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Reports of Chagas disease are increasing in non-endemic populations across the globe. Apart from vector eradication and prevention efforts by public health organizations, current pharmacological interventions are sparse and show important side effects. In this issue of the BJP, Andrade et al. elegantly demonstrate a new pharmacological paradigm whereby Trypanosoma cruzi host cell invasion requires significant cross-talk between receptors for kinins and endothelins. It is shown, for example, that acting via both ETA and ETB receptors, endothelin-1 (ET-1) cooperates with the (TLR2/CXCR2/B2 kinin receptor) complex to activate inflammatory processes in response to invading trypomastigotes. This study by Andrade et al. prompts, however, several important questions, summarized in this Commentary, such as the putative role of chymase-dependent production of ET-1, the contentious protective role of ACE inhibitors in Chagasic patients, the unexplored role of de novo formed B1 receptors for kinins triggered by cytokines and the putative role of compartmentalized calcium pools in host cell invasion by trypomastigotes. LINKED ARTICLE This article is a commentary on Andrade et al., pp. 1333-1347 of this issue. To view this paper visit [ABSTRACT FROM AUTHOR]

Unlabelled: Sarcoplasmic reticular (SR) Ca(2+) -ATPase (SERCA2a) is central to cardiac electrophysiological and mechanical function. It ensures full diastolic relaxation minimizing delayed after-potentials that would otherwise compromise membrane electrophysiological stability, and optimizes SR Ca(2+) refilling and systolic contraction. Previous studies demonstrated that the small molecule agent istaroxime stimulates SERCA2a-ATPase activity, restoring its function in failing hearts, and enhancing indices of mechanical, and SR Ca(2+) release and re-uptake, activity. Ferrandi et al (2013) now elegantly demonstrate its ability to dissociate the phospholamdan (PB) bound to cardiac SERCA2a, thereby removing the inhibitory effect of PB on SERCA2a. This effect was independent of the cAMP/PKA system and modified a specific SERCA2a reaction step. They used SERCA-enriched SR preparations from a rigorously validated and realistic physiological, canine model of cardiac failure with established Na(+) -K(+) -ATPase sensitivity to cardiac glycosides and SR Ca(2+) handling features. These findings potentially translate into a novel management of the major and increasingly important public health challenge of chronic cardiac failure., Linked Article: This article is a commentary on Ferrandi et al., pp. 1849-1861 of volume 169 issue 8. To view this paper visit http://dx.doi.org/10.1111/bph.12278., (© 2013 The British Pharmacological Society.)

1. The aim of this study in circular smooth muscle of rat distal colon was to determine whether Ca(2+) desensitisation, in addition to mechanisms lowering cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), was involved in the relaxation elicited by nitric oxide (NO). Changes in isometric tension and [Ca(2+)](cyt) were recorded simultaneously in fura-2-loaded strips. 2. In methacholine (10(-5) M)-precontracted preparations, exogenous NO (10(-4) M), adenosine 5'-triphosphate (ATP; 10(-3) M) and electrical field stimulation (EFS; 1 ms, 40 V, 4 Hz, 1 min) induced a decrease in smooth muscle tension, which was accompanied by a fall in [Ca(2+)](cyt). 3. The sarcoplasmic/endoplasmic reticulum Ca(2+) ATP-ase (SERCA) inhibitor thapsigargin (10(-6) M) did not exert an influence on the decrease in tension produced by exogenous NO, but significantly attenuated the fall in [Ca(2+)](cyt). Both the relaxation and the fall in [Ca(2+)](cyt) to ATP and EFS were unaffected by thapsigargin. 4. Calyculin-A (10(-6) M), a myosin light chain phosphatase (MLCP) inhibitor, significantly reduced the decrease in tension elicited by exogenous NO, but did not alter the fall in [Ca(2+)](cyt) to exogenous NO. Inactivating RhoA by exoenzyme C3 (2 mug ml(-1)) or inhibiting Rho-kinase with (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632; 10(-5) M) had no effect on the decrease of both tension and [Ca(2+)](cyt) generated by exogenous NO. 5. This paper demonstrates that a RhoA/Rho-kinase independent Ca(2+) desensitisation pathway contributes to the relaxation by NO in circular smooth muscle strips of rat distal colon.

