bluuebunny/crossref_metadata_2025_split huggingface dataset:
NO synthase in cholinergic nerves and NO‐induced relaxation in the rat isolated corpus cavernosum
Authors: Hedlund Petter , Alm Per , Andersson Karl‐Erik 
Published: January 2005

In the rat corpus cavernosum (CC), the distribution of immunoreactivity for neuronal and endothelial NO synthase (nNOS and eNOS), and the pattern of NOS‐immunoreactive (‐IR) nerves in relation to some other nerve populations, were investigated. Cholinergic nerves were specifically immunolabelled with antibodies to the vesicular acetylcholine transporter protein (VAChT).
In the smooth muscle septa surrounding the cavernous spaces, and around the central and helicine arteries, the numbers of PGP‐ and tyrosine hydroxylase (TH)‐IR terminals were large, whereas neuropeptide Y (NPY)‐, VAChT‐, nNOS‐, and vasoactive intestinal polypeptide (VIP)‐IR terminals were found in few to moderate numbers.
Double immunolabelling revealed that VAChT‐ and nNOS‐IR terminals, VAChT‐ and VIP‐IR terminals, nNOS‐IR and VIP‐IR terminals, and TH‐ and NPY‐IR terminals showed coinciding profiles, and co‐existence was verified by confocal laser scanning microscopy. TH immunoreactivity was not found in VAChT‐, nNOS‐, or VIP‐IR nerve fibres or terminals.
An isolated strip preparation of the rat CC was developed, and characterized. In this preparation, cumulative addition of NO to noradrenaline (NA)‐contracted strips, produced concentration‐dependent, rapid, and almost complete relaxations. Electrical field stimulation of endothelin‐1‐contracted preparations produced frequency‐dependent responses: a contractile twitch followed by a fast relaxant response. After cessation of stimulation, there was a slow relaxant phase. Inhibition of NO synthesis, or blockade of guanylate cyclase, abolished the first relaxant phase, whereas the second relaxation was unaffected.
The results suggest that in the rat CC, nNOS, VAChT‐, and VIP‐immunoreactivities can be found in the same parasympathetic cholinergic neurons. Inhibitory neurotransmission involves activation of the NO‐system, and the release of other, as yet unknown, transmitters.

British Journal of Pharmacology (1999) 127, 349–360; doi:10.1038/sj.bjp.0702556
Expression of P2Y receptors in cell lines derived from the human lung
Authors: Communi Didier , Paindavoine Pascale , Place Graham A , Parmentier Marc , Boeynaems Jean‐Marie 
Published: January 2005

Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo− and 16HBE14o− epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU‐3 serous cells, the 6CFSMEo− submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC‐1. These experiments revealed that P2Y2 and P2Y6 mRNA are co‐expressed in the 1HAEo−, 16HBE14o− and A549 epithelial cell lines. The CFPAC‐1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors.
We have then performed RT–PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo−, 16HBE14o−, 6CFSMEo−, HASMSC1 and CFPAC‐1. P2Y1 mRNA was only detected in the CALU‐3 cell line.
Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo− and the 16HBE14o− airway epithelial cell lines which co‐express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo− submucosal cells expressed a UTP‐specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

British Journal of Pharmacology (1999) 127, 562–568; doi:10.1038/sj.bjp.0702560
Structural and ionic determinants of 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid block of the CFTR chloride channel
Authors: Walsh Kenneth B , Long Kathryn J , Shen Xufeng 
Published: January 2005

The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding.
Wild‐type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild‐type CFTR current by 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) occurred in a voltage‐dependent manner with preferential inhibition of the inward currents (Kd=166 μM at −90 mV).
Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2‐butylamino‐5‐nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd=243 μM), while addition of an extra phenyl ring at this position (5‐nitro‐2‐(3,3‐diphenylpropylamino)‐benzoic acid) increased drug potency (Kd=58 μM). In contrast, removal of the benzoate ring (2‐amino‐4‐phenylbutyric acid) or the 5‐nitro group (2‐(3‐phenylpropylamino)‐benzoic acid) of NPPB severely limited drug block of the wild‐type channel.
NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage‐dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 μM, for the K335E and R347E mutants, respectively.
NPPB block of the inward wild‐type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN−.
These studies present the first step in the development of high affinity probes to the CFTR channel.

