bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of gender and the oestrous cycle on in vitro contractile responses of the rat urinary bladder to cholinergic stimulation
Authors: Longhurst Penelope A , Levendusky Mark
Published: January 2005

Experiments were done to determine the influence of gender and the oestrous cycle on rat urinary bladder contractility in response to cholinergic stimulation.

Bladder strips from female rats responded to high frequency stimulation with smaller contractile responses than did strips from males, and to low concentrations of carbachol with greater responses. The decreased responsiveness of bladder strips from female rats to electrical field stimulation can be primarily attributed to the rats in the oestrous stage of the oestrous cycle.

Bladder strips from female rats in all stages of the oestrous cycle were more sensitive to carbachol than those from males, but there were no differences in sensitivity to electrical field stimulation.

The contractile responses of strips from both male and female rats to carbachol were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: 4‐DAMP>>pirenzepine>methoctramine, suggesting that the receptor mediating contraction was the M3 subtype. There were no differences in pA2 values between bladder strips from male and female rats.

The data indicate that responsiveness of bladder strips to electrical field stimulation and carbachol is altered in female rats in the oestrous stage of the oestrous cycle. Furthermore, gender influences the sensitivity of rat bladder to muscarinic stimulation.

British Journal of Pharmacology (2000) 131, 177–184; doi:10.1038/sj.bjp.0703551
Participation of thromboxane A2 in the cough response in guinea‐pigs: antitussive effect of ozagrel
Authors: Shinagawa Kazuhiko , Kojima Masami , Ichikawa Kiyoshi , Hiratochi Masahiro , Aoyagi Shigemi , Akahane Masuo
Published: January 2005

The purpose of this study was to investigate the involvement of thromboxane A2 (TXA2) in the cough response in a guinea‐pig cough model. Here, we describe results obtained using a selective TXA2 synthetase inhibitor, ozagrel, and a selective TXA2 agonist, U‐46619.

Guinea‐pigs were anaesthetized and exposed to an aerosol of capsaicin (100 μM) to elicit coughing. The number of coughs was 20.0±5.8 during capsaicin provocation (5 min), but only 2.8±0.4 during a 5‐min inhalation of phosphate‐buffered saline (PBS) (P<0.05).

TXB2 levels in BAL were 101.4±8.0 and 58.4±8.7 pg ml−1 following capsaicin and PBS inhalation, respectively (P<0.01), but there was no intergroup difference in the cell populations in BAL.

Inhalation of U‐46619 did not induce a cough response by itself at concentrations of 100 ng ml−1 to 10 μg ml−1. However, it caused a 2 fold increase in the number of capsaicin‐induced coughs.

To explore the source of the TXA2, BAL cells were stimulated with capsaicin and the supernatants collected for analysis. The TXB2 concentration in BAL was increased dose‐dependently, indicating that TXA2 is released from BAL cells in response to capsaicin.

Ozagrel was administered orally 1 h before a 5 min capsaicin provocation and the number of coughs was counted during the capsaicin inhalation. Ozagrel decreased the number of coughs dose‐dependently (ED50 value, 26.3 mg kg−1).

These results show that TXA2 modulates the capsaicin‐induced cough response by increasing capsaicin‐sensitivity.

British Journal of Pharmacology (2000) 131, 266–270; doi:10.1038/sj.bjp.0703553
A novel positive allosteric modulator of the GABAA receptor: the action of (+)‐ROD188
Authors: Thomet Urs , Baur Roland , Razet Rodolphe , Dodd Robert H , Furtmüller Roman , Sieghart Werner , Sigel Erwin
Published: January 2005

(+)‐ROD188 was synthesized in the search for novel ligands of the GABA binding site. It shares some structural similarity with bicuculline. (+)‐ROD188 failed to displace [3H]‐muscimol in binding studies and failed to induce channel opening in recombinant rat α1β2γ2 GABAA receptors functionally expressed in Xenopus oocytes.

(+)‐ROD188 allosterically stimulated GABA induced currents. Displacement of [3H]‐Ro15‐1788 indicated a low affinity action at the benzodiazepine binding site. In functional studies, stimulation by (+)‐ROD188 was little sensitive to the presence of 1 μM of the benzodiazepine antagonist Ro 15‐1788, and (+)‐ROD188 also stimulated currents mediated by α1β2, indicating a major mechanism of action different from that of benzodiazepines.

Allosteric stimulation by (+)‐ROD188 was similar in α1β2N265S as in unmutated α1β2, while that by loreclezole was strongly reduced.

(+)‐ROD188 also strongly stimulated currents elicited by either pentobarbital or 5α‐pregnan‐3α‐ol‐20‐one (3α‐OH‐DHP), in line with a mode of action different from that of barbiturates or neurosteroids as channel agonists.

Stimulation by (+)‐ROD188 was largest in α6β2γ2 (α6β2γ2>>α1β2γ2=α5β2γ2>α2β2γ2= α3β2γ2), indicating a unique subunit isoform specificity.

Miniature inhibitory postsynaptic currents (mIPSC) in cultures of rat hippocampal neurons, caused by spontaneous release of GABA showed a prolonged decay time in the presence of 30 μM (+)‐ROD188, indicating an enhanced synaptic inhibitory transmission.

British Journal of Pharmacology (2000) 131, 843–850; doi:10.1038/sj.bjp.0703558
Induction by inhibitors of nitric oxide synthase of hyperresponsiveness in the human nasal airway
Authors: Turner P J , Maggs J R L , Foreman J C
Published: January 2005

The effects of inhibitors of nitric oxide synthase (NOS) on the responsiveness of the human nasal airway were investigated, by measuring the nasal response to histamine and bradykinin.

Repeated intranasal administration of NG‐nitro‐L‐arginine methyl ester (L‐NAME) or NG‐monomethyl‐L‐arginine (L‐NMMA), 1 μmol per nostril every 30 min for 6 h, increased the nasal obstruction induced by histamine, 50–500 μg, and bradykinin, 200 μg per nostril. A single administration of L‐NAME, 1 μmol per nostril did not induce hyperresponsiveness to histamine.

