Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells

Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.     

Quantal calcium release by purified reconstituted inositol 1,4,5-trisphosphate receptors.

Release of intracellular Ca2+ by inositol 1,4,5-trisphosphate (InsP3) occurs through specific receptor proteins which are ligand-activated Ca2+ channels. Changes in intracellular Ca2+ regulate many cellular functions. This Ca2+ release is a discontinuous quantal process in which successive increments of InsP3 transiently release precise amounts of Ca2+ (refs 4-6). Possible explanations of quantal Ca2+ release have included rapid degradation of InsP3, reciprocity of Ca2+ release and sequestration, desensitization of InsP3 receptors, or actions of InsP3 on discrete compartments of Ca2+ with variable sensitivity to InsP3 (ref. 4). We successfully reconstituted InsP3-induced Ca2+ flux in vesicles containing only purified InsP3 receptor protein. The reconstituted vesicles retain the regulatory features of the InsP3 receptor, including phosphorylation sites and modulation of Ca2+ release by adenine nucleotides. Using these reconstituted vesicles, we show here that quantal flux of Ca2+ elicited by InsP3 is a fundamental property of its receptor.     

Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate

D-myo-inositol-1,4,5-trisphosphate (InsP3) is a putative intracellular second messenger for the mobilization of Ca2+ from intracellular stores, in particular, the endoplasmic reticulum. Specific binding sites on the endoplasmic reticulum may participate in the InsP3-induced release of Ca2+ from the Ca2+ pool. To examine the specific binding sites on the endoplasmic reticulum, we synthesized an arylazide derivative of InsP3 for photoaffinity labelling; InsP3 coupled to p-azidobenzoic acid (InsP3-pAB) using N,N'-carbonyldiimidazole (CDI) was obtained at a 9-11% yield. Here, we report that InsP3-pAB, but not an arylazide derivative of inositol-1,4-bisphophate (Ins(1,4)P2), causes the irreversible inhibition of InsP3-induced release of Ca2+ in saponin-permeabilized photo-irradiated macrophages. The irreversible inhibition by InsP3-pAB after photo-irradiation was prevented by a 10-fold excess of unmodified InsP3.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2+ release.

The temporal and spatial distribution of increases in intracellular Ca2+ concentration is an important factor in cellular signal transduction. Inositol 1,4,5-trisphosphate (InsP3) plays a key part in agonist-induced Ca2+ release, which can take place abruptly and in a confined space by a mechanism that is not fully understood. Here we analyse the kinetics of InsP3-induced Ca2+ release following flash photolysis of caged InsP3 or caged Ca2+, and demonstrate that Ca(2+)-dependent immediate feedback control is an important determinant of the time course of Ca2+ release. The positive feedback mechanism is also important for the 'loading dependence' of InsP3-induced Ca2+ release. Furthermore, our results support the operation of positive cooperativity in channel opening and feedback control augments the steep InsP3 concentration-Ca2+ release relation. These inherent properties of InsP3-induced Ca2+ release are expected to give rise to temporally abrupt and/or spatially confined Ca2+ release within the cell.     

Ca2+ release from endoplasmic reticulum is mediated by a guanine nucleotide regulatory mechanism

Ca2+ accumulation and release from intracellular organelles is important for Ca2+-signalling events within cells. In a variety of cell types, the active Ca2+-pumping properties of endoplasmic reticulum (ER) have been directly studied using chemically permeabilized cells. The same preparations have been extensively used to study Ca2+ release from ER, in particular, release mediated by the intracellular messenger inositol 1,4,5-trisphosphate (InsP3). So far, these studies and others using microsomal membrane fractions have revealed few mechanistic details of Ca2+ release from ER, although a recent report indicated that InsP3-mediated Ca2+ release from liver microsomes may be dependent on GTP. In contrast to the latter report, we describe here the direct activation of a specific and sensitive guanine nucleotide regulatory mechanism mediating a substantial release of Ca2+ from the ER of cells of the neuronal cell line N1E-115. These data indicate the operation of a major new Ca2+ gating mechanism in ER which is specifically activated by GTP, deactivated by GDP, and which appears to involve a GTP hydrolytic cycle.     

The second messenger linking receptor activation to internal Ca release in liver

The increase in cytosolic [Ca2+] induced by Ca-mobilizing hormones in liver is mainly due to release of Ca from intracellular stores. For Ca to be released from internal sites a messenger must be formed at the plasma membrane which diffuses into the cytosol to signal Ca release from the intracellular organelles. One of the first actions of these hormones is to cause breakdown of the polyphosphoinositides to form soluble inositol phosphates. Some evidence for the idea that these substances could be the second messenger has been obtained in pancreatic acinar cells. Here we have found that hormone activation of hepatocytes causes rapid breakdown of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] to form inositol trisphosphate ( InsP3 ). When applied to permeabilized hepatocytes, InsP3 releases Ca from non-mitochondrial ATP-dependent pools. This suggests that InsP3 could be the messenger linking Ca-mobilizing receptor activation to intracellular Ca release in liver.     

