thapsigargin and chelerythrine

Norepinephrine (NE) activates adrenergic receptors (ARs) in the hypothalamic paraventricular nucleus (PVN) to increase excitatory currents, depolarise neurones and, ultimately, augment neuro-sympathetic and endocrine output. Such cellular events are known to potentiate intracellular calcium ([Ca

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Benzophenanthridines; Cadmium Chloride; Calcium; Clonidine; Cytosol; Estrenes; Macrocyclic Compounds; Male; Neurons; Norepinephrine; Oxazoles; Paraventricular Hypothalamic Nucleus; Phenylephrine; Prazosin; Pyrrolidinones; Rats; Receptors, Adrenergic, alpha-1; Thapsigargin

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Carrier Proteins; Caspases; Cell Line, Tumor; Chromatin; Colchicine; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Microfilament Proteins; Necrosis; Neurons; Nocodazole; Peptidomimetics; Quinolines; Rotenone; Signal Transduction; Staurosporine; Thapsigargin

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.

Topics: Benzophenanthridines; Boron Compounds; Calcium Channel Blockers; Calcium Signaling; Cell Line, Tumor; Cell Membrane; Chelating Agents; Cytoplasm; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation; Humans; Leukocyte Elastase; Mucin 5AC; Piperazines; Protein Kinase C; Protein Transport; Receptor, PAR-2; RNA, Messenger; Thapsigargin

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Benzophenanthridines; Binding Sites; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Humans; Isoquinolines; Kinetics; Molecular Docking Simulation; Structure-Activity Relationship

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET(A )receptor antagonist) or BQ-788 (ET(B )receptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET(B )receptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET(A )and ET(B )receptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET(A )and ET(B )receptor activation in murine small intestinal ICCs and ET(A )receptor activation in murine large intestinal ICCs by external Ca(2+) influx and internal Ca(2+) release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.

Topics: Animals; Benzophenanthridines; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Endothelin-1; Endothelin-2; Endothelin-3; Interstitial Cells of Cajal; Intestine, Large; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred BALB C; Naproxen; Oligopeptides; Patch-Clamp Techniques; Peptides, Cyclic; Piperidines; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Thapsigargin

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

Topics: Animals; Benzophenanthridines; Calcium; Calcium Signaling; Cell Proliferation; Cells, Cultured; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Group VI Phospholipases A2; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Protein Kinase C-epsilon; Pyrones; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.

Topics: 1-Butanol; Alkaloids; Androstadienes; Animals; Benzophenanthridines; Cell Membrane; Colchicine; Cytochalasin B; Cytoskeleton; Estrenes; Exocytosis; Flatfishes; Fluorescent Dyes; Hepatocytes; Indomethacin; Ionomycin; Isoquinolines; Masoprocol; Osmotic Pressure; Phenanthridines; Pyridinium Compounds; Pyrrolidinones; Quaternary Ammonium Compounds; Quinacrine; Signal Transduction; Sulfonamides; Thapsigargin; Wortmannin

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Botulinum Toxins; Botulinum Toxins, Type A; Brefeldin A; Calcium; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Furosemide; Glyburide; Glycyrrhetinic Acid; Guanosine Triphosphate; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Phenanthridines; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Synthesis Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Schwann Cells; Sciatic Nerve; Sulfonamides; Suramin; Thapsigargin; Time Factors; Type C Phospholipases; Uridine Triphosphate

This study investigates the exocytic responses of invertebrate hemocytes to pathogen-associated antigens. It demonstrates that a homologue of complement component C3, a key defensive protein of the innate immune system, is expressed by phagocytic hemocytes (non-refractile vacuolated cells) of the tunicate, Styela plicata. C3-like molecules are localized in sub-cellular vesicles and are rapidly exocytosed after stimulation with bacterial, fungal or algal cell surface molecules. Signal transduction analysis indicated that the induced secretion of C3-like molecules is mediated by a G-protein dependent signaling pathway, which modulates tubulin microtubules. All of this evidence indicates that hemocytes can contribute to host defense responses by rapidly exocytosing C3-like proteins at sites of infection.

