Platelet Membrane Potential: Simultaneous Measurement of diSC3(5) Fluorescence and Optical Density

1985 ◽  
Vol 54 (03) ◽  
pp. 645-649 ◽  
Author(s):  
Eva Pipili
Keyword(s):  
Membrane Potential ◽  
Time Course ◽  
Shape Change ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Receptor Interaction ◽  
Na Channel ◽  
Similar Time ◽  
High K ◽  
Contrast Exposure

SummaryThe role of membrane potential in the activation of human platelets by thrombin, ADP and PAF was assessed, using the fluorescent probe diSC3(5). Thrombin, ADP and PAF transiently depolarised the platelet membrane by 6-8 mV from its resting level (—70 mV). This depolarisation had a similar time course to that of shape change. The ionophores valinomycin and gramicidin hyperpolarised and depolarised the platelets respectively but did not activate them. In contrast, exposure of platelets to high K+ media both depolarised and caused them to change shape. Removal of Na+ from the suspension media abolished the depolarisation induced by thrombin, ADP and PAF but the platelets under these conditions were still capable of changing shape and aggregating. This result indicates that the observed depolarisation depends on Na+ fluxes. Amiloride or tetrodotoxin did not mimic the effect of Na+ removal suggesting that any Na+ movement involved does not go through the classic “Na+ channel”. Thrombin, ADP and PAF still depolarised the platelet membrane in the absence of added Ca+ +. Under these conditions, however, the membrane did not repolarise. It is evident that all three agents, thrombin, ADP and PAF, change the membrane potential of human washed platelets through a similar mechanism and this change seems to be a consequence of stimulus-receptor interaction (and platelet activation?). A causal relationship however between these events cannot be clearly shown.

The Role of Platelet Membrane Potential in the Initiation of Platelet Aggregation

1982 ◽  
Vol 47 (01) ◽  
pp. 022-026 ◽  
Author(s):  
D E MacIntyre ◽  
T J Rink
Keyword(s):  
Membrane Potential ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Null Point ◽  
Platelet Membrane ◽  
Resting Potential ◽  
Permselective Membrane ◽  
High K ◽  
Transduction Mechanisms

SummaryThe membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the non-covalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-permselective membrane. The resting potential calculated from the “valinomycin null point” (the K+ concentration gradient at which valinomycin did not change the potential) was approximately – 60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl− permeability, demonstrated by replacing Cl− with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.

scholarly journals The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin

1984 ◽  
Vol 221 (3) ◽  
pp. 897-901 ◽  
Author(s):  
T J Hallam ◽  
N T Thompson ◽  
M C Scrutton ◽  
T J Rink
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Similar Time ◽  
Transient Rise ◽  
Thromboxane Production

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

scholarly journals Regulation of renal Na transporters in response to dietary K

2018 ◽  
Vol 315 (4) ◽  
pp. F1032-F1041 ◽  
Author(s):  
Lei Yang ◽  
Shuhua Xu ◽  
Xiaoyun Guo ◽  
Shinichi Uchida ◽  
Alan M. Weinstein ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Protein Levels ◽  
Phosphorylated Form ◽  
High K ◽  
Enhanced Expression ◽  
Males And Females ◽  
Epithelial Na Channel

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved γ-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by ~50% and ~80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle’s loop and distal convoluted tubule along with Cl−, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis.

Fibrinogen Induces Changes In Platelet Membrane Potential

1981 ◽  
Author(s):  
H Vainer ◽  
A Caprani
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Human Albumin ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Limiting Current ◽  
Transmembrane Transport ◽  
Membrane Glycoproteins ◽  
Rotating Disc ◽  
Experimental Conditions

