ryanodine and 2-aminoethoxydiphenyl-borate

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10

Topics: Animals; Blood Pressure; Boron Compounds; Calcium Channels; Estrenes; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Microdomains; Mesenteric Arteries; Olive Oil; Paxillin; Pertussis Toxin; Phenol; Phenols; Potassium Channels; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Ryanodine; Ryanodine Receptor Calcium Release Channel; Type C Phospholipases; Vasodilation; Verapamil

Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol triphosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely.. We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine, 2-aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U- 73122).. Ryanodine decreases paired-pulse facilitation for intervals <20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals of <20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired-pulse depression for intervals <20 ms.. Ryanodine, IP3 receptors, and phospholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets for improving the safety factor of neuromuscular transmission during repetitive activity of the neuromuscular junction.

Topics: Animals; Anura; Biophysics; Boron Compounds; Calcium; Dose-Response Relationship, Drug; Electric Stimulation; Electrophysiology; Estrenes; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Neuromuscular Junction; Neuronal Plasticity; Phosphodiesterase Inhibitors; Pyrrolidinones; Ryanodine; Ryanodine Receptor Calcium Release Channel

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.

Topics: Action Potentials; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channels, L-Type; Carbachol; Female; Inositol 1,4,5-Trisphosphate Receptors; Large-Conductance Calcium-Activated Potassium Channels; Male; Muscle, Smooth; Mycotoxins; Myocytes, Smooth Muscle; Peptides; Potassium; Rabbits; Receptor, Muscarinic M3; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetracaine; Urethra

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca(2+) signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca(2+) oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs. LTD4-induced Ca(2+) oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca(2+) entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca(2+) oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP(3)R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca(2+) oscillations. Ca(2+) oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP(3)R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3. We conclude that IL-13 enhances frequency of LTD4-induced Ca(2+) oscillations in human ASMCs, which may be cooperatively modulated by IP(3)R, RyR systems and possibly by SOCE.

Topics: ADP-ribosyl Cyclase 1; Aged; Boron Compounds; Calcium; Calcium Signaling; Cell Count; Female; Gene Expression Regulation; Humans; Inositol 1,4,5-Trisphosphate Receptors; Interleukin-13; Leukotriene D4; Lung; Macrocyclic Compounds; Male; Myocytes, Smooth Muscle; Oxazoles; Receptors, Leukotriene; RNA, Messenger; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; TRPC Cation Channels

While glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels in the cell membrane of the pancreatic β-cell, there is also ample evidence for an important role of intracellular Ca(2+) stores in insulin secretion, particularly in relation to drug stimuli. We report here that thiopental, a common anesthetic agent, triggers insulin secretion from the intact pancreas and primary cultured rat pancreatic β-cells. We investigated the underlying mechanisms by measurements of whole cell K(+) and Ca(2+) currents, membrane potential, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and membrane capacitance. Thiopental-induced insulin secretion was first detected by enzyme-linked immunoassay, then further assessed by membrane capacitance measurement, which revealed kinetics distinct from glucose-induced insulin secretion. The thiopental-induced secretion was independent of cell membrane depolarization and closure of ATP-sensitive potassium (K(ATP)) channels. However, accompanied by the insulin secretion stimulated by thiopental, we recorded a significant intracellular [Ca(2+)] increase that was not from Ca(2+) influx across the cell membrane, but from intracellular Ca(2+) stores. The thiopental-induced [Ca(2+)](i) rise in β-cells was sensitive to thapsigargin, a blocker of the endoplasmic reticulum Ca(2+) pump, as well as to heparin (0.1 mg/ml) and 2-aminoethoxydiphenyl borate (2-APB; 100 μM), drugs that inhibit inositol 1,4,5-trisphosphate (IP(3)) binding to the IP(3) receptor, and to U-73122, a phospholipase C inhibitor, but insensitive to ryanodine. Thapsigargin also diminished thiopental-induced insulin secretion. Thus, we conclude that thiopental-induced insulin secretion is mediated by activation of the intracellular IP(3)-sensitive Ca(2+) store.

Topics: Anesthetics, Intravenous; Animals; Boron Compounds; Calcium; Estrenes; Glucose; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Insulin; Insulin Secretion; Insulin-Secreting Cells; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels; Pyrrolidinones; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Thiopental

The kidney is a major organ involved in calcium (Ca(2+)) metabolism. Ca(2+) is transported through renal tubular epithelial cells. The intracellular free calcium concentration ([Ca(2+)](i)) is tightly controlled at a low concentration, but transient increases and oscillations in [Ca(2+)](i) are induced by various conditions. In this study, we investigated the mechanisms underlying the spontaneous [Ca(2+)](i) oscillations observed in MDCK cells.. [Ca(2+)](i) was monitored in fura-2-loaded Madin-Darby canine kidney (MDCK) cells using a calcium imaging system. We investigated the mechanism by which [Ca(2+)](i) changed by applying drugs or by changing the extracellular Ca(2+) concentration.. Spontaneous [Ca(2+)](i) oscillations occurred in MDCK cells. The oscillations occurred irregularly and were not transmitted to neighboring cells. Spontaneous [Ca(2+)](i) oscillations in MDCK cells were initiated by Ca(2+) release from ryanodine/IP(3)-sensitive intracellular calcium stores, and their frequency was largely unaffected by the extracellular Ca(2+) concentration. Moreover, the frequency of the oscillations was increased by extracellular nucleotide, but was decreased when the nucleotides were removed.. Our study suggested that [Ca(2+)](i) release from ryanodine/IP(3)-sensitive intracellular calcium stores mediates spontaneous [Ca(2+)](i) oscillations in MDCK cells. Calcium oscillations may be associated with the function of the renal tubular epithelial cells.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Calcium Signaling; Cell Line; Dogs; Epithelial Cells; Estrenes; Fura-2; Inositol 1,4,5-Trisphosphate; Kidney Tubules; Probenecid; Pyrrolidinones; Ryanodine

We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.

Topics: Animals; Arteries; Arterioles; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Mice; Mice, Inbred C57BL; Muscle Development; Myocytes, Smooth Muscle; Oxazoles; Potassium Channels, Calcium-Activated; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetracaine; Type C Phospholipases; Vasomotor System

In endothelial cells there remain uncertainties in the details of how Ca(2+) signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), in contributing to the components of agonist-induced signals is unclear. The aim of this work was to increase understanding of the detailed mechanism of Ca(2+) signalling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries in response to muscarinic stimulation. In particular we have focused on the role of SERCA, and its interplay with capacitative Ca(2+) entry (CCE) and ER Ca(2+) release and uptake. We have determined its contribution to the Ca(2+) signal and how it varies with different physiological stimuli, including single and repeated carbachol applications and brief and prolonged exposures. In agreement with previous work, carbachol stimulated a rise in intracellular Ca(2+) in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca(2+) were superimposed, followed by a decline to basal Ca(2+) levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca(2+) spike and early oscillations are limited by SERCA activity, thus both are increased if SERCA is inhibited. (ii) SERCA activity is such that brief applications of carbachol do not trigger CCE, presumably because the fall in luminal Ca(2+) is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca(2+) entry occurs via STIM-mediated CCE and SERCA contributes to the cessation of CCE. In conclusion our data show how SERCA function is crucial to shaping endothelial cell Ca signals and its dynamic interplay with both CCE and ER Ca releases.

