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Biomedical subjects

W C Horne

Publications and source records attributed to W C Horne.

14 recordsLinked to original sources
PubMed1992

Activators of protein kinase C decrease serotonin transport in human platelets.

Treatment of human platelets with activators of protein kinase C (PKC) for 5-20 min resulted in substantial reductions in the rate of platelet serotonin (5-HT) transport. The mean Vmax observed after 5 min treatment with 1 microM 4-beta-12-tetradecanoylphorbol 13-acetate (beta-TPA) was 66% (n = 16, P = 0.0001) of the control value. 5 min of treatment with 1 microM mezerein reduced uptake to 78% (n = 3, P = 0.01) of control. Both beta-TPA and mezerein had little effect on the Km of transport and had EC50 values of approx. 100 mM when a 20-min treatment period was used. The maximum effects of both were reached at approx. 20 min and could be blocked with staurospine. The beta-TPA effect was stereospecific, as alpha-TPA did not alter platelet 5-HT uptake. Although the PKC activators may have altered transmembrane ion-gradients for Na+ and Cl-, which are co-transported with 5-HT, minimizing ion-gradient changes had little effect on the observed reductions in transport. The PKC activators also had little or no effect on platelet 5-HT release or on the number (Bmax) of 5-HT transporters expressed at the platelet surface. The data indicate that PKC activation may down-regulate the activity of the 5-HT transporter in platelets. Apparently, most of this effect is mediated through mechanisms other than changes in ion-gradients, reductions in the number of available transporters, or increased 5-HT release. The apparent regulation of 5-HT transport by PKC may have important implications in platelet and neuronal functioning.

Alkaloids↗
PubMed1992

Osteoclasts express high levels of pp60c-src in association with intracellular membranes.

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.

Animals↗
PubMed1991

Reproducibility and temporal stability of ADP-induced platelet aggregation: comparison of the anticoagulants sodium citrate and D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone.

We compared the reproducibility and temporal stability of ADP-induced aggregation of platelet-rich plasma anticoagulated with citrate or with D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK), a thrombin inhibitor. Citrate- or PPACK-platelet-rich plasma (PRP) was stored at room temperature in capped plastic tubes or in plastic syringes from which all air was expelled. At intervals over 1-5 hr after venipuncture, platelet aggregation was induced by 1-1.5 microM ADP. Initially, the aggregation of PPACK-PRP was nearly twice that of citrate-PRP. The response of PPACK-PRP stored in the syringe remained essentially constant over the interval of study, in contrast to the responses of the other samples which declined with time. The improved stability of the response obtained from samples anticoagulated with PPACK was due to the absence of citrate, since PRP containing both citrate and PPACK became less responsive over time in a manner similar to PRP which contained only citrate. Anticoagulation with PPACK rather than citrate results in a more reproducible and stable aggregation response and more closely reproduces the in vivo environment of the platelet.

Adenosine Diphosphate↗
PubMed1990

Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1.

In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem. 260, 9073-9076). The 10 kDa domain also contains the spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase inhibits the binding of protein 4.1 to spectrin and actin (Ling, E., Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In this study, we have identified two sites on protein 4.1 which account for 80% of the phosphate incorporated into protein 4.1 during metabolic labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More than 95% of the 32P incorporated into protein 4.1 was in the form of phosphoserine. Reverse-phase HPLC of the peptides generated by digestion of the isolated protein with trypsin or endoproteinase lysine C produced two major radioactive peaks. The phosphorylation sites, identified by gas phase sequencing of the purified phosphopeptides and confirmed by determining the residues converted to S-ethylcysteine by reacting the phosphopeptides with ethanethiol under alkaline conditions, were Ser-331, in the 16 kDa domain and Ser-467, in the 10 kDa domain.

Amino Acid Sequence↗
PubMed1990

The hyperserotonemia of autism.

The planned and ongoing studies of platelet function and composition should allow us to better define the alteration which we presume to be present in platelets of autistic subjects. Although much of the research focuses on serotonergic aspects, the more general research should permit a better delineation of the extent of the alteration and will protect against a premature narrowing of the inquiry. The methodological development which has been a necessary aspect of the work should contribute to an improved understanding of platelet function and composition, as well as result in improved clinical tools for the assessment of platelet functioning in neuropsychiatric disorders and hematology. As an example, improvements in short-term in vitro storage conditions to stabilize aggregation and shape change responses over time were found to be necessary, and are probably critical to an optimal comparison of these phenomena across groups. The identification of the platelet alteration which is responsible for the hyperserotonemia of autism should prove useful in several ways. It would be expected that assessment of the altered function would provide a marker with less overlap with the normal population than the multidetermined measure of blood 5-HT. Determination of the specific protein(s) involved in the altered platelet should lead directly to gene probes and chromosomal location. These, in turn, should prove useful for neonatal screening, subtyping, and more powerful genetic and family studies. Work of this sort might also allow early intervention and improved treatment. Finally, characterization of the physiological alteration would provide a basis for focusing studies of brain neurochemistry and should, as well, suggest modes of neuropharmacological intervention. The confidence that one can have in the basic finding of hyperserotonemia in autism and the potential benefits to be derived from its explication make further research in this area of great interest.

