Inhibition Of Intracellular Calcium Release Research Articles

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Synthesis, characterization and biological evaluation of indomethacin derived thioureas as purinergic (P2Y1, P2Y2, P2Y4, and P2Y6) receptor antagonists

G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.

Abstract 14219: Oxidation of PKA-RIα Protects Against Ischemia-reperfusion Injury by Inhibiting Lysosomal-triggered Calcium Release

Introduction: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, the regulatory Iα subunit of Protein Kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulfide bonds within the holoenzyme complex. However, the impact of disulfide formation on kinase function, and its influence on PKA signaling in the context of heart disease remains unknown. Methods & Results: Myocardial ischemia-reperfusion (I/R) was found to be a potent inducer of PKARIα disulfide formation in vivo , both in mice and in humans. Using imaging modalities with high spatial and temporal resolution, we found that this conformation did not increase intrinsic PKA catalytic activity, but rather facilitated enhanced AKAP-dependent compartmentation of PKARIα in the adult mouse left ventricular (LV) myocyte, with preferential localization to the lysosome under oxidized conditions (n=38-41 myocytes, N=3 animals, p<0.01). Investigations in isolated LV myocytes revealed disulfide-modified PKARIα to be a significant regulator of lysosomal two pore channel (TPC)-dependent calcium-induced calcium release, with myocytes from ‘redox dead’ PKARIα mice (Cys17Ser) displaying spontaneous sarcoplasmic reticulum calcium release events and pronounced intracellular calcium oscillations. These events were prevented by ryanodine receptor blockade (1 mM tetracaine; n=14, p<0.01), acute depletion of lysosomal calcium stores (100 nM bafilomycin; n=7; p<0.01), or TPC inhibition (5 μM Ned-19; n=9; p<0.05). Absence of I/R-induced disulfide formation in “redox dead” PKARIα mouse hearts resulted in larger infarcts (2-fold increase, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which could be fully prevented by administering the TPC inhibitor, Ned-19, at the time of reperfusion. Conclusions: Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKA oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics.

Potent Thiophene Antagonists of Human Complement C3a Receptor with Anti-Inflammatory Activity.

Structure-activity relationships for a series of small-molecule thiophenes resulted in potent and selective antagonism of human Complement C3a receptor. The compounds are about 100-fold more potent than the most reported antagonist SB290157. A new compound JR14a was among the most potent of the new antagonists in vitro, assessed by (a) inhibition of intracellular calcium release (IC50 10 nM) induced in human monocyte-derived macrophages by 100 nM C3a, (b) inhibition of β-hexosaminidase secretion (IC50 8 nM) from human LAD2 mast cells degranulated by 100 nM C3a, and (c) selectivity for human C3aR over C5aR. JR14a was metabolically stable in rat plasma and in rat liver microsomes and efficacious in rats when given orally to suppress rat paw inflammation, macrophage and mast cell activation, and histopathology induced by intraplantar paw administration of a C3aR agonist. Potent C3aR antagonists are now available for interrogating C3a receptor activation and suppressing C3aR-mediated inflammation in mammalian physiology and disease.

2161Redox-mediated PKA-RIalpha localisation to the lysosome inhibits myocardial calcium release and robustly reduces myocardial injury

