Receptor Antagonist Heparin Research Articles

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons.

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.

Glucagon-like peptide-1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate-dependent intracellular Ca 2+ mobilization

The signal transduction pathway of the cloned human glucagon-like peptide-1 (GLP-1) receptor was studied in voltage-clamped Xenopus oocytes. Binding of GLP-1(7–36)amide was associated with cAMP production, increased [Ca 2+] i and activation of Ca 2+-dependent Cl − current. The effect of GLP-1(7–36)amide reflects intracellular Ca 2+ mobilization and was suppressed by injection of the Ca 2+ chelator BAPTA and the inositol trisphosphate receptor antagonist heparin. The responses were not mimicked by the adenylate cyclase activator forskolin and unaffected by the protein kinase A (PKA) inhibitor Rp-cAMPS. We conclude that GLP-1 receptor expression in Xenopus oocytes evokes inositol trisphosphate-dependent intracellular Ca 2+ mobilization independent of the cAMP/PKA signaling pathway.

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells.

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.

Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca channels

Mechanisms underlying the 5-HT2A receptor induction of intracellular Ca2 mobilization and Ca2 influx in type I astroglial cells in primary culture from newborn rat cerebral cortex were evaluated. The 5-HT-evoked Ca2-transients, inhibited by the 5-HT2A antagonists ketanserin or 4-(4-fluorobenzoyl)-1-(4-phenylbutyl) piperidine oxalate, consisted of an initial peak caused by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2 release from internal stores, and a second sustained part which was due to Ca2 transport over the plasma membrane. The responses were pertussis toxin-insensitive, suppressed by the phospholipase C inhibitor neomycin and were inhibited by the Ca2-ATPase inhibitor thapsigargin. Furthermore, the responses were inhibited by the IP3 receptor antagonist heparin. When the second sustained part of the 5-HT-evoked response was studied, it was concluded that Ca2 influx was not a result of opening of voltage operated calcium channels of either L, N or T-type. Instead it appeared that Ca2 entered the cells through specialized voltage independent Ca2 channels which were dependent of the IP3 production and subsequent Ca2 release from internal stores. From this, we conclude that 5-HT opens Ca2 channels in astrocytes which closely resemble depletion-operated Ca2 channels (DOCCs).

Signal transduction in Jurkat T lymphocytes. Evidence for early Ca2+ movements between the cytoplasm and the nucleoplasm in activated cells.

Spatial analyses of the distribution of Ca2+ in resting and activated T and B lymphocytes have shown that the bulk of increased [Ca2+]i appears to be associated with the nuclear region. These observations suggest that Ca2+ is released from the perinuclear space or that it diffuses to the nucleoplasm, or both. We have used laser scanning confocal microscopy to assess whether cytoplasmic diffusion of Ca2+ could contribute to the rise in nuclear Ca2+. We found that the activation of individual Jurkat cells by use of an anti-Ti (beta-subunit) mAb induced a nucleus-associated increase in [Ca2+]i. In cells loaded with the InsP3 receptor antagonist heparin, the nuclear Ca2+ response was abolished but not the response to thapsigargin. Evidence for a cytoplasmic Ca2+ response was obtained by loading Jurkat cells with a cytoplasm-restricted Ca2+ probe (Calcium Green-1-Dextran). These observations suggested that a process of diffusion of cytoplasmic Ca2+ contributed to the rise of nuclear Ca2+ in Jurkat T cells. This interpretation was supported by the findings (1) that rapid scanning of thapsigargin-released Ca2+ showed an inverse relationship between the levels of cytoplasmic and nuclear Ca2+ and (2) that modulation of the external concentration of Ca2+ in thapsigargin-treated Jurkat cells showed a time-dependent decrease of fluorescence from the nucleoplasm that was reversed by raising the concentration of external Ca2+. We conclude that Ca2+ can rapidly diffuse between the cytoplasm and the nucleoplasm in activated Jurkat T lymphocytes and that hydrophilic Ca2+ probes largely partition to the nucleoplasm, thus giving rise to distorted nucleus-to-cytoplasm fluorescence ratios.

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus.

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

Endothelin-activated calcium signaling in enteric glia derived from neonatal guinea pig.

