Mikio Marumo

TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+ entry channel to intracellular alkalosis.

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG–01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.

Attenuated phagocytic activity of monocytes in type 2 diabetic Goto-Kakizaki rats.

The aim of this study was to determine whether phagocytic activity of leukocytes is altered in type 2 diabetes. Goto-Kakizaki (G-K) rats, a genetic model for type 2 diabetes, and Wistar rats (control) were used to analyze the immunological status of phagocytes. Direct analysis of phagocytes was performed using peripheral whole blood. Phagocytic activity of monocytes induced by Escherichia coli BioParticles was significantly lower in G-K rats than in the control rats, whereas no significant differences in phagocytic activity of granulocytes and lymphocytes were found between G-K and control rats. Monocytes of G-K rats showed significantly lower CD11b/c expression compared with that in monocytes of control rats. However, lipopolysaccharide-stimulated activation of extracellular signal-regulated kinase and nuclear factor-κB in monocytes was not significantly different between G-K and control rats. Restriction of diet in G-K rats greatly improved their hyperglycemic status, but did not restore the levels of phagocytic activity and CD11b/c expression in monocytes of G-K rats to the levels observed in control rats. The results suggest that the phagocytic activity of monocytes is attenuated in G-K rats and that this attenuation is independent of blood glucose levels and is partly explained by a decrease in CD11b/c expression in G-K rats.

Diverse effects of ethanol on Ca2+ entry and subsequent aggregation of platelets

Although alcohol is known to inhibit platelet aggregation, and transplasmalemmal Ca(2+) entry is profoundly involved in platelet aggregation, there has been limited knowledge about the relationship between alcohol and Ca(2+) entry. The purpose of this study was to determine whether and how ethanol in vitro affects Ca(2+) entry through different pathways and the subsequent aggregation of platelets. Thapsigargin, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and thrombin were used to stimulate human platelets. Ca(2)(+) entry and the subsequent aggregatory responses of platelets were measured by spectrofluorometry using fura-2/AM as an indicator and the light transmission method, respectively. Thapsigargin-induced Ca(2+) entry and the following platelet aggregation were significantly inhibited by ethanol at concentrations of 0.5% or more. OAG-induced Ca(2+) entry was significantly augmented by ethanol at concentrations of 0.5% or more, whereas platelet aggregation by OAG was significantly inhibited by ethanol at concentrations of 0.5 % or more. Thrombin-induced Ca(2+) entry was not significantly affected by ethanol up to 2%, whereas platelet aggregation by thrombin was markedly inhibited by ethanol at concentrations of 0.5% or more. Thrombin-induced Ca(2+) entry in the presence of SKF-96365 was augmented by pretreatment with ethanol. Ethanol in vitro showed diverse effects on the different Ca(2+) entry pathways of platelets, whereas aggregatory responses induced by activation of the different Ca(2+) entry pathways of platelets were all inhibited by ethanol. These results suggest that ethanol inhibits platelet aggregation mainly via a mechanism(s) other than transplasmalemmal Ca(2+) entry.

Intracellular alkalinization augments capacitative Ca2+ entry in platelets

In order to elucidate the significance of intracellular alkalinization in signal transduction of platelets, we investigated the effects on capacitative Ca(2+) entry (CCE) of intracellular alkalinization that was induced by NH(4)Cl. Addition of NH(4)Cl (10 mM) to the medium resulted in an elevation of intracellular pH by about 0.35, which was eliminated by simultaneous addition of propionate (20 mM), an inducer of intracellular acidification, to the medium. CCE was induced by an extracellular addition of Ca(2+) to platelets in which Ca(2+) stores had been depleted by stimulation with thapsigargin in nominally Ca(2+)-free medium. NH(4)Cl markedly augmented CCE and subsequent platelet aggregation, both of which were abolished in the presence of SKF-96365, an inhibitor of capacitative Ca(2+) entry in non-excitable cells such as platelets. The augmentation of CCE and subsequent aggregation by NH(4)Cl was not observed in the presence of propionate or SKF-96365. Extracellular alkalosis induced by Tris also markedly augmented CCE and subsequent aggregation. These augmenting effects of extracellular alkalosis by Tris were significantly but incompletely inhibited by simultaneous addition of propionate (20 mM), which completely eliminated elevation of intracellular pH elicited by Tris. Thus, the augmenting effect of extracellular alkalosis on CCE was in part mediated by intracellular alkalosis. These findings suggest that intracellular alkalinization is a potent signal that augments CCE in platelets.

Inhibition of thrombin‐induced Ca2+ influx in platelets by R59949, an inhibitor of diacylglycerol kinase

Objectives  The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca2+ influx of platelets.

Sensitivity of thrombin-induced platelet aggregation to inhibition by ethanol.