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

Background and purpose: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. Experimental approach: Vascular reactivity to ET-1 and ETA and ETB receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ETA and ETB receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [125I]-ET-1. Key results: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ETA or ETB receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ETA but not of ETB receptors abolished enhancement in HHcy tissues. ETA and ETB receptor gene expressions were not up-regulated. ETA receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A2 receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. Conclusions and implications: Increased ETA receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ETA receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility. British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009 This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Background and purpose: Vascular ATP-sensitive potassium (KATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca2+-dependent phosphatase, inhibits KATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular KATP channel (KIR6.1/SUR2B). The effect of intracellular Ca2+ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aα but not Aβ significantly attenuated KATP currents activated by low intracellular Ca2+, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal KATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca2+ (equivalent to increasing calcineurin activity), almost completely inhibited KATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and implications: Calcineurin Aα regulates KIR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the KATP channel. British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009 This article is commented on by Tammaro, pp. 551–553 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

1. The importance of Ca2+ release from the sarcoplasmic reticulum (SR) in excitation contraction (EC) coupling in human detrusor muscle remains controversial. In this paper the contribution of Ca2+ release to agonist induced contraction is assessed. 2. Dose response curves to carbachol (0.01 - 10 microM) were constructed before and after exposure to 200 nM Thapsigargin (Tg). Tg pre-treatment reduced the force of contraction at all agonist concentrations however, the reduction was dose dependent. At 0.1 microM the contractions were reduced to 14.5 +/- 7% (mean +/- s.e.mean) of controls (n = 8) while at 10 microM the contractions were only reduced to 92 +/- 3% of controls (n = 10). 3. The role of external Ca2+ was examined by measuring the magnitude of contraction to low and high doses of agonist in the presence and absence of external Ca2+. With (0.1-0.3 microM) carbachol the contractions in nominally Ca2+ free media were 4+/-4% of controls (n = 7) whilst with (1 - 10 microM) carbachol the contractions were 36 +/- 8% of controls (n=7) suggesting that at low agonist concentrations the release of Ca2+ has a requirement for external Ca2+. 4. Pre-treatment of muscle strips with the Ca2+ channel blocking agent diltiazem reduced the contractile responses to carbachol. Contractions induced by 0.1 microM were reduced to 29+/-11% (P<0.05) of controls while those activated by 10 microM were reduced to 86+/-6% (P= 0.1) of controls (n = 4) suggesting the Ca2+ influx needed to activate internal store release at low agonist stimulation is through L-type Ca2+ channels. 5. These observations confirm the importance of thapsigargin sensitive intracellular Ca2+ store release in the activation of contraction of detrusor smooth muscle and suggest the overall contribution of this store depends upon the magnitude of the agonist stimulation.

NAADP has been shown to be a potent calcium-releasing second messenger in a wide variety of cell types to date. However, research has been hampered by a lack of pharmacological agents, with which to investigate NAADP-induced calcium release, and by the molecular identity of its cellular target protein being unknown.In the present paper, the sea urchin egg model was used to investigate whether triazine dyes, which can act as nucleotide mimetics, can bind to the NAADP receptor, induce Ca2+ release and be used for affinity chromatography of the receptor.Indeed, all the triazine dyes tested (Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Green 5 (RG5), Cibacron Blue 3GA and Reactive Yellow 86) displayed micromolar affinities, except for Reactive Orange 14. Furthermore, unlike NAADP, RR120, RG19 and RG5 did not bind in an irreversible manner.The compound that displayed the highest affinity, RR120, was tested in a 45Ca2+ efflux assay. This compound released Ca2+ via the NAADP receptor, as shown by the ability of subthreshold NAADP concentrations to inhibit this release. Furthermore, heparin and ruthenium red were unable to block RR120-induced Ca2+ release.We have also shown that RG5 and RG19, immobilised on resins, retain the ability to bind to the receptor, and that this interaction can be disrupted by high salt concentrations. As a proof of principle, we have shown that this can be used to partially purify the NAADP receptor by at least 75-fold.In conclusion, triazine dyes interact with the NAADP receptor, and this could be exploited in future to create a new generation of pharmacological tools to investigate this messenger and, in combination with other techniques, to purify the receptor.British Journal of Pharmacology (2004) 142, 1241–1246. doi:10.1038/sj.bjp.0705886 [ABSTRACT FROM AUTHOR]

