Ionic regulation of human basophil releasability. I. Inhibitory effect of copper

The effects of copper (CuSO₄ and CuCl₂) onin vitro histamine release from human basophils stimulated by anti-IgE and Ca²⁺ ionophore A23187 were evaluated. Both CuSO₄ and CuCl₂ caused a dose-related inhibition of histamine release, which was more pronounced on anti-IgE-than on Ca²⁺ ionophore-induced histamine release. The concentration which produced 50% inhibition of anti-IgE-induced histamine release was 1.3 M for CuSO₄ and 1.5 M for CuCl₂; the maximal inhibition of Ca²⁺ ionophore-induced histamine release was 33% for CuCl₂ (4 M) and 51% for CuSO₄ (16 M). The inhibitory effect on anti-IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before stimulation, whereas no inhibition of Ca²⁺ ionophore-induced histamine release was found when extracellular Cu²⁺ was removed. The activity of Cu²⁺ was independent of any effects of deuterium oxide and colchicine, two agents known to interact with microtubules. Increased extracellular Ca²⁺ concentrations reduced the inhibitory effect of CuCl₂ on Ca²⁺ ionophore-induced histamine release, and Schild plot analysis demonstrated that Cu²⁺ ions are competitive antagonists of Ca²⁺ ions.These results indicate that Cu²⁺ ions in the micromolar range down-regulate anti-IgE- and Ca²⁺ ionophore-induced histamine release. Since Cu²⁺ concentration in human plasma is in the micromolar range (30 M with 10–30% of free Cu²⁺), it is conceivable that Cu²⁺ ions contribute to thein vivo regulation of histamine release from human basophils.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Effects of ATP and related compounds on the Ca-induced Ca release mechanism of theXenopus SR

The effects of ATP and related compounds on the Ca²⁺ release mechanism of the sarcoplasmic reticulum (SR) was studied by using skinned skeletal muscle fibers ofXenopus laevis. ATP evoked marked Ca²⁺ release at very low level of Mg²⁺. , -Methylene analogue of ATP was almost as effective as ATP, which suggests Ca²⁺ release evoked by ATP is elicited without ATP hydrolysis. ADP and AMP also evoked Ca²⁺ release from the SR, but the effect of them became gradually weaker than that of ATP as the number of phosphates decreased. CTP, UTP and ITP were less potent than ATP. Adenosine also evoked more effective Ca²⁺ release than inosine. The compounds with adenine base, therefore, seem to elicit more potent Ca²⁺ release than those which have the same number of phosphates but do not consist of adenine base. AMP and Ca²⁺ ion evoked Ca²⁺ release synergistically, and the Ca²⁺ release responses evoked by ATP and related compounds showed the same pharmacological characteristics as Ca-induced Ca release. So, these Ca²⁺ release responses are construed as the manifestation of the same mechanism as Ca-induced Ca release. Effective concentration range of ATP and the effect of pyrophosphate on Ca²⁺ release evoked by ATP suggest that neither the high affinity ATP catalytic site of (Ca²⁺+Mg²⁺) ATPase of the SR nor the low affinity ATP binding site, reported by Dupont (1977), is implicated in the enhancement of these Ca²⁺ release responses from the SR.     

Actions of lead on transmitter release at mouse motor nerve terminals

The actions of lead (Pb²⁺) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb²⁺ in a dose-related manner consistent with inhibition of Ca²⁺ entry into nerve terminals, with a half-maximal effect at 1.4 M (in 0.5 mM Ca²⁺ and 2 mM Mg²⁺). Pb²⁺ also inhibited the increased frequency of MEPPs (f _MEPP where MEPPs denotes miniature EPPs) produced by Ba²⁺ in the presence of raised K⁺, blocking the calculated Ba²⁺ entry half-maximally at 170 M. However, at concentrations of 50–200 nM, Pb²⁺ often increased f _MEPP in 20 mM K⁺ in the presence of Ca²⁺ and acted to promote the irreversible effect of lanthanum (La³⁺) to raise f _MEPP. In nominally Ca²⁺-free solution with 20 mM K⁺, brief (1 min) application of Pb²⁺ (20–320 M) caused rapid dose-dependent reversible rises in f _MEPP. With prolonged exposure to Pb²⁺,f _MEPP rose and then slowly declined; after removal of Pb²⁺, once f _MEPP had fallen to low levels, f _MEPP responded nearly normally to Ca²⁺ or ethanol, but not to Pb²⁺ itself. In 5 mM K⁺, 0 mM Ca²⁺ and varied [Pb²⁺] (where [ ] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in f _MEPP graded with [Pb²⁺] that persisted as a tail; results were consistent with growth f _MEPP with the 4th power of intracellular Pb²⁺ and removal of intracellular Pb²⁺ with a time constant of about 30 s. These results suggest that Pb²⁺ acts to block the entry of Ca²⁺ and Ba²⁺ into the terminal via voltage-gated Ca²⁺ channels through which Pb²⁺, at higher concentrations, also penetrates and then acts as an agonist at intracellular sites that govern transmitter release.     

