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T J Rink

Publications and source records attributed to T J Rink.

At least 73 records · Page 4Linked to original sources
PubMed1987

Stimulated calcium efflux from fura-2-loaded human platelets.

1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.

Anti-Bacterial Agents↗
PubMed1986

Free Ca2+ requirements of agonist-induced thromboxane A2 synthesis in human platelets.

In quin2-loaded human platelets ionomycin raised cytosolic free calcium to greater than 1 microM and generated less than 1 ng thromboxane. Collagen alone or in the presence of EPO92 generated up to 32 and 16 ng thromboxane respectively; in the latter case at calcium levels around 120 nM. Thrombin maximally raised calcium to greater than 1 microM and generated up to 27 ng thromboxane, although in the presence of 1 mM EGTA these calcium and thromboxane levels were reduced to 200 nM and 5 ng respectively.

Anti-Bacterial Agents↗
PubMed1986

Thrombin and ionomycin can raise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using fura-2.

In the presence of 1 mM EGTA, the addition of the calcium ionophore ionomycin to human platelets loaded with 30 microM fura-2 could elevate [Ca2+]i from less than 100 nM to a maximum of greater than 3 microM, presumably by discharge of Ca2+ from internal stores. Under the same conditions thrombin could maximally increase [Ca2+]i to a peak of greater than 1 microM which then declined to near resting levels within 3-4 minutes; by contrast in platelets loaded with 1 mM quin2 thrombin could raise [Ca2+]i to only about 200 nM. In the presence of 1 mM Ca2+ the peak response to thrombin in fura-2-loaded platelets was higher (1.4 microM) than that observed in the presence of EGTA (1.1 microM) and the elevation in [Ca2+] was prolonged, presumably by Ca2+ influx. These results with fura-2-loaded platelets indicate that mobilisation of internal Ca2+ can contribute a substantial proportion of the early peak [Ca2+]i evoked by thrombin directly confirming the deductions from previous work with different loadings of quin2. Under natural conditions the major role of Ca2+ influx may be to prolong the [Ca2+]i rise rather than to make it larger.

Aminoquinolines↗
PubMed1986

Effects of ionic substitution on [Ca2+]i rises evoked by thrombin and PAF in human platelets.

We have investigated the effects of substituting extracellular Na+ by choline or K+ on responses of quin2- and fura-2-loaded human platelets to thrombin and platelet-activating factor (PAF). Na+ substitution by choline did not affect the extent of the rise in [Ca2+]i evoked by either agonist. The response to thrombin, but not PAF, was slightly slowed. High K+ did not activate the cells, but the rises in [Ca2+]i evoked by both agonists were slowed and reduced. Shape change evoked by both agonists was little affected by either substitution. Aggregation evoked by PAF was reduced in high K+ but unaffected in choline. Thrombin-induced aggregation was unaffected by either substitution, even when the rise in [Ca2+]i was markedly reduced. The results suggest that the mechanism which generates Ca2+ fluxes in platelets is not voltage-dependent; but high K+ appears to interfere with the influx mechanism.

Blood Platelets↗
PubMed1986

Low concentrations of the stable prostaglandin endoperoxide U44069 stimulate shape change in quin2-loaded platelets without a measurable increase in [Ca2+]i.

Dose-response relationships for raised cytoplasmic free calcium concentration, [Ca2+]i, and shape change were measured simultaneously in quin2-loaded human platelets. With the calcium ionophore ionomycin the threshold [Ca2+]i for shape change was 300 nM with a maximal response at 800 nM. With 1 mM external Ca2+ the U44069 concentrations required to stimulate half-maximal shape change and an increase in [Ca2+]i were 2 and 41 nM, respectively. For PAF these values were 8.7 and 164 pg/ml, respectively. Low concentrations of U44069 and PAF evoked substantial shape change without any rise in [Ca2+]i. In the absence of external Ca2+, U44069 stimulated half-maximal shape change at 2 nM, and half-maximal elevation of [Ca2+]i at 69 nM: here, increased [Ca2+]i never reached the threshold [Ca2+]i for shape-change derived with ionomycin. These results suggest that some transduction mechanism other than elevated [Ca2+]i, as yet unidentified, can cause shape change.

