Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes

Injection of inositol trisphosphate (IP3) into oocytes of Xenopus laevis induces the appearance of a transient inward (Tin) current on hyperpolarization of the membrane. This current is carried largely by chloride ions, but is shown to depend on extracellular calcium, because it is abolished by removal of calcium in the bathing fluid or by addition of manganese. Recordings with aequorin as an intracellular calcium indicator show that a calcium influx is activated by hyperpolarization after intracellular injection of IP3 as well as after activation of neurotransmitter receptors thought to mediate a rise in IP3. Furthermore, by substituting barium for calcium in the bathing solution, inward barium currents can be recorded during hyperpolarization. We conclude that intracellular IP3 modulates the activity of a class of calcium channels, so as to allow an influx of calcium on hyperpolarization. In normal Ringer solution this then leads to the generation of a chloride current, because of the large numbers of calcium-dependent chloride channels in the oocyte membrane.     

Bcl-2 overexpression prevents calcium overload and subsequent apoptosis in dystrophic myotubes

Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.     

Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Pathways involved in RGD-mediated calcium transients in mature bovine oocytes

An arginine-glycine-aspartic acid (RGD)-containing peptide has been reported to generate calcium transients in bovine oocytes similar to those observed at fertilization. The research objective herein was to evaluate the response of bovine oocytes to an RGD peptide after injection with known antagonists of calcium releasing mechanisms in order to determine the initial calcium releasing pathway. Oocytes were injected with either heparin, an inhibitor of inositol 1,4,5-trisphosphate (IP3) induced calcium response, or procaine, which inhibits calcium release through the ryanodine receptor. Oocytes injected with heparin prior to RGD exposure did not display a calcium response. Oocytes injected with procaine prior to RGD exposure did exhibit a calcium response. Electroporation of IP3, caffeine, or exposure to RGD alone elicited a calcium response for each treatment group. Injection of heparin, procaine, vehicle medium (VM), or exposure to a non-RGD-containing peptide alone failed to elicit a calcium response. The data indicates that the RGD peptide is able to induce calcium transients in oocytes inhibited with procaine, but not those inhibited with heparin. These data suggest the pathway whereby the RGD peptide induces the first intracellular calcium transient in bovine oocytes is through IP3-mediated stores.     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitation and adaptation of Limulus ventral photoreceptors by inositol 1,4,5 triphosphate result from a rise in intracellular calcium

Single pressure injections of 1-10 pl of inositol 1,4,5 triphosphate (IP3) or inositol 4,5 bisphosphate [I(4,5)P2] excite Limulus ventral photoreceptors by inducing rapid bursts of inward current. After excitation by IP3, responses to subsequent injections of IP3 or light flashes are often reversibly diminished (adapted). Single injections of IP3 and I(4,5)P2 are effective at concentrations in the injecting pipette of 20 microM to 1 mM. Single injections of inositol 1,4 bisphosphate are ineffective at concentrations of 100-500 microM. Excitation by IP3 or I(4,5)P2 is accompanied by a rise in intracellular free calcium, as indicated by aequorin luminescence. Prior injection of calcium buffer solutions containing 100 mM EGTA greatly diminishes the total charge transferred across the plasma membrane during excitation by IP3 or I(4,5)P2, which suggests that a rise in Cai is necessary for excitation by the inositol polyphosphates. Adaptation of the response to light by IP3 is also abolished by prior injection of EGTA. In the same cells, the response to brief light flashes is slowed and diminished in amplitude by the injection of calcium buffer, but the charge transferred during the response is not significantly diminished. This suggests that light has access to a pathway of excitation in the presence of EGTA that is not accessible to intracellularly injected IP3.     

Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors : Detection with Aequorin

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca_i. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca_o reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Ca_o increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Ca_i may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Ca_i is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

Simple luminometric assay to detect phosphoinositol-linked receptor expression in Xenopus oocytes.

