Changes in the concentration of extracellular calcium can affect the balance between proliferation and differentiation in several cell types, including keratinocytes, breast epithelial cells, and fibroblasts. This report demonstrates that elevation of extracellular calcium stimulates proliferation-associated signaling pathways in rat fibroblasts and implicates calcium-sensing receptors (CaR) as mediators of this response. Rat-1 fibroblasts express CaR mRNA and protein and respond to known agonists of the CaR with increased IP 3 production and release of intracellular calcium. Agonists of the CaR can stimulate increased c-SRC kinase activity and increased extracellular signal-regulated kinase 1/mitogen-activated protein kinase activity. Both of the increases in SRC activity and mitogen-activated protein kinase activation are blocked in the presence of a nonfunctional mutant of the CaR, R796W. Proliferation of wild-type Rat-1 cells is sensitive to changes in extracellular calcium, but expression of the nonfunctional CaR mutant or inhibition of the calcium-dependent increase in SRC kinase activity block the proliferative response to calcium. These results provide evidence of a novel signal transduction pathway modulating the response of fibroblasts to extracellular calcium and imply that calciumsensing receptors may play a role in regulating cell growth in response to extracellular calcium, in addition to their well known function in systemic calcium homeostasis.

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Glucose transport into nonneuronal brain cells uses differently glycosylated forms of the glucose transport protein, GLUT1. Microvascular GLUT1 is readily seen on immunocytochemistry, although its parenchymal localization has been difficult. Following ischemia, GLUT1 mRNA increases, but whether GLUT1 protein also changes is uncertain. Therefore, we examined the immunocytochemical distribution of GLUT1 in normal rat brain and after transient global forebrain ischemia. A novel immunocytochemical finding was peptide-inhibitable GLUT1 immunoreactive staining in parenchyma as well as in cerebral microvessels. In nonischemic rats, parenchymal GLUT1 staining co-localizes with glial fibrillary acidic protein (GFAP) in perivascular foot processes of astrocytes. By 24 h after ischemia, both microvascular and nonmicrovascular GLUT1 immunoreactivity increased widely, persisting at 4 days postischemia. Vascularity within sections of brain similarly increased after ischemia. Increased parenchymal GLUT1 expression was paralleled by staining for GFAP, suggesting that nonvascular GLUT1 overexpression may occur in reactive astrocytes. A final observation was a rapid expression of inducible heat shock protein (HSP)70 in hippocampus and cortex by 24 h after ischemia. We conclude that GLUT1 is normally immunocytochemically detectable in cerebral microvessels and parenchyma and that parenchymal expression occurs in some astroglia. After global cerebral ischemia, GLUT1 overexpression occurs rapidly and widely in microvessels and parenchyma; its overexpression may be related to an immediate early-gene form of response to cellular stress.

Near-normalization of glycemia reduces the risks of chronic diabetic complications but increases the risk of serious hypoglycemia. Hypoglycemia can impair neuronal function in the brain and diminish awareness of subsequent hypoglycemic episodes, yet little is known about how neurons adapt to hypoglycemia. This study tests the hypothesis that isoform-specific alterations in brain glucose transport proteins occur in response to chronic hypoglycemia. To study this, groups of rats were injected with approximately 25 U/kg ultralente insulin daily at 1700 for 8 days to maintain hypoglycemia. Vascular-free and microvessel membrane fractions from brain were prepared for immunoblot analysis of GLUT-1 and GLUT-3 by use of isoform-specific antisera. Insulin treatment reduced blood glucose levels from 4.0 +/- 0.1 (vehicle-injected controls) to 1.7 +/- 0.1 mmol/l on day 8 (P < 0.001) and increased GLUT-3 protein expression (175.6% of control; P < 0.05). Microvascular GLUT-1 (55 kDa) tended to increase (195.6% of controls; P = 0.08) variably, whereas nonvascular GLUT-1 (45 kDa) was unchanged. We conclude that neuronal glucose transport protein (GLUT-3) expression adapts to chronic hypoglycemia. This adaptation may spare neuronal energy metabolism but could dampen neuronal signaling of glucose deprivation.

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