Investigation of the effects of the novel anticonvulsant compound carisbamate (RWJ-333369) on rat piriform cortical neurones in vitro

Background and purpose:

Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices.

Experimental approach:

Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings.

Key results:

Carisbamate (50–400 µmol·L⁻¹) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na⁺ channel block; 150–400 µmol·L⁻¹ carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl⁻ loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100–400 µmol·L⁻¹) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl⁻ conductance. Lidocaine (40–320 µmol·L⁻¹) mimicked carisbamate, implying similar modes of action. Carisbamate (300–600 µmol·L⁻¹) had no effect on spontaneous GABA_A miniature inhibitory postsynaptic currents and at lower concentrations (50–200 µmol·L⁻¹) inhibited Mg²⁺-free or 4-aminopyridine-induced seizure-like discharges.

Conclusions and implications:

Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na⁺ channels and increased Cl⁻ conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.     

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid,an endogenous end product of thyroid hormone metabolism

BACKGROUND AND PURPOSE

3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects.

EXPERIMENTAL APPROACH

TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 μg·kg⁻¹) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H₁ receptor antagonist pyrilamine (10 mg·kg⁻¹) or the H₂ receptor antagonist zolantidine (5 mg·kg⁻¹). TA1 (1.32 and 4 μg·kg⁻¹) was also given i.c.v. to mice lacking histidine decarboxylase (HDC^−/−) and the corresponding WT strain.

KEY RESULTS

TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 μg·kg⁻¹), stimulation of learning (1.32 and 4 μg·kg⁻¹), hyperalgesia (0.4, 1.32 and 4 μg·kg⁻¹) and hyperglycaemia (1.32 and 4 μg·kg⁻¹). All these effects were modulated by pyrilamine and zolantidine. In HDC^−/− mice, TA1 (1.32 and 4 μg·kg⁻¹) did not increase plasma glucose or induce hyperalgesia.

CONCLUSIONS AND IMPLICATIONS

Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.     

Antithrombotic effect of Z4A5 on coronary thrombosis in a canine model of acute unstable angina

Background and Purpose

The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina.

Experimental Approach

Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 μg·kg⁻¹ + 1 μg·kg⁻¹·min⁻¹, 30 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹ or 300 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively.

Key Results

The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion.

Conclusions and Implications

Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes.     

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.     

FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

Aim:

FTY720, a new immunomodulatory drug with low cytotoxicity, is currently used to treat multiple sclerosis. In this study, we investigated the effects of FTY720 on inflammatory cell infiltration in albumin overload-induced nephropathy of rats.

Methods:

Male Wistar rats were subjected to right-side nephrectomy and divided into 3 groups. One week after the surgery, albumin overload (AO) group was treated with BSA (5 g·kg⁻¹·d⁻¹, ip) for 9 weeks; AO+FTY720 group was given BSA (5 g·kg⁻¹·d⁻¹, ip) plus FTY720 (0.5 g·kg⁻¹·d⁻¹, ip) for 9 weeks; and control group received daily ip injection of equivalent volume of saline. All rats were killed 9 weeks after nephrectomy.

Results:

AO rats exhibited gradually increased urinary protein excretion accompanied by elevated urinary N-acetyl-β-O-glucosaminidase activity, and both reached their peak values at week 7. Furthermore, AO significantly increased lymphocytes and monocytes in circulation and the inflammatory cells recruited to tubulointerstitium, and the expression of inflammatory cytokines MCP-1, TNF-α and IL-6, as well as sphingosine 1-phosphate (S1P) receptors S1pr1 and S1pr3, and S1P-synthesizing enzyme sphingosine kinase 1 (Sphk1) in the kidney. Concomitant administration of FTY720 significantly attenuated all the AO-induced pathological changes.

Conclusion:

FTY720 alleviates tubulointerstitium inflammation in an AO rat model of nephropathy via down-regulation of the Sphk1 pathway.     

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.     

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide,in the regulation of rat gastric motor functions

Background and purpose:

Vgf gene expression has been detected in various endocrine and neuronal cells in the gastrointestinal tract. In this study we investigated the pharmacological activity of different VGF-derived peptides. Among these, TLQP-21, corresponding to the 556–576 fragment of the protein was the unique active peptide, and its pharmacological profile was further studied.