Aminoquinolines↗
PubMed1986

Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2-loaded human platelets.

The rapid (less than 1 sec) time course of [Ca2+]i rises was measured in fura-2-loaded, aspirin treated human platelets by stopped flow fluorimetry. With thrombin, which is known to cause substantial, rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, the mean delay before a detectible rise in [Ca2+]i in medium containing 1 mM Ca2+ o was 250 +/- 10 msec (S.E.M., n = 11). With ADP, which is reported not to stimulate phosphatidylinositol-4,5-bisphosphate hydrolysis, the delay under the same conditions was only 20 +/- 10 msec (S.E.M., n = 26). In the absence of external Ca2+, with 1 mM EGTA, the measured delays were 300 +/- 20 msec for thrombin and 210 +/- 10 msec for ADP. Times to peak were also faster for ADP than thrombin. These results suggest that thrombin and ADP promote Ca2+ influx in different ways. It is also possible that the process generating Ca2+ influx differs from that which triggers release from internal stores.

Adenosine Diphosphate↗
PubMed1986

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

Aminoquinolines↗
PubMed1986

Platelet activating factor raises intracellular calcium ion concentration in macrophages.

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.

Animals↗
PubMed1986

Haemodynamic responses to stimulation of the splanchnic and cardiac sympathetic nerves in the anaesthetized cat.

The changes in cardiac output and mean right atrial pressure (R.A.P.) evoked at different circulating blood volumes by stimulation of the splanchnic sympathetic nerves were investigated in adrenalectomized cats under chloralose anaesthesia, with unopened chests and spontaneous respiration and with active vascular reflexes. The cardiac autonomic nerves were cut or blocked pharmacologically. Stimulation of the distal ends of the splanchnic nerves at 4 Hz caused aortic pressure and R.A.P. to rise to maximum values at 2 min before declining slowly. Cardiac output rose more slowly to a steady state at 3 min; at higher circulating volumes it fell initially. Although the output increments were slower in development they were better sustained than those in total peripheral resistance. The proportionate output increments were largest and the R.A.P. increments least at low circulating volumes whereas at high volumes the R.A.P. increments were large but the output changes were small or negative; the pattern of changes resembled that resulting from infusion of blood. Stimulation of the cardiac sympathetic nerves evoked a rise in output and a fall in R.A.P. related in magnitude to the initial value of R.A.P. On simultaneous stimulation of the splanchnic and cardiac sympathetic nerves the changes in output combined whereas the R.A.P. changes cancelled, to give output increments of 25-50% with little change in R.A.P. at all circulating volumes. At high circulating volumes infusion of blood did not usually alter output or aortic pressure, but splanchnic nerve stimulation increased peripheral resistance and aortic pressure and commonly evoked a rise in left ventricular stroke work which could not be accounted for by known adrenergic mechanisms or by elevation of left ventricular end-diastolic pressure. Portal venous pressure was consistently elevated by splanchnic nerve stimulation; it rose more slowly than did aortic pressure or R.A.P. and was independent of a changing central venous pressure provided this did not exceed +5 mmHg. The cardiac output increments were not related to changes in the ratio between the input and output resistances of the portal vein and it is concluded that displacement blood from the peripheral to the central vasculature was induced by contraction capacitance vessels.

Adrenalectomy↗
PubMed1985

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.

Aminoquinolines↗
PubMed1985

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets.

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Agonists stimulate divalent cation channels in the plasma membrane of human platelets.