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Western; Calcimycin; Carrageenan; Cell Survival; Cholera Toxin; Colchicine; Colforsin; Complement C3; Cytochalasin D; Cytoplasmic Vesicles; Enzyme-Linked Immunosorbent Assay; Exocytosis; Hemocytes; Immunohistochemistry; Kinetics; Lipopolysaccharides; Mannans; Microscopy, Electron; Phenanthridines; Staurosporine; Tetradecanoylphorbol Acetate; Thapsigargin; Urochordata

In small segments of circular smooth muscle bundle isolated from the guinea-pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5-10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium-ATPase at internal stores), 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of inositol 1,4,5-trisphosphate (IP3)-receptor-mediated Ca2+ release), and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2-APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol-12-myristate-13-acetate, a selective stimulant of PKC, at low concentrations (< 10 nM) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration-dependent differences in the actions of phorbol ester on the translocation of PKC was considered.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Boron Compounds; Calcium; Calcium Signaling; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinogens; Electric Stimulation; Enzyme Inhibitors; Evoked Potentials; Guinea Pigs; Indoles; Ionophores; Lanthanum; Male; Maleimides; Muscle, Smooth; Phenanthridines; Protein Kinase C; Pyloric Antrum; Tetradecanoylphorbol Acetate; Thapsigargin

1. The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)-induced contraction in cat detrusor muscle cells 2. Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. 3. The maximal contraction to ACh was generated at 10(-11) M. This response was preferentially antagonized by M3 muscarinic receptor antagonist rho-fluoro-hexahydrosiladifenidol (rhoF-HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G-proteins (Gq/11), (Gs), (G0), (Gi1), (Gi2) and (Gi3) in the bladder detrusor muscle. ACh-induced contraction was selectively inhibited by (Gq/11) antibody but not to other G subunit. 4. The phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor neomycin reduced ACh-induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh-induced contraction. ACh-induced contraction was inhibited by antibody to PLC-beta1 but not PLC-beta3 and PLC-gamma. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh-induced contraction. Inositol 1,4,5-triphosphate IP3 receptor inhibitor heparin reduced ACh-induced contraction. 5. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor-dependent activation of Gq/11 and PLC-beta1 and IP3-dependent Ca(2+) release.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Calcium Channels; Cats; Cell Size; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; GTP-Binding Proteins; Heparin; Inositol 1,4,5-Trisphosphate Receptors; Isoenzymes; Male; Muscarinic Antagonists; Muscle, Smooth; Neomycin; p-Chloromercuribenzoic Acid; Phenanthridines; Phospholipases; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; Receptors, Muscarinic; Signal Transduction; Strontium; Thapsigargin; Type C Phospholipases; Urinary Bladder

Many excitatory neurotransmitters and neuropeptides regulate the activity of neuronal and endocrine cells by modulating voltage-operated Ca2+ channels. Paradoxically, however, excitatory neuromediators that provoke mobilization of intracellular calcium from inositol trisphosphate (IP3)-sensitive stores usually inhibit voltage-gated Ca2+ currents. We have recently demonstrated that neurotensin (NT) stimulates the electrical and secretory activities of frog pituitary melanotrophs, and increases intracellular calcium concentration in these cells. In the present study, we have investigated the effects of NT on Ca2+ currents in cultured frog melanotrophs by using the perforated patch-clamp technique. Frog neurotensin (f NT) reduced the amplitude and facilitated the inactivation of both L- and N-type Ca2+ currents. Application of the membrane-permeant Ca2+ chelator BAPTA-AM, the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, or the IP3 receptor antagonist 2-APB suppressed the reduction of Ca2+ currents induced by f NT. Incubation of melanotrophs with the diacylglycerol analogue PMA, which causes desensitization of protein kinase C (PKC), or with the PKC inhibitors chelerythrine and calphostin C, reduced the inhibitory effect of f NT. The NT-induced action potential waveforms, applied as voltage-clamp commands, decreased the amplitude of Ca2+ currents, and enhanced Ca2+ influx by increasing the Ca2+ spike frequency. Altogether, these data indicate that the inhibitory effect of f NT on Ca2+ currents results from activation of the IP3/PKC pathway. The observation that NT controls Ca2+ signalling through both amplitude and frequency modulations of Ca2+ currents suggests that NT might induce spacial and temporal changes of intracellular Ca2+ concentration leading to stimulation of exocytosis.