Adolescent↗
PubMed1989

Calcium-dependent proteolysis of actin during storage of platelet concentrates.

In an ongoing study of the changes that occur in platelet concentrates during storage, we investigated two 28-26-Kd proteins designated SP-1 and SP-2, respectively, which increase markedly during blood-bank storage of platelet concentrates at room temperature. Formation of SP-1 and SP-2 was inhibited by storage at 4 degrees C as well as by treatment of the concentrates with leupeptin, N-ethylmaleimide, and EDTA; DFP and PPACK had no effect. The calcium ionophore A23187 markedly stimulated production of SP-1 and SP-2. After partial purification, the two proteins were found to be associated with platelet cytoskeletal protein. Two-dimensional peptide mapping and amino acid sequencing identified SP-1 and SP-2 as fragments of actin formed by cleavage on the N-terminal side of residues Thr-106 and Ala-114, respectively. Generation of SP-1 and SP-2 during storage of platelet concentrate is likely attributable to calcium-dependent neutral protease degradation of actin and may have implications for development of the platelet storage lesion.

Actins↗
PubMed1988

Erythrocyte membrane skeleton phosphoproteins: identification of two unrelated phosphoproteins in band 4.9.

Human erythrocyte membrane band 4.9 is phosphorylated by several erythrocyte protein kinases. Chromatography of erythrocyte membrane skeleton proteins on DEAE-Sephacel produces two proteins with relative mobilities, on gel electrophoresis, similar to that of band 4.9. The first, with a molecular mass of 49 kDa, is quite basic (pI greater than 8) while the second, 50.5 kDa, is slightly acidic (pI = 6.2). Comparative two-dimensional peptide mapping reveals that both proteins are present in band 4.9 on one-dimensional gels of total erythrocyte membrane proteins and membrane skeleton proteins. The 49 kDa protein, but not the 50.5 kDa protein, binds to actin filaments in a sedimentation assay. In intact erythrocytes metabolically labeled with [32P]orthophosphate, the 49 kDa protein is phosphorylated by protein kinase C, cAMP-dependent protein kinase, and protein kinases which are active in the absence of exogenous kinase activators. In contrast, the 50.5 kDa protein is phosphorylated by protein kinase C but not by the other protein kinases examined. Finally, two-dimensional peptide mapping was employed to compare the 49 kDa protein and a 57 kDa protein which copurifies with, and has many characteristics of, the 49 kDa protein. Significant similarities were found in both 125I-labeled chymotryptic peptide maps and 32P-labeled tryptic peptide maps, suggesting that the 49 kDa and 57 kDa proteins are closely related.

Blood Proteins↗
PubMed1985

Differential phosphorylation of multiple sites in protein 4.1 and protein 4.9 by phorbol ester-activated and cyclic AMP-dependent protein kinases.

The phosphorylation of the membrane skeleton components protein 4.1 and protein 4.9 in intact erythrocytes is shown to increase in the presence of either 1 microM 12-O-tetradecanoyl phorbol 13-acetate or 2 mM dibutyryl cAMP. The phosphorylation induced by these protein kinase activators is compared by two-dimensional tryptic peptide mapping. In both proteins, the pattern of peptides phosphorylated in the presence of 12-O-tetradecanoyl phorbol 13-acetate differs from the pattern of peptides phosphorylated in the presence of dibutyryl cAMP. The relative locations of the phosphorylated sites on protein 4.1 have been determined using limited proteolysis by alpha-chymotrypsin.

Blood Proteins↗
PubMed1978

Probes of transmembrane potentials in platelets: changes in cyanine dye fluorescence in response to aggregation stimuli.

A noncovalent fluorescent probe that responded to changes in transmembrane potential was used to study the response of washed human platelets to aggregating agents. Concentration-dependent changes in the fluorescence were observed in response to ADP and to thrombin. No such changes were observed in response to collagen fibrils. Thus there was an indication that platelet membrane potential changed in response to aggregating stimuli, supporting the hypothesis that the mechanisms of platelet aggregation resembled the mechanisms of other systems that show stimulus-response coupling (e.g., muscle, adrenal chromaffin cells). The different responses to specific agents indicate that the agents may trigger platelet aggregation through different mechanisms.

Adenosine Diphosphate↗
PubMed1975

Fluorescent labeling of human platelets.

Noncovalently bound fluorescent probes have been used to study changes in the platelet which may occur during platelet aggregation. Platelets were exposed to either N-phenyl-naphthylamine (NPN) or 8-anilino-1-naphthalene-sulfonic acid (ANS). Both dyes were bound by the platelet, and platelet aggregation by collagen or thrombin was unaffected by the presence of the label. No change in fluorescence intensity or wavelength of maximum intensity was observed during platelet aggregation.

1-Naphthylamine↗