The ability of guinea pig enteric glia to respond to endothelins was examined using fura 2-based digital microscopy in glial cells derived from guinea pig taenia coli. Each isoform of endothelin (ET-1, ET-2, ET-3) evoked dose-dependent and equipotent increases in intracellular Ca2+ concentration ([Ca2+]i) and in percentage of cells responding, 4alaEt-1, an ETB receptor agonist, elicited similar [Ca2+]i increments. BQ-788, an ETB antagonist, inhibited [Ca2+]i responses to endothelin. Preincubation of glia with U-73122 a phospholipase C inhibitor, abolished the [Ca2+]i response to ET-3 exposure. Thapsigargin also eliminated ET-3-evoked Ca2+ signaling. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, introduced into glial cells by radio frequency electroporation, blocked [Ca2+]i responses to ET-3 (100 nM) in 63% of glia. Sustained elevation in [Ca2+]i was abolished by removal of Ca2+ from the buffer and inhibited 85. -3% by Ni2+ (1 mM). Preincubation of glia with 100 nM phorbol 12-myristate 13-acetate (24 h) also inhibited sustained increments in [Ca2+]i by 87%. The presence of IP3 receptors in enteric glia was confirmed by immunofluorescent confocal microscopy.

IP 3 receptor antagonist heparin uncompetitively inhibits [ 3H](+)-SKF-10047 binding to σ receptors

Interaction of σ receptors with intracellular Ca 2+ channel blocker and modulators was examined. Ryanodine and inositol 1,4,5-trisphosphate (IP 3) did not inhibit [ 3H](+)- N-allylnormetazocine ([ 3H](+)-SKF-10047) binding to σ receptors from either brain microsomal fractions or liver membrane extracts of the rat. However, the IP 3 receptor antagonist heparin inhibited [ 3H](+)-SKF-10047 to σ receptors in an uncompetitive manner with a K i of 93 μM. These results suggest that σ receptors may bear some relationship with IP 3 receptor associated proteins or channels.

Regulation of inositol trisphosphate-induced membrane currents in Xenopus oocytes by a Jurkat cell calcium influx factor.

The functional interactions of a Jurkat cell-derived calcium influx factor (CIF) with Ins(1,4,5)P3 were examined by microinjection and voltage-clamp recording of current responses in Xenopus oocytes. CIF, which stimulates Ca2+ entry directly on microinjection, was active at dilutions at which it had no direct effect by augmenting both initial rapid Ins(1,4,5)P3-mediated Ca2+ discharge-activated currents and later sustained Ca2+ entry-activated currents. Augmented initial membrane currents were 3-5-fold greater in peak amplitude than currents evoked by injection of the same dose of Ins(1,4,5)P3 alone. The augmented initial response was not decreased by removal of extracellular Ca2+, suggesting that there is potentiation of Ins(1,4,5)P3-mediated discharge from intracellular Ca2+ stores. However, the augmentation of Ins(1,4,5)P3-mediated discharge cannot be due to an enhanced production of endogenous Ins(1,4,5)P3 because maximal Ins(1,4,5)P3-activated currents saturate (approx. 500 nA) with supramaximal levels of Ins(1,4,5)P3 (10-50 microM). Depletion of Ca2+ stores, by pretreatment with thapsigargin or by prior injection with the Ins(1,4,5)P3 receptor antagonist heparin, abolished membrane currents elicited by Ins(1,4,5)P3/CIF co-injection, further suggesting that the Ins(1,4,5)P3 receptor was the target for the initial-current potentiating actions of CIF. In this regard, CIF also induced augmented initial currents with co-injection of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. The augmentation of Ins(1,4,5)P3-mediated currents by CIF was bell-shaped with regard to Ins(1,4,5)P3 concentration, reminiscent of the regulatory influence of Ca2+ on Ins(1,4,5)P3 responses. Co-injection of Ins(1,4,5)P3 and CIF also augmented (2-3-fold) later current responses arising from sustained Ca2+ entry. The augmented late-current responses were not due to enhanced Ca2+ store depletion because supramaximal levels of Ins(1,4,5)P3 (50 microM) or injection of the poorly metabolized Ins(1,4,5)P3 analogue, Ins(2,4,5)P3, cannot activate the same magnitude of Ca(2+)-entry-dependent currents. These results suggest that CIF at low levels interacts with Ins(1,4,5)P3 to sensitize two pathways of Ca2+ signalling: initial discharge and later Ca2+ entry. Thus under physiological conditions CIF might be more potent as a co-messenger than as a direct Ca2+ entry signal and might provide a novel type of direct feedback regulation between the stores-activated influx pathway and the Ins(1,4,5)P3 receptor. Moreover these results suggest that CIF modulation of the receptor for Ins(1,4,5)P3 may underlie control of both augmentation of discharge and Ca2+ entry, as has been predicted from the conformational coupling model of Ca2+ entry.