Topics: Alkaloids; Animals; Benzophenanthridines; Boron Compounds; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Cell Culture Techniques; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Inositol 1,4,5-Trisphosphate Receptors; Male; Naphthalenes; Neurotensin; Patch-Clamp Techniques; Phenanthridines; Pituitary Gland; Protein Kinase C; Rana ridibunda; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thapsigargin

Although the effects of mechanical loading on chondrocyte metabolic activities have been extensively characterized, the sequence of events through which extracellular mechanical signals are transduced into chondrocytes and ultimately modulate cell activities is not well understood. Here, studies were performed to map out the sequential intracellular signalling pathways through which compression-induced signals modulate aggrecan mRNA levels in bovine articular chondrocytes. Bovine articular cartilage explants were subjected to a compressive stress of 0.1 MPa for 1 h in the presence or absence of inhibitors or antagonists of the phosphoinositol and Ca(2+)/calmodulin signalling pathways in order to determine the roles of second messengers and effector molecules of these pathways in transducing the compression-induced signals. In the absence of the inhibitors, aggrecan mRNA levels were stimulated by compression 2-4-fold relative to levels in tare-loaded (see below) explants. Treatment of the explants with graded levels of the protein kinase C inhibitor chelerythrine or bisindolylmaleimide I, followed by 1 h compressive loading, did not significantly alter the load-induced elevation of aggrecan mRNA levels. In contrast, thapsigargin, which depletes the Ins(1,4,5)P3-sensitive intracellular Ca(2+) stores, completely blocked the load response without significantly altering aggrecan mRNA levels in tare-loaded explants. Similarly, antagonists of the Ca(2+)/calmodulin signalling pathway dose-dependently or completely blocked the load-response. The results obtained demonstrate that transduction of the compression-induced aggrecan mRNA-regulating signals requires Ins(1,4,5)P3- and Ca(2+)/calmodulin-dependent signalling processes in bovine articular chondrocytes.

Topics: Aggrecans; Alkaloids; Animals; Benzophenanthridines; Benzylamines; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cartilage; Cattle; Chondrocytes; Cyclic AMP; Cyclosporine; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix Proteins; Indoles; Inositol 1,4,5-Trisphosphate; Lectins, C-Type; Maleimides; Models, Biological; Phenanthridines; Protein Kinase C; Proteoglycans; RNA, Messenger; Signal Transduction; Sulfonamides; Thapsigargin; Time Factors

Suppression of the voltage-activated, noninactivating K(+) conductance (M conductance; g(M)) by muscarinic agonists, P(2Y) agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/(11) class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) but not by the inactive isomer, U73343. The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) failed to affect ATP-induced g(M) suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that "lipid-signaling" mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g(M) by ATP in amphibian sympathetic neurons.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Benzophenanthridines; Calcium; Calcium Channels; Culture Techniques; Electric Conductivity; Enzyme Inhibitors; Estrenes; Female; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Male; Neural Inhibition; Neurons; Phenanthridines; Protein Kinase C; Pyrrolidinones; Rana catesbeiana; ras Proteins; Receptors, Cytoplasmic and Nuclear; Sympathetic Nervous System; Thapsigargin; Type C Phospholipases

Oestrogen plays an essential role in regulating growth and differentiation in the human endometrium which undergoes dynamic morphological and functional changes during the menstrual cycle in preparation for implantation. In this tissue, it has been suggested that intracellular calcium could be a key signal in transducing early responses to steroid hormones. Here, we have investigated the rapid effects of 17beta-oestradiol on [Ca2+]i in a human endometrial cell line (RL95-2). Using confocal imaging microscopy, we show that physiological concentrations of 17beta-oestradiol trigger rapid and transient increases in [Ca2+]i. Our results demonstrate that 17beta-oestradiol-induced [Ca2+]i variations are critically dependent on calcium influx via lanthanum-sensitive calcium channels. Moreover, the 17beta-oestradiol-induced Ca2+ influx is significantly increased by the depletion of intracellular stores by thapsigargin and decreased by chelerythrine chloride, an inhibitor of protein kinase C. These data indicate a non-genomic action of 17beta-oestradiol to stimulate capacitative Ca2+ entry through store-operated calcium channels via a PKC-sensitive pathway.

