Spontaneous release of acetylcholine from autonomic nerves in the bladder

Background and purpose:

Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach:

Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results:

Hyoscine (3 µmol·L⁻¹), but not tetrodotoxin (TTX, 1 µmol·L⁻¹), reduced basal tension, distension-evoked contractile activity and physostigmine (1 µmol·L⁻¹)-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. ω-Conotoxin GVIA (0.3 µmol·L⁻¹) did not affect physostigmine-induced contractions when given either alone or in combination with ω-agatoxin IVA (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5–500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 µmol·kg⁻¹ i.v.).

Conclusions and implications:

The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca²⁺ channels.British Journal of Pharmacology (2009) 157, 607–619; doi:10.1111/j.1476-5381.2009.00166.x; published online 3 April 2009     

Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium

Background and purpose:

Thromboxane A₂ and endothelial dysfunction are implicated in the development of pulmonary hypertension. The receptor-transduction pathway for U46619 (9,11-dideoxy-9α, 11α-methanoepoxy prostaglandin F_2α)-induced contraction was examined in endothelium-intact (E+) and denuded (E−) rat pulmonary artery rings.

Experimental approach:

Artery rings were mounted on a wire myograph under a tension of 7–7.5 mN at 37°C and gassed with 95% O₂/5% CO₂. Isometric recording was made by using Powerlab data collection and Chart 5 software.

Key results:

Both E+ and E− contractile responses were sensitive to Rho-kinase inhibition and the chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid]. The E+ response was sensitive to the store-operated calcium channel blockers SKF-96365 {1-[B-[3-(4-methoxyphenyl)propoxy]-4-methoxy-phenethyl]-1H-imidazole hydrochloride} and 2-APB (2-amino ethoxy diphenylborate) (75–100 µmol·L⁻¹). The E− response was sensitive to 2-APB (10–30 µmol·L⁻¹), a putative IP₃ receptor antagonist, and the calcium and chloride channel blockers nifedipine, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulphonic acid) and niflumic acid but was insensitive to SKF-96365. Inhibiting K_V with 4-AP in E+ rings exposed a contraction sensitive to nifedipine, DIDS and niflumic acid, whereas inhibiting BK_Ca exposed a contraction sensitive to mibefradil, DIDS and niflumic acid. This indicates that removal of the endothelium allows the TP receptor to inhibit K_V, which may involve coupling to phospholipase C, because inhibition of phospholipase C with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-y]amino]hexyl]– 1H-pyrrole-2,5-dione) switched the E− pathway to the E+ pathway.

Conclusions and implications:

The results from this study indicate that distinct transduction pathways can be employed by the TP receptor to produce contraction and that the endothelium is able to influence the coupling of the TP receptor.British Journal of Pharmacology (2009) 157, 581–596; doi:10.1111/j.1476-5381.2008.00084.x; published online 22 April 2009This 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: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Background and purpose:

The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach:

Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results:

Icilin (3 or 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29°C), 3 µmol·L⁻¹ icilin (EC₅₀ 1.5 µmol·L⁻¹) and menthol (EC₅₀ 263 µmol·L⁻¹) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 µmol·L⁻¹ icilin (EC₅₀ 79 µmol·L⁻¹), allyl isothiocyanate (EC₅₀ 226 µmol·L⁻¹), cinnamaldehyde (EC₅₀ 38 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) increased miniature EPSC rate. The response to 100 µmol·L⁻¹, but not 3 µmol·L⁻¹ icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 µmol·L⁻¹, but not to 100 µmol·L⁻¹ icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 µmol·L⁻¹ icilin and innocuous cold were located more superficially than those responding to 100 µmol·L⁻¹ icilin.

Conclusions and implications:

Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.     

Piceatannol, a derivative of resveratrol, moderately slows INa inactivation and exerts antiarrhythmic action in ischaemia-reperfused rat hearts

Background and purpose:

Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts.

Experimental approach:

Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca²⁺ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts.

