Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

New insights into maitotoxin action

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5-min delay following toxin addition. Maitotoxin-induced 45Ca2+ influxes could not be blocked by either 1 microM verapamil, 1 microM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50 = 41 microM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+, the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 microM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose-response curves for H+ ion inhibition of maitotoxin-induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3- and HCO3-/Cl- ions. It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore-forming or channel-forming activity of a non-classical type. Some properties of maitotoxin resemble those of alpha-latrotoxin, others those of pore-forming agents such as melittin or alpha-toxin of Staphylococcus aureus.     

Pertussis toxin pretreatment abolishes dihydropyridine inhibition of calcium flux in the 235-1 pituitary cell line

In the present study we used 235-1 cells, a prolactin secreting clone derived from a pituitary tumor. In these cells maitotoxin, a calcium channels activator, likely acting on voltage sensitive calcium channels, increases intracellular free calcium measured by Quin 2 technique. Maitotoxin stimulation of calcium flux was inhibited both by nicardipine and verapamil in a dose dependent manner. Pertussis toxin pretreatment does not modify maitotoxin activation of calcium channels, while completely abolishes nicardipine inhibition of maitotoxin induced voltage sensitive calcium channels activation, without affecting verapamil effect. These results suggest a possible involvement of a pertussis toxin sensitive G protein in dihydropyridine inhibition of voltage sensitive calcium channels.     

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.     

Ca2+ channel activating function of maitotoxin, the most potent marine toxin known, in clonal rat pheochromocytoma cells

Actions of maitotoxin, the most potent marine toxin known obtained from toxic dinoflagellate, Gambier-discus toxicus, were studied using clonal rat pheochromocytoma cells (PC12), rat liver mitochondria and liposomes. Maitotoxin induced a profound release of norepinephrine and dopamine from PC12 cells and the molar ratio of norepinephrine to dopamine was almost the same as that stored in the cells. This releasing action was apparent at a concentration of 5 X 10(-10) g/ml or more, the releasing rate increased with an increase in the concentration of applied maitotoxin and attained maximum at about 10(-6) g/ml. The [3H]norepinephrine release induced by maitotoxin was abolished in the absence of external Ca2+ and increased with increasing concentration of external Ca2+ up to 10 mM. The release gradually decreased as the external Na+ concentrations were reduced from 130 to 20 mM, but maitotoxin is still able to induce a profound release in the absence of external Na+. The releasing action of maitotoxin was markedly suppressed by various Ca2+ channel blockers, such as Mn2+, verapamil, and nicardipine, and by a local anesthetic, tetracaine. The inhibitory actions of Ca2+ channel blockers were antagonized by external Ca2+ and became less obvious in the higher Ca2+ concentration range. Maitotoxin did not exhibit any ionophoretic activities on rat mitochondrial and liposomal membranes. These results suggest that maitotoxin has the ability to activate voltage-dependent Ca2+ channels of PC12 cells.     

Maitotoxin-induced free Ca changes in single rat hepatocytes

We report the results using bioluminescent and fluorescent indicators to investigate maitotoxin-induced free Ca changes in single rat hepatocytes. Maitotoxin generated a steadily rising free Ca increase after a long lag period. The free Ca increase was dependent on extracellular calcium and could be antagonised by chelation of extracellular calcium or the inclusion of nickel in the superfusate. Manganese-induced quench of cytoplasmic Fura2 dextran revealed an accelerated rate of calcium entry during the final period of the lag phase, immediately prior to the free Ca increase. Imaging experiments demonstrated a markedly different part of free Ca mobilisation compared with glycogenolytic stimuli. Moreover, the use of a combination of hormonal stimuli and maitotoxin revealed that some cells could exhibit free Ca oscillations despite steadily rising intracellular free Ca level. The significance of these observations in terms of the mechanism of action of maitotoxin and the mechanism of free Ca transient generation is discussed.     

