Increasing Temperature Results in Higher Allocation of Energy to Protein Synthesis in Sea Urchin Larvae (Lytechinus pictus).

AbstractIt is well established that metabolic processes change with temperature and size. Yet the underlying physiological mechanisms are less well understood regarding how such processes covary within a species and particularly so for developmental stages. Physiological analysis of larvae of the sea urchin Lytechinus pictus revealed that protein was the major biochemical substrate supporting metabolism. The complex dynamics of protein synthesis, turnover, and accretion changed during growth, showing a sevenfold decrease in the ratio of protein accretion to protein synthesis (protein depositional efficiency). To test hypotheses of physiological variation with rising temperature, larvae were reared over a temperature range experienced by this species in its ambient habitat. The thermal sensitivity of protein synthesis was greater than respiration (thermal sensitivity values of 3.7 and 2.4, respectively). Bioenergetic calculations revealed a disproportionate increase in energy allocation toward protein synthesis with rising temperature. These differential temperature sensitivities result in metabolic trade-offs of energy acquisition and expenditure, thereby altering physiological homeostasis. Such insights are of value for improving predictions about limits of biological resilience in a warming ocean.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells.

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species - Arbacia lixula and Lytechinus variegatus - and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Sea urchin reproductive performance in a changing ocean: poor males improve while good males worsen in response to ocean acidification.

Ocean acidification (OA) is predicted to be a major driver of ocean biodiversity change. At projected rates of change, sensitive marine taxa may not have time to adapt. Their persistence may depend on pre-existing inter-individual variability. We investigated individual male reproductive performance under present-day and OA conditions using two representative broadcast spawners, the sea urchins Lytechinus pictus and Heliocidaris erythrogramma. Under the non-competitive individual ejaculate scenario, we examined sperm functional parameters (e.g. swimming speed, motility) and their relationship with fertilization success under current and near-future OA conditions. Significant inter-individual differences in almost every parameter measured were identified. Importantly, we observed strong inverse relationships between individual fertilization success rate under current conditions and change in fertilization success under OA. Individuals with a high fertilization success under current conditions had reduced fertilization under OA, while individuals with a low fertilization success under current conditions improved. Change in fertilization success ranged from -67% to +114% across individuals. Our results demonstrate that while average population fertilization rates remain similar under OA and present-day conditions, the contribution by different males to the population significantly shifts, with implications for how selection will operate in a future ocean.

Effects of ocean warming and acidification on fertilization success and early larval development in the green sea urchin Lytechinus variegatus.

Ocean acidification and warming are predicted to affect the early life of many marine organisms, but their effects can be synergistic or antagonistic. This study assessed the combined effects of near-future (2100) ocean acidification (pH 7.8) and warming (+3 °C) on the fertilization, larval development and growth of the green sea urchin, Lytechinus variegatus, common in tropical reefs of Florida and the Caribbean. Acidification had no effect on fertilization, but delayed larval development, stunted growth, and increased asymmetry. Warming decreased fertilization success when the sperm:egg ratio was higher (1847:1), accelerated larval development, but had no effect on growth. When exposed to both acidification and warming, fertilization rates decreased, larval development accelerated (due to increased respiration/metabolism), but larvae were smaller and more asymmetric, meaning acidification and warming had additive effects. Thus, climate change is expected to decrease the abundance of this important herbivore, exacerbating macroalgal growth and dominance on coral reefs.

The Role of Male Variation in Fertilization Success in Determining the Costs and Benefits of Polyandry in the Broadcast Spawning Urchin Lytechinus variegatus.

Although the benefits to males mating with multiple females have been well documented, the benefits to females mating with multiple males (polyandry) are less studied, particularly the mechanism that might drive these potential benefits. Benefits of polyandry might stem from increasing the chance of mating with a high-quality or compatible male or stem from the ability of multiple males to fertilize more eggs than any single male. We examine the fertilization consequences of polyandry in the sea urchin Lytechinus variegatus. This species has variation in spine color, and we conducted matings between individual and pooled sperm from two males that matched or mismatched in color. The results indicate that (1) males with white spines achieved higher fertilization and were more likely to cause polyspermy than males with purple spines, and there was no effect of female spine color on fertilization; (2) when comparing the average success of individual matings with pooled-sperm matings, there was a net benefit to polyandry when purple-spine males were pooled, a net cost when white-spine males were pooled, and no difference when mismatched spine color males were pooled; and (3) the success under pooled-sperm trials, with any of the spine color combinations, never exceeded the success of the more successful male in the individual-male trials. Together these results suggest that the consequences of polyandry depend on the relation between sperm availability and the sensitivity of eggs to sperm limitation and polyspermy with respect to the specific set of available males. The potential fertilization consequences of a female spawning with multiple males might be associated primarily with increasing the amount of sperm available to fertilize her eggs and secondarily with increasing the chances of mating with a higher-quality or more compatible male, as opposed to a diversity of males.

Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus.

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been recorded with increasing frequency, intensity and geographic distribution. This dinoflagellate produces potent toxins that may cause mortality of marine invertebrates. Adults of sea urchins are commonly affected by O. cf. ovata exposure with evidence of spines loss and high mortality during periods of high dinoflagellate abundances. Here, we report on the effects of the toxic dinoflagellate O. cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus, a key ecological herbivore. Lytechinus variegatus eggs and sperm were experimentally exposed to different concentrations of Ostreopsis cf. ovata (4, 40, 400, and 4000 cells ml-1) to test the hypothesis that fertilization success, embryonic and larval development of the sea urchin are negatively affected by the toxic dinoflagellate even at low abundances. Reduced fertilization, developmental failures, embryo and larval mortality, and occurrence of abnormal offspring were evident after exposure to O. cf. ovata. Fertilization decreased when gametes were exposed to high O. cf. ovata abundances (400 and 4000 cells ml-1), but just the exposure to the highest abundance significantly reduced fertilization success. Sea urchin early development was affected by O. cf. ovata in a dose-dependent way, high dinoflagellate abundances fully inhibited the early development of L. variegatus. Ostreopsis cf. ovata significantly increased the mortality of sea urchin eggs and embryos in the first hours of exposure (∼1-3 h), regardless of dinoflagellate abundance. Abundances of 400 and 4000 O. cf. ovata cells ml-1 induced significantly higher mortality on sea urchin initial stages in the first hours, and no egg or embryo was found in these treatments after 18 h of incubation. The early echinopluteus larva was only reached in the control and in treatments with low Ostreopsis cf. ovata abundances (4 and 40 cells ml-1). The exposure to O. cf. ovata led to significantly higher occurrence of skeletal anomalies in the early larva of L. variegatus. Interactions of sea urchin gametes and Ostreopsis cells may naturally occur in coastal areas due to the match between O. cf. ovata blooms and L. variegatus reproductive period. Reduced larval density and increased larval abnormalities were observed even at low abundances (4 and 40 cells ml-1) frequently found in tropical environments all year round. The chronic exposure to O. cf. ovata could significantly impact larval fitness, thus compromising recruitment success, and highlight the negative effects of benthic HABs on sea urchin populations and its possible broader ecological implications.

Individual female differences in chemoattractant production change the scale of sea urchin gamete interactions.

Sperm selection by females is an important process influencing fertilization and, particularly in broadcast-spawning organisms, often occurs before sperm reach the egg. Waterborne sperm chemoattractants are one mechanism by which eggs selectively influence conspecific sperm behavior, but it remains an open question whether the eggs from different females produce different amounts of sperm chemoattractant, and how that might influence sperm behavior. Here, we quantify the differences in attractant production between females of the sea urchin species Lytechinus pictus and use computational models and microfluidic sperm chemotaxis assays to determine how differences in chemoattractant production between females affects their ability to attract sperm. Our study demonstrates that there is significant individual female variation in egg chemoattractant production, and that this variation changes the scope and strength of sperm attraction. These results provide evidence for the importance of individual female variability in differential sperm attraction and fertilization success.

Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter.

The rising concentration of atmospheric CO2 by anthropogenic activities is changing the chemistry of the oceans, resulting in a decreased pH. Several studies have shown that the decrease in pH can affect calcification rates and reproduction of marine invertebrates, but little attention has been drawn to their immune response. Thus this study evaluated in two adult tropical sea urchin species, Lytechinus variegatus and Echinometra lucunter, the effects of ocean acidification over a period of 24h and 5days, on parameters of the immune response, the extracellular acid base balance, and the ability to recover these parameters. For this reason, the phagocytic capacity (PC), the phagocytic index (PI), the capacity of cell adhesion, cell spreading, cell spreading area of phagocytic amebocytes in vitro, and the coelomic fluid pH were analyzed in animals exposed to a pH of 8.0 (control group), 7.6 and 7.3. Experimental pH's were predicted by IPCC for the future of the two species. Furthermore, a recovery test was conducted to verify whether animals have the ability to restore these physiological parameters after being re-exposed to control conditions. Both species presented a significant decrease in PC, in the pH of coelomic fluid and in the cell spreading area. Besides that, Echinometra lucunter showed a significant decrease in cell spreading and significant differences in coelomocyte proportions. The recovery test showed that the PC of both species increased, also being below the control values. Even so, they were still significantly higher than those exposed to acidified seawater, indicating that with the re-establishment of the pH value the phagocytic capacity of cells tends to restore control conditions. These results demonstrate that the immune system and the coelomic fluid pH of these animals can be affected by ocean acidification. However, the effects of a short-term exposure can be reversible if the natural values ​​are re-established. Thus, the effects of ocean acidification could lead to consequences for pathogen resistance and survival of these sea urchin species.

