Sea Urchin Egg Homogenates Research Articles

Differential Effect of Glycolytic Intermediaries upon Cyclic ADP-ribose-, Inositol 1′,4′,5′-Trisphosphate-, and Nicotinate Adenine Dinucleotide Phosphate-Induced Ca2+ Release Systems

We investigated the effect of glycolytic pathway intermediaries upon Ca2+ release induced by cyclic ADP-ribose (cADPR), inositol 1′,4′,5-trisphosphate (IP3), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6,-diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca2+ release by itself in sea urchin egg homogenate. However, FDP potentiates Ca2+ release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca2+ release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca2+ release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca2+ release induced by IP3 or NAADP had no effect upon Ca2+ release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca2+ release induced by both IP3 and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca2+ homeostases by switching preferential effects from one intracellular Ca2+ release channel to another.

The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs

The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs

Potentiation of cADPR-induced Ca(2+)-release by methylxanthine analogues.

Caffeine and other methylxanthines are known to induce Ca(2+)-release from intracellular stores via the ryanodine receptor. In the present work, a range of caffeine analogues, in which methyl groups at the 1 and 7 positions were replaced with alkyl chains containing different functional groups (oxo, hydroxyl, propargyl, ester, and acids), were synthesized. These compounds were then screened for their ability to potentiate Ca(2+)-release induced by cADPR (an endogenous modulator of ryanodine receptors) in sea urchin egg homogenates. Two of the synthesized methylxanthines, 1, 3-dimethyl-7-(7-hydroxyoctyl)xanthine (37) and 3-methyl-7-(7-oxooctyl)-1-propargylxanthine (66), were shown to be more potent than caffeine in potentiating cADPR-induced Ca(2+)-release, while 1,3-dimethyl-7-(5-ethylcarboxypentyl)xanthine (14) was shown to be more efficacious. The development of new methylxanthine analogues may lead to a better understanding of ryanodine receptor function and could possibly provide novel therapeutic agents.

The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs

Cytosolic extracts of boar sperm contain a soluble phospholipase C (PLC) activity that induces Ca2+ release in sea-urchin (Lytechinus pictus) egg homogenates and an uncharacterized protein factor that causes Ca2+ oscillations when injected into mammalian eggs. In the present study we fractionated boar sperm extracts on three different FPLC chromatographic columns and found that the fractions that caused maximal Ca2+ release in sea-urchin egg homogenates were also the ones that triggered Ca2+ oscillations in mouse eggs. Our data suggests that the sperm factor which triggers Ca2+ oscillations in eggs contains a PLC and not the 33 kDa glucosamine deaminase previously suggested to be one its components.

Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems.

We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.

A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca 2+ release in sea urchin egg homogenates

Injection of sperm extracts triggers Ca 2+ oscillations in mammalian eggs similar to those seen at fertilisation. Here, we show that addition of sperm extracts to sea urchin egg homogenates causes Ca 2+ release and inositol 1,4,5-trisphosphate (InsP 3) production. Furthermore depleting homogenates of phosphatidylinositol lipids using a phosphatidylinositol-specific phospholipase C blocked the sperm extract from causing InsP 3 production and a Ca 2+ rise. A response could be recovered by the addition of phosphatidylinositol 4,5-bisphosphate to either sperm extracts or egg homogenates. These data indicate that sperm extracts contain an InsP 3-generating phospholipase C which may play a role in Ca 2+ release at fertilisation.

Fluorescent analogs of NAADP with calcium mobilizing activity

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca 2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca 2+ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1, N 6-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1, N 6-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1, N 6-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3–4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca 2+ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 μM and 5 μM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca 2+ release with a half-maximal concentration of 60–80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca 2+ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca 2+ stores that are sensitive to NAADP in live cells.

Detection and functional characterization of ryanodine receptors from sea urchin eggs.