Topics: Animals; Arteries; Boron Compounds; Calcium; Calcium Signaling; Carbachol; Cell Shape; Endoplasmic Reticulum; Endothelial Cells; Estrenes; Extracellular Space; Indoles; Lanthanum; Membrane Glycoproteins; Microscopy, Confocal; Pyrrolidinones; Rats; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Stromal Interaction Molecule 1; Time Factors; Type C Phospholipases

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(²+) sparks and Ca(²+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(²+) and constricted the arteries. The blockade of IP₃Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(²+) sparks. Importantly, the IP₃Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(²+) waves: Ca(²+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(²+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(²+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(²+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.

Topics: Animals; Arteries; Arterioles; Boron Compounds; Calcium Signaling; Cricetinae; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Mesocricetus; Models, Animal; Muscle, Skeletal; Oxazoles; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetracaine

Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.

Topics: Afferent Pathways; Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cerebral Cortex; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Ion Channel Gating; Neurons; Nifedipine; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Ryanodine; Thalamus; Thapsigargin

The dominant mode of intracellular Ca(2+) release in adult mammalian heart is gated by ryanodine receptors (RyRs), but it is less clear whether inositol 1,4,5-trisphosphate (IP(3))-gated Ca(2+) release channels (IP(3)Rs), which are important during embryogenesis, play a significant role during early postnatal development. To address this question, we measured confocal two-dimensional Ca(2+) dependent fluorescence images in acutely isolated neonatal (days 1 to 2) and juvenile (days 8-10) rat cardiomyocytes, either voltage-clamped or permeabilized, where rapid exchange of solution could be used to selectively activate the two types of Ca(2+) release channel. Targeting RyRs with caffeine produced large and rapid Ca(2+) signals throughout the cells. Application of ATP and endothelin-1 to voltage-clamped, or IP(3) to permeabilized, cells produced smaller and slower Ca(2+) signals that were most prominent in subsarcolemmal regions and were suppressed by either the IP(3)R-blocker 2-aminoethoxydiphenylborate or replacement of the biologically active form of IP(3) with its L-stereoisomer. Such IP(3)R-gated Ca(2+) releases were amplified by Ca(2+)-induced Ca(2+) release (CICR) via RyRs since they were also reduced by compounds that block the RyRs (tetracaine) or deplete the Ca(2+) pools they gate (caffeine, ryanodine). Spatial analysis revealed both subsarcolemmal and perinuclear origins for the IP(3)-mediated Ca(2+) release events RyR- and IP(3)R-gated Ca(2+) signals had larger magnitudes in juvenile than in neonatal cardiomyocytes. Ca(2+) signaling was generally quite similar in atrial and ventricular cardiomyocytes but showed divergent development of IP(3)-mediated regulation in juveniles. Our data suggest that an intermediate stage of Ca(2+) signaling may be present in developing cardiomyocytes, where, in addition to RyR-gated Ca(2+) pools, IP(3)-gated Ca(2+) release is sufficiently large in magnitude and duration to trigger or contribute to activation of CICR and cardiac contraction.

Topics: Adenosine Triphosphate; Aging; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Calcium Signaling; Inositol 1,4,5-Trisphosphate Receptors; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Ryanodine; Ryanodine Receptor Calcium Release Channel

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca(2+) entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca(2+) elevation and is responsible for imparting extracellular Ca(2+) influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca(2+) pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca(2+) entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca(2+) entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca(2+) channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca(2+) mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca(2+) influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca(2+) mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca(2+) release from ER stores and extracellular Ca(2+) entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca(2+) entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca(2+) signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Signaling; Cattle; Dihydropyridines; Inositol Phosphates; Norepinephrine; PC12 Cells; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Isoforms; Rats; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Ryanodine

Sustentacular cells have structural features that allude to functions of secretion, absorption, phagocytosis, maintenance of extracellular ionic gradients, metabolism of noxious chemicals, and regulation of cell turnover. We present data detailing their dynamic activity. We show, using a mouse olfactory epithelium slice model, that sustentacular cells are capable of generating two types of calcium signals: intercellular calcium waves where elevations in intracellular calcium propagate between neighboring cells, and intracellular calcium oscillations consisting of repetitive elevations in intracellular calcium confined to single cells. Sustentacular cells exhibited rapid, robust increases in intracellular calcium in response to G-protein coupled muscarinic and purinergic receptor stimulation. In a subpopulation of sustentacular cells, oscillatory calcium transients were evoked. We pharmacologically characterized the properties of purinergic-evoked increases in intracellular calcium. Calcium transients were elicited by release from intracellular stores and were not dependent on extracellular calcium. BAPTA-AM, a cytosolic calcium chelator, and cyclopiazonic acid, an endoplasmic reticulum Ca(2+)-ATPase inhibitor irreversibly blocked the purinergic-induced calcium transient. Phospholipase C antagonist U73122 inhibited the purinergic-evoked calcium transient. 2-Aminoethoxydiphenyl borate, an inositol-1,4,5-trisphosphate (IP(3)) receptor antagonist, and the ryanodine receptor (RyR) antagonists tetracaine and ryanodine, inhibited the UTP-induced calcium transients. Collectively, these data suggest that activation of the phospholipase C pathway, IP(3)-mediated calcium release, and subsequent calcium-induced-calcium release is involved in ATP-elicited increases in intracellular calcium. Our findings indicate that sustentacular cells are not static support cells, and, like glia in the central nervous system, have complex calcium signaling.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Signaling; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Space; Mice; Mice, Inbred C57BL; Olfactory Mucosa; Phosphodiesterase Inhibitors; Pyrrolidinones; Receptors, G-Protein-Coupled; Receptors, Muscarinic; Receptors, Purinergic; Ryanodine; Tetracaine; Uridine Triphosphate

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1). The pharmacological inhibition of InsP(3) receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP(3)R1, which may play an important role in the regulation of neurite outgrowth.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Cells, Cultured; Chick Embryo; Fluorescent Antibody Technique; Ganglia, Spinal; Growth Cones; Immunoblotting; Immunohistochemistry; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Confocal; Microscopy, Fluorescence; Neurites; Neuronal Calcium-Sensor Proteins; Neuropeptides; Pseudopodia; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors

The relative contribution of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and ryanodine receptors (RyRs) to agonist-induced Ca(2+) signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca(2+) oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca(2+) oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca(2+) waves generated by the photolytic release of IP(3). However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP(3)-induced Ca(2+) oscillations or Ca(2+) wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca(2+) oscillations in a concentration-dependent manner. However, tetracaine did not affect IP(3)-induced Ca(2+) release or wave propagation nor the Ca(2+) content of SMC Ca(2+) stores as evaluated by Ca(2+)-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca(2+) oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca(2+) oscillations of SMCs were also observed at 37 degrees C. In Ca(2+)-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca(2+) levels indicating that relaxation also resulted from a reduction in Ca(2+) sensitivity. These results indicate that agonist-induced Ca(2+) oscillations in mouse small airway SMCs are primary mediated via IP(3)Rs and that tetracaine induces relaxation by both decreasing Ca(2+) sensitivity and inhibiting agonist-induced Ca(2+) oscillations via an IP(3)-dependent mechanism.

Topics: Anesthetics, Local; Animals; Boron Compounds; Bronchoconstriction; Bronchoconstrictor Agents; Calcium; Calcium Signaling; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lung; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Muscle Contraction; Myocytes, Smooth Muscle; Organ Culture Techniques; Permeability; Potassium Chloride; Ryanodine; Ryanodine Receptor Calcium Release Channel; Temperature; Tetracaine

Overactivation of ionotropic glutamate receptors induces a Ca(2+) overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca(2+) homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca(2+) release through ryanodine (RyR) and IP(3) (IP(3)R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca(2+) release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP(3)R inhibitors respectively, attenuated NMDA-triggered intracellular Ca(2+) increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca(2+) homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2alpha phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca(2+) release inhibitors. These results demonstrate that Ca(2+) release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.

