Changes In Intracellular Free Ca2 Research Articles

Perampanel: A novel, orally active, noncompetitive AMPA-receptor antagonist that reduces seizure activity in rodent models of epilepsy

To assess the pharmacology of perampanel and its antiseizure activity in preclinical models. Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile] is a novel, orally active, prospective antiepileptic agent currently in development for refractory partial-onset seizures. Perampanel pharmacology was assessed by examining changes in intracellular free Ca(2+) ion concentration ([Ca(2+) ](i) ) in primary rat cortical neurones, and [(3) H]perampanel binding to rat forebrain membranes. Antiseizure activity of orally administered perampanel was examined in amygdala-kindled rats and in mice exhibiting audiogenic, maximal electroshock (MES)-induced, pentylenetetrazole (PTZ) -induced, or 6 Hz-induced seizures. In cultured rat cortical neurones, perampanel inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced increases in [Ca(2+) ](i) (IC(50) 93 nm vs. 2 μm AMPA). Perampanel had a minimal effect on N-methyl-d-aspartate (NMDA)-induced increases in [Ca(2+) ](i) , and only at a high concentration (30 μm). [(3) H]Perampanel binding to rat forebrain membranes was not significantly displaced by glutamate or AMPA but was displaced by the noncompetitive AMPA receptor antagonists CP465022 (K(i) 11.2 ± 0.8 nm) and GYKI52466 (K(i) 12.4 ± 1 μm). In mice, perampanel showed protective effects against audiogenic, MES-induced, and PTZ-induced seizures (ED(50) s 0.47, 1.6, and 0.94 mg/kg, respectively). Perampanel also inhibited 6 Hz electroshock-induced seizures when administered alone or in combination with other antiepileptic drugs (AEDs). In amygdala-kindled rats, perampanel significantly increased afterdischarge threshold (p<0.05 vs. vehicle), and significantly reduced motor seizure duration, afterdischarge duration, and seizure severity recorded at 50% higher intensity than afterdischarge threshold current (p<0.05 for all measures vs. vehicle). Perampanel caused dose-dependent motor impairment in both mice (TD(50) 1.8 mg/kg) and rats (TD(50) 9.14 mg/kg), as determined by rotarod tests. In mice, the protective index (TD(50) in rotarod test/ED(50) in seizure test) was 1.1, 3.8, and 1.9 for MES-induced, audiogenic, and PTZ-induced seizures, respectively. In rat, dog, and monkey, perampanel had a half-life of 1.67, 5.34, and 7.55 h and bioavailability of 46.1%, 53.5%, and 74.5%, respectively. These data suggest that perampanel is an orally active, noncompetitive, selective AMPA receptor antagonist with potential as a broad spectrum antiepileptic agent.

Calcium signaling dysfunction in heart disease

In the heart, Ca(2+) is crucial for the regulation of contraction and intracellular signaling, processes, which are vital to the functioning of the healthy heart. Ca(2+) -activated signaling pathways must function against a background of large, rapid, and tightly regulated changes in intracellular free Ca(2+) concentrations during each contraction and relaxation cycle. This review highlights a number of proteins that regulate signaling Ca(2+) in both normal and pathological conditions including cardiac hypertrophy and heart failure, and discusses how these pathways are not regulated by the marked elevation in free intracellular calcium ([Ca(2+) ](i)) during contraction but require smaller sustained increases in Ca(2+) concentration. In addition, we present published evidence that the pool of Ca(2+) that regulates signaling is compartmentalized into distinct cellular microdomains and is thus distinct from that regulating contraction.

Sevoflurane inhibits angiotensin II-induced Rho kinase-mediated contraction of vascular smooth muscle from spontaneously hypertensive rat