1. Standard renal clearance techniques were used to assess the dose-response relationship between acute gentamicin infusion and the magnitude of hypercalciuria and hypermagnesiuria in the anaesthetized Sprague-Dawley rat. Also investigated were whether these effects occurred independently of renal tubular cell injury. 2. Acute gentamicin infusion was associated with a significant hypercalciuria and hypermagnesiuria evident within 30 min of drug infusion. The magnitude of these responses was related to the dose of drug infused (0.14-1.12 mg kg(-1) min[-1]). Increased urinary electrolyte losses resulted from a decreased tubular reabsorption of calcium and magnesium. 3. A rapid dose-related increase in urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion was also observed in response to gentamicin infusion. However, there was no evidence of renal tubular cell injury and no myeloid bodies were observed within the lysosomes of the proximal tubular cells. Gentamicin may thus interfere with the mechanisms for cellular uptake and intracellular processing of NAG causing increased NAG release into the tubular lumen. 4. The absence of changes in renal cellular morphology indicates that the excessive renal losses of calcium and magnesium were an effect of gentamicin per se and not the result of underlying renal tubular injury. The renal effects described in this paper were apparent after administration of relatively low total drug doses, and with plasma concentrations calculated to be within the clinical range. These findings suggest that disturbances of plasma electrolyte homeostasis could occur in the absence of overt renal injury in patients receiving aminoglycoside antibiotics.

Background and Purpose: Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+‐activated Cl− channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+. Experimental Approach: We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. Key Results: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh‐A01. Acute addition of niclosamide directly increased intracellular Ca2+, an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh‐A01 did not elevate intracellular Ca2+, thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. Conclusions and Implications: Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools. [ABSTRACT FROM AUTHOR]

Background and Purpose: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma.Experimental Approach: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish).Key Results: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish.Conclusion and Implications: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma. [ABSTRACT FROM AUTHOR]

Background and Purpose: α4β2 nicotinic ACh receptors (nAChRs) comprise the most abundant class of nAChRs in the nervous system. They assemble in two stoichiometric forms, each exhibiting distinct functional and pharmacological signatures. However, whether one or both forms are modulated by calcium or magnesium has not been established. Experimental Approach To assess the functional consequences of calcium and magnesium, each stoichiometric form was expressed in clonal mammalian fibroblasts and single‐channel currents were recorded in the presence of a range of ACh concentrations. Key Results: In the absence of divalent cations, each stoichiometric form exhibits high unitary conductance and simple gating kinetics composed of solitary channel openings or short bursts of openings. However, in the presence of calcium and magnesium, the conductance and gating kinetics change in a stoichiometry‐dependent manner. Calcium and magnesium reduce the conductance of both stoichiometric forms, with each cation producing an equivalent reduction, but the reduction is greater for the (α4)2(β2)3 form. Moreover, divalent cations promote efficient channel opening of the (α4)3(β2)2 stoichiometry, while minimally affecting the (α4)2(β2)3 stoichiometry. For the (α4)3(β2)2 stoichiometry, at high but not low ACh concentrations, calcium in synergy with magnesium promote clustering of channel openings into episodes of many openings in quick succession. Conclusion and Implications: Modulation of the α4β2 nAChR by divalent cations depends on the ACh concentration, the type of cation and the subunit stoichiometry. The functional consequences of modulation are expected to depend on the regional distributions of the stoichiometric forms and synaptic versus extrasynaptic locations of the receptors. [ABSTRACT FROM AUTHOR]

Electrogenic ion transport across the epithelium lining the descending colon of male Sprague-Dawley rats has been measured under short-circuit conditions. Responses to kallidin (lysylbradykinin) were inhibited by 70% if calcium was removed from the solution bathing the basolateral aspect of the tissue. Under identical conditions responses to prostaglandin E1 and dibutyryl cyclic adenosine monophosphate were not changed. Forskolin, which directly activates the catalytic subunit of adenylate cyclase, was inhibited by 35% by calcium removal, whereas responses to the phosphodiesterase inhibitor isobutylmethylxanthine were inhibited by 45% by the same procedure. In the absence of calcium, strontium could substitute in promoting the chloride secretory events triggered by kallidin. Magnesium ions antagonized the effects of the kinin in the presence of calcium ions in the bathing solution. The effects of kallidin were partially antagonized by verapamil and trifluoperazine and were potentiated by isobutylmethylxanthine. These results, together with earlier evidence, suggest that kinin elicits a chloride secretory response in this epithelium by stimulating the formation of prostaglandins which then activate adenylate cyclase. Extracellular calcium ions appear to have an important role in the proximal part of this cascade for prostaglandin generation. However, biochemical correlates of these biophysical responses presented in the following paper indicate a more complex role for calcium in the genesis of the kinin response.

