Distinct effects of CGRP on typical and atypical smooth muscle cells involved in generating spontaneous contractions in the mouse renal pelvis

Background and purpose:

We investigated the cellular mechanisms underlying spontaneous contractions in the mouse renal pelvis, regulated by calcitonin gene-related peptide (CGRP).

Experimental approach:

Spontaneous contractions, action potentials and Ca²⁺ transients in typical and atypical smooth muscle cells (TSMCs and ATSMCs) within the renal pelvis wall were recorded separately using tension and intracellular microelectrode recording techniques and Fluo-4 Ca²⁺ imaging. Immunohistochemical and electron microscopic studies were also carried out.

Key results:

Bundles of CGRP containing transient receptor potential cation channel, subfamily V, member 1-positive sensory nerves were situated near both TSMCs and ATSMCs. Nerve stimulation reduced the frequency but augmented the amplitude and duration of spontaneous phasic contractions, action potentials and Ca²⁺ transients in TSMCs. CGRP and agents increasing internal cyclic adenosine monophosphate (cAMP) mimicked the nerve-mediated modulation of TSMC activity and suppressed ATSMCs Ca²⁺ transients. Membrane hyperpolarization induced by CGRP or cAMP stimulators was blocked by glibenclamide, while their negative chronotropic effects were less affected. Glibenclamide enhanced TSMC Ca²⁺ transients but inhibited ATSMC Ca²⁺ transients, while both 5-hydroxydecanoate and diazoxide, a blocker and opener of mitochondrial ATP-sensitive K⁺ channels, respectively, reduced the Ca²⁺ transient frequency in both TSMCs and ATSMCs. Inhibition of mitochondrial function blocked ATSMCs Ca²⁺ transients and inhibited spontaneous excitation of TSMCs.

Conclusions and implications:

The negative chronotropic effects of CGRP result primarily from suppression of ATSMC Ca²⁺ transients rather than opening of plasmalemmal ATP-sensitive K⁺ channels in TSMCs. The positive inotropic effects of CGRP may derive from activation of TSMC L-type Ca²⁺ channels. Mitochondrial Ca²⁺ handling in ATSMCs also plays a critical role in generating Ca²⁺ transients.     

Towards selective antagonists of T-type calcium channels: design, characterization and potential applications of NNC 55-0396

NNC 55-0396 is a structural analog of mibefradil (Ro 40-5967) that inhibits both T-type and high-voltage-activated (HVA) Ca2+ channels with a higher selectivity for T-type Ca2+ channels. The inhibitory effect of mibefradil on HVA Ca2+ channels can be attributed to a hydrolyzed metabolite of the drug: the methoxy acetate side chain of mibefradil is removed by intracellular enzymes, thus it forms (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl hydroxy dihydrochloride (dm-mibefradil), which causes potent inhibition of HVA Ca2+ currents. By replacing the methoxy acetate chain of mibefradil with cyclopropanecarboxylate, a more stable analog was developed (NNC 55-0396). The acute IC50 of NNC 55-0396 to block recombinant Cav3.1 T-type channels expressed in HEK293 cells is approximately 7 muM, whereas 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents in INS-1 cells. Block of T-type Ca2+ current was partially reduced by membrane hyperpolarization and was enhanced at high stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channel channels, thus rendering it more selective to T-type Ca2+ channels.     

Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens

Background and purpose:

In rat vas deferens, nerve mediated-contractions to a single electrical stimulus consist of an early purinergic and a later adrenergic component with differing sensitivities to L-type calcium channel blockers. We have investigated the effects of the T-type calcium channel blockers mibefradil and (1S, 2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylic dihydrochloride (NNC 55-0396) against contractions in rat vas deferens. In addition, the actions of thalidomide were examined.

Experimental approach:

Prostatic and epididymal portions of rat vas deferens were stimulated with a single electrical stimulus every 5 min, and mouse whole vas deferens was stimulated with 40 pulses at 10 Hz every 5 min.

Key results:

Both mibefradil and NNC 55-0396 (100 µM) produced inhibition of contractions of epididymal portions (42 ± 13%, n= 7, and 43 ± 4%, n= 15, of control respectively). However, both agents produced small inhibitions of responses in prostatic portions, presumably by L-type calcium channel block. Thalidomide (100 µM) inhibited contractions in epididymal (55 ± 4% of control, n= 17) but not in prostatic portions of rat vas deferens. Thalidomide (10–100 µM) also inhibited contractions in mouse vas deferens.

Conclusions and implications:

The T-type calcium channel blockers mibefradil and NNC 55-0396 block particularly the adrenoceptor-mediated, nifedipine-resistant response to nerve stimulation in rat vas deferens, and this may suggest that this component involves T-type calcium channels. In addition, thalidomide has actions that resemble those of the T-type calcium channel blockers, in that it blocks nifedipine-resistant contractions in epididymal portions.     

Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves

BACKGROUND AND PURPOSE

The acute effects of PGE₂ on bladder smooth muscle and nerves were examined to determine the origin of PGE₂-induced spontaneous rhythmic contractions.

EXPERIMENTAL APPROACH

Contraction studies, confocal Ca²⁺ imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE₂ on bladder smooth muscle and efferent nerves.

KEY RESULTS

PGE₂ (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca²⁺ imaging showed that PGE₂ increased the frequency of whole-cell Ca²⁺ transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s⁻¹ to 0.90·s⁻¹. Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca²⁺ channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE₂-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE₂, implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged.

CONCLUSIONS AND IMPLICATIONS

It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE₂ to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves.     

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

Background and Purpose

Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca²⁺-activated K⁺ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function.

Experimental Approach

We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function.

Key Results

We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions.

Conclusions and Implications

Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity.     

The effect of Mn2+, Zn2+, Ba2+ and Ca2+ on spontaneous motility in sheep duodenum in vitro

• 1.1. The effects of several ions, Mn²⁺, Zn²⁺, Ba²⁺ and Ca²⁺, on spontaneous motility were investigated in longitudinal smooth muscle strips from sheep duodenum, in vitro
• 2.2. Mn²⁺ (0.5–1.5 mM) and Zn²⁺ (0.5–5 mM) inhibited both the amplitude and frequency of motility in Krebs solution and in Ca²⁺-free medium.
• 3.3. Ba²⁺(0.5–5 mM) evoked three types of contractile responses: (i) an increase in the frequency and a reduction of the amplitude of spontaneous contractions; (ii) a slight increase in muscle tone of the phasic contractions; and (iii) a rapid initial phasic contraction followed by slowly fading contraction. Ca²⁺ induced two kinds of responses in spontaneous motility: (i) a fast phasic contraction, followed by an increase in the amplitude and frequency of phasic contractions with no changes in its tone; and (ii) an increase in the amplitude of contractions.
• 4.4. The Ba²⁺-induced contractions were inhibited by EDTA, verapamil and diltiazem, but were not modified by sodium nitroprusside. The Ca²⁺-induced contractions were reduced by verapamil and diltiazem.
• 5.5. Our results show that Mn²⁺ and Zn²⁺ behave as inhibitors of sheep duodenum motility. In contrast, Ba²⁺ and Ca²⁺ stimulate motility. It is suggested that Ba²⁺ can penetrate the cells through voltage-dependent Ca²⁺ channels and behave as a partial substitute for Ca²⁺.

     

Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

BACKGROUND AND PURPOSE

Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca_v3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H₂S, we asked whether TRPA1 channels, in addition to Ca_v3.2 channels, contribute to the H₂S-induced mechanical hyperalgesia and allodynia in mice.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca_v3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice.

KEY RESULTS

Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55–0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca_v3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca_v3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons.

CONCLUSIONS AND IMPLICATIONS

Mechanical hyperalgesia and allodynia induced by NaHS/H₂S required activation of both Ca_v3.2 and TRPA1 channels in mice.     

Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis

BACKGROUND AND PURPOSE

The modulation of the spontaneous electrical and Ca²⁺ signals underlying pyeloureteric peristalsis upon nicotinic receptor activation located on primary sensory afferents (PSAs) was investigated in the mouse renal pelvis.

EXPERIMENTAL APPROACH

Contractile activity was followed using video microscopy, electrical and Ca²⁺ signals in typical and atypical smooth muscle cells (TSMCs and ASMCs) within the renal pelvis were recorded separately using intracellular microelectrodes and Fluo-4 Ca²⁺ imaging.

KEY RESULTS

Nicotine and carbachol (CCh; 1–100 μM) transiently reduced the frequency and increased the amplitude of spontaneous phasic contractions in a manner unaffected by muscarininc antagonists, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) and pirenzipine (10 nM) or L-NAME (L-Nω-nitroarginine methyl ester; 200 μM), inhibitor of NO synthesis, but blocked by the nicotinic antagonist, hexamethonium or capsaicin, depletor of PSA neuropeptides. These negative chronotropic and delayed positive inotropic effects of CCh on TSMC contractions, action potentials and Ca²⁺ transients were inhibited by glibenclamide (Glib; 1 μM), blocker of ATP-dependent K (KATP) channels. Nicotinic receptor-evoked inhibition of the spontaneous Ca²⁺ transients in ASMCs was prevented by capsaicin but not Glib. In contrast, the negative inotropic and chronotropic effects of the non-selective COX inhibitor indomethacin were not prevented by Glib.