Pretreatment with L‐arginine, 30 μmol, abolished the hyperresponsiveness to histamine caused by L‐NAME, 1 μmol. Pretreatment with NG‐nitro‐D‐arginine methyl ester (D‐NAME), 1 μmol, did not induce hyperresponsiveness to histamine.

Repeated administration of L‐NAME, 1 μmol, caused a significant reduction in the amount of nitric oxide measured in the nasal cavity.

Neither L‐NMMA, 1 μmol, nor L‐arginine, 30 μmol, altered the nasal hyperresponsiveness induced by platelet activating factor (PAF), 60 μg. PAF did not alter the levels of nitric oxide in the nasal cavity.

The results suggest that inhibition of nitric oxide synthase induces a hyperresponsiveness in the human nasal airway, and that this occurs by a mechanism different from that involved in PAF‐induced hyperresponsiveness.

British Journal of Pharmacology (2000) 131, 363–369; doi:10.1038/sj.bjp.0703561
Differential role of angiotensin II receptor subtypes on endothelial superoxide formation
Authors: Sohn Hae Young , Raff Ulrike , Hoffmann Anke , Gloe Torsten , Heermeier Kathrin , Galle Jan , Pohl Ulrich
Published: January 2005

The physiological role of the angiotensin II AT2 receptor subtype is not fully characterized. We studied whether AT2 receptor could antagonize AT1 mediated superoxide formation in endothelial cells.

In quiescent human umbilical vein endothelial cells (HUVEC) superoxide formation was measured after long‐term incubation (6 h) with angiotensin II in the presence or absence of its receptor blocker candesartan (AT1) or PD123319 (AT2) using the cytochrome c assay. In separate experiments, the effects of AT2 mediated effects on activities of cellular phosphates including the src homology 2 domain containing phosphatases (SHP‐1) was studied.

The basal superoxide formation (0.19±0.03 nmol superoxide mg protein−1 min−1) in HUVEC was increased by 37.1% after exposure to angiotensin II (100 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 μM) as well as the tyrosine kinase inhibitor genistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the superoxide formation by 73.7% in intact cells. Stimulation of AT2 went along with an increased activity of tyrosine phosphatases in total cell lysates (29.8%) and, in particular, a marked stimulation of src homology 2 domain containing phosphatases (SHP‐1, by 293.4%). The tyrosine phosphatase inhibitor vanadate, in turn, prevented the AT2 mediated effects on superoxide formation. The expression of both angiotensin II receptor subtypes AT1 and AT2 was confirmed by RT–PCR analysis.

It is concluded that AT2 functionally antagonizes the AT1 induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.

British Journal of Pharmacology (2000) 131, 667–672; doi:10.1038/sj.bjp.0703566
Methyl p‐hydroxybenzoate (E‐218) a preservative for drugs and food is an activator of the ryanodine receptor Ca2+ release channel
Authors: Cavagna Dario , Zorzato Francesco , Babini Elena , Prestipino Gianfranco , Treves Susan
Published: January 2005

Haloperidol is a drug used in the management of several psychotic disorders and its use has been linked to Neuroleptic Malignant Syndrome. In the present study we have investigated the effect of a commercial preparation of haloperidol, Serenase, on skeletal muscle sarcoplasmic reticulum.

Addition of Serenase to isolated terminal cisternae caused a rapid release of calcium. We tested whether the active Ca2+‐releasing substance was haloperidol or another compound present in the preparation.

Our results show that methyl p‐hydroxybenzoate, one of the preservatives and a commonly used anti‐microbial agent (E‐218) is an activator of Ca2+ release (E.C.50=2.0 mM), mediated by a ruthenium red‐sensitive Ca2+ release channel present in skeletal muscle terminal cisternae.

British Journal of Pharmacology (2000) 131, 335–341; doi:10.1038/sj.bjp.0703571
A novel, competitive mGlu5 receptor antagonist (LY344545) blocks DHPG‐induced potentiation of NMDA responses but not the induction of LTP in rat hippocampal slices
Authors: Doherty A J , Palmer M J , Bortolotto Z A , Hargreaves A , Kingston A E , Ornstein P L , Schoepp D D , Lodge D , Collingridge G L
Published: January 2005

We have investigated the pharmacological properties of LY344545, a structurally related epimer of the broad spectrum competitive metabotropic glutamate receptor antagonist, LY341495. We have found that LY344545 also antagonizes competitively nearly all mGlu receptor subtypes, but with a wide spectrum of activity. The order of potency for the human receptor isoforms was mGlu5a (IC50 of 5.5±0.6 μM)>mGlu2=mGlu3>mGlu1α=mGlu7>mGlu6=mGlu8. No significant mGlu4 receptor antagonist activity was detected at the highest concentration used (100 μM). 100 μM LY344545 displaced 50±5% of [3H]‐CGP39653 binding, but less than 30% of [3H]‐kainate or [3H]‐AMPA in radioligand binding assays.

LY344545 antagonized L‐glutamate stimulated Ca2+ release in CHO cells transfected with mGlu receptors in a concentration dependent manner with a 10 fold higher affinity for the rat mGlu5a receptor (Ki=2.1±0.6 μM) compared to the rat mGlu1α receptor (Ki=20.5±2.1 μM). 50 μM (1S, 3R)‐ACPD‐induced Ca2+ rises in hippocampal CA1 neurones were also antagonized (IC50=6.8±0.7 μM).

LY344545 antagonized 10 μM (S)‐3,5‐DHPG‐induced potentiation of NMDA depolarizations in CA1 neurones (EC50=10.6±1.0 μM). At higher concentrations (100 μM), LY344545 was an NMDA receptor antagonist.