1 A number of studies in isolated tissues have shown that mu-opioid-receptor-mediated agonism can be augmented or potentiated with reduction in extracellular calcium concentration [( Ca2+]o). These effects have been ascribed to alterations in post-receptor coupling but the nature of the changes involved have not been quantitatively elucidated. 2 In this paper, logistic curve-fitting and operational model-fitting (Black & Leff, 1983) were used to analyse the effects of variations in [Ca2+]o on the mu-receptor-mediated effects of [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGOL) in the isolated, coaxially-stimulated ileum of the guinea-pig. At each value of [Ca2+]o, the effects of irreversible receptor alkylation by beta-chlornaltrexamine (beta-CNA) were also investigated. 3 From these analyses it is concluded that with reduction in [Ca2+]o the efficacy of DAGOL in this system is increased and the sensitivity of the transducer relation is also enhanced, the latter indicating a trend towards positive co-operativity. Reduction of [Ca2+]o also appeared to produce a reduction in agonist affinity. 4 Experimental manipulation of [Ca2+]o may provide a useful means of enhancing mu-agonist efficacy, allowing detection of agonism in compounds with low intrinsic efficacies. However, the accompanying change in co-operativity of the transducer relation must be considered when quantifying agonism under these conditions.

1 The compound Org 6001 (3alpha-amino-2beta-hydroxy-5alpha-androstan-17-one hydrochloride) was found in recent experiments to exhibit anti-arrhythmic activity. Evidence is presented in this paper concerning its mode of action. 2 Org 6001 was 1.8 times more potent than procaine as a local anaesthetic on desheathed frog nerve. 3 Org 6001 had no effect on the resting potential of isolated cardiac muscle of rabbit, but greatly reduced the maximum rate of depolarization tion (MRD). The action potential duration TAPD) WAS MARGINALLY PROLONGED IN ATRIAL AND VENTRICULAR MUSCLE. 4 Org 6001 preferentially shortened APD in that part of the Purkinje system in which APD is normally longer than elsewhere, so that APD

Background and Purpose: As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca(v)1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca(v)1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca(v)1.2 channels, predominant in cardiac and vascular smooth muscles.Experimental Approach: Cardiac and smooth muscle isoforms of Ca(v)1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.Key Results: Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca(v)1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca(v)1.2SM and Ca(v)1.2b. Ca(v)1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca(v)1.2CM channels. When we modified alternatively spliced exons of cardiac Ca(v)1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.Conclusions and Implications: Alternative splicing of Ca(v)1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels. [ABSTRACT FROM AUTHOR]

Background and Purpose: The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.Experimental Approach: Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine).Key Results: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release.Conclusions and Implications: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC. [ABSTRACT FROM AUTHOR]

Background and Purpose: SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents.Experimental Approach: Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed.Key Results: SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca(V)1, Ca(V)2 and Ca(V)3) at concentrations typically utilized to assay TRPC function (10 microM). Particularly, human Ca(V)3.1 T-type Ca channels were more potently inhibited by SKF (IC(50) approximately 560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca(V)3.1 T-type currents in a rat cerebellar PC slice preparation.Conclusions and Implications: SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues. [ABSTRACT FROM AUTHOR]

Background and Purpose: Levosimendan is a novel, short half-life calcium sensitizer used as pharmacological inotropic support in acute decompensated heart failure. After oral administration, levosimendan is metabolized to OR-1855, which, in rats, is further metabolized into OR-1896. OR-1896 is a long-lasting metabolite of levosimendan sharing the pharmacological properties of the parent compound.Experimental Approach: Effects of oral OR-1896 treatment on post-infarct heart failure and cardiac remodelling were assessed in diabetic Goto-Kakizaki (GK) rats, an animal model of type II diabetes. Myocardial infarction (MI) was produced to GK rats by coronary ligation. Twenty-four hours after MI or sham operation, the rats were randomized into four groups: (i) MI; (ii) MI + OR-1896 treatment; (iii) sham; and (iv) sham + OR-1896. Cardiac function and markers of cardiac remodelling were assessed 1, 4 and 12 weeks after MI.Key Results: OR-1896 increased ejection fraction and fractional shortening in GK rats with MI. OR-1896 ameliorated post-infarct cardiac hypertrophy, and prevented the MI-induced increase in cardiac mRNA for atrial natriuretic peptide, monocyte chemoattractant protein-1 and connective tissue growth factor, markers of pressure/volume overload, inflammation and fibrosis respectively. OR-1896 also suppressed mRNA for senescence-associated p16(INK4A) and p19(ARF). The beneficial effects of OR-1896 were more prominent at week 12 than at week 4. OR-1896 did not influence systolic blood pressure, blood glucose level, myocardial infarct size or cardiovascular mortality.Conclusions and Implications: Oral treatment with calcium sensitizer OR-1896 protects against post-infarct heart failure and cardiac remodelling in experimental model of type II diabetes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity.Experimental Approach: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries.Key Results: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers.Conclusions and Implications: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction. [ABSTRACT FROM AUTHOR]