CONCLUSIONS AND IMPLICATIONS

The negative chronotropic effect of nicotinic receptor activation results from the release of calcitonin gene-related peptide (CGRP) from PSAs, which suppresses Ca²⁺ signalling in ASMCs. PSA-released CGRP also evokes a transient hyperpolarization in TSMCs upon the opening of KATP channels, which reduces contraction propagation but promotes the recruitment of TSMC Ca²⁺ channels that underlie the delayed positive inotropic effects of CCh.     

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder

Background and Purpose

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca²⁺ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Experimental Approach

The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca²⁺ dynamics were visualized using Fluo-4 fluorescence Ca²⁺ imaging.

Key Results

A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca²⁺ transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca²⁺ transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca²⁺ transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca²⁺ transients rely on Ca²⁺ release from the endoplasmic reticulum via the InsP₃ receptor and also require Ca²⁺ influx through store-operated Ca²⁺ channels.

Conclusions and Implications

The pericytes in mouse bladder can generate spontaneous Ca²⁺ transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.     

Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABA_A) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30 mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30 min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (K_i: 0.95 and 0.29 μM respectively), and also less potent at inhibiting testosterone 6-β-hydroxylation (K_i: 0.71 and 0.12 μM respectively). In the GABA_A α1-null model, NNC 55-0396 but not mibefradil, (each at 20 mg/kg), suppressed tremor while NNC 55-0396 at 12.5 mg/kg suppressed harmaline-induced tremor by half by 20–100 min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.     

Effect of calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens

1 The present study was carried out to look at the effect of different calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens considering that the response consisted of three components (I) the phasic response (II) the tonic response and (III) the spikes (rhythmic contractions). 2 Nifedipine (3 × 10^??9–1 × 10^??7 m ) inhibited all the components at the same concentration range, verapamil (1 × 10^??7–1 × 10^??5 m ) inhibited the phasic and tonic response but not the rhythmic activity. This latter component, at a certain concentration range and especially in the prostatic portion was markedly potentiated. Diltiazem and flunarizine lay in an intermediate position. 3 Papaverine, a Ca²⁺ antagonist that acts mainly intracellularly, inhibited preferentially the tonic component; ryanodine was practically inactive. 4 Cromakalim inhibited only partially the phasic and tonic components but totally inhibited the rhythmic contractions. 5 These results can be explained by postulating two types of calcium channels opened by α-adrenoceptor stimulation. The first one is verapamil- and nifedipine-sensitive and allows the entry of Ca²⁺ directly available for the contraction and responsible for the phasic and partially responsible for the tonic component. The second channel is merely nifedipine-sensitive and allows the entry of Ca²⁺ trigger which can release Ca²⁺ from intracellular sites: the mobilized Ca²⁺ is able to sustain the tonic component and is the main one responsible for the rhythmic activity. There is the possibility that this second channel is associated with ATP-sensitive K⁺ channels.     

A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

BACKGROUND AND PURPOSE

ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺-dependent chloride ion current (I_Cl,Ca), Ca_v1.2 ion channels and Ca²⁺ influx.

EXPERIMENTAL APPROACH

Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph.

KEY RESULTS

The I_Cl,Ca blockers, niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and the Ca_v1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45–50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50–70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

CONCLUSIONS AND IMPLICATIONS

I_Cl,Ca and Ca²⁺ influx via Ca_v1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.     

Large conductance Ca-activated K channel activation with NS1619 decreases myogenic and neurogenic contractions of rat detrusor smooth muscle

Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips pre-contracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20 mM). In the presence of elevated KCl (60 mM KCl), the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5–50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

Alzheimer's disease therapeutic candidate SAK3 is an enhancer of T-type calcium channels

Low-threshold Ca²⁺ spikes are mediated by T-type Ca²⁺ channels, which have electrophysiological properties of fast inactivation and slow deactivation kinetics. A low membrane potential of approximately ?60 mV is sufficient to trigger channel opening. We recently introduced a novel T-type Ca²⁺ channel enhancer that improves cognition and inhibits amyloid beta aggregation in an Alzheimer's disease (AD) mouse model. The enhancer stimulates ACh release, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activity, and neurogenesis in the hippocampus. Then, we discuss how T-type Ca²⁺ channel enhancer improves cognition and impaired neurogenesis and how CaMKII signaling in neurodegenerative diseases reduces amyloid beta aggregation. We provide a perspective of the potential AD therapies to target CaMKII signaling. In this context, we overview our attempts leading to the development of a T-type Ca²⁺ channel enhancer as cognitive enhancer, the action of which has been associated with CaMKII and presumably proteasome activity.     

