IP3-sensitive Pools Research Articles

IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-fos expression in hippocampal neurons

Ca2+ and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca2+ and cAMP signals, including some that are Ca2+-responsive, some that are cAMP-responsive and some that detect coincident Ca2+ and cAMP signals. Because Ca2+ and cAMP can influence each other’s amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca2+ are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca2+ buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP3 levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements – the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP3 metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca2+ stores. Our data indicate that Ca2+ release from IP3-sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA-dependent mechanism.

Astroglial cells synthesize and release endozepines, neuropeptides that are related to the octadecaneuropeptide ODN. Glial cells also express PACAP/VIP receptors. We have investigated the possible effect of PACAP on the release of ODN-like immunoreactivity (ODN-LI) by cultured rat astrocytes. Administration of PACAP27 and PACAP38 induced a concentration-dependent increase in secretion of ODN-LI whereas VIP was approximately 1000-fold less potent. The maximum effect of PACAP38 occurred after 5 min, then gradually declined during the next 10 min. The stimulatory effects of PACAP and VIP were abrogated by the PACAP antagonist PACAP6-38. PACAP38 stimulated cAMP formation, activated polyphosphoinositide turnover, and provoked calcium mobilization from IP3-sensitive pools. The PKA inhibitor H89 suppressed PACAP-induced secretion of ODN-LI, whereas PLC inhibitor U73122 and the PKC inhibitor chelerythrine had no effect. In contrast, U73122 restored the stimulatory action of PACAP on ODN-LI release and cAMP formation during prolonged (15 min) incubation with the peptide, and this effect was prevented by PMA. The present results demonstrate that PACAP stimulates endozepine release through activation of PAC1 receptors coupled to the AC/PKA pathway. Our data also show that activation of the PLC/PKC pathway down-regulates the effect of PACAP on endozepine release.

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity.

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.

Multiple effects of caffeine on Ca2+release and influx in human B lymphocytes

Caffeine has been used as a pharmacological tool to study the ryanodine receptor (RYR)-mediated Ca2+release from caffeine-sensitive, inositol 1,4,5,-trisphosphate (IP3)-insensitive pools. In the present study, we demonstrate multiple effects of caffeine on Ca2+homeostasis in human B lymphocytes. Although B cells express a functional RYR, which can be activated by 4-chloro-m-cresol following depletion of IP3-sensitive pools, caffeine does not activate RYR-mediated Ca2+release. Instead, caffeine dose-dependently inhibited IP3receptor (IP3R)-mediated Ca2+release, RYR-mediated Ca2+release and B cell receptor-initiated Ca2+influx, while high concentrations of caffeine (≥ 25mM) induced a Ca2+influx. In contrast with its ability to suppress receptor-stimulated Ca2+influx, caffeine had no significant effect on the store-operated Ca2+(SOC) channel-dependent Ca2+influx induced by thapsigargin. Thus, caffeine may act as an inhibitor on a single or multiple site(s) responsible for regulating the IP3R channel, RYR channel and presumably the receptor-mediated SOC channel. The present report may be the first demonstration of multiple effects of caffeine on Ca2+mobilization in single cell type. Our results suggest the need for caution regarding use of caffeine simply as a RYR-activator to study Ca2+homeostasis in eucaryotic cells.

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca 2+] i) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca 2+] i in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca 2+] i. ATP (1 mM) doubled the [Ca 2+] i after a few seconds and further induced a sustained increase of the [Ca 2+] i. Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca 2+] i. Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT–PCR analysis revealed that two metabotropic receptors (P2Y 1 and P2Y 2) and two ionotropic receptors (P2X 4 and P2X 7) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP 3-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X 7 receptors, which could be deleterious to these cells.

High concentrations of tricyclic antidepressants increase intracellular Ca2+ in cultured neural cells.

We examined the effect of tricyclic antidepressants on intracellular Ca2+ signalling in cultured cells of neuronal and glial origin. High concentrations of amitriptyline and desipramine increased the intracellular Ca2+ in PC-12 and U-87 MG cells. In PC-12 cells amitriptyline induced a biphasic rise in intracellular Ca2+. A rapid and transient increase due to release of Ca2+ from intracellular pools was followed by sustained elevation of [Ca2+]i due to influx from the extracellular medium. Desipramine evoked the Ca2+ release from intracellular pools but the influx of Ca2+ was not elicited. In U-87 MG cells both the drugs induced Ca2+ release from intracellular pools, however amitriptyline also induced a transient influx of Ca2+. To delineate the mechanisms involved in mobilization of Ca2+ by the drugs pharmacological agents that inhibit IP3 formation in cells and Ca2+ channel blockers were used and changes in [Ca2+]i and membrane potential were monitored. The results show that both the drugs release Ca2+ from IP3 sensitive pools by activation of phospholipase C and amitriptyline in addition activates a non specific cation channel in the plasma membrane of cells. Paradoxically at relatively lower concentrations (< 50 microM) amitriptyline and desipramine inhibited the Ca2+ signal induced by adenosine triphosphate in both the cell types. Our data demonstrate that tricyclic antidepressants at different doses may have inhibitory or stimulatory effects on cellular Ca2+ signalling.

