ryanodine and 2-5-di-tert-butylhydroquinone

Modulation of Ca(2+) stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca(2+) store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca(2+) imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca(2+) signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca(2+) signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 microM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca(2+) channels or sarco(endo)plasmic reticulum Ca(2+)-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca(2+) stores found in endoplasmic reticulum.

Topics: Animals; Caffeine; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cells, Cultured; Central Nervous System Stimulants; Cyclic AMP-Dependent Protein Kinases; Dantrolene; Enzyme Inhibitors; Goldfish; Gonadotropins, Pituitary; Hydroquinones; Isoquinolines; Muscle Relaxants, Central; Pituitary Gland; Potassium Channel Blockers; Potassium Channels; Ryanodine; Sulfonamides; Tetraethylammonium

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

Topics: Adenosine Triphosphate; Animals; Anura; Calcium; Cell Membrane; Chlorides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroquinones; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Kidney Tubules, Distal; Osmolar Concentration; Ryanodine; Sodium

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.

Topics: Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Female; Hydroquinones; Male; Mammals; Membrane Potentials; Neurons, Afferent; Rabbits; Ryanodine

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.

Topics: Animals; Antifungal Agents; Biological Transport; Caffeine; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cells, Cultured; Central Nervous System Stimulants; Enzyme Inhibitors; Estrenes; Extracellular Space; Fetus; Hydroquinones; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Isoenzymes; Lanthanum; Magnesium; Mice; Neural Crest; Neurons; Phosphodiesterase Inhibitors; Phospholipase C gamma; Pyrrolidinones; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thimerosal; Type C Phospholipases

1. Standard intracellular recording techniques with 'sharp' micropipettes were used to evoke action potentials (APs) in acutely dissociated adult nodose neurones. 2. APs induced a transient increase in [Ca2+]i (a calcium transient), recorded with fura-2, that was dependent upon [Ca2+]o and the number of APs. Over the range of one to sixty-five APs, the relation between the amplitude of the calcium transient and the number of APs was well fitted by a rectangular hyperbola (chi 2 = 3.53, r = 0.968). From one to four APs, the calcium transient-AP relation can be described by a line with a slope of 9.6 nM AP-1 (r = 0.999). 3. Charge movement corresponding to Ca2+ influx evoked by a single AP was 39 +/- 2.8 pC (mean +/- S.E.M.) and did not change significantly during trains of one to thirty-one APs (P < 0.05). 4. Caffeine (10 mM), a known agonist of the ryanodine receptor, produced an increase in [Ca2+]i. The caffeine-induced rise in [Ca2+]i was attenuated (by > 90%) by lowering [Ca2+]o, and by ryanodine (10 microM), 2,5-di(t-butyl)hydroquinone (DBHQ, 10 microM), or thapsigargin (100 nM). 5. Neurones incubated with ryanodine, DBHQ or thapsigargin required at least eight APs to evoke a detectable calcium transient. These reagents did not significantly affect Ca2+ influx (P < 0.05). In the presence of these inhibitors, the calcium transient-AP relation exhibited slopes of 1.2, 1.1 and 1.9 nM AP-1 for ryanodine, DBHQ and thapsigargin, respectively. When compared with the slope of 9.6 nM AP-1 in non-treated neurones, it appears that Ca2+ influx produced by a single AP is amplified by ca 5- to 10-fold.

Topics: Action Potentials; Animals; Cadmium; Caffeine; Calcium; Calcium-Transporting ATPases; Central Nervous System Stimulants; Electrophysiology; Enzyme Inhibitors; Female; Hydroquinones; Male; Neurons, Afferent; Nodose Ganglion; Rabbits; Ryanodine; Thapsigargin

Force of contraction (Fc) of isolated human and rat atrial myocardium shows characteristic patterns of mechanical restitution when single test intervals are interposed in regular stimulation. With several pharmacological agents that modify the function of the sarcoplasmic reticulum we have investigated the role of the sarcoplasmic reticulum in mechanical restitution in these two species. Caffeine, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) were used to reduce Ca2+ uptake, ryanodine to open Ca2+ release channels, and forskolin to stimulate Ca2+ uptake. Under control conditions, Fc recovered rapidly with test intervals shorter than steady-state, and was potentiated with longer than steady-state intervals. In human atrial tissue the maximum potentiation factor was 1.26 +/- 0.03 after a mean test interval of 9.70 +/- 1.55 s (n = 43) as compared to 3.07 +/- 0.45 after 30 sec. in rat atria (n = 48). Caffeine (3 mM) did not significantly affect steady-state Fc but abolished post-rest potentiation in human and rat preparations. Forskolin (1 microM) enhanced and accentuated the mechanical restitution curve in particular for short test intervals. In the presence of thapsigargin (10 microM), steady-state Fc and mechanical restitution could not be distinguished from time-matched controls exposed to solvent only, indicating that this agent is ineffective in human and rat atrial tissue. In contrast, the putative Ca2+ uptake inhibitor BHQ (100 microM) strongly reduced steady-state Fc and decreased potentiation at all intervals in human muscle, but shifted the mechanical restitution curve in parallel to lower values in rat atria. Ryanodine (10 nM) induced post-rest decay in human and depressed both steady-state Fc and post-rest potentiation in rat atrial muscle. From these results it is concluded that human and rat atrial muscle differ in the Ca2+ handling by the sarcoplasmic reticulum during mechanical restitution.

Topics: Animals; Binding, Competitive; Caffeine; Calcium; Calcium Channels; Colforsin; Enzyme Inhibitors; Humans; Hydroquinones; Myocardial Contraction; Phosphodiesterase Inhibitors; Rats; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin

To study 8-(N,N-Diethylamino)n-octyl-3,4,5-trimethoxybenzoate (TMB), a potent Ca(2+)-antagonist, actions on cellular calcium dynamics in vascular smooth muscle cell (VSMC) cultures.. A7r5 VSMC were cultured with Fura-2 measurements of intracellular Ca2+ concentration, [Ca2+]i.. TMB reduced [Ca2+]i from control levels and blocked [Ca2+]i increase caused by norepinephrine (NE) and 2,5-di (t-butyl)-1,4-benzohydroquinone (BHQ). [Ca2+]i reduction by TMB was further enhanced by ryanodine.. TMB is an effective agent for blocking the [Ca2+]i increase caused by NE and BHQ and for enhancing the [Ca2+]i reduction caused by ryanodine.

Topics: Animals; Aorta, Thoracic; Calcium; Calcium Channel Blockers; Cells, Cultured; Drug Synergism; Embryo, Mammalian; Gallic Acid; Hydroquinones; Muscle, Smooth, Vascular; Norepinephrine; Rats; Ryanodine

The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+]i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 microM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+]i fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hyperpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca(2+)-ATPase of endoplasmic reticulum and by affecting Ca(2+)-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-cresol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i. It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin.

Topics: Animals; Biological Clocks; Bombesin; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Cell Line; Central Nervous System Stimulants; Chelating Agents; Circadian Rhythm; Cresols; Cricetinae; Cytosol; Diazoxide; Egtazic Acid; Fura-2; Glucose; Hydroquinones; Membrane Potentials; Ryanodine; Verapamil