Experimental approach:

The effects of TLQP-21 were examined in vitro by smooth muscle contraction in isolated preparations from the rat gastrointestinal tract and, in vivo, by assessing gastric emptying in rats. Rat stomach tissues were also processed for immunohistochemical and biochemical characterization.

Key results:

In rat longitudinal forestomach strips, TLQP-21 (100 nmol·L⁻¹–10 µmol·L⁻¹) concentration-dependently induced muscle contraction (in female rats, EC₅₀ = 0.47 µmol·L⁻¹, E_max: 85.7 ± 7.9 and in male rats, 0.87 µmol·L⁻¹, E_max: 33.4 ± 5.3; n = 8), by release of prostaglandin (PG)E₂ and PGF_2a from the mucosal layer. This effect was significantly antagonized by indomethacin and selective inhibitors of either cyclooxygenase-1 (S560) or cyclooxygenase-2 (NS398). Immunostaining and biochemical studies confirmed the presence of VGF in the gastric neuronal cells. TLQP-21, injected i.c.v. (2–32 nmol per rat), significantly decreased gastric emptying by about 40%. This effect was significantly (P < 0.05) blocked by i.c.v. injection of indomethacin, suggesting that, also in vivo, this peptide acts in the brain stimulating PG release.

Conclusions and implications:

The present results demonstrate that this VGF-derived peptide plays a central and local role in the regulation of rat gastric motor functions.     

3-iodothyroacetic acid,a metabolite of thyroid hormone,induces itch and reduces threshold to noxious and to painful heat stimuli in mice

Background and purpose

Itch is associated with increased sensitization to nociceptive stimuli. We investigated whether 3-iodothyroacetic acid (TA1), by releasing histamine, induces itch and increases sensitization to noxious and painful heat stimuli.

Experimental Approach

Itch was evaluated after s.c. administration of TA1 (0.4, 1.32 and 4 μg·kg⁻¹). Mice threshold to noxious (NHT) and to painful heat stimuli were evaluated by the increasing-temperature hot plate (from 45.5 to 49.5°C) or by the hot plate (51.5°C) test, respectively, 15 min after i.p. injection of TA1 (0.4, 1.32 and 4 μg·kg⁻¹). Itch, NHT and pain threshold evaluation were repeated in mice pretreated with pyrilamine. Itch and NHT were also measured in HDC^+/+ and HDC^−/− following injection of saline or TA1 (1.32, 4 and 11 μg·kg⁻¹; s.c. and i.p.). pERK1/2 levels were determined by Western blot in dorsal root ganglia (DRG) isolated from CD1 mice 15 min after they received (i.p.): saline, saline and noxious heat stimulus (46.5°C), TA1 (0.1, 0.4, 1.32, 4 μg·kg⁻¹) or TA1 1.32 μg·kg⁻¹ and noxious heat stimulus.

Key Results

TA1 0.4 and 1.32 μg·kg⁻¹ induced itch and reduced NHT; pyrilamine pretreatment prevented both of these effects. TA1 4 μg·kg⁻¹ (i.p.) reduced pain threshold without inducing itch or modifying NHT. In HDC^−/− mice, TA1 failed to induce itch and to reduce NHT. In DRG, pERK1/2 levels were significantly increased by noxious heat stimuli and by TA1 0.1, 0.4 and 1.32 μg·kg⁻¹; i.p.

Conclusions and Implications

Increased TA1 levels induce itch and an enhanced sensitivity to noxious heat stimuli suggesting that TA1 might represent a potential cause of itch in thyroid diseases.     

Pharmacological characterization of recombinant NR1/NR2A NMDA receptors with truncated and deleted carboxy termini expressed in Xenopus laevis oocytes

Background and purpose:

The carboxy terminal domain (CTD) of NR2 N-methyl-d-aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A-containing NMDARs are influenced by this key functional domain.

Experimental approach:

Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg²⁺ and memantine and potentiation of NMDAR-mediated responses by chelating contaminating divalent cations.