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

The effect of acetylcholine on inositol lipid metabolism and intracellular calcium concentrations in bovine anterior pituitary cells

Acetylcholine (ACh) increased the intracellular calcium concentration in bovine anterior pituitary cells. In the presence of the calcium channel antagonists verapamil (20 microM) or nitrendepine (1 microM) the increase in calcium was partially inhibited but showed both transient and sustained components. In the presence of EGTA (2.5 mM) only the transient component was observed. ACh also decreased inositol radioactivity in phosphatidylinositides and increased it in inositol phosphates. It is concluded that the increase in calcium caused by acetylcholine requires both the entry of external calcium and mobilisation of internal calcium. Replacement of external sodium by N-methyl-D-glucamine inhibited the rises in calcium and inositol phosphate labelling in response to ACh. Tetrodotoxin (3 microM) or ouabain (50 microM) did not affect either response to ACh. Verapamil did not affect the calcium rise induced by ACh in the absence of external sodium. The phorbol ester PMA (10 nM) caused a transient rise in calcium and inhibited the calcium rise caused by acetylcholine: it did not modify the effect of acetylcholine on inositol phosphates. The dependence of the stimulation of external calcium entry and inositol phosphate production on external sodium ions and protein kinase C is discussed.     

Agents that stimulate phosphoinositide turnover also elevate cAMP in SK-N-SH human neuroblastoma cells.

Stimulation of m1 and of m3 muscarinic receptors has previously been shown to increase intracellular cAMP levels in a variety of cells. Although the mechanism underlying this response is not fully understood, it has been hypothesized to be secondary to the IP3-mediated rise in intracellular calcium. In order to determine whether other means of elevating intracellular calcium also raise cAMP levels, we stimulated SK-N-SH human neuroblastoma cells with bradykinin or with maitotoxin. Both of these agents stimulated phospholipase C, stimulated inositol phosphate release and elevated cAMP levels, thus demonstrating that this cAMP response is not unique to muscarinic receptor stimulation.     

Regulation of the intracellular calcium concentration in MMQ pituitary cells by dopamine and protein kinase C.

The MMQ pituitary cell line, which expresses a membranal dopamine receptor, was used to examine the individual contributions of dopamine and protein kinase C (PKC) to control of the intracellular calcium concentration. The calcium concentrations, monitored with the fluorescent dye Indo-1, increased in response to elevated K+, BAY K8644, and maitotoxin, implicating the presence of voltage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 60 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8644 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent D-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8644 or maitotoxin.     

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa

Maitotoxin, a presumed activator of the voltage-sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca²⁺ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage-sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.     

Accumulations of cyclic AMP and inositol phosphates in guinea pig cerebral cortical synaptoneurosomes: enhancement by agents acting at sodium channels

Activation of alpha 1-adrenergic receptors by norepinephrine in guinea pig cortical synaptoneurosomes augments accumulations of cyclic AMP elicited by 2-chloroadenosine and concomitantly increases formation of inositol phosphates. Various agents that affect calcium channels or sites of action of calcium have little or no effect on cyclic AMP accumulation elicited either with 2-chloroadenosine, or with a 2-chloroadenosine/norepinephrine combination, nor did they markedly affect formation of inositol phosphates elicited by norepinephrine. However, EGTA reduces both cyclic AMP accumulation and inositol phosphate formation. Agents such as batrachotoxin, scorpion (Leiurus) venom and pumiliotoxin B that are active at voltage-dependent sodium channels enhance accumulations of cyclic AMP and inositol phosphates. These effects are blocked by tetrodotoxin. It is proposed that enhanced influx of sodium ions increases phosphatidylinositol metabolism, resulting in formation of diacylglycerols and inositol phosphates, and that the former, through activation of protein kinase, causes an enhancement of cyclic AMP accumulations in brain tissue.     

Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. Evidence for an inhibition not mediated by voltage-dependent Ca2+ channels.

