In middle-aged subjects with no apparent heart disease, all measures of increased HR were associated with increased mortality and CV risk. However, night-time HR was the only parameter with prognostic importance after multivariable adjustment.

Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t 1 ⁄2 Ϸ5 h), which is dependent on plasma membrane cholesterol but independent of NH 4 Cl treatment of cells; NH 4 Cl impairs entry via clathrin-coated pits.

Type III sodium-dependent phosphate (NaP i ) cotransporters, Pit1 and Pit2, have been assigned housekeeping P i transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaP i cotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu 55 and Glu 575 , with glutamine or with lysine severely impaired or knocked out, respectively, P i transport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaP i cotransporters, in fungal NaP i cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaP i cotransport. Their putative transmembranic positions together with the presented data are consistent with Glu 55 and Glu 575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 P i transport function. Inorganic phosphate (P i )1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as P i uptake across the plasma membrane in animal cells have been identified (1-3), namely the sodium-dependent phosphate (NaP i ) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4)) and Pit2 (human Pit2 formerly GLVR2 (5)). Both proteins are characterized as type III NaP i cotransporters (6) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7). Furthermore, low extracellular P i levels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1,8). These observations strongly suggest that the major cellular P i demand in mammalian cells is handled by type III NaP i cotransporters (1). However, recent results also point at type III transporters as playing specific roles in chondroblastic and osteoblastic mineralization (9, 10) as well as being critically involved in vascular calcification under hyperphosphatemic conditions, which are often present in diabetic patients and individuals with renal failure (11). The mechanisms underlying the bone-forming roles of type III NaP i transporters are presently not known. Recent results, however, showed that high extracellular P i levels can induce expression of the gene for osteopontin and that the induction is dependent on Na ϩ -dependent P i uptake across the p...

Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms. Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50 % of the families reported worldwide. Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.