The release of intracellular Ca2+ in lacrimal acinar cells by α-, β-adrenergic and muscarinic cholinergic stimulation: the roles of inositol trisphosphate and cyclic ADP-ribose

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the -adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1–4 phosphate groups, resulting in release of Ca²⁺ from intracellular stores. Stimulation with the -adrenergic agonist phenylephrine, however, causes a release of Ca²⁺ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca²⁺ rise was completely inhibited by 100 M ryanodine. Adrenaline (causing activation of both - and -adrenergic receptors) induces a Ca²⁺ release with inositol phosphate synthesis identical to that occuring in the -adrenergic response. Thus, the signalling pathway for -adrenergic stimulation occurs via a path different from that which releases Ca²⁺ via muscarinic cholinergic and -adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] cause release of Ca²⁺ from intracellular stores. The Ca²⁺ release evoked by cADP-ribose, but not by Ins(1,4,5)P ₃, was abolished by 100 M ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca²⁺ signalling. When the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca²⁺]_i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.     

Interactions of bradykinin,calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells

Rise in free cytosolic calcium concentrations [Ca²⁺]_i in response to bradykinin and guanosine 5-O-thiotriphosphate (GTPS) was related to the action of phospholipase A₂ (arachidonic acid release). At 900 M extracellular CaCl₂, bradykinin induced a typical Ca²⁺ movement consisting of an initial [Ca²⁺]_i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca²⁺ level at approximately 290 nM. As the extracellular CaCl₂ concentration was reduced to 100 M, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca²⁺ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase [Ca²⁺]_i and saponin treated cells exhibited a very similar pattern of Ca²⁺ mobilization in response to bradykinin. However, with saponin treatment, GTPS (100 M) increased [Ca²⁺]_i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in [Ca²⁺]_i was observed in cells stimulated with both 100 M GTPS and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTPS. Pertussis toxin (PTX) did not affect the bradykinin induced Ca²⁺ mobilization. However, as we showed previously [1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A₂ (PLA₂) activation and Ca²⁺ mobilization but likely by different G subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTPS stimulated release of arachidonic acid appears to be only partially dependent on [Ca²⁺]_i. For example, 10 M ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl₂ concentrations below 300 M while GTPS stimulated a greater release of arachidonic acid at 300 and 100 M CaCl₂ than at 900 M CaCl₂. However, at 100 M CaCl₂, ionomycin increased [Ca²⁺]_i to the same level as bradykinin or GTPS stimulated cells incubated in 900 M CaCl₂.In previously published experiments [1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in [Ca²⁺]_i and occurred in the absence of any added extracellular CaCl₂. TPA did not induce an increase in [Ca²⁺]_i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7.     

Histamine release from mast cell granules

Rat peritoneal mast cells were separated from other cells by differential centrifugation in concentrated serum albumin. Granules were isolated from these cells by ultrasonic disintegration and subsequent centrifugation. 60–80% of the granules had intact membranes and retained their histamine store in a physiological salt solution. Histamine was released from the granules with intact membranes by Ca²⁺, 10 mM, in the presence of phosphatidyl serine, 25–50 g/ml. The release was initiated in 15 sec and completed in 16 min. Other divalent cations in 10 mM concentration, viz. Mg²⁺, Ba²⁺, Sr²⁺, Ni²⁺ and Mn²⁺, also released histamine from the granules in the presence of phosphatidyl serine. When histamine release was induced by calcium and phosphatidyl serine the calcium uptake in the granules was remarkably increased. Ca²⁺ could thus displace histamine from the granule matrix. The possibility that calcium with phosphatidyl serine may change the granule membrane permeability is discussed.     

Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion

Zn²⁺ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca²⁺ independent; in fact, in the absence of extracellular Ca²⁺, the secretory effects of Zn²⁺ were enhanced. At low concentrations (3–10 M), Zn²⁺ enhanced the secretory responses to 10-s pulses of 100 M 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K⁺. In the presence of DMPP, secretion was increased 47% above controls and in high-K⁺ solutions, secretion increased 54% above control. These low concentrations of Zn²⁺ did not facilitate the whole-cell Ca²⁺ (I _Ca) or Ba²⁺ (I _Ba) currents in patch-clamped chromaffin cells. Higher Zn²⁺ concentrations inhibited the currents (IC₅₀ values, 346 M for I _Ca and 91 M for I _Ba) and blocked DMPP- and K⁺-evoked secretion (IC₅₀ values, 141 and 250 M, respectively). Zn²⁺ permeated the Ca²⁺ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba²⁺, Ca²⁺, Sr²⁺ and Zn²⁺ were 991, 734, 330 and 7.4 pA, respectively. Zn²⁺ entry was also evidenced using the fluorescent Ca²⁺ probe fura-2. This was possible because Zn²⁺ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca²⁺, i.e. 360 nm. There was a slow resting entry of Zn²⁺ which was accelerated by stimulation with DMPP or high-K⁺ solution. The entry of Zn²⁺ was concentration dependent, slightly antagonized by 1 mM Ca²⁺ and completely blocked by 5 mM Ni²⁺. The entry of Ca²⁺ evoked by depolarization with high-K⁺ solution was antagonized by Zn²⁺. We conclude that inhibition by Zn²⁺ of evoked catecholamine secretion is associated with blockade of Ca²⁺ entry through Ca²⁺ channels recruited by DMPP or K⁺. However, the facilitation of secretion observed at low Zn²⁺ concentrations, or in the absence of Ca²⁺, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn²⁺ permeates the bovine chromaffin cell Ca²⁺ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn²⁺ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn²⁺ to fura-2 and the modification of its fluorescence spectrum.     

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on muscarinic receptor-induced Ca2+ mobilization in a human salivary epithelial cell line

We have investigated the effect of W-7, a calmodulin (CaM) antagonist, on Ca²⁺ mobilization in a human salivary epithelial cell line, HSG-PA, after muscarinic receptor stimulation. In a medium containing 1.5 mmol/l Ca²⁺, W-7 reduced both the maximum peak increase in cytosolic Ca²⁺ ([Ca²⁺]_i) which follows stimulation by carbachol (Cch, 100 mol/l) and the sustained nature of the response. Using an experimental approach which allows separate visualization of the intracellular Ca²⁺ release and extracellular Ca²⁺ entry phases, W-7 was shown preferentially to inhibit Ca²⁺ release. At 100 mol/l W-7, Cch-induced Ca²⁺ release was completely inhibited, but Cch-induced Ca²⁺ entry was partially (40%) maintained. This W-7 residual Ca²⁺ entry response was abolished when cells were depolarized with high K⁺ or gramicidin D. W-7 also substantially inhibited Cch-induced inositol trisphosphate (IP₃) production (75%). W-5, a less potent CaM antagonist than W-7, had markedly smaller effects on Cch-induced Ca²⁺ mobilization and IP₃ formation. W-7 (100 mol/ l) completely blocked (comparable to 10 mol/l atropine) the binding of the muscarinic antagonist [³H] quinuclidinyl benzilate (QNB) to muscarinic receptors on cell membranes, whereas Cch (at 100 mol/l) had minimal effects on ligand binding. W-7 and W-5 were equipotent in their ability to inhibit [³H] QNB binding. These results suggest that W-7 reduces Ca²⁺ mobilization in HSG-PA cells by a mechanism which likely involves the antagonism of a CaM regulatory step(s) but may also involve at least a partial blockade of the muscarinic receptor. In addition, in the presence of W-7, Ca²⁺ entry can occur via a receptor-operated Ca²⁺ pathway which is modulated by membrane potential.     

