thapsigargin and gadolinium-chloride

Odontoblasts, with their strategic arrangement along the outermost compartment of the dentin-pulp complex, have been suggested to have sensory function. In addition to their primary role in dentin formation, growing evidence shows that odontoblasts are capable of sensing mechanical stimulation. Previously, we found that most odontoblasts express TRPM7, the nonselective mechanosensitive ion channel reported to be critical in Mg

Topics: Animals; Fingolimod Hydrochloride; Gadolinium; Immunohistochemistry; Ionomycin; Male; Mechanotransduction, Cellular; Naltrexone; Odontoblasts; Radiometry; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin; Transient Receptor Potential Channels; TRPM Cation Channels

Arginine vasopressin (AVP) causes increase in intracellular Ca(2+) concentration with an oscillatory pattern. Ca(2+) mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca(2+) oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca(2+) oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca(2+) oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca(2+) oscillations and endogenous cADPR plays a major role in the AVP-induced Ca(2+) oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP(3)) induced Ca(2+) release but did not maintain sustained Ca(2+) oscillations. Removal of extracellular Ca(2+) halted ongoing AVP-mediated Ca(2+) oscillation, suggesting that it requires extracellular Ca(2+) entry. AVP-induced Ca(2+) oscillation was unaffected by nifedipine. Intracellular Ca(2+) store depletion induced by 20 μM thapsigargin in Ca(2+)-free medium triggered store-operated Ca(2+) entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl(3) and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca(2+)-free medium, application of extracellular Ca(2+) also triggered Ca(2+) influx, which was sensitive to GdCl(3) and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca(2+) oscillations in IMCD are mediated by the interplay of Ca(2+) release from RyRs and a Ca(2+) influx mechanism involving nonselective cation channels that resembles SOCE.

Topics: Animals; Arginine Vasopressin; Calcium; Calcium Channel Blockers; Calcium Signaling; Cyclic ADP-Ribose; Gadolinium; Imidazoles; Inositol 1,4,5-Trisphosphate; Kidney Medulla; Kidney Tubules, Collecting; Male; Nifedipine; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Thapsigargin

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca(2+) stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca(2+) imaging, we found that the depletion of ER/SR Ca(2+) stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca(2+) ([Ca(2+)](i)), followed by sustained increase depending on extracellular Ca(2+). But, TRP channels inhibitor (SKF96365), not L-type channels or the Na(+)/Ca(2+) exchanger inhibitors, inhibited [Ca(2+)](i) relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl(3)) or by an increased extracellular Ca(2+)([Ca(2+)](o)) increased the concentration of intracelluar Ca(2+), whereas, the sustained elevation of [Ca(2+)](i) was reduced in the presence of SKF96365. Similarly, the duration of [Ca(2+)](i) increase was also shortened in the absence of extracellular Ca(2+). Western blot analysis showed that GdCl(3) increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl(3). Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca(2+)-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

Topics: Animals; Apoptosis; Calcium; Calcium Channel Blockers; Gadolinium; Heart Ventricles; Imidazoles; Muscle Cells; Rats; Rats, Wistar; Receptors, Calcium-Sensing; Thapsigargin; TRPC Cation Channels

The effects of diethylstilbestrol (DES) on steady-state intracellular calcium concentration ([Ca(2+)](i)) and resting Ca(2+) influx were examined in primary cultures of bovine lens epithelial cells using conventional fluorometric techniques (Fura-2). At low concentrations (10 microM), DES usually induced relatively rapid increases in [Ca(2+)](i) that occurred over an interval of 10-50 s and that persisted for several minutes in the continued presence of the drug. In about 10% of the cells, cyclic oscillations in [Ca(2+)](i) were seen after adding 10 microM DES. At higher concentrations (100 microM), the drug induced more prolonged increases in [Ca(2+)](i) lasting several minutes. DES did not affect Mn(2+) quench determinations of resting Ca(2+) influx, and neither 100 microM GdCl(3), which blocked resting Ca(2+) influx, nor low [Ca(2+)](o) solutions substantially diminished the influence of DES on [Ca(2+)](i). Pretreatment of cells with the smooth endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitors cyclopiazonic acid (CPA) or thapsigargin completely abolished the effect of 10 microM DES on [Ca(2+)](i), while the IP(3) receptor blocker 2-aminoethoxydiphenyl borane (2-APB) had no effect. These results indicate that DES releases CPA-sensitive stores of intracellular Ca(2+), perhaps by inhibiting SERCA-dependent Ca(2+) sequestration.

