Rise In Ca2 Research Articles

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

Spatial and Temporal Resolution of Serotonin-Induced Changes in Intracellular Calcium in a Cultured Arterial Smooth Muscle Cell Line

Ca2+ transients (1-2 microM) evoked by serotonin (5-HT) in cultured A7r5 cells were studied using fura-2 and digital imaging microscopy. Fura-2 was introduced into cells either by incubation with its acetoxymethyl ester analogue fura-2/AM or by transient ATP-induced permeabilization of the sarcolemma such that the free fura-2 entered the cell directly. The distribution of cytoplasmic Ca2+ in unstimulated cells loaded by the former method was heterogeneous, reflecting, in part, separate pools of Ca2+ in the cytosol and sarcoplasmic reticulum (SR). In contrast, the distribution of Ca2+ was uniform in cells loaded with fura-2 by transient permeabilization; this reflected the restriction of fura-2 to the cytosol. Average Ca2+ in these cells was substantially lower than that in fura-2/AM-loaded cells, because SR Ca2+ influences the fura-2 signal from fura-2/AM-loaded cells, but not from cells loaded with free fura-2. The differences in the Ca2+ distribution measured by the two loading methods were also evident during the course of 5-HT-evoked Ca2+ transients. Spatial and temporal resolution of the rising phase of 5-HT-evoked Ca2+ transients in fura-2/AM-loaded cells revealed that the onset of the Ca2+ transients was first manifested as small regions of elevated Ca2+ that subsequently expanded until peak apparent intracellular Ca2+ levels were present in virtually all of the nonnuclear regions of the cells. The rate of rise of Ca2+ varied in different cell regions with the nucleus responding the slowest.

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylase a and Labile Metabolites During Anoxia: Correlation to Membrane Fluxes of K+ and Ca2+

The objective of the present study was to explore mechanisms responsible for activation of ion conductances in the initial phases of brain ischemia, particularly for the early release of K+ that precedes massive cell depolarization, and rapid downhill fluxes of K+, Na+, Cl-, and Ca2+. As it has been speculated that a K+ conductance can be activated either by an increase in the free cytosolic calcium concentration (Ca2+i) or by a fall in ATP concentration, the question arises whether the early increase in extracellular K+ concentration (K+e) is preceded by a rise in Ca2+i and/or a fall in ATP content. In the present experiments, ischemia was induced in rats by cardiac arrest, the time courses of the rise in K+e and cellular depolarization were determined by microelectrodes, and the tissue was frozen in situ through the exposed dura for measurements of levels of labile metabolites. including adenine nucleotides and cyclic AMP (cAMP), after ischemic periods of 15, 30, 60, and 120 s. Conversion of phosphorylase b to a was assessed, because it depends, among other things, on changes in Ca2+i. The K+e value rose within a few seconds following induction of ischemia, but massive depolarization (which is accompanied by influx of calcium) did not occur until after approximately 65 s. Activation of phosphorylase was observed already after 15 s and before glycogenolysis had begun. At that time, 3',5'-cAMP concentrations were unchanged, and total 5'-AMP concentrations were only moderately increased. The results demonstrate that a K+ conductance is activated at a time when the overall ATP concentration remains at 95% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of intracellular calcium by ATP in isolated outer hair cells of the guinea‐pig cochlea.

