Changes In Intracellular Free Ca2 Research Articles

An improved method for the purification of basophilic granulocytes from human blood

Studies on human basophils are hampered by the low number of basophils in peripheral blood. Here we describe the purification of human basophilic granulocytes with immunomagnetic beads to improve an already established method of purification. A 70% pure basophil suspension was incubated with monoclonal antibodies (CD2, CD14, CD16 and CD19) recognizing the contaminating cells. After incubation with magnetic beads coated with goat anti-mouse IgG, the bead-cell rosettes were removed by a magnet. In this way, the basophil purity increased to 94%. The loss of basophils during the bead procedure was about 20%. The amount of histamine per basophil and the spontaneous release of histamine during subsequent incubation of the cells was not altered by the purification procedure. The kinetics and the dose response of histamine release after the addition of anti-IgE or FMLP was also unchanged. Binding of CD63 was not altered, indicating that the purification procedure did not result in activation of the basophils. This improved method for the purification of human basophils should permit the measurement of non-basophil-specific parameters, such as changes in intracellular free Ca 2+, without the problem of interference from contaminating cells.

The relationship of KCl‐ and prostaglandin F2α‐mediated increases in tension of the porcine coronary artery with changes in intracellular Ca2+measured with fura‐2

1. Changes in intracellular free Ca2+ concentration ([Ca2+]i) were measured simultaneously with changes in muscle tension by use of the fluorescent Ca2+ indicator, fura-2, in coronary arterial rings of the pig. 2. Changes in [Ca2+]i were measured by monitoring the ratio of fluorescence due to excitation at 340 nm (F340) to that at 380 nm (F380). 3. Increases in tension of the porcine coronary artery induced by prostaglandin F2 alpha (PGF2 alpha) (2-30 microM) and KCl (25-70 mM) were accompanied by increases in the F340/F380 fluorescence ratio of fura-2. 4. KCl-induced increases in muscle tension, equivalent to those produced by PGF2 alpha, were observed to occur in the presence of a higher [Ca2+]i. 5. Nearly complete relaxation of KCl-induced contractions by sodium nitroprusside (SNP) was accompanied by only a partial reversal of the increase in [Ca2+]i that occurred during contraction. 6. Complete relaxation of the PGF2 alpha-contracted coronary artery by cromakalim (BRL 34915) was accompanied by a nearly complete reversal of the increase in [Ca2+]i caused by the contractile agent. 7. The contractile state of smooth muscle is not an indicator of [Ca2+]i. The [Ca2+]i-tension relationship is dependent upon the type of pharmacological agent that is used to change muscle tension.

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.

Effect of extracellular ATP level on flow-induced Ca ++ response in cultured vascular endothelial cells

Cultured vascular endothelial cells loaded with the highly fluorescent Ca(++)-sensitive dye Fura-2 were exposed to the flow of a fluid containing various concentrations of ATP (0, 0.5, 1, 5 microM) in an apparatus designed on the basis of fluid dynamics, and simultaneous changes in intracellular free Ca++ concentration were monitored by photometric fluorescence microscopy. The flow rate of the perfusate was altered from 0 to 6.3 to 22.8 to 39.0 cm/sec, inducing shear stress on the cell surface of 0, 2.9, 10.4, and 17.9 dynes/cm2, respectively. Although no significant change in intracellular Ca++ level was observed at ATP levels below 100 nM, at an ATP level of 500 nM, the intracellular Ca++ level increased together with an increase in the flow rate of the perfusate. At this level of ATP, the intracellular Ca++ levels at flow rates of 0, 6.3, 22.8, and 39.0 cm/sec were 44.8 +/- 7.3, 60.3 +/- 10.7, 74.0 +/- 5.8 and 89.4 +/- 6.4 nM (mean +/- SD; n = 8), respectively. At ATP levels over 1 microM, the flow-rate dependency of Ca++ response became less clear than that observed at the ATP level of 500 nM. These Ca++ responses to changes in flow rate disappeared when extracellular Ca++ was chelated by adding 2 mM of EGTA to the perfusate. These results suggest that the vascular endothelial cell has a mechanism that elevates the intracellular Ca++ level in accord with the flow rate at appropriate ATP concentrations, and that changes in intracellular Ca++ level under this mechanism seem to be chiefly caused by the influx of extracellular Ca++ into cells.