Angiotensin II (Ang II)-induced vasoconstriction is mediated by changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity. Protein kinase C- and Rho kinase-mediated signaling pathways are proposed for the regulation of the Ca(2+) sensitization mechanisms. We have demonstrated that sevoflurane inhibits Rho kinase-mediated contraction of isolated rat aortic smooth muscle. A recent study demonstrated that Rho-kinase mediated Ca(2+) sensitization was involved in the pathophysiology of hypertension. This study was designed to investigate the effects of sevoflurane on Ang II-induced Rho kinase-mediated vascular contraction in spontaneously hypertensive rats (SHR). The effects of sevoflurane on vasoconstriction, increase in [Ca(2+)](i), and membrane translocation of Rho kinase in response to Ang II were investigated in normotensive Wistar-Kyoto rats (WKY) and SHR, using an isometric force transducer, a fluorometer, and Western blotting, respectively. The inhibitory effects of sevoflurane on Ang II (10(-7)M)-induced contraction were greater (P < 0.05) in SHR than in WKY at the highest concentration of sevoflurane (5.1%). Y27632 (3 × 10(-7)M), a specific inhibitor of Rho kinase, inhibited the Ang II-induced contraction in SHR, but not in WKY. Sevoflurane did not affect the increases in [Ca(2+)](i) in response to Ang II in either strain. Ang II stimulated Rho kinase activity in SHR, which was almost abolished by sevoflurane at a concentration of 5.1% (P < 0.05). These findings suggest that the inhibition of the Ang II-induced contraction by sevoflurane in SHR may be, at least in part, due to the attenuation of the Rho kinase-mediated signaling pathway.

Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes

Neonatal rat ventricular cardiomyocytes were used to investigate mechanisms underlying transient changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by pulsed infrared radiation (IR, 1862 nm). Fluorescence confocal microscopy revealed IR-evoked [Ca2+]i events with each IR pulse (3-4 ms pulse⁻¹, 9.1-11.6 J cm⁻² pulse⁻¹). IR-evoked [Ca2+]i events were distinct from the relatively large spontaneous [Ca2+]i transients, with IR-evoked events exhibiting smaller amplitudes (0.88 ΔF/F0 vs. 1.99 ΔF/F0) and shorter time constants (τ =0.64 s vs. 1.19 s, respectively). Both IR-evoked [Ca2+]i events and spontaneous [Ca2+]i transients could be entrained by the IR pulse (0.2-1 pulse s⁻¹), provided the IR dose was sufficient and the radiation was applied directly to the cell. Examination of IR-evoked events during peak spontaneous [Ca2+]i periods revealed a rapid drop in [Ca2+]i, often restoring the baseline [Ca2+]i concentration, followed by a transient increase in [Ca2+]i.Cardiomyocytes were challenged with pharmacological agents to examine potential contributors to the IR-evoked [Ca2+]i events. Three compounds proved to be the most potent, reversible inhibitors: (1) CGP-37157 (20 μM, n =12), an inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX), (2) Ruthenium Red (40 μM, n =13), an inhibitor of the mitochondrial Ca2+ uniporter (mCU), and (3) 2-aminoethoxydiphenylborane (10 μM, n =6), an IP3 channel antagonist. Ryanodine blocked the spontaneous [Ca2+]i transients but did not alter the IR-evoked events in the same cells. This pharmacological array implicates mitochondria as the major intracellular store of Ca2+ involved in IR-evoked responses reported here. Results support the hypothesis that 1862 nm pulsed IR modulates mitochondrial Ca2+ transport primarily through actions on mCU and mNCX.

A quantitative description of active force generation in gastrointestinal smooth muscle

AbstractA mathematical model to describe the relationship between the intracellular Ca2+ concentration and active force production in a gastrointestinal (GI) smooth muscle cell (SMC) has been developed. Here the model has been constructed in terms of two modules, the first describing the activation of myosin light chain kinase (MLCK) through its interactions with calmodulin and Ca2+ ions, and the second consisting of a four state scheme describing myosin phosphorylation and cross‐bridge formation between actin and myosin. A prescribed Ca2+ transient, representing the dynamic changes in intracellular free Ca2+ that accompanies GI SMC excitation, was used as the input signal. Simulations demonstrated that at physiological Ca2+ levels, a 33% increase in peak Ca2+ concentration resulted in a 93% increase in myosin phosphorylation. This can possibly be attributed to the steep relationship between Ca2+ and MLCK activation over the normal Ca2+ range. The total number of cross‐bridges (sum of cycling cross‐bridges and latch‐bridges) was used to predict the active force generated in response to a phasic Ca2+ signal. The predicted forces were in qualitative agreement with experimental data from a canine antral smooth muscle strip. The development of this model represents a first step towards a greater understanding of the mechanisms that underlie GI motility. Copyright © 2010 John Wiley &amp; Sons, Ltd.