1. Intracellular calcium levels were monitored in isolated outer hair cells of the guinea-pig cochlea using the calcium-sensitive dye Fura-2. 2. The calcium in the cells was studied during application of ATP externally applied from a pipette. ATP induced a rise of intracellular calcium which could be separated into two components: a rapid rise, peaking in 20 s, localized around the apical end of the cell, and a slower rise, peaking in 50-150 s but spread throughout the cell. The effects were observed with 5, 25 and 100 microM-ATP concentrations. 3. In the absence of external Ca2+, ATP was still able to trigger a rise in Ca2+, but with a longer delay. Under these conditions, the cells did not show the initial rapid Ca2+ rise. The result suggests that ATP can mobilize intracellular stores. 4. A rise in intracellular Ca2+ was also observed when 5 mM-caffeine was applied to the bath. 5. Simultaneous measurements were made of whole-cell currents and intracellular calcium. ATP activated an inward current at resting potentials of -60 mV. Internal Ca2+ levels increased during the inward current. In current-clamped cells Ca2+ levels also increased during the associated depolarization produced by ATP. 6. Adenosine (150 microM) did not produce any measurable inward current. Acetylcholine (ACh, 100 microM-1 mM) produced only a small rise in Ca2+. However, applied simultaneously with ATP, ACh suppressed the rise in intracellular Ca2+ produced by ATP, with the kinetics of a competitive antagonist. 7. Intracellular Ca2+ increased with step depolarizations of the cell above -20 mV during whole-cell clamp. Large rises in Ca2+ were also observed on depolarizing the cell with isotonic KCl. 8. Calcium levels in supporting cells of the organ of Corti were sensitive to ATP. In these cells, rises in intracellular Ca2+ did not require the presence of extracellular Ca2+. 9. It is concluded that the organ of Corti contains receptors for ATP on a variety of the cells. ATP controls a direct entry of Ca2+ through the membrane and also may mobilize intracellular stores.

Long-term depression but not potentiation is induced in Ca2+-chelated visual cortex neurons

An entry of Ca2+ into postsynaptic sites may play a role in the induction of long-term potentiation (LTP) of synaptic transmission in the visual cortex. To test this hypothesis, a Ca(2+)-chelator was injected into layer II/III neurons of sliced visual cortex obtained from young rats, and excitatory postsynaptic potentials (EPSPs) of these cells to test stimulation of the white matter were observed before and after tetanic stimulation of the same site. To confirm the effectiveness of the tetanus, field potentials reflecting the activities of many cells were recorded with another extracellular electrode. The chelator injection led to long-term depression (LTD) of EPSPs following tetanic stimuli which simultaneously induced LTP of field potentials derived from unchelated cells in most of the slices tested. This suggests that a low concentration of post-synaptic, free Ca2+, when associated with tetanic inputs, may lead to LTD while a rise of Ca2+ may lead to LTP.

Effects of low extracellular Ca2+ on cytosolic free Ca2+, Na+, and pH of MDCK cells

The effects of lowering the Ca2+ electrochemical potential (delta mu Ca2+) by removing extracellular calcium (Ca2+o) was studied in Madin-Darby canine kidney (MDCK) cells. We measured cytosolic free calcium (Ca2+i), intracellular Na+ (Na+i), and intracellular pH (pHi) as well as Ca2+ and Na+ influx and efflux and H+ secretion. As soon as Ca2+o was lowered to zero, Ca2+ efflux increased 2-fold, and Na+ influx increased 4.3-fold. As a result, Ca2+i fell 71% from 120 to 34.7 nM, and Na+i rose 56% from 16.7 to 26.1 mM. At the same time, H+ secretion was depressed 38%, and pHi fell from 7.32 to 7.22. When Ca2+o was restored to normal (1.3 mM), there was a sharp rise in Ca2+i from 34.7 to 243 nM (+600%) caused by an immediate increase in Ca2+ influx (+112%). At the same time, Na+ efflux increased 20%. Ten to fifteen minutes after restoring Ca2+o to 1.3 mM, Ca2+i, Na+i, and pHi were back to control levels. These results suggest that reducing delta mu Ca2+ activates the forward mode of Na(+)-Ca2+ exchange which leads to a fall in Ca2+i and a decreased delta mu Na+ by increasing Na+i. The fall in delta mu Na+ secondarily depresses Na(+)-H+ exchange which results in a reduced H+ secretion and a fall in pHi. When Ca2+o is restored to 1.3 mM, the reverse mode of Na(+)-Ca2+ exchange is activated, since delta mu Ca2+ is larger than normal because of the low Ca2+i, and delta mu Na+ is reduced because of the high Na+i.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for Involvement of Protein Kinase C and Cyclic Adenosine 3’,5’ Monophosphate-Dependent Protein Kinase in the 1,25-Dihydroxy-Vitamin D3-Mediated Rapid Stimulation of Intestinal Calcium Transport, (Tr anscaltachia)*