1. Immunoblot analysis, [3H]ryanodine binding, and planar lipid bilayer techniques were used to identify and characterize the functional properties of ryanodine receptors (RyRs) from Lytechinus pictus and Strongylocentrotus purpuratus sea urchin eggs. 2. An antibody against mammalian skeletal RyRs identified an approximately 400 kDa band in the cortical microsomes of sea urchin eggs while a cardiac-specific RyR antibody failed to recognize this protein. [3H]Ryanodine binding to cortical microsomes revealed the presence of a high-affinity (Kd = 13 nM), saturable (maximal density of receptor sites, Bmax = 1.56 pmol (mg protein)-1) binding site that exhibited a biphasic response to Ca2+. 3. Upon reconstitution of cortical microsomes into lipid bilayers, only sparse and unstable openings of a high-conductance cation channel were detected. Addition of crude sea urchin egg homogenate to the cytosolic (cis side) of the channel increased the frequency of openings and stabilized channel activity. The homogenate-activated channels were Ca2+ sensitive, selective for Ca2+ over Cs+, and driven by ryanodine into a long-lived subconductance state that represented approximately 40 % of the full conductance level. Homogenate dialysed in membranes with a molecular weight cut-off <= 2000 lacked the capacity to increase the frequency of RyR openings and to stabilize channel activity. 4. Direct application of cyclic adenosine diphosphoribose (cADPR) or photolysis of NPE-cADPR ('caged' cADPR) by ultraviolet laser pulses produced transient activation of sea urchin egg RyRs. Calmodulin (CaM) failed to activate reconstituted RyRs; however, channel activity was inhibited by the CaM blocker trifluoroperazine, suggesting that CaM was necessary but not sufficient to sustain RyR activity. 5. These findings suggest that a functional Ca2+ release unit in sea urchin eggs is a complex of several molecules, one of which corresponds to a protein functionally similar to mammalian RyRs.

Preparative electrophoresis in "sieving media" of subcellular-sized particles.

The commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE-1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11-15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE-1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by "fluorescence reduction". The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr = 10(6)) solution in 25-50% yield (0.24-4 microg/8 lanes of the HPGE-1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients, KR = d(log mobility)/d(polymer concentration), are not ascertained.

Cyclic ADP-ribose activates caffeine-sensitive calcium channels from sea urchin egg microsomes.

Adenosine 5'-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca(2+)-selective single channels with conductances of 155.0 +/- 8.0 pS in asymmetric Cs+ solutions and 47.5 +/- 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca(2+)-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca(2+)-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.

Thio-NADP is not an antagonist of NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca2+ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995] J. Biol. Chem. 270, 3216-3223). Its effects in sea-urchin egg homogenates were investigated. At 50 microM, thio-NADP activates partial Ca2+ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca2+ releasing activity of 50 microM thio-NADP and reduces the subsequent inhibition by 73.7 +/- 1.3%. The residual inhibitory effect is no more than that exerted by 50 microM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by 32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific 32P-NAADP binding to egg microsomes with an IC50 of 40 microM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 microM, similar to the extent of inhibition effected by 40 microM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca2+ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca2+ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.

Cyclic GMP-dependent and -independent Effects on the Synthesis of the Calcium Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 microM cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

A Cytosolic Sperm Protein Factor Mobilizes Ca2+ from Intracellular Stores by Activating Multiple Ca2+ Release Mechanisms Independently of Low Molecular Weight Messengers

