scholarly journals Phorbol 12,13-dibutyrate binding to intact human platelets. The role of cytosolic free Ca2+

1991 ◽  
Vol 278 (2) ◽  
pp. 411-415 ◽  
Author(s):  
J Takaya ◽  
M Kimura ◽  
N Lasker ◽  
A Aviv
Keyword(s):  
Binding Capacity ◽  
Human Platelets ◽  
Significant Decline ◽  
Hill Coefficient ◽  
Primary Role ◽  
Kinase C ◽  
Equilibrium Dissociation ◽  
The Hill ◽  
Pdbu Binding

The role of Ca2+ was examined in regulating the binding of phorbol 12,13-dibutyrate (PdBu) to intact human platelets. Alterations in the cytosolic free Ca2+ concn. [(Ca2+]i), but not extracellular Ca2+, substantially influenced the binding parameters of the phorbol ester. Ca(2+)-depleted platelets demonstrated a significant decline in the maximal binding capacity (Bmax), an increase in equilibrium dissociation constant (Kd) and a decrease in the Hill coefficient (h), suggesting the presence of Ca(2+)-sensitive and Ca(2+)-insensitive populations of PdBu-binding sites. In 1 mM-Ca2+ buffer, thrombin (0.1 NIH unit/ml) and ionomycin (0.5 microM) evoked a rise in [Ca2+]i to approx. 300-500 nM, associated with a significant decline in Kd, but without an apparent effect on Bmax. No effect of thrombin was observed on PdBu binding in Ca(2+)-depleted platelets. Inhibition of protein kinase C (PKC) by H7 was associated with a greater thrombin-evoked [Ca2+]i transient and a decline in Kd. Staurosporine also decreased the Kd for PdBu binding. We propose that this effect of the PKC inhibitors on the Kd was also [Ca2+]i-dependent. These observations in intact platelets indicate that the primary role of agonist- or non-agonist-induced rise in [Ca2+]i is to increase the affinity of PKC for PdBu and, presumably, endogenous diacylglycerol. However, in itself a rise in [Ca2+]i does not increase the Bmax, for PdBu binding.

Hypersensitivity to Thromboxane A2 in Cholesterol-Rich Human Platelets

1990 ◽  
Vol 64 (04) ◽  
pp. 594-599 ◽  
Author(s):  
Takuya Tomizuka ◽  
Kyohei Yamamoto ◽  
Aizan Hirai ◽  
Yasushi Tamura ◽  
Sho Yoshida
Keyword(s):  
Calcium Concentration ◽  
Binding Capacity ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Cholesterol Content ◽  
Human Platelets ◽  
Dissociation Rate ◽  
Platelet Membrane ◽  
Maximal Concentration ◽  
Equilibrium Dissociation

SummaryThe effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,ll-epithio-ll,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

Intracellular ADP-ribose inhibits ATP-sensitive K+ channels in rat ventricular myocytes

1996 ◽  
Vol 271 (2) ◽  
pp. C464-C468 ◽  
Author(s):  
Y. G. Kwak ◽  
S. K. Park ◽  
U. H. Kim ◽  
M. K. Han ◽  
J. S. Eun ◽  
...  
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Physiological Role ◽  
Hill Coefficient ◽  
K Channel ◽  
Channel Activity ◽  
Rat Ventricular Myocytes ◽  
Cyclic Adp Ribose ◽  
The Hill

Cyclic ADP-ribose (cADPR), an NAD metabolite, has been shown to be a messenger for Ca2+ mobilization from intracellular Ca2+ stores. However, the physiological role of ADP-ribose (ADPR), another metabolite of NAD, is not known. We examined the effects of cADPR and ADPR on the ATP-sensitive K+ channel (KATP) activity in rat ventricular myocytes by use of the inside-out patch-clamp configuration. ADPR, but not cADPR, inhibited the channel activity at micromolar range with an inhibitor constant (Ki) of 38.4 microM. The Hill coefficient was 0.9. ATP inhibited the K+ channel with a Ki of 77.8 microM, and the Hill coefficient was 1.8. Single-channel conductance was not affected by ADPR. These findings strongly suggest that ADPR may act as a regulator of KATP channel activity.