Vascularly perfused duodenal loops from normal vitamin D-replete chicks were used to obtain insight with regards to the possible mechanism(s) by which 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] rapidly stimulates intestinal Ca2+ transport (transcaltachia). The phorbol ester, 12-o-tetradecanoyl phorbol-13 acetate (TPA) (100 nM), and the adenylate cyclase activator, forskolin (10 microM), were found to stimulate Ca2+ transport from the lumen to the vascular effluent to the same extent that physiological levels of 1,25(OH)2D3 achieve. The effects of both substances exhibited concentration dependence. Similarly to 1,25(OH)2D3, addition of either TPA or forskolin to the lumenal compartment of normal chicks or vascular perfusion of duodena from vitamin D-deficient chicks failed to stimulate Ca2+ transport. Also and analogously to 1,25(OH)2-D3, TPA and forskolin-enhanced duodenal Ca2+ transport was abolished by the Ca2(+)-channel antagonists nifedipine (1 microM) and verapamil (30 microM). In addition, the protein kinase C inhibitor, staurosporine, totally abolished the rise in Ca2+ transport caused by 130 pM 1,25(OH)2D3. The synthetic peptide IP20, a well characterized cAMP-dependent protein kinase inhibitor, was also effective in suppressing 1,25(OH)2D3-dependent stimulation of duodenal Ca2+ transport. Collectively these results suggest that protein kinase C and cAMP-dependent protein kinase mediate 1,25(OH)2D3 activation of basal lateral membrane Ca2(+)-channels as an early effect in the transcaltachic response.

Calcium released by photolysis of DM‐nitrophen stimulates transmitter release at squid giant synapse.

1. Transmitter release at the squid giant synapse was stimulated by photolytic release of Ca2+ from the 'caged' Ca2+ compound DM-nitrophen (Kaplan & Ellis-Davies, 1988) inserted into presynaptic terminals. 2. Competing binding reactions cause the amount of Ca2+ released by DM-nitrophen photolysis to depend on the concentrations of DM-nitrophen, total Ca2+, Mg+, ATP and native cytoplasmic Ca2+ buffer. Measurements of presynaptic [Ca2+] changes by co-injection of the fluorescent indicator dye Fura-2 show that DM-nitrophen photolysis causes a transient rise in Ca2+ followed by decay within about 150 ms to an increased steady-state level. 3. Rapid photolysis of Ca2(+)-loaded nitrophen within the presynaptic terminal was followed in less than a millisecond by depolarization of the postsynaptic membrane. As with action potential-evoked excitatory postsynaptic potentials (EPSPs), the light-evoked response was partially and reversibly blocked by 1-3 mM-kainic acid which desensitizes postsynaptic glutamate receptors. 4. Release was similar in magnitude and rate to normal action potential-mediated EPSPs. 5. The release of transmitter by photolysis of Ca2(+)-loaded DM-nitrophen was not affected by removal of Ca2+ from the saline or addition of tetrodotoxin. Photolysis of DM-nitrophen injected into presynaptic terminals without added Ca2+ did not stimulate release of transmitter nor did it interfere with normal action potential-mediated release. 6. Stimulation of presynaptic action potentials in Ca2(+)-free saline during the light-evoked response did not elicit increased release of transmitter if the ganglion was bathed in Ca2(+)-free saline, i.e. in the absence of Ca2+ influx. Increasing the intensity of the light or stimulating presynaptic action potentials in Ca2(+)-containing saline increased the release of transmitter. Therefore the failure of presynaptic voltage change to increase transmitter release resulting from release of caged Ca2+ was not due to saturation or inhibition of the release mechanism by light-released Ca2+. 7. Decreasing the temperature of the preparation increased the delay to onset of the light-evoked response and reduced its amplitude and rate of rise to an extent similar to that observed for action potential-evoked EPSPs.

