Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors.

Capacitative Ca2+ entry (CCE) is a universal mechanism for refilling intracellular Ca2+ stores in electrically non-excitable cells. The situation in excitable cells is less clear, however, since they may rely on other entry mechanisms for Ca2+-store refilling. In the present study we investigated CCE in intact PC12 cells, using acetylcholine to bring about activation of InsP3 receptors (InsP3Rs), caffeine to activate ryanodine receptors (RyRs) and thapsigargin to inhibit sarco/endoplasmic reticulum Ca2+-ATPase pumps. We found that depletion of the InsP3-, caffeine- or thapsigargin-sensitive stores promoted Ca2+ entry, suggesting that stimulation of either InsP3Rs or RyRs can activate CCE. The CCE pathways activated by InsP3Rs, RyRs and thapsigargin appeared to be independent at least in part, since their effects were found to be additive. However, CCE triggered by caffeine, acetylcholine or thapsigargin progressively diminished with time. The decay of CCE caused by one agent also inhibited subsequent responses to the others, suggesting that some component of the CCE pathway is common to all intracellular Ca2+ stores. The magnitude of CCE stimulated by InsP3Rs or RyRs was related to the size of the stores; the InsP3-sensitive store was smaller than the RyR-sensitive store and triggered a smaller entry component. However, both stores filled with a similar half time (about 1 min), and both could be filled more rapidly by depolarization-induced Ca2+ entry through voltage-operated channels. A significant basal Ca2+ influx was apparent in PC12 cells. The basal entry component may be under the control of the InsP3-sensitive Ca2+ store, since short incubations in Ca2+-free medium depleted this store.

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.

Expression and function of ryanodine receptors in nonexcitable cells.

We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses.

Calcium signalling. Cracking ICRAC in the eye.

Capacitative Ca(2+) entry produces the very rapid light response in Drosophila photoreceptors; studies using this amenable system may help unravel the machinery and mechanisms that underlie this Ca(2+)-entry pathway.

Histamine-induced Ca2+ entry precedes Ca2+ mobilization in bovine adrenal chromaffin cells.

The relationship between histamine-induced Ca2+ mobilization and Ca2+ entry in bovine adrenal chromaffin cells has been investigated. Stopped-flow fluorimetry of fura-2-loaded chromaffin cell populations revealed that 10 microM histamine promoted entry of Ca2+ or Mn2+ without measurable delay (< or = 20 ms), through a pathway that was insensitive to the dihydropyridine antagonist nifedipine. In the absence of extracellular Ca2+, or in the presence of 100 microM La3+, a blocker of receptor-mediated Ca2+ entry, 10 microM histamine triggered an elevation in intracellular calcium concentration ([Ca2+]i), but only after a delay of approx. 200 ms, which presumably represented the time required to mobilize intracellular Ca2+. These data suggested that histamine-induced bivalent-cation entry precedes extensive Ca2+ mobilization in chromaffin cells. In order to confirm that histamine can promote Ca2+ entry largely independently of mobilizing intracellular Ca2+, the ability of histamine to promote Ca2+ entry into cells whose intracellular Ca2+ store had been largely depleted was assessed. Fura-2-loaded chromaffin cells were treated with 10 microM ryanodine together with 40 mM caffeine, to deplete the hormone-sensitive Ca2+ store. This resulted in an approx. 95% inhibition of histamine-induced Ca2+ release. Under these conditions, histamine was still able to promote an entry of Ca2+ that was essentially indistinguishable from that promoted in control cells. In single cells, introduction of heparin (100 mg/ml), but not de-N-sulphated heparin (100 mg/ml), abolished the histamine-induced rise in [Ca2+]i. All these data suggest that histamine can induce G-protein- or inositol phosphate-dependent rapid (< or = 20 ms) Ca2+ entry without an extensive intracellular mobilization response in chromaffin cells, which points to activation of an entry mechanism distinct from the Ca(2+)-release-activated Ca2+ channel found in non-excitable cells.

Smoothly graded Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores.