Topics: Animals; Boron Compounds; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cattle; Diethylstilbestrol; Endoplasmic Reticulum, Smooth; Epithelial Cells; Fura-2; Gadolinium; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Lens, Crystalline; Receptors, Cytoplasmic and Nuclear; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.

Topics: 3T3 Cells; Animals; Calcium Channel Blockers; Calcium Signaling; Enzyme Inhibitors; Gadolinium; Gene Expression Regulation; Imidazoles; Intracellular Fluid; JNK Mitogen-Activated Protein Kinases; Kinetics; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Models, Biological; Oscillometry; Osteoblasts; Osteopontin; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA, Messenger; Sialoglycoproteins; Stress, Mechanical; Thapsigargin; Transcription, Genetic

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Animals, Newborn; Biological Transport; Calcium; Calcium Channel Blockers; Cell Membrane Permeability; Cells, Cultured; Cytosol; Dihydropyridines; Diltiazem; Egtazic Acid; Gadolinium; Hydrogen Peroxide; Isradipine; Kidney Cortex; Kidney Tubules, Collecting; Kinetics; Lanthanum; Nifedipine; Protamines; Rats; Rats, Sprague-Dawley; Thapsigargin; Verapamil

Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Calcium; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Fluorescent Dyes; Gadolinium; Lysophospholipids; Microscopy, Confocal; Stress, Mechanical; Thapsigargin; Time Factors; Xanthenes

The objective of the present study was to further investigate the ionic mechanism of the action of GHRP-6 on male rat pituitary cells in culture. A synthetic hexapeptide, GHRP-6 stimulates the secretion of growth hormone both in vivo and in vitro. It is generally accepted that Ca2+ and protein kinase C but not cAMP are involved in the signal transduction pathway of the action of GHRP-6. Ca2+-influx through voltage-gated Ca2+ channels and mobilization of internal stored Ca2+ are thought to be responsible for an increase in cytosolic Ca2+ concentration. For activation of the voltage-gated Ca2+ channels, however, it is not determined whether the membrane Na+ permeability plays a role. To answer this question, we measured intracellular Na+ concentration of the pituitary cells with ion imaging technique. We found that GHRP-6 increased [Na+]i; the Na+ response depended on the presence of extracellular Na+ and was blocked by Gd3+, known as a blocker of nonselective cation channels but not by tetrodotoxin, a blocker of the voltage-gated Na+ channel; thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, had no effect on the response; Ca2+ chelating agent, BAPTA had no inhibitory effect on the response; ouabain, an inhibitor of Na+-K+ ATPase, did not block the rise in [Na+]i induced by GHRP-6; somatostatin, which hyperpolarizes the cells by activating K+ channels, suppressed the response. These data clearly showed that GHRP-6 increased [Na+]i in the rat pituitary cells including somatotrophs. The rise in [Na+]i is likely to be due to an increase in the membrane Na+ permeability which should depolarize the cells, thereby activating the voltage-gated Ca2+ channels. This process leads to an influx of Ca2+ and subsequent increase in [Ca2+]i which results in an exocytotic release of GH.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Extracellular Space; Gadolinium; Hormones; Intracellular Fluid; Male; Oligopeptides; Ouabain; Pituitary Gland, Anterior; Rats; Rats, Wistar; Sodium; Somatostatin; Tetrodotoxin; Thapsigargin