Metabotropic Purinergic Receptors in Satellite Glial Cells

Objective: The dorsal root ganglia (DRG) contains the pseudo-unipolar neurons of sensory input. Neuron somata is enveloped by satellite glial cells (SGC) whose functions is still unknow. To further unveil the sinalization between neurons and glia in DRG we have investigated the expression of purinergic metabotropic receptors (P2Y) by SGC of DRG. Methods: DRG from newborn Wistar rats were treatead with trypsin and mechanically dissociated. The cells were cultivated on poli-L-lysine-treated glass slips. The culture was kept in DMEM with 5% fetal bovine serum, at 37°C and under 5% CO2 atmosphere. Changes in intracellular free-Ca2+ concentration ([Ca2+]i) were evaluted in a confocal laser scanning microscope (Zeiss, LSM 510), using Fluo-4 (Molecular Probes) as a calcium indicator. Time series were recorded in control conditions and during the exposure to the putative agonist. Results: ATP 0.1 mM promoted transitory increases in the [Ca2+]i in SGC (94.4%, n=18) even in free-Ca2+ medium (81,3%, n=32). The P2Y agonists ADP 0.1 mM, UTP 1 mM and UDP 1 mM promoted oscillations in 93.1% (n=54), 15.6% (n=32) and 38.1% (n=42) of the SGC, respectively. Like ADP, the MRS2365 0.1 mM, a selective P2Y1 agonist, promoted Ca2+ increase (91.6 %, n=12). Previous application of MRS2365 blunts the response of the SGC to BzATP 25μM (n=6), an agonist of P2X7 receptor. Conclusions: The SGC from DRG express P2Y and P2X7 receptors. The ADP sensitive subtype (P2Y1) predominates. A fraction of the observed SGC expresses the UDP sensitive subtype (P2Y6), and a yet smaller fraction expresses the UTP sensitive subtype (P2Y2 and/or P2Y4). Previous activation of the P2Y1 receptor drastically reduces cell responses to BzATP, probably by downregulation of P2X7 ionotropic receptor.

Vascular and Cellular Calcium in Normal and Hypertensive Pregnancy

Normal pregnancy is associated with significant hemodynamic changes in the cardiovascular system in order to meet the metabolic demands of mother and fetus. These changes include increased cardiac output, decreased vascular resistance, and vascular remodeling in the uterine and systemic circulation. Preeclampsia (PE) is a major complication of pregnancy characterized by proteinuria and hypertension. Several risk factors have been implicated in the pathogenesis of PE including genetic and dietary factors. Ca2+ is an essential dietary element and an important regulator of many cellular processes including vascular function. The importance of adequate dietary Ca2+ intake during pregnancy is supported by many studies. Pregnancy-associated changes in Ca2+ metabolism and plasma Ca2+ have been observed. During pregnancy, changes in intracellular free Ca2+ concentration ([Ca2+](i)) have been described in red blood cells, platelets and immune cells. Also, during pregnancy, an increase in [Ca2+](i) in endothelial cells (EC) stimulates the production of vasodilator substances such as nitric oxide and prostacyclin. Normal pregnancy is also associated with decreased vascular smooth muscle (VSM) [Ca2+](i) and possibly the Ca2+-sensitization pathways of VSM contraction including protein kinase C, Rho-kinase, and mitogen-activated protein kinase. Ca2+-dependent matrix metalloproteinases could also promote extracellular matrix degradation and vascular remodeling during pregnancy. Disruption in the balance between dietary, plasma and vascular cell Ca2+ may be responsible for some of the manifestation of PE including procoagulation, decreased vasodilation, and increased vasoconstriction and vascular resistance. The potential benefits of Ca2+ supplements during pregnancy, and the use of modulators of vascular Ca2+ to reduce the manifestations of PE in susceptible women remain an important area for experimental and clinical research.

N-Methyl-d-aspartate Receptors Mediate the Phosphorylation and Desensitization of Muscarinic Receptors in Cerebellar Granule Neurons

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-d-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the Gq/11-coupled M3-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M3-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M3-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.

TCF7L2 controls insulin gene expression and insulin secretion in mature pancreatic β-cells

Genetic studies have linked the risk of Type 2 diabetes with SNPs (single nucleotide polymorphisms) in the gene encoding the Wnt signalling-associated transcription factor, TCF7L2 (T-cell factor 7-like 2). The risk alleles have been associated with reduced glucose and GLP-1 (glucagon-like peptide 1)-stimulated insulin secretion. Recent evidence has suggested that inheritance of the at-risk T allele at SNP rs7903146 may increase the expression of TCF7L2 in adult human islets. However, the cellular mechanisms by which changes in TCF7L2 levels may affect insulin secretion are unclear. In the present paper, we describe the use of RNA silencing to investigate the role of TCF7L2 on insulin secretion and gene expression in rodent islets. We find that reduced TCF7L2 expression reduces glucose-simulated insulin secretion and insulin gene expression while slightly potentiating glucose stimulated changes in intracellular free Ca(2+) concentrations.