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Two polyphosphatidylinositide metabolites control two K+ currents in a neuronal cell

Hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) produces two prospective intracellular messengers: inositol 1,4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores; and diacylglycerol (DG), which activates protein kinase C. Here we show how the formation of these two substances triggered by one external messenger, bradykinin, leads to the appearance of two different sequential membrane conductance changes in the neurone-like NG108-15 neuroblastoma-glioma hybrid cell line. In these cells bradykinin rapidly hydrolyses PtdIns(4,5)P2 to InsP3 and DG, raises intracellular Ca2+ and hyperpolarizes then depolarizes the cell membrane. By voltage-clamp recording we show that the hyperpolarization results from the activation pharmacologically-identifiable species of Ca2+-dependent K+ current. This is also activated by intracellular injections of Ca2+ or InsP3 so may be attributed to the formation and action of InsP3. The subsequent depolarization results primarily from the inhibition of a different, voltage-dependent K+ current, the M-current that is also inhibited by DG activators. Hence we describe for the first time a dual, time-dependent role for these two intracellular messengers in the control of neuronal signalling by a peptide.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

Rapid mobilization of Ca2+ from rat insulinoma microsomes by inositol-1,4,5-trisphosphate

Several hormones and neurotransmitters raise the cytosolic free Ca2+ concentration by stimulating the influx of Ca2+ and/or by mobilizing stored Ca2+. However, the link between the agonist receptor on the cell surface and the organelle(s) from which Ca2+ is mobilized is unknown. One feature of the agonists that increase cytosolic Ca2+ is their rapid induction of phosphatidylinositol turnover and polyphosphoinositide hydrolysis; in some tissues this leads, within seconds, to a marked accumulation of the water-soluble products, inositol 1,4-bisphosphate ( Ins1 , 4P2 ) and inositol-1,4,5- trisphosphate ( Ins1 ,4, 5P3 ), suggesting that these might mediate Ca2+ mobilization from internal pools. Such an action of Ins1 ,4, 5P3 has recently been inferred from studies with permeabilized pancreatic acinar cells and hepatocytes. Here we show directly that Ins1 ,4, 5P3 rapidly releases Ca2+ from a microsomal fraction of rat insulinoma but not from mitochondria or secretory granules. Moreover, this response is transient and desensitizes the microsomes to subsequent Ins1 ,4, 5P3 additions. These results suggest that Ins1 ,4, 5P3 functions as a cellular messenger inducing early mobilization of Ca2+ from the endoplasmic reticulum.     

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.     

Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors.

Calcium release from intracellular stores is a point of convergence for a variety of receptors involved in cell signaling. Consequently, the mechanism(s) by which cells differentiate between individual receptor signals is central to transmembrane communication. There are significant differences in timing and magnitude of Ca2+ release stimulated by the m2 and m3 muscarinic acetylcholine receptors. The m2 receptors couple to a pertussis toxin-sensitive G protein to activate phosphatidyl inositol hydrolysis weakly and to stimulate small, delayed and oscillatory chloride currents. In contrast, m3 receptors potently activate phosphatidyl inositol hydrolysis and stimulate large, rapid and transient chloride currents by a pertussis toxin-insensitive G protein pathway. Using confocal microscopy, we now show that the m2- and m3-coupled Ca2+ release pathways can also be spatially distinguished. At submaximal acetylcholine concentrations, both receptors stimulated pulses of Ca2+ release from discrete foci in random, periodic and frequently bursting patterns of activity. But maximal stimulation of m2 receptors increased the number of focal release sites, whereas m3 receptors invariably evoked a Ca2+ wave propagating rapidly just beneath the plasma membrane surface. Analysis of pertussis toxin sensitivity and hybrid m2-m3 muscarinic acetylcholine receptors confirmed that these Ca2+ release patterns represent distinct cell signalling pathways.     

Possible association of alcohol tolerance with increased synaptic Ca2+ sensitivity

The inhibitory effect of ethanol on neurotransmitter release has been suggested to be due to either reduced Ca2+ entry or increased removal of free intracellular Ca2+ from the synapse. The use of the Ca2+ ionophore, A23187, to allow direct access of external Ca2+ to the presynaptic interior should help to determine which of these two factors is the more important, as ethanol should inhibit A23187-induced release of transmitter only if increased Ca2+ removal from the synapse is important. Here we show in rat striatal slices that, although 3H-dopamine release evoked by depolarization with 40 mM K+ is inhibited by 50 mM ethanol, the release evoked by A23187 is enhanced by the presence of ethanol in vitro. The results suggest that ethanol reduces depolarization-induced transmitter release by reducing Ca2+ entry to the presynaptic terminal. However, for brain slices taken from rats made tolerant to ethanol, 3H-dopamine release in the absence of ethanol showed altered characteristics; both K+ depolarization and A23187 released a significantly greater fraction of 3H-dopamine from these slices than from controls. Thus tolerance to the inhibitory effect of ethanol on release may develop by a mechanism involving increased sensitivity of the terminal to Ca2+ entry.