British Journal of Pharmacology (1999) 127, 369–376; doi:10.1038/sj.bjp.0702562
Neuropeptide Y is a prejunctional inhibitor of vagal but not sympathetic inotropic responses in guinea‐pig isolated left atria
Authors: Serone Adrian P , Angus James A 
Published: January 2005

The effects of NPY and related peptides were examined on basal contractile force and nerve‐mediated inotropic responses to electrical field stimulation of the guinea‐pig isolated left atrium.
Electrical field stimulus (EFS)‐inotropic response curves were constructed by applying 1–64 trains of four field pulses (200 Hz, 0.1 ms duration, 100 V) across isolated left atria (paced at 4 Hz, 2 ms, 1–4 V) within the atrial refractory period. Curves were constructed in presence of vehicle, propranolol (1 μM) or atropine (1 μM) to determine appropriate stimulus conditions.
The effects of PYY (1–10,000 nM), NPY (0.01–10 μM), N‐Ac‐[Leu28,31]NPY(24–36) (N‐A[L]NPY(24–36); 0.01–10 μM) and clonidine (0.1–1000 nM) were examined on the positive and negative inotropic responses to EFS (eight trains, four pulses per refractory period).
NPY‐related peptides had no effect on basal force of contraction nor on the inotropic concentration‐response curves to bethanechol or isoprenaline. All three peptides inhibited vagally‐mediated negative inotropic responses; rank order of potency PYY>NPYN‐A[L]NPY(24–36) was consistent with an action at prejunctional Y2‐receptors. Clonidine concentration‐dependently inhibited sympathetic inotropic responses. However, PYY, NPY and N‐A[L]NPY(24–36) failed to mediate any significant inhibition of the positive inotropic response to EFS.
These data demonstrate that NPY is an effective inhibitor of vagal but not sympathetically‐mediated inotropic responses in the guinea‐pig isolated left atria. This may suggest that endogenously co‐released NPY is important in mediating cross talk between efferent components of the autonomic nervous system modulating cardiac contractility, acting overall to sustain positive inotropic responses.

British Journal of Pharmacology (1999) 127, 383–390; doi:10.1038/sj.bjp.0702565
Hyperosmolarity reduces the relaxing potency of nitric oxide donors in guinea‐pig trachea
Authors: Hjoberg Josephine , Högman Marieann , Hedenstierna Göran 
Published: January 2005

Non‐responders to inhaled nitric oxide treatment have been observed in various patient groups. The bronchodilatory effect of inhaled nitric oxide was attenuated when the airway lumen was rendered hyperosmolar in an in vivo study on rabbits. We used a guinea‐pig tracheal perfusion model to investigate the effects of increased osmolarity (450 mOsm, NaCl added) on the relaxing potency of the nitric oxide donors sodium nitroprusside (SNP) and (±)‐S‐nitroso‐N‐acetylpenicillamine (SNAP).
Under iso‐osmolar conditions SNP relaxed the carbachol (CCh, 1 μM) contracted trachea by 83±3%. After pretreatment with intraluminal hyperosmolarity SNP relaxed the CCh‐contracted trachea by only 31±7% (P<0.05). When the trachea was contracted to the same extent under untreated and hyperosmolar conditions, the untreated trachea was completely relaxed by SNP but, after hyperosmolar pretreatment, SNP could no longer relax the trachea.
SNAP relaxed the CCh contracted trachea by 27±5%. After pretreatment with intraluminal hyperosmolarity, SNAP relaxed the trachea by 11±4%, which was less than in the iso‐osmolar control (P<0.05).
Extraluminal hyperosmolarity did not affect carbachol elicited contraction, and SNP administered externally during extraluminal hyperosmolarity was able to relax the trachea (P<0.05).
The cell permeable guanosine 3′5′‐cyclic monophosphate analogue 8‐Br‐cGMP relaxed the CCh contracted trachea in both iso‐osmolar (P<0.05) and hyperosmolar conditions (P<0.05).
The relaxant effect of nitric oxide donors on tracheal smooth muscle is markedly reduced when the airway epithelium is exposed to hyperosmolar solution.

