Mechanisms involved in the regulation of bovine pulmonary vascular tone by the 5-HT1B receptor

Background and purpose:

5-HT_1B receptors may have a role in pulmonary hypertension. Their relationship with the activity of BK_Ca, a T-type voltage-operated calcium channel (VOCC) and cyclic nucleotide-mediated relaxation was examined.

Experimental approach:

Ring segments of bovine pulmonary arteries were mounted in organ baths in modified Krebs–Henseleit buffer (37^oC) under a tension of 20 mN and gassed with 95% O₂/5% CO₂. Isometric recordings were made using Chart 5 software.

Key results:

Contractile responses to 5-HT (10 nM–300 µM) were inhibited similarly by the 5-HT_1B receptor antagonist SB216641 (100 nM) and the T-type VOCC blockers mibefradil (10 µM) and NNC550396 (10 µM) with no additive effect between SB216641 and mibefradil. Inhibition by SB216641 was prevented by the potassium channel blocker, charybdotoxin (100 nM). 5-HT_1B receptor activation and charybdotoxin produced a mibefradil-sensitive potentiation of responses to U46619. Bradykinin (0.1 nM–30 µM), sodium nitroprusside (0.01 nM–3 µM), zaprinast (1 nM–3 µM), isoprenaline (0.1 nM–10 µM) and rolipram (1 nM–3 µM) produced 50% relaxation of arteries constricted with 5-HT (1–3 µM) or U46619 (30–50 nM) in the presence of 5-HT_1B receptor activation, but full relaxation of arteries constricted with U46619, the 5-HT_2A receptor agonist 2,5 dimethoxy-4 iodoamphetamine (1 µM) or 5-HT in the presence of 5-HT_1B receptor antagonism. Enhanced relaxation of 5-HT-constricted arteries by cGMP-dependent pathways, seen in the presence of the 5-HT_1B receptor antagonist, was reversed by charybdotoxin whereas cAMP-dependent relaxation was only partly reversed by charybdotoxin.

Conclusions and implications:

5-HT_1B receptors couple to inhibition of BK_Ca, thus increasing tissue sensitivity to contractile agonists by activating a T-type VOCC and impairing cGMP-mediated relaxation. Impaired cAMP-mediated relaxation was only partly mediated by inhibition of BK_Ca.     

Bradykinin and B₂ receptor antagonism in rat and human articular chondrocytes

BACKGROUND AND PURPOSE

In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis.

EXPERIMENTAL APPROACH

BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8.

KEY RESULTS

Density of [³H]-BK binding sites was higher (13–30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B₂ receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B₁ receptor antagonist Lys-[Leu⁸][desArg⁹]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798 196, 200 nM; L-161 982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB.

CONCLUSION AND IMPLICATIONS

BK mediated inflammatory changes and cartilage degradation and B₂ receptor blockade would, therefore, be a potential treatment for OA.     

Effects of aldosterone and related steroids on LPS-induced increased expression of inducible NOS in rat aortic smooth muscle cells

BACKGROUND AND PURPOSE

Expression of inducible NOS (iNOS) is important in certain inflammatory diseases. We determined if the hormone aldosterone, a mineralocorticoid receptor (MR) agonist, affects LPS activation of iNOS expression in rat aortic smooth muscle cells (RASMC).

EXPERIMENTAL APPROACH

Cultured RASMC were treated with LPS, with or without agonists/antagonists of steroid receptors. iNOS expression was determined by nitrite assays on culture medium removed from treated cells and by immunoblotting of cell protein extracts.

