Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women

This review is aimed at providing readers with a comprehensive reference article about the distribution and function of P2 receptors in all the organs, tissues, and cells in the body. Each section provides an account of the early history of purinergic signaling in the organ/cell up to 1994, then summarizes subsequent evidence for the presence of P2X and P2Y receptor subtype mRNA and proteins as well as functional data, all fully referenced. A section is included describing the plasticity of expression of P2 receptors during development and aging as well as in various pathophysiological conditions. Finally, there is some discussion of possible future developments in the purinergic signaling field.

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Cellular distribution and functions of P2 receptor subtypes in different systems.

THE ability of cells to interact and communicate is a biological phenomenon that developed with the evolution of multicellular organisms. The cell-cell interactions that have evolved are essential for the survival of both simple organisms such as dictyostelium and complex organisms such as mammals. These cellular associations allow an organism to develop capacities that are greater than the simple sum of their individual parts. As early as 1878, Claude Bernard proposed that the “milieu interieur” (i.e. internally produced fluid environment) and a cybernetic-like control system (i.e. cell-cell interactions and communication) in multicellular organisms are needed to adaptively regulate the growth, development, and maintenance of normal tissue function (1, 2). The experimental analysis of cellular interactions was initiated with the investigation of cell aggregation in simple organisms such as sponges (3, 4) and progressed into the areas of embryology (5) and cell biology (6, 7). Analysis of cell biology on ...

Cell-Cell Interactions in the Testis

The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.

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Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.

Vitamin E and heart disease: basic science to clinical intervention trials.

Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate.

After selective destruction of Leydig cells in mature rats with ethylene dimethane sulfonate (EDS), repopulation of Leydig cells occurs. This repopulation process was studied in normal and sterile (prenatally irradiated) rats using morphological and histochemical techniques and by measuring hormone concentrations. Three days after administration of EDS to normal rats, extensive Leydig cell degeneration had occurred, testosterone concentrations were decreased to less than 10% of the normal value, and no 3 beta-hydroxysteroid dehydrogenase activity or pregnenolone production could be detected in isolated interstitial cells. Seven days after EDS administration, no cells with the appearance of Leydig cells were observed, and steroidogenic activities were still absent. After 14 days, single or paired Leydig cells were present again in the interstitium, but only after 21 days an increase in the plasma testosterone concentration and LH-dependent pregnenolone production was observed. On day 35, numerous Leydig cells were present, and testosterone levels were restored to normal. The depletion and repopulation of Leydig cells after administration of EDS to sterile rats showed a somewhat different pattern. Three days after administration of EDS, testosterone concentrations were decreased to less than 10% of the normal value, and isolated interstitial cells showed no steroidogenic activities as in normal rats, but a small number of Leydig cells was still present. A similar picture was observed between 4 and 9 days after EDS administration. This indicates that some Leydig cells from sterile rats, unlike Leydig cells from normal rats, were resistant to EDS. The repopulation of Leydig cells in sterile rats was faster than in normal rats. After 14 days, many groups of Leydig cells were present in the interstitium, and the plasma testosterone concentration and pregnenolone production in vitro were significantly increased. Normal plasma testosterone levels were restored on day 21. Serum LH and FSH were decreased immediately after EDS administration, but during the next days a sharp rise was observed in both normal and sterile rats. The rise in LH correlated with the decrease in testosterone, and restoration of LH levels took place when testosterone levels increased. FSH levels changed similarly, but were delayed, in comparison to LH. In rats with testosterone implants that suppressed LH levels to less than 2 ng/ml and maintained normal FSH levels, ranging from 150-340 ng/ml, as well as in hypophysectomized rats, no repopulation of Leydig cells could be observed until 35 days after EDS treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Repopulation of Leydig Cells in Mature Rats after Selective Destruction of the Existent Leydig Cells with Ethylene Dimethane Sulfonate Is Dependent on Luteinizing Hormone and Not Follicle-Stimulating Hormone

Publisher Summary This chapter discusses relevant observations on the preparation, purification, and characterization of isolated Leydig cells and explains that different in vitro preparations of Leydig cells have been used for studies on the biochemical mechanisms involved in regulation of steroidogenesis. The chapter also explains that several alternative methods for cell isolation and characterization have been developed and the techniques have been used to isolate Leydig cells from porcine and mouse testes and Leydig cell tumors. A careful investigation and standardization of the methods appears necessary, because for Leydig cells isolated from adult rats, a large variation in the capacity for steroid production has been observed. The recovery of Leydig cells after the isolation procedure is low, and there are indications that extensive cell damage can occur during tissue dispersion. When isolated cells are used for investigations on the biochemical mechanisms involved in the regulation of steroidogenesis, it is essential to work with homogeneous preparations of viable cells and prevent various degrees of functional heterogeneity and other artifacts, which may be caused by the isolation procedure.

[22] Preparation of isolated leydig cells

Three different collagenase dispersion techniques for isolation of Leydig cells from testes of mature rats have been compared with respect to the yield and quality of the isolated cells. No Leydig cells could be isolated after perfusion of the testis with collagenase (10 mg/ml) followed by incubation at 37°C without shaking, whereas Leydig cells were obtained after incubation of decapsulated testis tissue with collagenase (1 mg/ml) and shaking (80 cycles/min or 1500 cycles/min). Of the total phenylesterase activity, which is localized in the endoplasmic reticulum of interstitial cells, 50 and 85% was released after shaking at 80 and 1500 cycles/, respectively. After sedimentation of the cells 14 and 56% of the microsomal esterase activity was present in the supernatant, whilst 6 and 10% of the esterase activity could be recovered in Ficoll-purified cells. The high esterase activity in the supernatant and the low esterase activity in the purified cells indicated the presence of many broken cells. Testosterone production by cells prepared with the low shaking frequency could be stimulated 9-fold by LH, whereas cells prepared with the high shaking frequency did not respond to LH. Cell preparations which could be stimulated with LH could also be stimulated (3-fold) with NADPH, indicating the presence of damaged cells in addition to intact cells. The percentage of damaged cells in the preparations was estimated using an NADH-dependent histochemical test for mitochondrial diaphorase activity, which shows activity when cell membranes are damaged. Using this criterion, it was found that about 30% of ficoll-purified cells were damaged, whereas only 2% of cells which were pre-incubated and attached to plastic appeared to be damaged. In***

R. Molenaar

Papers

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate.

Repopulation of Leydig Cells in Mature Rats after Selective Destruction of the Existent Leydig Cells with Ethylene Dimethane Sulfonate Is Dependent on Luteinizing Hormone and Not Follicle-Stimulating Hormone

[22] Preparation of isolated leydig cells

The steroidogenic activity of isolated Leydig cells from mature rats depends on the isolation procedure

Peroxidative stress and in vitro ageing of endothelial cells increases the monocyte-endothelial cell adherence in a human in vitro system