Effects of vasoactive agonists on the membrane potential of cultured bovine aortic and guinea‐pig coronary endothelium.

1. The effects of bradykinin, ATP, adenosine, histamine and thrombin on the membrane potential of confluent monolayers of cultured bovine aortic endothelial cells (BAECs) and guinea-pig coronary endothelial cells (GCECs) were studied at 37 degrees C using the whole-cell mode of the patch-clamp technique. 2. The amplitude histogram of the resting potentials of BAEC monolayers showed a bimodal distribution with one peak around -25 mV and another peak around -85 mV. Transitions from one potential level to the other were observed. The bistable membrane potential can be explained by an N-shaped current-voltage relation of the endothelial cell membrane. 3. When BAECs with a low resting potential (-10 to -30 mV) were superfused with maximally effective concentrations of ATP (2-10 microM) an initial hyperpolarization of -80 to -90 mV was observed which decayed to a plateau of about -60 mV within 1 min. When ATP was removed after 2-3 min the membrane potential returned to control level within 1 min. This was followed by a second hyperpolarization of 10-20 mV, which decayed within 15 min. 4. In the absence of extracellular calcium, ATP produced only a brief transient hyperpolarization in aortic endothelium. The plateau and the secondary hyperpolarization were abolished. These findings are consistent with the idea that the changes in membrane potential reflect changes in intracellular free Ca2+ and that the initial peak is due to release of Ca2+ from intracellular stores, whereas the plateau and the secondary hyperpolarization depend on transmembrane Ca2+ influx. 5. Bradykinin evoked potential changes similar to ATP in BAECs, except that the secondary hyperpolarization during wash-out was absent. When the membrane potential was more negative than -80 mV, ATP and bradykinin induced only a small initial hyperpolarization followed by a depolarization of up to 20 mV. 6. In aortic endothelium, ADP (10 microM) evoked a much smaller response than ATP. Adenosine (10 microM), thrombin (2 units/ml), acetylcholine (10 microM) and histamine (10 microM) had only a very small effect on the membrane potential, if any. 7. The amplitude histogram of the membrane potential of GCECs showed only one peak around -35 mV. In coronary endothelium, application of bradykinin, ATP, histamine, thrombin, acetylcholine and adenosine all evoked a transient hyperpolarization of 10-40 mV lasting 1 min or less, which then turned into a depolarization. 8. The K+ channel openers cromakalim (BRL 34915) and lemakalim (BRL 38227) did not affect the membrane potential of GCECs or BAECs.(ABSTRACT TRUNCATED AT 400 WORDS)

Diphenylamine-2-carboxylate (DPC) reduces calcium influx in a mouse mandibular cell line (ST 885)

Non-selective cation channels are found in many diverse cell types and have been proposed as a potential entry path for Ca 2+. ST 885 cells contain large numbers of these channels which are active in the resting cell. We have used Fura-2 to monitor changes in intracellular free Ca 2+ ([Ca 2+] i) in response to step changes in extracellular Ca 2+ ([Ca 2+] o). We found that DPC, a blocker of the non-selective cation channel in these cells, caused a reduction of approximately 50% in the rate of rise in [Ca 2+] i following a step increase in [Ca 2+] o. Since experiments demonstrate that this phenomenon is not due to DPC blockade of Cl − channels, the Na + Ca 2+ exchanger or cyclooxygenase, we conclude that it is attributable to a direct effect of DPC on the non-selective cation channel. It thus appears that the non-selective cation channel is a significant pathway for basal Ca 2+ entry in these cells.

Presynaptic Mechanism of Action of 4‐Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B‐50 (GAP‐43) Phosphorylation

Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca2+. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca2+ concentration ([Ca2+]i) and B-50 phosphorylation in synaptosomes. 4-AP (100 microM) elevates the [Ca2+]i (as determined with fura-2) to approximately the same extent as depolarization with 30 mM K+ (from an initial resting level of 240 nM to approximately 480 nM after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K+ still evoked an increase in [Ca2+]i, which was additive to the elevation caused by 4-AP. Several Ca2+ channel antagonists (CdCl2, LaCl3, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca2+]i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na+ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca2+ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na+ channel activity.