Stimulation of cells with Ca(2+)-mobilizing hormones often leads to the generation of temporally and spatially complex changes in the intracellular Ca2+ ion concentration ([Ca2+]i). To understand the mechanisms regulating Ca2+ release from intracellular stores more clearly, we investigated the ability of histamine to release Ca2+ stores under different experimental conditions, using video imaging of single Fura-2-loaded HeLa cells. In Ca(2+)-free medium, stepwise increases in histamine concentration released an increasing proportion of the intracellular Ca2+ pool. This pattern of Ca2+ release is analogous to the "quantal" release of Ca2+ previously observed using permeabilized cells. Quantal Ca2+ release was observed at both 20 and 37 degrees C and was not due to inactivation or desensitization of the Ca2+ release mechanism, since application of histamine in a pulsatile manner, which avoided desensitization of the Ca(2+)-release mechanism, still produced a quantal response. In Ca(2+)-containing medium at both 20 and 37 degrees C, stepwise increases in histamine concentration evoked [Ca2+]i responses where the amplitude was smoothly graded in direct proportion to the histamine concentration. Similar smoothly graded responses were observed from HeLa cells in Ca(2+)-free medium. These data indicate that hormone-evoked Ca2+ release from intracellular stores is limited by the hormone concentration, and that the mechanisms underlying complex [Ca2+]i signals do not lead to an all-or-none release of Ca2+ from the entire intracellular Ca2+ pool. We suggest that the hormone-sensitive intracellular Ca2+ pool is composed of functionally discrete units that are recruited by agonists in a concentration-dependent manner.

Quantal Ca2+ mobilization by ryanodine receptors is due to all-or-none release from functionally discrete intracellular stores.

Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.

A caffeine- and ryanodine-sensitive intracellular Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells.

We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded cells, caffeine cause a concentration-dependent increase in [Ca2+]i that was saturable, reversible and inhibited in a use-dependent fashion by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, with an EC50 of 13 mM caffeine and a Hill coefficient (h) of 2.7, indicating that the release mechanism was co-operative. Pretreatment of intact cell populations with increasing concentrations of caffeine in nominally Ca(2+)-free medium inhibited the subsequent Ca2+ response to a maximal concentration of ATP, in a dose-dependent manner. In permeabilized cells, a maximal concentration (40 microM) of InsP3 still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffeine, whereas caffeine was unable to release Ca2+ after the InsP3-sensitive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP3-sensitive Ca2+ store, and a store that is sensitive to both InsP3 and caffeine. Depletion of the caffeine-sensitive Ca2+ store by caffeine and ryanodine pretreatment in intact cells attenuated the Ca2+ response to ATP, but not to 55 mM K+, suggesting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source after ATP stimulation, but not after depolarization with 55 mM K+. Pretreatment of intact cells with ATP and ryanodine resulted in a use-dependent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodine receptors. The rate of recovery, but not the magnitude or rate of onset, of the depolarization-induced [Ca2+]i transient was modulated by the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unable to refill. We conclude that the caffeine- and ryanodine-sensitive Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells, and that its role may in part be governed by the nature of the stimulating agent.

Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems.

A cloned seven transmembrane-spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster ovary cell line, both inhibits adenylate cyclase activity and leads to the elevation of intracellular Ca2+ levels by separate G-protein-coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

Extracellular ATP triggers two functionally distinct calcium signalling pathways in PC12 cells.

We have investigated the effects of extracellular ATP on Ca2+ signalling, and its relationship to secretion in rat pheochromocytoma (PC12) cells. In single cells, extracellular ATP evoked two very distinct subcellular distributions of intracellular calcium concentration ([Ca2+]i), only one of which could be mimicked by the pyrimidine nucleotide UTP, suggesting the involvement of more than one cell surface receptor in mediating the ATP-induced responses. ATP and UTP were equipotent in activating a receptor leading to inositol phosphate production and the mobilisation of intracellular Ca2+. In some cells (19%) this rise in [Ca2+]i initiated at a discrete site and then propagated across the cell in the form of a Ca2+ wave. In addition to mobilising intracellular Ca2+ through a 'nucleotide' receptor sensitive to ATP and UTP, the results indicate that ATP also activates divalent cation entry through an independent receptor-operated channel. Firstly, ATP-induced entry of Ca2+ or Mn2+ was independent of Ca2+ mobilisation, as prior treatment of cell populations with UTP abolished the ATP-evoked release of intracellular Ca2+ stores, but left the Ca(2+)- and Mn(2+)-entry components uneffected. Secondly, although UTP and ATP were equally effective in generating inositol phosphates, only ATP stimulated divalent cation entry, indicating that ATP-activated influx was independent of phosphoinositide turnover. Thirdly, single cell experiments revealed a subpopulation of cells that responded to ATP with divalent cation entry without mobilising Ca2+ from intracellular stores. Lastly, the dihydropyridine antagonist, nifedipine, reduced the ATP-induced rise in [Ca2+]i by only 24%, suggesting that Ca2+ entry was largely independent of L-type voltage-operated Ca2+ channels. The Ca2+ signals could also be distinguished at a functional level. Activation of ATP-induced divalent cation influx was absolutely required to evoke transmitter release, because ATP triggered secretion of [3H]dopamine only in the presence of external Ca2+, and UTP was unable to promote secretion, irrespective of the extracellular [Ca2+]. The results suggest that the same extracellular stimulus can deliver different Ca2+ signals into the same cell by activating different Ca2+ signalling pathways, and that these Ca2+ signals can be functionally distinct.