KEY RESULTS

LPS (1 µg·mL⁻¹) increased nitrite and iNOS protein above that in control (untreated) cells. These effects of LPS were reduced by aldosterone (0.1–10 µM). The MR antagonists, eplerenone (10 µM) and spironolactone (10 or 50 µM), did not inhibit these actions of 1 µM aldosterone, but the latter were prevented by 10 µM mifepristone, a glucocorticoid (GR) and progestogen receptor (PR) antagonist. Mifepristone also prevented the reduction of LPS-induced nitrite increase produced by 1 µM dexamethasone (GR agonist) and 10 µM progesterone (PR agonist). Spironolactone (10–50 µM) by itself decreased LPS-induced increases in nitrite and iNOS protein. Mifepristone (10 µM) partially reversed these effects of 10 µM spironolactone, but not those of 50 µM; the effects of 50 µM spironolactone were also unchanged when mifepristone was increased to 50 µM.

CONCLUSIONS AND IMPLICATIONS

This pharmacological profile suggests that aldosterone, and possibly 10 µM spironolactone, use mechanisms that are dependent on PR and/or GR, but not MR, to inhibit iNOS induction in RASMC. With 50 µM spironolactone, other inhibitory mechanisms requiring further investigation may become predominant.     

Differential regulation of β2 -adrenoceptor-mediated inotropic and lusitropic response by PDE3 and PDE4 in failing and non-failing rat cardiac ventricle

BACKGROUND AND PURPOSE

β-Adrenoceptors play a major role in regulating myocardial function through cAMP-dependent pathways. Different phosphodiesterases (PDEs) regulate intracellular cAMP-pools and thereby contribute to the compartmentalization of cAMP-dependent effects. We explored the involvement of PDEs in limiting the β₂ adrenoceptor-mediated positive inotropic (PIR) and lusitropic (LR) responses in sham-operated (Sham) and failing rat hearts.

EXPERIMENTAL APPROACH

Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Rats developing heart failure were studied 6 weeks after surgery. Contractility was measured in left ventricular strips from failing and Sham hearts. cAMP was quantified by RIA.

KEY RESULTS

In ventricular strips, stimulation of β₂-adrenoceptors with (-)-adrenaline (300 nM CGP20712A present) exerted a small PIR and LR. In Sham hearts, β₂-adrenoceptor-mediated as well as β₁-adrenoceptor-mediated PIR and LR were increased by selective inhibition of either PDE3 (1 µM cilostamide) or PDE4 (10 µM rolipram). In failing rat hearts, PDE3 inhibition enhanced PIR and LR to both β₁- and β₂-adrenoceptor stimulation while PDE4 inhibition had no effect on these responses despite a significant increase in cAMP levels. Combined PDE3/4 inhibition further enhanced the PIR and LR of β₂- and β₁-adrenoceptor activation both in Sham and failing hearts, compared with PDE3 inhibition alone. PDE4 enzyme activity was reduced in failing hearts.

CONCLUSIONS AND IMPLICATIONS

Both PDE3 and PDE4 attenuated β₂- and β₁-adrenoceptor-mediated contractile responses in Sham hearts. In failing hearts, these responses are attenuated solely by PDE3 and thus even selective PDE3 inhibitors may provide a profound enhancement of β-adrenoceptor-mediated responses in heart failure.     

Sildenafil restores cognitive function without affecting β-amyloid burden in a mouse model of Alzheimer's disease

BACKGROUND AND PURPOSE

Inhibitors of phosphodiesterase 5 (PDE5) affect signalling pathways by elevating cGMP, which is a second messenger involved in processes of neuroplasticity. In the present study, the effects of the PDE5 inhibitor, sildenafil, on the pathological features of Alzheimer''s disease and on memory-related behaviour were investigated.

EXPERIMENTAL APPROACH

Sildenafil was administered to the Tg2576 transgenic mouse model of Alzheimer''s disease and to age-matched negative littermates (controls). Memory function was analysed using the Morris water maze test and fear conditioning tasks. Biochemical analyses were performed in brain lysates from animals treated with saline or with sildenafil.

KEY RESULTS

Treatment of aged Tg2576 animals with sildenafil completely reversed their cognitive impairment. Such changes were accompanied in the hippocampus by a reduction of tau hyperphosphorylation and a decrease in the activity of glycogen synthase kinase 3β (GSK3β) and of cyclin-dependent kinase 5 (CDK5) (p25/p35 ratio). Moreover, sildenafil also increased levels of brain-derived neurotrophic factor (BDNF) and the activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus without any detectable modification of brain amyloid burden.