Endothelin mediates Ca influx and release in porcine coronary smooth muscle cells

Endothelin (ET)-induced changes in intracellular free Ca (Cai) in freshly dispersed coronary artery smooth muscle cells were determined using fura-2 microfluorometry to differentiate the action of ET on Ca influx vs. release from internal stores. Comparison was made with caffeine (CAF)-induced Ca release from the sarcoplasmic reticulum (SR) to determine whether ET acts on the same Ca store. In physiological external solution, ET (5 x 10(-8) M) induced a rapid (within 90 s), transient (less than 2.5-min duration) 70% increase in Cai above baseline (n = 20). Pretreatment with diltiazem (10(-4) M; n = 10) did not change the amplitude or shape of the ET-induced Cai transient. In Ca-free solution, ET elicited a Cai response similar in duration but smaller (P less than 0.05) in peak magnitude (31% increase; n = 7). CAF (5 x 10(-3) M) also elicited a rapid (less than 60 s), transient 82% increase in Cai (n = 7). In the continued presence of CAF, ET caused no change in Cai. In contrast, ET elicited a transient 69% increase in Cai (n = 8), and in the continued presence of ET, CAF caused a 24% increase in Cai. Ryanodine (5 x 10(-5) M) suppressed the subsequent ET-induced Cai transient. These data on porcine cells suggest ET induces a rapid release of Ca from a CAF- and ryanodine-sensitive store and causes rapid influx of Ca, which is different from bovine smooth muscle cells. The return of Cai to baseline values in the continued presence of ET suggests the ET-sensitive store is depleted and increased Ca efflux matches Ca influx.

Inositol phosphate formation and [Ca2+]i in secretagogue-stimulated rabbit gastric mucous cells

The formation of inositol phosphates and the changes in free intracellular Ca2+ ([Ca2+]i) in isolated rabbit gastric mucous cells during cholinergic stimulation were examined and the potential role of inositol phosphate turnover and [Ca2+]i in gastric mucus secretion evaluated. Rabbit chief and parietal cells were studied for comparison. The formation of [3H]inositol phosphates in mucous, chief, and parietal cells was stimulated in a time- and concentration-dependent fashion by acetylcholine (ACh). The ACh-induced initial [Ca2+]i peak was maximally (10(-4) M ACh) 199 +/- 8% of basal in mucous cells, 427 +/- 20% in chief, and 455 +/- 31% in parietal cells and was followed by a lower-level plateau in mucous and parietal cells but by a more rapid decline in chief cells. As in parietal and chief cells, the initial [Ca2+]i peak occurred in mucous cells in the absence of external Ca2+. ACh stimulated a mucous cell membrane Ca2(+)-entry mechanism in addition to release of Ca2+ from intracellular stores. The concentration-response relationships for the production of [3H]-inositol phosphates, the initial rise in [Ca2+]i, and the stimulation of glycoprotein secretion by ACh were virtually identical. Suppression of the [Ca2+]i rise by the intracellular Ca2(+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the secretory response. As with many other secretory cells, gastric mucous cells possess cholinergic receptors that upon stimulation mediate the hydrolysis of phosphoinositides, a release of Ca2+ from intracellular stores, and a stimulation of Ca2+ influx through the plasma membrane.

The immunoglobulin light chain related protein λ5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transducing molecule

