Among the key effects of fluid shear stress on vascular endothelial cells is modulation of gene expression. Promoter sequences termed shear stress response elements (SSREs) mediate the responsiveness of endothelial genes to shear stress. While previous studies showed that shear stress responsiveness is mediated by a single SSRE, these endogenous promoters often encode for multiple SSREs. Moreover, hybrid promoters encoding a single SSRE rarely respond to shear stress at the same magnitude as the endogenous promoter. Thus, to better understand the interplay between the various SSREs, and between SSREs and endothelial-specific sequences (ESS), we generated a series of constructs regulated by SSREs cassettes alone, or in combination with ESS, and tested their response to shear stress and endothelial specific expression. Among these constructs, the most responsive promoter (NR1/2) encoded a combination of two GAGACC/SSREs, the Sp1/Egr1 sequence, as well as a TPA response element (TRE). This construct was four- to five-fold more responsive to shear stress than a promoter encoding a single SSRE. The expression of constructs containing other SSRE combinations was unaffected or suppressed by shear stress. Addition of ESS derived from the Tie2 promoter, either 5' or 3' to NR1/2 resulted in shear stress transcriptional suppression, yet retained endothelial specific expression. Thus, the combination and localization order of the various SSREs in a single promoter is crucial in determining the pattern and degree of shear stress responsiveness. These shear stress responsive cassettes may prove beneficial in our attempt to time the expression of an endothelial transgene in the vasculature.
In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre-load), glass microspheres simulating afterload will stimulate the secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 μm, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen-coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT-PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro-Electrode-Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin-2 (60%), transforming growth factor-β (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias.
The presence of an uptake system and a functional glycine receptor in adrenal medulla chromaffin cells was investigated using an autoradiographic technique in adrenal gland slices. Specific 3[H]glycine binding was observed in both adrenal cortex and medulla slices, while only specific binding of [3H]strychnine was seen only in chromaffin cells and was not associated with cortical cells. [3H]Glycine binding sites in the cortex are apparently different from those of [3H]strychnine binding sites in the medulla since excess strychnine does not displace [3H]glycine from adrenal cortex but does so from medulla. This difference supports biochemical evidence for glycine transport into medulla cells and glycine receptor sites on the chromaffin cell membrane.
The combined effect of calcitonin and triamcinolone acetonide on Ca2+ content in cultured bone cells was studied. Cultures were first preincubated with 45Ca for 24 hrs thus achieving a steady state between the extracellular and intracellular 45Ca2+. Calcitonin was added for 24 hrs and subsequently, triamcinolone acetonide was added for time intervals ranging from 1 to 24 hrs. Calcitonin induced a significant increase in the content of cellular exchangeable Ca2+. When triamcinolone was added to cultures pretreated with calcitonin a marked efflux activity was noted. Concomitant ultrastructural histochemical examinations, using the K-pyroenthymonate fixation method, further substantiated the above finding: calcitonin treated cells revealed increased number of Ca-pyroenthymonate precipitates, whereas the combined treatment led to the disappearance of such Ca-aggregates.
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