Ca2+ oscillations can be induced in mammalian eggs and somatic cells by microinjection of a cytosolic sperm protein factor. The nature of the sperm factor-induced Ca2+ signaling was investigated by adding sperm protein extracts to homogenates of sea urchin eggs, which contain multiple classes of Ca2+ release mechanisms. We show that the sperm factor mobilizes Ca2+ from non-mitochondrial Ca2+ stores in egg homogenates after a distinct latency. This latency is abolished by preincubation of sperm extracts with egg cytosol. The preincubation step is highly temperature-dependent and generates a high molecular weight, protein-based Ca2+-releasing agent that can also mobilize Ca2+ from purified egg microsomes. This Ca2+ release appears to be mediated via both inositol 1,4,5-trisphosphate and ryanodine receptors, since homologous desensitization of these two release mechanisms by their respective agonists inhibits further release by the sperm factor. However, sperm factor-induced Ca2+ release by these channels is independent of inositol 1,4, 5-trisphosphate or cADPR since antagonists of either of these two messengers did not block the Ca2+ release effected by the sperm factor. The sperm protein factor may cause Ca2+ release via an enzymatic step that generates a protein-based Ca2+-releasing agent.

Roles for adenosine ribose hydroxyl groups in cyclic adenosine 5'-diphosphate ribose-mediated Ca2+ release.

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring and potent Ca2+-mobilizing agent. Structural analogues are currently required as pharmacological tools for the investigation of this topical molecule, but modifications to date have concentrated primarily upon the purine ring. Two novel dehydroxylated analogues of cADPR have now been prepared from chemically synthesized nicotinamide adenine dinucleotide (NAD+) precursors modified in the ribose moiety linked to adenine. ADP-ribosyl cyclase of Aplysia californica catalyzed the conversion of 2'A-deoxy-NAD+ and 3'A-deoxy-NAD+ into the corresponding 2'A-deoxy-cADPR and 3'A-deoxy-cADPR analogues, respectively. These analogues were used to assess the effect of 2'- and 3'-hydroxyl group deletion in the adenosine ribose moiety of cADPR on the Ca2+-releasing potential of cADPR. These compounds were found to have comparatively markedly different activities as agonists for Ca2+ mobilization in sea urchin egg homogenate. 2'A-Deoxy-cADPR is similar to cADPR, whereas 3'A-deoxy-cADPR is at least 100-fold less potent, indicating that the 3'A-hydroxyl group, but not the 2'A-hydroxyl group, is essential for calcium releasing activity. EC50 values recorded were 32 nM, 58 nM, and 5 microM for cADPR, 2'A-deoxy-cADPR, and 3'A-deoxy-cADPR, respectively. Moreover, 200 nM 2'A-deoxy-cADPR was required to desensitize the cADPR-sensitive Ca2+ channel to a subsequent addition of 100 nM cADPR, but 20 microM 3'A-deoxy-cADPR was required to produce the same desensitizing effect. This is in accordance with the 100-fold lower potency exhibited by the latter analogue. To further investigate the importance of the 3'-hydroxyl group, we have also synthesized 3'A-O-methyl-cADPR, in which the 3'-hydroxyl group of adenosine has been methylated and its ability potentially to donate a hydrogen atom in a hydrogen bond has been removed. Although inactive in releasing Ca2+, 3'A-O-methyl-cADPR inhibited cADPR-induced Ca2+ release in a dose-dependent manner with an approximate IC50 value of 5 microM, whereas 3'-O-methyladenosine had no effect. This further supports the requirement of a 3'-OH group for Ca2+ releasing activity. In addition, however, it suggests that this group may not be crucial for ligand-receptor recognition. Thus, replacement of the hydrogen atom of the hydroxyl with a methyl group effects a change of activity from an agonist to an antagonist of cADPR-induced Ca2+ release. Two other analogues with modifications in the 2' and/or 3' positions, 3'-cADPR phosphate and 2',3'-cyclic-cADPR phosphate, were synthesized and tested for their Ca2+-mobilizing activity in sea urchin egg homogenates. Both analogues were inactive with respect to both agonistic and antagonistic activities on the cADPR-sensitive Ca2+ release mechanism. These are the first steps toward a wider structure-activity relationship for cADPR, and this is the first study to implicate a crucial role for the adenosine ribose hydroxyl groups of cADPR in the biological activity of this cyclic nucleotide. Additionally, this is the first report of a cADPR receptor antagonist that is not modified at the 8-position of the purine ring.