Serotonin regulates gap junction coupling in the developing rat somatosensory cortex.

To further elucidate the role of the neuromodulatory transmitter serotonin (5-HT) during early postnatal development of the neocortex, we investigated the effects of 5-HT on gap junction coupling in the somatosensory cortex of rats aged between postnatal days 7 and 10. The gap junction-permeable tracer neurobiotin was injected into single neurons via microelectrodes or patch pipettes. Under control conditions, clusters of about 25 tracer-coupled neurons were observed. Serotonin reduced dye-coupling between lamina II/III pyramidal cells in a concentration-dependent and reversible manner. The 1,4,5-inositol triphosphate (IP3) receptor antagonist heparin as well as the protein kinase C inhibitor NPC 15437 suppressed the uncoupling action of 5-HT, suggesting that the serotonergic effect involved IP3 receptor-mediated release of calcium ions from intracellular stores. In contrast, the 5-HT-induced reduction in gap junction coupling was not antagonized by Rp-adenosine-3',5'-cyclic monophosphothionate, an inhibitor of cAMP dependent protein kinase. The uncoupling effect of 5-HT was mimicked by 5-HT2 receptor agonists and antagonized by the 5-HT2 receptor antagonist ritanserin, indicating that 5-HT suppressed gap junction coupling via activation of 5-HT2 class receptors. Our results suggest that the developmental functions of 5-HT not only involve the modulation of chemical synaptic transmission but also include the regulation of the gap junctional communication system during differentiation of the neocortex.

ATP causes release of intracellular Ca2+ via the phospholipase C beta/IP3 pathway in astrocytes from the dorsal spinal cord

Calcium signaling within astrocytes in the CNS may play a role comparable to that of electrical signaling within neurons. ATP is a molecule known to produce Ca2+ responses in astrocytes, and has been implicated as a mediator of intercellular Ca2+ signaling in other types of nonexcitable cells. We characterized the signal transduction pathway for ATP-evoked Ca2+ responses in cultured astrocytes from the dorsal spinal cord. Nearly 100% of these astrocytes respond to extracellularly applied ATP, which causes release of Ca2+ from an intracellular pool that is sensitive to thapsigargin and insensitive to caffeine. We found that intracellular administration of IP3 also caused release of Ca2+ from a thapsigargin-sensitive intracellular pool, and that IP3 abolished the response to ATP. The ATP-evoked Ca2+ response was blocked by the IP3 receptor antagonist heparin, applied intracellularly, but not by N-desulfated heparin, which is not an antagonist at these receptors. The Ca2+ response caused by ATP was also blocked by a phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. Increases in [Ca2+]i were elicited by intracellular application of activators of heterotrimeric G-proteins, GTP gamma S and AIF4-. On the other hand, [Ca2+], was unaffected by a G-protein inhibitor, GDP beta S, but it did abolish the Ca2+ response to ATP. Pretreating the cultures with pertussis toxin did not affect responses to ATP. Our results indicate that in astrocytes ATP-evoked release of intracellular Ca2+ is mediated by IP3 produced as a result of activating phospholipase C coupled to ATP receptors via a G-protein that is insensitive to pertussis toxin. ATP is known to be released under physiological and pathological circumstances, and therefore signaling via the PLC-IP3 pathway in astrocytes is a potentially important mechanism by which ATP may play a role in CNS function.

Inositol 1,4,5-trisphosphate activates receptor-mediated calcium entry by two different pathways in hepatocytes.

