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.     

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca2+ influx in motoneuron development raises important questions about the regulation of Ca2+ signalling induced by voltage-dependent Ca2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca2+ current activated at 0 mV from a holding potential of -100 mV, approximately 45% was omega-conotoxin-GVIA (1 microM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca2+ currents. Based on this pharmacology, changes in intracellular free Ca2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K+ depolarization, the intracellular Ca2+ transient induced by the activation of each type of Ca2+ channel appeared to be quantitatively proportional to their Ca2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K+-induced intracellular free calcium concentration transients indicating both the absence of Ca2+-gated Ca2+-release channels and of a Ca2+-induced Ca2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca2+ channels that function as important regulators of intracellular Ca2+ signalling and may be involved in their development.     

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H- imidazole hydrochloride) stimulates phosphoinositide hydrolysis in human U373 MG astrocytoma cells

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imi dazole hydrochloride) stimulated the accumulation of [3H]inositol monophosphates ([3H]IP1) in human U373 MG astrocytoma cells prelabelled with [3H]inositol (EC50 15 +/- 1 microM, Hill coefficient 3.8 +/- 0.4). SK&F 96365-induced accumulation of [3H]IP1 increased linearly with time, but there was no initial rapid formation of [3H]IP3. SK&F 96365 also stimulated [3H]IP1 accumulation in human HeLa cells, but only to a small extent in slices of rat cerebral cortex and guinea-pig cerebellum. SK&F 96365-induced accumulation of [3H]IP1 in U373 MG cells increased as extracellular Ca2+ was increased from nominally zero to 4 mM, but there was no evidence that SK&F 96365 induced any marked entry of Ca2+ into cells; only an inhibition of store-refilling-induced Ca2+ entry was apparent. Further, the response to SK&F 96365 was additive with that to the Ca2+ ionophore ionomycin. Depolarization of the cells with raised K+ produced only a small stimulation of phosphoinositide hydrolysis. SK&F 96365 caused the release of Ca2+ from intracellular stores in U373 MG cells (EC50 26 +/- 14 microM), but thapsigargin induced only a small accumulation of [3H]IP1. Miconazole, another N-substituted imidazole, also stimulated [3H]IP1 accumulation in U373 cells.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 microM, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (> or = 5 microM), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 microM, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 microM, it blocks that current. AA (1-10 microM) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 microM, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 microM, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.     

Intracellular calcium release channel expression during embryogenesis

The release of intracellular calcium (Ca2+) via either inositol 1,4, 5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) activates a wide variety of signaling pathways in virtually every type of cell. In the present study we demonstrate that at early stages of development IP3R mRNA and functional IP3-gated Ca2+ release channels are widely expressed in virtually all tissues in murine embryos. As organogenesis proceeds, more specialized RyR channels are expressed in many cell types and the triggering mechanisms for intracellular Ca2+ release become more diverse to include IP3-dependent and voltage-dependent and Ca2+-induced Ca2+ release. As development proceeds virtually all cell types continue to express IP3R channels but in excitable cells including skeletal and cardiac muscles the major Ca2+ release channels are RyRs. This developmental switch from predominantly IP3-mediated to both IP3-mediated and IP3-independent pathways for intracellular Ca2+ release is consistent with data showing that IP3R plays an important regulatory role in cellular proliferation and apoptosis, whereas RyR is required for other cellular functions including muscle contraction.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

Cd2+ and Co2+ at micromolar concentrations mobilize intracellular Ca2+ via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd2+- and Co2+-induced intracellular Ca2+ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd2+, Co2+ and methacholine significantly increased the generation of 1,4,5-IP3. The results suggest that Cd2+ and Co2+ mobilize Ca2+ from IP3-sensitive Ca2+ stores, possibly through the presumptive Cd2+ receptor.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Dual effect of the anti-allergic astemizole on Ca2+ fluxes in rat basophilic leukemia (RBL-2H3) cells: release of Ca2+ from intracellular stores and inhibition of Ca2+ release-activated Ca2+ influx

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.     

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca(2+)-ATPases and Ca2+ release channels

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebrum.     

Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca2+ safeguards against spontaneous activity

BACKGROUND: Ca2+ waves allow effective delivery of intracellular Ca2+ signals to cytosolic targets. Propagation of these regenerative Ca2+ signals probably results from the activation of intracellular Ca2+ channels by the increase in cytosolic [Ca2+] that follows the opening of these channels. Such positive feedback is potentially explosive. Mechanisms that limit the spontaneous opening of intracellular Ca2+ channels are therefore likely to have evolved in parallel with the mechanism of Ca2+-induced Ca2+ release. RESULTS: Maximal rates of 45Ca2+ efflux from permeabilised hepatocytes superfused with medium in which the [Ca2+] was clamped were cooperatively stimulated by inositol 1,4,5-trisphosphate (IP3). A minimal interval of approximately 400 msec between IP3 addition and the peak rate of Ca2+ mobilisation indicate that channel opening does not immediately follow binding of IP3. Although the absolute latency of Ca2+ release was unaffected by further increasing the IP3 concentration, it was reduced by increased [Ca2+]. CONCLUSIONS: We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activation; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provided by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind before the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate.     