Background and Purpose: K(+) channels play a role in the proliferation of cancer cells. We have investigated the effects of specific K(+) channel inhibitors on basal and oestrogen-stimulated proliferation of breast cancer cells.Experimental Approach: Using the mammary adenocarcinoma cell line MCF-7 we assayed cell proliferation by radiolabelled thymidine incorporation in the absence or presence of various K(+) channel inhibitors with or without 17beta-oestradiol.Key Results: Inhibitors of K(v)10.1 and K(Ca)3.1 K(+) channels suppressed basal proliferation of MCF-7 cells, but not oestrogen-stimulated proliferation. TRAM-34, a specific inhibitor of K(Ca)3.1 channels increased or decreased cell proliferation depending on the concentration. At intermediate concentrations (3-10 microM) TRAM-34 increased cell proliferation, whereas at higher concentrations (20-100 microM) TRAM-34 decreased cell proliferation. The enhancement of cell proliferation caused by TRAM-34 was blocked by the oestrogen receptor antagonists ICI182,780 and tamoxifen. TRAM-34 also increased progesterone receptor mRNA expression, decreased oestrogen receptor-alpha mRNA expression and reduced the binding of radiolabelled oestrogen to MCF-7 oestrogen receptor, in each case mimicking the effects of 17beta-oestradiol.Conclusions and Implications: Our results demonstrate that K(+) channels K(v)10.1 and K(Ca)3.1 play a role in basal, but not oestrogen-stimulated MCF-7 cell proliferation. TRAM-34, as well as inhibiting K(Ca)3.1, directly interacts with the oestrogen receptor and mimics the effects of 17beta-oestradiol on MCF-7 cell proliferation and gene modulation. Our finding that TRAM-34 is able to activate the oestrogen receptor suggests a novel action of this supposedly specific K(+) channel inhibitor and raises concerns of interpretation in its use. [ABSTRACT FROM AUTHOR]

Background and Purpose: Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca2+ channel currents (I(Ca)). However, little is known about which receptor subtype(s) mediate this effect.Experimental Approach: The effect of carbachol on I(Ca) was studied in single intestinal myocytes from M2 or M3 muscarinic receptor knockout (KO) and wild-type (WT) mice.Key Results: In M2KO cells, carbachol (100 microM) induced a sustained I(Ca) suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I(Ca) in M3KO cells, but with a phasic time course. In M2/M3-double KO cells, carbachol had no effect on I(Ca). The extent of the suppression in WT cells was greater than the sum of the I(Ca) suppressions in M2KO and M3KO cells, indicating that it is not a simple mixture of M2 and M3 receptor responses. The G(i/o) inhibitor, Pertussis toxin, abolished the I(Ca) suppression in M3KO cells, but not in M2KO cells. In contrast, the G(q/11) inhibitor YM-254890 strongly inhibited only the I(Ca) suppression in M2KO cells. Suppression of I(Ca) in WT cells was markedly reduced by either Pertussis toxin or YM-254890.Conclusion and Implications: In intestinal myocytes, M2 receptors mediate a phasic I(Ca) suppression via G(i/o) proteins, while M3 receptors mediate a sustained I(Ca) suppression via G(q/11) proteins. In addition, another pathway that requires both M2/G(i/o) and M3/G(q/11) systems may be operative in inducing a sustained I(Ca) suppression. [ABSTRACT FROM AUTHOR]