Key results:

Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC₅₀ = 2.2 µmol·L⁻¹, NR2A(trunC); 2.7 µmol·L⁻¹, NR2A(delC) compared with 3.3 µmol·L⁻¹, NR2A(WT)] but did significantly increase glycine potency [EC₅₀ = 500 nmol·L⁻¹, NR2A(trunC); 900 nmol·L⁻¹, NR2A(delC) compared with 1.3 µmol·L⁻¹, NR2A(WT)]. Voltage-dependent Mg²⁺ block of NR2A(WT)- and NR2A(trunC)-containing NMDARs was similar but low concentrations of Mg²⁺ (1 µmol·L⁻¹) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA-induced potentiation was similar at each of the three NMDAR constructs.

Conclusions and implications:

Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD-mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling.     

Inotropy and L-type Ca2+ current,activated by β1- and β2-adrenoceptors,are differently controlled by phosphodiesterases 3 and 4 in rat heart

Background and purpose

β₁- and β₂-adrenoceptors coexist in rat heart but β₂-adrenoceptor-mediated inotropic effects are hardly detectable, possibly due to phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide (300 nmol·L⁻¹) and the PDE4 inhibitor rolipram (1 µmol·L⁻¹) on the effects of (−)-catecholamines.

Experimental approach

Cardiostimulation evoked by (−)-noradrenaline (ICI118551 present) and (−)-adrenaline (CGP20712A present) through β₁- and β₂-adrenoceptors, respectively, was compared on sinoatrial beating rate, left atrial and ventricular contractile force in isolated tissues from Wistar rats. L-type Ca²⁺-current (I_Ca-L) was assessed with whole-cell patch clamp.

Key results

Rolipram caused sinoatrial tachycardia. Cilostamide and rolipram did not enhance chronotropic potencies of (−)-noradrenaline and (−)-adrenaline. Rolipram but not cilostamide potentiated atrial and ventricular inotropic effects of (−)-noradrenaline. Cilostamide potentiated the ventricular effects of (−)-adrenaline but not of (−)-noradrenaline. Concurrent cilostamide + rolipram uncovered left atrial effects of (−)-adrenaline. Both rolipram and cilostamide augmented the (−)-noradrenaline (1 µmol·L⁻¹) evoked increase in I_Ca-L. (−)-Adrenaline (10 µmol·L⁻¹) increased I_Ca-L only in the presence of cilostamide but not rolipram.

Conclusions and implications

PDE4 blunts the β₁-adrenoceptor-mediated inotropic effects. PDE4 reduces basal sinoatrial rate in a compartment distinct from compartments controlled by β₁- and β₂-adrenoceptors. PDE3 and PDE4 jointly prevent left atrial β₂-adrenoceptor-mediated inotropy. Both PDE3 and PDE4 reduce I_Ca-L responses through β₁-adrenoceptors but the PDE3 component is unrelated to inotropy. PDE3 blunts both ventricular inotropic and I_Ca-L responses through β₂-adrenoceptors.     

Effect of low doses of cannabidiolic acid and ondansetron on LiCl-induced conditioned gaping (a model of nausea-induced behaviour) in rats

Background and Purpose

To determine the minimally effective dose of cannabidiolic acid (CBDA) that effectively reduces lithium chloride (LiCl)-induced conditioned gaping reactions (nausea-induced behaviour) in rats and to determine if these low systemic doses of CBDA (5–0.1 μg·kg⁻¹) relative to those of CBD could potentiate the anti-nausea effects of the classic 5-hydroxytryptamine 3 (5-HT₃) receptor antagonist, ondansetron (OND).

Experimental Approach

We investigated the efficacy of low doses of CBDA to suppress acute nausea, assessed by the establishment of conditioned gaping to a LiCl-paired flavour in rats. The potential of threshold and subthreshold doses of CBDA to enhance the reduction of nausea-induced conditioned gaping by OND were then determined.

Key Results

CBDA (at doses as low as 0.5 μg·kg⁻¹) suppressed nausea-induced conditioned gaping to a flavour. A low dose of OND (1.0 μg·kg⁻¹) alone reduced nausea-induced conditioned gaping, but when it was combined with a subthreshold dose of CBDA (0.1 μg·kg⁻¹) there was an enhancement in the suppression of LiCl-induced conditioned gaping.