Topics: Animals; Apoptosis; Boron Compounds; Calcium; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Eukaryotic Initiation Factor-2; Heat-Shock Proteins; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Mitochondria; N-Methylaspartate; Neurons; Oxidative Stress; Rats; Ryanodine

The cellular mechanism of the voltage-dependent properties of slow potentials were investigated in single bundles of circular smooth muscle isolated from the gastric corpus of guinea-pig using conventional microelectrode recordings. Hyperpolarization of the membrane by current injection decreased the frequency and increased the amplitude of slow potentials linearly. At potentials negative of -80 mV, slow potential generation was abolished and a periodic generation of clustered unitary potentials was evident. Application of cyclopiazonic acid (CPA, 20 microM) or thapsigargin (1 microM; inhibitors of Ca(2+)-ATPase), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 0.1 microM; mitochondrial protonophore) or 2-aminoethoxydiphenyl borate (2-APB, 20 microM; inhibitor of IP(3) receptor-mediated Ca(2+) release) depolarized the membrane and reduced or inhibited the amplitude and frequency of slow potentials: repolarization of the membrane to the resting level by current injection resulted in a recovery of the amplitude of slow potentials in the presence of CPA or CCCP, but not 2-APB. The slow potentials abolished by thapsigargin did not recover upon membrane repolarization. The altered frequency of slow potentials by 2-APB, CPA or CCCP was not reversed by membrane repolarization to control potentials. Depolarization of the membrane by about 10 mV with high-potassium solution also reduced the amplitude and increased the frequency of slow potentials in a manner restored by repolarization to control potentials upon current injection, suggesting that membrane depolarization did not affect the voltage dependency of pacemaker activity. The results indicate that in corpus circular muscles the voltage dependency of the frequency and amplitude of slow potentials requires a functional Ca(2+) store and mitochondria.

Topics: Animals; Boron Compounds; Calcium Signaling; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Electrophysiology; Guinea Pigs; In Vitro Techniques; Indoles; Ionophores; Male; Membrane Potentials; Muscle, Smooth; Potassium; Ryanodine; Stomach; Thapsigargin

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Boron Compounds; Calcium; Dose-Response Relationship, Drug; Female; Juvenile Hormones; Moths; omega-Conotoxins; Ovary; Protein Kinase Inhibitors; Ryanodine; Thapsigargin

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca(2+) sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca(2+) released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca(2+) entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca(2+)-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca(2+) release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca(2+) entry through L-type Ca(2+).

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amiloride; Animals; Aorta, Thoracic; Autonomic Denervation; Blood Pressure; Boron Compounds; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Chloride Channels; Dose-Response Relationship, Drug; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Transport Modulators; Nitrobenzoates; Periodicity; Phenylephrine; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium Channel Blockers; Sodium-Calcium Exchanger; Thapsigargin; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Verapamil

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and I

Topics: Animals; Boron Compounds; Calcium; Coronary Vessels; Inositol 1,4,5-Trisphosphate; Myocytes, Smooth Muscle; Protein Kinase C; Ryanodine; Signal Transduction; Swine; Type C Phospholipases; Uridine Triphosphate

Administration of the Group 1 metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) facilitates ("primes") subsequent long-term potentiation (LTP) through a phospholipase C signaling cascade that may involve release of Ca2+ from the endoplasmic reticulum (ER). We investigated the intracellular calcium pathways involved in this priming effect, recording field potentials from area CA1 of rat hippocampal slices before and after high-frequency stimulation. The priming of LTP by DHPG was prevented by co-administration of cyclopiazonic acid, which depletes ER Ca2+ stores. The priming effect was also blocked by the ryanodine receptor (RYR) antagonist ryanodine (RYA, 100 microM). In contrast, a low dose of RYA (10 microM) which opens the RYR channel, by itself primed LTP. In addition to RYR activation, entry of extracellular calcium through store-operated channels appears necessary for priming, since diverse treatments known to impede store-operated channel activity completely blocked both RYA and DHPG priming effects. Thus, RYR activation plays a critical role in the priming of LTP by Group 1 mGluRs, and this effect is coupled to the entry of extracellular calcium, probably through store-operated calcium channels.

Topics: Animals; Boron Compounds; Calcium; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Indoles; Long-Term Potentiation; Male; Methoxyhydroxyphenylglycol; Nitriles; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tyrphostins

Spontaneous local Ca(2+) release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca(2+) release events, we sought to investigate the signaling pathway for spontaneous local Ca(2+) release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca(2+) release events, indicating that these events are RyR-mediated Ca(2+) sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP(3)Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca(2+) sparks. Under patch clamp conditions, dialysis of IP(3) to activate IP(3)Rs increased the activity of local Ca(2+) events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca(2+) sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca(2+) sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca(2+) sparks, whereas the PKC inhibitor chelerythrine, PKCvarepsilon inhibitory peptide, or PKCvarepsilon gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca(2+) sparks in airway SMCs through two distinct signaling pathways: a positive IP(3)-IP(3)R pathway and a negative diacylglycerol-PKCvarepsilon pathway.

Topics: Alkaloids; Animals; Benzophenanthridines; Boron Compounds; Caffeine; Calcium; Cells, Cultured; Diglycerides; Estrenes; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Mice; Myocytes, Smooth Muscle; Oxazoles; Pyrrolidinones; Respiratory System; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Type C Phospholipases

Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, resulted in increases in intracellular Ca(2+) ([Ca(2+)](i)). In M. sexta, Gi proteins are involved in the mastoparan-stimulated increase in [Ca(2+)](i). However, there is no involvement of Gi proteins in the mastoparan-stimulated increase in [Ca(2+)](i) in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic glands mastoparan increases [Ca(2+)](i) even in the absence of extracellular Ca(2+). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan's first site of action is influx of Ca(2+) through plasma membrane Ca(2+) channels while in B. mori prothoracic glands, mastoparan's first site of action is mobilization of Ca(2+) from intracellular stores. In M. sexta, the combined results indicate the presence of mastoparan-sensitive plasma membrane Ca(2+) channels, distinct from those activated by prothoracicotropic hormone or the IP(3) signalling cascade, that coordinate spatial increases in [Ca(2+)](i) in prothoracic gland cells. We propose that in B. mori, mastoparan stimulates Ca(2+) mobilization from ryanodine-sensitive intracellular Ca(2+) stores in prothoracic gland cells.

