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T J Rink

Publications and source records attributed to T J Rink.

At least 109 records · Page 6Linked to original sources
PubMed1981

Free calcium ions in neurones of Helix aspersa measured with ion-selective micro-electrodes.

1. Intracellular free calcium concentration, [Ca2+]i, was measured in giant neurones of the sub-oesophageal ganglia of Helix aspersa, using Ca-selective micro-electrodes containing a PVC-gelled, neutral-ligand sensor. 2. In calibration solutions the electrodes had a virtually ideal, Nernstian, response down to 1 microM-Ca2+ in the presence of 0.125 M-K+, 18-24 mV from 1 to 0.1 microM-Ca2+ and 8-14 mV from 0.1 to 0.01 microM-Ca2+. Interference from H+ and Mg2+ was negligible. The small response to Na+ at sub-micromolar Ca2+ was taken into account, when necessary, in measurement of [Ca2+]i. 3. Measurements of basal [Ca2+]i were made in ganglia from animals kept only a few weeks in captivity, in a bathing solution equilibrated with air and containing 2 mM-Ca2+. In thirteen measurements from impalements which met stringent criteria for electrode performance and cell viability, the mean basal pCa (--log10[Ca2+]) was 6.77 +/- 0.07 (S.E.), corresponding to a mean free Ca2+ concentration of 0.17 microM. 4. The basal [Ca2+]i in neurones from a group of snails kept hibernating for several months was higher, mean pCa 6.15, for ganglia handled in 2 mM-Ca2+ solution. 5. Intracellular injections of Ca2+ or EGTA raised and lowered, respectively, the indicated basal [Ca2+]i, showing that the electrodes responded appropriately inside the cells and that unknown or untested components of cytoplasm were not significantly interfering with the Ca-sensor. 6. Altering the external Ca2+ concentration between 0.1 and 10 mM usually produced only small, +/- 0.1 pCa units, changes in basal [Ca2+]i of satisfactorily impaled, quiescent cells. 7. In cell 1F, which has repetitive spikes with a substantial Ca current, changes in Ca gradient or blockade of voltage-dependent Ca channels sometimes markedly altered [Ca2+]i, showing that Ca entry with the spikes was elevating [Ca2+]i. 8. Replacing external Na+ with Li+ or bis(2-hydroxyethyl)dimethylammonium had little effect on [Ca2+]i. 9. Elevating CO2 to 5% or 79% lowered [Ca2+]i by an average of 0.16 and 0.26 pCa units respectively.

Animals↗
PubMed1980

Free calcium in heart muscle at rest and during contraction measured with Ca2+ -sensitive microelectrodes.

Direct measurements of free Ca2+ in heart cells are needed for an understanding of the regulation of contractility. We developed and used Ca2+ -sensitive microelectrodes with fine tips, stable properties and ample sensitivity to free Ca2+ in the sub-micromolar range. In quiescent ventricular muscle, measurements which passed tests for electrode sealing and cell viability gave a mean free Ca2+ concentration of 0.26 microM. During contractures, we recorded Ca2+ transients rising as high as 10 microM. In studying the effects of catecholamines on free Ca2+ and force, we found evidence that adrenaline can reduce myofibrillar Ca2+ sensitivity in intact heart muscle.

Animals↗
PubMed1980

Free calcium in Xenopus embryos measured with ion-selective microelectrodes.

A regulatory role for intracellular free calcium has been suggested in cell division and intercellular communication via gap junctions. Previous measurements of cytoplasmic free Ca2+ during cell division and uncoupling have been made with the Ca2+-sensitive photoprotein aequorin. We now report experiments using Ca2+-sensitive microelectrodes to monitor free Ca2+ in the cells and intercellular fluid of early embryos of the amphibian Xenopus laevis. In addition to measuring basal levels, we have looked for changes in free Ca2+ during cell division and during electrical uncoupling of the normally coupled embryonic cells, induced by acidification of the intracellular medium.

Animals↗
PubMed1980

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium.

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca2+] levels. Pipettes of tip outer diameter down to 0.4 micron were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204-206) but with very hydrophobic cations replacing Na+ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca2+] levels. Lowering the Ca2+ concentration in the internal electrolyte also increased electrode lifetime. Electrodes showed a Nernstian response to [Ca2+] down to 1 micro M free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca2+. Selectivity for Ca2+ over Mg2+ and H+ was sufficiently high that typical free intracellular levels of Mg2+ and H+ caused negligible interference. Selectivity for Ca2+ over Na+ was adequate for cells with 10(-2) M free Na+, but higher levels could raise significantly the detection limit for Ca2+. Preliminary measurements of [free Ca2+] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca2+ microelectrodes and optical indicators are discussed.

Animals↗
PubMed1980

Lymphocyte membrane potential assessed with fluorescent probes.

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.