Potential Roles of Angiotensin Receptor-Activating Autoantibody in the Pathophysiology of Preeclampsia

Preeclampsia affects ≈7% of first pregnancies and is one of the leading causes of maternal and neonatal mortality and morbidity in the United States and the world.1–3 The clinical hallmarks of the disorder include hypertension, proteinuria, hypercoagulability, edema, and placental abnormalities. In advanced stages, clinical symptoms include cerebral edema, renal failure, and the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. The clinical management of preeclampsia is hampered by the lack of reliable diagnostic tests and effective therapy for the disorder. In some cases, termination of pregnancy is the only available option to prevent further deterioration of the fetus and mother. Fifteen percent of all preterm births are indicated early deliveries for preeclampsia.3 The resulting preterm births and the associated increased infant morbidity and mortality are especially disheartening consequences of preeclampsia. Despite being one of the leading causes of maternal death and a major contributor to maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are poorly understood. Roberts and colleagues4,5 were among the first to propose that alterations in endothelial cell function by activating agents produced by the placenta initiate the clinical syndromes of preeclampsia. Circulating factors, such as inflammatory cytokines, endothelin, and soluble vascular endothelial growth factor receptor termed soluble fms-like tyrosine kinase-1 (sFlt-1), are elevated in preeclamptic women and are proposed to be important links between placental ischemia and endothelial dysfunction.5–9 In particular, recent studies have shown that preeclampsia is associated with the presence of maternal autoantibodies capable of binding to and activating the angiotensin (Ang) receptor type-1 (AT1).10–15 AT1 receptor agonistic antibodies, herein termed AT1-AA, are rarely seen in normotensive pregnant women.10,11 Since the initial discovery of these autoantibodies, considerable evidence supporting a pathophysiological role of AT1-AA in preeclampsia has …

Glucose-Dependent Regulation of γ-Aminobutyric Acid (GABAA) Receptor Expression in Mouse Pancreatic Islet α-Cells

The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, gamma-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABA(A) receptor (GABA(A)R) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABA(A)R alpha4, beta3, and gamma2 subunit mRNAs were detected in mouse islets and clonal alphaTC1-9 cells, and immunocytochemistry confirmed the presence of GABA(A)Rs at the plasma membrane of primary alpha-cells. Glucose dose-dependently increased GABA(A)R expression in both islets and alphaTC1-9 cells such that mRNA levels at 16 mmol/l glucose were approximately 3.0-fold (alpha4), 2.0-fold (beta3), or 1.5-fold (gamma2) higher than at basal glucose concentrations (2.5 or 1.0 mmol/l, respectively). These effects were mimicked by depolarizing concentrations of K(+) and reversed by the L-type Ca(2+) channel blocker nimodipine. We conclude that 1) release of GABA from neighboring beta-cells contributes substantially to the acute inhibition of glucagon secretion from mouse islets by glucose and 2) that changes in GABA(A)R expression, mediated by changes in intracellular free Ca(2+) concentration, may modulate this response in the long term.

Simultaneous monitoring of three key neuronal functions in primary neuronal cultures

The coupling of Ca2+ influx to synaptic vesicle (SV) recycling in nerve terminals is essential for neurotransmitter release and thus neuronal communication. Both of these parameters have been monitored using fluorescent reporter dyes such as fura-2 and FM1-43 in single central nerve terminals. However, their simultaneous monitoring has been hampered by the proximity of their fluorescence spectra, resulting in significant contamination of their signals by bleedthrough. We have developed an assay that simultaneously monitors both SV recycling and changes in intracellular free Ca2+ ([Ca2+]i) in cultured neurons using the reporter dyes FM4-64 and fura-2AM. By monitoring both fura-2 and FM4-64 emission in the far red range, we were able to visualize functionally independent readouts of both SV recycling and [Ca2+]i independent of fluorescence bleedthrough. We were also able to incorporate an assay of cell viability without any fluorescence bleedthrough from either fura-2 or FM4-64 signals, using the dye SYTOX Green. We propose that this assay of three key neuronal functions could be simply translated into a high content screening format for studies investigating small molecule inhibitors of these processes.