Topics: Animals; Bombyx; Boron Compounds; Calcium; Calcium Signaling; Dose-Response Relationship, Drug; Gadolinium; Guanosine Diphosphate; Intercellular Signaling Peptides and Proteins; Manduca; Peptides; Pertussis Toxin; Ryanodine; Thapsigargin; Thionucleotides; Time Factors; Wasp Venoms

Experiments were performed to determine if capacitative Ca(2+) entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP(3) receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP(3)- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca(2+) stores in these cells.. Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca(2+) were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca(2+).. Treating myocytes with 10 microM cyclopiazonic acid (CPA), removing extracellular Ca(2+), and briefly applying 10 mM caffeine and 10 microM 5-hydroxytryptamine (5-HT) depleted SR Ca(2+) stores. Extracellular Ca(2+) reintroduction caused cytosolic [Ca(2+)] to elevate above baseline signifying CCE. The InsP(3) receptor inhibitors 2-aminobiphenylborate (50-75 microM; 2-APB) and xestospongin-C (20 microM; XeC) abolished CCE. Yet, CCE was unaffected by 10 microM or 300 microM ryanodine or 10 microM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 microM), however, can inhibit both ryanodine receptors and InsP(3) receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 microM).. The results provide evidence that CCE activated by depletion of both InsP(3) and ryanodine SR Ca(2+) stores in canine PASMCs is dependent on functional InsP(3) receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP(3) receptors.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Hypoxia; Cytosol; Dantrolene; Dogs; Enzyme Inhibitors; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oxazoles; Pulmonary Artery; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Serotonin; Time Factors

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca(2+)](i) (Ca(2+) transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca(2+) transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca(2+) transients synchronously with each other, but did not often show a close temporal relationship with Ca(2+) transients in USMCs. Nicardipine (1 microm) suppressed Ca(2+) transients in USMCs but not in ICC-LCs. Ca(2+) transients in ICC-LCs were abolished by cyclopiazonic acid (10 microm), ryanodine (50 microm) and caffeine (10 mm) or by removing extracellular Ca(2+), and inhibited by 2-aminoethoxydiphenyl borate (50 microm) and 3-morpholino-sydnonimine (SIN-1; 10 microm), but facilitated by increasing extracellular Ca(2+) or phenylephrine (1-10 microm). These results indicated that Ca(2+) transients in urethral ICC-LCs in situ rely on both Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through non-L-type Ca(2+) channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.

Topics: Adrenergic alpha-Agonists; Animals; Boron Compounds; Caffeine; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Signaling; Calcium-Transporting ATPases; Enzyme Inhibitors; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Potentials; Molsidomine; Muscle Contraction; Myocytes, Smooth Muscle; Nicardipine; Nitric Oxide; Nitric Oxide Donors; Phenylephrine; Rabbits; Receptors, Adrenergic, alpha; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors; Urethra

Spontaneous transient currents, due to activation of Ca(2+)-dependent K(+) and Cl(-) channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca(2+) events responsible for triggering Ca(2+)-dependent Cl(-) channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca(2+) events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca(2+) (Ca(2+) sparks) in 57% of cells. There was an average of 6.4 +/- 0.5 release sites/cell with a frequency of 0.9 +/- 1 Hz/cell and peak amplitude DeltaF/F(o) of 67 +/- 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP(3)) receptors. Caffeine caused either a global Ca(2+) rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP(3) receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca(2+) sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca(2+) channels, consistent with Ca(2+) influx regulating Ca(2+) release from stores. We establish for the first time that Ca(2+) sparks occur in CCSMC and arise from Ca(2+) release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca(2+) influx in CCSMC.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Calcium Signaling; Cells, Cultured; Chloride Channels; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Male; Membrane Potentials; Myocytes, Smooth Muscle; Nifedipine; Patch-Clamp Techniques; Penis; Potassium; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors

Few experimental models produce spontaneous tachycardia in normal left atria to allow the study of the cellular mechanisms underlying this contributor to atrial fibrillation. We reported 2-aminoethoxydiphenyl borate (2-APB) that provokes sporadic spontaneous mechanical activity and calcium leak in isolated rat left atria. Since sarcoplasmic reticulum calcium leak in the presence of high calcium load may trigger tachyarrhythmias, we tested how conditions that increase calcium load affect 2-APB-induced ectopic activity. Exposing superfused rat left atria to (i) 30 nM isoproterenol, (ii) 3 microM forskolin, (iii) 300 nM (-)BayK 8644 ((4S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluormethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), (iv) 300 nM FPL-64176 (2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester) or (v) 120 microM ouabain increases their force of contraction, evidence of calcium loading, but does not produce ectopic activity. Spontaneous mechanical activity occurs in left atria superfused with 20 microM 2-APB at 47+/-6 contractions/min in the absence of pacing. Any of these five agents increase rates of 2-APB-induced spontaneous mechanical activity to >200 contractions/min in the absence of pacing. Washing tachycardic left atria with superfusate lacking 2-APB restores normal function, demonstrating the reversibility of these effects. Decreasing superfusate sodium reverses this tachycardia and two hyperpolarization-activated current (I(f)) inhibitors blunt this ectopic activity. Thus conditions that increase atrial calcium load increase the frequency of spontaneous mechanical activity. Decreasing extracellular sodium and I(f) inhibitors suppress this spontaneous tachycardia suggesting forward-mode sodium-calcium exchange and I(f)-like activities underlie this activity. This model may help define cell pathways that trigger atrial tachyarrhythmias.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Benzazepines; Boron Compounds; Calcium; Calcium Channel Agonists; Cardiotonic Agents; Cyclic Nucleotide-Gated Cation Channels; Heart Atria; In Vitro Techniques; Male; Ouabain; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; RNA, Messenger; Ryanodine; Tachycardia

We report in this study a 2-aminoethoxydiphenyl borate (2-APB) activated Ca2+ pathway in NG115-401L (401L) neuronal cells bearing resemblance to hormonal and ryanodine receptor activated pathways. We observed that 2-APB, in contrast to much earlier work, did not inhibit store operated Ca2+ channel (SOC) function, but rather induced potent Ca2+ discharge responses that robustly activated SOC-mediated Ca2+ influx. Further, these studies intriguingly revealed that the 2-APB-induced Ca2+ release pathway likely couples conformationally to targets in the plasma membrane, as membrane permeabilization or actin perturbation abolished the ability of the compound to stimulate Ca2+ signals. These findings suggest that conformationally sensitive complexes form between endoplasmic reticulum and plasma membrane components that not only regulate Ca2+ influx, previously proposed as the conformational coupling hypothesis, but are also required to promote Ca2+ release from intracellular stores. These observations further characterize the 401L neuronal cell line as having unique characteristics that may prove useful in gaining insight into the nature of the coupling mechanism linking Ca2+ release to Ca2+ influx.

Topics: Animals; Blotting, Northern; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cell Line; Drug Interactions; Enzyme Inhibitors; Mice; Neurons; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ryanodine

The Na(+)/Ca(2+) exchanger (NCX) in plasma membranes either moves Ca(2+) out of (forward mode) or into (reverse mode) cells depending on the electrochemical gradient of these ions across the membrane. In this report, we characterize the sources responsible for the elevation in [Ca(2+)](i) elicited by reverse mode NCX activity. The elevation in [Ca(2+)](i) elicited by reverse mode NCX activity was significantly diminished by thapsigargin. KB-R7943 could only partially suppress the [Ca(2+)](i) change. Measurement of the [Ca(2+)](i) concurrent with reverse mode NCX current by perforated whole-cell patch showed that elevation in [Ca(2+)](i), but not the current, was inhibited by thapsigargin. The change in [Ca(2+)](i) response elicited by nicotinic acetylcholine receptor agonist was inhibited by thapsigargin. These suggest the importance of intracellular Ca(2+) stores in facilitating the [Ca(2+)](i) elevation elicited by reverse mode NCX activity under physiological condition.

