bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of anaesthetic and non‐anaesthetic effects on depolarization‐evoked glutamate and GABA release from mouse cerebrocortical slices
Authors: Liachenko Serguei , Tang Pei , Somogyi George T. , Xu Yan
Published: January 2005

Investigation with substances that are similar in structure, but different in anaesthetic properties, may lead to further understanding of the mechanisms of general anaesthesia.

We have studied the effects of two cyclobutane derivatives, the anaesthetic, 1‐chloro‐1,2,2‐trifluorocyclobutane (F3), and the non‐anaesthetic, 1,2‐dichlorohexafluorocyclobutane (F6), on K+‐evoked glutamate and γ‐aminobutyric acid (GABA) release from isolated, superfused, cerebrocortical slices from mice, by use of h.p.l.c. with fluorescence detection for quantitative analysis.

At clinically relevant concentrations, the anaesthetic, F3, inhibited 40 mM K+‐evoked glutamate and GABA release by 72% and 47%, respectively, whereas the structurally similar non‐anaesthetic, F6, suppressed evoked glutamate release by 70% but had no significant effects on evoked GABA release. A second exposure to 40 mM KCl after a ∼30 min washout of F3 or F6 showed recovery of K+‐evoked release, suggesting that F3 and F6 did not cause any non‐specific or irreversible changes in the brain slices.

Our findings suggest that suppression of excitatory neurotransmitter release may not be directly relevant to the primary action of general anaesthetics. A mechanism involving inhibitory postsynaptic action is implicated, in which a moderate suppression of depolarization‐evoked GABA release by the anaesthetic may be consistent with the enhancement of postsynaptic GABAergic activities.

British Journal of Pharmacology (1998) 123, 1274–1280; doi:10.1038/sj.bjp.0701728
Evidence that inducible nitric oxide synthase is involved in LPS‐induced plasma leakage in rat skin through the activation of nuclear factor‐κB
Authors: Iuvone Teresa , D'Acquisto Fulvio , Van Osselaer Nancy , Di Rosa Massimo , Carnuccio Rosa , Herman Arnold G.
Published: January 2005

Rats challenged with lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO) following the induction of the inducible NO‐synthase (iNOS) in several tissues and organs. Recent studies have shown that the expression of iNOS is regulated at the transcriptional level by a transcription nuclear factor‐κB (NF‐κB). In this study we investigated the role of NO in a model of LPS‐induced plasma‐leakage in rat skin and the involvement of NF‐κB.

Plasma leakage in the rat skin was measured over a period of 30 min to 2 h as the local accumulation of intravenous (i.v.) injection of [125I]‐human serum albumin ([125I]‐HSA) in response to intradermal (i.d.) injection of LPS. LPS (1, 10, 100 μg/site) produced a dose‐related increase in plasma extravasation (18.2±3.2, 27.2±2.9, 40.4±9.6 μl/site) as compared to saline control (11.4±2.2 μl/site). This increase was maximal after 2 h; therefore this time point and the dose of LPS 10 μg/site was used in all the successive experiments.

To investigate the role of NO in LPS‐induced plasma leakage in rat skin, the non‐selective NOS inhibitor NGnitro‐l‐arginine‐methyl ester (l‐NAME) or the more selective iNOS inhibitor S‐methyl‐isothiourea (SMT) was injected i.d. with LPS. l‐NAME and SMT (0.01, 0.1 and 1 μmol/site) inhibited LPS‐induced plasma leakage in a dose‐related fashion (l‐NAME: 26.0±5.5, 20.2±1.6, 18.0±2.0 μl/site; SMT: 19.5±1.5, 17.0±1.6, 15.0±2.6 μl/site) as compared to LPS alone (27.2±2.9 μl/site). At the lowest concentration used (0.01 μmol/site), SMT significantly reduced plasma leakage by 30%±0.7 while l‐NAME (0.01 μmol/site) was not effective.

Treatment with increasing concentrations of pyrrolidinedithyocarbamate (PDTC) (0.01, 0.1, 1 μmol/site), an inhibitor of NF‐κB activation, injected i.d. 30 min before LPS challenge, inhibited in a concentration‐dependent fashion LPS‐induced plasma leakage by 9.0±0.6, 33±4.0, 51±2.0% respectively. Moreover, PDTC (0.1, 1 μmol/site) suppressed LPS‐induced NF‐κB DNA‐binding.