Conclusions and Implications

CBDA potently reduced conditioned gaping in rats, even at low doses and enhanced the anti-nausea effect of a low dose of OND. These findings suggest that combining low doses of CBDA and OND will more effectively treat acute nausea in chemotherapy patients.     

Myoendothelial coupling in the mesenteric arterial bed; segmental differences and interplay between nitric oxide and endothelin-1

Background and purpose:

We tested the hypothesis that activated arterial smooth muscle (ASM) stimulates endothelial vasomotor influences via gap junctions and that the significance of this myoendothelial coupling increases with decreasing arterial diameter.

Experimental approach:

From WKY rats, first-, second-, third-and fourth-order branches of the superior mesenteric artery (MA1, MA2, MA3 and MA4 respectively) were isolated and mounted in wire-myographs to record vasomotor responses to 0.16–20 µmol·L⁻¹ phenylephrine.

Key results:

Removal of endothelium increased the sensitivity (pEC₅₀) to phenylephrine in all arteries. The nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) (100 µmol·L⁻¹) did not modify pEC₅₀ to phenylephrine in all denuded arteries, and increased it in intact MA1, MA2 and MA3 to the same extent as denudation. However, in intact MA4, the effect of L-NAME was significantly larger (ΔpEC₅₀ 0.57 ± 0.02) than the effect of endothelium removal (ΔpEC₅₀ 0.20 ± 0.06). This endothelium-dependent effect of L-NAME in MA4 was inhibited by (i) steroidal and peptidergic uncouplers of gap junctions; (ii) a low concentration of the NO donor sodium nitroprusside; and (iii) by the endothelin-receptor antagonist bosentan. It was also observed during contractions induced by (i) calcium channel activation (BayK 8644, 0.001–1 µmol·L⁻¹); (ii) depolarization (10–40 mmol·L⁻¹ K⁺); and (iii) sympathetic nerve stimulation (0.25–32 Hz).

Conclusions and implications:

These pharmacological observations indicated feedback control by endothelium of ASM reactivity involving gap junctions and a balance between endothelium-derived NO and endothelin-1. This myoendothelial coupling was most prominent in distal resistance arteries.     

Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta

Background and purpose:

Nebivolol is a highly selective β₁-adrenoceptor antagonist with β₃-adrenoceptor agonist properties and is a racemate mixture of D- and L-enantiomers. However, the cellular mechanisms of the effects of each enantiomer are not yet clear and are a matter for debate. The aim of the present experiments was to determine the adrenoceptors involved in the vascular effects of D- and L-enantiomers of nebivolol in rat thoracic aorta.

Experimental approach:

Responses to nebivolol enantiomers were evaluated in rings of thoracic aorta from male Sprague-Dawley rats.

Key results:

D-nebivolol (0.1–10 µmol·L⁻¹), but not L-nebivolol, significantly shifted to the right the concentration-response curve to phenylephrine, an α₁-adrenoceptor agonist, in a concentration-dependent manner. For the following experiments, aortic rings were constricted with endothelin 1 and now both enantiomers produced an endothelium-dependent relaxation of the rings involving the nitric oxide pathway. This relaxation was not modified by 1 µmol·L⁻¹ CGP 20,712A (β₁-adrenoceptor antagonist), but significantly blunted by 7 µmol·L⁻¹ L-74,8337 (β₃-adrenoceptor antagonist). However, only the vasorelaxation induced by D-nebivolol was significantly reduced by 1 µmol·L⁻¹ ICI 118,551 (β₂-adrenoceptor antagonist).

Conclusions and implications:

Our results suggest that the nebivolol enantiomers act on different targets. D-nebivolol induced vasorelaxation by activating β₂- and β₃-adrenoceptors and antagonizing α₁-adrenoceptors. L-nebivolol induced vasorelaxation by activating only β₃-adrenoceptors in our model. Our results emphasize that nebivolol is a β₁-adrenoceptor antagonist with several important pharmacological differences from other β₁-adrenoceptor antagonists.     

Ouabain attenuates cardiotoxicity induced by other cardiac steroids

Background and purpose:

All cardiac steroids have a similar structure, bind to and inhibit the ubiquitous transmembrane protein Na⁺, K⁺-ATPase and increase the force of contraction of heart muscle. However, there are diverse biological responses to different cardiac steroids both at the cellular and at the molecular level. Moreover, we have recently shown that ouabain inhibits digoxin- and bufalin-induced changes in membrane traffic. The present study was designed to test the hypothesis that ouabain also has an inhibitory effect on cardiotoxicity induced by other cardiac steroids.