Topics: Animals; Boron Compounds; Calcium; Cattle; Cells, Cultured; Chromaffin Cells; Intracellular Fluid; Ryanodine; Sodium-Calcium Exchanger; Thapsigargin

Advances in our understanding of murine airway physiology have been hindered by the lack of suitable, ex vivo, small airway bioassay systems. In this study, we introduce a novel small murine airway bioassay system that permits the physiological and pharmacological study of intrapulmonary bronchial smooth muscle via a bronchial ring (BR) preparation utilizing BR segments as small as 200 microm in diameter. Using this ex vivo BR bioassay, we characterized small airway smooth muscle contraction and relaxation in the presence and absence of bronchial epithelium. In control BRs, the application of mechanical stretch is followed by spontaneous bronchial smooth muscle relaxation. BRs pretreated with methacholine (MCh) partially attenuate this stretch-induced relaxation by as much as 42% compared with control. MCh elicited a dose-dependent bronchial constriction with a maximal tension (E(max)) of 8.7 +/- 0.2 mN at an EC(50) of 0.33 +/- 0.02 microM. In the presence of nifedipine, ryanodine, 2-aminoethoxydiphenyl borate, and SKF-96365, E(max) to MCh was significantly reduced. In epithelium-denuded BRs, MCh-induced contraction was significantly enhanced to 11.4 +/- 1.0 mN with an EC(50) of 0.16 +/- 0.04 microM (P < 0.01). Substance P relaxed MCh-precontracted BR by 62.1%; however, this bronchial relaxation effect was completely lost in epithelium-denuded BRs. Papaverine virtually abolished MCh-induced constriction in both epithelium-intact and epithelium-denuded bronchial smooth muscle. In conclusion, this study introduces a novel murine small airway BR bioassay that allows for the physiological study of smooth muscle airway contractile responses that may aid in our understanding of the pathophysiology of asthma.

Topics: Animals; Biological Assay; Boron Compounds; Bronchi; Bronchoconstrictor Agents; Calcium Channel Blockers; Dose-Response Relationship, Drug; Imidazoles; In Vitro Techniques; Methacholine Chloride; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nifedipine; Papaverine; Potassium Chloride; Respiratory Mucosa; Ryanodine; Stress, Mechanical; Substance P; Vasodilator Agents

To determine the Ca2+ source and cellular mechanisms of spontaneous Ca2+ oscillations in hippocampal astrocytes.. The cultured cells were loaded with Fluo-4 AM, the indicator of intracellular Ca2+, and the dynamic Ca2+ transients were visualized with confocal laser-scanning microscopy.. The spontaneous Ca2+ oscillations in astrocytes were observed first in co-cultured hippocampal neurons and astrocytes. These oscillations were not affected by tetrodotoxin (TTX) treatment and kept up in purity cultured astrocytes. The spontaneous Ca2+ oscillations were not impacted after blocking the voltage-gated Ca2+ channels or ethylenediamine tetraacetic acid (EDTA) bathing, indicating that intracellular Ca2+ elevation was not the result of extracellular Ca2+ influx. Furthermore, the correlation between the spontaneous Ca2+ oscillations and the Ca2+ store in endoplasmic reticulum (ER) were investigated with pharmacological experiments. The oscillations were: 1) enhanced when cells were exposed to both low Na+ (70 mmol/L) and high Ca2+ (5 mmol/L) solution, and eliminated completely by 2 micromol/L thapsigargin, a blocker of sarcoplasmic reticulum Ca2+-ATPase; and 2) still robust after the application with either 50 micromol/L ryanodine or 400 micromol/L tetracaine, two specific antagonists of ryanodine receptors, but depressed in a dose-dependent manner by 2-APB, an InsP3 receptors (InsP3R) blocker.. InsP3R-induced ER Ca2+ release is an important cellular mechanism for the initiation of spontaneous Ca2+ oscillation in hippocampal astrocytes.

Topics: Animals; Animals, Newborn; Astrocytes; Boron Compounds; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cells, Cultured; Edetic Acid; Endoplasmic Reticulum; Hippocampus; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Confocal; Neurons; Rats; Rats, Sprague-Dawley; Ryanodine; Tetracaine; Tetrodotoxin; Thapsigargin

To investigate the developmental regulation of intracellular Ca2+ transients, an essential event in excitation-contraction coupling, during cardiomyocyte differentiation.. Using the embryonic stem (ES) cell in vitro differentiation system and pharmacological intervention, we investigated the molecular and functional regulation of Ca2+ handling proteins on the Ca2+ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM).. Nifedipine, a selective antagonist of L-type Ca2+ channels, totally blocked Ca2+ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca2+ transients of ESCM were also inhibited by both ryanodine [an inhibitor of ryanodine receptors (RyRs)] and 2-aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors (IP3Rs)]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased with the differentiation. Thapsigargin, an inhibitor of SR Ca2+-pump ATPase, inhibited Ca2+ transients equally at three differentiation stages that matched the expression profile. Na+ free solution, which inhibits Na+-Ca2+ exchanger (NCX) to extrude Ca2+ from cytosol, did not affect the amplitude of Ca2+ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca2+ concentration.. The Ca2+ transients in ESCM depend on both the sarcolemmal Ca2+ entry via L-type Ca2+ channels and the SR Ca2+ release from RyRs and IP3Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca2+ transients until the latter differentiation stage.

Topics: Animals; Boron Compounds; Calcium; Calcium Channels, L-Type; Calcium Signaling; Cell Differentiation; Cells, Cultured; Embryonic Stem Cells; Inositol 1,4,5-Trisphosphate Receptors; Mice; Myocytes, Cardiac; Nifedipine; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Sodium-Calcium Exchanger; Thapsigargin

Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with D-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 microm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release.

Topics: Anesthetics, Local; Animals; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Estrenes; Female; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Potentials; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pyrrolidinones; Rabbits; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetracaine; Type C Phospholipases; Urethra

We investigated K+ currents and their regulation by the sarcoplasmic reticulum in mesenteric arterial smooth muscle cells of the spontaneously hypertensive rat (SHR). Using perforated patch-clamp technique, we found the overall K+ current density was significantly lower in adult SHR compared to adult Wistar-Kyoto rats (WKY). The K+ currents were almost exclusively of large-conductance Ca2+-dependent (BK(Ca)) variety in SHR, but largely of voltage-gated (Kv) variety in WKY. Western blot assay showed parallel findings. These differences were not observed in pre-hypertensive rats. Depleting the intracellular Ca2+ store inhibited the K+ currents in adult SHR. Ryanodine augmented the K+ current at 1 microM, but suppressed it at 10 microM; 2-aminoethoxydiphenyl borate demonstrated concentration-dependent inhibition. We conclude that an alteration of membrane K+ channel composition has resulted in lower overall K+ current density. The changes in K+ current type may indicate an underlying defect in Ca2+-handling that predisposes smooth muscle cells to the hypertensive phenotype.

Topics: Animals; Blotting, Western; Boron Compounds; Caffeine; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Hypertension; Indoles; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Ryanodine; Sarcoplasmic Reticulum; Tetraethylammonium; Time Factors

The aim of this study was to investigate the signal transduction mechanisms underlying the inhibitory effect induced by pituitary adenylate cyclase activating peptide (PACAP-27) on the spontaneous contractile activity of longitudinal muscle of mouse ileum. Mechanical activity of ileal segments was recorded isometrically in vitro. PACAP-27 produced apamin-sensitive reduction of the amplitude of the spontaneous contractions. 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), adenylate cyclase inhibitor, or genistein and tyrphostin 25, tyrosine kinase inhibitors, had negligible effects on PACAP-27-induced inhibition. PACAP-27 effects were significantly inhibited by U-73122, phopholipase C (PLC) inhibitor, by 2-aminoethoxy-diphenylborate (2-APB), permeable blocker of inositol 1,4,5-triphosphate (IP3) receptors and by depletion of Ca2+ stores with cyclopiazonic acid or thapsigargin. Ryanodine did not reduce PACAP-27-inhibitory responses. We suggest that, in mouse ileum, the inhibitory responses to PACAP-27 involve stimulation of PLC, increased production of IP3 and localised Ca2+ release from intracellular stores, which could provide the opening of apamin-sensitive Ca2+-dependent K+ channels.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Apamin; Boron Compounds; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Genistein; Ileum; In Vitro Techniques; Indoles; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein-Tyrosine Kinases; Pyrrolidinones; Ryanodine; Thapsigargin; Tyrphostins; Vasodilator Agents

The Ca(2+) that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca(2+)channels. Using electrophysiology and Ca(2+) imaging, we show that, in amacrine cell dendrites, at least some of the Ca(2+) that triggers transmitter release comes from endoplasmic reticulum Ca(2+) stores. We show that both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca(2+)] during the brief depolarization of a dendrite. Only the Ca(2+) released through IP(3)Rs, however, seems to promote the release of transmitter. Antagonists for the IP(3)R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca(2+) from internal stores, enhanced both spontaneous and evoked transmitter release.