Abelson Leukemia Virus-transformed mouse cell lines with an early pre-B phenotype carry partially rearranged or unrearranged Ig-H genes and consequently do not express intact IgM-H protein (mu protein). Such early mu- pre-B cells express an intracellular protein complex of the pre-B cell specific 22 kDa protein lambda 5 and a 16 kDa protein designated p16. Late pre-B cell lines which carry a rearranged IgM-H chain gene in which a continuous translational reading frame has been established in the fused V-D-J element express intact mu-protein, which forms an intracellular complex with lambda 5 and p16. We show here that both the lambda 5/p16 or the mu/lambda 5/p16 complexes can be immunoprecipitated from lysates of cells surface labeled with 125I. Thus early pre-B cells express the lambda 5/p16 complex on the cell surface in the absence of mu protein, while mu+ late pre-B cells express a surface mu/lambda 5/p16 complex. To investigate a possible signal transduction function of the lambda 5/p16 and mu/lambda 5/p16 complexes on the surface of pre-B cell lines we measured the changes in intracellular free Ca2+ after treatment of cells with anti-lambda 5 or anti-mu antibodies. Two mu- early pre-B cell lines showed a rapid and transient increase in intracellular free Ca2+ when incubated with anti-lambda 5 antibodies but not when incubated with anti-mu, while the mu+ late pre-B cell line CB32 showed a rapid and transient increase in intracellular Ca2+ after incubation with anti-lambda 5 or anti-mu. These results show that both the lambda 5/p16 and the mu/lambda 5/p16 cell surface protein complexes can transduce an external signal to the inside of the cell, which implicates these complexes in the regulation of pre-B cell physiology.

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site

A novel modulatory site on the N-methyl- d-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca 2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.

Platelet-derived growth factor stimulates non-mitochondrial Ca2+ uptake and inhibits mitogen-induced Ca2+ signaling in Swiss 3T3 fibroblasts.

Changes in intracellular free Ca2+ concentration [( Ca2+]i) were used to study the interaction between mitogens in Swiss 3T3 fibroblasts. Platelet-derived growth factor (PDGF) produced an increase in [Ca2+]i and markedly decreased the increases in [Ca2+]i caused by subsequent addition of bradykinin and vasopressin. If the order of the additions was reversed the [Ca2+]i response to PDGF was not inhibited by bradykinin or vasopressin. Inhibition of protein kinase C by staurosporine or chronic treatment of the cells with phorbol 12-myristate 13-acetate prevented the inhibitory effect of PDGF on the [Ca2+]i response to vasopressin but not bradykinin. PDGF did not decrease the receptor binding of bradykinin and produced only a small decrease in the receptor binding of vasopressin. PDGF decreased the rise in [Ca2+]i caused by the Ca2+ ionophores 4-bromo-A23187 and ionomycin and by a membrane perturbing ether lipid, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, both in the presence and absence of external Ca2+. There was no change in cell 45Ca2+ influx caused by PDGF, vasopressin, or bradykinin. 45Ca2+ efflux from cells exposed to PDGF and vasopressin mirrored the changes in [Ca2+]i caused by the agents, that is, PDGF added after vasopressin produced a further increase in 45Ca2+ efflux but vasopressin did not increase 45Ca2+ efflux after PDGF. PDGF but not vasopressin caused an increase in the uptake of 45Ca2+ into an inositol 1,4,5-trisphosphate-insensitive non-mitochondrial store in permeabilized cells. The results suggest that the decreased [Ca2+]i response to mitogens after PDGF represents an action of PDGF at a point beyond the release of intracellular Ca2+ and the influx of external Ca2+, caused by an increase in the rate of removal of cytoplasmic free Ca2+. This increased removal of cytoplasmic Ca2+ by PDGF is not due to the increased export of Ca2+ from the cell but results from increased Ca2+ uptake into non-mitochondrial stores.

Participation of both intracellular free Ca 2+ and protein kinase C in tonic vasoconstriction induced by prostaglandin F 2α

The roles of intracellular free Ca 2+ and protein kinase C in the tonic contraction induced by prostaglandin were studied. Prostaglandin F 2α induced tonic contraction of rat thoracic aorta in both control and Ca 2+-free solution. Close correlations were observed between the contractile response of aortic strips and the changes in intracellular free Ca 2+ concentration in vascular smooth muscle cells assessed with the fluorescent Ca 2+ indicator fura 2, both in control and Ca 2+-free solutions. Prostaglandin F 2α also enhanced the production of inositol 1,4,5-triphosphate in vascular smooth muscle cells before the rise of the intracellular free Ca 2+ concentration. Moreover, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, inhibited the tonic contractions induced by PGF 2α and 12-O-tetradecanoyl phorbol-13-acetate, a direct activator of protein kinase C, at similar concentrations. These results suggest that both intracellular free Ca 2+ and protein kinase C participate in prostaglandin F 2α-induced tonic contraction.