Abstract Background Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulphide bonds within the holoenzyme complex. However, the effect of PKARIα oxidation on downstream signalling in the heart, particularly under states of oxidative stress, remains unexplored. Purpose To determine the direct functional consequences of PKARIα oxidation in the heart and investigate their impact on ischaemia/reperfusion (I/R) injury. Methods and results Experiments using the AKAR3ev FRET biosensor in murine left ventricular (LV) myocytes and Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged wild-type (WT) and mutant RIα proteins expressed in RIα-null fibroblasts showed that PKARIα oxidation does not increase the kinases' catalytic activity, but enhances its binding to A-kinase anchoring proteins (AKAP; n=30–39/N=3, p<0.01). Super-resolution microscopy revealed localisation of oxidised PKARIα to lysosomes in WT myocytes, which was completely absent in “redox dead” Cys17Ser PKARIα knock-in mice (KI; panel A; n=38–41/N=3, p<0.01) and reduced when AKAP binding was prevented using the RIAD disruptor peptide (30.6±5.1% reduction; n=35–37/N=3, p<0.01). Displacement of PKARIα from lysosomes resulted in spontaneous sarcoplasmic reticulum calcium release and dramatic calcium oscillations in KI LV myocytes (panel B), which were preventable by ryanodine receptor blockade (1 mM tetracaine; n=14, p<0.01), acute depletion of endolysosomal calcium stores (100 nM bafilomycin; n=7; p<0.01), or lysosomal two-pore channel inhibition (5 μM Ned-19; n=9; p<0.05). I/R (secondary to cardiopulmonary bypass) was found to induce PKARIα oxidation in the myocardium of patients undergoing cardiac surgery (panel C; n=18, p<0.05). Absence of this response in KI mouse hearts resulted in 2-fold larger infarcts (p<0.01) and a concomitant reduction in LV contractile recovery (final LVDP of 55.9±8.6 vs 82.5±7.1 mmHg in WT; n=7–8, p<0.05), both which were prevented by addition of Ned-19 at the time of reperfusion (panel D; n=4, p<0.01). Conclusions Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKARIα oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics. Acknowledgement/Funding British Heart Foundation CH/12/3/29609, RG/16/12/32451; Garfield-Weston Foundation MPS/IVIMS-11/12-4032; Wellcome Trust Fellowship 0998981Z/12/Z

Juglone as antihypertensive agent acts through multiple vascular mechanisms

ABSTRACTBackground: Juglone, a natural phenolic compound obtained from the walnut tree, is known for its wide range of biological activities. However, it has yet to be tested for its effects on hypertension and vascular tone. This investigation was aimed to explore the antihypertensive effect and the nature of vascular reactivity of juglone in rat models.Methods: Juglone was tested in in vivo and in vitro experiments in rats. The responses were analyzed and recorded through a PowerLab data acquisition system.Results: Intravenous injection of juglone significantly decreased the mean arterial blood pressure (MAP) in normotensive and hypertensive rats (Max. fall, 43.50 ± 2.96 vs 49.66 ± 3.28 mmHg). In rats pretreated with Nω-Nitro l-arginine methyl ester (L-NAME), the effect of juglone on MAP was reduced as compared to the control. However, in rats pretreated with atropine the fall in MAP by juglone was not altered. Juglone induced relaxation in the phenylephrine, K+ (80 mM), and angiotensin II pretreated isolated rat aortic rings. This vasorelaxant effect was reduced with L-NAME pretreatment. Atropine pretreatment did not modify the vasorelaxant effect of juglone. Pre-incubation with juglone attenuated the intracellular Ca2+ release by suppressing phenylephrine peak formation and also shifted CaCl2 concentration–response curves (CRCs) to the right. Of note, combined treatment with 4-aminopyridine and barium chloride also reduced juglone-mediated vasorelaxation suggesting a role of K+-channels as well.Conclusion: In conclusion, juglone exerts its antihypertensive effect through vasorelaxation, which is mediated by nitric oxide, inhibition of intracellular calcium release and opening of K+-channels.

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells

IntroductionProstaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. MethodsPrimary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate–activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. ResultsPGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. ConclusionsThese results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention.

Activation of Cold-Sensing Transient Receptor Potential Melastatin Subtype 8 Antagonizes Vasoconstriction and Hypertension Through Attenuating RhoA/Rho Kinase Pathway

Environmental cold is a nonmodifiable hypertension risk factor. Transient receptor potential melastatin subtype 8 (TRPM8) is a cold-sensing cation channel that can be activated by menthol, a compound with a naturally cold sensation in mint. Little is known about the effect of TRPM8 activation on vascular function and blood pressure. Here, we report that TRPM8 is abundantly expressed in the vasculature. TRPM8 activation by menthol attenuated vasoconstriction via RhoA/Rho kinase pathway inhibition in wild-type mice, but the effect was absent in TRPM8(-/-) mice. Chronic dietary menthol blunted mesenteric arterial constriction and lowered blood pressure in genetic hypertensive rats via inhibition of RhoA/Rho kinase expression and activity in the vivo study. TRPM8 effect was associated with inhibition of intracellular calcium release from the sarcoplasmic reticulum, RhoA/Rho kinase activity, and sustained arterial contraction in the vitro study. Importantly, 8-week chronic menthol capsule treatment moderately lowered systolic blood pressure and diastolic blood pressure in prehypertensive individuals compared with the placebo group. Furthermore, chronic menthol capsule administration also improved flow-mediated dilatation in prehypertensive individuals, but not in the placebo group. In conclusion, our study demonstrates that TRPM8 activation by menthol benefits vascular function and blood pressure by inhibiting calcium signaling-mediated RhoA/Rho kinase activation in the vasculature. These findings add to the evidence that long-term dietary menthol treatment had favorable effects on hypertension treatment.