LY344545 also blocked the induction, but not the expression, of LTP at CA3 to CA1 synapses with an IC50>300 μM. This effect is consistent with its weak activity at NMDA receptors.

These results demonstrate that the binding of ligands to mGlu receptor subtypes is critically dependent on the spatial orientation of the same molecular substituents within a given chemical pharmacophore. The identification of LY344545 as the first competitive antagonist to show selectivity towards mGlu5 receptors supports the potential to design more selective and potent competitive antagonists of this receptor.

These results further indicate that mGlu receptor‐mediated potentiation of NMDA responses is not essential for the induction of LTP.

British Journal of Pharmacology (2000) 131, 239–244; doi:10.1038/sj.bjp.0703574
Arachidonic acid‐mediated cooxidation of all‐trans‐retinoic acid in microsomal fractions from human liver
Authors: Nadin Louise , Murray Michael
Published: January 2005

The quantitative importance of prostaglandin H synthase (PGHS)‐mediated cooxidation of all‐trans‐retinoic acid (ATRA) was evaluated in human liver microsomes (n=17) in relation to CYP‐dependent ATRA 4‐hydroxylation.

Observed rates of ATRA cooxidation (4.6–20 pmol mg protein−1 min−1) and 4‐hydroxylation (8.7–45 pmol mg protein−1 min−1) were quantitatively similar and exhibited similar individual variation (4 and 5 fold, respectively).

From kinetic studies cooxidation was an efficient process in human hepatic microsomes (VmaxKm−1=0.25) compared with NADPH‐ and NADH‐mediated 4‐hydroxylation by CYP (VmaxKm−1=0.14 and 0.02, respectively).

The capacity of lipid hydroperoxide metabolites of arachidonic acid to mediate ATRA oxidation was established directly, but downstream products (D, E, F and I‐series prostaglandins) were inactive.

cDNA‐expressed CYPs supported ATRA oxidation by lipid hydroperoxides. Whereas CYPs 2C8, 2C9 and 3A4, but not CYPs 1A2 or 2E1, were effective catalysts of the NADPH‐mediated reaction, cooxidation supported by 15(S)‐hydroperoxyeicosatetraenoic acid was mediated by all five CYPs. The cooxidation reaction in human hepatic microsomes was inhibited by the CYP inhibitor miconazole.

These findings indicate that ATRA oxidation is quantitatively significant in human liver. Lipid hydroperoxides generated by intracellular enzymes such as prostaglandin synthase and lipoxygenases are sources of activated oxygen for CYP‐mediated deactivation of ATRA to polar products.

British Journal of Pharmacology (2000) 131, 851–857; doi:10.1038/sj.bjp.0703579
Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum
Authors: Chen Lan‐Ying , Chen Xi , Tian Xiao‐Li , Yu Xiao‐Hong
Published: January 2005

To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR).

Calcium uptake was monitored with a dual‐wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively.

128 μmol l−1 Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un‐competitively inhibited SR Ca2+,Mg2+‐ATPase activity, causing the stoichiometric ratio of SR Ca2+/ATP to decrease to 1.43 from 2.0 of control.

Inhibitory rates on SR Ca2+,Mg2+‐ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l−1 ATP to 60% in 5 mmol l−1 ATP.

Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)‐modified protein fluorescence of SR labelled with N‐(3‐pyrene)‐maleimide.

Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7‐ or 12‐(9‐anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1,6‐diphenyl‐1,3,5‐hexatrine (DPH).

These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca2+,Mg2+‐ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca2+]i and myocardial contractility.

British Journal of Pharmacology (2000) 131, 530–536; doi:10.1038/sj.bjp.0703582
Cefaclor, a cephalosporin antibiotic, delays gastric emptying rate by a CCK‐A receptor‐mediated mechanism in the rat
Authors: Bozkurt Ayhan , Deniz Mustafa , Yegen Berrak Ç
Published: January 2005

Studies in vitro suggest that cephalosporin antibiotics release the gut hormone cholecystokinin. Cholecystokinin is known to inhibit gastric emptying. Here we examine the effects of cefaclor on gastric emptying and intestinal motility.

Male Sprague‐Dawley rats were fitted with gastric cannulas. Following a 3‐week recovery, the rate of gastric emptying of saline, peptone (4.5%) or cefaclor was determined after instillation into the gastric cannula, while intestinal transit was measured by using the propagation of arabic gum + charcoal mixture given intraduodenally.

Gastric emptying of saline was significantly delayed by the addition of cefaclor (3, 10, 30 or 100 mM). The CCK‐A antagonist SR‐27897B (1 mg kg−1, i.p.) reversed the delay induced by 10 mM cefaclor, whereas the CCK‐B antagonist CI‐988 (1 mg kg−1, i.p.) had no significant effect. In capsaicin‐treated rats, 10 mM cefaclor emptied more rapidly than in vehicle‐treated animals.

Thirty‐minute intestinal transit was increased at 30 and 100 mM of cefaclor, while the gastric acid secretion following cefaclor instillation was no different than the group which received saline.

The cephalosporin antibiotic cefaclor appears to be a potent stimulant of CCK release from gut endocrine cells, resembling the effects of peptone. Cefaclor delays gastric emptying via capsaicin‐sensitive afferent pathways, which involve CCK‐A receptor interaction.

British Journal of Pharmacology (2000) 131, 399–404; doi:10.1038/sj.bjp.0703585
The effects of caffeine on ATP‐sensitive K+ channels in smooth muscle cells from pig urethra
Authors: Teramoto Noriyoshi , Yunoki Takakazu , Tanaka Kiyoshi , Takano Makoto , Masaki Ichiro , Yonemitsu Yoshikazu , Sueishi Katsuo , Ito Yushi
Published: January 2005

The effects of caffeine on both levcromakalim‐induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch‐clamp techniques (conventional whole‐cell configuration and cell‐attached configuration). The effects of caffeine were also examined on currents in inside‐out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2ΔC36) which form ATP‐sensitive K+ channels (KATP channels).