Background and Purpose: The endocannabinoid, anandamide, has anti-arrhythmic effects. The aim of the present study was to explore the electrophysiological effects of anandamide on rat myocardium.Experimental Approach: Evoked action potentials (APs) were recorded using intracellular recording technique in rat cardiac papillary muscles. In addition, L-type Ca2+ current was measured and analysed using whole-cell patch-clamp recording technique in isolated rat cardiac ventricular myocytes.Key Results: In cardiac papillary muscles, anandamide (1, 10, 100 nM) decreased AP duration in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased AP amplitude, overshoot and Vmax in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB1 receptors, but not AM630 (100 nM), a CB2 receptor antagonist. Furthermore, an agonist of L-type Ca2+ channels, Bay K 8644 (0.5 microM), a K+ channel blocker tetraethylammonium chloride (20 mM) and the nitric oxide synthase inhibitor L-NAME (1 mM) had no effect on anandamide-induced decrease in AP duration. In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca2+ current concentration-dependently, and shifted the current-voltage relationship curve of the Ca2+ current. Anandamide (100 nM) shifted the steady-state inactivation curve to the left and the recovery curve to the right. Blockade of CB1 receptors with AM251 (100 nM), but not CB2 receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca2+ currents.Conclusions and Implications: These data suggest that anandamide suppressed AP and L-type Ca2+ current in cardiac myocytes through CB1 receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: The effects of hydrogen peroxide (H(2)O(2)) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca(2+))-induced] and the effect of such treatment on anti-oxidative enzyme activities.Experimental Approach: Uteri were isolated from virgin Wistar rats and suspended in an organ bath. Uteri were allowed to contract spontaneously or in the presence of Ca(2+) (6 mM) and treated with H(2)O(2) (2 microM-3 mM) over 2 h. Anti-oxidative enzyme activities (manganese superoxide dismutase-MnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT, glutathione peroxidase-GSHPx and glutathione reductase-GR) in H(2)O(2)-treated uteri were compared with those in uteri immediately frozen after isolation or undergoing spontaneous or Ca(2+)-induced contractions, without treatment with H(2)O(2). The effect of inhibitors (propranolol, methylene blue, L-NAME, tetraethylamonium, glibenclamide and 4-aminopyridine) on H(2)O(2)-mediated relaxation was explored.Key Results: H(2)O(2) caused concentration-dependent relaxation of both spontaneous and Ca(2+)-induced uterine contractions. After H(2)O(2) treatment, GSHPx and MnSOD activities were increased, while CuZnSOD and GR (In Ca(2+)-induced rat uteri) were decreased. N(omega)-nitro-L-arginine methyl ester antagonized the effect of H(2)O(2) on Ca(2+)-induced contractions. H(2)O(2)-induced relaxation was not affected by propranolol, potentiated by methylene blue and antagonized by tetraethylamonium, 4-aminopyridine and glibenclamide, with the last compound being the least effective.Conclusions and Implications: H(2)O(2) induced dose-dependent relaxation of isolated rat uteri mainly via changes in voltage-dependent potassium channels. Decreasing generation of reactive oxygen species by stimulation of anti-oxidative pathways may lead to new approaches to the management of dysfunctional uteri. [ABSTRACT FROM AUTHOR]

Background and Purpose: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved.Experimental Approach: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied.Key Results: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied.Conclusions and Implications: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-activated Cl(-) channel (Cl(Ca)) blocker niflumic acid (NFA) produces a paradoxical dual effect on I(Cl(Ca)), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I(Cl(Ca)) were modulated by phosphorylation.Experimental Approach: I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 microM).Key Results: Dephosphorylation enhanced the ability of 100 microM NFA to inhibit I(Cl(Ca)). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, approximately 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I(Cl(Ca)) in the range of 0.1-250 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP.Conclusions and Implications: Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl(Ca) channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site. [ABSTRACT FROM AUTHOR]

Background and Purpose: The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP(3)R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR).Experimental Approach: Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca(2+)](cyto) measured using fluo-3. IP(3)Rs were activated by photolysis of caged IP(3) and RyRs activated by hydrostatic application of caffeine.Key Results: FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca(2+)](cyto) rise evoked by activation of either RyR or IP(3)R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP(3)-evoked [Ca(2+)](cyto) rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP(3)-evoked [Ca(2+)](cyto) release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP(3)R-mediated Ca(2+) release. The mTOR inhibitor LY294002, like rapamycin, decreased IP(3)-evoked Ca(2+) release.Conclusions and Implications: Ca(2+) release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP(3)R-mediated Ca(2+) release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP(3)-mediated Ca(2+) release may be explained by mTOR inhibition. [ABSTRACT FROM AUTHOR]

Background and Purpose: Coordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β-glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers.Experimental Approach: We investigated the effects of CBX and 18βGA on intercellular Ca2+ waves, evoked by inositol 1,4,5-trisphosphate (IP3 ) in the endothelium of intact mesenteric resistance arteries.Key Results: Acetycholine-evoked IP3 -mediated Ca2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca2+ release. Localised photolysis of caged IP3 (cIP3 ) was used to provide precise spatiotemporal control of site of cell activation. Local cIP3 photolysis generated reproducible Ca2+ increases and Ca2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca2+ waves in a time-dependent manner by inhibiting the initiating IP3 -evoked Ca2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K+ -channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential.Conclusion and Implications: CBX and 18βGA inhibit IP3 -mediated Ca2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell-cell communication by acting at sites that are unrelated to gap junctions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca2+ as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere-length dependence of Ca2+ -sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism.Experimental Approach: Multicellular preparations from the left ventricular-free wall of hearts from organ donors were chemically permeabilized and Ca2+ activated in the presence or absence of 0.5-μM mavacamten at 1.9 or 2.3-μm sarcomere length (37°C). Isometric force and frequency-dependent viscoelastic myocardial stiffness measurements were made.Key Results: At both sarcomere lengths, mavacamten reduced maximal force and Ca2+ sensitivity of contraction. In the presence and absence of mavacamten, Ca2+ sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca2+ sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5).Conclusion and Implications: Mavacamten did not eliminate sarcomere length-dependent increases in the Ca2+ sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies. [ABSTRACT FROM AUTHOR]