Animals↗
PubMed1980

Triggering of lymphocyte capping appears not to require changes in potential or ion fluxes across the plasma membrane.

Capping induced by anti-Ig antibody on mouse spleen lymphocytes was found to proceed normally over a wide range of membrane potentials from approx. 0 to -65 mV, as estimated with fluorescent probes. The potential was manipulated by ionic substitution in the medium and/or application of gramicidin. Various agents which inhibit capping had differing effects on the membrane potential, some producing no measurable change, others depolarising the cells. In particular valinomycin (10-7 M) was found to inhibit capping in cells both slightly hyperpolarised from the normal resting potential, and fully depolarised. Valinomycin was found to deplete the lymphocytes markedly of ATP and this effect was sufficient to account for the inhibition of capping. Capping occurred in a simplified (sucrose) medium lacking Na+, K+ and Ca2+, suggesting that fluxes across the plasma membrane of these ions are not required. It is concluded that after ligand binding, some reorganisation of receptor protein at the inner face of the membrane is the sufficient stimulus for the intracellular rearrangements involved in capping.

Animals↗
PubMed1980

Haemodynamic responses to stimulation of the cardiac autonomic nerves in the anaesthetized cat with closed chest.

1. The changes in cardiac output and mean right atrial pressure (R.A.P.) evoked by stimulation of the cardiac autonomic nerves were investigated in cats under chloralose anaesthesia, with unopened chests and spontaneous respiration, and with active vascular reflexes. Cardiac output was measured by thermal dilution; the technique used was calibrated against the direct Fick method.2. The initial values of R.A.P. and output were varied by infusion of dextran-saline solution followed by withdrawal of blood. At positive values of R.A.P. withdrawal of blood caused a fall in R.A.P. with no change in cardiac output. At negative R.A.P. blood withdrawal caused a fall in output with little change in R.A.P.: the linear regression coefficient for output on R.A.P. was 48.2 ml./min.kg.mmHg (S.E. 2.06, n = 63, nine cats).3. Stimulation of the right cardiac sympathetic nerve increased heart rate by 69.2 beats/min (S.E. 4.0) from the resting rate of 158 beats/min (S.E. 6.3, ten cats). The acceleration was accompanied in most instances by a rise in cardiac output and a fall in R.A.P. and the magnitude of the rise in output was related to that of the fall in R.A.P.4. In no experiment could R.A.P. be reduced below -2.5 mmHg either by withdrawal of blood or by sympathetic stimulation. At negative values of R.A.P. the fall in R.A.P. and rise in output evoked by sympathetic stimulation were small; substantial changes could be obtained only from positive initial values of R.A.P. The proportional increase in output evoked by a given proportional increase in heart rate during near-maximal sympathetic stimulation had a linear relationship to the initial value of R.A.P. over the range -2 to +8 mmHg. The output increment was less than proportional to the rate increment at all values of R.A.P. below +3 mmHg.5. In five experiments stimulation of the left cardiac sympathetic nerve evoked a greater increase in output for a given increase in heart rate than did stimulation of the right nerve; on the other hand both nerves gave similar increments of output for a given fall in R.A.P.6. Stimulation of the distal end of the right vagus nerve slowed the heart and caused a fall in cardiac output and a rise in R.A.P. The change in output associated with a given change in R.A.P. was significantly greater (P = 0.05) during sympathetic than during vagal stimulation in 14 out of 18 tests; the difference increased as circulating volume was reduced.7. It is concluded that the relationship between cardiac output and R.A.P. during sympathetic and vagal stimulation is consistent with the hypothesis that neurally evoked changes in cardiac performance vary output mainly, but not exclusively, by inducing changes in R.A.P. which alter the pressure gradient for the return of blood to the right atrium from the periphery.

Animals↗
PubMed1978

Osmotic behaviour of human red blood cells: an interpretation in terms of negative intracellular fluid pressure.

1. The observation that human red blood cells do not shrink in hypertonic media as much as expected for ideal osmometers has previously been explained in terms of either a marked increase in the osmotic coefficient of the cell contents or an increase in the chloride content of the cells.2. Changes in suspension pH and haematocrit have been observed when the concentration of the unbuffered NaCl medium was doubled. The small increases in external pH, and the size of the volume decreases, are inconsistent with variations in the Cl content as a significant factor in the non-ideal osmotic responses.3. Membrane potentials of red cells in buffered media were followed using the fluorescent dye, diS-C(3)-(5). On shrinking at pH 7.4, the cells hyperpolarized ca. 5 mV as predicted if changes in the osmotic coefficient rather than in Cl content explained the osmotic behaviour.4. Regarding haemoglobin in concentrated solution as a solute with high osmotic coefficient is formally correct but is little help in understanding the properties of the solution. We have found it useful to consider separately haemoglobin and the rest of the contents of the cell. The haemoglobin then supports part of the total hydrostatic pressure on the cell leaving the crystalloid solution to experience a reduced fluid pressure. In greatly shrunken cells the contents act like a gel with the matrix of haemoglobin under compression and the fluid which fills the spaces within the matrix under tension.