Polarized secreted IL-17C is a mediator of respiratory epithelial innate immune response

The IL-17 family of cytokines consists of at least six members (IL-17A to -F). IL-17 directly activates epithelial cells leading to the expression of inflammatory mediators and antimicrobial factors. In this study, our aim was to examine the expression and function of IL-17C in respiratory epithelial cells during bacterial infection. In vitro studies showed that common bacterial pathogens, such as Pseudomonas aeruginosa and Haemophilus influenzae, and ligands of Toll-like receptors 3 and 5 (flagellin, polyI:C) induced the expression and release of IL-17C in cultured human bronchial epithelial cells. Differentiated bronchial epithelial cells secreted IL-17C into the basolateral compartment and the secretion of IL-17C was increased in response to basolateral stimulation as compared to apical stimulation with bacterial stimuli. Pharmacological inhibition of intracellular calcium release and calcium chelators inhibited the secretion of IL-17C whereas the release of the inflammatory mediator IL-6 was not affected. IL-17C enhanced inflammatory responses of respiratory epithelial cells infected with P. aeruginosa. These data show that calcium-dependent signaling cascades mediate the polarized secretion of IL-17C and that IL-17C mediates innate immune responses of respiratory epithelial cells.

Dual effects of daily FTY720 on human astrocytes in vitro: relevance for neuroinflammation

BackgroundFTY720 (fingolimod, Gilenya™) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1β.MethodsAstrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1β-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA.ResultsWe observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1β. Recurrent FTY720 treatments did not inhibit serum- or IL-1β-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1β were unaffected by FTY720 treatment(s).ConclusionOur results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.

Muscarinic Type 3 Receptor Induces Cytoprotective Signaling in Salivary Gland Cells through Epidermal Growth Factor Receptor Transactivation

Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.

Androgens Rapidly Activate Nuclear Factor-Kappa B via Intracellular Ca2+ Signalling in Human Vascular Endothelial Cells

There exists a striking gender difference in the incidence of atherosclerosis. Androgen exposure may predispose men to earlier onset atherosclerosis. We previously demonstrated that the potent androgen, dihydrotestosterone, enhanced the binding of monocytes to endothelial cells, via androgen receptor/nuclear factor kappa B-dependent expression of the cell adhesion molecules, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1. We now show that testosterone and dihydrotestosterone can also induce a novel, non-genomic pathway that leads to the rapid activation of nuclear factor-kappa B via intracellular Ca2+ signalling, initiated at the plasma membrane. Human umbilical vein endothelial cells exposed to 6-60 nM testosterone or dihydrotestosterone showed a rapid increase in intracellular calcium levels. The testosterone or dihydrotestosterone effect on increased intracellular calcium could not be abrogated by pre-incubation with androgen receptor antagonist, hydroxyflutamide, or by U73122, an inhibitor of intracellular calcium release from endoplasmic reticulum stores. However, pre-incubation with both Ni2+or an extracellular Ca2+ chelator blocked the testosterone-induced intracellular Ca2+ surge. Testosterone conjugated to bovine serum albumin was equal to free testosterone in its ability to induce the intracellular Ca2+ surge. Binding studies showed testosterone does bind to the plasma membrane, however, classical androgen receptor was unable to be detected in the plasma membrane of human umbilical vein endothelial cells. Testosterone was found to rapidly increase nuclear factor-kappa B activity, an effect that was blocked when cells were incubated in calcium-free media. This study demonstrates for the first time that testosterone induces a non-genomic membrane-initiated Ca2+ dependent signalling pathway that leads to the rapid activation of nuclear factor-kappa B.