Quantal Ca2+ release from caffeine-sensitive stores in adrenal chromaffin cells.

In populations of fura-2-loaded chromaffin cells, caffeine caused a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), in the presence or absence of external Ca2+ ([Ca2+]o), that was saturable, reversible, and inhibited in a use-dependent fashion by ryanodine. These data confirm that caffeine mobilizes Ca2+ from the ryanodine-sensitive intracellular stores in chromaffin cells. In nominally Ca(2+)-free medium, sustained stimulation of cell populations or single cells with low caffeine concentrations failed to completely empty the caffeine-sensitive stores. In each case, there was a transient [Ca2+]i elevation, but a subsequent challenge with a higher caffeine concentration evoked a further [Ca2+]i rise, indicating that Ca2+ stores within individual cells were heterogeneous in their sensitivities to caffeine and that caffeine-induced Ca2+ release was quantal. The heterogeneous sensitivity was also demonstrated using ryanodine; pretreatment of cell populations with increasing caffeine concentrations with a constant ryanodine concentration, caused a dose-dependent irreversible inhibition of the response to the subsequent addition of a maximal caffeine concentration. We conclude that, within single chromaffin cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to caffeine and the fraction of Ca2+ stores mobilized by caffeine increases in direct proportion to the caffeine concentration.

Stimulus-secretion coupling in excitable cells: a central role for calcium.

Secretion of vesicular contents by exocytosis is a common feature of neuroendocrine secretory cells such as adrenal chromaffin cells and PC12 cells. Although it is clear that in these cells an elevation in intracellular calcium concentration, [Ca2+]i, is the triggering event that induces secretion, recent studies using video-imaging, patch-clamp and flash photolysis techniques have all indicated that the Ca2+ signal that triggers secretion is in fact very complex, with the subcellular distribution of Ca2+ being of particular importance along with the magnitude of the rise. It has become evident that Ca2+ signals with different spatial profiles can be triggered in the same cell by a given stimulus, depending upon the nature of the Ca2+ signalling pathway activated, and that this ability to be able to vary the method of delivery of Ca2+ into the cell is important physiologically, because it provides a means of obtaining differential activation of Ca(2+)-dependent processes.

Exocytosis in adrenal chromaffin cells.

Recent advances have led to an increased understanding of the Ca(2+)-signalling pathway leading to exocytosis in bovine adrenal chromaffin cells. Video-imaging studies have allowed the temporal and spatial aspects of the Ca2+ signal to be investigated in detail. Ca2+ entry at the plasma membrane appears to be crucial for the activation of exocytosis. Ca2+ can enter through the nicotinic channel or characterised voltage-activated channels, or through other poorly defined pathways due to a variety of agonists. Emptying of internal Ca2+ stores is sufficient to activate a Ca2+ entry pathway. The elevation of cytosolic Ca2+ concentration leads to a reorganisation of the cortical actin network and to the triggering of exocytosis. Studies on permeabilised chromaffin cells have resulted in the identification of some of the proteins that control Ca(2+)-dependent exocytosis. These include the peripheral plasma membrane protein annexin II and the cytosolic proteins, protein kinase C and 14-3-3 proteins (Exo1).

Fertilisation and thimerosal stimulate similar calcium spiking patterns in mouse oocytes but by separate mechanisms.