Topics: Amacrine Cells; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium-Binding Proteins; Central Nervous System Stimulants; Chick Embryo; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Endoplasmic Reticulum; Evoked Potentials; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Glycine; Heparin; Immunohistochemistry; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Microscopy, Confocal; Organic Chemicals; Patch-Clamp Techniques; Phenylacetates; Receptors, Cytoplasmic and Nuclear; Retina; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Topics: Animals; Boron Compounds; Calcium; Calcium Channels; Carbachol; Dose-Response Relationship, Drug; Estrenes; Gastric Fundus; Gastrointestinal Motility; Guinea Pigs; Image Processing, Computer-Assisted; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Microscopy, Confocal; Myocytes, Smooth Muscle; Neomycin; Oxazoles; Phosphodiesterase Inhibitors; Pyrrolidinones; Receptors, Cytoplasmic and Nuclear; Ryanodine; Software; Tissue Distribution; Type C Phospholipases

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.

Topics: Boron Compounds; Calcium; Calcium Channels, L-Type; Calcium Signaling; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Gingiva; Humans; Mitochondria; Neomycin; Receptor, PAR-1; Ryanodine; Sarcoplasmic Reticulum; Sulfonamides; Thapsigargin; Thrombin; Verapamil

Intracellular Ca(2+) ([Ca(2+)](i)) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca(2+) release from intracellular Ca(2+) stores produces [Ca(2+)](i) oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca(2+)](i) oscillations in ICCs are preserved in the presence of dihydropyridine Ca(2+) antagonists, which suppress Ca(2+) activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca(2+)](i) oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca(2+)](i) oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP(3)Rs. The reconstituted [Ca(2+)](i) oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

Topics: Anesthetics, Local; Animals; Biological Clocks; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Cell Line; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Fura-2; Humans; Ileum; Immunohistochemistry; Kinetics; Macrocyclic Compounds; Mice; Microscopy, Fluorescence; Muscle, Smooth; Nifedipine; Oxazoles; Proto-Oncogene Proteins c-kit; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tacrolimus; Tetracaine

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been found within mammalian intracardiac ganglia, but the cellular effects of these neuropeptides remain poorly understood. Fluorometric calcium imaging and whole cell patch clamp recordings were used to examine the effects of PACAP and VIP on [Ca2+]i and neuroexcitability, respectively, in intracardiac neurons of neonatal rats. PACAP and VIP evoked rapid increases in [Ca2+]i that exhibited both transient and sustained components. Pharmacological experiments using PAC1 and VPAC receptor-selective antagonists demonstrated that the elevations in [Ca2+]i result from the activation of VPAC receptors. The transient increases in [Ca2+]i were shown to be the product of Ca2+ mobilization from caffeine/ryanodine-sensitive intracellular stores and were not due to inositol 1,4,5-trisphosphate-mediated calcium release. In contrast, the sustained [Ca2+]i elevations were dependent on extracellular Ca2+ and were blocked by the transient receptor channel antagonist, 2-aminoethoxydiphenyl borate, which suggests that they are due to Ca2+ entry via store-operated channels. In addition to elevating [Ca2+]i, both PACAP and VIP depolarized intracardiac neurons, and PACAP was further shown to augment action potential firing in these cells. Depolarization of intracardiac neurons by the neuropeptides was dependent on activation of VPAC receptors and the concomitant increases in [Ca2+]i. Although activation of PAC1 receptors alone had no direct effects on neuroexcitability, PAC1 receptor stimulation potentiated the VPAC receptor-induced depolarizations. Furthermore, enhanced action potential firing was only observed upon concurrent stimulation of PAC1 and VPAC receptors, which indicates that these receptors act synergistically to enhance neuroexcitability in intracardiac neurons.

Topics: Animals; Animals, Newborn; Boron Compounds; Caffeine; Calcium; Cell Membrane; Central Nervous System Stimulants; Electrophysiology; Fluorometry; Heart; Membrane Potentials; Neurons; Peptides; Rats; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; Ryanodine; Time Factors

In mammals, endothelin receptors are sub-classified into ET(A) receptors (ET(A)R), which are purely constrictive in vascular smooth muscle (VSM), and ET(B)R, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ET(B)R. The Ca(2+) signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ET(B)R agonists IRL 1620 and sarafotoxin S6c (SRX) increase [Ca(2+)](i) in VSM to the same extent as ET-1 and ET(B)R appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP(3)) receptors (IP(3)R), we utilized two inhibitors of the mammalian IP(3)R, TMB-8 and 2-APB. In Ca(2+)-free Ringer, these agents inhibit the response to ET(B)R agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca(2+)](i), (calcium-induced calcium release) or by cyclic adeninediphosphate ribose (cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red, 8-Br cADPR and high concentrations of ryanodine; these agents blocked the [Ca(2+)](i) response to ET(B)R agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark, ET(B)R activation stimulates both IP(3)R and RyR, and that cADPR is involved in RyR activation.

Topics: Analysis of Variance; Animals; Boron Compounds; Calcium; Calcium Channels; Cyclic ADP-Ribose; Endothelins; Fluorescence; Gallic Acid; Inositol 1,4,5-Trisphosphate Receptors; Maine; Muscle, Smooth, Vascular; Peptide Fragments; Receptor, Endothelin B; Receptors, Cytoplasmic and Nuclear; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Squalus acanthias; Time Factors; Viper Venoms

Transiently local release of Ca(2+) from the sarcoplasmic reticulum (SR) activates nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO(-)) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca(2+) mobilization was involved in STOCs alteration by ONOO(-). STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca(2+); by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca(2+) channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO(-) donor, at concentrations of 20-200 microM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca(2+) (200 nM), ryanodine (10 microM), or nifedipine (10 microM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 microM) had little effect on ONOO(-) -induced STOCs activation. These findings suggest that complex Ca(2+)-mobilizing pathways, including external Ca2+ influx through VGCCs activation and subsequent internal Ca(2+) release through RyRs but not IP3Rs, are involved in ONOO(-)mediated STOCs enhancement in ASMCs.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Electrophysiology; Female; Male; Membrane Potentials; Models, Biological; Molsidomine; Muscle, Smooth; Myocytes, Smooth Muscle; Nifedipine; Patch-Clamp Techniques; Peroxynitrous Acid; Potassium; Rats; Rats, Wistar; Ryanodine; Signal Transduction; Time Factors

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 +/- 9 microm) and La3+ (IC50 = 7.2 +/- 3 microm), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Glutamic Acid; Lanthanum; Magnesium; Mitochondria; Neurons; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Ryanodine; Thapsigargin