BACKGROUND The purpose of this study was to determine the concentration of ethanol required for inhibition of thrombin-induced aggregation of human platelets in vitro. METHODS Three methods, light transmission method, particle counting method using light scattering and screen filtration pressure method using whole blood, were used for evaluation of platelet aggregation. RESULTS Thrombin (0.2 U/ml)-induced platelet aggregation was significantly inhibited by ethanol, and the threshold ethanol concentrations for inhibition determined by measurements using the light transmission method, particle counting method and screen filtration pressure method were 0.5%, 0.125% and 2%, respectively. Platelet aggregation induced by a lower concentration (0.05 U/ml) of thrombin was detected by the screen filtration pressure method but not by the light transmission method or particle counting method. The threshold ethanol concentration for the inhibition of thrombin (0.05 U/ml)-induced platelet aggregation was 0.125% according to results obtained by using the screen filtration pressure method. CONCLUSIONS Sensitivities for detection of thrombin-induced platelet aggregation and for detection of inhibition of platelet aggregation by ethanol were high in the screen filtration pressure method and the particle counting method, respectively. The threshold concentration of ethanol to inhibit thrombin-induced platelet aggregation is about 0.125% (approximately 21 mmol/l), which is easily attainable after drinking alcohol.

Intracellular alkalinization augments capacitative Ca2+ entry in vascular smooth muscle cells

Agonist-induced Ca2+ influx of vascular smooth muscle cells is thought to be triggered by depletion of intracellular Ca2+ stores. This study investigated the effects of intracellular alkalinization on capacitative Ca2+ entry in A7r5 rat aortic smooth muscle cells. Intracellular alkalinization was induced by NH4Cl. Transplasmalemmal Ca2+ influx due to Ca2+ store depletion induced by thapsigargin, which was abolished by pretreatment of the cells with SKF-96365 but not affected by that with verapamil, was significantly increased by pretreatment with NH4Cl. Neither 5-hydroxytryptamine-induced inositol monophosphate accumulation nor intracellular Ca2+ release from its stores was affected by NH4Cl. These results suggest that intracellular alkalinization acts on the process(es) after depletion of Ca2+ stores and facilitates capacitative Ca2+ entry in vascular smooth muscle cells.

Association Between Serum Long-Chain n-3 and n-6 Polyunsaturated Fatty Acid Profiles and Glomerular Filtration Rate Assessed by Serum Creatinine and Cystatin C Levels in Japanese Community-Dwellers

Background Plasma concentration of n-3 polyunsaturated fatty acids (PUFAs) has been reported to be associated with renal function in Western populations. However, few studies have investigated the association between serum long-chain n-3 and n-6 PUFA profiles and renal function in a Japanese population with high marine-derived long-chain n-3 PUFA intake. Methods A cross-sectional study was performed in 549 Japanese rural community-dwellers aged 40 to 64 years. In adjusted analysis of covariance, we assessed the relationship between estimated glomerular filtration rate (eGFR) and tertiles of serum long-chain n-3 and n-6 PUFA profiles ([eicosapentaenoic acid {EPA} + docosahexaenoic acid {DHA}]:arachidonic acid [AA]). GFR was estimated by Japanese specific equations using serum creatinine and cystatin C (eGFRcre and eGFRcys). Using multivariate-adjusted linear regression models, we also assessed the relationships between eGFRs and several n-3 and n-6 PUFAs, which have been suggested to be associated with renal function. Results In all participants, higher dietary fish intake as assessed by a semi-quantitative questionnaire was associated with higher serum value of (EPA+DHA):AA. Participants in the higher (EPA+DHA):AA tertiles had non-significantly higher eGFRcre and significantly higher eGFRcys (P = 0.016). In addition, eGFRcys in T2+T3 of (EPA+DHA):AA was significantly higher than that in T1 (adjusted mean eGFRcys, T1: 87 ml/min/1.73 m2, T2+T3: 91 ml/min/1.73 m2; P < 0.01). Among the PUFAs, only (EPA+DHA) was significantly associated with eGFRcys. Conclusions Serum (EPA+DHA):AA, which reflects an individual’s fish intake, might be associated with eGFRcys in Japanese community-dwellers.

Inverse associations of HDL cholesterol and oxidized HDL with d-dimer in patients with type 2 diabetes mellitus

Please cite this article as: Shigeyuki Ebara, Mikio Marumo, Chika Yamabata, Ikumi Nishibe, Jun-ichi Soneda, Jun Mukai, Makoto Ohki, Kagehiro Uchida, Ichiro Wakabayashi , Inverse associations of HDL cholesterol and oxidized HDL with d-dimer in patients with type 2 diabetes mellitus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tr(2017), doi: 10.1016/ j.thromres.2017.04.018

In vitro inhibitory effect of sarpogrelate hydrochloride on serotonin-induced platelet small-aggregate formation

Abstract Although sarpogrelate hydrochloride is known to be a selective S2-serotonergic receptor antagonist for platelets, the direct effect of sarpogrelate on serotonin-induced platelet activation has not been evaluated previously. The purpose of this study was to examine the in vitro effect of sarpogrelate on small-aggregate formation induced by serotonin by means of a particle-counting method using light scattering. Both 1 μM and 10 μM of serotonin-induced small-aggregate formations in platelet-rich plasma obtained from healthy volunteers were dose-dependently inhibited by the addition of 0.1 to 10 μM of sarpogrelate. The concentration of sarpogrelate causing 50% inhibition of the control response was calculated as 4.6 μM and 7.4 μM in 1 μM and 10 μM of serotonin-induced small-aggregate formation, respectively. Results of this study suggest that sarpogrelate inhibits serotonin-induced platelet activation and may thus be a useful antiplatelet agent.