263 Junctional tachycardia in the mouse can be prevented by pacemaker channel (If) blockade with Ivabradine hydrochloride

Atrioventricular nodal re-entrant tachycardia (AVNRT) and junctional tachycardia (JT) are common, electrocardiographically similar, arrhythmias. AVNRT is described as re-entry conduction between the fast (FP) and slow (SP) AV nodal pathways. JT occurring following cardiac surgery in neonates increases mortality risk by 5.6-14%. JT mechanisms are unknown, but may include autonomic nervous system involvement. Pacemaking mechanisms utilize a membrane clock, involving the pacemaker funny channel (If), and an intracellular calcium clock involving rhythmic oscillations in calcium release from ryanodine receptors. The SP, formed by the inferior extension of the compact AV node (INE), contains the dominant AVN pacemaker, expresses If, and may be involved in JT. We investigated the mechanisms of junctional arrhythmia in male wild type C57BL/6 mice. Studies utilized an intracardiac octapolar electrode catheter in anaesthetized mice. Programmed electrical stimulation elicited antegrade AVN conduction and SP conduction in 8/18 mice, single re-entrant beats in 3/18, but rarely AVNRT (1/18). Rapid ventricular pacing (RVP, 50 Hz, 400 ms, 10-30 bursts), induced junctional arrhythmia in 14/18 mice (83%) producing a 32% increase in rate (325 ± 15 vs 429 ± 19 BPM, P < 0.001). Response of arrhythmia to single premature atrial stimuli distinguish AVNRT from JT. Premature FP activation did not terminate the arrhythmia (0/14) indicating JT. RVP caused decreases (46 ± 2.6%) in minimum end-diastolic blood pressure and enhanced nodal conduction following 10 bursts (pre-pacing AH = 31 ± 1ms vs post-pacing AH = 28 ± 1ms, P < 0.001, n = 14) prior to JT initiation, suggesting involvement of a baroreflex response. Vagal nerve stimulation (5/5, X2 = 10, P < 0.002) and propranolol (7/7, X2 = 14, P < 0.001) prevented JT induction, confirming a role for the autonomics. Ivabradine hydrochloride, a selective If blocker, prevented JT (8/9, X2 = 14.4, P < 0.001). Ruthenium red, a potent inhibitor of intracellular calcium release also prevented JT (5/5, X2 = 10,P < 0.002). Forskolin (an adenylyl cyclase activator) and caffeine (a stimulator of intracellular calcium release) together produced long lasting (>2 mins) spontaneous JT (5/5, X2 = 10, P < 0.002) that was terminated by Ivabradine (5/5, X2 = 10,P < 0.002). AVNRT was infrequent, but the mouse is susceptible to JT. JT may rely on an interaction of membrane and intracellular events which can be prevented by If channel blockade. Ivabradine might be useful in neonatal patients with post-operative JT.

Vascular and renal function of cGMP signalling

Results For the functional analysis of cGMP signalling, transgenic murine mutants were used. cGKI signalling pathways include interaction of the cGKIb-isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). NO/cGMP and ANP/cGMP-dependent relaxation of aortic smooth muscle was strongly affected in IRAG-deficient mice. NO/ ANP-dependent inhibition of intracellular calcium release was suppressed in IRAG-deficient vascular smooth muscle cells (VSMC). Furthermore, reduction of store operated calcium entry by cGMP was affected in IRAG-KO VSMC. Basal mean arterial blood pressure, heart rate and activity were not changed in IRAG knockout mice. However, under pathophysiological conditions like sepsis (induced by E. coli lipopolysaccharide application) IRAG knockout mice were resistant to blood pressure reduction.