British Journal of Pharmacology (1999) 127, 391–396; doi:10.1038/sj.bjp.0702569
Pharmacological characterization of the muscarinic receptor antagonist, glycopyrrolate, in human and guinea‐pig airways
Authors: Haddad El‐Bdaoui , Patel Hema , Keeling Joelle E , Yacoub Magdi H , Barnes Peter J , Belvisi Maria G 
Published: January 2005

In this study we have evaluated the pharmacological profile of the muscarinic antagonist glycopyrrolate in guinea‐pig and human airways in comparison with the commonly used antagonist ipratropium bromide.
Glycopyrrolate and ipratropium bromide inhibited EFS‐induced contraction of guinea‐pig trachea and human airways in a concentration‐dependent manner. Glycopyrrolate was more potent than ipratropium bromide.
The onset of action (time to attainment of 50% of maximum response) of glycopyrrolate was similar to that obtained with ipratropium bromide in both preparations. In guinea‐pig trachea, the offset of action (time taken for response to return to 50% recovery after wash out of the test antagonist) for glycopyrrolate (t1/2 [offset]=26.4±0.5 min) was less than that obtained with ipratropium bromide (81.2±3.7 min). In human airways, however, the duration of action of glycopyrrolate (t1/2 [offset]>96 min) was significantly more prolonged compared to ipratropium bromide (t1/2 [offset]=59.2±17.8 min).
In competition studies, glycopyrrolate and ipratropium bromide bind human peripheral lung and human airway smooth muscle (HASM) muscarinic receptors with affinities in the nanomolar range (Ki values 0.5–3.6 nM). Similar to ipratropium bromide, glycopyrrolate showed no selectivity in its binding to the M1–M3 receptors. Kinetics studies, however, showed that glycopyrrolate dissociates slowly from HASM muscarinic receptors (60% protection against [3H]‐NMS binding at 30 nM) compared to ipratropium bromide.
These results suggest that glycopyrrolate bind human and guinea‐pig airway muscarinic receptors with high affinity. Furthermore, we suggest that the slow dissociation profile of glycopyrrolate might be the underlying mechanism by which this drug accomplishes its long duration of action.

British Journal of Pharmacology (1999) 127, 413–420; doi:10.1038/sj.bjp.0702573
The effects of verapamil and diltiazem on N‐, P‐ and Q‐type calcium channels mediating dopamine release in rat striatum
Authors: Dobrev Dobromir , Milde Alexander S , Andreas Klaus , Ravens Ursula 
Published: January 2005

The putative inhibitory effects of verapamil and diltiazem on neuronal non‐L‐type Ca2+ channels were studied by investigating their effects on either K+‐ or veratridine‐evoked [3H]‐dopamine ([3H]‐DA) release in rat striatal slices. Involvement of N‐, P‐ and Q‐type channels was identified by sensitivity of [3H]‐DA release to ω‐conotoxin GVIA (ω‐CTx‐GVIA), ω‐agatoxin IVA (ω‐Aga‐IVA) and ω‐conotoxin MVIIC (ω‐CTx‐MVIIC), respectively.
KCl (50 mM)‐evoked [3H]‐DA release was abolished in the absence of Ca2+, and was insensitive to dihydropyridines (up to 30 μM). It was significantly blocked by ω‐CTx‐GVIA (1 μM), ω‐Aga‐IVA (30 nM) and was confirmed to be abolished by ω‐CTx‐MVIIC (3 μM), indicating involvement of N‐, P‐ and Q‐type channel subtypes.
Verapamil and diltiazem inhibited K+‐evoked [3H]‐DA release in a concentration‐dependent manner. The inhibitory effects of verapamil or diltiazem (each 30 μM) were fully additive to the effect of ω‐CTx‐GVIA (1 μM), whereas co‐application with ω‐Aga‐IVA (30 nM) produced similar effects to those of ω‐Aga‐IVA alone.
As shown previously, veratridine‐evoked [3H]‐DA release in Ca2+ containing medium exclusively involves Q‐type Ca2+ channels. Here, diltiazem (30 μM) did not inhibit veratridine‐evoked [3H]‐DA release, whereas verapamil (30 μM) partially inhibited it, indicating possible involvement of Q‐type channels in verapamil‐induced inhibition. However, verapamil (30 μM) inhibited this release even in the absence of extracellular Ca2+, suggesting that Na+ rather than Q‐type Ca2+ channels are involved.
Taken together, our results suggest that verapamil can block P‐ and at higher concentrations possibly N‐ and Q‐type Ca2+ channels linked to [3H]‐DA release, whereas diltiazem appears to block P‐type Ca2+ channels only.

