thapsigargin and adenosine-3--5--cyclic-phosphorothioate

An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.

Topics: 1-Methyl-3-isobutylxanthine; Calcium; Calcium-Transporting ATPases; Cyclic AMP; Cytoplasm; Egtazic Acid; Enzyme Inhibitors; Humans; Male; Phosphorylation; Phosphotyrosine; Protein Kinase Inhibitors; Proteins; Signal Transduction; Sperm Capacitation; Spermatozoa; Thapsigargin; Thionucleotides

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

Topics: Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dideoxyadenosine; Diglycerides; Enzyme Inhibitors; Estrenes; Grasshoppers; Inositol 1,4,5-Trisphosphate; Isoquinolines; Muscarine; Muscarinic Agonists; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Receptors, Muscarinic; Second Messenger Systems; Sphingosine; Sulfonamides; Thapsigargin; Thionucleotides; Type C Phospholipases

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

beta-Receptor desensitisation, low basal cAMP, and a negative force-frequency relationship are characteristic changes in human heart failure. Isolated cardiomyocytes from noradrenaline-treated guinea pigs also show these features. We tested the hypothesis that low basal cAMP underlies the loss of contractile response to increasing stimulation frequency in this model.. Isolated cardiomyocytes were obtained from noradrenaline-treated (NA) and sham-operated (SHAM) guinea pigs. They were stimulated from 0.1-2 Hz and contraction amplitude was monitored with a video edge-detection system.. NA cells had less positive amplitude-frequency responses (AFR) compared to SHAMs at 2 mM (P = 0.002, n = 17), or midrange Ca2+ concentrations (EC40-EC60) (P < 0.001, n = 13). When the cAMP agonist, 8-CPT-cAMP (CPT, 10 microM) or high Ca2+ (above EC75) was added to NA cells the AFR was normalised to that of SHAM myocytes (NA vs. SHAM P = ns). In control experiments the cAMP antagonists, Rp-cAMPS (Rpc) and Rp-8-CPT-cAMPS (Rp8, 100 microM), blocked the positive inotropic effects of CPT at 0.5 Hz (control pD2 = 4.36 +/- 0.06, Rp8 pD2 = 3.68 +/- 0.08, P < 0.0001), n = 6 paired). Rpc (100 microM) completely but reversibly blocked the effect of maximal isoprenaline in control experiments (P < 0.0001). Neither antagonist reduced the AFR compared to time-matched controls (P = ns, n = 6). Blockade of SERCA2a with thapsigargin resulted in a significant reduction in the AFR (ANOVA P < 0.0001).. The results are consistent with sarcoplasmic reticulum (SR) function being a more important determinant of the amplitude-frequency relationship than tonic levels of cAMP under basal conditions. Reversal of AFR depression by CPT may result from stimulation of SR Ca2+ uptake.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Calcium-Transporting ATPases; Cell Size; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Guinea Pigs; Heart Failure; Isoproterenol; Male; Myocardial Contraction; Myocardium; Norepinephrine; Thapsigargin; Thionucleotides

In this study, we examined the influence of high levels of cytosolic calcium on phosphorylation status and function of GLUT-4 in isolated rat adipocytes. Intracellular calcium was elevated by exposing adipocytes to either extracellular ATP (1.6 mM) or thapsigargin (100 nM). Both agents increased cytosolic calcium 2-3 fold. While basal glucose uptake was unaffected, both ATP and thapsigargin reduced insulin-stimulated glucose transport by 40-70% (p < 0.05). Neither ATP nor thapsigargin affected GLUT-4 content or its translocation from the low density microsomes to the plasma membrane (PM). In contrast, GLUT-4 immunoprecipitated from the PM of adipocytes exposed to either ATP or thapsigargin was phosphorylated to a greater extent than the GLUT-4 isolated from control cells. ATP and thapsigargin also abolished insulin-stimulated dephosphorylation of GLUT-4. At the same time, GLUT-4 intrinsic activity was significantly reduced in adipocytes with high levels of cytosolic calcium (p < 0.05). Preincubation of adipocytes with cAMP antagonist, RpcAMP (10(-4) M), and calcium channel blocker, nitrendipine (30 microM), improved the ability of insulin to dephosphorylate GLUT-4 and restored insulin-stimulated GLUT-4 intrinsic activity. We conclude that elevated levels of cytosolic calcium interfere with insulin's ability to dephosphorylate GLUT-4, thus reducing its intrinsic activity.

Topics: Adenosine Triphosphate; Adipose Tissue; Animals; Biological Transport; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Cyclic AMP; Deoxyglucose; Glucose; Insulin; Kinetics; Male; Monosaccharide Transport Proteins; Nitrendipine; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin; Thionucleotides