Western blot analysis showed significant levels of iNOS proteins in the skin samples of LPS‐treated rats, as compared to basal levels present in saline‐injected rat skin. PDTC (0.1, 1.0 μmol/site) dose‐dependently decreased the amount of iNOS protein expression induced by LPS.

Our results indicate that LPS‐induced plasma leakage in rat skin is modulated by NO mainly produced by the inducible isoform of NOS. Furthermore, the suppression of plasma leakage by PDTC, an inhibitor of NF‐κB activation, is correlated to the inhibition of iNOS protein expression.

British Journal of Pharmacology (1998) 123, 1325–1330; doi:10.1038/sj.bjp.0701730
Quantification and distribution of α1‐adrenoceptor subtype mRNAs in human proximal urethra
Authors: Nasu Kimio , Moriyama Nobuo , Fukasawa Ritsu , Tsujimoto Gozoh , Tanaka Teruo , Yano Junichi , Kawabe Kazuki
Published: January 2005

We performed RNase protection assays and in situ hybridization to investigate the ratio of the three α1‐adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross‐sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra.

In situ hybridization studies showed no significant differences in the cross‐sectional distribution of α1‐adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense.

Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α1 drugs, α1‐selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.

British Journal of Pharmacology (1998) 123, 1289–1293; doi:10.1038/sj.bjp.0701731
ATP stimulation of Ca2+‐dependent plasminogen release from cultured microglia
Authors: Inoue Kazuhide , Nakajima Kazuyuki , Morimoto Takako , Kikuchi Yoshiaki , Koizumi Schuichi , Illes Peter , Kohsaka Shinichi
Published: January 2005

ATP (10–100 μm), but not glutamate (100 μm), stimulated the release of plasminogen from microglia in a concentration‐dependent manner during a 10 min stimulation. However, neither ATP (100 μm) nor glutamate (100 μm) stimulated the release of NO. A one hour pretreatment with BAPTA‐AM (200 μm), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 μm). The Ca2+ ionophore A23187 induced plasminogen release in a concentration‐dependent manner (0.3 μm to 10 μm).

ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration‐dependent manner which was very similar to the ATP‐evoked plasminogen release, whereas glutamate (100 μm) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μm) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells).

The ATP‐evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2‐Methylthio ATP was active (7 out of 7 cells), but α,β‐methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 μm) was shown not to inhibit the ATP‐evoked increase in [Ca2+]i (20 out of 20 cells). 2′‐ and 3′‐O‐(4‐Benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long‐lasting increase in [Ca2+]i even at 1 μm, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′‐triphosphate‐2′, 3′‐dialdehyde (oxidized ATP, 100 μm), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 μm).

These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.

British Journal of Pharmacology (1998) 123, 1304–1310; doi:10.1038/sj.bjp.0701732
Desensitization and resensitization of δ‐opioid receptor‐mediated Ca2+ channel inhibition in NG108‐15 cells
Authors: Morikawa Hitoshi , Fukuda Kazuhiko , Mima Hiroyuki , Shoda Takehiro , Kato Shigehisa , Mori Kenjiro
Published: January 2005

To approach the mechanisms underlying desensitization of the opioid receptor‐mediated Ca2+ channel inhibition, the effects of prolonged application of [D‐Ala2, D‐Leu5]enkephalin (DADLE) on Ba2+ currents (IBa) through Ca2+ channels were analysed in NG108‐15 neuroblastoma × glioma hybrid cells.

Inhibition of IBa by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min.

Maximal desensitization of the δ‐opioid receptor‐Ca2+ channel coupling was attained by 1 μM DADLE. The EC50 value for desensitization was estimated to be 78 nM.

RNA blot hybridization analysis and immunoblot analysis revealed the expression of β‐adrenoceptor kinase‐1 (βARK1) in NG108‐15 cells.

Heparin, an inhibitor of βARK, significantly reduced the magnitude and rate of desensitization, whereas Rp‐cyclic AMPS and PKI (14‐24)amide, inhibitors of cyclic AMP‐dependent protein kinase (PKA), or long‐term treatment with phorbol 12‐myristate 13‐acetate to induce down‐regulation of protein kinase C (PKC) had no significant effect.

Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization.