Key results:

In rat ventricular cells, piceatannol (3–30 µmol·L⁻¹) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V_max) without altering Ca²⁺ transients. Piceatannol decreased peak I_Na and slowed I_Na inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L⁻¹) decreased peak I_Na without slowing I_Na inactivation. The inhibition of peak I_Na or V_max was associated with a negative shift of the voltage-dependent steady-state I_Na inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L⁻¹, piceatannol could inhibit I_Ca,L, I_to, I_Kr, Ca²⁺ transients and Na⁺-Ca²⁺ exchange except I_K1. Piceatannol (1–10 µmol·L⁻¹) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury.

Conclusions and implications:

The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I_Na inhibition and uniquely slowing I_Na inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L⁻¹.     

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ��-cells against oxidative damage via the ERK1/2 pathway

BACKGROUND AND PURPOSE

Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H₂O₂). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

EXPERIMENTAL APPROACH

Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay.

KEY RESULTS

Quercetin (20 µmol·L⁻¹) potentiated both glucose (8.3 mmol·L⁻¹)- and glibenclamide (0.01 µmol·L⁻¹)-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L⁻¹), protected β-cell function and viability against oxidative damage induced by 50 µmol·L⁻¹ H₂O₂ and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective.

CONCLUSION AND IMPLICATIONS

Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected β-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing β-cell dysfunction associated with diabetes deserves further investigation.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Differential effect of opioid and cannabinoid receptor blockade on heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats

BACKGROUND AND PURPOSE

Opioids and cannabinoids interact in drug addiction and relapse. We investigated the effect of the opioid receptor antagonist naloxone and/or the cannabinoid CB₁ receptor antagonist rimonabant on cannabinoid-induced reinstatement of heroin seeking and on cannabinoid substitution in heroin-abstinent rats.

EXPERIMENTAL APPROACH

Rats were trained to self-administer heroin (30 µg·kg⁻¹ per infusion) under a fixed-ratio 1 reinforcement schedule. After extinction of self-administration (SA) behaviour, we confirmed the effect of naloxone (0.1–1 mg·kg⁻¹) and rimonabant (0.3–3 mg·kg⁻¹) on the reinstatement of heroin seeking induced by priming with the CB₁ receptor agonist WIN55,212-2 (WIN, 0.15–0.3 mg·kg⁻¹). Then, in a parallel set of heroin-trained rats, we evaluated whether WIN (12.5 µg·kg⁻¹ per infusion) SA substituted for heroin SA after different periods of extinction. In groups of rats in which substitution occurred, we studied the effect of both antagonists on cannabinoid intake.

KEY RESULTS

Cannabinoid-induced reinstatement of heroin seeking was significantly attenuated by naloxone (1 mg·kg⁻¹) and rimonabant (3 mg·kg⁻¹) and fully blocked by co-administration of sub-threshold doses of the two antagonists. Moreover, contrary to immediate (1 day) or delayed (90 days) drug substitution, rats readily self-administered WIN when access was given after 7, 14 or 21 days of extinction from heroin, and showed a response rate that was positively correlated with the extinction period. In these animals, cannabinoid intake was increased by naloxone (1 mg·kg⁻¹) and decreased by rimonabant (3 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Our findings extend previous research on the crosstalk between cannabinoid and opioid receptors in relapse mechanisms, which suggests a differential role in heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Evodiamine improves congnitive abilities in SAMP8 and APP(swe)/PS1(ΔE9) transgenic mouse models of Alzheimer's disease

Aim:

To investigate the effect of evodiamine (a quinolone alkaloid from the fruit of Evodia rutaecarpa) on the progression of Alzheimer''s disease in SAMP8 and APP^swe/PS1^ΔE9 transgenic mouse models.

Methods:

The mice at age of 5 months were randomized into the model group, two evodiamine (50 mg·kg⁻¹·d⁻¹ and 100 mg·kg⁻¹·d⁻¹) groups and an Aricept (2 mg·kg⁻¹·d⁻¹) group. The littermates of no-transgenic mice and senescence accelerated mouse/resistance 1 mice (SAMR1) were used as controls. After 4 weeks of treatment, learning abilities and memory were assessed using Morris water-maze test, and glucose uptake by the brain was detected using positron emission tomography/computed tomography (PET/CT). Expression levels of IL-1β, IL-6, and TNF-α in brain tissues were detected using ELISA. Expression of COX-2 protein was determined using Western blot.