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Topics: Animals; Anti-Infective Agents, Local; Anticoagulants; Boron Compounds; Calcium; Calcium Channels; Extracellular Space; Feedback, Physiological; Guinea Pigs; Heparin; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Male; Membrane Potentials; Myocytes, Smooth Muscle; Receptors, Cytoplasmic and Nuclear; Ryanodine; Sarcoplasmic Reticulum; Thimerosal; Vas Deferens

To investigate the role of ryanodine receptors in glomerular arterioles, experiments were performed using an isolated perfused hydronephrotic kidney model. In the first series of studies, BAYK-8644 (300 nM), a calcium agonist, constricted afferent (19.6 +/- 0.6 to 17.6 +/- 0.5 microm, n = 6, P < 0.01) but not efferent arterioles. Furthermore, BAYK-8644 elicited afferent arteriolar oscillatory movements. Subsequent administration of nifedipine (1 microM) inhibited both afferent arteriolar oscillation and constriction by BAYK-8644 (to 19.4 +/- 0.5 microm). In the second group, although BAYK-8644 constricted afferent arterioles treated with 1 microM of thapsigargin (19.7 +/- 0.6 to 16.8 +/- 0.6 microm, n = 5, P < 0.05), it failed to induce rhythmic contraction. Removal of extracellular calcium with EGTA (2 mM) reversed BAYK-8644-induced afferent arteriolar constriction (to 20.0 +/- 0.5 microm). In the third series of investigations, ryanodine (10 microM) but not 2-aminoethoxyphenyl borate (100 microM) abolished afferent arteriolar vasomotion by BAYK-8644. In the fourth series of experiments, in the presence of caffeine (1 mM), the stronger activation of voltage-dependent calcium channels by higher potassium media resulted in greater afferent arteriolar constriction and faster oscillation. Our results indicate that L-type calcium channels are rich in preglomerular but not postglomerular microvessels. Furthermore, the present findings suggest that either prolonged calcium influx through voltage-dependent calcium channels (BAYK-8644) or sensitized ryanodine receptors (caffeine) is required to trigger periodic calcium release through ryanodine receptors in afferent arterioles.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Arterioles; Boron Compounds; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Enzyme Inhibitors; Hydronephrosis; Kidney Glomerulus; Male; Nifedipine; Ouabain; Periodicity; Phosphodiesterase Inhibitors; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Renal Circulation; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tetraethylammonium; Thapsigargin; Vasoconstriction

Astrocytes in the rat thalamus display spontaneous [Ca(2+)](i) oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca(2+)](i) oscillations using slices loaded with Fluo-4 AM (5 microM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca(2+)](i) oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca(2+)](i) increases are not due to membrane-coupled PLC activation. Spontaneous [Ca(2+)](i) increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca(2+)](i) oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca(2+) entry. Increasing [Ca(2+)](o) increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca(2+) ATPase by cyclopiazonic acid also induced [Ca(2+)](i) transients in astrocytes indicating a role for cytoplasmic Ca(2+) in the induction of spontaneous oscillations. Incubation with 20 microM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca(2+) has a role in triggering Ca(2+) release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca(2+)](i) oscillations.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aniline Compounds; Animals; Animals, Newborn; Anti-Infective Agents, Local; Astrocytes; Boron Compounds; Caffeine; Calcium; Calcium Channel Agonists; Calcium Signaling; Dioxolanes; Drug Interactions; Enzyme Inhibitors; In Vitro Techniques; Methoxyhydroxyphenylglycol; Microscopy, Confocal; Patch-Clamp Techniques; Purines; Rats; Ryanodine; Thalamus; Thimerosal; Xanthenes

Ca2+ and Na+ play important roles in neurons, such as in synaptic plasticity. Their concentrations in neurons change dynamically in response to synaptic inputs, but their kinetics have not been compared directly. Here, we show the mechanisms and dynamics of Ca2+ and Na+ transients by simultaneous monitoring in Purkinje cell dendrites in mouse cerebellar slices. High frequency parallel fibre stimulation (50 Hz, 3-50-times) depolarized Purkinje cells, and Ca2+ transients were observed at the anatomically expected sites. The magnitude of the Ca2+ transients increased linearly with increasing numbers of parallel fibre inputs. With 50 stimuli, Ca2+ transients lasted for seconds, and the peak [Ca2+] reached approximately 100 microm, which was much higher than that reported previously, although it was still confined to a part of the dendrite. In contrast, Na+ transients were sustained for tens of seconds and diffused away from the stimulated site. Pharmacological interventions revealed that Na+ influx through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Ca2+ influx through P-type Ca channels were essential players, that AMPA receptors did not operate as a Ca2+ influx pathway and that Ca2+ release from intracellular stores through inositol trisphosphate receptors or ryanodine receptors did not contribute greatly to the large Ca2+ transients.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Anesthetics, Local; Animals; Animals, Newborn; Boron Compounds; Calcium; Calcium Channel Blockers; Cells, Cultured; Cerebellum; Dendrites; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Female; Fluorescent Dyes; Glutamic Acid; Glycine; In Vitro Techniques; Iontophoresis; Male; Mice; omega-Conotoxin GVIA; Patch-Clamp Techniques; Purkinje Cells; Ryanodine; Sodium; Stimulation, Chemical; Tetrodotoxin; Time Factors

We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized Ca(2+) release events (sparks) and large-conductance Ca(2+)-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 microM). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 +/- 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway.

Topics: Aniline Compounds; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Cholecystokinin; Enzyme Inhibitors; Fluorescent Dyes; Gallbladder; Guinea Pigs; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle, Smooth; Nifedipine; Patch-Clamp Techniques; Peptides; Phosphodiesterase Inhibitors; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Thapsigargin; Xanthenes

In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) by release of Ca(2+) from intracellular stores. The ROS-induced Ca(2+) signals showed large variability in shape and time-course and resembled in part Ca(2+) signals in response to physiological secretagogues. ROS-induced Ca(2+) mobilization started at the luminal cell pole and spread towards the basolateral side in a wave manner. ROS-evoked Ca(2+) responses were not inhibited by the phospholipase C (PLC) inhibitor U73122 (10 microM). Neither 2-aminoethoxy-diphenylborate (2-APB) (70 microM) nor ryanodine (50 microM) suppressed ROS-evoked Ca(2+) release. ROS still released Ca(2+) when the endoplasmic reticulum Ca(2+)-ATPase was blocked with thapsigargin (1 microM), or when rotenone (10 microM) was added to release Ca(2+) from mitochondria. Our results suggest that pancreatic acinar cells ROS do not unspecifically affect Ca(2+) homeostasis. ROS primarily affect Ca(2+) stores located in the luminal cell pole, which is also the trigger zone for agonist-induced Ca(2+) signals. Release of Ca(2+) induces Ca(2+) waves carried by Ca(2+)-induced Ca(2+) release and produces thereby global Ca(2+) signals. Under oxidative stress conditions, the increase in [Ca(2+)](i) could be one mechanism contributing to an overstimulation of the cell which could result in cell dysfunction and cell damage.