Exposure of freshly ovulated mouse oocytes to a fertilising spermatozoon, thimerosal, Sr2+ or acetylcholine induced similar Ca2+ spiking responses. We propose that each of the four agents reduces the threshold for Ca2+ release from internal stores, but by different mechanisms. All agents except thimerosal stimulated oocyte activation, but thimerosal caused dissassembly of the meiotic spindle and thus prevented progress into interphase. Dithiothreitol (DTT) completely blocked and reversed the spiking responses induced by thimerosal, but facilitated and accelerated those induced by spermatozoa, Sr2+ and acetylcholine. The stimulatory effect of DTT was not simply a consequence of progress into interphase, but was attributable, at least in part, to an enhancement of divalent cation entry, as measured by Mn2+ quench analysis of fura-2 in both fertilised and unfertilised oocytes. Possible mechanisms by which DTT might achieve its effects are discussed.

Spatial localization of agonist-induced Ca2+ entry in bovine adrenal chromaffin cells. Different patterns induced by histamine and angiotensin II, and relationship to catecholamine release.

The spatial organization of agonist-induced Ca2+ entry in single bovine adrenal chromaffin cells has been investigated using video-imaging techniques to visualize fura-2 quenching by the Ca2+ surrogate, Mn2+. The potent secretagogue histamine, in addition to releasing Ca2+ from intracellular stores, resulted in a large influx of external Mn2+ that occurred over the entire surface of the cell. The influx of Ca2+ that this mirrors was found to be an obligatory requirement for the triggering of catecholamine release by histamine, which suggests that such a global influx of Ca2+ into the cell probably underlies the ability of this agonist to stimulate a large secretory response. By contrast, the weaker secretagogue angiotensin II, which also acts through the second messenger inositol trisphosphate, produced a localized entry of external Mn2+ in 64% of cells. In these cells, localized Mn2+ entry always occurred at the pole of the cell in which the angiotensin II-induced rise in [Ca2+]i was largest. Since exocytosis in response to angiotensin II has previously been shown to be restricted to this same pole of the cell (Cheek et al. (1989). J. Cell Biol. 109, 1219-1227), these results suggest that localized influx of Ca2+ in response to angiotensin II could underlie the polarized exocytotic response observed with this stimulus. These results directly demonstrate that different agonists can induce different patterns of divalent cation influx in the same cells and, furthermore, suggest how these different patterns can have a direct influence on cellular function.

Ca2+ influx induced by the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone and thapsigargin in bovine adrenal chromaffin cells.

We have characterized the effect of the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) and thapsigargin on the concentration of cytosolic Ca2+ in single bovine adrenal chromaffin cells by video-imaging of fura-2-loaded cells. Addition of either inhibitor released Ca2+ from internal stores in the absence of external Ca2+. tBHQ was unable to stimulate further Ca2+ release after addition of thapsigargin, but thapsigargin could do so after release by tBHQ, indicating that the tBHQ-sensitive stores are a sub-set of those sensitive to thapsigargin. Angiotensin II was able to elicit Ca2+ release after application of tBHQ, indicating that at least part of the tBHQ-sensitive stores were distinct from those discharged by Ins(1,4,5)P3. In the presence of external Ca2+, both Ca(2+)-ATPase inhibitors produced a more prolonged rise in cytosolic Ca2+ consistent with stimulated Ca2+ entry. The ability of the inhibitors to activate a Ca(2+)-entry pathway was confirmed by monitoring quenching of fura-2 after stimulated entry of the Ca2+ surrogate Mn2+. These findings indicate that bovine adrenal chromaffin cells possess a mechanism by which Ca2+ entry can be activated, following emptying of certain internal stores, independently of receptor occupation.

Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium.

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

Calcium signalling and the triggering of secretion in adrenal chromaffin cells.

The pivotal intracellular message for triggering catecholamine release from bovine adrenal chromaffin cells is an elevation in the concentration of cytosolic free Ca2+ ([Ca2+]i). Studies using video-imaging techniques have shown that a rise in [Ca2+]i at the cell periphery, that is due to Ca2+ entry, is the major activating signal for exocytosis. The cytoskeleton has been identified as a major regulatory site of exocytosis, with Ca(2+)-induced disruption of the cortical actin network being required in order that previously restrained granules may have access to their exocytotic sites. The Ca(2+)- and phospholipid-dependent annexin protein, calpactin, has been strongly implicated in a late stage of interaction between granules and the plasma membrane by both ultrastructural and biochemical studies.