Experimental approach:

The hypothesis was tested in isolated heart muscle preparations and in an in vivo model of cardiotoxicity in guinea pigs.

Key results:

Ouabain at a low dose attenuated the toxicity induced by bufalin and digoxin in heart muscle preparations. In addition, ouabain at the low dose (91 ng·kg⁻¹·h⁻¹), but not at a higher dose (182 ng·kg⁻¹·h⁻¹), delayed the development of digoxin-induced (500 µg·kg⁻¹·h⁻¹) cardiotoxicity in anaesthetized guinea pigs, as manifested by delayed arrhythmia and terminal ventricular fibrillation, as well as a reduced heart rate. In addition, as observed with ouabain, the phosphoinositide 3-kinase inhibitor wortmannin (100 µg·kg⁻¹·h⁻¹) delayed the digoxin-induced arrhythmia in anaesthetized guinea pigs.

Conclusions and implications:

The present study demonstrates the inhibitory effect, probably through signal transduction pathways, of ouabain on digoxin- and bufalin-induced cardiotoxicity in guinea pigs. Further understanding of this phenomenon could be beneficial for increasing the therapeutic window for cardiac steroids in the treatment of chronic heart failure.     

Dalcetrapib: no off-target toxicity on blood pressure or on genes related to the renin-angiotensin-aldosterone system in rats

Background and purpose:

The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model.

Experimental approach:

Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg·kg⁻¹·day⁻¹; dalcetrapib 100, 300 or 500 mg⁻¹·kg·day⁻¹; or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days'' treatment with torcetrapib 40 mg·kg⁻¹·day⁻¹, dalcetrapib 500 mg·kg⁻¹·day⁻¹ or vehicle was measured by quantitative polymerase chain reaction.

Key results:

Torcetrapib transiently increased mean AP in normotensive rats (+3.7 ± 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 ± 0.6 mmHg with 40 mg·kg⁻¹·day⁻¹ at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas.

Conclusions and implications:

In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.     

Facilitation of central imidazoline I1-site/extracellular signal-regulated kinase/p38 mitogen-activated protein kinase signalling mediates the hypotensive effect of ethanol in rats with acute renal failure

Background and purpose:

This study investigated the role of central sympathetic activity and related mitogen-activated protein kinase (MAPK) signalling in the cardiovascular effects of ethanol in a model of acute renal failure (ARF).

Experimental approach:

The effects of pharmacological interventions that inhibit peripheral or central sympathetic activity or MAPK on the cardiovascular actions of ethanol in rats with ARF induced by glycerol were evaluated.

Key results:

Glycerol (50%, 10 mL·kg⁻¹, i.m.) caused progressive increases and decreases in blood pressure (BP) and heart rate (HR) respectively. Subsequent i.v. ethanol (0.25 or 1 g·kg⁻¹) elicited dose-related changes in BP (decreases) and HR (increases). These effects were replicated after intracisternal (i.c.) administration of ethanol. Blockade of nicotinic cholinoceptors (nAChR, hexamethonium, 20 mg·kg⁻¹) or α₁-adrenoceptors (prazosin, 1 mg·kg⁻¹) attenuated cardiovascular effects of ethanol. Ethanol hypotension was also attenuated after the centrally acting sympatholytic drug moxonidine (selective I₁-site agonist, 100 µg·kg⁻¹ i.v.), but not guanabenz (selective α₂-receptor agonist, 30 µg·kg⁻¹, i.v.), suggesting involvement of central circuits of I₁ sites in ethanol-evoked hypotension. Selective blockade I₁ sites (efaroxan) but not α₂ (yohimbine) adrenoceptors abolished the hypotensive response to ethanol. Intracisternal administration of PD98059 or SB203580, inhibitors of extracellular signal-regulated kinase (ERK 1/2) and p38 MAPK, respectively, reduced the hypotensive action of moxonidine or ethanol. When used simultaneously, the two MAPK inhibitors produced additive attenuation of ethanol hypotension.