Intracellular citrate induces regenerative calcium release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum was studied in voltage-clamped guinea-pig atrial myocytes. Cells were dialysed with a pipette solution containing the Ca²⁺ indicator 1- [2-amino-5-(6-carboxyindol-2-yl) phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid](Indo-1, 100 M) and as main anion either chloride or the low-affinity Ca²⁺ buffer citrate. Intracellular Ca²⁺ transients (Ca_i transients) were elicited by depolarizations from a holding potential of –50 mV. In chloride-dialysed cells, Ca_i transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. In citratedialysed cells, membrane depolarizations were associated with a small rise in [Ca²⁺]_i. These small changes in [Ca²⁺]_i were either followed by a large Ca_i. transient or failed to induce large changes in [Ca²⁺]_i. The peak amplitude of the large Ca_i transient did not vary with the amplitude of the depolarizing pulse. These results demonstrate that in the presence of intracellular chloride, Ca²⁺ release in atrial cells is a graded process triggered by Ca²⁺ influx. Using citrate as the main intracellular anoin, Ca²⁺ release triggered by Ca²⁺ entry was no longer graded but occurred in a regenerative manner. The results are discussed in terms of two models in which citrate, affects the spatial distribution of [Ca²⁺]_i or the loading state of the sarcoplasmic reticulum.     

Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones

The afterhyperpolarization (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca²⁺-sensitive K⁺ conductances following Ca²⁺ influx via Ca²⁺ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05±3.76% of those evoked at 90-s stimulus intervals (n=35). Since there was no consistent effect of ryanodine (5 M), ruthenium red, (300 M) or dantrolene Na (35 M) on this frequency dependence, it is unlikely to result from release of Ca²⁺ from intracellular stores. Ca²⁺ currents (I _Ca, studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca²⁺-induced I _Ca inactivation and I _Ca run-down. There was excellent correlation (r=0.964) between the estimated changes in Ca²⁺ influx and the expected activation of the Ca²⁺-sensitive K⁺ current, I _AHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of I _Ca.     

Agonist-induced calcium flux,phosphoinositide metabolism,aggregation and enzyme secretion in human neutrophils

Formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and leukotriene B₄ (LTB₄) are potent activators of human neutrophils. Using human neutrophils prelabelled with the fluorescent indicator dye, Quin 2, or with [³²P]-orthophosphate, we examined the effects of these stimuli on intracellular free calcium concentration, [Ca²⁺]i, and, on various indices of phosphoinositide metabolism, including [³²P]-phosphatidic acid (PtdA) formation. The concentration-dependence of the observed changes in [Ca²⁺]i or [³²P]-PtdA were then compared to stimulus-induced aggregation and enzyme release (-N-acetylglucosaminidase (NAG) and lysozyme).FMLP, PAF and LTB₄ caused a concentration-dependent elevation of [Ca²⁺]i, aggregation and enzyme release. However, unlike FMLP and PAF, LTB₄ (2.5 M) did not cause significant formation of [³²P]-PtdA. The concentration response curves for agonist-induced elevation of [Ca²⁺]i lie to the left of those for aggregation and enzyme release. FMLP and PAF also caused an elevation of [Ca²⁺]i at concentrations lower than those required to elicit [³²P]-PtdA formation.These observations suggest that [Ca²⁺]i elevationper se cannot mediate human neutrophil functional responses to FMLP, PAF and LTB₄. Consequently there may exist other mediator(s) that act in concert with [Ca²⁺]i or are triggered by [Ca²⁺]i elevation to promote human neutrophil activation. Both the elevation of [Ca²⁺]i and the formation of these putative mediator(s) in response to LTB₄ apparently occur independently of inositol phospholipid hydrolysis.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

At low extracellular calcium concentration platelet activating factor induces beta-thromboglobulin release from human platelets through thromboxane-endoperoxides formation