Changes in Intracellular Free Ca2+ Concentration by Activation of α-Adrenoceptors in Rat Tail Artery

Changes in intracellular free Ca2+ concentration ([Ca2+]1) by activation of α1- and α2-adrenoceptors were microfluorometrically measured in rat tail arteries loaded with the Ca2+-sensitive fluorescent dye fura-2. Under the normal condition (1.8 m M Ca2+), stimulation with the selective α1-adrenoceptor agonist phenylephrine very rapidly raised [Ca2+]i to the peak concentration, plateauing at this level, whereas the stimulation with the selective α2-adrenoceptor agonist UK-14,304 gradually increased [Ca2+]i. In Ca2+-free solution, the stimulation of αα-adrenoceptors caused a transient increase in [Ca2+]i, whereas the stimulation of α2-adrenoceptors had no effect. These results directly prove that α1-adrenoceptor activation induces both the release of intracellular Ca2+ and the influx of extracellular Ca2+, whereas activation of α2-adrenoceptors evokes only the influx of extracellular Ca2+. Plotting the increase in [Ca2+]i against the developed contraction revealed that it was always linearly related regardless of the type of α-adrenoceptor stimulated. Therefore, the intracellular mechanisms after the increase in [Ca2+]i are probably common to the vasocontractile responses mediated by α1 - and α2-adrenoceptors.

Changes in intracellular free Ca2+ concentration by activation of .ALPHA.-adrenoceptors in rat tail artery.

Changes in intracellular free Ca2+ concentration ([Ca2+]i) by activation of alpha 1- and alpha 2-adrenoceptors were microfluorometrically measured in rat tail arteries loaded with the Ca2(+)-sensitive fluorescent dye fura-2. Under the normal condition (1.8 mM Ca2+), stimulation with the selective alpha 1-adrenoceptor agonist phenylephrine very rapidly raised [Ca2+]i to the peak concentration, plateauing at this level, whereas the stimulation with the selective alpha 2-adrenoceptor agonist UK-14,304 gradually increased [Ca2+]i. In Ca2(+)-free solution, the stimulation of alpha 1-adrenoceptors caused a transient increase in [Ca2+]i, whereas the stimulation of alpha 2-adrenoceptors had no effect. These results directly prove that alpha 1-adrenoceptor activation induces both the release of intracellular Ca2+ and the influx of extracellular Ca2+, whereas activation of alpha 2-adrenoceptors evokes only the influx of extracellular Ca2+. Plotting the increase in [Ca2+]i against the developed contraction revealed that it was always linearly related regardless of the type of alpha-adrenoceptor stimulated. Therefore, the intracellular mechanisms after the increase in [Ca2+]i are probably common to the vasocontractile responses mediated by alpha 1- and alpha 2-adrenoceptors.

Complement C5b-9 complex activates phospholipases in glomerular epithelial cells

In rat membranous nephropathy, formation of the C5b-9 membrane attack complex (MAC) leads to proteinuria in association with glomerular visceral epithelial cell (GEC) injury. These alterations in GEC function and morphology might result from changes in intracellular free Ca2+ concentration [( Ca2+]i) and activation of phospholipases. We demonstrate that in cultured rat GEC, antibody-directed formation of noncytolytic amounts of the MAC induced a rapid and sustained increase in [Ca2+]i that was partly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The MAC elevated levels of inositol bis- (IP2) and trisphosphate (IP3), as well as 1,2-diacylglycerol (DAG) and phosphatidic acid (PA). In permeabilized GEC, IP3 released Ca2+ from intracellular stores. Cellular 45Ca2+ uptake was also increased by the MAC. Thus, in GEC, the MAC induced Ca2+ mobilization from intracellular stores secondary to activation of phospholipase C and production of IP3, as well as enhanced Ca2+ influx. In addition, C5b-9 stimulated release of arachidonic acid (AA), prostaglandin F2 alpha, and thromboxane A2. Indomethacin partially inhibited the increase in DAG levels observed with the MAC, whereas the prostaglandin H2/thromboxane A2 analogue U46619 elevated DAG, suggesting that an eicosanoid product of MAC-induced AA release may enhance the activation of phospholipase C. Activation of phospholipases by the MAC may lead to altered GEC function and thereby contribute to the pathophysiological changes that characterize complement-dependent rat membranous nephropathy.