A simple and sensitive method to measure the expression of phosphoinositol-linked receptors in Xenopus laevis oocytes is described. Oocytes are co-injected with the calcium photoprotein aequorin and RNA, encoding the receptor of interest. The binding of ligand to the expressed receptor increases intracellular calcium that induces the aequorin to luminesce. With an autosampler-equipped luminometer, this provides a fully automated assay of receptor expression of oocytes. This method was applied to cloning the bombesin/GRP receptor expressed in Swiss 3T3 fibroblasts. Oocytes expressing the cloned BR showed up to a 10,000-fold increase in light emission in response to bombesin. The sensitivity of this procedure allowed detection of positive luminometer signals in single oocytes injected with RNA transcribed from cDNA pools as large as 25,000 clones. These findings show the potential value of this procedure for rapid screening of expression libraries, structure/function analysis of receptors and analysis of receptor antagonists or agonists.     

A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors

Inositol-trisphosphate was pressure-injected into Limulus ventral photoreceptors; these injections induced electrical responses that mimic several aspects of the electrical responses induced by light. Single cells were also injected with aequorin. Injections of inositol-trisphosphate into such cells induced an increase in luminescence from the intracellular aequorin, even in the absence of extracellular calcium ions. These aequorin responses show directly that inositol-trisphosphate induces an increase in ionized calcium concentration within intact and functioning cells that arises from release of calcium ions from intracellular stores.     

Dynamic changes to the inositol 1,4,5-trisphosphate and ryanodine receptors during maturation of bovine oocytes

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR) have been identified as two ligand-gated calcium channels which play a critical role in mediating calcium release in many different types of cells and tissues. The physiological significance of the two receptors in regulation of intracellular calcium during meiotic maturation and fertilization in the bovine oocyte was evaluated. Metabolic labeling of bovine oocytes by Met-Cys 35S during early and late maturation was followed by immunoprecipitation of both RyR and IP3R using specific antibodies against these two receptors. Results indicate that IP3R is translated throughout the maturation period; in contrast, RyR is only translated during the late maturation period of bovine oocytes. In addition, the experiments reported here investigate the temporal and spatial relationships between these calcium channels and the endoplasmic reticulum (ER) and cortical granules (CG). Immunocytochemistry, fluorescence staining and confocal microscopy were applied at four oocyte developmental stages: the germinal vesicleintact (GV-intact), metaphase I (MI) and metaphase II (MII) stages of maturation and the fertilized egg at 6 h post insemination (hpi). Although oocytes demonstrated some differences in staining patterns and localization, both receptor types showed apparent dynamic changes during meiotic maturation and dramatic decreases in signals after insemination. These results indicate the changes in the number and distribution of IP3R and RyR may account for the increased intracellular calcium responsiveness at fertilization. The IP3R appears to associate with the ER at the sub-vitelline membrane cortex in bovine oocytes. In addition, RyR appears to associate with the CG. In conclusion, although these two receptors may have different functional roles in regulation of calcium release during meiotic maturation and fertilization, it appears that both IP3R and RyR contribute to the significant increase of intracellular calcium during fertilization and activation in the bovine oocyte.     

Types of muscarinic response in xenopus oocytes

Four distinct types of membrane response of $\text{Xenopus laevis}$ oocytes to the application of acetylcholine (ACh) are described. The responses of single oocytes in summer are different from those in winter. The four response types differ in their dependence on the dose of acetylcholine and on the presence of calcium ions, and may appear solely or in various combinations in single oocytes. All response types can be elicited by oxotremorine and blocked by atropine.     

Second messenger-controlled membrane conductance in locust (Locusta migratoria) olfactory neurons

The membrane conductance of olfactory neurons of Locusta migratoria was examined using the whole-cell configuration of the patch-clamp technique. Intracellular application of the second messenger inositol 1,4,5 trisphosphate (IP(3)) via a dual pipette technique elicited a clear increase in the membrane conductance. The IP(3)-induced conductance increased due to a rise in the extracellular concentration of calcium from 100&mgr;M to 4mM. Micromolar concentrations of ruthenium red partially blocked the IP(3)-induced increase in membrane conductance. Stimulating olfactory neurons with odour (hexenoic acid) resulted in an increase in the membrane conductance partially similar to that mediated IP(3). These findings suggest that stimulation with appropriate odours as well as intracellular application of IP(3) activate the same calcium-permeable ion channels in the plasma membrane of insect olfactory neurons.     