Erythrocytes↗
PubMed1977

The influence of sodium on calcium movements and catecholamine release in thin slices of bovine adrenal medulla.

1. 45Ca efflux and uptake, net Ca movements, and catecholamine secretion were studied in thin slices of bovine adrenal medulla. 2. There was a slow component of 45Ca wash-out which is attributed to efflux to intracellular Ca. This efflux was strongly influenced by cations in the external solution, being reduced by 50% when Mg replaced Ca, and the residual efflux being reduced by 80% when choline replaced Na. 3. None of the substitutes tested, choline, K, Li, Tris or sucrose, could replace Na in maintaining 45Ca efflux into Ca-free solution. The Na-dependent Ca efflux showed sigmoidal activation by Na, indicating a requirement for the co-operative action of two or more Na ions in the extrusion of each Ca. 4. In the presence of 3-6 mM-Ca, Na deprivation failed to reduce 45Ca efflux. When K, Li and choline replaced Na the rate constant of 45Ca efflux increased. 5. Ca-dependent 45Ca efflux was studied by adding back Ca to Ca-free solutions. Its activation by Ca was hyperbolic indicating that one external Ca is involved in the extrusion of each Ca, consistent with a Ca:Ca exchange process. The apparent affinity for Ca and the maximal efflux were different in the presence of different monovalent cations. 6. 45Ca uptake was increased when Li or choline replaced Na in a solution containing 3-6 mM-Ca. Net Ca uptake also increased, but to a much smaller extent, supporting the idea of a Ca:Ca exchange process. 7. Mg had little effect in activating or inhibiting 45Ca efflux. Co appeared to act as a weak agonist and weak inhibitor of Ca-dependent 45Ca efflux. Ba strongly activated 45Ca efflux.8. Elevation of [Na]t, with ouabain treatment, did not appreciably affect Na-dependent 45Ca efflux. This may indicate that while this Ca efflux is dependent on external Na, it is not dependent on the Na gradient. Elevation of [Na]i had rather little effect on 45Ca uptake. 9 Exposure of slices to Li or choline solutions evoked a Ca-dependent increase in catecholamine output. This could be attributed to the observed increase in net Ca uptake in these conditions. Sucrose and Tris solutions produced Ca-independent secretory responses with quite different time courses. These results emphasize the importance of the Na substitute used in determining the secretory effect of Na deprivation. 10. Elevation of [Na]i did not change basal catecholamine release, nor did it greatly affect the secretory response to Na deprivation. 11. Only some of the secretory effects of NA deprivation can be attributed to the influence of Na on Ca movements.

Adrenal Medulla↗
PubMed1977

Membrane potential of guinea-pig spermatozoa.

The membrane potential of guinea-pig spermatozoa was investigated by using the fluorescent dye, diS-C3-(5). The potential did not vary when K replaced Na in the suspending medium, and in a physiological salt solution the resting potential was found to be about +13 mV.

Animals↗
PubMed1976

Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential.

1. The mechanism by which the fluorescent, cationic dye diS-C3-(5) responds to the membrane potential of red blood cells has been investigated. 2. The dye in aqueous solution absorbs most strongly at 650 nm. Addition of white, haemoglobin-free membranes red shifts the absorption maximum ca. 20 nm, while addition of membrane-free cell lysate results in the appearance of a new dye absorption peak at 590 nm. Thus the dye binds both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialysable, presumably haemoglobin. 3. Dye added to a suspension of intact cells shows a strong absorption at 590 nm indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. 4. The amount of dye which partitions into (and on to) the cells can be determined, as reported by Sims, Waggoner, Wang & Hoffman (1974), from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. 5. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, ai/ao, is determined by the membrane potential according to the Nernst relation. Driving the membrane potenial negative then increases the cell associated dye by increasing the activity ratio and hence ai and the amount of dye bound to cell contents. 6. At the known Donnan equilibrium potential the internal dye activity can be calculated from the external activity. An empirical relation between cell associated dye and internal activity has been determined by measuring the dye partition between cells and medium at different external activities. 7. Using this empirial relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity can be estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. 8. The membrane potenial of cells has been altered by adding valinomycin in the presence of different K gradients. Under the conditions used, the 'constant field' permeability for K-Val is 15-20 times that of Cl. 9. Dye binding to haemoglobin is influenced by pH and thus dye partitioning into cells changes with intracellular pH. Increasing intracellular pH increases the amount of dye partitioned, while decreasing pH decreases this amount. 10. When large potentials are produced with valinomycin there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and the Cl concentration ratio and not by the membrane potentials. 11. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes...

Chlorides↗