We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca 2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca 2÷ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend ~12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca 2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca 2÷ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca ~+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.The muscle fiber contains an intricate membraneous network that controls muscle contraction and relaxation by regulating the intracellular calcium concentration. The plasma membrane or plasmalemma invaginates transversely into the muscle sarcoplasm to form transverse tubules, which are connected to an internal reticular membrane system, the sarcoplasmic reticulum (SR). 1 The SR surrounds the sarcomere in Abbreviation used in this paper. SR, sarcoplasmic reticulum. a sleevelike manner, and is composed of two distinct portions: (a) the terminal cisternae which are junctionally associated with the transverse tubule, and (b) the longitudinal cisternae or longitudinal SR, which connect medially with the two term...

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2 H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2 H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0 -200 g) to ovariectomized rats increased mammary epithelial cell proliferation, according to a doseresponse relationship up to the 100 g dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose-response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2 H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270 -400 days and die-away values after 2 H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1-1.5% new cells per day, whereas obese ad libitum-fed ob͞ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2 H2O enrichments in body water were achieved by daily 2 H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2 H2O enrichment (Ϸ3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2 H2O was 0.056 (CD4 ؉ ) and 0.043 (CD8 ؉ ) (replacement rate <0.1% per day). In summary, 2 H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells. deuterated water ͉ cell proliferation ͉ DNA synthesis ͉ adipogenesis ͉ vascular smooth muscle cell proliferation