Effect of PDBu on [alpha]1‐adrenoceptor‐mediated contraction and changes of intracellular free Ca2+ concentration in uterine artery of pregnant sheep

PKC activation by phorbol 12, 13-dibutyrate (PDBu) shows inhibitory effects on phenylephrine (PE)-induced contractions in pregnant uterine arteries. We hypothesize that PDBu inhibits PE-induced increases of intracellular free Ca2+ concentration ([Ca2+]i). Uterine arteries were isolated from near term pregnant ewes. Vascular smooth muscle [Ca2+]i was measured simultaneously with muscle contraction in the same tissue loaded with Fura-2 by using a CAF-110 intracellular Ca2+ analyzer. PE-induced contraction and simultaneously measured [Ca2+]i were compared between before and after PDBu treatment. Compared with before PDBu treatment, both 0.1 and 1 μM PDBu produced a significant decrease in PE-induced contraction and completely blocked PE-induced increases of [Ca2+]i as indicated by no changes of Rf340/380. These results suggested that PKC activation in pregnant uterine artery plays an inhibitory role in α1-adrenoceptor-mediated contraction through blocking the availability of free Ca2+ to the contractile proteins. (Supported in part by NIH grants HL57787 and HD 31226)

Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca2+ entry into the cells; however, a direct link between HBP and intracellular Ca2+ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP-N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine (O-GlcNAc) on ANG II-mediated changes in intracellular free Ca2+ concentration ([Ca2+]i). We found that glucosamine blocked ANG II-induced [Ca2+]i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca2+]i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca2+]i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca2+]i homeostasis in the heart.

Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus

Dynamic changes in intracellular free Ca 2+ concentration play a crucial role in various neural functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca 2+-induced Ca 2+-release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K +-evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration–response curves for Ry on GABA release was lower than that for glutamate in both basal and K +-evoked conditions, suggesting that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca 2+ release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death.

Progesterone regulation of catecholamine secretion from chromaffin cells

Stress stimulates the adrenal medulla to rapidly secrete catecholamines (CAs), and the adrenal cortex to release progesterone (PROG), which may locally regulate stress-induced CA release. We used bovine chromaffin cells to investigate the effects of PROG on CA secretion. PROG dose-dependently inhibited CA secretion induced by nicotinic acetylcholine receptor (nAChR) agonist 1,1-dimethyl-4-phenlypiperazinium iodide (DMPP) up to 77%. Pre-incubation with PROG up to 1 h increased this inhibition. 3α,5α-Tetrahydroprogesterone (3α,5α-THP) and dexamethasone were less potent inhibitors. Patch-clamp techniques revealed that PROG co-applied with DMPP inhibited peak DMPP-induced current up to 68% and with 3 min pre-incubation inhibited both peak and integrated current up to ∼95%. Monitoring of FURA-2 showed that PROG similarly inhibited parallel changes in intracellular-free Ca ++ concentration. PROG also inhibited CA secretion elicited by elevated K + (38%), and, in single cells, suppressed Ca ++ current evoked by step depolarization, inhibiting amplitude by 15%, and reducing the time constant of current decay during depolarization by 57%. In contrast to the immediate inhibition of nicotinic current, inhibition of Ca ++ current became statistically significant only after 1 min exposure to PROG. PROG did not inhibit secretion stimulated by high Ca ++ perfusion of permeabilized cells. These data suggest that PROG inhibits CA secretion from chromaffin cells predominantly by rapidly inhibiting nAChRs, and by gradually enhancing the inactivation of voltage-dependent Ca ++ channels (VDCCs), but not by affecting secretory processes downstream of Ca ++ influx. This study supports a role for adrenocortical PROG in the regulation of CA secretion during stress.

Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands

The spatiotemporal changes in intracellular free Ca 2+ concentration ([Ca 2+] i) as well as fluid secretion and exocytosis induced by acetylcholine (ACh) in intact acini of guinea pig nasal glands were investigated by two-photon excitation imaging. Cross-sectional images of acini loaded with the fluorescent Ca 2+ indicator fura-2 revealed that the ACh-evoked increase in [Ca 2+] i was immediate and spread from the apical region (the secretory pole) of acinar cells to the basal region. Immersion of acini in a solution containing a fluorescent polar tracer, sulforhodamine B (SRB), revealed that fluid secretion, detected as a rapid disappearance of SRB fluorescence from the extracellular space, occurred exclusively in the luminal region and was accompanied by a reduction in acinar cell volume. Individual exocytic events were also visualized with SRB as the formation of Ω-shaped profiles at the apical membrane. In contrast to the rapidity of fluid secretion, exocytosis of secretory granules occurred with a delay of ∼70 s relative to the increase in [Ca 2+] i. Exocytic events also occurred deep within the cytoplasm in a sequential manner with the latency of secondary exocytosis being greatly reduced compared with that of primary exocytosis. The delay in sequential compound exocytosis relative to fluid secretion may be important for release of the viscous contents of secretory granules into the nasal cavity.

A Study of the Cardioprotective Effect of Breviscapine During Hypoxia of Cardiomyocytesin vitroand During Myocardial Infarctionin vivo

Breviscapine is a flavonoid extracted from Erigeron breviscapus. Hand.-Mazz, and it has been reported that breviscapine can activate K+ channels and block Ca2+ channels. In this paper, we studied the cardioprotective effects of breviscapine on electrocardiogram (ECG) changes (ST-segment elevation), infarction size in dog heart subjected to myocardial infarction caused by left coronary artery ligation and lactate dehydrogenase (LDH) leakage, changes of intracellular free Ca2+ levels, apoptosis and necrosis in cultured neonatal rat cardiomyocytes subjected to hypoxia. Additionally, the effect of breviscapine on myocardial oxygen consumption was detected in dog myocardium in vitro. The results showed that breviscapine treatment (1 mg/kg, 2 mg/kg and 4 mg/kg) significantly reduced ST-segment elevation and infarction size in hearts subjected to myocardial infarction, that breviscapine treatment (14.29 microg/mL, 28.57 microg/mL and 57.14 microg/mL) significantly decreased oxygen consumption in myocardium, and that breviscapine treatment (5 microg/mL, 10 microg/mL and 20 microg/mL) significantly reduced LDH leakage, intracellular free Ca2+ levels, apoptosis and necrosis in cardiomyocytes subjected to hypoxia. In conclusion, the present study indicates that breviscapine is in favor of myocardial protection.

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools

Spontaneous Ca2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca2+ pool. This report describes spontaneous Ca2+ oscillations dependent on two separate Ca2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca2+ were monitored with ratiometric Ca2+- fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca2+ (less than 1 microM)decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca2+ stores that was greater in the nuclear compartment (4.20 +/- 0.23 ratio units, n = 56) than cytoplasm (2.54 +/- 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca2+ transient in the cytoplasm (4.99 +/- 0.66, n = 17) than in the nucleus (2.67 +/- 0.29). Pre-treatment with Ca2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca2+ most diminished. Depletion of internal Ca2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca2+ influx.

Effects of Infrasound on Changes of Intracellular Calcium Ion Concentration and on Expression of RyRs in Hippocampus of Rat Brain

In the present study, we investigated the molecular biological effect of infrasound on changes of intracellular free Ca2+ concentration ([Ca2+]i) and on expression of ryanodine receptors (RyRs) in the hippocampus of rats. 60 SD rats were randomized into ten groups and examined for [Ca2+]i and RyRs expression after exposure to 8Hz 90dB infrasound at days 1, 7, 14 and 21. Groups exposed to infrasound were exposed to 8Hz 90dB infrasound for 2 hours once daily. Acute cell separation, fluo-3/AM fluorescence technique and laser scanning confocal microscope (LSCM) were used for measurement of the [Ca2+]i level. There was no significant difference of [Ca2+]i at 1d and 7d infrasound exposure groups compared to control, but there was a significant incease at 14d group (P&lt;0.01) and 21d group (P&lt;0.05). Immunohistochemistry indicated the expression of RyRs in CA1 and CA3 pyramidal cells of the hippocampus showed no difference at 1d and 7d groups compared to control, whereas significant decrease was observed at 14d group (P&lt;0.01) and 21d group (P&lt;0.05). The data suggest that infrasound of 8Hz 90dB can induce [Ca2+]i increase and decrease the expression of RyRs in CA1 and CA3 regions of the hippocampus after exposure to infrasound for 14 and 21 days.