EXTENDING THE VIABILITY OF SPERMATOZOA AND EGGS OF THE SEA URCHIN LYTECHINUS VARIEGATUS.

BACKGROUND: The storage of spermatozoa and eggs of the sea urchin Lytecninus variegatus can meet the demand of different human activities. OBJECTIVE: To develop a protocol easy to reproduce for spermatozoa cryopreservation and cooling of the eggs of the sea urchin. MATERIALS AND METHODS: Different formulations of artificial sea water were tested for their effectiveness in the freezing of sea urchin spermatozoa and storage of the eggs. RESULTS: Protocol for freezing of spermatozoa in liquid nitrogen presented the positive results when the cryoprotectant solution was diluted in artificial seawater free of calcium and magnesium. For the conservation of the eggs by cooling, the calcium-free artificial sea water, the calcium- and magnesium-free sea water, and the low-sodium water proved more efficient in preserving the integrity of the eggs. CONCLUSION: The results showed success in the freezing protocol of spermatozoa and cooling of the eggs mainly in artificial calcium- and magnesium-free sea water.

Direct relationship between osmotic and ionic conforming behavior and tissue water regulatory capacity in echinoids.

Echinoderms are considered marine osmoconforming invertebrates. However, many are intertidal or live next to estuaries, tolerating salinity changes and showing extracellular gradients to dilute seawater. Three species of echinoids - Lytechinus variegatus, which can occur next to estuarine areas, the rocky intertidal Echinometra lucunter, and the mostly subtidal Arbacia lixula - were submitted to a protocol of stepwise (rate of 2-3 psu/h) dilution, down to 15 psu, or concentration, up to 45 psu, of control seawater (35 psu). Coelomic fluid samples were obtained every hour. The seawater dilution experiment lasted 8h, while the seawater concentration experiment lasted 6h. Significant gradients (40-90% above value in 15 psu seawater) for osmolality, sodium, magnesium, and potassium were shown by L. variegatus and E. lucunter. A. lixula showed the smallest gradients, displaying the strongest conforming behavior. The esophagus of the three species was challenged in vitro with 20 and 50% osmotic shocks (hypo- and hyperosmotic). A. lixula, the most "conforming" species, showed the highest capacity to avoid swelling of its tissues upon the -50% hyposmotic shock, and was also the species less affected by salinity changes concerning the observation of spines and ambulacral feet movement in the whole-animal experiments. Thus, the most conforming species (A. lixula) displayed the highest capacity to regulate tissue water/volume, and was also the most euryhaline among the three studied species. In addition, tissues from all three species swelled much more than they shrank under osmotic shocks of same magnitude. This distinct trend to gain water, despite the capacity to hold some gradients upon seawater dilution, helps to explain why echinoderms cannot be fully estuarine, or ever enter fresh water.

Acetyl cholinesterase activity and muscle contraction in the sea urchin Lytechinus variegatus (Lamarck) following chronic phosphate exposure.

The common shallow-water sea urchin Lytechinus variegatus is capable of surviving inorganic phosphate exposures as high as 3.2 mg L(-1) and organic phosphate exposures of 1000 mg L(-1) . Nonetheless, chronic exposure to low, medium, and high-sublethal concentrations of organic phosphate inhibits the muscle enzyme acetyl cholinesterase (AChE), responsible for the break down of the neurotransmitter acetylcholine, as well as inhibiting contractions in the muscles associated with the Aristotle's lantern. AChE activity, measured in both a static enzyme assay and by vesicular staining, displayed concentration-dependent declines of activity in individuals maintained in organic phosphate for 4 weeks. The activity of AChE was not adversely affected by exposure to inorganic phosphate or seawater controls over the same time period. Maximum force of muscle contraction and rates of muscle contraction and relaxation also decreased with chronic exposure to increasing concentrations of organic phosphate. Chronic exposure to inorganic phosphates elicited no response except at the highest concentration, where the maximum force of muscular contraction increased compared to controls. These findings indicate that shallow-water populations of Lytechinus variegatus subjected to organic phosphate pollutants may display impaired muscular activity that is potentially related to the inhibition of the muscle relaxant enzyme AChE, and subsequently muscular overstimulation, and fatigue.

Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus.