Mitogen-induced [Ca2+]i changes in individual human fibroblasts: Image analysis reveals asynchronous responses which are characteristic for different mitogens

Abstract Temporal changes in intracellular free Ca2+ concentration ([Ca2+]i) in cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca2+]i in each individual cell within a microscopic field. Responses to BK are heterogeneous with respect to the shapes of the [Ca2+]i time courses. Furthermore, the onsets of these responses follow a variable lag period such that the individual cell responses occur asynchronously. The asynchrony and heterogeneity of individual cell responses are not related to cell cycle differences since noncycling cells respond in a similar manner. When cells are ranked according to order of an initial response to BK (the first cell to respond is ranked 1, the second to respond is 2, etc.), restimulation of the same cells with BK elicits a similar order of cell responses, and the shape of the [Ca2+]i time course of an individual cell is similar for both responses to BK. If cells that were stimulated with BK are washed and restimulated with thrombin (which produces [Ca2+]i changes similar to those induced by BK), the response order to thrombin does not correspond to the response order following BK stimulation. These data suggest that the asynchrony of [Ca2+]i changes induced by BK or thrombin is characteristic for each mitogen and may be determined by cell-to-cell variation in receptor number.

Contribution of external and internal Ca 2+ to changes in intracellular free Ca 2+ produced by mitogens in swiss 3T3 fibroblasts: The role of dihydropyridine sensitive Ca 2+ channels

Changes in intracellular free Ca 2+ concentration ([Ca 2+] i ) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca 2+ in the external medium by using EGTA had no significant effect on the increase in [Ca 2+] i produced by vasopresin, bradykinin, bombesin or prostaglandin E 2, but reduced the increase in [Ca 2+] i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E 1 44% and by prostaglandin F 2α 47%. The dihydropyridine Ca 2+-channel antagonist nifedipine at 10 μM inhibited the [Ca 2+] i response to PDGF by 41% in both the presence of and in the absence of external Ca 2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (BAY K8644), a Ca 2+-channel agonist, at 10 μM produced an increase in [Ca 2+] i and decreased the [Ca 2+] i response to PDGF by 39%. Nifedipine did not block 45Ca 2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca 2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca 2+] i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca 2+ through dihydropyridine sensitive Ca 2+ channels, as well as release of internal Ca 2+.

Spontaneous and beta-adrenergic receptor-mediated taurine release from astroglial cells are independent of manipulations of intracellular calcium

Stimulation of beta-adrenergic receptors on LRM55 astroglial cells results in cAMP-dependent release of taurine. We have previously demonstrated that extracellular Ca2+ is not required for either spontaneous or receptor-mediated taurine release (Martin et al., 1988b). In the present series of experiments we investigated the relationship between changes in intracellular free Ca2+ ([Ca2+]i) and taurine release. [Ca2+]i was measured using the fluorescent probe fura-2 and was manipulated by changing the concentration of Ca2+ in the incubation medium and by using the Ca2+ ionophore ionomycin. [Ca2+]i was reduced from 150 +/- 95 nM (n = 46) in control medium (containing 1.1 mM CaCl2) to 46 +/- 10 nM (n = 43) in saline containing no CaCl2 and 10 microM EGTA. [Ca2+]i was rapidly elevated to greater than or equal to 1 microM in medium containing 100 microM CaCl2 and 10 microM ionomycin. Taurine release, either spontaneous or stimulated by isoproterenol, was not significantly affected by these manipulations of [Ca2+]i. [Ca2+]i did not change when cells were stimulated with 100 nM isoproterenol in either control saline containing 1.1 mM CaCl2 or in CaCl2-free saline containing 10 microM EGTA. Other secretogogs (serotonin and ethanol) did not cause changes in [Ca2+]i. These data indicate that neither spontaneous or receptor-mediated taurine release from astroglial cells is Ca2+ dependent. However, when cells were preloaded with Ca2+, allowed to recover briefly, and then stimulated with isoproterenol, it was possible to demonstrate transient increases in Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of neuronal signal transduction systems by extracellular ATP.

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)