In conventional whole‐cell configuration, the levcromakalim (100 μM)‐induced inward current (symmetrical 140 mM K+ conditions) was inhibited by caffeine (1 mM) at a holding potential of −50 mV. In contrast, ryanodine (10 μM) caused no significant inhibitory effect on the gradual decay of the levcromakalim‐induced current at −50 mV.

The amplitude of the 30 μM levcromakalim‐induced current was enhanced by 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM).

In cell‐attached configuration, the levcromakalim‐induced K+ channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at −50 mV.

Reverse transcriptase‐polymerase chain reaction (RT–PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra.

Caffeine (3 mM) inhibited the channel activity of Kir6.2ΔC36 expressed in COS7 cells (3 mM caffeine, 65±6%, n=4; 10 mM caffeine, 29±2%, n=4).

These results suggest that caffeine can inhibit the activity of KATP channels through a direct blocking effect on the pore‐forming Kir subunit.

British Journal of Pharmacology (2000) 131, 505–513; doi:10.1038/sj.bjp.0703586
Airway reactivity, inflammatory cell influx and nitric oxide in guinea‐pig airways after lipopolysaccharide inhalation
Authors: Toward Toby J , Broadley Kenneth J
Published: January 2005

The aim of this study was to investigate the relationship between airway reactivity, leukocyte influx and nitric oxide (NO), in conscious guinea‐pigs after aerosolized lipopolysaccharide (LPS) exposure.

Inhaled histamine (1 mM, 20 s), causing no bronchoconstriction before LPS exposure (30 μg ml−1, 1 h), caused bronchoconstriction at 0.5 and 1 h (P<0.02) after LPS exposure. This airway hyperreactivity (AHR) recovered by 2 h. In contrast, 48 h after LPS exposure, the response from a previously bronchoconstrictor dose of histamine (3 mM, 20 s) was attenuated (P<0.01) i.e. airway hyporeactivity (AHOR).

Investigation of the cellular content of bronchoalveolar lavage fluid (BALF) from these animals revealed a rapid (0.5 h: 691 fold increase) and progressive neutrophil influx after LPS exposure (24 h: 36.3±2.3×106 cells per sample), that subsided 48 h later. Macrophages and eosinophils also time‐dependently increased (0.5 h: 4.6±0.4 and 0.1±0.05; 48 h: 31.0±6.0 and 1.8±0.3×106 cells per sample, respectively) after LPS, compared to vehicle exposure (24 h: neutrophils, eosinophils and macrophages: 0.28±0.19, 0.31±0.04 and 4.96±0.43×106 cells per sample, respectively).

The combined NO metabolites in BALF, after vehicle (1 h), or LPS (1 h: AHR and 48 h: AHOR) exposure, were respectively increased (41%, P<0.01), decreased (47%, P<0.01) and further increased (80%, P<0.001), compared with naïve animals.

Inhaled No‐nitro‐L‐arginine methyl ester (L‐NAME: 1.2 and 12 mM, 15 min), reduced BALF NO metabolites 2 h later, but did not cause AHR to histamine (P>0.05). When L‐NAME inhalation followed LPS, AHR was prolonged from 1 h to at least 4 h (P<0.01).

In summary, aerosolized LPS inhalation caused neutrophil and macrophage airways infiltration, and an early development of AHR followed 48 h later by AHOR to histamine. AHR and AHOR coincided with a respective reduction and elevation in airways NO (metabolites). Thus, NO may aid recovery from AHR, as inhibition of its production prolongs AHR. However, NO deficiency alone is not responsible for LPS‐induced AHR.

British Journal of Pharmacology (2000) 131, 271–281; doi:10.1038/sj.bjp.0703589
Dual modulation by thrombin of the motility of rat oesophageal muscularis mucosae via two distinct protease‐activated receptors (PARs): a novel role for PAR‐4 as opposed to PAR‐1
Authors: Kawabata Atsufumi , Kuroda Ryotaro , Kuroki Naoko , Nishikawa Hiroyuki , Kawai Kenzo
Published: January 2005

Since protease‐activated receptors (PARs) are distributed throughout the gastrointestinal tract, we investigated the role of PARs in modulation of the motility of the rat oesophageal muscularis mucosae.

Thrombin produced contraction of segments of the upper and lower part of the smooth muscle. Trypsin contracted both the muscle preparations only at high concentrations. SFLLR‐NH2 and TFLLR‐NH2 (PAR‐1‐activating peptides), but not the PAR‐1‐inactive peptide FSLLR‐NH2, evoked a marked contraction. In contrast, the PAR‐2 agonist SLIGRL‐NH2 and the PAR‐4 agonist GYPGKF‐NH2 caused no or only a negligible contraction.

In oesophageal preparations precontracted with carbachol, thrombin produced a dual action i.e. relaxation followed by contraction. TFLLR‐NH2 further contracted the precontracted preparations with no preceding relaxation. GYPGKF‐NH2, but not the inactive peptide GAPGKF‐NH2, produced marked relaxation. Trypsin or SLIGRL‐NH2 caused no relaxation.

The PAR‐1‐mediated contraction was completely abolished in Ca2+‐free medium and considerably attenuated by nifedipine (1 μM) and in a low Na+ medium. The PAR‐4‐mediated relaxation was resistant to tetrodotoxin (10 μM), apamin (0.1 μM), charybdotoxin (0.1 μM), L‐NG‐nitroarginine methyl ester (100 μM), indomethacin (3 μM), propranolol (5 μM) or adenosine 3′,5′‐cyclic monophosphorothioate, 8‐bromo, Rp‐isomer (30 μM).

