显增加（P<0.01）。结论:β磷酸甘油能诱导体外培养的大鼠主动脉平滑肌细胞的钙化,钙化能促进平滑肌细胞的增殖。     

Calcium induced reversible alterations in excitation-contraction coupling in verapamil treated rat myocardium

We studied the effects of a calcium channel blocking agent, verapamil (V) (2 to 10 micrograms/ml), in the presence of increasing external calcium on simultaneously recorded transmembrane electrophysiological properties and mechanical function of rat myocardium. Left ventricular papillary muscles from male Fischer 344 rats were studied electrically, by standard microelectrode techniques, and mechanically in an isolated tissue bath at 30 degrees C. Control (0 micrograms/ml V + 2.4 mM Ca2+) = C, action potential duration at 50% and 75% repolarization (D50ap and D75ap) recorded from papillary muscles were short (14.1 +/- 0.75 ms; 33.3 +/- 2.7 ms) compared with recordings from papillary muscles subjected to increasing doses of verapamil (2, 4, 6, 8, or 10 micrograms/ml) + 2.4 mM Ca2+ = V, (17.3 +/- 0.77 ms; 121.4 +/- 8.9 ms: 10 micrograms/ml) (P less than 0.001). Upon augmentation of external calcium [10 micrograms/ml Verapamil + augmented Ca2+ (4.8, 7.2, or 9.6 mM] = VCa, D50ap and D75ap decreased but still remained significantly longer than control D50ap and D75ap (15.1 +/- 0.77 ms; 110.1 +/- 7.9 ms). Developed tension (Td), time to peak developed tension (TPT), time to one-half relaxation (T1/2R) and resting tension (Tr) decreased as a function of verapamil concentration. Although TPT and T1/2R returned toward C values when external calcium was increased, Tr continued to decrease while Td increased above control levels. A significant correlation was found between measured parameters of contraction and transmembrane action potential for C and VCa muscles. However, in V muscles no significant correlation was observed between these same mechanical and electrical parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Models to understand contractile function in the airways

Although the role of contractile function in the airways is controversial, there is general consensus on the importance of airway smooth muscle (ASM) as a therapeutic target for diseases characterized by airway obstruction, such as asthma or chronic obstructive pulmonary disease. Indeed, the use of bronchodilators to relax ASM is the most common and effective practice to treat airflow obstruction. Excessive pathologic bronchoconstriction may originate from primary alterations of ASM mechanical function and/or from the effects exerted on ASM function by disease processes, such as inflammation and remodeling. An in depth knowledge of the potentially multiple mechanisms that distinctively regulate primary and secondary alterations in ASM contractile function would be essential for the development of new therapeutic approaches aimed at preventing the occurrence or reducing the severity of bronchoconstriction. The present review discusses studies that have addressed the mechanisms of altered ASM contractile function in models of airway hyperresponsiveness. Although not comprehensively, in the present review, animal models of intrinsic airway hyperresponsiveness, normal ontogenesis, and allergic sensitization are analyzed in the attempt to summarize the current knowledge on regulatory mechanisms of ASM contractile function in health and disease. Studies in human ASM and the need for additional models to understand contractile function in the airways are also discussed.     

Aging affects passive stiffness and spindle function of the rat soleus muscle

Aging affects many motor functions, notably the spinal stretch reflexes and muscle spindle sensitivity. Spindle activation also depends on the elastic properties of the structures linked to the proprioceptive receptors. We have calculated a spindle efficacy index, SEI, for old rats. This index relates the spindle sensitivity, deduced from electroneurograms recording (ENG), to the passive stiffness of the muscle. Spindle sensitivity and passive incremental stiffness were calculated during ramp and hold stretches imposed on pseudo-isolated soleus muscles of control rats (aged 4 months, n=12) and old rats (aged 24 months, n=16). SEI were calculated for the dynamic and static phases of ramp (1-80 mm/s) and for hold (0.5-2mm) stretches imposed at two reference lengths: length threshold for spindle afferents discharges, L(n) (neurogram length) and slack length, L(s). The passive incremental stiffness was calculated from the peak and steady values of passive tension, measured under the stretch conditions used for the ENG recordings, and taking into account the muscle cross-sectional area. The pseudo-isolated soleus muscles were also stretched to establish the stress-strain relationship and to calculate muscle stiffness constant. The contralateral muscle was used to count muscle spindles and spindle fibers (ATPase staining) and immunostained to identify MyHC isoforms. L(n) and L(s) lengths were not significantly different in the control group, while L(n) was significantly greater than L(s) in old muscles. Under dynamic conditions, the SEI of old muscles was the same as in controls at L(s), but it was significantly lower than in controls at L(n) due to increased passive incremental stiffness under the stretch conditions used to analyze the ENG. Under static conditions, the SEI of old muscles was significantly lower than control values at all the stretch amplitudes and threshold lengths tested, due to increased passive incremental stiffness and decreased spindle sensitivity at L(s). The muscle stiffness constant values were greater in old muscles than in controls, confirming the changes in elastic properties under passive conditions due to aging. Aging also altered the intrafusal fibers: it increased the mean number of intrafusal fibers and the contents in the slow, neonatal and developmental isoforms intrafusal of MyHC have been modified. These structural modifications do not seem great enough to counteract the loss of the spindle sensitivity or the spindle efficacy under passive conditions and after the nerve was severed. However, they may help to maintain the spindle afferent message under natural conditions and under fusimotor control.     