Cyclic ADP-ribose induces Ca2+ release from caffeine-insensitive Ca2+ pools in canine salivary gland cells.

Cyclic ADP-ribose (cADPR), a novel putative messenger of the ryanodine receptor, was examined regarding its ability to mobilize Ca2+ from intracellular Ca2+ stores in isolated cells of parotid and submandibular glands of the dog. cADPR induced a rapid and transient Ca2+ release in the digitonin-permeabilized cells of salivary glands. cADPR-induced Ca2+ release was inhibited by ryanodine receptor antagonists ruthenium red, ryanodine, benzocaine, and imperatoxin inhibitor but not by the inositol 1,4,5-trisphosphate (IP3)-receptor antagonist heparin. Thapsigargin, at a concentration of 3 to 30 microM, inhibited IP3-induced Ca2+ release, while higher concentrations were required to inhibit cADPR-induced Ca2+ release. Cross-potentiation was observed between cADPR and ryanodine or SrCl2, suggesting that cADPR sensitizes the Ca2+-induced Ca2+ release mechanism. Cyclic AMP plays a stimulatory role on cADPR- and IP3-induced Ca2+ release in digitonin-permeabilized cells. Calmodulin also potentiated cADPR-induced Ca2+ release, but inhibited IP3-induced Ca2+ release. Acetylcholine and ryanodine caused the rise in intracellular free Ca2+ concentration ([Ca2+]i) in intact submandibular and parotid cells. Caffeine did not produce any increase in Ca2+ release or [Ca2+]i rise in any preparation. ADP-ribosyl cyclase activity was found in the centrifuged particulate fractions of the salivary glands. These results suggest that cADPR serves as an endogenous modulator of Ca2+ release from Ca2+ pools through a caffeine-insensitive ryanodine receptor channel, which are different from IP3-sensitive pools in canine salivary gland cells. This system is positively regulated by cyclic AMP and calmodulin.

Hypocalcemia modifies the intra-cellular calcium response to the α1-adrenergic agent phenylephrine in rat hepatocytes

In vivo, extracellular calcium ([Ca 2+] e) homeostasis is maintained within a very narrow range by the calcium regulating hormones. At the cellular level, the response to many agents is transduced by changes in cytosolic Ca 2+ ([Ca 2+] i) which involves both mobilization of cellular pools and entry of [Ca 2+] e through plasma membrane channels. To investigate the cellular effects of chronic hypocalcemia (Ca-) on [Ca 2+] i homeostasis, hepatocytes, a cell type well characterized for its [Ca 2+] i response, were used. Data indicate that Ca- leads to a significant shift to the left in the basal resting cytosolic Ca 2+ concentration distribution curve with half-maximum cumulative frequency of 119 versus 149 nM in Ca- and normal rats (N) respectively ( P < 0.0001). The response to the α 1-adrenergic agonist phenylephrine (Phe) was also influenced by Ca- with a dampening of the dose-response curve, a significant decrease in the frequency of sustained responses ( P < 0.001), and significant changes in the oscillation pattern. Indeed, hepatocytes obtained from Ca- exhibited a higher frequency of large amplitude, low frequency oscillations than N most particularly at the 2 and 5 μM Phe dose while N predominantly exhibited low amplitude, high frequency oscillations on sustained plateaus ( P < 0.001). IP 3 receptor (IP 3R) binding studies and Ca 2+ mobilization from IP 3-sensitive pools showed that IP 3R was highly sensitive to the prevailing Ca 2+ with, in the range of resting (Ca 2+] i R affinity significantly lower in Ca- than in N. Upon exposure of permeabilized cells to 25 μM IP 3, Ca 2+ mobilization from the IP 3-sensitive intracellular pool was significantly reduced by Ca- ( P < 0.05) suggesting a decrease in the IP 3-mobilizable Ca 2+ pool in Ca-. Our results indicate that hypocalcemia significantly alters [Ca 2+] i signalling by perturbing the initial response to agonist and the [Ca 2+] i response pattern. In addition, the decrease in Ca 2+-mobilization from IP 3-sensitive pools suggests that hypocalcemia may also lead to a decrease in the Ca 2+ content of intracellular pools.