Pharmacological inhibition of intracellular calcium release blocks acid-induced bone resorption

In vivo chronic metabolic acidosis induces net Ca2+ efflux from bone, and incubation of neonatal mouse calvariae in medium simulating physiological metabolic acidosis induces bone resorption. It appears that activation of the proton (H+) receptor OGR1 in the osteoblast leads to an increase in intracellular Ca2+, which is associated with an increase in cyclooxygenase 2 (COX2) and PGE2-induced receptor activator of NF-κB ligand (RANKL) and H+-induced osteoclastic bone resorption. To support this hypothesis, we tested whether intracellular Ca2+ signaling was integral to H+-induced bone resorption by determining whether 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and 2-aminoethoxydiphenyl borate (2-APB), inhibitors of inositol trisphosphate-mediated Ca2+ signaling, would block H+-induced bone resorption in cultured neonatal calvariae and, if so, would do so by inhibiting H+-induced stimulation of COX2 and RANKL in osteoblastic cells. We found that H+-induced bone resorption is significantly inhibited by TMB-8 and 2-APB. Both compounds also inhibit H+-induced stimulation of COX2 protein in calvariae and COX2 mRNA and protein levels in primary osteoblasts. H+-induced stimulation of RANKL in calvarial cultures, as well as primary cells, is also completely inhibited by TMB-8 and 2-APB. These results support the hypothesis that H+ stimulation of net Ca2+ efflux from bone, mediated by COX2- and subsequent PGE2-induced RANKL production, is initiated in the osteoblast via activation of Ca2+ signaling.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCdelta inhibitor rottlerin, and downregulation of PKCdelta was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCdelta in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCdelta siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCdelta-dependent manner.

Cyclic AMP-Independent CGRP8–37-Sensitive Receptors Mediate Adrenomedullin-Induced Decrease of CaCl2-Contraction in Pregnant Rat Mesenteric Artery

Objectives: We tested the hypothesis that adrenomedullin reduces calcium influx independent of potassium channels in depolarized endothelium-denuded mesenteric artery from pregnant rats. Results: Adrenomedullin reduced the CaCl₂-induced contraction, while the receptor antagonist calcitonin gene-related peptide (CGRP)_8–37, but not adrenomedullin_22–52, reversed these effects. Adenylate cyclase inhibition by SQ22536 did not prevent adrenomedullin effects on CaCl₂-induced contraction. Adrenomedullin did not inhibit depolarization-induced calcium entry to isolated vascular smooth muscle. Inhibition of myosin light-chain (MLC) phosphatase by calyculin A reversed the effects of adrenomedullin on contraction caused by submillimolar concentrations of CaCl₂, while adrenomedullin still inhibited contraction caused by higher concentrations of CaCl₂. However, the ratio of phosphorylated to total myosin phosphatase target 1, the regulatory subunit of MLC phosphatase, did not change with adrenomedullin, indicating a lack of MLC phosphatase activation. Interestingly, sodium fluoride, a nonspecific protein phosphatase inhibitor, completely blocked the effect of adrenomedullin on CaCl₂-induced contraction. Adrenomedullin inhibited calcium mobilization from intracellular stores induced by thapsigargin. Conclusion: Adrenomedullin inhibits CaCl₂-induced contraction, without affecting calcium influx, through a CGRP_8–37-sensitive receptor, but not using the cyclic adenosine monophosphate pathway, probably through activation of protein phosphatases. Inhibition of intracellular calcium release is an additional role played by adrenomedullin in calcium homeostasis in vascular smooth muscle.

VEGF Inhibits PDGF-Stimulated Calcium Signaling Independent of Phospholipase C and Protein Kinase C