Age and hypertrophy related changes in contractile post-rest behavior and action potential properties in isolated rat myocytes

“Physiological” aging as well as early and progressive cardiac hypertrophy may affect action potential (AP) pattern, contractile function, and Ca²⁺ handling. We hypothesize that contractile function is disturbed in hypertrophy from early stages and is differently affected in aged myocardium. In vivo function, cardiomyocyte contractile behavior and APs were compared in Wistar-Kyoto (WIS) rats and spontaneously hypertensive rats (SHR) at different ages and degrees of hypertrophy (3–4, 9–11, 20–24 months). Post-rest (PR) behavior was used to investigate the relative contribution of the sarcoplasmic reticulum (SR) and the Na/Ca exchanger (NCX) to cytosolic Ca²⁺ removal. APs were recorded by whole-cell current-clamp and sarcomere shortening by video microscopy. Cyclopiazonic acid was used to suppress Ca²⁺ ATPase (SERCA) function. Heart weight/body weight ratio was increased in SHR versus WIS within all age groups. Myocyte steady state (SS) shortening amplitude was reduced in young SHR versus WIS. Aging led to a significant decay of SS contractile amplitude and relengthening velocity in WIS, but the PR potentiation was maintained. In contrast, aging in SHR led to a decrease of PR potentiation, while SS contraction and relengthening velocity increased. APD_50% was always prolonged in SHR versus WIS. With aging, APD_50% increased in both WIS and SHR, but was still shorter in WIS. However, in old WIS the late AP portion (APD_90%) was prolonged. Ca²⁺ handling and AP properties are disturbed progressively with aging and with increasing hypertrophy. Decreased amplitude of shortening and velocity of relengthening in aged WIS may be attributed to reduced SERCA function. In SHR, an increase in SR leak and shift towards transmembraneous Ca handling via NCX may be responsible for the changes in contractile function. A prolonged APD_90% in aged WIS may be an adaptive mechanism to preserve basal contractility. Therefore, the effects on contractile parameters and AP are different in hypertrophy and aging.     

哮喘豚鼠模型支气管平滑肌钙通道特性的变化

目的探讨钙离子通道变化与支气管哮喘豚鼠模型气道高反应性的关系.方法利用膜片钳技术细胞贴附式方法测定哮喘A组(4只,22个细胞)和正常对照A组(5只,25个细胞)支气管平滑肌细胞(BSMCs)电压依赖性钙离子通道动力学变化,全细胞式测定哮喘B组(5只,25个细胞)和正常对照B组(6只,24个细胞)BSMCs内向峰值钙电流.结果(1)BSMCs钙通道开放时间哮喘A组为(1.70±0.40)ms,正常对照A组为(0.70±0.10)ms,两组开放时间比较差异有显著性(P＜0.01);哮喘A组BSMCs钙通道关闭时间为(5.7±0.6)ms,正常对照A组为(7.9±0.4)ms,两组比较差异有显著性(P＜0.01);开放概率哮喘A组为(0.40±0.10)%,正常对照A组为(0.20±0.20)%,哮喘A组较正常对照A组增加了46%,两者比较差异有显著性(P＜0.01);(2)哮喘B组BSMCs平均内向峰值钙电流为(-54±23)PA(微微安培),正常对照B组为(-25±8)PA,哮喘B组较正常对照B组增加了1.17倍,两组比较差异有显著性(P＜0.01).结论哮喘豚鼠BSMCs钙离子通道活性增加,可能是气道高反应性主要原因之一.     

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.     

自发性高血压大鼠大脑中动脉血管肌源性紧张度的时程变化

目的观察自发性高血压大鼠(SHR)大脑中动脉血管肌源性紧张度的时程变化。方法选择SHR 32只,随机分为8、12、16、20周龄组,每组8只;另选Wistar大鼠32只按照SHR分组原则分为8、12、16、20周龄对照组,每组8只。测量不同管腔压力下主动态血管内径和被动态血管内径。观察各周龄组大鼠血管肌源性紧张度变化与血压的关系。结果与不同周龄对照组比较,不同周龄SHR组尾动脉压、血管肌源性紧张度明显升高。8、12、1 6周龄SHR组血管肌源性紧张度随大鼠周龄增加而缓慢单调增加,20周龄SHR组较16周龄SHR组明显下降;不同周龄SHR组尾动脉压随大鼠周龄增加而缓慢单调增加,与16周龄SHR组比较,20周龄SHR组尾动脉压明显增加(P<0.01)。结论血管肌源性紧张度作用参与了高血压发生、发展的整个过程。     

