Cytoplasmic Calcium Ion Concentration Research Articles

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin.

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin.

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.

Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes.

In the stressed animal, the vasoactive hormones vasopressin and angiotensin-II and the neurotransmitter noradrenaline induce liver cells to release glucose from glycogen. The intracellular signal that links the cell-surface receptors for noradrenaline (alpha 1) and vasoactive peptides to activation of glycogenolysis is known to be a rise in the cytoplasmic concentration of free calcium ions (free Ca). The receptors for these agonists induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a minor plasmalemma lipid, to produce inositol trisphosphate and diacylglycerol. Inositol trisphosphate has been shown to mobilize intracellular calcium in hepatocytes. We show here, by means of aequorin measurements in single, isolated rat hepatocytes, that the free Ca response to these agonists consists of a series of transients. Each transient rose within 3 s to a peak free Ca of at least 600 nM and had a duration of approximately 7 s. The transients were repeated at intervals of 0.3-4 min, depending on agonist concentration. Between transients, free Ca returned to the resting level of approximately 200 nM. Clearly, the mechanisms controlling free Ca in hepatocytes are more complex than hitherto suspected.

Calcium and the cytoskeleton.

The shape of eukaryotic cells in maintained by a structure known as the cytoskeleton, comprising three different type of cytoplasmic filaments: microtubules, microfilaments (actin-containing filaments) and intermediate filaments. The cytoskeleton in therefore expected to be a traget for intracellular second messengers that promote changes in cell shape. These include elevation of the cytoplasmic concentration of calcium ions. Most of the known cytoskeletal receptors for calcium are associated with the actin filament system and these may be important in regulating many types of cell motility, including locomotion, phagocytosis and secretion.

Concurrent measurement of platelet ionized calcium concentration and aggregation: Studies with the Lumiaggregometer

Platelet aggregation and aequorin-indicated cytoplasmic ionized calcium concentration ([Ca i 2+]) were measured simultaneously using a modification of a commercially available instrument, the Chronolog Whole Blood LumiaggregometerR. The [Ca i 2+] rise in response to arachidonic acid, the Ca 2+-ionophore A23187, collagen, and epinephrine occurred either before or simultaneously with platelet shape change or aggregation. PGD 2 caused concentration-dependent inhibition of both the [Cai 2+] rise and aggregation. However, suppression of the [Ca 2+] rise alone did not fully account for the inhibition of aggregation due to the elevated cAMP produced by PGD2. Agonist-induced elevation of [Ca 2+] did not depend on platelet aggregation, but was enhanced by close platelet-to-platelet contact. Concurrent measurement of platelet aggregation and [Ca 2+] may further clarify pathways to platelet activation.

The effects of some non-steroidal anti-inflammatory agents on membrane-associated calcium in rabbit peritoneal neutrophils

Several non-steroidal anti-inflammatory agents (NSAIAs) (flufenamate, flurbiprofen, indomethacin, phenylbutazone, piroxicam and salicylate), one anti-inflammatory steroid (hydrocortisone) and three compounds known to affect cellular calcium metabolism [nifedipine, calcium ionophore A23187, 8-(diethylamino)octyl 3,4,5,-trimethoxy benzoate hydrochloride (TMB-8)] were tested for their effects on membrane-associated calcium in rabbit peritoneal neutrophils using the fluorescent probe chlortetracycline (CTC). The NSAIAs reduced the level of fluorescence attained by incubating neutrophils with CTC, and caused an immediate reduction in the fluorescence of CTC-loaded neutrophils, both effects being concentration-related. Nifedipine, A23187 and TMB-8 also reduced the fluorescence of CTC-loaded cells. However, these measured reductions in fluorescence were due in whole or in part to a drug-induced drop in the autofluorescence of the neutrophils. After applying a correction factor which took account of this effect there was still an immediate concentration-related reduction in CTC-dependent fluorescence after the addition of all the NSAIAs. A23187 also caused a reduction in CTC fluorescence, but nifedipine, TMB-8 and hydrocortisone were inactive. Flufenamate, indomethacin and piroxicam reduced the drop in fluorescence brought about by N-formyl-methionyl-leucyl-phenylalanine (FMLP). These findings suggest that NSAIAs and A23187 displace membrane-associated calcium. Electron microscopical findings confirm that indomethacin and salicylate both affect membrane-associated calcium. Using the fluorescent probe Quin 2, an attempt was made to determine whether or not the displacement of membrane-associated calcium by NSAIAs was accompanied by changes in cytoplasmic calcium ion concentration. This was unsuccessful, however, since the effects of the NSAIAs on the fluorescence of Quin 2-loaded neutrophils was accounted for almost entirely by their effects on the autofluorescence of the cells.

Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores.

Antibodies against the T3-antigen receptor complex can activate the human T cell line, Jurkat, to produce interleukin 2 (2-5). This activation is initiated by a receptor-mediated increase in the concentration of free cytoplasmic calcium ions [Ca2+]i (3, 4). In this communication, we investigate the mechanism by which the receptor complex increases [Ca2+ )i in Jurkat cells. The initial receptor-mediated change in [Ca2+]i can occur when extracellular Ca2+ is depleted by EGTA. Perturbation of the T cell antigen receptor, therefore, generates a signal which mobilizes Ca2+ from intracellular stores. As inositol trisphosphate appears to function as such a signal for certain hormone receptors, we measured the levels of inositol trisphosphate and of the other inositol phosphate compounds in Jurkat. Antibodies to either the antigen receptor heterodimer or T3 determinants result in marked elevations of all three inositol phosphates. These changes in inositol phosphates are not secondary to the receptor-mediated increases in [Ca2+]i as demonstrated by the inability of the Ca2+ ionophore, ionomycin, to affect the levels of any of these compounds. In concentrations between 0.1 and 1 microM, purified inositol trisphosphate releases Ca2+ from permeabilized Jurkat cells. Taken together, these data indicate that, during activation, perturbation of the T3-antigen receptor complex generates inositol trisphosphate. This compound functions as an intracellular signal to release Ca2+ from intracellular stores, leading to increases in [Ca2+]i.

Stimulation of the Na/H exchanger of sea urchin eggs by phorbol ester.

On fertilization of a sea urchin egg, marked changes occur in the cytoplasmic concentration of calcium and hydrogen ions. These ionic signals represent the necessary and sufficient stimuli for the increased metabolism, protein synthesis and DNA synthesis that constitute egg activation. Cytoplasmic alkalinization, the major immediate cause of the increased rate of protein synthesis which occurs at fertilization, arises because the sperm-induced intracellular calcium transient activates a coupled flux of sodium ions and hydrogen ions across the oolemma. The experiments reported here suggest that the second messenger which links the activation of the Na/H exchange to the calcium transient may be a substance which stimulates protein kinase C8, as 12-O-tetradecanoyl phorbol acetate (TPA), a known activator of protein kinase C9, appears to stimulate protein synthesis by turning on the Na/H exchanger and causing a cytoplasmic alkalinization. Our data indicate that one consequence of treating other tissues with TPA, a tumour promoter, may be an increase in intracellular pH.

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2.

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.

Calcium ions, drug action and the heart—with special reference to calcium antagonist drugs

Calcium antagonists, of which the best known are verapamil, nifedipine and diltiazem, are a powerful group of cardioactive agents with a clinical spectrum of indications rather similar to those of beta-adrenoceptor blockade, including angina of effort angina at rest, hypertension and supraventricular tachycardias (nifedipine is ineffective for the latter). In angina caused by coronary spasm, calcium antagonists are preferred to beta-blockade. Calcium antagonists have a basically different mode of action from beta-adrenoceptor blockade, although both ultimately act on the free cytoplasmic calcium ion concentration. Critical differences between the calcium antagonists are dependent on the individual properties of the calcium antagonists concerned. Different binding sites on the sarcolemma have been identified for nifedipine-like agents and verapamil, but with a different interaction with the nifedipine site. None of these sites might be relevant to the binding of calcium antagonists to the tissue of their therapeutic site of action (arterial smooth muscle for all; atrioventricular node for verapamil and diltiazem). As a group, calcium antagonists cause vascular dilation and do not cause bronchial constriction, in contrast to the beta-adrenoceptor blocking agents. In many patients, these diverse properties allow safe combination of calcium antagonists and beta-dadrenoceptor blockers if due care is observed, especially in the case of nifefipine. The clinical differences between the effects of various calcium antagonists reflect: (i) the greater vasodilator capacity of nifedipine, so that at a given concentration the afterload effect dominates over possible effects on the nodal or myocardial tissue; (ii) the greater inhibition of vagal tone by nifedipine than by verapamil or diltiazem; and (iii) the greater inhibition of the atrioventricular node by verapamil and diltiazem. In angina of effort, calcium antagonists are now becoming the agents of first choice in some centers. Experimental use of calcium antagonists include the possible prevention of ventricular fibrillation, the inhibition of ischemic injury, the prevention of catecholamine mediated injury to the nyocardium and decreased arterial calcinosis.

