calcimycin and maitotoxin

Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target.

Topics: Animals; Calcimycin; Calpain; Cells, Cultured; Chromatography, Liquid; Marine Toxins; Mice; Mice, Transgenic; Molecular Weight; N-Methylaspartate; Nanotechnology; Neurons; Oxocins; Peptides; Phosphorylation; Primary Cell Culture; Proteolysis; Rats; Spectrometry, Mass, Electrospray Ionization; Staurosporine; Tandem Mass Spectrometry; tau Proteins

1. The aim of the present study was to determine the effects of maitotoxin on nerve growth factor production and the Ca2+ influx in clonal rat glioma cells (C6-BU-1). 2. Maitotoxin (1 - 10 ng ml-1) induced a profound increase in 45Ca2+ influx in an extracellular Ca2+-dependent manner. However, high KCl had no effect at all. These effects were supported by the results from the analysis of intracellular Ca2+ concentration using fura 2. 3. The maitotoxin-induced 45Ca2+ influx was inhibited by inorganic Ca2+ antagonists, such as Mg2+, Mn2+ and Co2+. The inhibitory effect of Co2+ was antagonized by increasing the extracellular Ca2+ concentrations. 4. Maitotoxin (3 ng ml-1) as well as A-23187 (1microM) and dibutyryl cyclic AMP (0.5 mM) caused an acceleration of nerve growth factor (NGF) production in C6-BU-1 cells, as determined by NGF enzyme immunoassay. 5. Reverse transcription polymerase chain reaction (RT - PCR) analysis showed that maitotoxin (10 ng ml-1) enhanced the expression of NGF mRNA, which was abolished by the removal of extracellular Ca2+. A-23187 also accelerated its expression. 6. These results suggest that maitotoxin activates a voltage-insensitive Ca2+ channel and accelerates NGF production mediated through a Ca2+ signalling pathway in C6-BU-1 glioma cells.

Topics: Animals; Brain Neoplasms; Bucladesine; Calcimycin; Calcium Channel Agonists; Calcium Channels; Cell Line; Fluorescent Dyes; Fura-2; Glioma; Immunoenzyme Techniques; Marine Toxins; Neoplasm Proteins; Nerve Growth Factors; Oxocins; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Fish Diseases; Fishes; Hemolysis; Hydrogen-Ion Concentration; Lethal Dose 50; Magnesium; Marine Toxins; Mice; Oxocins; Quinacrine

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.

Topics: Astrocytoma; Brain Neoplasms; Calcimycin; Calcium; Chelating Agents; Egtazic Acid; Extracellular Space; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Ionophores; Marine Toxins; Oxocins; Phosphatidylinositols; Tumor Cells, Cultured

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Fetus; Kainic Acid; Kinetics; Marine Toxins; N-Methylaspartate; Neurons; Neurotoxins; Nitric Oxide Synthase; Okadaic Acid; Oxocins; Rats; Rats, Sprague-Dawley; Spectrin

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.

Topics: Activins; Alkaloids; Animals; Calcimycin; Calcium; Female; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Inhibins; Intracellular Membranes; Luteinizing Hormone; Marine Toxins; Oxocins; Protein Kinase C; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate

Maitotoxin (MTX) increases formation of [3H]inositol phosphates from phosphoinositides and release of [3H]arachidonic acid from phospholipids in pheochromocytoma PC12 cells. Formation of [3H]inositol phosphates is detected within 1 min of incubation even with concentrations as low as 0.3 ng/ml (90 pm) MTX, whereas release of [3H]arachidonic acid is not detected until 20 min even with concentrations as high as 1 ng/ml (300 pm) MTX. Stimulation of arachidonic acid release can be detected at 0.03 ng/ml (9 pm) MTX, whereas 0.1 ng/ml (30 pm) MTX is the threshold for detection of phosphoinositide breakdown. Organic and inorganic calcium channel blockers, except Cd2+ and a high concentration of Mn2+, have no effect on MTX-elicited phosphoinositide breakdown, whereas inorganic blockers (e.g., Co2+, Mn2+, Cd2+), but not organic blockers (nifedipine, verapamil, diltiazem), inhibit MTX-stimulated arachidonic acid release. All calcium channel blockers, however, inhibited MTX-elicited influx of 45Ca2+ and the MTX-elicited increase in internal Ca2+ measured with fura-2 was markedly reduced by nifedipine. MTX-elicited phosphoinositide breakdown and arachidonic acid release are abolished or reduced, respectively, in the absence of extracellular calcium plus chelating agent. The calcium ionophore A23187 has little or no effect alone but, in combination with MTX, A23187 inhibits MTX-elicited phosphoinositide breakdown and enhances arachidonic acid release, the latter even in the absence of extracellular calcium. The results suggest that different sites and/or mechanisms are involved in stimulation of calcium influx, breakdown of phosphoinositides, and release of arachidonic acid by MTX.