British Journal of Pharmacology (1999) 127, 576–582; doi:10.1038/sj.bjp.0702574
Role of potassium channels and nitric oxide in the relaxant effects elicited by β‐adrenoceptor agonists on hypoxic vasoconstriction in the isolated perfused lung of the rat
Authors: Dumas Jean‐Paul , Goirand Françoise , Bardou Marc , Dumas Monique , Rochette Luc , Advenier Charles , Giudicelli Jean‐François 
Published: January 2005

The aims of this study were to compare, in the rat isolated perfused lung preparation, the antagonist effects of a nonselective β‐adrenoceptor agonist (isoprenaline), a selective β2‐adrenoceptor agonist (salbutamol) and a selective β3‐adrenoceptor agonist (SR 59104A) on the hypoxic pulmonary pressure response, and to investigate the role of K+ channels, endothelium derived relaxing factor and prostaglandins in these effects. K+ channels were inhibited by glibenclamide, charybdotoxin or apamin, NO synthase and cyclo‐oxygenase were inhibited by NG‐nitro‐L‐arginine methyl ester (L‐NAME) and indomethacin, respectively.
Hypoxic ventilation produced a significant increase in perfusion pressure (+65%, P<0.001) and L‐NAME significantly increased this response further (+123%, P<0.01). After apamin, L‐NAME, indomethacin, post‐hypoxic basal pressure did not return to baseline values (P<0.001).
Glibenclamide partially inhibited the relaxant effects of isoprenaline (P<0.05) and salbutamol (P<0.001) but not that of SR 59104A. In contrast, charybdotoxin and apamin partially inhibited the relaxant effects of SR 59104A (P=0.053 and <0.01, respectively) but did not modify the effects of isoprenaline and salbutamol. L‐NAME partially inhibited the dilator response of salbutamol (P<0.01) and SR 59104A (P<0.05) but not that of isoprenaline.
We conclude that (a) EDRF exerts a significant inhibition of the hypoxic pulmonary response, (b) SKCa channel activation, EDRF and prostaglandins contribute to the reversal of the hypoxic pressure response, (c) the vasodilation induced by isoprenaline is mediated in part by activation of KATP channels, that of salbutamol by activation of KATP channels and EDRF. In contrast, SR 59104A partly operates through BKCa, SKCa channels and EDRF activation, differing in this from the β1 and β2‐adrenoceptor agonists.

British Journal of Pharmacology (1999) 127, 421–428; doi:10.1038/sj.bjp.0702575
Interleukin‐6 production by activated human monocytic cells is enhanced by MK‐571, a specific inhibitor of the multi‐drug resistance protein‐1
Authors: Vellenga Edo , Tuyt Leonore , Wierenga Bart‐Jan , Müller Michael , Dokter Wim 
Published: January 2005

The intracellular transport of leukotriene C4 (LTC4) in hematopoietic cells such as human monocytes is controlled by an ATP dependent carrier encoded by the multidrug resistance protein1 (MRP1) gene whose function can be blocked by the compound MK‐571. Since LTs play a major role in control of cytokine expression in monocytes, we questioned whether blocking of the MRP1 mediated function by MK‐571 might affect cytokine production.
MK‐571 strongly enhanced IL‐6 expression at mRNA and protein level in lipopolysaccharide (LPS) and interleukin‐1 (IL‐1) stimulated human monocytes giving rise to 2.0±0.4 (x±s.d.) and 5.7±3.5 fold induction of IL‐6 protein secretion. The increase in IL‐6 secretion was accompanied by an enhanced phosphorylation of p38 but not of c‐Jun‐N terminal kinase.
The involvement of the kinase signalling pathways was further analysed by using SB203580 and PD98059, specific inhibitors of the p38 and ERK1/2 signalling route. MK‐571 mediated upregulation of IL‐6 in the presence of IL‐1 was partially attenuated by SB203580 and PD98059. Electrophoretic mobility shift assays demonstrated that MK‐571 did not affect the IL‐1 induced DNA binding activity of Activator Protein‐1 and Nuclear Factor‐κB but rather enhanced the transactivational activity of an IL‐6 promoter construct. Finally it was shown that the MK‐571 mediated effects on IL‐6 secretion could not be inhibited by the LT synthesis inhibitor SB203347 or by the anti‐oxidant pyrrolidine dithiocarbamate (PDTC).
These results indicate that the membrane transporter MRP1 is involved in the regulation of IL‐6 expression in activated human peripheral blood monocytes.