Changes in platelet membrane potential (MP) reflecting ion transmembrane transport accompany the platelet activation by various stimuli. We have studied the platelet MP and the effect of fibrinogen on its expression, using an electro-chemical method. The MP determination is based on the evaluation of the transmembrane partition coefficient (PC) (Ci/Ce) of an electroactive tracer, such as the ferro- cyanid anion. Washed human platelets are incubated with the tracer solution ; the amount of tracer in the supernatant (Ce) and in the acellular lysate of SDS-treated platelet pellet (Ci) is measured as the diffusion limiting current corresponding to the tracer oxidation at the surface of a rotating disc electrode. The kinetics for the PC evaluation have been studied in order to standardize the experimental conditions which allow to express the MP by the Nernst equation : Vi-Ve = RT/4F. Log Ci/Ce.The mean value ± SD (N = 9) of the platelet MP in saline is -5.5 ± 0.5 mV (PC = 0.445 ± 0.020). When fibrinogen replaces the saline, a MP of -8.83 mV (PC = 0.257) is observed. While it contrasts sharply with the MP of platelets in saline, it is comparable to the MP of platelets in plasma, which is : -8.63 mV (PC=0.266). We have also measured the effect of human albumin and gamma globulins on the MP ; both these plasmatic proteins cause a much smaller decrease of MP, as compared to fibrinogen (or plasma), all used at physiological concentrations in saline and without the platelet pretreatment with any agent.The results suggest that a relationship might exist between the marked decrease of platelet MP caused by fibrinogen - possibly via mediators of the transmembrane transport- and/or some of the platelet membrane glycoproteins which appear to function as binding sites for fibrinogen and must undergo (conformational) changes that allow fibrinogen to bind.

Reversal of thromboxane A2/prostaglandin H2 and ADP-induced calcium release in intact platelets

1985 ◽  
Vol 249 (1) ◽  
pp. H8-H13
Author(s):  
L. D. Brace ◽  
D. L. Venton ◽  
G. C. Le Breton
Keyword(s):  
Receptor Antagonist ◽  
Calcium Release ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Mobilization ◽  
Receptor Interaction ◽  
Calcium Sequestration ◽  
Platelet Shape

We previously demonstrated that thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2), ADP, and A23187 cause calcium mobilization in intact human platelets. Other studies have also shown that platelet shape change and aggregation induced by a variety of platelet agonists can be reversed by specific antagonists. In the present study, we used the fluorescent calcium probe chlortetracycline to evaluate whether the reversal of platelet activation involves a resequestration of intraplatelet calcium. It was found that the TXA2/PGH2 receptor antagonist 13-azaprostanoic acid (13-APA) reversed calcium mobilization and shape change induced by AA but not that induced by ADP. A similar specificity of action was observed using the specific ADP receptor antagonist, ATP, in that ATP only reversed ADP-induced calcium release and shape change. In contrast, prostacyclin reversed both AA and ADP-induced calcium redistribution and shape change. In the latter experiments, a net calcium sequestration was actually observed on prostacyclin addition. These findings indicate that the resequestration of released calcium leads to platelet deactivation. Furthermore, there appear to be at least two mechanisms by which a reduction in cytosolic calcium can be produced: specific interruption of the agonist-receptor interaction, for example, 13-APA antagonism of TXA2/PGH2; and stimulation of platelet adenosine 3',5'-cyclic monophosphate production by prostacyclin and consequent calcium sequestration.

scholarly journals Interaction of Human Platelets with the 2′, 3′-Dialdehyde Derivatives of ADP (o-ADP) and ATP (o-ATP) as Potential Affinity Labels for the ADP Receptor

1977 ◽  
Author(s):  
P. H. Pearce ◽  
M. C. Scrutton
Keyword(s):  
Time Course ◽  
Shape Change ◽  
Periodate Oxidation ◽  
Human Platelets ◽  
Affinity Labels ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Derivatives Of ◽  
Platelet Shape ◽  
Stable Incorporation

In order to characterise the receptor responsible for mediating platelet aggregation and secretion induced by ADP we have synthesised oADP and oATP by periodate oxidation of ADP and ATP. oADP induces platelet shape change but inhibits aggregation competitively with respect to ADP. The properties of inhibition of aggregation induced by adrenaline, collagen or thrombin are also consistent with the conclusion that the effects of oADP result from interaction with the ADP receptor(s). The effects of oATP generally resemble those of oADP except that oATP inhibits, rather than induces, shape change and causes this effect at a concentration in excess of that required for inhibition of aggregation. ,Brief exposure of intact platelets to [3H]-oADP or oATP causes stable incorporation of [3H] into the membrane proteins. Analysis of [3H] distribution by SDS gel electrophoresis after prolonged dialysis against SDS/mercaptoethanol shows predominant incorporation of [3H]-OATP into a fraction at 28000 dal tons over a concentration range consistent with that required for inhibition of aggregation. When [3H]-OADP is used, incorporation occurs predominantly into 4 fractions at 28000, 45000 (probably actin), 65000 and 220000 daltons. The [3H] incorporation/ [oADP] relationship and the time course of incorporation suggest that the 28000 and 65000 dalton fractions are likely candidates for identification as ADP receptors. Our data suggest a tentative identification of the 28000 dalton fraction as the receptor responsible for mediating aggregation induced by ADP and also indicate the possibility that different receptors may be involved in induction of shape change and aggregation by this agonist.

