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Biomedical subjects

T J Rink

Publications and source records attributed to T J Rink.

At least 91 records · Page 5Linked to original sources
PubMed1984

Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol.

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.

Blood Platelets↗
PubMed1984

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

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.

Aminoquinolines↗
PubMed1984

Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

The role of changes in cytoplasmic free calcium, [Ca2+]i, in the responses to platelet-activating factor (PAF) was studied in human platelets loaded with the fluorescent calcium indicator, quin2. In the presence of 1 mM external calcium, PAF raised [Ca2+]i 8-10-fold in a few seconds to peak near 1 microM. [Ca2+]i then declined over several minutes towards the basal level. In the absence of external calcium there was a much smaller increase in [Ca2+]i of similar pattern. These findings suggest that PAF increases [Ca2+]i partly by discharge of internal Ca2+, but mainly by stimulated influx. Blockade of cyclo-oxygenase with aspirin only slightly reduced the [Ca2+]i changes, indicating that thromboxane A2 is not a major mediator of the calcium movements. In control conditions PAF could stimulate shape-change, aggregation and secretion. Aggregation and secretion were roughly halved by blockade of cyclo-oxygenase. Shape-change and secretion still occurred under conditions where the [Ca2+]i rise was small or suppressed, indicating a role for intracellular activators other than Ca2+. The possible involvement of products of phosphoinositide breakdown is discussed.

Aminoquinolines↗
PubMed1984

Intracellular free magnesium in neurones of Helix aspersa measured with ion-selective micro-electrodes.

Cytoplasmic free Mg2+ concentration, [Mg2+]i, was measured in identified neuronal cell bodies of the suboesophageal ganglia of Helix aspersa, using Mg2+-selective micro-electrodes. In calibration solutions, the electrodes showed significant interference from K+, but not from Na+, or Ca2+, at concentrations found intracellularly. Therefore, in order to calibrate the electrodes properly, it was necessary first to obtain an accurate value for intracellular free K+ concentration [( K+]i). The mean value for [K+]i was 91 mM (S.E. of the mean +/- 2.2 mM, n = 8), measured with K+-sensitive 'liquid ion exchanger micro-electrodes'. In seven experiments, which met stringent criteria for satisfactory impalement and electrode calibration, the mean [Mg2+]i was 0.66 mM (S.E. of the mean +/- 0.05 mM). The mean [Mg2+]i in cells that had spontaneous spike activity was not significantly different from that in quiescent cells. If Mg2+ was in electrochemical equilibrium, the ratio [Mg2+]i/[Mg2+]o would be about 55. Mg2+ is therefore not passively distributed across the neuronal membrane and an outwardly directed extrusion mechanism must exist to keep [Mg2+]i low and constant, even in cells undergoing spike activity.

Action Potentials↗
PubMed1984

Aspects of the regulation of cell volume.

It is generally thought that osmoregulation is aimed not at osmotic pressure, or extracellular Na concentration per se, but at the regulation of cell volume. Tacit assumptions often made in considering body fluid distribution include the following: cell membranes support no significant hydrostatic pressure gradient; cell membranes are much more permeable to water than to the major solutes; therefore intracellular and extracellular osmotic pressure are equal; cell solute content is stable and not related to cell volume or osmotic pressure; accordingly cell volume varies inversely with osmotic pressure. In fact, counter examples are known to each of these and the one generalisation that seems to hold is that there are no water pumps and that water rapidly attains thermodynamic equilibrium between interstitial and intracellular fluid. Three topics will be discussed that illustrate some of these points. Even in the simplest model cells are not expected to shrink in direct proportion to increasing osmotic pressure, since they contain 20 to 30% solids. However, after allowing for this, cells still shrink less than predicted. One way of interpreting this retention of water is in terms of an increasing negative intracellular fluid pressure that results from the gelled nature of cytoplasm (Hladky and Rink, 1978). Many cell types respond to osmotic swelling or shrinking by subsequent adjustment of their solute content to return the volume towards normal. Human lymphocytes can do this remarkably quickly, via large increases in permeability to K and Cl. The mechanisms that produce these changes have possible implications for the functioning of osmoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase.