CONCLUSIONS AND IMPLICATIONS

Sildenafil improved cognitive functions in Tg2576 mice and the effect was not related to changes in the amyloid burden. These data further strengthen the potential of sildenafil as a therapeutic agent for Alzheimer''s disease.     

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

BACKGROUND AND PURPOSE

To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.

EXPERIMENTAL APPROACH

Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs.

KEY RESULTS

18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.

CONCLUSION AND IMPLICATIONS

When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.     

Safety, tolerability and pharmacokinetics of an intravenous bolus of sildenafil in patients with pulmonary arterial hypertension

AIMS

To assess pharmacokinetics and pharmacodynamics of a 10 mg intravenous sildenafil bolus in pulmonary arterial hypertension (PAH) patients stabilized on 20 mg sildenafil orally three times daily.

METHODS

Pharmacokinetic parameters were calculated using noncompartmental analysis.

RESULTS

After an acute increase, plasma concentrations stabilized within the range reported previously for a 20 mg oral tablet. At 0.5 h, mean ± SD changes from baseline were −8.4 ± 11.7 mmHg (systolic pressure), −2.6 ± 7.3 mmHg (diastolic pressure) and −3.5 ± 10.4 beats min⁻¹ (heart rate). There was no symptomatic hypotension.

CONCLUSIONS

Although further research is warranted, a 10 mg sildenafil intravenous bolus appears to provide similar exposure, tolerability and safety to the 20 mg tablet.     

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5

BACKGROUND AND PURPOSE

By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5.

EXPERIMENTAL APPROACH

Using fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed.

KEY RESULTS

The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1 µM cGMP after approximately 10 min.

CONCLUSIONS AND IMPLICATIONS

We conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals.     

Quercetin and its principal metabolites, but not myricetin, oppose lipopolysaccharide-induced hyporesponsiveness of the porcine isolated coronary artery

BACKGROUND AND PURPOSE

Quercetin is anti-inflammatory in macrophages by inhibiting lipopolysaccharide (LPS)-mediated increases in cytokine and nitric oxide production but there is little information regarding the corresponding effect on the vasculature. We have examined the effect of quercetin, and its principal human metabolites, on inflammatory changes in the porcine isolated coronary artery.

EXPERIMENTAL APPROACH

Porcine coronary artery segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1 µg·mL⁻¹ LPS. Some segments were also co-incubated with quercetin-related flavonoids or Bay 11-7082, an inhibitor of NFκB. Changes in isometric tension of segments to vasoconstrictor and vasodilator agents were recorded. Nitrite content of the incubation solution was estimated using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically.

KEY RESULTS

Lipopolysaccharide reduced, by 35–50%, maximal contractions to KCl and U46619, thromboxane A₂ receptor agonist, and impaired endothelium-dependent relaxations to substance P. Nitrite content of the incubation medium increased 3- to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. Quercetin (0.1–10 µM) opposed LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. Similarly, 10 µM Bay 11-7082, 10 µM quercetin 3′-sulphate and 10 µM quercetin 3-glucuronide prevented LPS-induced changes, while myricetin (10 µM) was inactive. Myricetin (10 µM) prevented quercetin-induced modulation of LPS-mediated nitrite production.

CONCLUSION AND IMPLICATIONS

Quercetin, quercetin 3′-suphate and quercetin 3-glucuronide, exerted anti-inflammatory effects on the vasculature, possibly through a mechanism involving inhibition of NFκB. Myricetin-induced antagonism of the effect of anti-inflammatory action of quercetin merits further investigation.     

Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladder cancer cells

BACKGROUND AND PURPOSE

Sorafenib is an inhibitor of several intracellular signalling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in tumour cells. Sorafenib is used in the therapy of advanced renal cell carcinoma, and several phase II clinical trials are being carried out in patients with urothelial carcinomas.

