Lack of effect of ouabain on calcium homeostasis in rat platelets: comparative study with human platelets

1994 ◽  
Vol 266 (3) ◽  
pp. R651-R657
Author(s):  
T. Oshima ◽  
T. Ishida ◽  
H. Matsuura ◽  
M. Ishida ◽  
K. Ishibashi ◽  
...  
Keyword(s):  
Wistar Rats ◽  
Time Course ◽  
Calcium Concentration ◽  
Sodium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Time Dependent ◽  
Maximal Dose ◽  
Peak Response ◽  
Rat Platelets

The precise mechanisms of Ca2+ handling in rat platelets are not fully understood. We sought to determine whether rat platelets possess a Na(+)-Ca2+ exchanger. First, we investigated the time course of the effect of ouabain (10(-4) M) on cytosolic sodium concentration ([Na+]i) and Ca2+ homeostasis in platelets from Wistar rats in comparison with those from humans. Ouabain increased platelet [Na+]i in both rat and human platelets. Whereas ouabain induced a time-dependent increase in basal and thrombin-stimulated (0.3 units/ml) cytosolic calcium concentration ([Ca2+]i) in human platelets, no change was found in rat platelets. Furthermore, 90-min pretreatment of rat platelets with ouabain did not affect the [Ca2+]i response to thrombin (0.1-1.0 units/ml) or a maximal dose of ionomycin (5 microM). Also the decline in [Ca2+]i after the peak response evoked by these agonists in the absence of extracellular Ca2+ was not changed by ouabain pretreatment. Similarly, replacement of extracellular Na+ had no influence on any of these determinations. Thus decreasing the plasma membrane Na+ gradient did not affect basal [Ca2+]i, thrombin-induced mobilization of Ca2+ from intracellular stores, internal Ca2+ discharge capacity, or Ca2+ extrusion from cytosol of rat platelets. In contrast to human platelets, a Na(+)-Ca2+ exchange mechanism does not appear to play a significant role in Ca2+ homeostasis of rat platelets.

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.

scholarly journals Synthetic lipopeptide Pam3CysSer(Lys)4 is an effective activator of human platelets

1994 ◽  
Vol 266 (6) ◽  
pp. C1684-C1691 ◽  
Author(s):  
M. Berg ◽  
S. Offermanns ◽  
R. Seifert ◽  
G. Schultz
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Cellular Effects ◽  
Prostacyclin Receptor ◽  
Serotonin Secretion ◽  
Peptide Moiety ◽  
Lipid Moiety ◽  
Molecular Masses ◽  
Aggregation Of Platelets

Lipopeptide analogues of the NH2-terminus of bacterial lipoprotein are known to induce activation of macrophages, neutrophils, and lymphocytes. We studied the effect of the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine [Pam3CysSer(Lys)4] on several functions of human platelets. Pam3CysSer(Lys)4 led to the aggregation of platelets and induced the secretion of serotonin with an effectiveness similar to thrombin. These cellular effects of Pam3CysSer(Lys)4 were concentration dependent, being half maximal at 2-3 microM and maximal at 10-30 microM. Another lipopeptide also induced platelet aggregation and serotonin secretion but was less potent and less effective than Pam3CysSer(Lys)4. The lipid moiety and the peptide moiety of Pam3CysSer(Lys)4 alone were without any effect. Lipopeptides also stimulated tyrosine phosphorylation of several proteins with molecular masses similar to those found to be tyrosine phosphorylated in response to thrombin, and Pam3CysSer(Lys)4 led to an increase in the cytosolic calcium concentration. All studied responses of platelets to lipopeptides were inhibited by the prostacyclin receptor agonist cicaprost. Taken together, our data show that lipopeptides are effective activators of human platelets and that this activation is susceptible to the action of physiological platelet inhibitors.

The Effect of Changed Extracellular Calcium and Sodium Concentration on the Electroretinogram of the Crayfish Retina

1980 ◽  
Vol 35 (3-4) ◽  
pp. 308-318 ◽  
Author(s):  
H. Stieve ◽  
I. Claßen-Linke
Keyword(s):  
Dark Adaptation ◽  
Time Course ◽  
Calcium Concentration ◽  
Light Adaptation ◽  
Sodium Concentration ◽  
Strong Increase ◽  
Calcium Stores ◽  
Half Time ◽  
Astacus Leptodactylus ◽  
External Calcium

