Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin

A rise in cytosolic free calcium ([Ca2+]i) is thought to be the principal mediator in vascular smooth muscle contraction. Quantitative changes of [Ca2+]i in response to two vasoconstrictor peptide hormones, angiotensin II and vasopressin, were directly measured in monolayers of adherent cultured rat aortic smooth muscle cells loaded with the fluorescent calcium indicator Quin 2. Angiotensin II induced rapid, concentration-dependent rises in [Ca2+]i from 1.53 +/- 0.27 X 10(-7) (n = 16) up to 1.2 X 10(-6) M, with ED50 of 0.45 X 10(-9) M, an effect which was blocked by the antagonist analogue [Sar1, Ala8]angiotensin II. Vasopressin also elicited transient rises in [Ca2+]i to peak levels of about 8 X 10(-7) M, with ED50 of 1.05 X 10(-9) M, and this response was completely abolished by a vasopressor antagonist. In calcium-free medium, basal [Ca2+]i levels fell to 0.92 +/- 0.24 X 10(-7) M (n = 4), and both hormones were still able to raise [Ca2+]i, although to a lesser extent. Readdition of extracellular calcium following the [Ca2+]i transient induced a second, slower [Ca2+]i rise. In calcium-containing medium, lanthanum ion (2 X 10(-5) M) reduced peptide-evoked [Ca2+]i rises to the values observed in calcium-free medium. Stimulation with each peptide completely desensitized the smooth muscle cells to a subsequent identical challenge, with little crosstachyphylaxis. Potassium ion (50 mM) only minimally affected [Ca2+]i levels. The calcium channel blocker nifedipine (10(-6) M) did not prevent the [Ca2+]i rises induced by angiotensin II, vasopressin, or potassium. These findings indicate that the two physiologically important vasoconstrictor hormones angiotensin II and vasopressin rapidly raise [Ca2+]i in cultured vascular smooth muscle cells, in part by mobilizing calcium from intracellular pools and in part through activation of receptor-operated calcium channels.     

Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell

Effects of adrenocorticotropin (ACTH) on cytoplasmic free calcium concentration, [Ca2+]c, have been measured in adrenal glomerulosa cells using a calcium-sensitive photoprotein, aequorin. ACTH causes a rapid and transient increase in [Ca2+]c. Dose response study demonstrates that 1 pM ACTH induces an elevation of [Ca2+]c and that effect of ACTH appears to be saturated at 100 pM. ACTH action is greatly inhibited but not abolished by removal of extracellular calcium and is completely blocked in medium containing no added calcium and 1 mM EGTA. Under similar conditions, angiotensin II induces a remarkable rise in [Ca2+]c. ACTH action is not affected by pretreatment with dantrolene, which considerably decreases angiotensin II action on [Ca2+]c. One micromolar forskolin, which mimics 1 nM ACTH-mediated elevation of intracellular cAMP, does not increase [Ca2+]c nor modulates changes in [Ca2+] induced by a low dose of ACTH. One hundred micromolar forskolin or 1 mM 8-bromo-cAMP, however, increases [Ca2+]c even in calcium-free medium containing 1 mM EGTA. When glomerulosa cells are co-loaded with aequorin and quin2, angiotensin II-induced change in aequorin signal is greatly reduced, and ACTH-induced change is abolished. Quin2 loading results in accumulation of calcium in the cell under both unstimulated and stimulated conditions. These results indicate that ACTH increases [Ca2+]c by cAMP-independent mechanism, that ACTH action on [Ca2+]c is exclusively dependent on extracellular calcium, and that quin2 is unable to detect the rapid change in [Ca2+]c because of its calcium chelating activity.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading

The Ca concentration ([Ca2+]i) in single rat peritoneal mast cells was measured by means of the new fluorescent Ca-indicator dye fura-2. Dye-loaded cells were made to degranulate with either antigen or compound 48/80. In cells loaded with extracellularly applied, membrane-permeant fura-2 ester, degranulation was accompanied by a permanent loss of 40-60% of the fluorescence, but comparison of fluorescence at different wavelengths indicated no or only small changes in [Ca2+]i. When cells were loaded by microinjection of the impermeant potassium salt of the dye, degranulation resulted in no permanent loss of fluorescence, but instead was preceded by transient fluorescence changes that indicate a rapid, large and transient increase in [Ca2+]i. We suggest that ester-loaded fura-2 accumulates to a significant degree in the secretory granules and is lost from the cell during exocytosis.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Evidence that phorbol ester interferes with stimulated Ca2+ redistribution by activating Ca2+ efflux in neutrophil leucocytes.