EXPERIMENTAL APPROACH

Using a panel of human bladder cancer cell lines (RT4, T24, J82), we characterized systematically the effects of sorafenib on intracellular signalling, migration, proliferation and apoptosis.

KEY RESULTS

We demonstrated that at low concentrations (<1 µM), sorafenib is capable of significantly stimulating migration and proliferation of the bladder cancer cells. We hypothesize that these stimulatory effects on tumour cell functions might be explained by an activation of the Ras/ERK-1/2 signal transduction pathway. In addition, the comparison of different bladder cancer cell lines not only revealed a different biology (e.g. cell migration), but also a differential susceptibility to the anti-apoptotic effects of sorafenib. Finally, we confirmed in different bladder cancer cell lines the known inhibitory actions of sorafenib in pharmacological concentrations (≥3 µM) on ERK-1/2 phosphorylation, migration and proliferation, as well as the pro-apoptotic effects of the compound.

CONCLUSIONS AND IMPLICATIONS

Taken together, these findings suggest that although sorafenib has the potential to be used in the treatment of urothelial carcinoma, this compound might also activate bladder cancer cells at low concentrations. This should be relevant for dosing regiments to optimize the treatment with this promising anti-tumour drug.     

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.     

Release of ATP from rat urinary bladder mucosa: role of acid,vanilloids and stretch

Background and purpose:

ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs).

Experimental approach:

Rat mucosal strips (containing urothelium and lamina propria) in Perspex microbaths were superfused with Krebs solution. ATP was measured after exposure of matched strips to acid (pH 6.6–5.0), capsaicin (0.1–10 µM), EFS or stretch (150% of original length).

Key results:

Median basal ATP release was 3.46 nmol·g⁻¹. The mucosal strips responded to stimuli with potency order (median, IQR): acid (pH 5.6–6.0) 286 (103–555) > 10 µM capsaicin 188 (117–431) > 10 Hz EFS 63.0 (13.3–96.4) > stretch 24.4 (6.73–55.1) nmol ATP g⁻¹. ATP release in response to acid was pH dependent (P < 0.05). Responses to capsaicin did not desensitize nor were they concentration dependent. TRPV1 antagonist, capsazepine (10 µM) abolished capsaicin-evoked ATP release, and reduced acid-evoked (pH 6.5) release to 30% (P < 0.001). The ASIC channel antagonists gadolinium (0.1 mM) and amiloride (0.3 µM) reduced (P < 0.05) the acid-evoked (pH 6.5) release to 40 and 6.5% respectively. ASIC (ASIC1, ASIC2a, ASIC2b, ASIC3) and two TRPV1 gene products were detected in mucosal and detrusor extracts.

Conclusions and implications:

Capsaicin (at TRPV1) and acid (at both TRPV1 and ASIC) induce ATP release from the rat bladder mucosa. This ATP appears to be principally of urothelial origin. This study highlights the importance of ATP and acid as signalling molecules in modulating bladder function.     

Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta

Background and purpose:

Previous work has shown that N^G-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N^G, N^G-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.

Experimental approach:

The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.

Key results:

ADMA (100 µM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300–1000 µM) had no greater effect. Although ADMA (100 µM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 µM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 µM), protected ACh-induced relaxation against blockade by l-NAME (30 µM).

Conclusions and implications:

In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA.     

Riluzole protects against cardiac ischaemia and reperfusion damage via block of the persistent sodium current

Background and purpose:

Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP).

Experimental approach:

Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1–10 µM) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+]_i were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 µM were assessed in rat isolated myocytes using patch clamp techniques.

Key results:

In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 µM and produced rapid and almost complete recovery upon reperfusion from 3 and 10 µM. In isolated myocytes, riluzole at 3 and 10 µM greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+]_i and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC₅₀ of 2.7 µM, whereas the block of the transient sodium current was only apparent at concentrations above 30 µM.