The Sources of Ca2+ for Muscarinic Receptor-induced Contraction in the Rat Ileum

The contractile responses obtained by activation of different muscarinic receptor subtypes in the longitudinal muscle of the rat ileum and especially the responses of this muscle to acetylcholine in a Ca²⁺-free medium have been investigated. In Ca²⁺-containing solution, acetylcholine elicited similar concentration-dependent contractile responses in the duodenum, jejunum and ileum strips of the rat intestine. The response to a maximal concentration of the agonist (1 μM) consisted of a rapid phasic response followed by a slower tonic one. Nifedipine completely relaxes or inhibits the sustained response and only partially diminishes the phasic one, which suggests that the phasic contraction depends on the release of internal Ca²⁺ as well as Ca²⁺ entry from the extracellular space through voltage-dependent Ca²⁺ channels, but that the tonic contraction only depends on the influx of the external ion. In Ca²⁺-free medium, acetylcholine (1 μM) induced phasic contractions that depend on the release of this ion from internal stores. Participation of different subtypes of receptors (M₁, M₂ and M₃) in these responses depends on the inhibitory action shown by methoctramine, 4?DAMP and atropine but not by pirenzepine in two different experimental models.     

Myoplasmic [Ca2+], Crossbridge Phosphorylation and Latch in Rabbit Bladder Smooth Muscle

Tonic smooth muscle exhibit the latch phenomenon: high force at low myosin regulatory light chains (MRLC) phosphorylation, shortening velocity (Vo), and energy consumption. However, the kinetics of MRLC phosphorylation and cellular activation in phasic smooth muscle are unknown. The present study was to determine whether Ca²⁺-stimulated MRLC phosphorylation could suffice to explain the agonist- or high K⁺-induced contraction in a fast, phasic smooth muscle. We measured myoplasmic [Ca²⁺], MRLC phosphorylation, half-time after step-shortening (a measure of Vo) and contractile stress in rabbit urinary bladder strips. High K⁺-induced contractions were phasic at both 22℃ and 37℃: myoplasmic [Ca²⁺], MRLC phosphorylation, 1/half-time, and contractile stress increased transiently and then all decreased to intermediate values. Carbachol (CCh)-induced contractions exhibited latch at 37℃: stress was maintained at high levels despite decreasing myoplasmic [Ca²⁺], MRLC phosphorylation, and 1/half-time. At 22℃ CCh induced sustained elevations in all parameters. 1/half-time depended on both myoplasmic [Ca²⁺] and MRLC phosphorylation. The steady-state dependence of stress on MRLC phosphorylation was very steep at 37℃ in the CCh- or K⁺-depolarized tissue and reduced temperature flattend the dependence of stress on MRLC phosphorylation compared to 37℃. These data suggest that phasic smooth muscle also exhibits latch behavior and latch is less prominent at lower temperature.     

Inhibition of T-type Ca²⁺ channels by endostatin attenuates human glioblastoma cell proliferation and migration

BACKGROUND AND PURPOSE

Endostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions.

EXPERIMENTAL APPROACH

Using cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca²⁺ channels.

KEY RESULTS

Extracellular application of ES reversibly inhibited T-type Ca²⁺ channel currents (T-currents) in U87 cells, whereas L-type Ca²⁺ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three α₁ subunits of T-type Ca²⁺ channels (Ca_V3), α_1G (Ca_V3.1), α_1H (Ca_V3.2) and α_1I (Ca_V3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only Ca_V3.1 and Ca_V3.2, but not Ca_V3.3 or Ca_V1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca²⁺ channel blocker mibefradil occluded these inhibitory effects of ES.

CONCLUSION AND IMPLICATIONS

This study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca²⁺ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells.

LINKED ARTICLE

This article is commented on by Santoni et al., pp. 1244–1246 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01908.x     

Cardiomyocyte Ca<Superscript>2+</Superscript> overload in atrial tachycardia: is blockade of L-type Ca<Superscript>2+</Superscript> channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca²⁺ overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca²⁺ overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca²⁺ channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca²⁺ channel current (I_Ca,L) that may reinforce Ca²⁺ overload thereby promoting AF in the atrium. Ca²⁺ channel blockers which target T-type Ca²⁺ channels in addition to I_Ca,L (for instance, efonidipine) may be more efficient at preventing Ca²⁺ overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca²⁺ channel blockers that additionally target T-type Ca²⁺ channels.     

Relaxant Effect of Spermidine on Acethylcholine and High K+-induced Gastric Contractions of Guinea-Pig

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage-dependent calcium current (VDCC_L). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (I_Ba), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC₅₀=1.1±0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K⁺-induced contraction: Spermidine (5 mM) significantly reduced high K⁺ (50 mM)-induced contraction to 37±4.7% of the control (p<0.05), and inhibitory effect of spermidine on I_Ba was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca²⁺-induced Ca²⁺ release (CICR),'' was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCC_L and Ca²⁺ releasing mechanism in guinea-pig stomach.