Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.     

Combined effects of glucose and oxygen concentrations upon the ultrastructure of cultured islet cells and insulin accumulation in culture media

Rat pancreatic islets cultured for 6 days at 100 or 500 mg/dl glucose and 20 or 7% O2 were examined electron-microscopically, and insulin accumulation in the culture media was assayed immunologically. In the islets cultured at 500 mg/dl glucose and 20% O2, B cells exhibited hypertrophy of granular endoplasmic reticulum and Golgi apparatus, an abundance of free ribosomes, degranulation and the margination of secretory granules. In islets cultured at 500 mg/dl glucose and 7% O2, B cells exhibited dilatation of endoplasmic reticulum cisternae and dominance of Golgi vesicles in addition to the above-mentioned changes. These changes, together with the correlated data on insulin accumulation, are discussed with special reference to the effects of glucose and oxygen upon the synthesis and release of insulin in B cells.     

Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. I. Simultaneous binding of three sodium and two potassium or rubidium ions to the enzyme

We previously measured the amounts of Na+ and K+ ions bound to the Na+,K+-dependent ATPase [EC 3.6.1.3] purified from porcine kidney by a modified membrane filtration method [(1979) J. Biochem. 86, 509--523]. In this study, we improved the method for measuring the amount of the active site and measured the amount of Rb+ ions (a K+ congener) bound to the ATPase as well as those of Na+ and K+ ions to get more accurate information on the K+- and Na+-binding sites. The following results were obtained. Two kinds of cation-binding sites were found to exist on the ATPase molecule. One was the Na+-binding sites (3 mol per mol of active site). Na+ ions were bound to the sites cooperatively (Hill coefficient, 2.5--3), and the apparent dissociation constant was 0.20--0.32 mM. Three moles of Na+ ions bound to the sites was displaced by 1 mol of K+ ions bound to the ATPase (phi K, 24 microM). The other was the K+-binding sites (2 mol per mol of active site). Two moles of K+, Rb+, or Na+ ions was bound to the sites cooperatively (Hill coefficient, 1.5--2), and their apparent dissociation constants were 0.044, 0.024, and 2.2 mM, respectively. We measured the amounts of Na+ and Rb+ ions bound to the ATPase in the presence of 0.8 mM NaCl and 0.13 mM RbCl, and obtained unequivocal evidence for the simultaneous binding of 3 mol of Na+ ions and 2 mol of Rb+ ions per mol of active site of the ATPase.     

Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. III. Marked changes in affinities for monovalent cations induced by formation of an ADP-insensitive but no an ADP-Sensitive phosphoenzyme

We measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0 degrees C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.     

Cell-free plasma layer in cerebral microvessels.

Two diameters of vessel and red cell column in cerebral microvessels (> 29.8 microns in diameter) of cat were measured together with red cell velocity, using a two fluorescent tracer method. A fluorescein isothiocyanate (FITC)-labeled red cell was adopted as a flow tracer to measure the cell velocity with a dual window technique. Based on the fluorescence image, the red cell column diameter was measured. Plasma was stained with rhodamine-B isothiocyanate (RITC)-labeled dextran to measure the vessel diameter. The thickness of the cell-free plasma layer could be determined from the difference of the two diameters. The obtained thickness of the cell-free layer was not described by a simple function of vessel diameter or red cell velocity; it was dependent on the pseudo shear rate defined by the ratio of cell velocity to vessel radius. The layer thickness increased with a decrease in the pseudo shear rate.     

Development of a psoriasis-like syndrome following lithium therapy

A correlation between lithium and psoriasis has been observed. In this paper, the case of a 17-yr-old girl is reported who developed psoriatic lesions after administration of lithium carbonate. Further-more, serum lithium levels in some psoriatic patients are disclosed, and induction of psoriasis by lithium in experimental animals is described. Serum lithium levels in 27 patients were significantly higher (p<0.025) than those of controls. Uninvolved parts of skin tissues obtained from three cases of psoriasis were transplanted to nude mice. After supplementing lithium as the chloride, these skin grafts developed the histologic change characteristic of psoriasis. However, the lithium compound by itself did not increase superoxide production of polymorphonuclear leukocytes in psoriasis.     

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full-length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615-1628]. Alignment of the sequence for the N-terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3'-untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.