The quenching of fura-2 fluorescence by the influx of extracellular Mn2+ was measured to indicate the flux rates through receptor-operated calcium channels in the plasma membrane of rat hepatocytes. Neomycin, an inhibitor of phospholipase C, inhibited the vasopressin-induced influx of Mn2+. Thus, the agonist-induced entry of extracellular calcium into hepatocytes is linked to a phospholipase C-generated second messenger. Microinjection of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or 3-deoxy-3-fluoro-Ins(1,4,5)P3 revealed that Ins(1,4,5)P3 rather than Ins(1,3,4,5)P4 is responsible for calcium entry. The activation of phospholipase C by vasopressin produced an influx of Mn2+ independent of the depletion of intracellular calcium stores if this depletion was delayed by the Ins(1,4,5)P3 receptor antagonist heparin or by the use of a low agonist concentration. Thapsigargin, an inhibitor of the store calcium pump, leading to an Ins(1,4,5)P3-independent emptying of stores, gave a short living signal (less than 3 min) for calcium entry. We propose that Ins(1,4,5)P3 is able to stimulate calcium entry by two pathways. (a) Ins(1,4,5)P3 activates receptor-operated calcium channels in a direct manner. The calcium entry resulting from this is followed (b) by the Ins(1,4,5)P3-induced depletion of calcium stores, producing a store-dependent entry.

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine-sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP-ribose (cADP-ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP-ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.

Thapsigargin-induced Ca2+ mobilization in acutely isolated mouse lacrimal acinar cells is dependent on a basal level of Ins(1,4,5)P3 and is inhibited by heparin

The tumour-promoting agent thapsigargin has been shown to inhibit the microsomal Ca(2+)-ATPase and cause Ca2+ mobilization in a variety of cell types including exocrine acinar cells [Bird, Obie and Putney (1992) J. Biol. Chem. 267, 18382-18386]. When applied to acutely isolated lacrimal acinar cells, thapsigargin caused a slow biphasic activation of both the Ca(2+)-dependent K+ and Cl- currents measured using the whole-cell patch-clamp technique. If the only action of thapsigargin is to inhibit sequestration into Ca2+ pools, then Ca2+ mobilization following exposure to thapsigargin indicates that there is a significant 'leak' of Ca2+ into the cytoplasm, which is normally countered by Ca(2+)-ATPase activity. In the present study, we introduced the Ins(1,4,5)P3 receptor antagonist heparin (200 micrograms/ml) into lacrimal acinar cells via the patch-clamp pipette. Following a 5 min preincubation in the presence of heparin, neither acetylcholine (1 microM) nor thapsigargin (1 microM) caused any significant increase in either Ca(2+)-dependent current. Caffeine has been shown to suppress basal Ins(1,4,5)P3 levels in exocrine acinar cells [Toescu, O'Neill, Petersen and Eisner (1992) J. Biol. Chem. 267, 23467-23470]. Preincubation with caffeine (10 mM) also inhibited the response to subsequent exposure to thapsigargin. These data suggest that, in acutely isolated lacrimal cells, the source of the Ca2+ leak which gives rise to Ca2+ mobilization following inhibition of Ca2+ re-uptake by thapsigargin is Ca2+ release, from Ins(1,4,5)P3-dependent Ca2+ pools, caused by resting Ins(1,4,5)P3 levels.

Ca 2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca 2+ signals, since they are morphologically polarized and generate agonist-specific Ca 2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca 2+ signals and the Ca 2+-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca 2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca 2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimick these responses. The shortlasting local Ca 2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Calcium oscillations in pancreatic acinar cells, evoked by the cholecystokinin analogue JMV-180, depend on functional inositol 1,4,5-trisphosphate receptors.