Juvenile and adult immature and in vitro matured ovine oocytes evaluated in relation to membrane electrical properties, calcium stores, IP3 sensitivity and apoptosis occurrence in cumulus cells

The analysis of differences between juvenile and adult oocytes may provide useful information on the acquisition of meiotic and developmental competence of the female gamete. In oocytes collected from either ewes or 40-day-old lambs, we evaluated membrane electrical properties, such as resting potential, conductance, activation ion currents, L-type Ca(2+) currents as well as calcium stores and IP3 sensitivity; in addition, the incidence of apoptosis in cumulus cells in these two age categories was compared. The analysis was carried out in oocytes both prior to and after in vitro maturation. Significant differences were found in all the examined parameters in relation to maturational stages whereas minor differences were recorded in relation to age of the donor. IP3 sensitivity strongly increased after in vitro maturation following a dose-dependent pattern from 1 to 500 micromol/L with a significant interaction (P < 0.01) between dose and maturational stage. The incidence of apoptosis in cumulus cells strongly increased after in vitro maturation and was greater in adult than in juvenile cumulus cells (39.2 +/- 5.8% vs. 21.9 +/- 3.5%; P < 0.01). In conclusion, all the examined parameters were greatly affected by the maturational stage, whereas minor differences were due to age-related oocyte quality, that is, at plasma membrane levels to conductance, activation current peaks and calcium currents, at cytosol level to calcium stores and IP3 sensitivity, and to incidence of apoptosis in cumulus cells. These parameters were compared with previous data in bovine to analyze oocyte quality in juvenile and adult individuals or between species.     

Activation of in vitro matured pig oocytes using activators of inositol triphosphate or ryanodine receptors

In our study, we observed the activation of in vitro matured pig oocytes and their subsequent parthenogenetic cleavage after stimulation of ryanodine receptors (RyR) using ryanodine (Ry), caffeine or cyclic adenosine diphosphate ribose (cADPri) or after stimulation of inositol triphosphate receptors (IP(3)R) using D-myo-inositol 1,4,5-triphosphate (IP(3)). Heparin, a potent blocker of IP(3)R, prevented the activation of porcine oocytes using IP(3), but blockers of RyR (ruthenium red or procaine) prevented activation after stimulation by RyR and stimulation by IP(3)R using IP(3). The drugs were injected into oocytes matured to the stage of metaphase II and activation was determined by assessment of pronuclear formation. The activity of H1 kinase was determined and our results demonstrated a significant drop in H1 activity in the activated oocytes. The cleavage of parthenogenetic embryos progresses to more advanced stages after stimulation by IP(3)R than after stimulation by RyR. Our results could indicate that, in pig oocytes, the calcium released from IP(3)-sensitive stores triggers the calcium release from ryanodine-sensitive intracellular stores, which is necessary for oocyte activation. The calmodulin inhibitors ophiobolin A and W7 reduce the activation of oocytes induced by stimulation of RyR or IP(3)R.     

Inositol phosphate regulation of voltage-dependent calcium channels in cerebellar granule neurons.