Abstract The electroretinogram (ERG) of the isolated retina of the crayfish Astacus leptodactylus evoked by strong 10 ms light flashes at constant 5 min intervals was measured while the retina was continuously superfused with various salines which differed in Ca2+ -and Na+ -concentrations. The osmotic pressure of test- and reference-saline was adjusted to be identical by adding sucrose. Results: 1. Upon raising the calcium-concentration of the superfusate in the range of 20-150 mmol/l (constant Na+ -concentration: 208 mmol/l) the peak amplitude hmax and the half time of decay t2 of the ERG both decrease gradually up to about 50% in respect to the corresponding value in reference saline. 2. The recovery of the ERG due to dark adaptation following the “weakly light adapted state” is greatly diminished in high external [Ca2+]ex. 3. Lowering the external calcium-concentration (10 →1 mmol/l) causes a small increase in hmax and a strong increase of the half time of decay t2 (about 180%). Upon lowering the calcium concentration of the superfusate to about 1 nmol/l by 1 mmol/l of the calcium buffer EDTA, a slowly augmenting diminution of the ERG height hm SLX occurs. How­ever, a strong retardation of the falling phase of the ERG characterized by an increase in t2 occurs quickly. Even after 90 min stay in the low calcium saline the retina is still not inexcitable; hmax is 5 - 10% of the reference value. The diminution of hmax occurs about six-fold faster when the buffer concentration is raised to 10 mmol/l EDTA. 4. Additional lowering of the Na+ -concentration (208 →20.8 mmol/l) in a superfusate with a calcium concentration raised to 150 mmol/l causes a strong reduction of the ERG amplitude hmax to about 10%. 5. In a superfusate containing 1 nmol/l calcium such lowering of the sodium concentration (208 → 20.8 mmol/l) causes a diminution of the ERG height to about 40% and the shape of the ERG to become polyphasic; at least two maxima with different time to peak values are observed. Interpretation: 1. The similarity of effects, namely raising external calcium concentration and light adaptation on the one hand and lowering external calcium and dark adaptation on the other hand may indicate that the external calcium is acting on the adaptation mechanism of the photoreceptor cells, presumably by influencing the intracellular [Ca2+]. 2. The great tolerance of the retina against Ca2+ -deficiency in the superfusate might be effected by calcium stores in the retina which need high Ca2+ -buffer concentrations in the superfusate to become exhausted. 3. In contrast to the Limulus ventral nerve photoreceptor there does not seem to be an antagonis­ tic effect of sodium and calcium in the crayfish retina on the control of the light channels. 4. The crayfish receptor potential seems to be composed of at least two different processes. Lowering calcium-and lowering external sodium-concentration both diminish the height and change the time course of the two components to a different degree. This could be caused by in­ fluencing the state of adaptation and thereby making the two maxima separately visible.

scholarly journals Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets

1992 ◽  
Vol 283 (2) ◽  
pp. 379-383 ◽  
Author(s):  
J W Heemskerk ◽  
J Hoyland ◽  
W T Mason ◽  
S O Sage
Keyword(s):  
Calcium Concentration ◽  
Calcium Signalling ◽  
Peak Frequency ◽  
Cytosolic Calcium ◽  
Light Level ◽  
Optical Probe ◽  
Human Platelets ◽  
Free Calcium ◽  
Fura 2 ◽  
Subsequent Activation

Fura-2-loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free calcium concentration ([Ca2+]i) was measured in single platelets by low-light-level video-ratio image-processing of the optical probe signal. Some fibrinogen-bound platelets showed repetitive spiking in [Ca2+]i with a mean frequency of about 2/min, which increased to 5/min in the presence of ADP. Other cells showed no activity until the addition of agonist. When immobilized in the presence of prostaglandin I2 and the fibrinogen antagonist Arg-Gly-Asp-Ser, the platelets adhered less firmly to fibrinogen, and in many [Ca2+]i remained low and constant. Subsequent activation of such platelets with ADP evoked oscillations in [Ca2+]i with a peak frequency of about 5/min and which persisted for at least 5 min. These results indicate that human platelets, like many other non-excitable cells, have an elaborate system of calcium signalling involving spiking.

scholarly journals ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store

1990 ◽  
Vol 265 (3) ◽  
pp. 675-680 ◽  
Author(s):  
S O Sage ◽  
R Reast ◽  
T J Rink
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Second Phase ◽  
Stopped Flow ◽  
Bivalent Cation ◽  
Fura 2 ◽  
Fast Phase ◽  
Lower Temperature ◽  
Delayed Phase

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.

Plasmin Inhibition of Thrombin-induced Platelet Aggregation

1975 ◽  
Vol 33 (02) ◽  
pp. 286-309 ◽  
Author(s):  
Jonathan L Miller ◽  
Alfred J Katz ◽  
Maurice B Feinstein
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Calcium Concentration ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Platelet Protein ◽  
Platelet Receptor ◽  
Induced Aggregation ◽  
Plasmin Inhibitor ◽  
Maximal Block

SummaryThe effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10–20 μM, plasmin (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin’s inhibition of aggregation.Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction.Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin -treated platelets, upon centrifugation and treatment with SBTI.With the use of a “cold initiation” technique, the release by thrombin of 46.7 ± 6-7 (mean ± SEM) μg of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by SBTI) aggregated normally upon addition of as little as 10 μg human plasma fibrinogen per mg platelet protein.It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.