Topics: Adrenal Gland Neoplasms; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Radioisotopes; Drug Interactions; Enzyme Activation; Marine Toxins; Oxocins; Pheochromocytoma; Phosphatidylinositols; Phospholipases; Phospholipases A; Rats; Tumor Cells, Cultured; Type C Phospholipases

We have appraised the properties of steady state calcium exchange in cultured swine granulosa cells prelabeled to isotopic equilibrium with 45Ca2+. Subsequent steady state efflux parameters were assessed by nonlinear least-squares curve-fitting to a two-compartment model. The resultant mean rate constants were 0.27 +/- 0.07 min-1 and 0.030 +/- 0.0015 min-1, which correspond to respective half-lives of calcium exchange of 2.6 +/- 0.67 min and 23 +/- 1.1 min (means +/- SD, n = 18 experiments). The estimated sizes of the rapidly and slowly exchangeable calcium compartments in ovarian cells were 1.8 +/- 0.51 and 4.47 +/- 0.15 mumol calcium/mg protein, respectively. Equilibrium exchange of calcium was temperature dependent, and accelerated markedly by putative stimulators of calcium influx, such as maitotoxin, A23187, and BAY K 8644. Conversely, presumptive inhibitors of the calcium-magnesium dependent ATPase, such as trifluoperazine and orthovanadate, significantly diminished the size of the slowly exchangeable pool and its corresponding rate constant. A similar profound reduction in the rate of slow calcium exchange was induced by a phorbol ester and by an inhibitor of intracellular calcium mobilization, dantrolene. Physiological effector hormones also altered equilibrium calcium exchange in swine granulosa cells. Thus, FSH and prostaglandin E2 increased the rapid and slowly exchanging rate constants by factors of approximately 2.2 and 1.6, whereas prostaglandin F2 alpha increased the sizes of the slowly and rapidly exchangeable calcium compartments. In summary, the present work delineates properties of physiological steady state calcium exchange in swine granulosa cells. Physiological effector hormones, such as FSH, prostaglandin E2, and prostaglandin F2 alpha, have distinct effects on steady state calcium exchange in ovarian cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium; Dantrolene; Female; Granulosa Cells; In Vitro Techniques; Kinetics; Marine Toxins; Oxocins; Swine; Tetradecanoylphorbol Acetate; Trifluoperazine; Vanadates; Vanadium

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Drug Synergism; Female; Glycerides; Marine Toxins; Oxocins; Pituitary Gland, Anterior; Pituitary Hormones, Anterior; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Type C Phospholipases

Reserpine exerts direct effects on several tissues, including inhibition of hormone release from rat anterior pituitary cells. To test the hypothesis that reserpine may be acting as a calcium channel antagonist, normal or GH3 rat anterior pituitary cells were preincubated in reserpine or the conventional calcium channel blocker, D-600, followed by exposure to 45Ca2+ together with stimulants of calcium uptake: maitotoxin, a potent calcium channel activator; A23187, a calcium ionophore; or 50 mMK+. After incubation, the cells were harvested by vacuum filtration and cell-associated radioactivity determined. In normal cells, reserpine blocked both basal and K+-stimulated calcium uptake. Reserpine selectively blocked maitotoxin but not A23187-induced calcium uptake. In GH3 cells 9 microM reserpine and 30 microM D-600 were equally effective in blocking maitotoxin-stimulated calcium uptake. Reserpine appears to block voltage-dependent calcium channels in pituitary cells in a concentration-dependent manner but not calcium uptake caused nonspecifically by A23187.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Cell Line; Female; Gallopamil; Ion Channels; Marine Toxins; Oxocins; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Reserpine