Results:

In Morris water-maze test, evodiamine (100 mg·kg⁻¹·d⁻¹) significantly alleviated the impairments of learning ability and memory. Evodiamine (100 mg·kg⁻¹·d⁻¹) also reversed the inhibition of glucose uptake due to development of Alzheimer''s disease traits in mice. Furthermore, the dose of evodiamine significantly decreased the expression of IL-1β, IL-6, TNF-α, and COX-2 that were involved in the inflammation due to Alzheimer''s disease.

Conclusion:

The results indicate that evodiamine (100 mg·kg⁻¹·d⁻¹) improves cognitive abilities in the transgenic models of Alzheimer''s disease.     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

Reduced signal transduction by 5-HT4 receptors after long-term venlafaxine treatment in rats

BACKGROUND AND PURPOSE

The 5-HT₄ receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT₄ receptor-mediated signalling events.

EXPERIMENTAL APPROACH

The effects of 21 days treatment (p.o.) with high (40 mg·kg⁻¹) and low (10 mg·kg⁻¹) doses of venlafaxine, were evaluated at different levels of 5-HT₄ receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg⁻¹, 21 days) was also evaluated on 5-HT₄ receptor density.

KEY RESULTS

Treatment with a high dose (40 mg·kg⁻¹) of venlafaxine did not alter 5-HT₄ mRNA expression, but decreased the density of 5-HT₄ receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg⁻¹) while it was attenuated in rats treated with 40 mg·kg⁻¹ of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT₄ receptor density.

CONCLUSIONS AND IMPLICATIONS

Our data indicate a functional desensitization of 5-HT₄ receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake.     

A cannabinoid receptor, sensitive to O-1918, is involved in the delayed hypotension induced by anandamide in anaesthetized rats

Background and purpose:

Intravenous injection of the endocannabinoid anandamide induces complex cardiovascular changes via cannabinoid CB₁, CB₂ and vanilloid TRPV1 receptors. Recently, evidence has been accumulating that in vitro, but not in vivo, anandamide relaxes blood vessels, via an as yet unidentified, non-CB₁ vascular cannabinoid receptor, sensitive to O-1918 (1,3-dimethoxy-5-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene). We here examined whether the anandamide-induced hypotension in urethane-anaesthetized rats was also mediated via a non-CB₁ vascular cannabinoid receptor.

Experimental approach:

Effects of two antagonists (O-1918 and cannabidiol) of the non-CB₁ vascular cannabinoid receptor on anandamide-induced changes in mean, systolic and diastolic blood pressure (MBP, SBP, DBP), mesenteric (MBF) and renal (RBF) blood flow and heart rate (HR) in urethane-anaesthetized rats was examined.

Key results:

In anaesthetized rats, anandamide (1.5–3 µmol·kg⁻¹) and its stable analogue methanandamide (0.5 µmol·kg⁻¹) caused a delayed and prolonged decrease in MBP, SBP, DBP, MBF and RBF by about 10–30% of the respective basal values without changing HR. In pithed rats, anandamide (3 µmol·kg⁻¹) decreased blood pressure by about 15–20% of the basal value without affecting HR, MBF and RBF. All vascular changes were reduced by about 30–70% by cannabidiol and O-1918 (3 µmol·kg⁻¹, each).

Conclusions and implications:

Non-CB₁ cannabinoid vascular receptors, sensitive to O-1918, contribute to the hypotensive effect of anandamide in anaesthetized rats. Activation of these receptors may be therapeutically important as the endocannabinoid system could be activated as a compensatory mechanism in various forms of hypertension.     

Antibodies against the cardiac sodium/bicarbonate co-transporter (NBCe1) as pharmacological tools

BACKGROUND AND PURPOSE

Na⁺/HCO₃^- co-transport (NBC) regulates intracellular pH (pH_i) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein.

EXPERIMENTAL APPROACH

We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pH_i was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H⁺ flux, J_H, in mmol·L⁻¹ min⁻¹ at pH_i 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpH_i) respectively.

KEY RESULTS

The potassium pulse produced a pH_i increase of 0.18 ± 0.006 (n = 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased J_H by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pH_i increase than control,(0.25 ± 0.018) whereas the recovery of pH_i from acidosis was faster (J_H was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively.