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg.

1. The sea urchin egg homogenate is an ideal model to characterize Ca2+-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP3- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+-releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3. NAADP-induced Ca2+-release was blocked by L-type Ca2+-channel blockers and by Bay K 8644, while InsP3- and cyclic ADPR-induced Ca2+-release were insensitive to these agents. L-type Ca2+-channel blockers did not displace [32P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4. Similarly, a number of K+-channel antagonists blocked NAADP-induced Ca2+-release selectively over InsP3- and cyclic ADPR-induced Ca2+-release. Radioligand studies showed that these agents were not competitive antagonists. 5. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+-release mechanisms. 6. The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca2+-release. While the alkaline condition permitted InsP3 and cyclic ADPR-induced Ca2+-release, the acidic condition completely hampered both Ca2+-release mechanisms. 7. The present results characterize pharmacologically and biochemically the novel Ca2+-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

Cyclic ADP-ribose signaling in sea urchin gametes: metabolism in spermatozoa

The molecular mechanism that initiates Ca2+ signaling in sea urchin egg fertilization has not yet been clarified. To determine whether sea urchin sperm may generate and possibly supply cyclic ADP-ribose (cADPR) as a Ca2+-releasing factor in the course of sea urchin egg fertilization, we determined cADPR content and the capacity for cADPR synthesis in sea urchin sperm. cADPR content was determined using the sea urchin egg homogenate Ca2+-release bioassay combined with high-performance liquid chromatography (HPLC). We found that sperm homogenates synthesized cADPR from beta-NAD but did not synthesize cADPR when alpha-NAD was the substrate. The identity of cADPR generated by sperm homogenates was verified by HPLC analysis, use of specific Ca2+-release antagonists, and homologous desensitization of the sea urchin egg homogenate Ca2+-release bioassay. The ambient content of cADPR was approximately 0.3 nmol cADPR/g wet wt sea urchin sperm. Our results show that sperm can synthesize cADPR and that they contain cADPR levels comparable to other tissues.

Nicotinamide inhibits cyclic ADP-ribose-mediated calcium signalling in sea urchin eggs.

Cyclic ADP ribose (cADPR) is a potent Ca(2+)-releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. beta-NAD+, cGMP and nitric oxide (NO) have been reported to mobilize Ca2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca(2+)-mobilizing action of beta-NAD+, cGMP and NO, but has no effect on cADPR-induced Ca2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca2+, we have shown that nicotinamide inhibits cADPR-mediated Ca2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca2+ release by beta-NAD+, cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.

Regulation of cADP-ribose-induced Ca2+ Release by Mg2+ and Inorganic Phosphate

cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.

Nicotinate-adenine dinucleotide phosphate-induced Ca2+-release does not behave as a Ca2+-induced Ca2+-release system

We investigated the dependence of nicotinate-adenine dinucleotide phosphate (NAADP)-induced Ca2+ release from intracellular stores of sea urchin egg homogenates, upon extravesicular Ca2+. In contrast to the Ca2+ release induced inositol 1',4',5'-triphosphate (IP3) or cyclic ADP-ribose (cADPR), the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ over a wide range of concentrations (0-0.1 mM). The Ca2+ release triggered by either cADPR or IP3 was biphasically modulated by extravesicular Ca2+, and the Ca2+ release by these agents was abolished when the extravesicular Ca2+ was removed by chelation with 2 mM EGTA. On the other hand, NAADP-triggered Ca2+ release was not influenced by EGTA. These data indicate that while both cADPR and IP3 systems behave as functional Ca(2+)-induced Ca2+ release mechanisms, NAADP activates a Ca2+ release mechanism which is independent of the presence of extravesicular Ca2+. Therefore, the NAADP-sensitive Ca2+ release mechanisms may have a unique regulatory impact upon intracellular Ca2+ homoeostasis.

Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro

We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2 beta binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2 alpha binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2 beta. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.