Membrane Microviscosity and Plasma Triacylglycerols in the Rat

1998 ◽  
Vol 94 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Marie-Aude Devynck ◽  
J. Kuneš ◽  
Kim Hanh Le Quan Sang ◽  
J. Zicha
Keyword(s):  
Wistar Rats ◽  
Calcium Concentration ◽  
Lipid Membrane ◽  
Cytosolic Calcium ◽  
Calcium Handling ◽  
Erythrocyte Ghosts ◽  
Membrane Microviscosity ◽  
Genetically Hypertensive Rats ◽  
Multiple Cell ◽  
Outer Membrane Leaflet

1. Multiple cell membrane alterations have been described in humans and animals with various genetic forms of hypertension and/or dyslipidaemia. The aim of our study was to characterize membrane microviscosity, using two different fluorescent probes exploring either the outer membrane leaflet [trimethylamino-diphenylhexatriene (TMA-DPH)] or the lipid membrane core [diphenylhexatriene (DPH)], in platelets and erythrocytes of genetically hypertensive rats of the Prague hereditary hypertriglyceridaemic (HTG) strain. The relationships of membrane microviscosity to hypertension, hypertriglyceridaemia and cell calcium handling were also investigated. 2. Membrane microviscosity was similar in HTG and normotensive control Wistar rats when measured in platelets or erythrocyte ghosts incubated in Na+-containing medium. On the contrary, TMA-DPH fluorescence anisotropy was significantly reduced in HTG platelets incubated in Na+-free medium because external Na+ removal elicited a larger rise of TMA-DPH anisotropy in Wistar platelets. 3. Plasma triacylglycerols were associated positively with platelet TMA-DPH anisotropy and negatively with DPH anisotropy in both strains. The slopes of these relationships were reduced in HTG compared with Wistar rats. Platelet TMA-DPH anisotropy correlated positively and DPH anisotropy negatively with the cytosolic calcium concentration in unstimulated platelets, the slopes being almost identical in both strains. 4. Pulse pressure correlated negatively with TMA-DPH anisotropy and positively with DPH anisotropy found in erythrocyte ghosts. 5. The present results suggest that plasma triacylglycerols and cytosolic calcium are capable of modulating the membrane microviscosity in this new animal model of genetic hypertension associated with hypertriglyceridaemia.

Calcium Entry in Nonexcitable Cells: Lessons from Human Platelets

Physiology ◽  
1992 ◽  
Vol 7 (3) ◽  
pp. 108-113
Author(s):  
SO Sage ◽  
MP Mahaut-Smith ◽  
TJ Rink
Keyword(s):  
Calcium Signaling ◽  
Platelet Activation ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Entry ◽  
Cytosolic Calcium Concentration ◽  
Stimulus Response ◽  
Signaling Mechanisms

A rise in cytosolic calcium concentration plays an important role in platelet activation. As well as helping define stimulus-response coupling in these intriguing and clinically important cells, the study of calcium mobilisation in platelets serves as an important model for elucidating calcium signaling mechanisms in general.

Mass Contents of Inositol 1,4,5-Trisphosphate and 1,2-Diacylglycerol in Human Platelets Stimulated with a Thromboxane Analogue and Thrombin

1992 ◽  
Vol 68 (03) ◽  
pp. 341-345 ◽  
Author(s):  
A Suganuma ◽  
S Nakashima ◽  
Y Okano ◽  
Y Nozawa
Keyword(s):  
Time Course ◽  
Second Messengers ◽  
Human Platelets ◽  
Maximal Dose ◽  
Low Level ◽  
Inositol 1,4,5 Trisphosphate ◽  
Concentration Dependency

SummaryMass contents of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) were measured in U46619-stimulated human platelets. 1 µM of U46619 induced maximum responses in aggregation, 5-hydroxytryptamine (5HT) secretion and increase in intracellular free Ca2+ concentration ([Ca2+]i). Aggregation was almost comparable to that induced by maximal dose (1 U/ml) of thrombin, while 5HT release was almost half. The initial [Ca2+]i peak in response to U46619 was about half of thrombin stimulation. Production of IP3 and DG was, however, less than one tenth of that seen in thrombin stimulation. The profile (time course and concentration-dependency) of IP3 formation did not correlate with that of [Ca2+]i, suggesting that U46619 stimulates IPs-dependent and -independent Ca2+ mobilization. DG production was small but sustained for more than 5 min. These findings support the recent hypothesis that aggregation is regulated by a delayed accumulation of DG. The low level of 5HT secretion could be explained by the low production of second messengers, IP3 and DG.

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