Conclusions and implications:

Sympathoinhibitory pathways of central I₁-sites and downstream ERK/p38 MAPK signalling were involved in the hypotensive action of ethanol in ARF.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious,hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3–4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 μg kg⁻¹ h⁻¹, i.v.)
2. The early (1–2 h) hypotension in SD rats (−11±3 mmHg; n=7) was significantly less than that in TG rats (−35±3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108±3 mmHg; 24 h LPS, 112±4 mmHg; TG rats: baseline, 171±2 mmHg; 24 h LPS, 169±3 mmHg). At this stage in the SD rats there was a renal vasodilatation (Δ vascular conductance, 29±10 [kHz mmHg⁻¹]10³) but not in TG rats (Δ vascular conductance 2±3[kHz mmHg⁻¹]10³).
3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 μg kg⁻¹ bolus, 600 μg kg⁻¹ h⁻¹) between 24 and 31 h in SD rats caused a fall in MAP of 16±2 mmHg accompanied by hindquarters vasodilatation (Δ vascular conductance 11±3 (kHz mmHg⁻¹)10³). In TG rats, under the same conditions, the fall in MAP was −60±6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (Δ vascular conductance, 23±5, 32±7, and 14±4 (kHz mmHg⁻¹)10³, respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats.
4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was −17±4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5±3, −4±5, and 12±4 (kHz mmHg⁻¹)10³, respectively.
5. Administration of the angiotensin (AT₁)-receptor antagonist, losartan (10 mg kg⁻¹, i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (−12±3 and −14±4 mmHg, respectively).
6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.

     

Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Background and purpose

The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1–2 µmol·L⁻¹), on Ca²⁺-dependent pro-inflammatory activities, cytosolic Ca²⁺ fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µmol·L⁻¹) and platelet-activating factor (200 nmol·L⁻¹).

Experimental approach

Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B₄ and cAMP by competitive binding ELISA procedures, and Ca²⁺ fluxes by fura-2/AM-based spectrofluorimetric and radiometric (⁴⁵Ca²⁺) procedures.

Key results

Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B₄ by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 µmol·L⁻¹ (mean percentages of the control values of 30 ± 1, 12 ± 3 and 21 ± 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca²⁺ concentrations and store-operated Ca²⁺ influx. These montelukast-mediated alterations in Ca²⁺ handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.

Conclusions and implications

Montelukast, primarily a cysteinyl leukotriene (CysLT₁) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.     

Modelling of α1-adrenoceptor-mediated temporal dynamics of inotropic response in rat heart to assess ligand binding and signal transduction parameters

Background and purpose:

In order to use the transient response to an antagonist (prazosin) to evaluate properties of agonist interactions with the α₁-adrenoceptor system, an integrative mechanistic model of cardiac uptake of prazosin and its competitive interaction with phenylephrine at the receptor site was developed. Based on the operational model of agonism, the aim was to evaluate both the receptor binding and signal transduction process as determinants of the inotropic effect of phenylephrine.

Experimental approach:

In Langendorff-perfused rat hearts, prazosin outflow concentration and left ventricular developed pressure were measured, first in the presence of 12.3 µmol·L⁻¹ phenylephrine following a 1 min infusion of 1.27 nmol [³H]-prazosin, and second, when after 30 min the phenylephrine concentration in perfusate was reduced to 6.1 µmol·L⁻¹, the 1 min infusion of 1.27 nmol [³H]-prazosin was repeated.

Key results:

The kinetic model accounted for cardiac uptake and receptor binding kinetics of prazosin (dissociation constant, mean ± SD: 0.057 ± 0.012 nmol·L⁻¹), assuming that the competitive displacement of phenylephrine (dissociation constant: 101 ± 13 nmol·L⁻¹) reduced the receptor occupation by the agonist and, consequently, contractility. This competitive binding process appeared to be the rate-determining step in response generation. The relationship between receptor occupancy and inotropic response was described by an efficacy parameter (τ, ratio of receptor density and coupling efficiency) of 4.9.

Conclusions and implications:

Mechanistic pharmacodynamic modelling of the kinetics of antagonism by prazosin allows quantitative assessment of the α₁-adrenoceptor system both at the receptor and post-receptor levels.