T-type Ca2+ channels represent a specific channel family overexpressed in different types of tumours. Their involvement in controlling the proliferation, angiogenesis and invasion of tumour cells, has been partially clarified. The article by Zhang et al. in this issue of BJP provides the first evidence of anti-tumoural effects of endostatin (ES) in U87 glioma cells. He demonstrated that ES or mibefradil (a L/T-type calcium channel blocker), reduces the proliferation and migration of U87 glioma cells in a T-type Ca2+ channel-dependent manner. However, the difference in the blocking effect of mibefradil on T-type calcium channel expression as compared with its ability to inhibit proliferation and migration, supports the idea of a broader T/L-type-independent effect of the mibefradil blocker. Overall, these findings provide new insights for the future development of a novel class of anti-T-type calcium channel blockers in the therapy of glioblastoma. LINKED ARTICLE This article is a commentary on Zhang et al., pp. 1247-1260 of this issue. To view this paper visit [ABSTRACT FROM AUTHOR]

Background and Purpose: FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 receptors (IP3 R) and ryanodine receptors (RyR) to alter Ca2+ signalling in endothelial cells.Experimental Approach: We investigated the effects of FK506 and rapamycin on Ca2+ release via IP3 R and RyR in hundreds of endothelial cells, using the indicator Cal-520, in intact mesenteric arteries from male Sprague-Dawley rats. IP3 Rs were activated by acetylcholine or localised photo-uncaging of IP3 , and RyR by caffeine.Key Results: While FKBPs were present, FKBP modulation with rapamycin did not alter IP3 -evoked Ca2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP3 -evoked Ca2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP3 -evoked Ca2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP3 -evoked Ca2+ release. These findings suggest that IP3 -evoked Ca2+ release is not modulated by FKBP, but by FK506-mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca2+ signalling suggesting that RyR is not functional in native endothelium.Conclusion and Implications: The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca2+ release and altered FKBP binding to IP3 and RyR. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hypertension has been the leading preventable cause of premature death worldwide. The aim of this study was to design a more efficient vaccine against novel targets for the treatment of hypertension.Experimental Approach: The epitope CE12, derived from the human L-type calcium channel (CaV 1.2), was designed and conjugated with Qβ bacteriophage virus-like particles to test the efficacy in hypertensive animals. Further, the hepatitis B core antigen (HBcAg)-CE12-CQ10 vaccine, a bivalent vaccine based on HBcAg virus-like particles and targeting both human angiotensin AT1 receptors and CaV 1.2 channels, was developed and evaluated in hypertensive rodents.Key Results: The Qβ-CE12 vaccine effectively decreased the BP in hypertensive rodents. A monoclonal antibody against CE12 specifically bound to L-type calcium channels and inhibited channel activity. Injection with monoclonal antibody against CE12 effectively reduced the BP in angiotensin II-induced hypertensive mice. The HBcAg-CE12-CQ10 vaccine showed antihypertensive effects in hypertensive mice and relatively superior antihypertensive effects in spontaneously hypertensive rats and ameliorated L-NAME-induced renal injury. In addition, no obvious immune-mediated damage or electrophysiological adverse effects were detected.Conclusion and Implications: Immunotherapy against both AT1 receptors and CaV 1.2 channels decreased the BP in hypertensive rodents effectively and provided protection against hypertensive target organ damage without obvious feedback activation of renin-angiotensin system or induction of dominant antibodies against the carrier protein. Thus, the HBcAg-CE12-CQ10 vaccine may provide a novel and promising therapeutic approach for hypertension. [ABSTRACT FROM AUTHOR]

A conference paper about the effects of the Rho kinase (ROK) inhibitor Y-27632 on contractile responses to carbachol (CCh), thapsigargin (Tg) and high potassium Krebs is presented. It reveals that in intact tissues, CCh and Tg produce sustained contractions of similar magnitude. It concludes that results support a role for ROK in calcium sensitization in the mouse anococcygeus.