Summary The aim of this study was to investigate the mechanism involved in -thromboglobulin (BTG) release induced by platelet activating factor (PAF) in human platelet-rich plasma (PRP) and washed platelets (WP) during aggregation. PAF was used in PRP at increasing concentrations starting at its threshold concentration for irreversible aggregation (TAC: 90–150 nM). In citrated PRP, PAF induced release of BTG (80–95% of total content) and thromboxane B₂ (TXB₂) formation (30–40 pmol/ml). At low PAF concentrations aggregation and BTG release were blocked by apyrase (a scavenger of ADP), by ASA (an inhibitor of cyclooxygenase) and by BM 13177 (a thromboxane receptor antagonist). Higher concentrations of PAF overcame the effect of apyrase, but only induced reversible aggregation and minor release in the presence of ASA or BM 13177. In heparinized PRP, PAF induced full irreversible aggregation, but only very low BTG release (about 25% of total content) and thromboxane synthesis (2–3 pmol/ml). WP resuspended in the presence of 2 mmol/l Ca²⁺ seldom responded to PAF alone, as previously shown by others, but full aggregation could be induced by concomitant addition of subthreshold concentrations of PAF (25–50 nM) and epinephrine (1 M). In these conditions average BTG release from WP was less than 20% of the amount releasable by thrombin. In contrast, when WP were resuspended in the absence of Ca²⁺, stimulation by PAF+EPI induced sustained BTG release (40–50% of total content) and TXB₂ synthesis (15–20 pmol/ml). We conclude that at low Ca²⁺ concentration PAF induces BTG release mainly through thromboxane-endoperoxides formation. In contrast, when [Ca²⁺] is normal, PAF does not or weakly induces thromboxane formation and BTG release.     

Effect of free sarcoplasmic Ca2+ concentration on maximum unloaded shortening velocity: measurements on single glycerinated rabbit psoas muscle fibres

Summary Isotonic shortening velocity in tension-controlled quick releases was studied at different sarcomere lengths, loads and Ca²⁺ concentrations using single glycerinated rabbit psoas muscle fibres. During a single quick release shortening velocity decreases with increasing shortening. Length-velocity relations, that is, momentary shortening velocity as a function of the corresponding sarcomere length, of a single quick release yield straight lines when graphed on a logarithmic scale. This indicates an exponential decrease of the isotonic shortening velocity during a quick release. A given shortening affects the subsequent part of the length-velocity relation in a manner which can be simulated by loading the fibre. The decrease in shortening velocity during a quick release could therefore be interpreted as the result of a shortening-induced increase in contractile system load. Thetotal amount of this additional load is proportional to the amount of shortening but is independent of the Ca²⁺ concentration. It follows that the change in loadper crossbridge due to a given shortening l, is inversely proportional to the degree of activation of the contractile system, provided that the number of cycling crossbridges during isotonic shortening is dependent on sarcoplasmic Ca²⁺ concentration as is known for the isometric state. These shortening-induced changes in crossbridge load should be considered in interpreting Ca²⁺ effects on shortening velocity. One approach may be extrapolation of the length-velocity relation to the starting length of the respective quick release, that is, the point at which all length-velocity relations of different Ca²⁺ concentrations converge. Thus, when an increase in crossbridge load due to shortening is taken into account, an effect of the free Ca²⁺ concentration on maximum unloaded shortening velocity cannot be proved.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

Recognition of the ‘calcium paradox’ and the effects of verapamil and gallopamil in human adenoidal mast cells

Human mast cells from adenoids show when resuspended in medium containing 10^–3 M CaCl₂ after their temporary exposure to Ca²⁺-free saline for about 20 min an irreversible reduction of responsiveness to a variety of stimuli: The histamine release induced by concanavalin A or ionophore A 23187 is only 30–50% of the one obtained in cells which were kept in 10^–3 M Ca²⁺ throughout the experiment. This phenomenon called calcium paradox can be almost entirely avoided if the cells are temporarily exposed to 10^–4 M Ca²⁺ instead of Ca²⁺-free saline. Number yields, average histamine contents of mast cells and the rate of the spontaneous histamine release are not affected by the transitory lack of Ca²⁺, nor is the histamine release enhancing effect of adenosine.At 10^–3 M Ca²⁺ concentration the calcium antagonists verapamil or gallopamil cause a significant inhibition of the Con A-induced histamine release only at concentrations much higher (10^–4 M) than those effective in smooth muscle preparations. The actions of both calcium antagonists were not affected by the presence of added extracellular adenosine.To whom requests for reprints should be addressed.