It has been reported that the synthetic heptapeptide cholecystokinin (CCK) analogue JMV-180 evokes cytosolic Ca2+ signals in pancreatic acinar cells via mechanisms that do not include either the generation or action of inositol 1,4,5-trisphosphate (InsP3) (Saluja, A. K., Dawra, R. K., Lerch, M. M., and Steer, M. L. (1992) J. Biol. Chem. 267, 11202-11207; Yule, D. I., and Williams, J. A. (1992) J. Biol. Chem. 267, 13830-13835). We have now investigated the CCK- and JMV-180-evoked cytosolic Ca2+ oscillations by measurement of the Ca(2+)-sensitive ion currents in internally perfused mouse pancreatic acinar cells. We find that the InsP3 receptor antagonist heparin (500 micrograms/ml) blocks Ca2+ oscillations induced by both CCK (5-20 pM) and JMV 180 (10-40 nM), whereas de-N-sulfated heparin (500 micrograms/ml), which does not affect InsP3 binding to its receptor, fails to inhibit the responses to the two agonists. We conclude that the cytosolic Ca2+ oscillations evoked by both CCK and JMV-180 are dependent on functional InsP3 receptors.

Inositol trisphosphate-independent agonist-stimulated calcium influx in rat pancreatic acinar cells.

CCK-JMV-180 is a cholecystokinin analog that stimulates digestive enzyme secretion from pancreatic acinar cells but does not cause either generation of inositol 1,4,5-trisphosphate or depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool. We report that CCK-JMV-180 can accelerate Ca2+ influx into fura-2-loaded dispersed rat pancreatic acini and single acinar cells. Furthermore, CCK-JMV-180 accelerates Ca2+ influx into cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin and into acini loaded with the Ca(2+)-chelating agent BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate that agonist-stimulated Ca2+ influx can occur (a) without depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool, (b) without a rise in cytoplasmic free Ca2+ concentrations, and (c) after blockade of inositol 1,4,5-trisphosphate receptors. They suggest that depletion of an inositol 1,4,5-trisphosphate-independent intracellular Ca2+ storage pool and/or generation of a non-inositol 1,4,5-trisphosphate second messenger by CCK-JMV-180 may be a sufficient signal for acceleration of Ca2+ influx into rat pancreatic acinar cells.

Local and global cytosolic Ca 2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate

Submaximal stimulation with agonists generating inositol 1,4,5-trisphosphate (IP 3) evokes cytosolic Ca 2+ oscillations in many different cell types. In general, each Ca 2+ rise is initiated from a specific region near the plasma membrane and then spreads as a wave throughout the cell. We now demonstrate that low (physiological) agonist concentrations evoke local cytosolic Ca 2+ spikes in the secretory pole of single mouse pancreatic acinar cells that are particularly sensitive to blockade by the IP 3 receptor antagonist heparin. These spikes can occur alone or repetitively or can precede longer lasting Ca 2+ signals that spread throughout the cell. Intracellular IP 3 application mimics these agonist actions. The short-lasting local Ca 2+ spikes provide an economical signaling mechanism and are of physiological significance since they activate Ca 2+-dependent Cl − and cation currents important for control of fluid secretion.

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes.

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.

Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2.

Stimulation of cells with calcium-mobilizing agonists frequently results in inositol 1,4,5-trisphosphate (InsP3)-mediated discharge of Ca from an internal store. We report here a technique for directly monitoring Ca within this and other stores in gastric epithelial cells. This technique takes advantage of the propensity of the acetoxymethyl ester derivative of the fluorescent dye mag-fura-2 (which is sensitive to Ca concentrations above 5 microM) to accumulate in subcellular compartments where it can report changes in the free Ca concentration. Intact dye-loaded cells responded to cholinergic stimulation with a decrease in the 350 nm/385 nm excitation ratio, as measured in individual cells with a digital imaging microscope, consistent with reduced Ca concentration in one or more cellular compartments. When cells were permeabilized with digitonin and incubated in an "intracellular buffer," the cytoplasmic dye was released, leaving the mag-fura-2 in the internal store. InsP3 caused the ratio from the trapped indicator to decrease (i.e., Ca was released) in a dose-dependent manner, and this effect was blocked by the InsP3 receptor antagonist heparin. Ca sequestration into the internal store was ATP-dependent, and reuptake into the InsP3-sensitive pool was blocked by thapsigargin, a specific inhibitor of the Ca-ATPase of the internal store. We used this technique to investigate the role of Cl on the release and reloading of the InsP3-sensitive internal store and found that Ca uptake was reduced in Cl-free solutions, suggesting an important function for Cl in the refilling of this pool.