T‐Cell Activation. III. Attempts to Activate MHC Class I‐Negative and Class I‐Transfected EL4 T‐Lymphoma Cells by Immobilized Anti‐CD3 Antibody

The aim of this study was to examine whether the unresponsiveness of MHC class I-negative subclones of the EL4 thymoma to CD3 cross-linking can be restored by transfection of class I genes into the H-2-negative cells. Cell activation experiments with selected MHC class I-negative subclones and H-2b- and H-2Ld-positive transfectants showed that these cells are equally capable of secreting interleukin 2 (IL-2) after exposure to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and ionomycin. In contrast, only the parental H-2-positive EL4 cells are capable of responding to treatment with immobilized anti-CD3 antibody with IL-2 secretion and IL-2 receptor expression. Measurements of intracellular free Ca2+ (Ca2+i) following anti-CD3 antibody-induced cross-linking of parental EL4 cells and H-2-negative and H-2b gene-transfected subclones showed that the parental cells and two of the class I transfectants, one H-2-positive and one H-2-negative, responded with a slow rise in Ca2+i, whereas one H-2-positive transfected cell clone was completely refractory to CD3 cross-linking. Modulation experiments using parental EL4 cells, H-2-negative subclones and H-2-positive transfectants demonstrated that the CD3 and class I molecules of these different cells are modulated to the same extent after exposure to specific antibodies. The present findings thus indicate that the unresponsiveness of H-2-negative EL4 subclone cells to CD3 cross-linking is not functionally associated with a lack of class I surface expression.

Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones.

1. Impaired intracellular Ca2+ concentration ([Ca2+]i) regulation may underlie alterations in neuronal function during hypoxia or hypoglycaemia and may initiate cell damage. We have used the Ca2(+)-sensitive fluorophore, Fura-2, to study the regulation of [Ca2+]i in neurones isolated from mouse dorsal root ganglia. Mean resting [Ca2+]i was 163 +/- 11 nM (mean +/- S.E.M., n = 38). 2. Depolarization by exposure to 20 or 30 mM-K+ caused a rapid Co2(+)- and Cd2(+)-sensitive rise in [Ca2+]i, which subsequently declined with a time course usually fitted by the sum of two exponential functions. 3. Interference with mitochondrial function (by CN- or FCPP) or with glycolysis (by glucose removal) all raised [Ca2+]i by up to 220%. Addition of FCCP in the presence of CN- further increased [Ca2+]i. The response to CN- was still seen in the absence of extracellular Ca2+, although it attenuated rapidly, indicating release from an intracellular store. 4. Either CN- or glucose removal increased the rise in [Ca2+]i induced by K+ 2- to 3-fold and slowed recovery, suggesting interference with sequestration or extrusion of [Ca2+]i. 5. Resting [Ca2+]i rose when external Na+ was replaced by Li+ or N-methyl-D-glucamine, demonstrating the presence of a Na(+)-Ca2+ exchange process. However, Na+ replacement had only a slight effect on the handling of a Ca2+ load. 6. We conclude that (i) Ca2+ is released into the cytoplasm from intracellular organelles when energy supplies are reduced: (ii) that the extrusion or sequestration of Ca2+ entering the cell during electrical activity is rapidly impaired by interference with mitochondrial metabolism: and (iii) Na(+)-Ca2+ exchange makes only a small contribution to intracellular Ca2+ homeostasis. 7. [Ca2+]i would thus be expected to rise in vivo during hypoxia or hypoglycaemia and may initiate alterations in neuronal function. However, if a rise in Ca2+ is an important cause of cell damage in cerebral hypoxaemia, the combination of excitation and hypoxia will lead to the largest increases in [Ca2+]i, while hypoxia alone appears to cause only a small increase in [Ca2+]i in quiescent cells.