The cues triggering large-scale broadcast-spawning events in marine invertebrates are not fully understood. Using the sea urchin Lytechinus variegatus, we tested the effectiveness of a variety of potential spawning cues in eliciting a spawning response. In the laboratory, during two consecutive spawning seasons, about 400 isolated sea urchins were exposed to phytoplankton, sperm, or eggs, singly or in combination. The likelihood of spawning, time to spawning, and spawning behavior were recorded for both sexes. Sperm was most successful at inducing spawning. No response to eggs was noted. Phytoplankton alone did not trigger spawning, but when a phytoplankton cue was followed by the addition of sperm, spawning behavior was induced, the time between addition of sperm and spawning was reduced, and the variance among individuals in the time of spawning initiation was reduced. Males spawned sooner in response to cues than females and rarely spawned spontaneously in phytoplankton or control treatments. A semilunar pattern in the sensitivity to spawning cues was noted. During time periods when sea urchins were less ripe, the ratio of spawning males to spawning females increased. Our results indicate that seasonal and lunar cycles, together with the presence of phytoplankton, increase the sensitivity of these sea urchins to spawning cues and the precision of their responses to conspecific sperm.

Sniffing out new data and hypotheses on the form, function, and evolution of the echinopluteus post-oral vibratile lobe.

The performance requirements of ciliary band feeding explain the convoluted forms of many marine invertebrate larvae. Convolutions increase surface area and therefore feeding rates per unit body volume. We review recent advances in morphology, neural development, and behavior at settlement of the echinoid Lytechinus pictus and provide new ultrastructural and expression data on larvae of its congener, L. variegatus. Larvae of the echinometrid Colobocentrotus atratus contain neurons identified by their expression of nitric oxide synthase (NOS), indicating that this character is not unique to Lytechinus. We hypothesize that in some echinoids the convoluted shape of the post-oral vibratile lobe (POVL) covaries with the distribution of identified sensory neurons to enable olfaction during settlement. An analysis of variation in structural elaboration of the post-oral transverse ciliary band (PTB) within Echinoida and in feeding larvae of other echinoderm classes indicates that only echinoids, but not all echinoids, possess this novel character; larvae that do are distributed heterogeneously within the class. In recognition of this specialized function for the POVL and surrounding ectoderm, and because it is lobate and grows toward the mouth, we propose naming this structure the adoral lobe.

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos.

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.

Inhibition of sea urchin fertilization by plant lectins.

Effects of plant lectins on sea urchin (Lytechinus variegatus) fertilization and a partial characterization of lectin-binding involved in the process were evaluated. IC50 doses for inhibition of fertilization varied from 4.1 to 135.5 microg/ml when the lectins were pre-incubated with sperms and from 0.7 to 33.4 microg/ml when pre-incubated with eggs. Such effects were reversed when the lectins were heat inactivated. FITC-labeled lectins bound egg surfaces while their denatured forms did not. Glucose/mannose specific lectins bound weaker to eggs when pre-incubated with the glycoprotein bovine lactotransferrin. None of the glycoproteins assayed diminished FITC patterns of the Gal/GalNAc binding lectins. Pre-incubation of Glucose/mannose binding lectins with eggs did not alter binding of Gal/GalNAc lectins. Lectins with distinct competencies for binding monosaccharide and glycoconjugates were able to inhibit sea urchin fertilization.

Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin.

The egg jellies of sea urchins contain sulfated polysaccharides with unusual structures, composed of linear chains of l-fucose or l-galactose with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among sulfated polysaccharides from different species. These polysaccharides show species specificity in inducing the acrosome reaction, which is a critical event for fertilization. Females of the sea urchin Lytechinus variegatus spawn eggs containing a sulfated fucan with the repetitive sequence [3-alpha-L-Fucp-2(OSO(3))-1 --> 3-alpha-L-Fucp-4(OSO(3))-1 --> 3-alpha-L-Fucp-2,4(OSO(3))-1 --> 3-alpha-L-Fucp-2(OSO(3))-1](n). We now observe that, close to winter, a period of decreased fertility for the sea urchin, the females synthesize a distinct sulfated fucan with a simple structure, composed of 4-sulfated, 3-linked alpha-fucose residues. This sulfated fucan is inactive when tested in vitro for the acrosome reaction using homologous sperm. The amount of egg jellies spawned by females (and their constituent sulfated polysaccharides) varied greatly throughout the year. Apparently, there is a correlation between the temperature of the sea water and the expression of the 4-sulfated, 3-linked sulfated fucan. Overall, we described the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity and may be involved in the periodicity of the reproductive cycle of the invertebrate.

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo.

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation.

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.

Second harmonic imaging of exocytosis at fertilization.

Second harmonic generation offers an important alternative and complement to fluorescence for the imaging of cellular structure and function. Staining the eggs of the sea urchin, Lytechinus pictus, with the styryl dye di-8-ANEPPS, we have observed large changes in both second harmonic generation and two-photon fluorescence after fertilization, consistent with the dynamics of exocytosis of cortical granules. With nonlinear imaging on a scanning microscope, we are able to visualize the wave of exocytosis in real time.

Phosphoinositide metabolism at fertilization of sea urchin eggs measured with a GFP-probe.

Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.