Topics: Alkaloids; Benzophenanthridines; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Dose-Response Relationship, Drug; Endometrium; Estradiol; Female; Humans; Microscopy, Confocal; Phenanthridines; Protein Kinase C; Thapsigargin

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 microM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

Topics: Alkaloids; Animals; Benzophenanthridines; Brain; Calcium; CHO Cells; Cricetinae; Cytosol; Down-Regulation; Enzyme Activation; Glycerophospholipids; Phenanthridines; Phospholipase D; Protein Kinase C; Protein Kinase Inhibitors; Rats; Receptor, Cholecystokinin A; Receptors, Cholecystokinin; Recombinant Proteins; Sincalide; Tetradecanoylphorbol Acetate; Thapsigargin

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cells, Cultured; Enzyme Activation; Inositol 1,4,5-Trisphosphate; Ionomycin; Male; Naphthalenes; Phenanthridines; Phorbol Esters; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Submandibular Gland; Thapsigargin

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Topics: Alkaloids; Benzophenanthridines; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Humans; Ionophores; Liver Neoplasms; Models, Chemical; Oleic Acid; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Quinacrine; Thapsigargin; Tumor Cells, Cultured

Mobilization of intracellular Ca2+ stores is coupled to Ca2+ influx across the plasma membrane, a process termed capacitative Ca2+ entry. Capacitative Ca2+ entry was examined in cultured guinea pig enteric glia exposed to 100 microM ATP, an inositol trisphosphate-mediated Ca2+-mobilizing agonist, and to 1 microM thapsigargin, an inhibitor of microsomal Ca2+ ATPase. Both agents caused mobilization of intracellular Ca2+ stores followed by influx of extracellular Ca2+. This capacitative Ca2+ influx was inhibited by Ni2+ (88 +/- 1%) and by La3+ (87 +/- 1%) but was not affected by L- or N-type Ca2+ channel blockers. Pretreatment of glia with 100 nM phorbol 12-myristate 13-acetate for 24 h decreased capacitative Ca2+ entry by 48 +/- 2%. Chelerythrine (0.1-10 microM), a specific antagonist of protein kinase C (PKC), dose dependently inhibited capacitative Ca2+ entry. The nitric oxide synthase inhibitor NG-nitro-L-arginine (1 mM) decreased Ca2+ influx by 42 +/- 1%. Capacitative Ca2+ entry was inhibited to a similar degree by the guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Capacitative Ca2+ entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by PKC and nitric oxide.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Barium; Benzophenanthridines; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Enzyme Inhibitors; Guinea Pigs; Kinetics; Lanthanum; Microsomes; Myenteric Plexus; Neuroglia; Phenanthridines; Tetradecanoylphorbol Acetate; Thapsigargin

We have previously shown that ANG II increases microvascular permeability in normal dog lungs but not after pacing-induced heart failure. This study investigated how ANG II induces permeability in isolated blood-perfused canine lung lobes and what alterations occur during heart failure. In normal lobes, the protein kinase C (PKC) inhibitors staurosporine (500 nM) or chelerythrine (10 microM) did not modify ANG II-induced increases in the capillary filtration coefficient (Kf,c, ml . min-1 . cmH2O-1 . 100 g-1; an index of microvascular permeability), suggesting that PKC is not involved. Thapsigargin (150 nM) was used to stimulate capacitative Ca2+ entry in lobes from control dogs and dogs paced at 245 beats/min for 4 wk to induce heart failure. In control lobes, Kf,c rose after thapsigargin, from 0.06 +/- 0.01 to 0.17 +/- 0.03 ml . min-1 . cmH2O-1 . 100 g-1 (mean +/- SE, P < 0.05) but did not change in the paced group. A Ca2+ ionophore, A-23187, increased Kf,c in both control (10 microM; 0.05 +/- 0.01 to 0.17 +/- 0.05 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) and pace (5 microM; 0.06 +/- 0.01 to 0. 21 +/- 0.07 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) lobes, indicating that increasing intracellular Ca2+ is sufficient to induce pulmonary microvascular permeability after pacing. We conclude that during heart failure, Ca2+ signaling within the pulmonary microvascular endothelium is altered.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcimycin; Calcium; Capillary Permeability; Cardiac Output, Low; Cardiac Pacing, Artificial; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Ionophores; Lung; Phenanthridines; Protein Kinase C; Signal Transduction; Staurosporine; Thapsigargin; Vascular Resistance