The effects of intracellularly applied inositol phosphates on voltage-dependent calcium channel currents were assessed in rat cerebellar neurons using the whole-cell recording configuration of the patch-clamp technique. Intraneuronal perfusion of 10 microM inositol 1,4,5-trisphosphate (IP3) increased the amplitude of currents elicited by depolarization from a holding potential of -40 mV. IP3 did not modify current activation, but shifted the steady-state inactivation curve toward more positive values. The dose-response curve indicated an EC50 of 0.5 microM for IP3. Inositol 1,3,4,5-tetrakisphosphate (IP4), but not inositol 4,5,-bisphosphate, mimicked the effect of IP3. The effect of IP3 persisted in the presence of 100 micrograms/ml heparin and did not depend on intracellular calcium mobilization, as similar responses were not produced by 10 mM caffeine or by intrapipette calcium buffering at pCa 6 instead of pCa 7.7. Preincubation with omega-conotoxin led to a 55% inhibition of barium current; however, inhibition was reversed by IP3, which reestablished the control current amplitude. These results imply that IP3 and IP4 can elicit calcium entry by modifying both the gating characteristics and the pharmacological properties of voltage-dependent calcium channels.     

Acetylcholine receptors in monkey and rabbit oocytes

Membrane potential responses to acetylcholine (ACh, 10(-7)-10(-3 M) were investigated in monkey and rabbit ovarian oocytes. In monkey oocytes ACh most commonly elicited a short-latency hyperpolarization concomitant with a decreased membrane input resistance (Rin). Under voltage-clamp short-latency ACh currents had an equilibrium potential of approximately -40 mV. In rabbit oocytes responses to ACh consisted of an increase in Rin or of a depolarization with an equilibrium potential of approximately -15 mV. Curare, hexamethonium, and atropine (10(-5)-10(-3) M) did not block these ACh responses. Thus, the oocyte membrane in the rabbit contains ACh receptors that cannot be classified as either muscarinic or nicotinic.     

Inositol 1,4,5 trisphosphate releases calcium from specialized sites within Limulus photoreceptors

We have investigated the subcellular distribution and identity of inositol trisphosphate (InsP3)-sensitive calcium stores in living Limulus ventral photoreceptor cells, where light and InsP3 are known to raise intracellular calcium. We injected ventral photoreceptor cells with the photoprotein aequorin and viewed its luminescence with an image intensifier. InsP3 only elicited detectable aequorin luminescence when injected into the light-sensitive rhabdomeral (R)-lobe where aequorin luminescence induced by light was also confined. Calcium stores released by light and InsP3 are therefore localized to the R-lobe. Within the R-lobe, InsP3-induced aequorin luminescence was further confined around the injection site, due to rapid dilution and/or degradation of injected InsP3. Prominent cisternae of smooth endoplasmic reticulum are uniquely localized within the cell beneath the microvillar surface of the R-lobe (Calman, B., and S. Chamberlain, 1982, J. Gen. Physiol., 80:839-862). These cisternae are the probable site of InsP3 action.     

Injection of inositol 1,3,4,5-tetrakisphosphate into Xenopus oocytes generates a chloride current dependent upon intracellular calcium

Injection of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into voltage-clamped oocytes of Xenopus laevis elicited an oscillatory chloride membrane current. This response did not depend upon extracellular calcium, because it could be produced in calcium-free solution and after addition of cobalt to block calcium channels in the surface membrane. However, it was abolished after intracellular loading with the calcium chelating agent EGTA, indicating a dependence upon intracellular calcium. The mean dose of Ins(1,3,4,5)P4 required to elicit a threshold current was 4 x 10(-14) mol. In comparison, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) gave a similar oscillatory current with doses of about one twentieth as big. Hyperpolarization of the oocyte membrane during activation by Ins(1,3,4,5)P4 elicited a transient inward current, as a result of the opening of calcium-dependent chloride channels subsequent to the entry of external calcium. In some oocytes the injection of Ins(1,3,4,5)P4 was itself sufficient to allow the generation of the transient inward current, whereas in others a prior injection of Ins(1,4,5)P3 was required. We conclude that Ins(1,3,4,5)P4 causes the release of intracellular calcium from stores in the oocyte, albeit with less potency than Ins(1,4,5)P3. In addition, Ins(1,3,4,5)P4 activates voltage-sensitive calcium channels in the surface membrane, via a process that may require 'priming' by Ins(1,4,5)P3.