Thus, thrombin plays a dual role in modulating the motility of the oesophageal muscularis mucosae, producing contraction via PAR‐1 and relaxation via PAR‐4. The PAR‐1‐mediated effect appears to occur largely through increased Na+ permeability followed by activation of L‐type Ca2+ channels and subsequent influx of extracellular Ca2+. Our data could provide evidence for a novel role of PAR‐4 as opposed to PAR‐1, although the underlying mechanisms are still open to question.

British Journal of Pharmacology (2000) 131, 578–584; doi:10.1038/sj.bjp.0703590
Differences in the effects of urinary incontinence agents S‐oxybutynin and terodiline on cardiac K+ currents and action potentials
Authors: Jones Stephen E , Shuba Lesya M , Zhabyeyev Pavel , McCullough John R , McDonald Terence F
Published: January 2005

The cardiac electrophysiological effects of S‐oxybutynin, a single‐enantiomer drug under evaluation for the management of urinary incontinence, have been investigated and compared with those of terodiline, an incontinence agent withdrawn following reports of QT lengthening and ventricular tachyarrhythmia. Membrane currents were recorded from whole‐cell configured guinea‐pig and rabbit ventricular myocytes, and action potentials were recorded from guinea‐pig and rabbit papillary muscles.

L‐type Ca2+ current (ICa,L), rapidly‐activating K+ current (IKr) and slowly‐activating K+ current (IKs) were unaffected by submicromolar S‐oxybutynin and inhibited by higher concentrations; IC50 values were 17.8 μM for ICa,L, 12 μM for IKr, and 41 μM for IKs. Terodiline IC50 values were somewhat lower for ICa,L (15.2 μM) and IKs (30 μM), but 24 fold lower in the case of IKr (0.5 μM).

The durations of action potentials in guinea‐pig and rabbit papillary muscles driven at 1 Hz were unaffected or moderately shortened by 0.1–100 μM S‐oxybutynin, but lengthened by terodiline. Terodiline (10 μM) also depressed maximal upstroke velocity.

The action potential plateau shortened by an average of 23% when control rabbit papillary muscles were driven at 0.4 Hz instead of 1 Hz. Plateau shortening was significantly smaller in the presence of drugs (30 μM S‐oxybutynin, 3 and 30 μM terodiline), suggesting that they suppress the transient outward current (Ito) involved in rate‐dependent shortening. In experiments on rabbit ventricular myocytes, 3 and 30 μM S‐oxybutynin inhibited Ito by 9±2% and 35±3%, respectively, whereas 3 and 30 μM terodiline inhibited the current by 31±3% and 87±3%, respectively.

The results indicate that S‐oxybutynin has relatively weak non‐specific effects on cardiac ion channels, and that clinically relevant submicromolar concentrations are unlikely to have terodiline‐like proarrhythmic actions on the myocardium.

British Journal of Pharmacology (2000) 131, 245–254; doi:10.1038/sj.bjp.0703595
Influence of β‐adrenoceptor tone on the cardioprotective efficacy of adenosine A1 receptor activation in isolated working rat hearts
Authors: Ford William R , Jugdutt Bodh I , Lopaschuk Gary D , Schulz Rick , Clanachan Alexander S
Published: January 2005

This study investigated the role of β‐adrenoceptors in the cardioprotective and metabolic actions of adenosine A1 receptor stimulation.

Isolated paced (300 beats min−1) working rat hearts were perfused with Krebs‐Henseleit solution containing 1.2 mM palmitate. Left ventricular minute work (LV work), O2 consumption and rates of glycolysis and glucose oxidation were measured during reperfusion (30 min) following global ischaemia (30 min) as well as during aerobic conditions.

Relative to untreated hearts, N6‐cyclohexyladenosine (CHA, 0.5 μM) improved post‐ischaemic LV work (158%) and reduced glycolysis and proton production (53 and 42%, respectively). CHA+propranolol (1 μM) had similar beneficial effects, while propranolol alone did not affect post‐ischaemic LV work or glucose metabolism. Isoprenaline (10 nM) impaired post‐ischaemic function and after 25 min ischaemia recovery was comparable with 30 min ischaemia in untreated hearts (41 and 53%, respectively). Relative to isoprenaline alone, CHA+isoprenaline improved recovery of LV work (181%) and reduced glycolysis and proton production (64 and 60%, respectively).

In aerobic hearts, CHA, propranolol or CHA+propranolol had no effect on LV work or glucose oxidation. Glycolysis was inhibited by CHA, propranolol and CHA+propranolol (50, 53 and 52%, respectively). Isoprenaline‐induced increases in heart rate, glycolysis and proton production were attenuated by CHA (85, 57 and 53%, respectively).

The cardioprotective efficacy of CHA was unaffected by antagonism or activation of β‐adrenoceptors. Thus, the mechanism of protection by adenosine A1 receptor activation does not involve functional antagonism of β‐adrenoceptors.

British Journal of Pharmacology (2000) 131, 537–545; doi:10.1038/sj.bjp.0703597
Potent stimulation and inhibition of the CFTR Cl− current by phloxine B
Authors: Bachmann Alexander , Russ Ulrich , Waldegger Siegfried , Quast Ulrich
Published: January 2005

The effects of the fluoresceine derivative, phloxine B, on the Cl− current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR.

In whole oocytes, the CFTR Cl− current (ICFTR) was activated by superfusion with isobutylmethylxanthine and forskolin. ICFTR was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited ICFTR weakly.

In excised inside‐out macropatches, ICFTR was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01–3 μM), applied after activation, increased ICFTR within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations.

The effect of phloxine B after 30 s had a bell‐shaped concentration‐dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC50 value of ∼1 μM.

In the absence of cPKA, phloxine B did not stimulate ICFTR. In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM).

The data show that phloxine B modulates ICFTR by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell.

British Journal of Pharmacology (2000) 131, 433–440; doi:10.1038/sj.bjp.0703600
Orphan‐receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin‐1
Authors: Maguire Janet J , Kuc Rhoda E , Davenport Anthony P
Published: January 2005

We have determined the distribution of receptors for human urotensin‐II (U‐II) in human and rat CNS and peripheral tissues.