Induction of hippocampal long-term depression requires release of Ca2+ from separate presynaptic and postsynaptic intracellular stores

Studies have suggested that an increase in intracellular [Ca2+] is necessary for the induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission, and that release of Ca2+ from intracellular storage pools can be necessary to induce LTP. We investigated whether release of Ca2+ from intracellular stores also is required for the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Both thapsigargin (1 microM) and cyclopiazonic acid (1 microM), compounds that deplete all intracellular Ca2+ pools by blocking LTP-dependent Ca2+ uptake into intracellular compartments, blocked the induction, but not maintenance, of LTD by low-frequency stimulation (LFS) (1 Hz/15 min) without affecting baseline synaptic transmission. Washout of the reversible inhibitor cyclopiazonic acid restored the ability to induce LTD. In contrast, thapsigargin did not block depotentiation of LTP by 1 Hz LFS, suggesting that LTP causes a reduction in the threshold [Ca2+] necessary for LTD. Selective depletion of the ryanodine receptor-gated Ca2+ pool by bath application of ryanodine (10 microM) also blocked the induction of LTD, indicating a requirement for Ca(2+)-induced Ca2+ release. Impalement of CA1 pyramidal neurons with microelectrodes containing thapsigargin (500 nM to 200 microM) prevented the induction of LTD at synapses on that neuron without blocking LTD in the rest of the slice. In contrast, similar filling of CA1 pyramidal neurons with ryanodine (2 microM to 5 mM) did not block the induction of LTD. From these data, we conclude that the induction of LTD requires release of Ca2+ both from a presynaptic ryanodine-sensitive pool and from postsynaptic (presumably IP3-gated) stores.     

Ethanol administration delays recovery from acute pancreatitis induced by exocrine hyperstimulation

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.     

Signaltransduction in vascular endothelium: the role of intracellular calcium and ion channels

Endothelial cells (ECs) provide an ideal surface for blood flow. They inhibit the initiation of blood-clotting, but can also under certain conditions activate this process. ECs influence thrombolysis as well as thrombogenesis. They are "antigen-presenting cells" and play a key role in angiogenesis. In addition, ECs control the permeability of the barrier between bloodvessels and interstitium. One of their most important functions is the regulation of the diameter of the blood vessels and their adaptation to the demanded hemodynamic needs. The production and release of diverse compounds, which interfere with different neighboured target cells, initiate this plethora of functions. Ca2+ signals in endothelial cells play the key role in the release of NO, prostacyclin (PGI2), platelet activating factor (PAF), von Willebrand factor (vWF), tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI). Changes in the intracellular Ca2+ concentration ([Ca2+]i) are determined by release from intracellular stores and entry through the plasma membrane. The diversity of Ca2+ entry pathways and mechanisms of their control are described. At least two different types of Ca2+ entry channels exist: 1. typical highly Ca2+ selective ion channels which might be activated by depletion of intracellular Ca2+ stores (Ca2+ release-activated Ca2+ channels, CRAC), and 2. Non-selective Ca2+ permeable cation channels (NSC). The latter shares many features with an NSC induced by expression of the protein TRPC3. These channels are only weakly operated by store depletion and require a permissive Ca2+ and Ins(1,4,5)P3 concentration in the cytosol. CRAC channels are possible indirectly involved in Ca2+ entry during mechano-stimulation of ECs. After activation of these entry channels, influx of Ca2+ depends on the driving force. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identified as Kir2.1, a large-conductance, Ca2+ activated K+ channel (hslo) and at least two Cl- channels (a volume regulated Cl- channel, VRAC, and a Ca2+ activated Cl- channel, CaCC). It will be explained how these ion channels interact in the regulation of the long-lasting (plateau-type) increase in [Ca2+]i which mainly controls NO-synthesis and release. Furthermore, it will be demonstrated that Ca2+ oscillations depend on intracellular events rather than Ca2+ entry from the extracellular space.     

Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.     

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation-contraction coupling

BACKGROUND: Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells). RESULTS: In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2 microM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR. CONCLUSIONS: These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.     

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in a freshwater field crab, Barytelphusa guerini

The kinetics of efflux of calcium mobilized from intracellular stores following activation of human neutrophils with the synthetic chemotactic tripeptide, fMLP (1 microM), as well as that of the subsequent store-operated influx of this cation, has been measured by radiometric procedures using 45Ca. These procedures enabled distinction between net efflux and influx of 45Ca. Preincubation of neutrophils in medium containing 45Ca as the sole source of Ca2+, followed by activation with fMLP, resulted in a rapid efflux of the cation, which coincided with its release from intracellular stores. Efflux terminated at approximately 30 s after addition of fMLP to neutrophils and resulted in the loss of 42 +/- 3% (P < 0.005) of cell-associated 45Ca. Net influx of 45Ca, which was insensitive to the voltage-dependent Ca2+ channel blockading agent, verapamil (20 microM), could only be detected at 30-60 s after the addition of fMLP to neutrophils, and proceeded for about 5 min, resulting in intracellular concentrations of Ca2+ which were 27 +/- 3% (P<0.05) higher than preactivation levels. These results demonstrate that the efflux of cytoplasmic Ca2+ mobilized from intracellular stores during activation of neutrophils by fMLP, and the subsequent influx of extracellular Ca2+ to replete these stores, are chronologically distinct events in fMLP-activated neutrophils.     

The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl- secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 microM) induced both Ca2+ release from intracellular stores and plasmalemma Ca2+ influx. During NSP4-induced [Ca2+]i mobilization, [Na+]i homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+ entry into these cells. The ED50 of the NSP4 effect on peak [Ca2+]i mobilization was 4.6 +/- 0.8 nM. Pretreatment of cells with either 2.3 x 10(-3) units/ml trypsin or 4.4 x 10(-2) units/ml chymotrypsin for 1-10 min abolished the NSP4-induced [Ca2+]i mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+]i in human intestinal cells through receptor-mediated phospholipase C activation and IP3 production.     

Pituitary adenylate cyclase-activating polypeptide causes Ca2+ release from ryanodine/caffeine stores through a novel pathway independent of both inositol trisphosphates and cyclic AMP in bovine adrenal medullary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.