Conclusions and implications:

Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.     

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     

Desensitization of the soluble guanylyl cyclase/cGMP pathway by lipopolysaccharide in rat isolated pulmonary artery but not aorta

Background and purpose:

To investigate the function of soluble guanylyl cyclase (sGC)/3′,5′-cyclic guanosine monophosphate (cGMP) pathway in lipopolysaccharide (LPS)-induced changes in vascular reactivity of rat isolated pulmonary artery and aorta.

Experimental approach:

Nitric oxide (NO) production, contraction responses to endothelin-1 (ET-1), relaxation responses to sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY412272 and T-0156, SNP-induced cGMP production and expression of sGC_α1, sGC_β1 and 3′,5′-cyclic nucleotide phosphodiesterase-5 (PDE5) proteins were measured in LPS-treated pulmonary and aortic rings from male Wistar rats.

Key results:

In both vessels, LPS (10 μg mL⁻¹, 20 h) increased NO production, which was inhibited by the selective inducible NOS (iNOS) inhibitor 1400W (1 μM). In the aorta, LPS decreased ET-1-induced contractility and this decrease was inhibited by the selective sGC inhibitor ODQ (10 μM) but not by removal of endothelium, or inhibitors of cyclooxygenase (indomethacin, 10 μM) or iNOS (1400W, 1 μM). Furthermore, aortic relaxation responses to the direct sGC activator BAY412272 were enhanced. In the pulmonary artery, SNP (1 nM to 30 μM)-induced relaxation and cGMP production, BAY412272-induced relaxation and sGC_β1 protein expression were decreased, whereas relaxation responses to the PDE5-specific inhibitor T-0156 (0.1–100 nM) were enhanced. Relaxation responses to the phosphodiesterase-resistant cGMP analogue, 8-pCPT-cGMP, and protein expression levels of sGC_α1 and PDE5 were not altered in either vessel.

Conclusion and implications:

LPS caused a selective hypocontractility of rat aorta to ET-1 mediated mainly through NO-independent sGC activation, whereas in the pulmonary artery, the effect of sGC activation was reduced by a decreased protein expression of sGC_β1 together with increased PDE5 activity.     

Hyperlipidaemia induced by a high-cholesterol diet leads to the deterioration of guanosine-3′,5′-cyclic monophosphate/protein kinase G-dependent cardioprotection in rats

Background and purpose:

Hyperlipidaemia interferes with cardioprotective mechanisms, but the cause of this phenomenon is largely unknown, although hyperlipidaemia impairs the cardioprotective NO–cGMP system. However, it is not known if natriuretic peptide–cGMP–protein kinase G (PKG) signalling is affected by hyperlipidaemia. Therefore, we investigated the cardioprotective efficacy of cGMP-elevating agents in hearts from normal and hyperlipidaemic rats.

Experimental approach:

Male Wistar rats were rendered hyperlipidaemic by feeding with 2% cholesterol-enriched chow for 12 weeks. Hearts isolated from normal and hyperlipidaemic rats were perfused (Langendorff mode) and subjected to 30 min occlusion of the left main coronary artery, followed by 120 min reperfusion. 8-Br-cGMP (CG, 10 nM), B-type natriuretic peptide-32 (BNP, 10 nM), S-nitroso-N-acetyl-penicillamine (SNAP, 1 µM) were perfused from 10 min prior to coronary occlusion until the 15th min of reperfusion. Infarct size (% of ischaemic risk zone) was determined by triphenyltetrazolium staining.

Key results:

Treatment with CG, SNAP or BNP decreased infarct size significantly in normal hearts from its control value of 41.6 ± 2.9% to 15.5 ± 2.4%, 23.3 ± 3.0% and 25.3 ± 4.6%, respectively (P < 0.05). Protection by BNP was abolished by co-perfusion of PKG inhibitors KT5823 (600 nM) or Rp-8pCPT-PET-cGMPs (1 µM), confirming its PKG dependence. In hearts from hyperlipidaemic rats, CG, SNAP or BNP failed to decrease infarct size. Hyperlipidaemia did not alter basal myocardial PKG content, but decreased its activity as assessed by phosphorylation of cardiac troponin I.