Convergence of cAMP and phosphoinositide pathways during rat parotid secretion

Rat parotid acinar cells were employed to investigate the mechanism by which receptor agonists that activate the phosphoinositide pathway enhance the stimulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) on amylase secretion. Norepinephrine (NE), which activates both alpha- and beta-adrenoceptors, evoked a secretory response that was greater than the sum of the responses obtained when NE was employed as a beta-agonist (in the presence of prazosin) and as an alpha-agonist (in the presence of propranolol). The enhancement of amylase secretion induced by NE was accompanied by an augmented rise in Ca2+ influx, as determined by fura-2 analysis. NE-induced cAMP production was comparable to that evoked by NE as a beta-agonist, and the accumulation of [3H]inositol 1,4,5-trisphosphate (IP3) evoked by NE was comparable to that elicited by NE as an alpha-agonist. The beta-adrenoceptor agonist isoproterenol potentiated the rise in cytosolic Ca2+ elicited by the muscarinic agonist carbachol, while possessing no stimulatory effect of its own. Isoproterenol had no effect on carbachol-induced stimulation of [3H]IP3 or 1,3,4,5-[3H]inositol tetrakisphosphate accumulation. Ionomycin and dibutyryl cAMP in combination produced a similar enhancing effect on the Ca2+ signal and amylase release as adrenergic and muscarinic receptor agonists. These results suggest that the synergism between the phosphoinositide and cAMP-signaling systems in parotid cells resides in enhanced Ca2+ availability.

Voltage‐gated and agonist‐mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp.

1. Intracellular free calcium (Ca2+i) was estimated in single GH3 cells by dual wavelength emission spectrofluorimetry using the Ca2+ indicator dye Indo-1, while cells were held under voltage clamp using patch clamp techniques. 2. Depolarization of cells evoked a transient rise in Ca2+i that increased with increasing duration of depolarization to a peak at about 10 s. 3. Calcium transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. They were abolished in the absence of extracellular calcium and by application of 10 microM-nifedipine. 4. Thyrotrophin-releasing hormone (TRH) evoked a transient rise in Ca2+i that was followed by a more sustained period of elevated Ca2+i in some cells. The transient phase of the response but not the sustained phase was seen in the absence of extracellular calcium. 5. Ca2+i transients evoked by depolarization were not affected by pre-release of internal Ca2+ stores with TRH. 6. The results demonstrate that voltage-gated Ca2+ entry and Ca2+ store release can each elevate cytoplasmic free calcium in GH3 cells and may both be important for stimulus-secretion coupling. Non-voltage-gated Ca2+ entry is not a major source of Ca2+ under these conditions.

Effect of Granulocyte-Macrophage Colony-Stimulating Factor on Superoxide Production in Cytoplasts and Intact Human Neutrophils: Role of Protein Kinase and G-Proteins

Granulocyte-macrophage colony-stimulating factor, GM-CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet-Leu-Phe and platelet-activating factor, PAF, but not by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan. The potentiation is greatest in fMet-Leu-Phe-stimulated cells. This indicates that the actions of only certain receptors are potentiated by GM-CSF. Incubation of the cells with the protein kinase inhibitor H-7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM-CSF does not affect the observed potentiation. The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM-CSF. The data suggest that neither protein kinase C nor protein synthesis is necessary for GM-CSF action. On the other hand, no potentiation can be seen in the presence of cytochalasin B. Unlike intact cells, GM-CSF does not enhance superoxide production by cytoplasts stimulated with fMet-Leu-Phe. The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM-CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM-CSF. The amount of actin associated with the cytoskeleton under control of fMet-Leu-Phe-stimulated condition is the same in normal and GM-CSF-treated human neutrophils. Botulinum D toxin ADP-ribosylates a protein with a molecular weight of 22 kDa. This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM-CSF. Botulinum D toxin does not affect the basal or the fMet-Leu-Phe-induced rise in the intracellular concentration of free calcium in human neutrophils. GM-CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet-Leu-Phe. The increases are inhibited by pertussis toxin. Several important conclusion can be drawn from these data. 1) GM-CSF potentiates the rise in Ca2+i produced by PAF and fMet-Leu-Phe, and these potentiations are inhibited in pertussis-toxin-treated cells. 2) GM-CSF does not prime cytoplasts to stimulation by fMet-Leu-Phe. This suggests that the granules and/or nucleus are necessary for the priming action. 3) The priming by GM-CSF is not mediated by the H-7-sensitive protein kinase C, botulinum D-sensitive G-protein, or protein synthesis.

