NAADP induces pH changes in the lumen of acidic Ca2+ stores

NAADP (nicotinic acid-adenine dinucleotide phosphate)-induced Ca2+ release has been proposed to occur selectively from acidic stores in several cell types, including sea urchin eggs. Using fluorescence measurements, we have investigated whether NAADP-induced Ca2+ release alters the pH(L) (luminal pH) within these acidic stores in egg homogenates and observed their prompt, concentration-dependent alkalinization by NAADP (but not beta-NAD+ or NADP). Like Ca2+ release, the pH(L) change was desensitized by low concentrations of NAADP suggesting it was secondary to NAADP receptor activation. Moreover, this was a direct effect of NAADP upon the acidic stores and not secondary to increases in cytosolic Ca2+ as it was not mimicked by IP3 (inositol 1,4,5-trisphosphate), cADPR (cyclic adenine diphosphoribose), ionomycin, thapsigargin or by direct addition of Ca2+, and was not blocked by EGTA. The results of the present study further support acidic stores as targets for NAADP and for the first time reveal an adjunct role for NAADP in regulating the pH(L) of intracellular organelles.     

Multiplicity of Ca2+ messengers and Ca2+ stores: a perspective from cyclic ADP-ribose and NAADP

It is generally believed that multiple Ca2+ stores are present in cells, a notion that has now been made substantive by the discovery of multiple Ca2+ mobilizing messengers. Cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP) are two such messengers that are derived from NAD and NADP, respectively. A wide variety of cells, from plants to mammals, including human, have been shown to be responsive to these two novel Ca2+ messengers. Not only are their structures and mechanisms of action different, their targeted Ca2+ stores are also distinct and separable. This article explores the implications of the multiplicity of Ca2+ stores in cellular signaling. Special emphasis will be put on the recent progress in the understanding of the physiological functions of NAADP.     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

NAADP controls cross-talk between distinct Ca2+ stores in the heart

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation

In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.     

Coordination of Ca2+ signalling by NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.     

Intercellular Bridge Mediates Ca2+ Signals between Micropatterned Cells via IP3 and Ca2+ Diffusion

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca²⁺ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca²⁺ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca²⁺ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca²⁺ diffusion and endogenous Ca²⁺ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca²⁺ communication. Extracellular Ca²⁺ and the paracrine pathway have practically no effect on the endogenous Ca²⁺ response, demonstrated by application of Ca²⁺-free medium, exogenous ATP, and P2Y₁₃ receptor antagonist. In contrast, the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin and inositol trisphosphate (IP₃) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca²⁺ increase, which signifies involvement of IP₃-sensitive calcium store release. Notably, passive Ca²⁺ diffusion into the connected cell can clearly be detected when IP₃-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca²⁺ diffusion and IP₃-mediated endogenous Ca²⁺ response contribute to the Ca²⁺ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.     

Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitochondrial Ca2+ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called 'calciosome'. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of approximately 10(-7) mol/l to approximately 10(-6) mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-insensitive Ca2+ pools Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a Ca2+/H+ exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Ca2+ signalling: a new route to NAADP

The LKB1 serine/threonine kinase is a tumour suppressor responsible for the inherited familial cancer disorder Peutz-Jeghers syndrome and is inactivated in a large percentage of human lung cancers. LKB1 acts a master kinase, directly phosphorylating and activating a family of 14 AMPK (AMP-activated protein kinase)-related kinases which control cell metabolism, cell growth and cell polarity. In this issue of the Biochemical Journal, Hardie and colleagues discover an alternative splice form of LKB1 that alters the C-terminus of the protein containing a few known sites of post-translational regulation. Although widely expressed, the short isoform (LKB1(s)) is the sole splice isoform expressed in testes, and its expression peaks at the time of spermatid maturation. Male mice lacking the LKB1(s) isoform have dramatic defects in spermatozoa, resulting in sterility.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Rapidly exchanging Ca2+ stores: Role in the control of Ca2+ homeostasis and [Ca2+] in oscillations

The rapidly exchanging intracellular calcium stores play an important role in control of cytoplasmic calcium homeostasis and in generation of intracellular calcium signals. These stores are specific intracellular compartments which are able to accumulate and release calcium in response to appropriate stimuli. Two types of stores can be distinguished in nonmuscle cells based on substances discharging these stores: (1) Ca²⁺-sensitive and (2) inositol-1,4,5-trisphosphate-sensitive intracellular depots. These two depots can be either separate intracellular compartments or a single compartment that shares both releasing mechanisms. The state of the art of our understanding of the cytoplasmic calcium release is the focus of this review.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 9–15, January–February, 1994.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.     

Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks

Hormones that act through the calcium-releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular calcium oscillations, which have been ascribed to calcium feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic calcium concentration. To investigate the functional significance of this phenomenon, we have developed mathematical models of the interaction of both second messengers. The models account for both positive and negative feedbacks of calcium on IP3 metabolism, mediated by calcium activation of phospholipase C and IP3 3-kinase, respectively. The coupled IP3 and calcium oscillations have a greatly expanded frequency range compared to calcium fluctuations obtained with clamped IP3. Therefore the feedbacks can be physiologically important in supporting the efficient frequency encoding of hormone concentration observed in many cell types. This action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. The ectopic expression of an IP3 binding protein has been used to decrease the rate of IP3 turnover experimentally, resulting in a dose-dependent slowing and eventual quenching of the Ca2+ oscillations. These results are consistent with a model based on positive feedback of Ca2+ on IP3 production.