Changes in extracellular zinc concentration participate in modulating fundamental cellular processes such as proliferation, secretion, and ion transport in a mechanism that is not well understood. Here, we show that a micromolar concentration of extracellular zinc triggers a massive release of calcium from thapsigarginsensitive intracellular pools in the colonocytic cell line HT29. Calcium release was blocked by a phospholipase-C inhibitor, indicating that formation of inositol 1,4,5-triphosphate is required for zinc-dependent calcium release. Zinc influx was not observed, indicating that extracellular zinc triggered the release. The Ca i 2؉ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca 2؉ -sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Ca i 2؉ rise stimulated the activity of the Na ؉ ͞H ؉ exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn 2؉ -sensing receptor is functional in colonocytes. Because Ca i 2؉ rise is known to regulate key cellular and signal-transduction processes, the zinc-sensing receptor may provide the missing link between extracellular zinc concentration changes and the regulation of cellular processes.Z inc is an essential micronutrient involved in structural and regulatory cellular functions. Zinc interacts with zinc-finger domains and acts as a cofactor of numerous enzymes (1, 2). Zinc ions also specifically bind to many membrane receptors, transporters, and channels, thereby modulating their activity (3). Therefore, it is not surprising that zinc deficiency affects multiple organs, including the digestive (4), immune (5), and neuronal (2) systems. A severe lack of zinc also is linked to the attenuation of growth and sexual development (2). Conversely, an excess of extracellular zinc is considered toxic. Indeed, brain ischemia is accompanied by a massive release of synaptic zinc permeating into neurons, leading to neuronal cell death (6-8). Furthermore, striking changes in plasma-zinc concentration occur during diverse pathophysiological syndromes including myocardial infarction, hepatic renal failure, and neoplastic processes (9). Despite the large fluctuations in extracellular zinc concentrations and their subsequent clinical importance, little is known about cellular signaling mechanisms that sense changes in extracellular-zinc concentration.The calcium-sensing receptor serves as an example for the importance of an ion-sensing mechanism. It is a G proteincoupled receptor that senses changes in extracellular calcium and regulates diverse cellular functions (10, 11). Although the existence of other ion-sensing mechanisms have been suggested (12), none have been characterized fully yet.Gastrointestinal pathology, manifested in severe diarrhea, is an important hallmark of zinc deficiency. Indeed, zinc has a major role in the duration and severity of diarrheal diseases (4). The morbidity and mortality caused by diarrheal diseas...
The glucose transport properties of brush border membrane vesicles from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) of rabbit kidney were studied. In the outer cortical preparation the behavior of the sodium-dependent component of D-glucose flux indicated the presence of a low-affinity transport system with Km congruent to 6 mM and Vmax congruent to 10 nmol.min-1.mg protein-1 as measured under zero trans conditions at 40 mM NaCl and 17 degrees C. By contrast, in the outer medullary preparation this component of flux behaved as a high-affinity system with Km congruent to 0.35 mM and Vmax congruent to 4 nmol.min-1.mg protein-1. Differences in transport specificity between the two preparations were also indicated and glucose uptake by the outer cortical vesicles was significantly more sensitive to inhibition by phlorizin. These results suggest the existence of two distinct sodium-dependent D-glucose transport systems in the renal proximal tubule brush border membrane. The kinetic studies presented here were done under zero trans sodium and glucose conditions. The rationale and methodology for carrying out these measurements reliably are discussed in detail.
Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT-1 in reducing zinc-dependent cell damage in astrocytes was assessed. Zinc-dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of approximately 200 microM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc-pretreated cells mediated by L-type calcium channels. Heterologous expression of ZnT-1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT-1 expression. Immunohistochemical analysis demonstrated endogenous ZnT-1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4-fold increase in the expression of the putative zinc transporter ZnT-1 in astroglia as shown by immunoblot analysis. The elevated ZnT-1 expression following zinc priming or after heterologous expression of ZnT-1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT-1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.
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