Summary Although cultured vascular smooth muscle cells have been extensively characterized and investigated, there are very few studies
of cultured intestinal smooth muscle cells. The aim of this study was to culture colonic smooth muscle (CSM) cells from the
rabbit colon. Freshly isolated CSM cells from the circular muscle layer of the distal colon were prepared by collagenase digestion.
In primary culture, CSM cells attached to the culture vessels by 48 to 72 h, proliferated by 3 to 7 d, and reached confluency
by 14 to 17 d with a “hill-and-valley” pattern. Spontaneous contractions were not observed at any time at 21° or 37° C. Confluent
primary cultures were greater than 95% CSM cells, as identified by intensely positive immunofluorescent staining to smooth
muscle actin-specific CGA7 and muscle-specific HHF-35 monoclonal antibodies. Transmission electron microscopy of freshly isolated
and proliferating CSM cells revealed ultrastructural features consistent with smooth muscle cells. We successfully cultured
CSM cells of the rabbit from freshly isolated cells and validated these CSM cells by electron microscopy and immunocytochemical
staining. These highly pure primary cultures may be used to investigate numerous aspects of CSM cell metabolism and physiology.
These studies were supported by the National Institutes of Health grant to the Inflammatory Bowel Disease Center (Bethesda,
MD) P30-AM-32200 and R01-DK-31147. Dr. Kao is the recipient of a Research Career Development Award from the National Foundation
for Ileitis and Colitis, Inc. A preliminary report of this work was presented at the American Motility Society Meeting, Houston,
TX, in October 1986, and appeared in abstract form inGastroenterology 91: 1057; 1986. 相似文献
Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios PNa/PCl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for PBa/PCl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes. 相似文献
Summary Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca2+ and voltage activated, has a conductance of 221±7 pS, and can be inhibited by 10mm tetraethylammonium and by 1mm quinidine, but not by 4-aminopyridine, nor by 1mm Ba2+ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1±3.9 nSvs. a leak conductance of 1.01±0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5±1.4 pF. 相似文献
Abstract Modification of the ‘intracellular concentration of reduced glutathione’ (IC-GSH) affected the response of cultured rose cells (Rosa damascena) to ultraviolet radiation (UV)-induced leakage of K+. High IC-GSH induced by incubation of cells in 10 mol m?3 GSH (IC-GSH increased linearly with time from 20 to about 600 μmol g?1 in 61.2 ks) caused cells to become significantly less sensitive to UV. Low IC-GSH induced by treatment with 1 mol m?3 buthionine sulphoximine (BSO) plus 1 mol m?3 diethylmaleate (DEM) (IC-GSH decreased from 20 to about 3 μg g?1 in 61.2 ks) reduced, rather than increased, the UV-sensitivity of the cells. However, treatment with DEM also induced a large transient K+ leakage; and treatment with BSO induced a slight leakage. The K+ leaked was recovered by 3.24 ks. Following K+ recovery, the DEM-treated cells showed almost complete insensitivity to UV, and BSO-treated cells showed a slightly reduced sensitivity to UV. These results are in agreement with our previous findings that other treatments (heat, cycloheximide, UV), which also cause a transient leakage of K+, also reduce the induction of K+ leakage by a subsequent UV treatment. We conclude that high IC-GSH may play a role in protecting plant cells from UV-induced K+ leakage. Increased UV-sensitivity with low ICGSH was not observed, we believe, because of the transient K+ leakage, though the mechanism of reduced sensitivity to UV induced by transient leakage of K+ is not known at this time. Treatment with UV did not reduce the IC-GSH, showing that this is not the mechanism by which UV induces K+ leakage. 相似文献
Application of salicylate increased the concentration of metallothionein (MT) in liver of pregnant rats as well as of adult male rats, whereas in fetal liver, MT was reduced by salicylate. Induction of MT synthesis by salicylate is an indirect effect because in cultured hepatocytes salicylate did not induce MT synthesis. Salicylate increased MT also in adrenalectomized rats. Indomethacin induced the same concentration of MT in maternal liver as salicylate. However, indomethacin had no effect on MT in fetal liver. Induction of MT in adult liver by salicylate and indomethacin was independent of zinc. 相似文献
The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life. 相似文献
Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure. 相似文献
Summary Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent
cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport
process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface
facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show
domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone
(PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; c) high alkaline phosphatase activity; and d) Na+-dependent transport of phosphate (Pi) and α-methylglucoside (α-MG). The sugar uptake was selectively inhibited by phlorizin,
a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems
for Pi and α-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived
from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium.
Phorbol esters and PTH did not exert an effect on Pi and α-MG transport in mouse primary cultures. The present study demonstrates
that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function.
Cindy Bell was the recipient of an MRC Studentship Award. This work was supported by the MRC (Group in Medical Genetics).
This is publication number 88011 of the McGill University-Montreal Children's Hospital Research Institute. 相似文献
Confidence in the measurement of positive effects determined by monitoring of environmentally or occupationally exposed individuals can be enhanced by a knowledge of the normal variability in these endpoints in the general population. Confounding effects can be determined and study interpretation improved by correlation of this variability with various lifestyle factors such as sex and age of donor, smoking and drinking habits, viral infections, exposure to diagnostic X-rays, etc.
8 blood samples were taken from each of 24 male and 24 female volunteers over a period of 2 years. Questionnaires pertaining to lifestyle were completed at the time of each sampling. Whole blood was cultured and slides prepared for CA or SCE analysis. Separated mononuclear cells were cultured with a range of phytohaemagglutinin concentrations and the maximum level of mitogen-induced blastogenesis was determined by measurement of [3H]thymidine uptake.
There was a significant effect of both year and season of sampling for all 3 endpoints. No significant effects in any of the 3 endpoints were found with respect to sex or age of donor nor any of the other lifestyle factors, although SCE frequency and mitogen-induced blastogenesis were nearly always higher in females than males. These results point to the need for concurrent sampling of controls with exposed populations. 相似文献