Injection of guanosine and adenosine nucleotides into Limulus ventral photoreceptor cells.

1. Several nucleotide and nucleotide analogues had striking effects when pressure-injected into Limulus ventral photoreceptor cells. The poorly hydrolysable GTP analogues guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS), guanylyl imidodiphosphate (Gpp[NH]p) and guanylyl (beta, gamma methylene) diphosphonate (Gpp[CH(2)]p) produced large increases in the frequency of ;discrete events' that were recorded from photoreceptors in darkness. This effect was only observed after the injected cell was exposed to light. Injection of the ATP analogue ATPgammaS had effects similar to those of the GTP analogues.2. We conclude that GTPgammaS, Gpp[NH]p, Gpp[CH(2)]p and ATPgammaS act at a common site to cause a light-dependent, long-term activation of the excitation mechanism of the photoreceptor.3. Injection of GTP or GDP at pH 4.8 was followed by a smooth, transient depolarization that was observed neither when GTP at pH 7.5 was injected nor when ATP, 5'GMP or 2-[N-morpholino] ethane sulphonic acid (MES) were injected at pH 4.8. The reversal potential of the current induced by GTP injection was significantly more positive than the reversal potential of the light-induced current.4. We conclude that GTP injection induces changes of membrane conductance either in addition to, or different from, the light-induced change of membrane conductance.5. Injection of the ATP analogue adenylyl imidodiphosphate (App[NH]p), and the pyrophosphate analogue imidodiphosphate (p[NH]p) produced a drastic decrease in the sensitivity of photoreceptors to light. This decrease in sensitivity was partially reversed when the concentration of calcium ions in the bathing medium was reduced.6. We suggest that App[NH]p and p[NH]p injections act by increasing the cytoplasmic concentration of calcium ions.

A model for the mechanism of tip extension in pollen tubes

Three main mechanisms are proposed to account for the tip growth of pollen tubes. (1) The tip region is supported against the internal osmotic pressure of the cell by a fibrillar network, composed mainly of microfilaments, that is stabilized by calcium ions. Tip extension is promoted by a lowering of the local cytoplasmic calcium ion concentration, through uptake by the mitochondria and/or endoplasmic reticulum, which leads to a weakening of the fibrillar network. (2) Vesicles, derived from dictyosomes in the main body of the tube, fuse with the apical plasma membrane, providing new membrane and further carbohydrate for the wall. The rate of fusion is proportional to the rate of diffusion of calcium ion across the plasma membrane at the tip. (3) The callose lining present in the pollen tube wall, except at the tip, renders the wall impermeable and restricts entry of calcium ions to the apical plasma membrane. This restriction limits the rate of vesicle fusion, and tube growth, to the tip.This model is discussed in the light of previous observations on the growth and structure of pollen tubes under normal and experimental conditions.

Alveolar sacs of Tetrahymena: ultrastructural characteristics and similarities to subsurface cisterns of muscle and nerve.