In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s. The other was slower, starting after 1 min, and was mimicked by nimodipine, a dihydropyridine calcium channel antagonist. The effects of dopamine and nimodipine were not additive on both basal and TRH-stimulated inositol phosphate production. Furthermore, the dopamine inhibition in the presence of TRH was much higher than the inhibition induced by nimodipine. It is thus likely that calcium entry through voltage-dependent calcium channels triggers a positive feedback on TRH stimulation of phospholipase C. However, depolarizing concentrations of K+ or BAY-K-8644, a voltage-dependent calcium channel agonist, had no effect on inositol monophosphate and bisphosphate accumulation. Ionomycin, even at a very high concentration (10 microM), had only a slight and transient effect on inositol phosphate formation. In addition, these agents did not affect the TRH dose-dependent stimulation of inositol phosphate production. These results suggest that the intracellular calcium concentrations that we measured under basal and TRH-stimulated conditions are sufficient to allow the maximal activity of phospholipase C which can be obtained under these two experimental conditions. In contrast, any decrease in the intracellular calcium concentration by a dihydropyridine antagonist, suppression of extracellular calcium, or inactivation of a voltage-dependent calcium channel by long term depolarization with K+ decreased the phospholipase C activities measured under basal and TRH-stimulated conditions. From these data it can be concluded that dopamine inhibits inositol phosphate production by two distinct mechanisms. The slow dopamine-induced inhibition of TRH-stimulated inositol phosphate production which is mimicked by nimodipine is likely because of an inhibition of a voltage-dependent calcium channel. This is substantiated further by the fact that ionomycin (10 microM) was able to reverse the nimodipine inhibitions as well as this slow component of dopamine inhibition. The nature of the rapid inhibition of TRH-stimulated inositol phosphate production induced by dopamine, but not by nimodipine, remains to be determined. It is suppressed in the absence of extracellular Ca2+. This may suggest that this inhibition is related to blockade of non-dihydropyridine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A

The two mammalian neuropeptides substance P (SP) and neurokinin A (NKA) have been demonstrated to stimulate DNA synthesis in connective tissue cells, suggesting that peripheral neurons may play a role in development and tissue regeneration. In this study we have tried to identify intracellular messengers required for SP- and NKA-induced DNA synthesis. SP and NKA, as well as platelet-derived growth factor (PDGF) stimulated formation of inositol phosphates in smooth muscle cells (SMC), whereas no effect on inositol phosphates formation occurred in response to nonmitogenic neuropeptides. Pretreatment of the cells with pertussis toxin markedly decreased DNA synthesis induced by NKA. This toxin inhibits formation of inositol phosphates by acting on a regulatory G-protein. Calcium and calmodulin antagonists also inhibited NKA-induced DNA synthesis. These results imply that the mitogenic signal(s) produced by activated neuropeptide receptors involves formation of inositol phosphate and activation of a calcium/calmodulin dependent process. We further report that other neuropeptides occurring in peripheral neurons, i.e., vasoactive intestinal polypeptide, calcitonin gene-related peptide, neuropeptide Y, somatostatin, or cholecystokinin, are without growth-stimulatory effect on cultured SMC.     

Maitotoxin stimulates phosphoinositide breakdown in neuroblastoma hybrid NCB-20 cells

1. Maitotoxin (MTX) was an extraordinarily potent stimulant of phosphoinositide breakdown in the neuroblastoma hybrid NCB-20 cells. 2. Maximal responses were obtained at 0.25-0.5 ng MTX/ml, and resulted in increased formation of [3H]inositol mono-, bis-, and trisphosphates. Increased formation of [3H]inositol bis- and trisphosphate was observed as early as 15 sec after the addition of MTX. 3. MTX-induced phosphoinositide breakdown in NCB-20 cells was not antagonized by organic (nifedipine, methoxyverapamil) or inorganic (Mn2+, Co2+, Cd2+) calcium channel blockers. However, the response on phosphoinositide breakdown was completely eliminated in the absence of extracellular calcium. 4. The results suggest that MTX either directly stimulates phosphoinositide breakdown in a calcium-dependent manner or acts indirectly through calcium channels insensitive to organic/inorganic calcium channel blockers.     

Exogenous Gangliosides Stimulate Breakdown of Neuro-2A Phosphoinositides in a Manner Unrelated to Neurite Outgrowth

Gangliosides administered exogenously are well-known effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BBG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BBG concentration of 25 microM. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on ganglioside-stimulated neurite outgrowth. A similar result was obtained with psi-tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded.     

Microinjection of inositol 1,2-(cyclic)-4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium

Inositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium-regulated chloride channels but do not have a calcium-induced calcium release system. Microinjection of inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,2-(cyclic)-4,5-trisphosphate (cIP3), inositol 1,4,5-trisphosphate (IP3), or inositol 4,5-bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1-phosphate (IP1), inositol 1,3,4,5,6-pentakisphosphate (IP5), inositol 1,4-bisphosphate, or inositol 3,4-bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half-maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono- or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long-term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 much greater than IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.