CONCLUSIONS AND IMPLICATIONS

Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.     

Cannabinoid CB1 receptors mediate the effects of corticotropin-releasing factor on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization

BACKGROUND AND PURPOSE

The endocannabinoid and corticotropin-releasing factor (CRF) systems have been implicated in several long-lasting behavioural effects of prior cocaine experience. The present experiments were designed to probe functional interactions between endocannabinoids and CRF by testing the role of cannabinoid CB₁ receptors in cocaine-related behaviours induced or mediated by CRF.

EXPERIMENTAL APPROACH

In Experiment 1, rats trained to self-administer cocaine were pretreated with the CB₁ receptor antagonist, AM251 (0, 10, 100 or 200 µg, i.c.v.), before tests for reinstatement in response to CRF (0, 0.5 µg, i.c.v.), intermittent footshock stress (0, 0.9 mA) or cocaine (0, 10 mg·kg⁻¹, i.p.). In Experiment 2, rats pre-exposed to cocaine (15–30 mg·kg⁻¹, i.p.) or saline for 7 days were pretreated with AM251 (0, 10 or 100 µg, i.c.v.) before tests for locomotion in response to CRF (0.5 µg, i.c.v.), cocaine (15 mg·kg⁻¹, i.p.) or saline (i.c.v.).

KEY RESULTS

Pretreatment with AM251 selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine.

CONCLUSIONS AND IMPLICATIONS

These findings reveal a mediating role for CB₁ receptor transmission in the effects of CRF on cocaine-related behaviours.     

Prolonged pharmacodynamic effects of S-0139, an intravenously administered endothelin A (ETA) antagonist, in the human forearm blood flow model

AIMS

To estimate the pharmacologically active dose range of a new investigational compound S-0139, a selective endothelin A (ET_A) receptor antagonist, in man, and to examine the duration of its pharmacodynamic effect.

METHODS

Venous occlusion plethysmography was performed to assess changes in forearm blood flow following intra-brachial administration of endothelin-1 (ET-1). ET_A antagonists have been shown to block ET-1-induced vasoconstriction in this model. The study was conducted in three parts: (1) a pilot study to explore dose–response (dose range 0.08–13.33 µg kg⁻¹ min⁻¹), (2) a randomized study to confirm dose–response (placebo, 2.5, 6.67 and 15 µg kg⁻¹ min⁻¹), and (3) a delayed administration study (15.7 µg kg⁻¹ min⁻¹) to explore the duration of the pharmacodynamic effect. In all studies a 3-h infusion of S-0139 was given and during the last 90 min of the infusion, ET-1 was infused concurrently for 90 min. In study (3) a second ET-1 infusion was given starting 3 h after completion of the first.

RESULTS

Intravenously administered S-0139 resulted in significant inhibition of ET-1-induced vasoconstriction in the forearm (plasma concentration 800–2000 ng ml⁻¹). In the delayed administration study, the same extent of inhibition was still present when ET-1 was administered 3 h after the end of infusion of S-0139, even though the S-0139 plasma concentrations (mean 17 ng ml⁻¹) were well below pharmacologically active concentrations as determined in studies 1 and 2.

CONCLUSIONS

S-0139 dose-dependently blocks ET-1-mediated vasoconstriction in the forearm and has a prolonged duration of effect beyond that expected from its pharmacokinetic profile.     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

Ca2+-calmodulin can activate and inactivate cardiac ryanodine receptors

Background and purpose:

Ca²⁺-calmodulin (Ca²⁺CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.

Experimental approach:

We investigated the effects of low concentrations of Ca²⁺CaM (50–100 nmol·L⁻¹) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L⁻¹ CaM on sarcoplasmic reticulum (SR) Ca²⁺-release.

Key results:

Ca²⁺CaM activated or inhibited single RyR2, but activation was much more likely at low (50–100 nmol·L⁻¹) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L⁻¹ CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca²⁺-release events. Application of caffeine demonstrated that 50 nmol·L⁻¹ CaM reduced SR Ca²⁺ content.

Conclusions and implications:

We describe for the first time how Ca²⁺CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca²⁺-release and the use of caffeine suggests that this is a RyR2-mediated effect.