Effects of Na readmission on cellular45Ca fluxes in Na-Depleted guinea pig taenia coli

Summary The removal of Na from the medium causes a cellular Ca uptake in the smooth muscle of the guinea pig taenia coli which is rapidly reversed if medium Na is readmitted. This net extrusion was characterized in tissues which were first Na-depleted in a zero-Na (sucrose) solution. Li was able to substitute for Na in mediating this effect. K was also able to mimic Na in this respect if the depolarization-mediated Ca influx caused by the isotonic K solution was blocked with 10^–5 m D-600. The net Ca extrusion upon Na readmission was due to a small decrease in Ca influx, as well as a marked increase in the transmembrane Ca efflux rate, as revealed by⁴⁵Ca washout experiments. The increased⁴⁵Ca efflux upon Na readmission could be mimicked by Li, K, choline and tris. We conclude that the Na/Ca-exchange hypothesis is insufficient to explain these data, in that both Ca extrusion and⁴⁵Ca efflux can be stimulated in the absence of a Na gradient, or in the absence of any monovalent cationic gradient. These observations are discussed in terms of a possible intracellular competition of Ca and monovalent cations for anionic binding sites, as well as with regard to a possible direct stimulation of a plasmalemmal CaATPase by monovalent cations.     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Steric aspects of agonism and antagonism at beta-adrenoceptors: synthesis of and pharmacological experiments with the enantiomers of formoterol and their diastereomers.

The enantiomers of formoterol (R;R and S;S) and their diastereomers (R;S and S;R) were synthesized and purified using a new procedure which required the preparation of the (R;R)- and (S;S)-forms of N-(1-phenylethyl)-N-(1-(p-methoxyphenyl)-2-propyl)-amine as important intermediates. The enantiomeric purity obtained was greater than 99.3%, usually greater than 99.7%. The four stereoisomers were examined with respect to their ability to interact in vitro with beta-adrenoceptors in tissues isolated from guinea pig. The effects measured were (1) relaxation of the tracheal smooth muscle (mostly beta 2), (2) depression of subtetanic contractions of the soleus muscle (beta 2), and (3) increase in the force of the papillary muscle of the left ventricle of the heart (beta 1). All enantiomers caused a concentration-dependent and complete relaxation of the tracheal smooth muscle which was inhibited by propranolol. The order of potency was (R;R) much greater than (R;S) = (S;R) greater than (S;S). There was a 1,000-fold difference in potency between the most and the least potent isomer. The presence of the (S;S)-isomer did not affect the activity of the (R;R)-isomer on the tracheal smooth muscle. Also on the skeletal and cardiac muscles (R;R)-formoterol was more potent than its (R;S)-isomer. The selectivity for beta 2-adrenoceptors appeared to be slightly higher for the (R;R)-isomer than for the (R;S)-isomer. The potency of the (S;R)- and (S;S)-isomers on the papillary muscle was too low to be determined accurately.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

Plate-Induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes,chondrocytes, and fibroblasts

The BALB/3T3 clone A31 mouse embryo cell line has been used by many investigators as a model “normal” “fibroblast” line for a variety of in vitro studies. It has been shown, however, that these cells are not “normal” because they will produce tumors within 2–4 months if 3 × 10⁴ cells are implanted subcutaneously in BALB/c mice attached to 0.2 × 5 × 10-mm plastic plates. Previous studies also suggested that these cells were not fibroblasts because they gave rise to tumors with the characteristics of vascular endothelium not fibroblasts. We now report that BALB/3T3 (clone A31), BALB/3T3-T, a proadipocyte subclone of clone A31 cells, and six recent subclones of BALB/3T3-T cells show additional differentiation patterns when tumors derived by implantation of these cells attached to plastic plates are examined. Differentiation into pericytes, chondrocytes, and fibroblasts was observed. We conclude that the BALB/3T3 clone A31 cell line and related lines are multipotent mesenchymal cells which are capable of differentiation into a variety of cell types.     

Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

平滑肌22α蛋白在血管稳态和血管重构中的作用

有关血管稳态和重构的分子机制一直是近年来的研究热点,也被视为治疗血管损伤性疾病的突破点.大量研究证实,血管损伤修复及病理性重构过程与血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的表型转化、异常增殖与迁移、细胞衰老关系密切.平滑肌22α(smooth muscle 22α,SM2...     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子-1α和细胞增殖的关系

在低氧条件下,观察大鼠肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells,PASMCs）中活性氧（reactive oxygen species,ROS）的变化,探讨ROS的变化是否通过调控低氧诱导因子-4α（hypoxia-inducible factor 1α, HIF-1α）的表达影响PASMCs的增殖。采用组织块法原代培养大鼠PASMCs,分成3组：常氧组（21％O2,24h）,低氧组（5％O2,24h）,低氧＋Mn-TBAP组（5％O2,24h,Mn-TBAP是一种ROS清除剂）。用激光共聚焦显微镜荧光染色法检测细胞内ROS的变化;用RT-PCR和免疫组织化学方法分别测定HIF-1α mRNA和蛋白的表达;用MTT法检测细胞增殖程度。结果显示：（1）低氧组PASMCs内ROS水平明显高于常氧组（P〈0．05）,低氧＋Mn-TBAP组ROS水平明显低于低氧组（P〈0．05）,但仍高于常氧组（P〈0．05）;（2）低氧组及低氧＋Mn-TBAP组的HIF-1α mRNA和蛋白表达均高于常氧组（P〈0．05）,且低氧组表达高于低氧＋Mn-TBAP组（P〈0．05）;（3）低氧组细胞增殖明显高于常氧组和低氧＋Mn-TBAP组（P〈0．05）,低氧＋Mn-TBAP组细胞增殖高于常氧组（P〈0．05）。结果表明：在低氧条件下大鼠PASMCs中ROS水平明显升高,RROS的变化能够调节HIF-1α的表达,进而影响平滑肌细胞的增殖,提示ROS可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。