In rat, [125I]‐U‐II binding density was highest in the abducens nucleus of brainstem (139.6±14 amol mm−2). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22.5±6 amol mm−2).

In human tissues density was highest in skeletal muscle and cerebral cortex (∼30 amol mm−2), with lower levels (<15 amol mm−2) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma.

Receptor density was less in human coronary artery smooth muscle (14.6±3 amol mm−2, n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels.

In human skeletal muscle [125I]‐U‐II bound to a single receptor population with KD=0.24±0.17 nM and Bmax=1.97±1.1 fmol mg−1 protein (n=4).

U‐II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub‐nanomolar EC50 values. U‐II was 50 times more potent in arteries and <10 times more potent in veins than endothelin‐1 (ET‐1). The maximum response to U‐II (∼20% of control KCl) was significantly less than to ET‐1 (∼80% KCl). In contrast, in rat aorta, U‐II and ET‐1 were equipotent with similar maximum responses.

This is the first report of high affinity receptors for [125I]‐U‐II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U‐II in human physiology.

British Journal of Pharmacology (2000) 131, 441–446; doi:10.1038/sj.bjp.0703601
Vagal neurotransmission to the ferret lower oesophageal sphincter: inhibition via GABAB receptors
Authors: Smid S D , Blackshaw L A
Published: January 2005

GABAB receptors modulate the function of the lower oesophageal sphincter (LOS) in vivo by inhibiting neurotransmitter release in the vagal pathway controlling LOS relaxation. We aimed to determine whether this effect was mediated peripherally on vagal motor outflow to the ferret LOS in vitro.

The LOS, with intact vagal innervation, was prepared from adult ferrets and LOS tension measured. Vagal stimulation (0.5–10 Hz, 30 V) evoked a tetrodotoxin‐sensitive, frequency‐dependent relaxation.

Both GABA (3×10−4 M) and (±)baclofen (2×10−4 M) inhibited vagally‐stimulated LOS relaxation. The potent GABAB receptor‐selective agonist 3‐APPA dose‐dependently inhibited vagally‐stimulated LOS relaxation, with an EC50 value of 0.7 μM

Decreased responses following vagal stimulation in the presence of (±)baclofen or 3‐APPA were reversed with the potent GABAB receptor antagonist CGP 62349.

Neither CGP 62349 nor muscimol (GABAA receptor agonist) alone affected LOS responses following vagal stimulation.

Agonists of other G protein‐coupled receptors (clonidine (α2‐adrenoceptor) (5×10−6 M), U50488 (κ opioid) (10−5 M), neuropeptide Y (10−6 M)) did not affect vagally‐mediated LOS relaxation.

The present study supports a discrete presynaptic inhibitory role for GABAB receptors on vagal preganglionic fibres serving inhibitory motorneurones in the ferret LOS.

British Journal of Pharmacology (2000) 131, 624–630; doi:10.1038/sj.bjp.0703602
Pharmacological analysis by HOE642 and KB‐R9032 of the role of Na+/H+ exchange in the endothelin‐1‐induced Ca2+ signalling in rabbit ventricular myocytes
Authors: Wang Hao , Sakurai Kiyoharu , Endoh Masao
Published: January 2005

The role of Na+/H+ exchange in endothelin‐1 (ET‐1)‐induced increases in Ca2+ transients and cell shortening was studied in rabbit ventricular myocytes loaded with indo‐1/AM. Selective inhibitors of Na+/H+ exchange HOE642 (4‐isopropyl‐3‐methyl‐sulphonylbenzoyl guanidine methanesulphonate) and KB‐R9032 (N‐(4‐isopropyl‐2,2‐dimethyl‐3‐oxo‐3,4‐dihydro‐2H‐benzo‐[1,4]oxazine‐6‐carbonyl) guanidine methanesulphonate) were used as pharmacological tools for the analysis.

ET‐1 at 0.1 nM induced an increase in Ca2+ transients by 45.6%, while it increased cell shortening by 109.6%. For a given increase in cell shortening, the ET‐1‐induced increase in Ca2+ transients was much smaller than that induced by isoprenaline (ISO, 10 nM).

Pretreatment with HOE642 and KB‐R9032 (1 μM) inhibited the increase in cell shortening induced by 0.1 nM ET‐1 by 51 and 65.4%, respectively, without a significant alteration of ET‐1‐induced increase in Ca2+ transients. HOE642 and KB‐R9032 did not affect baseline levels of cell shortening and peak Ca2+ transients, and the effects of ISO (10 nM).

These results indicate that activation of Na+/H+ exchange by ET‐1 may play an important role in the positive inotropic effect and the ET‐1‐induced increase in myofilament Ca2+ sensitivity in rabbit ventricular myocytes.

British Journal of Pharmacology (2000) 131, 638–644; doi:10.1038/sj.bjp.0703608
The bee venom peptide tertiapin underlines the role of IKACh in acetylcholine‐induced atrioventricular blocks
Authors: Drici Milou‐Daniel , Diochot Sylvie , Terrenoire Cécile , Romey Georges , Lazdunski Michel
Published: January 2005

Acetylcholine (ACh) is an important neuromodulator of cardiac function that is released upon stimulation of the vagus nerve. Despite numerous reports on activation of IKACh by acetylcholine in cardiomyocytes, it has yet to be demonstrated what role this channel plays in cardiac conduction. We studied the effect of tertiapin, a bee venom peptide blocking IKACh, to evaluate the role of IKACh in Langendorff preparations challenged with ACh.

ACh (0.5 μM) reproducibly and reversibly induced complete atrioventricular (AV) blocks in retroperfused guinea‐pig isolated hearts (n=12).

Tertiapin (10 to 300 nM) dose‐dependently and reversibly prevented the AV conduction decrements and the complete blocks in unpaced hearts (n=8, P<0.01).