The in vitro effect of synthetic diacylglycerol (DG) and phorbol myristate acetate (PMA), potent stimulators of protein kinase C, was studied on prolactin release. These substances increased, in a concentration-dependent manner, prolactin release from primary cultures of anterior pituitary cells. Similarly, exposure of pituitary cells to phospholipase C, which liberates endogenous DG from various substrates, also enhanced prolactin release. The effect of Ca2+ mobilization on PMA-, synthetic DG- or phospholipase C-induced prolactin release was examined. A23187 at 400 nM or 2 ng/ml maitotoxin, a Ca2+ channel activator, did not affect prolactin release by themselves, but enhanced the release of prolactin induced by DG, PMA or phospholipase C. The stimulatory effects of DG, PMA and phospholipase C on prolactin release were reduced by co-incubation with dopamine. These results suggest that the presumed activation of protein kinase C by DG and mobilization of Ca2+ may be synergistically involved in the regulation of prolactin release. Dopamine appears to inhibit prolactin release at a point distal to the DG-enhanced stimulation of the process.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Diglycerides; Dopamine; Enzyme Activation; Female; Marine Toxins; Oxocins; Pituitary Gland, Anterior; Prolactin; Protein Kinase C; Protein Kinases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Type C Phospholipases

The rat pituitary tumor 7315a secretes PRL and ACTH. Although dopamine has no effect on unstimulated PRL release from this tumor, dopamine decreases the adenylate cyclase activity in tumor cell homogenates in a manner similar to that in normal pituitary tissue. However, it was observed that under basal conditions, 7315a tumor cells have an abnormal calcium metabolism because 1) basal PRL release from tumor cells is not modified by the calcium channel blocker D-600 and is only moderately decreased by low calcium, treatments that markedly decrease normal pituitary PRL release; 2) D-600 had no effect on basal 7315a tumor calcium uptake, but blocked the increase in calcium uptake due to the calcium channel activator maitotoxin; 3) increasing the medium Ca+2 concentration above 5 mM increases 7315a PRL release, whereas this treatment decreases PRL release from normal pituitary cells. Maitotoxin and the calcium ionophore A23187 increased 7315a tumor cell PRL release in a manner similar to that in normal pituitary cells. Because dopamine blocks PRL release induced by maitotoxin, A23187, or elevated medium calcium concentration in 7315a tumor cells, the refractoriness of basal 7315a tumor cell PRL release to dopamine may be due to the abnormal calcium balance of the tumor cells under basal conditions.

Topics: Adenylyl Cyclases; Animals; Calcimycin; Calcium; Cells, Cultured; Dopamine; Female; Gallopamil; Marine Toxins; Neoplasm Transplantation; Oxocins; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains

Maitotoxin (MTX), the most potent marine toxin, caused a dose-dependent contraction of the rabbit isolated aorta at concentrations of 10(-10)-3 X 10(-8) g/ml. The dose-contractile response curve for MTX was shifted to the right in a parallel manner by verapamil (10(-6) M), was slightly shifted to the right by phentolamine (10(-6) M) and was not or little affected by tetrodotoxin, methysergide, chlorpheniramine or indomethacin. The MTX-induced contraction was abolished by incubation in Ca2+-free medium and was increased in a linear fashion with Ca2+ concentrations between 0.03 and 1.2 mM. In Ca2+-free solution, the contractile responses produced by re-introduction of Ca2+, Sr2+ or Ba2+ were potentiated after treatment with MTX (10(-8) g/ml.) and a high concentration of KCl (4 X 10(-2) M). After treatment with verapamil (10(-7)-10(-6) M), the dose-contractile response curve for Ca2+, Sr2+ or Ba2+ in the presence of MTX or KCl was shifted to the right in a parallel manner, indicating competitive antagonism. But the dose-response curve for Ca2+ in the presence of A23187 (3 X 10(-5) M), a Ca ionophore, was not affected at all by verapamil (10(-6) M). The tissue Ca content of the aorta was increased 31% by treatment with MTX (10(-8) g/ml.). This effect of MTX was markedly inhibited in the presence of verapamil. On the basis of these results, it is suggested that the MTX-induced contraction of the aorta is caused mainly by a direct action on smooth muscle, possibly due to an increase in Ca2+ permeability which occurred through voltage-sensitive Ca2+ channels in the smooth muscle cell membrane.

Topics: Animals; Aorta, Thoracic; Calcimycin; Calcium; Calcium Chloride; Dose-Response Relationship, Drug; Magnesium; Magnesium Chloride; Male; Marine Toxins; Muscle Contraction; Muscle, Smooth, Vascular; Oxocins; Phentolamine; Potassium Chloride; Rabbits; Sodium-Potassium-Exchanging ATPase; Verapamil