ABSTRACT The ultrastructure of the layer of alveolar sacs of Tetrahymena thermophila and its relationship with the plasma membrane were examined in thin sections and freeze-fracture replicas. In thin sections, the plasma membrane and outer alveolar membrane are seen to parallel each other closely and are linked by struts 9 nm wide spaced at 9 nm intervals. The plasma and alveolar membranes have different ultrastructural characteristics. The unit-membrane structure of the plasma membrane is asymmetric and thicker than that of the alveolar membrane. The unit-membrane structure of the alveolar membrane is symmetric. The lateral borders of adjacent alveolar sacs are closely apposed and flattened against one another, forming alveolar sutures. The pellicle at the cell surface, consisting of the plasma membrane, the alveolar sacs and the underlying epiplasm, overlies a single layer of mitochondria and a single layer of flattened cisterns of rough-surfaced endoplasmic reticulum (RER). Examination of freeze-fracture replicas reveals that the plasma membrane and the inner and outer alveolar membranes each have a unique population of unit-membrane particles. This suggests that they are separate membrane domains. The appearance of alveolar sutures, i.e. the apposed lateral borders of adjacent alveolar sacs, suggests that the alveolar membranes in this region are more rigid than elsewhere and may be sealed together by an adhesive substance. Interruptions in the sutures are interpreted as indicating that adjacent alveolar sacs are connected by small isthmuses or channels, confirming earlier reports that alveolar sacs are large chambers within a continuous, interconnected system. Alveolar sacs, in their relationship with the plasma membrane, closely resemble the subsurface cisterns of nerve and muscle cells. In muscle cells, the cisterns participate in the regulation of the cytoplasmic calcium ion concentration that controls contraction of myofilaments. Several functions of the surface of Tetrahymena, e.g. secretion of mucocysts and ciliary motility, are believed to be calcium-dependent, and the alveolar sacs may play a role in mechanisms that control the concentration of this divalent cation in localized regions along the cell surface. The alveolar sacs contain laminated inclusion bodies that appear to be rich in phospholipid. The inclusion bodies may represent reserves of phospholipid material stored in preparation for membrane formation.

Residual Calcium Ions Depress Activation of Calcium-Dependent Current

Calcium ions enter and accumulate during depolarization of some cells, activating a potassium current, IK(Ca), that depends on the cytoplasmic concentration of calcium ions, [Ca]i. However, elevation of [Ca]i can depress IK(Ca) elicited by a subsequent membrane depolarization. The depression of IK(Ca) is ascribed here to a [Ca]i-mediated inactivation of the voltage-gated calcium conductance, which causes a net reduction in calcium ions available for the activation of IK(Ca). This suggests that other processes dependent on gated calcium entry may also be depressed by small background elevations in cytosolic free calcium ions.

The role of microvesicles in buffering [Ca2+]i in the neurohypophysis.

It is well established that hormone release from neurosecretory nerve terminals is triggered by an increase of the cytoplasmic ionized calcium concentration which occurs after the arrival of action potentials1. For the neurosecretory nerve terminals to recover their basal Ca2+ concentration, the calcium which has entered during depolarization must be extruded and/or accumulated in subcellular organelles or bound to some cytoplasmic proteins. Neurosecretory nerve endings are characterized by the presence of hormone-containing granules and microvesicles (earlier referred to as ‘synaptoid’ vesicles). The role of these microvesicles has been the subject of debate and has recently been reviewed2. One of the most attractive hypotheses has been that these organelles represent the retrieved membrane of the granule after exocytosis. However, on stimulation of hormone release, the number of microvesicles does not increase, suggesting that they do not participate in the endocytotic process3. Using stereology as a quantifying method, we recently demonstrated a redistribution of microvesicles towards the release site during potassium-stimulated hormone release3. It is thus possible that microvesicles in neurosecretory nerve terminals might function as calcium accumulating organelles as suggested for the coated vesicles isolated from the cerebral cortex4,5. We report here that a microvesicle-containing microsomal fraction isolated from neurosecretosomes (isolated neurosecretory nerve terminals) possesses an ATP-dependent calcium transporting system. This system enables the fraction to accumulate calcium ions when the external ionized calcium concentration is <1 µM, which is in the physiological range.

Platelet aggregation and the release reaction induced by ionophores for divalent cations

A comparison is made of the effects of ionophores X-537A and A 23187 on platelets. In the presence of calcium, the release reaction promoted by A 23187 is enhanced; the platelets take up calcium and they clump. By contrast, X-537A induces a release reaction independent of external calcium and does not mediate calcium-uptake by platelets or induce a platelet aggregation. The effect of X-537A on platelets may not result from its property as a calcium-ionophore. The release reaction induced by A 23187 is most likely mediated by an increase in the concentration of cytoplasmic calcium ions.