Na+-H+ and Cl(-)-OH-(HCO3-) exchange in gastric glands

1986 ◽  
Vol 250 (4) ◽  
pp. G524-G534 ◽  
Author(s):  
A. M. Paradiso ◽  
P. A. Negulescu ◽  
T. E. Machen
Keyword(s):  
Time Course ◽  
Gland Cell ◽  
Ringer Solution ◽  
Disulfonic Acid ◽  
Proton Conductance ◽  
Gastric Glands ◽  
Similar Time ◽  
High K ◽  
K Concentration ◽  
Intracellular Buffering Capacity

The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to measure intracellular (pHi) and pH electrode to measure extracellular pH (pHo) in suspensions of gastric glands isolated from rabbit stomachs. The fluorescence of BCECF-loaded glands was calibrated in terms of pHi by equilibrating pHo and pHi using ionophores or digitonin and titrating pHo to different values. An APPENDIX is included that covers details of dye calibration and interpretation of fluorescence signals. Glands incubated in NaCl Ringer solution had pHi 7.11. Na+-free Ringer solution caused pHi to decrease reversibly to 6.80. Na+-dependent alkalinization of pHi followed a similar time course to the acidification of pHo. These changes were blocked by 1 mM amiloride. When gland cells were acidified (using two different techniques) realkalinization was completely Na+ dependent but was independent of the presence of Cl-; also, neither high extracellular K+ concentration ([K+]o) nor high [K+]o plus 10(-5) M valinomycin affected the rates of Na+-dependent alkalinization. A neutral Na+-H+ exchanger was implicated. Glands also exhibited Cl(-)-dependent changes of pHi that were blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2 X 10(-4) M). A Cl(-)-OH-(HCO3-) exchanger was indicated. Other studies showed that intracellular buffering capacity was approximately 45 mM (pH-1) and that the apparent proton conductance of gland cell membranes was small.

Thrombin-Induced Responses In Human Platelets Differ In Their Requirement For Receptor Occupancy; Evidence For Tight Coupling Between Occupancy And Formation Of Phos- Phatidic Acid

1981 ◽  
Author(s):  
Holm Holmsen ◽  
Carol A Dangelmaier ◽  
Holm-Kjetil Holmsen
Keyword(s):  
Phosphatidic Acid ◽  
Time Course ◽  
Shape Change ◽  
Receptor Occupancy ◽  
Acid Synthesis ◽  
Human Platelets ◽  
Dense Granule ◽  
Cyclic Process ◽  
Tight Coupling ◽  
Acid Hydrolases

Thrombin causes secretion of dense granule constituents from platelets. The time course and extent of this response is unaltered if thrombin is rapidly neutralized after secretion has started, indicating that a sustained occupancy of the thrombin receptors is not required.Using a 50-fold excess of hirudin to neutralize thrombin, we show here that the shape change and aggregation responses also are independent on sustained receptor occupancy. In contrast, the secretion of acid hydrolases, liberation of [3H-]arachidonate (from phospholipids in platelets prelabeled with [3H]arachidonate) and formation of [32P]phosphatidic acid (in platelets prelabeled with 32P-orthophosphate) induced by thrombin were immedately stopped by hirudin. However, the incorporation of 32P into phosphatidylinositol and breakdown of [3H]phospha- tidyl inositol continued unaffected after thrombin removal. Thrombin-induced platelet responses can thus be subdivided into two classes according to their requirement for receptor occupancy: The first class (shape change, aggregation, dense granule secretion phosphatidylinositol breakdown and phosphatidylinositol synthesis) requires a short, initial occupancy, while the second class (acid hydrolase secretion, arachidonate liberation and phospha- tidic acid synthesis) requires an occupancy that is sustained as long as the response is executed. The increased turnover of phosphoinositides is a cyclic process, and our results show that only one step - the formation of phosphatidic acid from diacylglyceride and ATP - is tightly coupled to receptor occupancy by the agonist.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

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