Agonists such as thrombin, PAF (platelet-activating factor) and ADP are known to cause a larger elevation in [Ca2+]i in quin2-loaded platelets in the presence of extracellular Ca2+ than in its absence. The simplest interpretation of these observations is that in the presence of extracellular calcium there is an influx component across the cell surface. In the presence of Mn2+, a divalent cation which is known to avidly bind to quin2 and to quench its fluorescence, the agonists produce a small initial rise in quin2 fluorescence followed by a decrease in fluorescence to well below the resting level. The result indicates entry of Mn2+, presumably through some form of receptor-operated Ca2+ channel.

Adenosine Diphosphate↗
PubMed1985

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths.

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.

Animals↗
PubMed1985

Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2.

ADP produces a rapid elevation in the concentration of cytoplasmic free calcium, [Ca2+]i, in quin2-loaded human platelets which begins within 1 s of stimulation and peaks after 10 s. In the presence of 1 mM-extracellular calcium, [Ca2+]i peaks at 670 +/- 50 nM in the absence and 610 +/- 30 nM in the presence of a cyclo-oxygenase inhibitor. The production of prostaglandin endoperoxides and thromboxane A2 are not required for stimulation of Ca2+ fluxes by ADP but appear to have a supportive role. In the absence of extracellular calcium ions and with 1 mM-extracellular EGTA, stimulation with ADP caused [Ca2+]i to peak at 160 +/- 20 nM in the absence and 150 +/- 10 nM in the presence of a cyclo-oxygenase inhibitor. ADP can cause the discharge of calcium ions from internal stores and does not require the prior formation of prostaglandin endoperoxides or thromboxane A2. The rise in [Ca2+]i in the presence of extracellular Ca2+ is sixfold larger than in the absence of extracellular Ca2+. This suggests that the major component of the ADP-stimulated rise in [Ca2+]i is caused by the influx of Ca2+ ions across the plasma membrane. Diltiazem, D600, nimodipine and nifedipine had little or no effect on resting or ADP-stimulated [Ca2+]i levels. Depolarization with potassium-rich media alone or in conjunction with valinomycin had no effect on basal [Ca2+]i and only a partial inhibitory effect on ADP-stimulated increases in [Ca2+]i. Depolarization had no effect on the ADP-stimulated rise in [Ca2+]i in Ca2+-free media. Hyperpolarization had no marked effect on the rise in [Ca2+]i produced by ADP in the presence of extracellular calcium. These results are consistent with there being no voltage-dependent channels in the platelet plasma membrane. Using ionomycin, a selective Ca2+ ionophore, and measuring both quin2 fluorescence and optical density of the suspension simultaneously, the threshold [Ca2+]i for shape change was determined to be 300 nM with half-maximal effect at 500 nM and maximal shape change at 800 nM. ADP produced maximal shape change confirmed by scanning electron microscopy with corresponding [Ca2+]i at below 200 nM. The level of [Ca2+]i required to produce aggregation using ionomycin was approximately 1 microM. ADP alone, or following a smaller rise in [Ca2+]i produced by ionomycin to disguise the effect of ADP, produced an aggregatory response at concentrations below 1 microM. These data indicate that excitatory mechanisms are involved producing shape change and aggregation to ADP other than a stimulated rise in [Ca2+]i.

Adenosine Diphosphate↗
PubMed1984

TMB-8 inhibits secretion evoked by phorbol ester at basal cytoplasmic free calcium in quin2-loaded platelets much more effectively than it inhibits thrombin-induced calcium mobilisation.

TMB-8 is widely regarded as an 'intracellular calcium antagonist', supposedly inhibiting the mobilisation of intracellular calcium. Rarely, however, have the effects of this compound on Ca2+ movements been measured. We report here that TMB-8 is not very effective in inhibiting thrombin-induced Ca2+ influx or internal release in human platelets judged from the fluorescent signal of cytoplasmic quin2. Only approx. 40% inhibition was seen at 500 microns TMB-8. Somewhat lower concentrations blocked the secretory response to thrombin and also the secretion evoked at basal [Ca2+]i by phorbol ester and collagen. It is suggested that one target for TMB-8 may be the C-kinase pathway.

Aminoquinolines↗