Effects of activation on distribution of Ca2+ in single arterial smooth muscle cells. Determination with fura-2 digital imaging microscopy.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in mammalian vascular smooth muscle. We used the fluorescent calcium indicator fura-2 and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in arterial myocytes and the changes elicited by activation with norepinephrine (NE). Viable arterial myocytes were obtained from bovine tail arteries by enzymatic digestion. In modified Krebs' solution containing 1.8 mM Ca2+, these myocytes were relaxed and spindle-shaped. The cells contracted rapidly when exposed to NE or high-K+ solution ejected from a micropipette; they relaxed slowly when the activator was washed away. NE evoked a rise in Ca2+ concentration ([Ca2+]) in the cells within 100 msec, at a time when the cells had not yet begun to contract. Maximal [Ca2+] levels were attained within 600 msec, at which time the cells were substantially contracted. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed prior to activation, with an average resting level of 111 +/- 14 nM (n = 6). During NE-evoked contractions, intracellular [Ca2+] increased, and the distribution of [Ca2+] became much more heterogeneous. On recovery from activation, the cells relaxed, usually attaining less than 90% of their original resting length. In contrast to the relatively uniform Ca2+ distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. The large spatial variation of [Ca2+] after cell activation implies that Ca2+ was sequestered at localized sites in the cell during relaxation.

Calcium Fluxes, Calcium Antagonists, and Calcium-Related Pathology in Brain Ischemia, Hypoglycemia, and Spreading Depression: A Unifying Hypothesis

It has been commonly assumed that calcium, which normally serves important functions as a membrane stabilizer, metabolic regulator, and sec­ ond messenger, also can mediate anoxic and toxic cell death (Schanne et aI., 1979; Farber, 1981; Trump et aI., 1981). It is then postulated that when the plasma membrane becomes unduly permeable to calcium, the free cytosolic concentration (Ca2+) rises to toxic levels. As applied to the brain, this hypothesis predicts that loss of cellular calcium ho­ meostasis underlies selective neuronal vulnerability in ischemia, hypoglycemia, and epileptic seizures (Siesj6, 1981; Meldrum, 1983; Raichle, 1983; for further literature, see Siesj6 and Wieloch, 1985; Siesj6, 1988). It should be clearly understood that ischemia, particularly if dense, causes all cells to loose their calcium homeostasis. The hypothesis predicts, therefore, that some cells are more vulner­ able than others because they have a higher density of calcium channels in their plasma membranes. Presumably, this could lead to untolerable local in­ creases in calcium concentration. In a recent extension of the calcium hypothesis, it was speculated that increased calcium cycling across ischemia-damaged membranes leads to a sustained rise in Ca2 + j and slow calcium overload of mitochondria, thereby causing delayed neuronal death (Deshpande et aI., 1987; see also Martins et aI., 1988). Dux et aI. (1987), inducing transient isch­ emia in the gerbil, recently assessed the time course of mitochondrial calcium deposits in glia cells and in pyramidal cells of the hippocampus CAl sector and

Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo‐1 AM

The present report describes the increase in cytosolic-free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo-1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F-Met-Leu-Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide-angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin-1 alpha (IL-1 alpha), granulocyte-colony stimulating factor (G-CSF), nor granulocyte-macrophage-colony stimulating factor (GM-CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.

The consequences of simulated ischaemia on intracellular Ca2+ and tension in isolated ferret ventricular muscle.