The free calcium ion concentration, [Ca2+]i, in the cytoplasmic matrix of quin2-loaded neutrophil leucocytes increases rapidly after addition of concanavalin A. This increase is effectively abolished by a short (3 min) preincubation with 10 nM-TPA (12-O-tetradecanoylphorbol 13-acetate). TPA also inhibits a [Ca2+]i rise of similar magnitude induced by low concentrations (10 nM) of calcium ionophore A23187, suggesting that phorbol ester does not interfere with a physiological influx mechanism. To investigate the effects of TPA further, cells were depleted of Ca2+ during quin2 loading and then re-equilibrated with normal extracellular [Ca2+]. The return to a stable [Ca2+]i value was preceded by a transient overshoot in [Ca2+]i, implying delayed activation of an efflux mechanism by rising [Ca2+]i. TPA abolished the transient, suggesting preactivation by TPA of the efflux mechanism before Ca2+ influx. TPA also stimulates net Ca2+ efflux from neutrophils and neutrophil cytoplasts. These observations are consistent with the thesis that TPA stimulates a Ca2+-efflux mechanism in these cells.     

Inhibition of macrophage secretion by tetanus toxin is not directly linked to cytosolic calcium homeostasis

Tetanus toxin (TT) inhibits secretion of neurotransmitters from neurons and lysozyme from human macrophages (Mphi). Because these secretory events are associated with changes in cytosolic free calcium [Ca2+]i, we examined the effect of TT on Mphi calcium homeostasis and secretion in response to ionomycin and phorbol myristate acetate (PMA). Using Quin 2 to report [Ca2+]i, basal [Ca2]i was similar for control cells (133 nM) and Mphi treated with TT (127 nM). In response to ionomycin (50 nM) [Ca2+]i increased to 548 +/- 74 nM in control cells and to 357 +/- 36 nM in TT-treated Mphi (p less than 0.02, N = 12). Despite this rise in [Ca2+]i, neither control Mphi nor TT-treated Mphis secreted the lysosomal enzyme lysozyme in response to this concentration of ionomycin (50 nM). In both control and TT-treated Mphi, stimulation with a higher concentration of ionomycin (1000 nM) caused saturation of the quin 2 fluorescence signal. However, lysozyme secretion from TT-Mphi was inhibited. In response to the phorbol ester, PMA (3 uM), [Ca2+]i did not increase in either control Mphi or TT-treated Mphi. However, secretion of lysozyme from TT-treated Mphi was also inhibited in response to this stimulus (70.8% of control, p less than 0.02, N = 3). These data indicate that the ability of TT to inhibit secretion from Mphi is not directly linked to alterations of cytosolic calcium homeostasis.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

N-terminal amino acid sequence of an insect neurohormone, melanization and reddish coloration hormone (MRCH): heterogeneity and sequence homology with human insulin-like growth factor II

Catecholamine release from chromaffin cells in response to carbamylcholine and high K+ is transient. Monitoring intracellular free calcium ([Ca2+]i) using quin2 demonstrated a transient rise in [Ca2+]i in response to carbamylcholine. The termination of secretion due to carbamylcholine is probably a consequence of the return of [Ca2+]i to resting levels as the nicotinic receptors desensitise. Depolarisation with 55 mM K+ led to a long-lasting rise in [Ca2+]i which persisted after the secretory response had terminated. The maintained rise in [Ca2+]i appeared to be due to continued opening of verapamil-sensitive Ca2+ channels. These results suggest that inactivation of voltage-dependent Ca2+ channels does not account for the transient nature of the secretory response in chromaffin cells.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.     

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.     

Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria

The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.     

Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.