In summary, we have characterized the time course and concentration‐dependence of the desensitization of DADLE‐induced IBa inhibition in NG108‐15 cells. This desensitization was reversible after removal of DADLE. It is suggested that βARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

British Journal of Pharmacology (1998) 123, 1111–1118; doi:10.1038/sj.bjp.0701733
Heterogeneity and underlying mechanism for inotropic action of endothelin‐1 in rat ventricular myocytes
Authors: Katoh Hideki , Terada Hajime , Iimuro Masaru , Sugiyama Shiho , Qing Ke , Satoh Hiroshi , Hayashi Hideharu
Published: January 2005

To clarify the mechanisms underlying the positive inotropic action of endothelin‐1 (ET‐1), we investigated the effect of ET‐1 on twitch cell shortening and the Ca2+ transient in rat isolated ventricular myocytes loaded with a fluorescent Ca2+ indicator indo‐1.

There was a cell‐to‐cell heterogeneity in response to ET‐1. ET‐1 (100 nm) increased twitch cell shortening in only 6 of 14 cells (44 %) and the increase in twitch cell shortening was always accompanied by an increase in the amplitude of the Ca2+ transient.

The ETA‐ and ETB‐receptors antagonist TAK‐044 (100 nm) almost reversed both the ET‐1‐induced increases in twitch cell shortening and in the Ca2+ transient. In the ET‐1 non‐responding cells, the amplitude of the Ca2+ transient never increased.

Intracellular pH slightly increased (∼0.08 unit) after 30 min perfusion of ET‐1 in rat ventricular myocytes. However, ET‐1 did not change the myofilament responsiveness to Ca2+, which was assessed by (1) the relationship between the Ca2+ transient amplitude and twitch cell shortening, and by (2) the Ca2+ transient‐cell shortening phase plane diagram during negative staircase.

We concluded that there was a cell‐to‐cell heterogeneity in the positive inotropic effect of ET‐1, and that the ET‐receptor‐mediated positive inotropic effect was mainly due to an increase in the Ca2+ transient amplitude rather than to an increase in myofilament responsiveness to Ca2+.

British Journal of Pharmacology (1998) 123, 1343–1350; doi:10.1038/sj.bjp.0701743
Differential responses of pulmonary arteries and veins to histamine and 5‐HT in lung explants of guinea‐pigs
Authors: Shi Weibin , Wang Chong‐Gang , Dandurand Ron J. , Eidelman David H. , Michel René P.
Published: January 2005

The mechanisms by which histamine and 5‐HT differentially contract pulmonary arteries and veins are unclear. In lung explants from 26 guinea‐pigs, we compared responses of pulmonary arteries and vein to histamine, 5‐HT and KCl, and examined potential determinants for the differential responses. Lungs were filled with agarose, sectioned into ∼1 mm thick slices, and vascular luminal areas measured by image analysis.

Histamine and 5‐HT produced a concentration‐dependent constriction in arteries and veins, greater in the latter. KCl constricted arteries and veins equally.

The histamine H1 antagonist chlorpheniramine (10−4 M) abolished contractions to histamine; the H2 antagonist cimetidine enhanced maximal responses and sensitivity of arteries and veins to histamine, and diminished the differences between their maximal responses; the NO synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG) increased the maximal responses of arteries and veins, and the differences between their responses; indomethacin had no effect.

Contractions to 5‐HT were abolished in arteries and markedly reduced in veins by the 5‐HT2 antagonist ketanserin (10−4 M); L‐NOARG potentiated the maximal responses of arteries but not of veins; indomethacin increased the maximal responses of arteries but reduced them in veins.

By morphometry, arteries had a greater medial thickness and luminal diameter than veins.

The data suggest that in guinea‐pigs, H2 receptors are responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium‐derived vasoactive substances are responsible for their differential contractile responses to 5‐HT.

British Journal of Pharmacology (1998) 123, 1525–1532; doi:10.1038/sj.bjp.0701759
Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious, hypertensive, transgenic ((mRen‐2)27) rats
Authors: Gardiner S. M. , Kemp P. A. , March J. E. , Bennett T.
Published: January 2005

Age‐matched (3–4 months old) male, heterozygous, hypertensive, transgenic ((mRen‐2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague‐Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 μg kg−1 h−1, i.v.)

The early (1–2 h) hypotension in SD rats (−11±3 mmHg; n=7) was significantly less than that in TG rats (−35±3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre‐LPS value (SD rats: baseline, 108±3 mmHg; 24 h LPS, 112±4 mmHg; TG rats: baseline, 171±2 mmHg; 24 h LPS, 169±3 mmHg). At this stage in the SD rats there was a renal vasodilatation (Δ vascular conductance, 29±10 [kHz mmHg−1]103) but not in TG rats (Δ vascular conductance 2±3[kHz mmHg−1]103).