Tertiapin dose‐dependently blunted the ACh‐induced negative chronotropic response from an ACh‐induced decrease in heart rate of 39±16% in control conditions to 3±3% after 300 nM tertiapin (P=0.01). These effects were not accompanied by any significant change in QT intervals.

Tertiapin blocked IKACh with an IC50 of 30±4 nM with no significant effect on the major currents classically associated with cardiac repolarisation process (IKr, IKs, Ito1, Isus, IK1 or IKATP) or AV conduction (INa and ICa(L)).

In summary, tertiapin prevents dose‐dependently ACh‐induced AV blocks in mammalian hearts by inhibiting IKACh.

British Journal of Pharmacology (2000) 131, 569–577; doi:10.1038/sj.bjp.0703611
Cyclic GMP‐independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor
Authors: Homer Kerry L , Wanstall Janet C
Published: January 2005

In rat pulmonary artery pre‐contracted with phenylephrine, the mechanisms of relaxation to the nitric oxide (NO) donor, spermine NONOate, were investigated.

Responses to spermine NONOate were only partially blocked by the soluble guanylate cyclase inhibitor, ODQ (1H‐[1,2,4]Oxadiazolo‐[4,3,‐a]quinoxalin‐1‐one) at concentrations up to 30 μM. Ten μM ODQ gave maximal inhibition. Endothelium removal had no effect on the potency of spermine NONOate or its inhibition by ODQ.

The protein kinase G inhibitor, Rp‐8‐Br‐cGMPS (100 μM), caused minimal inhibition of spermine NONOate despite causing marked inhibition of glyceryl trinitrate and isosorbide dinitrate.

Spermine NONOate (100 μM) caused a 35 fold increase in guanosine 3′5′ cyclic monophosphate (cyclic GMP) above basal levels in pulmonary artery rings. ODQ (3 μM) abolished this cyclic GMP production but did not inhibit corresponding relaxant responses. Similar results were seen with another NONOate (MAHMA NONOate; 10 μM).

ODQ‐resistant relaxation to spermine NONOate (i.e. relaxation seen in the presence of 10 μM ODQ) was inhibited by potassium (80 mM), charybdotoxin (300 nM), iberiotoxin (300 nM), apamin (100 nM), ouabain (1 mM) or thapsigargin (100 nM) but not by 4‐aminopyridine (3 mM), glybenclamide (10 μM) or diltiazem (10 μM).

Potassium, charybdotoxin, ouabain and thapsigargin also inhibited ODQ‐resistant relaxation to FK409 ((±)‐E‐4‐ethyl‐2‐[E‐hydroxyimino]‐5‐nitro‐3‐hexenamide).

We conclude that, on rat pulmonary artery, spermine NONOate can produce cyclic GMP‐independent relaxation that involves, at least in part, activation of Na+/K+‐ATPase, sarco‐endoplasmic reticulum Ca2+‐ATPase and calcium‐activated potassium channels.

British Journal of Pharmacology (2000) 131, 673–682; doi:10.1038/sj.bjp.0703613
5‐HT4 receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle
Authors: Prins N H , Akkermans L M A , Lefebvre R A , Schuurkes J A J
Published: January 2005

5‐HT4 receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5‐HT4 receptor agonists in vivo. We set out to characterize 5‐HT4 receptor‐mediated effects in longitudinal muscle strips of canine and human large intestine.

Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L‐NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide‐induced relaxation to EFS.

The selective 5‐HT4 receptor agonist prucalopride (0.3 μM) enhanced EFS‐evoked contractions, that were antagonized in both preparations by the selective 5‐HT4 receptor antagonist GR 113808 (0.1 μM). The prucalopride‐induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 μM) or tetrodotoxin (0.3 μM) inhibited EFS‐induced contractions, which were then unaffected by prucalopride (0.3 μM) in both tissues.

In the presence of methysergide (3 μM; both tissues) and granisetron (0.3 μM; only human tissues), 5‐HT (0.3 μM) enhanced EFS‐induced contractions, an effect that was antagonized by GR 113808 (0.1 μM). In the presence of atropine or tetrodotoxin, EFS‐induced contractions were inhibited, leaving 5‐HT (0.3 μM) ineffective in both preparations.

This study demonstrates for the first time that in human and canine large intestine, 5‐HT4 receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5‐HT4 receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

British Journal of Pharmacology (2000) 131, 927–932; doi:10.1038/sj.bjp.0703615
Evidence that PAR‐1 and PAR‐2 mediate prostanoid‐dependent contraction in isolated guinea‐pig gallbladder
Authors: Tognetto Michele , Trevisani Marcello , Maggiore Barbara , Navarra Giuseppe , Turini Alessandro , Guerrini Remo , Bunnett Nigel W , Geppetti Pierangelo , Harrison Selena
Published: January 2005

We have investigated the ability of protease‐activated receptor‐1 (PAR‐1), PAR‐2, PAR‐3 and PAR‐4 agonists to induce contractile responses in isolated guinea‐pig gallbladder. Thrombin, trypsin, mouse PAR‐1 activating (SFLLRN‐NH2) peptide, and mouse PAR‐2 activating (SLIGRL‐NH2) and human PAR‐2 activating (SLIGKV‐NH2) peptides produced a concentration‐dependent contractile response.

Mouse PAR‐4 activating (GYPGKF‐NH2) peptide, the mouse PAR‐1 reverse (NRLLFS‐NH2) peptide, the mouse PAR‐2 reverse (LRGILS‐NH2) and human PAR‐2 reverse (VKGILS‐NH2) peptides caused negligible contractile responses at the highest concentrations tested.

An additive effect was observed following the contractile response induced by either trypsin or thrombin, with the addition of a different PAR agonist (SFLLRN‐NH2 and SLIGRL‐NH2, respectively). Desensitization to PAR‐2 activating peptide attenuated the response to trypsin but failed to attenuate the response to PAR‐1 agonists, and conversely desensitization to PAR‐1 attenuated the response to thrombin but failed to alter contractile responses to PAR‐2 agonists.