A conference paper about the responses to acetylcholine and oxotremorine sesquifumarate (Oxo-S) in isolated pulmonary artery smooth muscle cells is presented. It discusses the monitoring of changes in the fluorescence of the calcium indicator, fluo-4. It indicates that Oxo-S evoked calcium signals depend on G1 and calcium influx.

A conference paper that reports on experiments designed to characterize the calcium entry pathways in smooth muscle cells of guinea-pig aorta is presented. It discusses how these pathways contribute to sulprostone action, in which sulprostone potently contracts human pulmonary artery and guinea-pig aorta preparations. It concludes that the variable nifedipine sensitivity of contraction may be related to variation in the relative contributions of different calcium influx pathways.

Background and Purpose: Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins.Experimental Approach: We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis.Key Results: RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm.Conclusion and Implications: We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species. [ABSTRACT FROM AUTHOR]

Background and Purpose: Capsaicin-mediated modulation of taste nerve responses is thought to be produced indirectly by the actions of neuropeptides, for example, CGRP and substance P (SP), on taste cells implying they play a role in taste sensitivity. During the processing of gustatory information in taste buds, CGRP shapes peripheral taste signals via serotonergic signalling. The underlying assumption has been that SP exerts its effects on taste transmitter secretion in taste buds of mice.Experimental Approach: To test this assumption, we investigated the net effect of SP on taste-evoked ATP secretion from mouse taste buds, using functional calcium imaging with CHO cells expressing high-affinity transmitter receptors as cellular biosensors.Key Results: Our results showed that SP elicited PLC activation-dependent intracellular Ca2+ transients in taste cells via neurokinin 1 receptors, most likely on glutamate-aspartate transporter-expressing Type I cells. Furthermore, SP caused Type I cells to secrete GABA.Conclusion and Implications: Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the current results indicate that SP elicited secretion of GABA, which provided negative feedback onto Type II (receptor) cells to reduce taste-evoked ATP secretion. These findings are consistent with a role for SP as an inhibitory transmitter that shapes the peripheral taste signals, via GABAergic signalling, during the processing of gustatory information in taste buds. Notably, the results suggest that SP is intimately associated with GABA in mammalian taste signal processing and demonstrate an unanticipated route for sensory information flow within the taste bud. [ABSTRACT FROM AUTHOR]

Background and Purpose: Kv 7.4 and Kv 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV ) channels, with little inhibitory effect on Kv 7 family members at mmol concentrations. However, the effect of 4-AP on Kv 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv 7.4 and Kv 7.5 channels and characterize the effect of 4-AP on rat resistance arteries.Experimental Approach: Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing Kv 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments.Key Results: 4-AP increased the Kv 7.4-mediated current in oocytes and HEK cells but did not affect Kv 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K+ current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca2+ ]i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the Kv 7 channel blocker XE991 and BKCa channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation.Conclusions and Implications: These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of Kv 7.4 and BKCa channels and attenuating NA-mediated signalling. [ABSTRACT FROM AUTHOR]

Background and Purpose: Human parathyroid hormone (PTH) is critical for maintaining physiological calcium homeostasis and plays an important role in the formation and maintenance of the bone. Full-length PTH and a truncated peptide form are approved for treatment of hypoparathyroidism and osteoporosis respectively. Our initial goal was to develop an improved PTH therapy for osteoporosis, but clinical development was halted. The novel compound was then repurposed as an improved therapy for hypoparathyroidism.Experimental Approach: A longer-acting form of PTH was synthesised by altering the peptide to increase cell surface residence time of the bound ligand to its receptor. In vitro screening identified a compound, which was tested in an animal model of osteoporosis before entering human trials. This compound was subsequently tested in two independent animal models of hypoparathyroidism.Key Results: The peptide identified, LY627-2K, exhibited delayed internalization kinetics. In an ovariectomy-induced bone loss rat model, LY627-2K demonstrated improved vertebral bone mineral density and biomechanical properties at skeletal sites and a modest increase in serum calcium. In a Phase I clinical study, dose-dependent increases in serum calcium were reproduced. These observations prompted us to explore a second indication, hypoparathyroidism. In animal models of this disease, LY627-2K restored serum calcium, comparing favourably to treatment with wild-type PTH.Conclusions and Implications: We summarize the repositioning of a therapeutic candidate with substantial preclinical and clinical data. Our results support its repurposing and continued development, from a common indication (osteoporosis) to a rare disease (hypoparathyroidism) by exploiting a shared molecular target.Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc. [ABSTRACT FROM AUTHOR]