Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool

The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.     

Electrophysiological evidence that glycoprotein IIb-IIIa complex is involved in calcium channel activation on human platelet plasma membrane.

The involvement of platelet glycoprotein (GP) IIb-IIIa complex in calcium channel activity on the plasma membrane was investigated using an electrophysiological approach. Plasma membrane vesicles were prepared from thrombin-stimulated platelets and incorporated into planar lipid bilayers. Voltage-independent Ca2+ channel currents with a conductance of about 10 pS (in 53 mM Ba2+) were observed, in membranes derived from thrombin-stimulated, but not unstimulated platelet membranes. These channel activities were markedly reduced by exposure of membranes to EGTA at 37 degrees C. This reduction was specifically related to the dissociation of the GPIIb-IIIa complex since preincubation of the membranes with a monoclonal antibody to the GPIIb-IIIa complex (AP-2) could protect the channel activities from the effect of EGTA. Thrombasthenic platelets, which lack the GPIIb-IIIa complex, showed impaired channel activities characterized by decreased open probability and lowered conductance states. Furthermore, when platelets were stimulated by thrombin in the presence of EGTA, AP2, or the synthetic peptide RGDS, to prevent fibrinogen binding to the GPIIb-IIIa complex, open probabilities of the channel currents in these membrane vesicles were also decreased. These results suggest that the GPIIb-IIIa complex is involved in platelet Ca2+ channel activation and that ligand binding to the complex during platelet activation may modify the activation of Ca2+ channels.     

Parasite-induced processes for adenosine permeation in mouse erythrocytes infected with the malarial parasite Plasmodium yoelii.

In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.     

Agonists stimulate divalent cation channels in the plasma membrane of human platelets

Agonists such as thrombin, PAF (platelet-activating factor) and ADP are known to cause a larger elevation in [Ca2+]i in quin2-loaded platelets in the presence of extracellular Ca2+ than in its absence. The simplest interpretation of these observations is that in the presence of extracellular calcium there is an influx component across the cell surface. In the presence of Mn2+, a divalent cation which is known to avidly bind to quin2 and to quench its fluorescence, the agonists produce a small initial rise in quin2 fluorescence followed by a decrease in fluorescence to well below the resting level. The result indicates entry of Mn2+, presumably through some form of receptor-operated Ca2+ channel.     

Dissociation of store release from transmembrane influx of calcium in human neutrophils.

Release of Ca2+ from intracellular stores was visualised in individual neutrophils in the presence of the Mn2+ or SKF 96365. Influx of Mn2+ quenched fura-2 close to the plasma membrane but did not quench fura-2 at the site of store release. The size and location of the 'cloud' of elevated Ca2+ was unaffected by the channel blocker SKF 96365. Furthermore, the size and location was unaffected by the presence of extracellular Ca2+. This dissociation of transmembrane influx from store release demonstrates that the entry of Ca2+ into the cytosol of neutrophils occurs directly into the cytosol and not via the store site.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Receptor-operated calcium influx in rat hepatocytes. Identification and characterization using manganese

Agonist-stimulated divalent cation entry was studied in fura-2-loaded hepatocytes. In the presence of extracellular Mn2+, the Ca2(+)-mobilizing hormone vasopressin produced a severalfold stimulation of the basal rate of fura-2 fluorescence quenching as a result of Mn2+ influx; this effect was blocked by the presence of Ni2+ in the incubation medium. Half-maximum and maximum stimulation of Mn2+ influx was observed with 0.1 and 0.8 nM vasopressin, respectively. Agonist-stimulated Mn2+ influx was also seen with angiotensin II, ATP, phenylephrine, and the combination of AlCl3 and NaF. The stimulation of Mn2+ influx did not occur immediately after addition of Ca2(+)-mobilizing agents, but was characterized by a latency period of 20-30 s. In contrast to vasopressin, glucagon did not stimulate Mn2+ influx into hepatocytes, but produced both a 3-fold enhancement of the rate of vasopressin-stimulated Mn2+ entry and the abolishment of the latency period. The effects of glucagon were mimicked by forskolin and dibutyryl cAMP. Pretreatment of hepatocytes with pertussis toxin or depolarization of the cells altered neither the basal rate of Mn2+ entry nor the ability of vasopressin to stimulate this rate. Emptying of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by treatment with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) did not enhance Mn2+ entry into hepatocytes; however, exposure of the cells to tBuBHQ for 2 min markedly enhanced the ability of vasopressin, alone or in combination with glucagon, to increase the rate of Mn2+ influx. Furthermore, pretreatment with tBuBHQ for 2 min abolished the latency of vasopressin-stimulated Mn2+ influx. It is concluded that Ca2(+)-mobilizing hormones stimulate Ca2+ influx in hepatocytes, possibly through receptor-operated Ca2+ channels. The stimulation of divalent cation entry is transduced by a G protein, and the rate of influx appears to be controlled both by the intracellular level of cAMP and the empty state of an intracellular Ca2+ pool that may be inositol 1,4,5-trisphosphate-insensitive.     

