A Ca2+ Current Activated by Release of Intracellular Ca2+ Stores in Rat Basophilic Leukemia Cells (RBL-1)

We have characterized a Ca²⁺ current activated by depletion of intracellular Ca²⁺ stores (capacitative Ca²⁺ entry current) as a first step to investigate the mechanisms underlying communication between the intracellular Ca²⁺ stores and the plasma membrane Ca²⁺ permeability. Whole cell currents in response to voltage ramps from −125 to +60 mV from a holding potential of −40 mV were recorded in rat basophilic leukemia cells (RBL-1 cells) in solutions designed to optimize detection of a Ca²⁺ current. An inwardly rectifying current could be activated upon dialysis of the cell interior with pipette solutions devoid of Ca²⁺ and containing 20 mm BAPTA, a procedure expected to passively deplete intracellular Ca²⁺ stores. The current was maximally activated within 2 min, was sensitive to extracellular Ca²⁺ concentration and was abolished by removal of extracellular Ca²⁺. The current was markedly reduced in the presence of Ni²⁺ or La³⁺. The pathway activated by this protocol was permeant to Ba²⁺, displaying complex permeability characteristics at negative potentials. A small inward Mn²⁺ current consistent with a finite permeability of the pathway to Mn²⁺ was detected. In contrast Ni²⁺ displayed no detectable current carrying ability. Extracellular Na⁺ permeated the pathway in the absence of extracellular Ca²⁺. Under conditions designed to reduce passive depletion of intracellular Ca²⁺ stores, a Ca²⁺ current indistinguishable from that described above was activated by addition of ionomycin. This observation is consistent with the activation of the Ca²⁺ influx pathway occurring as a result of events associated with depletion of intracellular Ca²⁺ stores. Importantly, application of extracellular Ni²⁺ in the presence of ionomycin irreversibly inhibited the current. The presence of an inwardly rectifying K⁺ current in RBL cells could confound studies of the capacitative Ca²⁺ entry current when recorded using pipette solutions devoid of K⁺ since this current would be inward over the voltage range used to investigate the capacitative Ca²⁺ entry current. This study compares an inward rectifying K⁺ current and the capacitative Ca²⁺ entry current in RBL cells and highlights some similarities and differences between the two currents. The results demonstrate that caution should be exercised in interpreting recordings made using extracellular solutions containing even modest amounts of K⁺ when studying the capacitative Ca²⁺ entry current in RBL cells. Received: 12 September 1995/Revised: 18 June 1996     

Characterization of store-operated Ca channels in pancreatic duct epithelia

Store-operated Ca²⁺ channels (SOCs) are activated by depletion of intracellular Ca²⁺ stores following agonist-mediated Ca²⁺ release. Previously we demonstrated that Ca²⁺ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca²⁺ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca²⁺ influx as Ca²⁺ was released from intracellular stores. Direct measurement of [Ca²⁺] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP₃ or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La³⁺ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca²⁺ entry through SOCs fills internal Ca²⁺ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca²⁺ such as salt and mucin secretion from the exocrine pancreatic ducts.     

Store-Dependent Entry of Ca<Superscript>2+</Superscript> into Sensory Neurons of the Rat

We studied store-dependent (activated by depletion of the endoplasmic reticulum, ER, store) entry of Ca²⁺ from the extracellular medium into neurons of the rat spinal ganglia (small- and medium-sized cells; diameter, 18 to 36 μm). Activation of ryanodine-sensitive receptors of the ER in the studied neurons superfused by Tyrode solutions containing Ca²⁺ or with no Ca²⁺ was provided by application of 10 mM caffeine. The decay phase of caffeine-induced calcium transients in a Ca²⁺-containing solution was significantly longer than that in a Ca²⁺-free solution. This fact allows us to suppose that such a phenomenon is determined by Ca²⁺ entry into the neuron from the extracellular medium activated by caffeine-induced depletion of the ER store. Substitution of Ca²⁺-free extracellular solution by Ca²⁺-containing Tyrode solution, after depletion of the ER stores induced by applications of 100 nM ryanodine, 200 μM ATP, or 1 μM thapsigargin, resulted in increases in the concentration of intracellular Ca²⁺. These observations allow us to postulate that store-dependent Ca²⁺ entry into the studied neurons is activated after depletion not only of the inositol trisphosphate-sensitive ER store but also of the ryanodine-sensitive store. This entry also occurs after blocking of ATPases of the ER by thapsigargin. The kinetic characteristics of the rising phase of store-dependent Ca²⁺ entry induced by depletion of the ER stores under the influence of various agents are dissimilar; this can be related to different mechanisms of activation of such signals and/or to a compartmental organization of the ER. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 277–283, May–June, 2005.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

A Store-Operated Nonselective Cation Channel in Human Lymphocytes

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca²⁺ and Na⁺ influxes in lymphocytes. While Ca²⁺ influx is mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels, the pathway responsible for Na⁺ influx is largely unknown.2. We show that thapsigargin, ionomycin, ADP-ribose and IP₃ activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I–V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca²⁺ release-activated nonselective cation (CRANC) channel.3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity.4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca²⁺ entry.5. These results suggest that CRANC channels are responsible for the Na⁺ influx as well as a portion of the Ca²⁺ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca²⁺ stores; and that LTRPC2 protein is a likely component of the CRANC channel.     

Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

Synaptically Activated Ca<Superscript>2+</Superscript> Release From Internal Stores in CNS Neurons

Synaptically activated postsynaptic [Ca²⁺]_i increases occur through three main pathways: Ca²⁺ entry through voltage-gated Ca²⁺ channels, Ca²⁺ entry through ligand-gated channels, and Ca²⁺ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca²⁺ release from stores in CNS neurons primarily occurs as a result of IP₃ mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca²⁺ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca²⁺]_i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca²⁺]_i increases due to Ca²⁺ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons.     

Unraveling STIM2 function

The discovery of molecular players in capacitative calcium (Ca²⁺) entry, also referred to as store-operated Ca²⁺ entry (SOCE), supposed a great advance in the knowledge of cellular mechanisms of Ca²⁺ entry, which are essential for a broad range of cellular functions. The identification of STIM1 and STIM2 proteins as the sensors of Ca²⁺ stored in the endoplasmic reticulum unraveled the mechanism by which depletion of intracellular Ca²⁺ stores is communicated to store-operated Ca²⁺ channels located in the plasma membrane, triggering the activation of SOCE and intracellular Ca²⁺-dependent signaling cascades. Initial studies suggested a dominant function of STIM1 in SOCE and SOCE-dependent cellular functions compared to STIM2, especially those that participate in immune responses. Consequently, most of the subsequent studies focused on STIM1. However, during the last years, STIM2 has been demonstrated to play a more relevant and complex function than initially reported, being even important to sustain normal life in mice. These studies have led to reconsider the role of STIM2 in SOCE and its relevance in cellular physiology. This review is intended to summarize and provide an overview of the current data available about this exciting isoform, STIM2, and its actual position together with STIM1 in the mechanism of SOCE.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Histamine causes Ca entry via both a storemoperated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca²⁺ enters the cell through a (capacitative) Ca²⁺ entry pathway activated as a consequence of intracellular Ca²⁺ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 μM) caused a rapid but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, histamine caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Re-introduction of external Ca²⁺ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H₁-receptor antagonist, mepyramine (2 μM). A comparison was made between store-dependent Ca²⁺ entry consequent upon store mobilization with histamine in Ca²⁺-free medium and plateau phase Ca²⁺ entry resulting from stimulation with histamine in Ca²⁺-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P ? 0.0001) at the same concentration of histamine (10 μM). It is concluded that histamine causes Ca²⁺ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca²⁺ entry through other pathways.     

Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

Pore opening mechanism of CRAC channels

Three decades ago, James W. Putney Jr. conceptualized the idea of store-operated calcium entry (SOCE) to explain how depletion of endoplasmic reticulum (ER) Ca²⁺ stores evokes Ca²⁺ influx across the plasma membrane. Since the publication of this highly influential idea, it is now established that SOCE is universal among non-excitable and probably even many types of excitable cells, and contributes to numerous effector functions impacting immunity, muscle contraction, and brain function. The molecules encoding SOCE, the STIM and Orai proteins, are now known and our understanding of how this pathway is activated in response to ER Ca²⁺ store depletion has advanced significantly. In this review, we summarize the current knowledge of how Orai1 channels are activated by STIM1, focusing on recent work supporting a hydrophobic gating mechanism for the opening of the Orai1 channel pore.     

The Physiological Function of Store-operated Calcium Entry

Store-operated Ca²⁺ entry is a process whereby the depletion of intracellular Ca²⁺ stores signals the opening of plasma membrane Ca²⁺ channels. It has long been thought that the main function of store-operated Ca²⁺ entry was the replenishment of intracellular Ca²⁺ stores following their discharge during intracellular Ca²⁺ signaling. Recent results, however, suggest that the primary function of these channels may be to provide direct Ca²⁺ signals to recipients localized to spatially restricted areas close to the sites of Ca²⁺ entry in order to initiate specific signaling pathways.     

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.     

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.     

PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca2+ channels in PC12 cells

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca²⁺ entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca²⁺ elevation and is responsible for imparting extracellular Ca²⁺ influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca²⁺ pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca²⁺ entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca²⁺ entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca²⁺ channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca²⁺ mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca²⁺ influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca²⁺ mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca²⁺ release from ER stores and extracellular Ca²⁺ entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca²⁺ entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca²⁺ signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.