Effect of Linoleic Acid Hydroperoxide on Endothelial Cell Calcium Homeostasis and Phospholipid Hydrolysis

The relationship between intracellular free calcium ion concentrations ([Ca2+]i) and release of arachidonic acid from membrane phospholipids following peroxidation was examined in rabbit aortic endothelial cells treated with linoleic acid hydroperoxide (LOOH). LOOH (0.1–0.4) μmol/106cells) caused a rapid and dose-dependent transient increase in [Ca2+]iin the presence of extracellular Ca2+that remained elevated over baseline for 15 to 30 s. In the absence of extracellular Ca2+, LOOH also evoked a transient increase in [Ca2+]iof lesser magnitude which immediately returned to basal (or below basal) levels. In this regard, the rise in intracellular Ca2+after LOOH or vasopressin (AVP) treatments involved, at least in part, related intracellular pools that in each case was followed by influx of extracellular Ca2+. The intracellular membrane sources of Ca2+remain unidentified as common sources known to be affected by vasopressin were not directly involved. Most notably, the LOOH evoked rise in [Ca2+]iwas not associated with release of IP3, suggesting that the source of intracellular Ca2+is not IP3-sensitive pools. However, pretreatment with LOOH strongly inhibited the rise in [Ca2+]iupon subsequent addition of AVP or LOOH and the extent of such inhibition was dependent on the availability of free intracellular Ca2+and presence of extracellular Ca2+. These findings suggest that reuptake of Ca2+into intracellular membrane pools is reduced in the presence of LOOH and/or the availability of Ca2+from agonist-sensitive sites is inhibited by LOOH. An increase in free 20:4 levels was found after LOOH treatment that was only partly prevented using intracellular Ca2+chela tors which maintained [Ca2+]iat basal levels after LOOH treatment. These findings suggest that LOOH induction of phospholipid hydrolysis proceeds following small transients in [Ca2+]ithat are considerably less than that evoked by agents such as AVP, approximating basal Ca2+concentrations. Inhibition of LOOH-induced lipid peroxidation by vitamin E also prevented the rise in [Ca2+]iand 20:4 release indicating that phospholipid hydrolysis is dependent, at least in part, on membrane lipid peroxidation. Inhibition of protein kinase C (PKC) completely blocked LOOH-induced release of 20:4 but had little effect on the LOOH-induced rise in [Ca2+]i, suggesting an indirect rela- tionship between LOOH-induced membrane Ca2+signalling events, with intervention via PKC-mediated induction of phospholipid hydrolysis. A rapid and progressive translocation of PKC to the membrane fraction was evident after LOOH addition over the time course corresponding to the maximal release of 20:4 which was also inhibited by vitamin E. The findings are discussed in terms of possible mechanisms underlying the stimulation of phospholipase(s), purportedly phospholipase A2(PLA2) activity, by peroxidation of cell membrane phospholipids and enhancement of protein kinase activity.

Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch

Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (<1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca2+]i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura-2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induce no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca2+]i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 microM gadolinium ions, by 50 microM nifedipine, or 250 microM verapamil, suggesting an influx of calcium through stretch-activated (SA) channels and L-type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), indicating that IP3 sensitive pools, IP3 insensitive pools, GS alpha subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of Gi alpha and G(o) alpha subunits, completely abolished calcium transients and oscillations. These results indicate that Ca2+ flux due to mechanical stretching is likely mediated through SA ion channels and is dependent on tyrosine kinases, pertussis toxin-sensitive subunits of G-proteins, and actin filaments.

Stimulation of Ca2+ influx by endothelin-1 is subject to negative feedback by elevated intracellular Ca2+

Endothelin-1 (ET-1) has been shown to require Ca2+ influx for activation of vascular smooth muscle in vivo, but in vitro models show that ET-1 mobilizes intracellular Ca2+ and is independent of extracellular Ca2+. We present data that suggest ET-1 modulates cellular responses through a dual mechanism involving both phosphatidylinositol turnover and Ca2+ channel activation. Addition of low concentrations of ET-1 (less than 10(-9) M) to serum-deprived quiescent Rat-1 cells stimulated Ca2+ influx while having little effect on diacylglycerol (DG) release or intracellular Ca2+ levels. In contrast, higher concentrations of ET-1 (greater than 10(-9) M) stimulated intracellular Ca2+ transients and release of inositol trisphosphate (IP3) and DG but did not activate Ca2+ uptake. Stimulation of Ca2+ influx at low [ET-1] could not be accounted for by depletion of intracellular IP3-sensitive pools. Neither the stimulation of Ca2+ influx at low [ET-1] nor the inhibitory actions of high [ET-1] could be mimicked by the activation of protein kinase C. We tested the hypothesis that elevated intracellular Ca2+ was inhibitory for Ca2+ influx. When intracellular Ca2+ transients were maintained below approximately 165 nM by chelation with BAPTA or BAPTA derivatives with altered affinity for Ca2+, Ca2+ influx was stimulated over the entire range of ET-1 concentrations. In addition, experimentally elevating intracellular Ca2+ levels with the tumor promoter thapsigargin abolished ET-1-stimulated Ca2+ influx. These data suggest that the biological consequences of ET-1 release may be determined by local concentration differences. Thus in vascular smooth muscle cells ET-1 may act either to mobilize intracellular Ca2+ or to promote Ca2+ influx, depending on the distance from the endothelial cell source in the vascular wall. The activation of different processes by low and high ET-1 concentrations may determine the physiological response to ET-1 stimulation in vivo.