thapsigargin and alpha-beta-methyleneadenosine-5--triphosphate

The detection of focal Ca(2+) transients (called neuroeffector Ca(2+) transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca(2+) in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels, T-Type; Calcium Signaling; Chelating Agents; Electric Stimulation; Electrophysiology; In Vitro Techniques; Indoles; Male; Mibefradil; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Models, Biological; Muscle Contraction; Muscle, Smooth; Nickel; Prazosin; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Ryanodine; Thapsigargin; Vas Deferens

Integrins mediate cell-extracellular matrix connection and are particularly important during neuronal development. We here investigated the regulation of fibronectin (FN) matrix and neurotrophins on the embryonic synaptic transmission. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. The SSC increasing action of alpha,beta-methylene adenosine triphosphate was enhanced in neurons grown on FN substratum, which was further potentiated by chronic treatment with brain-derived neurotrophic factor (BDNF). The SSC increasing action of thapsigargin, carbonyl cyanide m-chlorophenylhydrazone and N-methyl-D-aspartate was also markedly potentiated in neurons grown on FN-coated glass coverslips and chronically treated with BDNF. FN matrix or BDNF alone only exerts slight potentiation on the SSC increasing action of these three drugs. Our results suggest that FN matrix can collaborate with neurotrophin in regulating synaptic transmission at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular junction.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Brain-Derived Neurotrophic Factor; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix; Fibronectins; Integrins; Membrane Potentials; N-Methylaspartate; Nerve Growth Factors; Neuromuscular Junction; Neurotransmitter Agents; Neurotrophin 3; Synaptic Transmission; Thapsigargin; Uncoupling Agents; Xenopus laevis

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

Topics: Acrylates; Adenosine Triphosphate; Animals; Bicuculline; Caffeine; Calcium; Capsaicin; Cell Line; Diazepam; Dihydropyridines; Dose-Response Relationship, Drug; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Ganglia, Spinal; Hybrid Cells; Mice; Mice, Inbred BALB C; Neurons; Potassium Chloride; Receptors, GABA-A; Receptors, GABA-B; Thapsigargin; Time Factors

1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage-operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U-73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC-induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling.

Topics: Adenosine; Adenosine Triphosphate; Animals; Aorta; Calcium; Calcium Channel Blockers; Calcium Signaling; Cells, Cultured; Cyclic AMP; Egtazic Acid; Estrenes; Imidazoles; Ionomycin; Manganese; Muscle, Smooth, Vascular; Phosphorylcholine; Pyrrolidinones; Sphingosine; Staurosporine; Swine; Thapsigargin; Thionucleosides; Thionucleotides; Virulence Factors, Bordetella

Adenosine-5'-triphosphate (ATP) released from damaged cells can affect functions of adjacent cells. Injuries of peripheral tissue stimulate nerves, but effect of ATP on the nerve bundles is still speculative. Peripheral nerves are surrounded by perineurium, therefore the response of perineurium may be a first event of nerve stimulation at tissue injuries. The aim of the present study is to clarify whether the perineurium responds to ATP. To this end, we analyzed the dynamics of the intracellular calcium concentration ([Ca2+]i) of perineurial cells by confocal microscopy. ATP induced a [Ca2+]i increase of perineurial cells. Ca2+ channel blockers and removing of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. This indicated that the [Ca2+]i increase was due to an influx of extracellular Ca2+. Adenosine-5'-diphosphate also elicited an increase of [Ca2+]i, but P1 receptor agonists had few effects on [Ca2+]i dynamics. Suramin (an antagonist of P2X and P2Y receptors) totally inhibited ATP-induced [Ca2+]i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5'-triphosphate (a P2Y receptor agonist) induced no significant change in [Ca2+]i, but alpha,beta-methylene ATP (a P2X receptor agonist) caused a [Ca2+]i increase. In conclusion, perineurial cells respond to extracellular ATP mainly via P2X receptors.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Anesthetics, Local; Animals; Antineoplastic Agents; Calcium; Enzyme Inhibitors; Glyburide; Hypoglycemic Agents; Lidocaine; Male; Microscopy, Confocal; Peripheral Nerves; Rats; Rats, Inbred WKY; Sciatic Nerve; Suramin; Tetraethylammonium; Tetrodotoxin; Thapsigargin; Triazines; Uridine Triphosphate

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Cell Line; DNA, Complementary; Humans; Kinetics; Magnesium; Muscle, Skeletal; Protein Binding; Rabbits; Recombinant Proteins; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Transfection; Tritium

The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2_, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Transporting ATPases; Crystallization; Kinetics; Models, Theoretical; Muscles; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Terpenes; Thapsigargin