调气中药对豚鼠体外胃平滑肌运动的影响

[目的]观察同归脾、胃经具有调理气机功能的莱菔子、大腹皮、白术、小茴香、莪术,香附、蒲公英等7味中药对豚鼠体外胃纵行肌条的收缩作用,探讨其促胃动力作用规律。[方法]制备豚鼠胃底、胃体平滑肌条,以0.85%氯化钠(NS)和乙酰胆碱(ACh)作对照,观察7味中药对豚鼠体外胃纵行平滑肌的收缩效应。[结果]7味中药对胃底胃体纵行肌条均有不同程度的兴奋作用。与NS组比较,差异有统计学意义(P<0.01)。以ACh标化实验数据,莱菔子的收缩作用最强,与大腹皮比较,差异无统计学意义(P>0.05),与其他5味中药比较,差异有统计学意义(P<0.05),白术、蒲公英、小茴香的作用次之,无论在胃体还是胃底,莪术和香附的收缩作用均稍弱,分别与其他5味药比较差异均有统计学意义(P<0.05)。[结论]7味中药均有较强的促胃动力作用,其中莱菔子、大腹皮作用显著,值得进一步开发研究。从脾胃论治,结合气机升降理论开展促胃肠动力中药筛选是一条有效途径。     

糖尿病大鼠胃血管病变和平滑肌收缩功能与胃动力变化的研究

目的探讨胃平滑肌的收缩功能及胃血管病变与糖尿病胃动力变化之间的关系。方法采用链脲佐菌素腹腔注射建立糖尿病大鼠模型。分别于造模后4周和24周时应用单光子发射断层显像仪(SPECT)技术检测大鼠胃排空时间和速率,观察胃平滑肌的收缩功能及胃血管变化。结果 24周模型组大鼠胃平滑肌的收缩振幅和频率显著下降,4周模型组轻度下降。显微镜下观察,4周模型组,胃黏膜微血管增生,内皮损害较轻,黏膜下及肌层血管轻度纤维化,胃排空加快;24周糖尿病组,胃黏膜微血管数目明显减少,内皮损伤明显,黏膜下及肌层血管纤维化加重,管径/管腔比值明显增加,胃排空减慢。结论胃平滑肌收缩功能的改变和血管病变可能是引起糖尿病胃动力障碍的原因。     

低氧致大鼠肺动脉平滑肌细胞中PTEN/Akt1表达的变化与细胞增殖的关系

目的 通过观察低氧致大鼠肺动脉平滑肌细胞(PASMC)与细胞张力蛋白同源在10号染色体有缺失的磷酸酶(PTEN)和丝氨酸/苏氨酸蛋白激酶1(Akt1)mRNA及其蛋白表达水平的变化与低氧PASMC增殖的关系,探讨PTEN/Akt1信号途径在低氧肺血管重建中的可能调控作用.方法 用组织块法培养PASMC.采用半定量逆转录-PCR技术检测常氧组、低氧2、8、12、24 h组PTEN、Akt1基因mRNA的表达水平,采用Western blot技术检测相应的蛋白表达水平.采用噻唑蓝比色法和氚-胸腺嘧啶脱氧核苷(3H-TdR)掺入法检测PASMC的增殖改变.数据用x±s表示,采用Excel 2003软件进行t检验,P<0.05为差异有统计学意义.结果低氧刺激PASMC不断增殖,3H-TdR法检测的吸光度值低氧12 h组(0.70±0.10)比常氧组(0.37±0.06)明显增高(t=14.29,P<0.01);噻唑蓝法检测的吸光度值低氧24 h组(11 208±679)比常氧组(8374±545)明显增高(t=19.56,P<0.01).各组均检测出PTEN、Akt1基因mRNA及蛋白表达的变化.常氧组Akt1的mRNA、总蛋白、磷酸化蛋白的吸光度值分别为0.76±0.09、25±6和48±8,随着低氧培养时间延长,吸光度值逐渐升高,低氧8 h组达到高峰,分别为1.05±0.09、41±7和79±14,与常氧组比较,差异均有统计学意义(t值为8.31～168.00,P<0.05和P<0.01),随后开始下降,低氧24 h组恢复至常氧组水平.常氧组PTEN的mRNA、磷酸化蛋白的吸光度值分别为0.25±0.06和98±8,并随着低氧培养时间延长而逐渐升高,低氧24 h组达到高峰,分别为0.38±0.05和232±12,与常氧组比较,差异均有统计学意义(t值分别为22.04和50.46,均P<0.01).结论 PTEN/Akt1的转录和激活与低氧肺血管重建的PASMC增殖密切相关.