Topics: Animals; Boron Compounds; Calcium; Cells, Cultured; Cytosol; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; Kinetics; Male; Mice; Pancreas; Pyrrolidinones; Reactive Oxygen Species; Ryanodine; Sincalide; Type C Phospholipases; Xanthine Oxidase

1. The role of internal Ca(2+) stores in the generation of the rhythmic electrical and contractile activity in the guinea-pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. 2. Ryanodine (30 microM) transiently increased contraction amplitude, while caffeine (0.5 - 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2-aminoethoxy-diphenylborate (2-APB 60 microM), xestospongin C (1 microM), U73122 (5 microM) and neomycin (4 mM), blockers of IP(3)-dependent release from Ca(2+) stores. 3. 60 mM K(+) saline-evoked contractions were reduced by caffeine (1 mM), U73122 (5 microM) and neomycin (4 mM), but little affected by ryanodine or 2-APB (60 microM). 4. Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6+/-1.0 min(-1)) in small urothelium-denuded strips of proximal renal pelvis. 5. Action potential discharge was blocked in 75 and 35% of cells by 2-APB (60 microM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. 6. Cyclopiazonic acid (CPA 10 microM for 10 - 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. 7. We conclude that the action potential in caffeine-sensitive cells and the shoulder component of caffeine-insensitive action potential arise from the entry of Ca(2+) through Ca(2+) channels. The inhibitory actions of modulators of internal Ca(2+) release were partially explained by a blockade of Ca(2+) entry.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Electric Conductivity; Electrophysiology; Estrenes; Guinea Pigs; Indoles; Kidney Pelvis; Macrocyclic Compounds; Muscle Contraction; Muscle, Smooth; Neomycin; Oxazoles; Pyrrolidinones; Ryanodine

Spontaneous electrical activity and internal Ca(2+) concentration ([Ca(2+)](i)) were measured simultaneously using conventional microelectrodes and fura-2 fluorescence, respectively, in isolated circular smooth muscle bundles of the guinea-pig gastric antrum. The smooth muscle bundles generated periodic slow potentials with accompanying spike potentials and associated transient increases in [Ca(2+)](i) (Ca(2+)-transients). Nifedipine abolished the spike potentials but not the slow potentials, and reduced the amplitude of associated Ca(2+)-transients. Caffeine, in the absence or presence of ryanodine, reduced resting [Ca(2+)](i) levels and abolished the slow potentials and associated Ca(2+)-transients. Depolarization elevated and hyperpolarization reduced resting [Ca(2+)](i) levels with associated changes in the frequency of slow potentials. The amplitude of Ca(2+)-transients changed in a bell-shaped manner with the membrane potential change. Slow potentials and associated Ca(2+)-transients were abolished if [Ca(2+)](i) levels were reduced by BAPTA-AM or if the internal Ca(2+) pump was inhibited by cyclopiazonic acid. 2-Aminoethoxy-diphenylborate (2-APB), a known inhibitor of inositol trisphosphate (IP(3))-mediated Ca(2+) release, also blocked slow potentials and Ca(2+)-transients. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial protonophore, depolarized the membrane, elevated [Ca(2+)](i) levels and abolished slow potentials and Ca(2+)-transients. Inhibition of mitochondrial ATP-sensitive K(+) channels by glybenclamide and 5-hydroxydecanoic acid (5-HAD) abolished slow potentials and Ca(2+)-transients, without altering the smooth muscle [Ca(2+)](i). It is concluded that in antrum circular muscles, the frequency of slow potentials is correlated with the level of [Ca(2+)](i). The slow potential is coupled to release of Ca(2+) from an internal store, possibly through the activation of IP(3) receptors; this may be initiated by the activation of ATP-sensitive K(+) channels in mitochondria following Ca(2+) handling by mitochondria.

Topics: Animals; Anti-Arrhythmia Agents; Biological Clocks; Boron Compounds; Caffeine; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chelating Agents; Decanoic Acids; Egtazic Acid; Electric Stimulation; Female; Glyburide; Guinea Pigs; Hydroxy Acids; Indoles; Male; Membrane Potentials; Mitochondria; Muscle, Smooth; Phosphodiesterase Inhibitors; Potassium Channels; Pyloric Antrum; Ryanodine; Uncoupling Agents

Calcium influx and elevation of intracellular free calcium (Ca2+i), with subsequent activation of degenerative enzymes is hypothesized to cause cell injury and death after trauma. We examined the effects of traumatic compressive injury on (Ca2+)i dynamics in spinal cord white matter. We conducted electrophysiological studies with ryanodine and inositol (1,4,5)-triphosphate (IP3) receptor agonists and antagonists in an in vitro model of spinal cord injury (SCI). A 25-30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (37 degrees C) and injured with a modified clip (2-g closing force) for 15 sec. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. The CAP decreased to 55.2+/-6.8% of control (p < 0.05) after spinal cord injury (SCI). Chelation of Ca2+i with BAPTA-AM (a high-affinity calcium chelator) promoted significantly greater recovery of CAP amplitude (83.2+/-4.2% of control; p < 0.05) after injury. Infusion of caffeine (1 and 10 mM) exacerbated CAP amplitude decline (45.1+/-5.9% of control; p < 0.05; 44.6+/-3.1% of control; p < 0.05) postinjury. Blockade of Ca2+i release through ryanodine-sensitive receptors (RyRs) with dantrolene (10 microM) and ryanodine (50 microM), conferred significant (p < 0.05) improvement in CAP amplitude after injury. On the other hand, blockade of Ca2+i with inositol (1,4,5)-triphosphate receptor (IP3Rs) blocker 2APB (10 microM) also conferred significant improvement in CAP amplitude after injury (82.9+/-7.9%; p < 0.05). In conclusion, the injurious effects of Ca2+i in traumatic central nervous system (CNS) white matter injury appear to be mediated both by RyRs and through IP3Rs calcium-induced calcium release receptors (CICRs).

Topics: Action Potentials; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium-Binding Proteins; Central Nervous System Stimulants; Chelating Agents; Dantrolene; Egtazic Acid; Electrophysiology; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Male; Models, Animal; Muscle Relaxants, Central; Neural Conduction; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord Injuries

The elasmobranch Squalus acanthias controls plasma osmolality and extracellular fluid volume by secreting a hypertonic fluid from its rectal gland. Because we found a correlation between extracellular Ca(2+) concentration and changes in cytosolic Ca(2+) ([Ca(2+)](i)), we sought the possible presence of a calcium-sensing receptor in rectal gland artery and tubules. Cytosolic Ca(2+) of both tissues responded to the addition of external Ca(2+) (0.8-5.3 mmol l(-1)) in a linear fashion. Spermine, Gd(3+) and Ni(2+), known agonists of the calcium-sensing receptor, increased [Ca(2+)](i). To assess the participation of inositol triphosphate (IP(3)) generation, sarcoplasmic/endoplasmic reticulum (SR/ER) Ca(2+) depletion, and activation of store-operated Ca(2+) entry, we utilized thapsigargin and ryanodine to deplete Ca(2+) SR/ER stores and the inhibitory reagents TMB-8 and 2-APB to block IP(3) receptors. In each case, these agents inhibited the [Ca(2+)](i) response to agonist stimulation by approximately 50 %. Blockade of L-channels with nifedipine had no significant effect. Increases in ionic strength are known to inhibit the calcium-sensing receptor. We postulate that the CaSR stimulates Ca(2+)-mediated constriction of the rectal gland artery and diminishes cyclic AMP-mediated salt secretion in rectal gland tubules during non-feeding conditions. When the shark ingests sea water and fish, an increase in blood and interstitial fluid ionic strength inhibits the activity of the calcium-sensing receptor, relaxing the rectal gland artery and permitting salt secretion by the rectal gland tubules.

Topics: Animals; Arteries; Boron Compounds; Calcium; Calcium Channel Blockers; Dogfish; Female; Gadolinium; Gallic Acid; In Vitro Techniques; Male; Nickel; Nifedipine; Receptors, Calcium-Sensing; Receptors, Cell Surface; Ryanodine; Salt Gland; Spermine; Thapsigargin

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (<100 ms in duration) and "slow" (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Cell Membrane; Female; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate; Male; Membrane Potentials; Muscle, Smooth; Nifedipine; Rabbits; Ryanodine; Urethra; Urinary Bladder