Trifluoperazine, chlorpromazine and other drugs known to inhibit calmodulin-dependent processes are also known to inhibit protein kinase C. The effect of these agents on secretion evoked by known activators of C-kinase has been studied in human platelets loaded with the fluorescent Ca indicator, quin2 and preincubated with aspirin. The secretory response stimulated by phorbol ester and exogenous diacylglycerol, at basal levels of cytoplasmic free Ca2+, [Ca2+]i, was suppressed by trifluoperazine, chlorpromazine and W-7, as was the secretion evoked by collagen that occurs without a change in [Ca2+]i. The response to thrombin, which is accompanied by elevated [Ca2+]i was barely affected. Modest elevation of [Ca2+]i by Ca ionophore was able to overcome the inhibitory effect of these drugs on the response to phorbol ester, diacylglycerol, and collagen.

Blood Platelets↗
PubMed1983

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium.

A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of beta-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B2 is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A2 aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI2 inhibits platelet aggregation by ADP and thrombin in a normal dose-related plasma, and PGI2 inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and beta-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Ca2+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.

Adolescent↗
PubMed1982

Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion.

Cytoplasmic free [Ca2+], [Ca2+]i, was measured in human platelets using the intracellularly-trapped, fluorescent indicator quin2. Basal [Ca2+]i with the Ca2+ -ionophore ionomycin revealed apparent thresholds for shape-change, 5-HT release and aggregation of approx. 0.5 microM, 0.8 microM and 2 microM. Thrombin raised [Ca2+]i to 3 microM fast enough for the Ca2+ to have triggered the cell activation. However, thrombin released more 5-HT than ionomycin could, and in Ca2+ -free medium thrombin evoked shape-change and secretion even when [Ca2+]i remained near basal levels throughout, suggesting the existence of alternative triggers for shape-change and secretory exocytosis.

Blood Platelets↗
PubMed1982

The role of platelet membrane potential in the initiation of platelet aggregation.

The membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the noncovalent, 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+-perm-selective 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.

Benzothiazoles↗
PubMed1982

Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.

A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

Aminoquinolines↗
PubMed1982

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Ig) on the cytoplasmic free calcium concentration, [Ca2+]i, measured with the fluorescent indicator quin2, and the relationship of [Ca2+]i to the capping of surface Ig. Anti-Ig causes a rapid rise of [Ca2+], which precedes capping. Assuming that only those 40-50% of the cells which can bind anti-Ig (the B cells) undergo a [Ca2+]i response, [Ca2+]i in those cells approaches 500 nM. It declines to resting levels over many minutes, roughly paralleling the formation of caps. Part of the [Ca2+]i signal is due to stimulated influx across the plasma membrane, since in Ca2+-free medium, anti-Ig gives a smaller and shorter [Ca2+]i rise. The amplitude of this reduced transient now varies inversely with quin2 content, as if some 0.25 mmol Ca per liter of cells was released into the cytoplasm from internal stores. These stores are probably sequestered in organelles since A23187 in Ca2+-free medium also causes a transient [Ca2+]i rise after which anti-Ig has no effect. These organelles seem not to be mitochondria because uncouplers have hardly any effect on [Ca2+]i. Though anti-Ig normally raises [Ca2+]i before causing capping, there seems to be no causal link between the two events. Cells in Ca2+-free medium whose stores have been emptied by A23187, still cap with anti-Ig even though there is no [Ca2+]i rise. Cells loaded with quin2 in the absence of external Ca2+ still cap anti-Ig normally even though their [Ca2+]i remains steady at below 30 nM, four times lower than normal resting [Ca2+]i.

Adenosine Triphosphate↗
PubMed1982

Cytoplasmic pH and free Mg2+ in lymphocytes.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.

Animals↗
PubMed1981

Ca2+-selective electrodes: a novel PVC-gelled neutral carrier mixture compared with other currently available sensors.

The great importance of cytoplasmic free Ca2+ in regulating cellular function has stimulated the development of several more-or-less calcium-selective sensor systems for microelectrodes. The known formulations each have serious deficiencies, particularly in fine microelectrodes. We have developed a new sensor which gives distinctly improved performance in submicron tips. This sensor combines the widely used neutral carrier ETH1001 (Ammann et al., 1979) with an intensely hydrophobic salt into a poly(vinyl-chloride) gel. This study compares the calibration responses of the various sensor systems under a uniform set of experimental conditions.

Animals↗