scholarly journals Phosphorylation-dependent and -independent pathways of platelet aggregation

1989 ◽  
Vol 258 (2) ◽  
pp. 479-485 ◽  
Author(s):  
S P Watson ◽  
S Hambleton
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Signalling Pathways ◽  
Ionophore A23187 ◽  
Phorbol Dibutyrate ◽  
Kinase C ◽  
Dependent Pathway

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

Modification of Na+/H+ Exchanger in Human Platelets by 1,2-Dioctanoylglycerol, a Protein Kinase C Activator

1990 ◽  
Vol 64 (01) ◽  
pp. 165-171 ◽  
Author(s):  
Yukio Ozaki ◽  
Yuki Mastsumoto ◽  
Yutaka Yatomi ◽  
Masaaki Higashihara
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Rate Constant ◽  
Human Platelets ◽  
Kinase C ◽  
Km Value ◽  
The Difference ◽  
The One ◽  
Protein Kinase C Activation

SummaryProtein kinase C activation in human platelets has a modulatory role in maintaining intracellular pH (pHi), by adjusting pHi at a particular value (7.22). Changes in pHi induced by protein kinase C appeared to be dependent upon the difference between H+ efflux catalyzed by the Na+/H+ exchanger and H+ production. The pHi recovery after acid loading was significantly facilitated by protein kinase C activation. Analysis of the rate constant for pHi recovery suggested that the turnover rate or the apparent affinity of the Na+/H+ exchanger for H+ was increased. Protein kinase C also decreased the Km value of the Na+/H+ exchanger for extracellular Na+. Thus, it is suggested that the role of protein kinase C in platelet pHi regulation is dual, adjusting the pHi value at a certain setpoint on the one hand, and increasing the rate constant of the Na+/H+ exchanger on the other.

PROTEIN KINASE C CONTROLS CA2+ MOBILIZATION IN HUMAN PLATELETS

1987 ◽  
Author(s):  
W Siffert ◽  
P Scheid ◽  
JW N Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Cytosolic Ph ◽  
Fluorescent Indicators ◽  
Kinase C ◽  
Deutsche Forschungsgemeinschaft ◽  
Ionophore Monensin ◽  
Stimulation Of

Platelet stimulation has been shown to result in a rise of cytosolic pH (pHi) as a result of an activation of a Na+/H+ antiport. We have investigated the role of pH in Ca2+ mobilization in human platelets. pHi and free Ca2+, {Ca2+)i, were measured in platelets loaded with the fluorescent indicators BCECF and quin2, respectively. Stimulation of platelets by either thrombin or OAG, an activator of protein kinase C (Pk-C), increased pHi. Pretreatment of platelets with inhibitors of Pk-C, trifluoperazine (TFP) or sphingosine (SPH), blocked the stimulus-induced rise in pHi, suggesting a role of Pk-C in the activation of Na+/H+ exchange. Blocking Na+/H+ exchange by an amiloride analogue or by TFP similarly suppressed the thrombin-induced increase in {Ca2*}i. This effect could be prevented by increasing pHi with the Na+/H+ ionophore monensin or with NH4Cl. The thrombin-induced (0.05 U/ml) rise in {Ca2+}i was more than 3-fold enhanced when the pH was raised from 6.8 to 7.4.Our results demonstrate that pHi controls Ca2+ mobilization in human platelets and suggest that Pk-C contributes to this control by activating the Na+/H+ exchanger.Supported by the Deutsche Forschungsgemeinschaft. No Sche 46/5-2.

Role of protein kinase C in the regulation of phospholipase A2 activity in human platelets

1990 ◽  
Vol 18 (3) ◽  
pp. 467-468 ◽  
Author(s):  
CAROLINE P. D. WHEELER-JONES ◽  
YATIN PATEL ◽  
VIJAY V. KAKKAR ◽  
SUSHILA KRISHNAMURTHI
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Human Platelets ◽  
Kinase C ◽  
Phospholipase A2 Activity

R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE

1987 ◽  
Author(s):  
S K Joseph ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Kinase Inhibitor ◽  
Human Platelets ◽  
Maximal Concentration ◽  
Inhibitory Effects ◽  
Kinase C ◽  
Granule Secretion ◽  
Granule Release

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.

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