British Journal of Pharmacology (1999) 127, 441–448; doi:10.1038/sj.bjp.0702577
Recovery of impaired K+ channels in mesenteric arteries from spontaneously hypertensive rats by prolonged treatment with cholecalciferol
Authors: Borges Antonio C R , Feres Teresa , Vianna Lucia M , Paiva Therezinha B 
Published: January 2005

The mechanism responsible for blood pressure reduction in spontaneously hypertensive rats (SHR) after prolonged cholecalciferol treatment was studied. Two‐week treatment of SHR with 0.125 mg cholecalciferol kg−1 body weight per day orally caused significant reductions of systolic blood pressure and of the resting perfusion pressure of the mesenteric vascular bed at constant flow.
In addition, the treated animals presented a normalization of the maximum vasoconstriction response to noradrenaline and a reduction of the maximum effect of the adrenaline concentration‐response curves. This latter effect probably was due to recovery of the impaired Ca2+‐dependent K+ channels coupled to α2‐adrenoceptors since it was prevented by apamin.
The treatment with cholecalciferol also normalized the smooth muscle cell membrane potential of de‐endothelialized mesenteric arteries of SHR and their hyperpolarizing responses to α2‐adrenergic agonists, which were depressed in untreated SHR.
In mesenteric rings with endothelium, α2‐adrenergic agonists caused similar hyperpolarizing responses in the SHR and in normotensive Wistar (NWR) and Wistar Kyoto (WKY). In non cholecalciferol‐treated SHR the hyperpolarizing mediator involved in this effect was NO, while in NWR it was the endothelium‐derived hyperpolarizing factor (EDHF). After cholecalciferol treatment, the hyperpolarization induced by α2‐adrenergic agonists in SHR smooth muscle cells was mediated by EDHF, as in NWR.
Our results indicate that the hypotensive effect of cholecalciferol in the SHR is probably due to the normalization of vascular reactivity, by restoring the functioning of apamin‐ and ATP‐sensitive K+ channels located in the vascular smooth muscle cell membrane, which are impaired in the SHR.

British Journal of Pharmacology (1999) 127, 772–778; doi:10.1038/sj.bjp.0702581
Protease‐activated receptor‐2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells
Authors: Hamilton Justin R , Chow Jean M , Cocks Thomas M 
Published: January 2005

Protease‐activated receptors (PARs) are activated by an irreversible proteolytic mechanism which renders cleaved receptors unresponsive to subsequent challenges with activating enzymes. Non‐specific proteolysis of PARs downstream of the activation site also prevents subsequent enzymic activation. Therefore, we investigated the effects of non‐activating amino‐terminal proteolysis with the bacterial protease thermolysin on PAR‐mediated relaxation of porcine coronary artery ring preparations contracted with the thromboxane A2 mimetic U46619 (1–10 nM).
Treatment of contracted artery ring segments with thermolysin (0.01–1 u ml−1, 20 min) caused no response, but abolished endothelium‐dependent relaxations induced by the enzymic activators of PAR‐1 and PAR‐2, thrombin (0.01–0.3 u ml−1) and trypsin (0.003–0.1 u ml−1) respectively. The same treatment, however, did not affect similar responses to the proteolysis‐independent PAR‐1 and PAR‐2 activating peptides, SFLLRN‐NH2 and SLIGRL‐NH2 respectively (0.1–10 μM).
The inhibition of responsiveness to trypsin after thermolysin treatment recovered in a time‐dependent manner, with maximal recovery (77.3±8.0% of time controls) occurring 150 min after thermolysin treatment. No recovery of responsiveness to thrombin after thermolysin treatment was observed within this time, however, the thrombin response returned to control levels after 20 h.
The recovery of responsiveness to trypsin was inhibited by the translation inhibitor cycloheximide (100 μM; 17.3±4.7%) and the protein trafficking inhibitor brefeldin A (10 μM; 12.1±4.8%) but was unaffected by the transcription inhibitor actinomycin D (2 μM; 65.1±3.6%), which did, however, abolish upregulation of B1‐kinin receptors in this preparation.
In conclusion, our findings indicate that activation‐independent amino‐terminal proteolysis of PARs stimulates selective recovery of endothelial cell PAR‐2 responsiveness, which appears to be regulated by translation. Such a novel mechanism for the maintenance of responsiveness to enzymic PAR‐2 activators may imply that these receptors play important roles in vascular homeostasis.

British Journal of Pharmacology (1999) 127, 617–622; doi:10.1038/sj.bjp.0702583