Despite advances in both open and endovascular techniques for treatment of arterial occlusive disease, restenosis because of neointimal hyperplasia continues to be a major cause of graft failure and restenosis. This phenomenon has been attributed to vascular smooth muscle cell (VSMC) activation by several potent mitogens including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) released at the site of injury. PDGF is known to stimulate calcium influx in VSMC that has been shown to be critical for VSMC migration and proliferation. We have previously shown that VEGF inhibits PDGF-stimulated VSMC proliferation. The objective of this set of experiments was to investigate whether VEGF modulated PDGF-stimulated Ca2+ influx in VSMC. Primary cultured human aortic SMC were grown to subconfluency and assigned to the following groups: no stimulation, stimulation with PDGF-BB (20 ng/ml), stimulation with VEGF165 (40 ng/ml), or a combination of PDGF-BB + VEGF165. Ca2+ influx was measured using a Fura-2 fluorescence assay. The intracellular Ca2+ fraction was assayed with the Fura-2 assay by using Ca2+-free media. Phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC), and Akt phosphorylation was assessed with standard immunoblotting techniques at 1, 5, and 10 min time points. Ca2+-calmodulin kinase II (CaMKII) activity was extrapolated from the phosphorylation of Phospholamban B (PLB), a well-known protein substrate, at 1, 5, and 10 min time points. PDGF stimulation resulted in a 328 +/- 9 nm total calcium influx in VSMC. The combination of VEGF + PDGF resulted in a 273 +/- 21 nm total calcium influx, an amount significantly less than with PDGF alone (P < 0.04). PDGF stimulation resulted in a 72 +/- 35 nm intracellular calcium release. The addition of VEGF to PDGF resulted in an intracellular calcium release of only 15 +/- 11 nm, a significant decrease compared to PDGF alone (P < 0.01). The phosphorylation of PLCgamma1, PKC, and Akt was equivalent at 1, 5, and 10 min between the PDGF and the PDGF + VEGF treatment groups. There was an increase in CaMKII activity at 1 and 5 min time points in both the PDGF and PDGF + VEGF treatment groups suggesting that extracellular calcium influx is sufficient for CaMKII activation. VEGF inhibits PDGF-stimulated total calcium influx and, in particular, PDGF-stimulated intracellular calcium release in VSMC. The equivalent phosphorylation of PLCgamma1, PKC, and Akt suggests that the inhibitory mechanism by VEGF on calcium influx occurs downstream of these proximal mediators. The inhibition of intracellular calcium release did not inhibit CaMKII activity. VEGF may play an important role in modulating PDGF induced VSMC proliferation by specifically inhibiting intracellular calcium release in response to PDGF.

Trans- andcis-resveratrol increase cytoplasmic calcium levels in A7r5 vascular smooth muscle cells

The effects of trans- and cis-resveratrol on cytosolic Ca2+ concentration ([Ca2+]i) were studied using fura-2 in vascular smooth muscle cells (A7r5). Both isomers of resveratrol caused a sustained elevation in [Ca2+]i, cis-resveratrol being significantly more effective than the trans-isomer. The resveratrol-induced increase in [Ca2+]i was significantly potentiated by the previous application of low concentrations of thapsigargin, partially inhibited by nifedipine or Ni2+, and not affected by SKF 96365. In the absence of extracellular Ca2+, both isomers of resveratrol induced a transient, slow increase in [Ca2+]i, which was inhibited by the previous depletion of intracellular stores with thapsigargin and completely blocked by preincubation with TMB-8, an inhibitor of intracellular calcium release. Reintroduction of Ca2+ in the external solution after the resveratrol-induced release of Ca2+ activated the Ca2+ influx through store-operated calcium channels. The resveratrol-induced increase in [Ca2+]i in the absence of extracelullar Ca2+ partially reduced the increase in [Ca2+]i evoked by the subsequent application of thapsigargin. Our results suggest that trans- and cis-resveratrol induce a depletion of Ca2+ from the same intracellular stores released by thapsigargin and subsequent capacitative influx of Ca2+. Additionally, a direct activation of transmembrane Ca2+ influx through another type of channel may be also implicated.

Dantrolene protects hippocampal cells from damage induced by TsTX, an α-scorpion toxin from Tityus serrulatus

We examined the effects of dantrolene, an inhibitor of intracellular calcium release, on alterations associated with the intrahippocampal injection of the TsTX scorpion toxin. Male Wistar rats (230–250 g) were injected with Ringer solution (1 μl; n=6); TsTX toxin (1 μg/μl; n=8); and dantrolene (10.0 mg/kg) plus TsTX toxin (1 μg/μl; n=6). After injection, electroencephalographic (EEG) recordings and observation of animals behaviour were performed continuously for 4 h. One week later, animals were submitted to histopathological analysis. TsTX caused electrographic seizure expressed by moderate or intense discharges and neuronal loss in hippocampal areas in all injected animals ( n=8). Dantrolene reduced the effect of TsTX. Thus, 67% of rats (four out of six) treated with toxin and dantrolene had electrographic convulsions, but only for 30 min after injection and none of them presented neuronal damage. Dantrolene or Ringer had no effects on the EEG.