1. In order to study cellular events occurring in ischaemia, we have developed a method for simulating ischaemia in an isolated papillary muscle. Muscles were suspended in a chamber and changed from conventional superfusion with Tyrode solution to gas perfusion with 95% N2/5% CO2 (N2 gas perfusion), thus simultaneously stopping oxygenation and flow. Surface cells of the preparation were injected with the photoprotein aequorin in order to monitor intracellular free calcium concentration [( Ca2+]i). 2. Gas perfusion with 95% O2/5% CO2 (O2 gas perfusion) had little effect on the tension or Ca2+ transients. Superfusion with Tyrode solution equilibrated with 95% N2/5% CO2 (N2 Tyrode) caused tension to decline to 30-40% of control, but had little effect on the amplitude of the Ca2+ transients. N2 gas perfusion caused tension to fall more rapidly and to a lower level than superfusion with N2 Tyrode. Ca2+ transients showed a small initial decline followed by a slowly developing increase in magnitude and duration. 3. Long exposures to N2 gas perfusion caused tension to decline to very low levels and Ca2+ transients to increase to a maximum. After a variable length of time, resting tension began to increase. At approximately the same time, Ca2+ transients began to decrease and eventually disappeared. Resting Ca2+ increased during N2 gas perfusion and remained elevated when the Ca2+ transients had declined. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with O2 gas. 4. N2 gas perfusion caused a depolarization of the resting potential and an abbreviation of the action potential. In a long exposure the action potential eventually failed. These changes could be reversed by restarting superfusion with standard Tyrode or by perfusion with O2 gas. 5. Many of the effects of N2 gas perfusion could be mimicked by the addition of 20 mM-lactic acid to the superfusing solution, which caused a profound reduction of tension and also an increase in the amplitude and duration of the Ca2+ transients. Calculation of the changes in intracellular pH caused by the addition of lactic acid suggest that the fall in intracellular pH produced by lactic acid was similar to that occurring in ischaemia. 6. Repeated exposures to N2 gas perfusion caused tension to fall more rapidly and an increased resting tension to develop more rapidly. The slowly developing rise in Ca2+ transients was abolished and a rise in resting Ca2+ occurred more quickly. 7. When muscles were quiescent, exposure to N2 gas perfusion caused an increase in resting light.(ABSTRACT TRUNCATED AT 400 WORDS)

Chymotryptic-type protease inhibitors block the increase in Ca2+ and Il-2 production in activated Jurkat T cells.

Several chymotryptic-type protease inhibitors were found to inhibit both anti-CD3 mAb- and PHA-induced rise in Ca2+ and IL-2 production in Jurkat T cells. The magnitude of inhibition was a function of the effectors used to stimulate Ca2+ entry and depended on the concentration of the inhibitors. Neither tryptic-type protease inhibitors nor an elastase substrate prevented anti-CD3 mAb- or PHA-induced Ca2+ rise in Jurkat cells. The inhibitory effect of N-alpha-p-tosyl-L-phenylalanine chloromethyl-ketone on anti-CD3 mAb- and PHA-induced rise in Ca2+ resulted from a rapid increase in Ca2+ efflux. The inhibitors which were effective on Ca2+ mobilization also inhibited IL-2 production initiated by an anti-CD3 mAb in the presence of 12-O-tetradecanoylphorbol-13-acetate, and to a lesser extent by PHA or the calcium ionophore A23187. No inhibition of IL-2 production was observed when tryptic-type protease inhibitors or the elastase inhibitor were used. In addition, membrane preparations from Jurkat cells were found to hydrolyze the chymotryptic substrate Suc-Ala-Ala-Phe-paranitroaniline, an effect markedly inhibited by N-alpha-p-tosyl-L-phenylalanine chloromethylketone. Moreover, this inhibitor protected one potential endogenous substrate (Mr 38 kDa) from proteolysis. Taken together, these observations show that chymotryptic-type protease inhibitors block the responses generated by the binding of anti-CD3 mAb to Jurkat cells, and suggest that a chymotryptic-like membrane protease contributes to T cell activation.

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sarcoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1 mM EGTA, resulted in a phasic increase in myofibrillar free Ca2+ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca2+ response was higher than the free Ca2+ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca2+ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca2+ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.