Calcium antagonists lower plasma levels of lipoproteins and suppress hepatic very low-density lipoprotein (VLDL) secretion. Similar effects have been observed with the calcium ionophore A23187. We studied further the effect of calcium on VLDL metabolism.. Hepatocytes from male Wistar rats were isolated and cultured in the presence or absence of calcium-mobilizing hormones, or compounds that either stimulate or inhibit the activity of protein kinase C. Secreted VLDL (d < 1.006 g mL-1) was isolated by centrifugation (145,000 x g), and lipids and apolipoprotein B were analysed.. VLDL secretion reached maximum in hepatocytes cultured in medium containing calcium 0.8-2.4 mmolL-1. Depleting the cells of calcium by incubating in calcium-free medium or by treating the cells with the Ca(2+)-ATPase inhibitor thapsigargin (5 x 10-7 molL-1) suppressed lipid secretion to less than 15% of control, and this was accompanied by an increase in cellular levels of triacylglycerol. Calcium loading (medium calcium > 2.4 mmolL-1) suppressed both lipoprotein secretion and cellular levels of lipids, suggesting a reduced overall rate of lipid synthesis. At an extracellular calcium concentration of 0.8 mmolL-1, angiotensin II, vasopressin, endothelin-1 (10(-7) molL-1) or phenylephrine (10(-4) molL-1) suppressed VLDL secretion (maximum to 37% of control), and elevated medium calcium attenuated this effect. The protein kinase C inhibitor chelerythrine (5 x 10(-5) molL-1) and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (10(-6) molL-1), suppressed VLDL secretion to 18% and 60% of control, respectively, whereas the protein kinase C-inactive 4 alpha-PMA was without an effect. No effect on ketogenesis was observed by these compounds, indicating that suppressed lipid secretion was not due to an enhanced oxidation of lipids.. Hepatic VLDL secretion can be related to changes in hepatocyte levels of calcium and the activity of protein kinase C.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Cholesterol; Culture Media; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Ketone Bodies; Lipid Metabolism; Lipoproteins, VLDL; Liver; Male; Phenanthridines; Phenylephrine; Protein Kinase C; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Thapsigargin

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.

Topics: Alkaloids; Animals; Benzophenanthridines; Calcimycin; Calcium; Carbachol; Cell Line; Corticotropin-Releasing Hormone; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Kinetics; Mice; Nifedipine; Phenanthridines; Protein Kinase C; Receptors, Corticotropin-Releasing Hormone; RNA, Messenger; Tetradecanoylphorbol Acetate; Thapsigargin; Transcription, Genetic

We have previously shown that chronic antagonism of metabotropic glutamate receptors in the brain attenuates naloxone-precipitated withdrawal symptoms in rats treated chronically with subcutaneous (s.c.) morphine. Several subtypes of metabotropic glutamate receptors are directly linked, through a guanine nucleotide regulatory protein, to the phosphatidylinositol (p.i.) second messenger system. In the present investigation, we assessed the effect of inhibiting the products of p.i. hydrolysis on the development of opioid dependence. Thus, concurrently with subcutaneous morphine, we infused intracerebroventricularly (i.c.v.) in rats, various doses of chelerythrine, which selectively inhibits the activation of protein kinase C, and thapsigargin, which inhibits the release of intracellular Ca2+ when given chronically. Both chelerythrine and thapsigargin reduced the severity of naloxone-precipitated abstinence symptoms when infused i.c.v. at a dose of 10 nmol/day. A single injection of either chelerythrine or thapsigargin immediately prior to the precipitation of withdrawal failed to decrease the severity of abstinence symptoms. Our results suggest that by chronically inhibiting activity of the phosphatidylinositol system, the development of morphine dependence can be attenuated.

Topics: Alkaloids; Analgesics, Opioid; Analysis of Variance; Animals; Behavior, Animal; Benzophenanthridines; Calcium-Transporting ATPases; Enzyme Activation; Hydrolysis; Male; Morphine; Morphine Dependence; Phenanthridines; Phosphatidylinositols; Protein Kinase C; Rats; Substance Withdrawal Syndrome; Thapsigargin