Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

The envelope glycoprotein of an amphotropic murine retrovirus binds specifically to the cellular receptor/phosphate transporter of susceptible species.

A rat cDNA (rRam-1), which was cloned on the basis that it enables Chinese hamster ovary (CHO) cells to be infected by amphotropic host range murine retroviruses, was recently found to encode a widely expressed Na(+)-phosphate symporter (M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994). CHO cells express the hamster homolog of Ram-1 but are resistant to amphotropic retroviruses. Although the amphotropic envelope glycoprotein gp70 bound weakly onto control CHO cells, CHO/rRam-1 cells had novel high-affinity binding sites, and the resulting strongly adsorbed gp70 was only slowly removed from cell surfaces, with a half-life of greater than 6 h. CHO/rRam-1 cells were also specifically and efficiently killed by exposure to amphotropic gp70 followed by antiserum to gp70 in the presence of complement. Infection with an appropriately pseudotyped form of amphotropic retrovirus 4070A did not perturb control CHO cells or inhibit their phosphate transport. In contrast, 4070A infection of CHO/rRam-1 cells caused major alterations including cell-cell fusions, a specific 40% down-modulation of the rRam-1 component of phosphate transport, and complete interference to super-infection by amphotropic viruses. The 4070A virus-infected CHO/rRam-1 cells retained a substantial cell surface pool of rRam-1 that functioned as a phosphate transporter but not as a viral receptor. We conclude that amphotropic gp70 binds more strongly to rRam-1 than to the homologous hamster protein and that this stable attachment is necessary for infection, interference, membrane fusion, and pathogenesis.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

Prostanoid synthesis by vascular slices and cultured vascular cells of piglet aorta

Biosynthesis of prostanoids was studies in vascular slices of human umbilical arteries, piglet aorta and vena cava as well as in cultured vascular cells of piglet aorta. After preincubation with radioactive labeled arachidonic acid, prostanoids in the incubation media of slices or cultured cells were measured by radioimmunoassay or by radioactivity determination of labeled compounds following separation on reserved-phase high performance liquid chromatography. In all vascular slices 6-keto-PGF1α was the main prostanoid found, followed by PGE2. Thromboxane B2 and PGF2α were also formed, but only in trace amounts. In cultured cells taken from the three layers of the vascular wall, the prostanoid profiles differed markedly from those obtained from vascular slices. Each cell strain showed a specific prostanoid pattern. Endothelial cells synthesized predominantly 6-keto-PGF1α and PGF2α. In smooth muscle cells no 6-keto-PGF1α could be detected; here the predominant prostanoid was PGE2. PGF2α was formed in smaller quantities. Fibroblasts synthesized all prostanoids (PGE2, PGF2α, TXB2, 6-keto-PGF1α), PGE2 and PGF2α being the major products. In vascular slices and in cultured endothelial cells, the predominant prostacyclin derivative detected was 6-keto-PGF1α; the enzymatic PGI2-metabolite, 6,15-diketo-PGF1α, could be detected only in piglet vena cava slices in small amounts.     

Radioimmunological and biological measurement of prostaglandins in rabbit urine : Decrease of PGE2 excretion at high NaCl intake

The estimation of prostaglandin (PG) E₂ and of PGF_2α by radioimmunoassay is described in detail. PGE₂ was measured after conversion to either PGB₂ or PGF_2α and the results compared to bioassay. The methods were used to follow the excretion of PGE₂ and PGF_2α after salt loading in rabbits. A marked reduction of PGE₂ levels was observed at high NaCl intake, while PGF_2α excretion remained unchanged.