Conclusions and implications:

This is the first demonstration that defects in the cardioprotective cGMP–PKG system could be a critical biochemical anomaly in hyperlipidaemia.     

Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway

BACKGROUND AND PURPOSE

Hydrogen sulphide (H₂S) is a novel neuromodulator. The present study aimed to investigate the protective effect of H₂S against cell injury induced by 6-hydroxydopamine (6-OHDA), a selective dopaminergic neurotoxin often used to establish a model of Parkinson''s disease for studying the underlying mechanisms of this condition.

EXPERIMENTAL APPROACH

Cell viability in SH-SY5Y cells was measured using MTT assay. Western blot analysis and pharmacological manipulation were employed to study the signalling mechanisms.

KEY RESULTS

Treatment of SH-SY5Y cells with 6-OHDA (50–200 µM) for 12 h decreased cell viability. Exogenous application of NaHS (an H₂S donor, 100–1000 µM) or overexpression of cystathionine β-synthase (a predominant enzyme to produce endogenous H₂S in SH-SY5Y cells) protected cells against 6-OHDA-induced cell apoptosis and death. Furthermore, NaHS reversed 6-OHDA-induced loss of tyrosine hydroxylase. Western blot analysis showed that NaHS reversed the down-regulation of PKCα, ε and Akt and the up-regulation of PKCδ in 6-OHDA-treated cells. Blockade of PKCα with Gö6976 (2 µM), PKCε with EAVSLKPT (200 µM) or PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM) reduced the protective effects of H₂S. However, inhibition of PKCδ with rottlerin (5 µM) failed to affect 6-OHDA-induced cell injury. These data suggest that the protective effects of NaHS mainly resulted from activation of PKCα, ε and PI3K/Akt pathway. In addition, NaHS-induced Akt phosphorylation was significantly attenuated by Gö6976 and EAVSLKPT, suggesting that the activation of Akt by NaHS is PKCα, ε-dependent.

CONCLUSIONS AND IMPLICATIONS

H₂S protects SH-SY5Y cells against 6-OHDA-induced cell injury by activating the PKCα, ε/PI3K/Akt pathway.     

Time-dependent interactions of the hypotensive effects of sildenafil citrate and sublingual glyceryl trinitrate

AIM

To investigate the time course of the hypotensive interaction between sildenafil and glyceryl trinitrate (GTN).

METHODS

Two double-blind, placebo-controlled, randomized, crossover studies were performed. Subjects were challenged with sublingual GTN 400 µg at different times after oral sildenafil 100 mg. After each GTN challenge frequent measures of blood pressure (BP) were made. In the first study GTN was given 1–48 h after sildenafil/placebo to 33 healthy men. In the second study GTN was given 1–8 h after sildenafil/placebo to 20 men with stable angina.

RESULTS

In healthy men there was a greater mean maximum reduction in BP with sildenafil/GTN than with placebo/GTN only at 1 h. In angina patients, there was a greater mean maximum reduction in BP with sildenafil/GTN than with placebo/GTN for up to 8 h. The mean (95% confidence interval) differences in maximum systolic BP reduction (mmHg) at 1, 4, 6 and 8 h were −16 (−12, −21), −12 (−4, −20), −6 (1, −12) and −9 (−3, −15), all P < 0.05 except at 6 h (NS). At 6 and 8 h the interaction was not more than additive, and hypotensive symptoms did not occur.

CONCLUSIONS

In men with angina there is an interaction on BP reduction between sildenafil and GTN for ≥ 8 h after sildenafil administration, but this is no more than additive from 6 h. These data may be helpful to clinicians who are considering the use of GTN in patients presenting with angina who have received sildenafil within 24 h.