The contractile responses produced by thrombin, trypsin, SFLLRN‐NH2 and SLIGRL‐NH2 were markedly reduced in the presence of the cyclo‐oxygenase inhibitor, indomethacin, whilst the small contractile response produced by NRLLFS‐NH2 and LRGILS‐NH2 were insensitive to indomethacin.

The contractile responses to thrombin, trypsin, SFLLRN‐NH2 and SLIGRL‐NH2 were unaffected by the presence of: the non‐selective muscarinic antagonist, atropine; the nitric oxide synthase inhibitor, L‐NAME; the sodium channel blocker, tetrodotoxin; the combination of selective tachykinin NK1 and NK2 receptor antagonists, (S)‐1‐[2‐[3‐(3,4‐dichlorphenyl)‐1 (3‐isopropoxyphenylacetyl) piperidin‐3‐yl] ethyl]‐4‐phenyl‐1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)‐N‐methyl‐N‐[4‐acetylamino‐4‐phenylpiperidino‐2‐(3,4‐dichlorophenyl)‐butyl] benzamide (SR48968), respectively.

The results indicate that PAR‐1 and PAR‐2 activation causes contractile responses in the guinea‐pig gallbladder, an effect that is mediated principally by prostanoid release, and is independent of neural mechanisms.

British Journal of Pharmacology (2000) 131, 689–694; doi:10.1038/sj.bjp.0703618
Correolide, a nor‐triterpenoid blocker of Shaker‐type Kv1 channels elicits twitches in guinea‐pig ileum by stimulating the enteric nervous system and enhancing neurotransmitter release
Authors: Vianna‐Jorge Rosane , Oliveira Cyntia Ferreira , Garcia Maria L , Kaczorowski Gregory J , Suarez‐Kurtz Guilherme
Published: January 2005

Correolide (1–10 μM), a nortriterpene purified from Spachea correae and a selective blocker of Kv1 potassium channels, elicits repetitive twitching in guinea‐pig ileum. This effect is not seen in guinea‐pig duodenum, portal vein, urinary bladder or uterine strips, nor in rat or mouse ileum.

The time course and amplitude of the correolide‐induced twitches in guinea‐pig ileum are similar to those elicited by electrical stimulation of the enteric nervous system.

The correolide‐induced twitching is not affected by pre‐treatment with capsaicin (1 μM), but is facilitated by the NO synthase inhibitor, NG‐nitro‐L‐arginine methyl esther (L‐NAME, 200 μM).

The correolide‐induced twitching is abolished by tetrodotoxin (1 μM) or hexamethonium (100 μM), and is markedly inhibited by nifedipine (0.3 μM) or atropine (0.2 μM). The atropine‐resistant component is inhibited by selective antagonists of NK1 and NK2 tachykinin receptors, namely GR 82334 and GR 94800 (1 μM each). The former compound is more effective in inhibiting the correolide‐induced, atropine‐resistant activity.

Correolide intensified the twitching of ileum segments exposed to saturating concentrations of margatoxin (MgTX), which suggests that Kv1 sub‐types other than Kv1.1 (Kv1.4 or Kv1.5) are involved in the relatively greater degree of stimulation of the enteric nervous system by correolide, as compared to MgTX.

We propose that blockade of Kv1 channels by correolide increases the excitability of intramural nerve plexuses promoting release of acetylcholine and tachykinins from excitatory motor neurons. This, in turn, leads to Ca2+‐dependent action potentials and twitching of the muscle fibres.

British Journal of Pharmacology (2000) 131, 772–778; doi:10.1038/sj.bjp.0703620
Short‐ and long‐term differential effects of neuroprotective drug NS‐7 on voltage‐dependent sodium channels in adrenal chromaffin cells
Authors: Yokoo Hiroki , Shiraishi Seiji , Kobayashi Hideyuki , Yanagita Toshihiko , Minami Shin‐ichi , Yamamoto Ryuichi , Wada Akihiko
Published: January 2005

In cultured bovine adrenal chromaffin cells, NS‐7 [4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy) pyrimidine hydrochloride], a newly‐synthesized neuroprotective drug, inhibited veratridine‐induced 22Na+ influx via voltage‐dependent Na+ channels (IC50=11.4 μM). The inhibition by NS‐7 occurred in the presence of ouabain, an inhibitor of Na+,K+ ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS‐7.

NS‐7 attenuated veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels (IC50=20.0 μM) and catecholamine secretion (IC50=25.8 μM).

Chronic (12 h) treatment of cells with NS‐7 increased cell surface [3H]‐STX binding by 86% (EC50=10.5 μM; t1/2=27 h), but did not alter the KD value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans‐Golgi network, but was not associated with increased levels of Na+ channel α‐ and β1‐subunit mRNAs.

In cells subjected to chronic NS‐7 treatment, 22Na+ influx caused by veratridine (site 2 toxin), α‐scorpion venom (site 3 toxin) or β‐scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS‐7, and veratridine‐induced 22Na+ influx remained depressed even at higher concentration of veratridine; however, either α‐ or β‐scorpion venom, or Ptychodiscus brevis toxin‐3 (site 5 toxin) enhanced veratridine‐induced 22Na+ influx as in nontreated cells.

These results suggest that in the acute treatment, NS‐7 binds to the site 2 and reversibly inhibits Na+ channels, thereby reducing Ca2+ channel gating and catecholamine secretion. Chronic treatment with NS‐7 up‐regulates cell surface Na+ channels via translational and externalization events, but persistently inhibits Na+ channel gating without impairing the cooperative interaction between the functional domains of Na+ channels.

British Journal of Pharmacology (2000) 131, 779–787; doi:10.1038/sj.bjp.0703622