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.     

Dynamics of Ca2+ transients in norepinephrine-stimulated individual H-35 hepatoma cells: fura-2 digital imaging microscopy and high time-resolution microspectrofluorometry

We investigated spatiotemporal changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10(-6) M) resulted in an initial transient increase in [Ca2+]i, followed by a small sustained [Ca2+]i plateau above the pre-stimulation level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an alpha-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca(2+)-free medium or by pre-treatment of cells with La3+. On the other hand, the sustained plateau was eliminated by Ca(2+)-free medium or La3+. Therefore, H-35 cells have a Ca(2+)-signaling pathway which is activated via alpha-adrenergic receptors. Mn2+ entered the cytosol after NE stimulation, as shown by quenching of fura-2. This indicates that H-35 hepatoma cells possess Mn(2+)-permeable Ca2+ channels at the plasma membrane. In addition, the Ca2+ efflux pattern from H-35 cells to the extracellular space during NE stimulation was visualized by digital imaging microscopy when free fura-2 was equilibrated between the cells and the extracellular space. The efflux of Ca2+ from H-35 begins between the initial [Ca2+]i transient and the sustained [Ca2+]i plateau.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry

Stimulation of platelets with thrombin or parotid acinar cells with carbachol results in an increase in [Ca2+]i which is due to both release from internal stores and influx across the plasma membrane. In platelets, thrombin also stimulates Mn2+ entry into the cytosol; this is seen as a stimulated quench of fura-2 fluorescence. In the parotid, however, carbachol does not stimulate Mn2+ entry. This result suggests different mechanisms of stimulated divalent cation entry in the two cell types and could have important implications in the study of receptor-mediated Ca2+ entry mechanisms.     

Mastoparan increases membrane permeability in rat parotid cells independently of action on G-proteins

Mastoparan, a peptide toxin from wasp venom, stimulated the accumulation of inositol phosphates in rat parotid acinar cells. Addition of this peptide to fura-2-loaded cells resulted in a rapid increase in the fura-2 fluorescence ratio (340 nm/380 nm), suggesting that mastoparan stimulates an increase in cytosolic Ca2+ concentration. However, this change in the ratio appears to be due, in part, to fura-2 leakage from the cells, because addition of Mn2+, which quenches extracellular fura-2 fluorescence, reduced the increased fluorescence ratio. In addition to the fura-2 leakage, mastoparan caused considerable leakage of lactate dehydrogenase, a cytosolic marker enzyme. Furthermore, mastoparan decreased the number of trypan blue-excluding cells, indicating a decrease in cell viability. These results suggest that mastoparan enhances the membrane permeability by a mechanism independent of the activation of G-proteins.     

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.     

Determination of platelet adhesion to collagen and the associated formation of phosphatidic acid and calcium mobilization

A method was developed to study the adhesion of platelets to fibrillar collagen at 37 degrees C in the absence of aggregation. Human platelets were labeled with [3H]-oleic acid, gel-filtered, and incubated with collagen in the presence of receptor antagonists to thromboxane A2, 5-hydroxytryptamine, and platelet-activating factor, as well as a fibrinogen/fibronectin inhibitor and an ADP-removing system. Those platelets that adhered to collagen were separated from those that did not by filtration through a 10-microns nylon mesh and the extent of platelet adhesion was quantitated by determination of the radioactivity retained by the mesh. The extent of platelet adhesion was proportional to the amount of collagen added up to 100 micrograms/ml and was essentially complete by 1 min. At least 80-90% of the platelets were capable of adhering to collagen. Adhesion was potentiated by the presence of extracellular Mg2+ and this potentiation was inhibited by extracellular Ca2+. Phosphatidic acid increased markedly in those platelets that adhered to collagen and this was associated with increases in cytosolic free Ca2+ levels that could be detected using the fluorescent Ca2+ indicator fura-2.     

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.