Co‐infusion of LPS and the non‐selective endothelin receptor antagonist, SB 209670 (600 μg kg−1 bolus, 600 μg kg−1 h−1) between 24 and 31 h in SD rats caused a fall in MAP of 16±2 mmHg accompanied by hindquarters vasodilatation (Δ vascular conductance 11±3 (kHz mmHg−1)103). In TG rats, under the same conditions, the fall in MAP was −60±6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (Δ vascular conductance, 23±5, 32±7, and 14±4 (kHz mmHg−1)103, respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats.

In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was −17±4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5±3, −4±5, and 12±4 (kHz mmHg−1)103, respectively.

Administration of the angiotensin (AT1)‐receptor antagonist, losartan (10 mg kg−1, i.v.) following co‐infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (−12±3 and −14±4 mmHg, respectively).

These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.

British Journal of Pharmacology (1998) 123, 1403–1408; doi:10.1038/sj.bjp.0701762
Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality
Authors: Im Haesook K. , Bin Im Wha , Pregenzer Jeffrey F. , Stratman Nancy C. , VonVoigtlander Philip F. , Jon Jacobsen E.
Published: January 2005

Imidazoquinoxaline PNU‐97775 and imidazoquinoline PNU‐101017 are benzodiazepine site ligands with a second low affinity binding site on GABAA receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.

In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU‐100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU‐100079 with a trifluoroethyl substituent produced a positive allosteric response.

These functional characteristics were monitored with Cl− currents measurements in cloned rat αxβ2γ2 subtypes of GABAA receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABAA receptors with binding of [35S]‐TBPS, which is a high affinity ligand specific for GABAA receptors with exquisite sensitivity to allosteric modulations.

This structure‐functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

British Journal of Pharmacology (1998) 123, 1490–1494; doi:10.1038/sj.bjp.0701763
Age and region‐dependent contraction to α‐adrenoceptor agonists in rat and guinea‐pig isolated trachea
Authors: Preuss Janet M. H. , Rigby Paul J. , Goldie Roy G.
Published: January 2005

The influence of age and of region on α‐adrenoceptor‐mediated contraction to (−)‐adrenaline and (−)‐noradrenaline was examined in rat (4–136 weeks) and guinea‐pig (2–156 weeks) isolated tracheal ring preparations with particular emphasis on the early (up to 12 weeks) maturation phase.

In rat tracheal rings, significant regional variation was observed with respect to maximal (−)‐adrenaline‐induced contraction, such that the greatest activity was seen in ring preparations from the laryngeal end of the trachea. Tracheal rings from the carinal end responded very poorly or were unresponsive to (−)‐adrenaline, depending on animal age. These regional differences were seen across the age range. The potencies of (−)‐adrenaline and (−)‐noradrenaline remained unchanged with respect to animal age, but the maximum contractile tension that developed in response to these agonists increased with increasing animal age in all regions of the trachea.

In guinea‐pig isolated tracheal tissue, maximum contractile responses (Emax) to (−)‐adrenaline and (−)‐noradrenaline remained unchanged with increasing animal age. In addition, there was no evidence for a region‐dependence in the responsiveness of tracheal tissue to α‐adrenoceptor‐mediated contraction in this species.

In both guinea‐pig and rat isolated tracheal tissue, α‐adrenoceptor‐mediated contraction appeared to involve the activation of α1‐adrenoceptors.

British Journal of Pharmacology (1998) 123, 1541–1548; doi:10.1038/sj.bjp.0701765
PYY‐preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats
Authors: Yang H. , Li W. P. , Reeve J. R. , Rivier J. , Taché Y.
Published: January 2005

Microinjection of peptide YY (PYY, 7–46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane‐anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY.

[Pro34]rat(r)/porcine(p)PYY and [Pro34]human(h)PYY (23–117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68±11 and 89±31 μmol 90 min−1 respectively.

Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27±8 and 23±8 μmol 90 min−1 respectively) and a higher dose (116 pmol) tended to reduce the response.

Pancreatic polypeptide (PP, 4–46 pmol), [Leu31,Pro34]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY3‐36, pNPY5‐36 and pNPY13‐36 (25–168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion.

The rank order of potency of PYY[Pro34]r/pPYY=[Pro34]hPYY>r/hNPY=pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu31,Pro34]NPY and C‐terminal NPY/PYY fragments suggest that a PYY‐preferring receptor subtype may be involved in mediating the stimulating effect.

British Journal of Pharmacology (1998) 123, 1549–1554; doi:10.1038/sj.bjp.0701767
Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle
Authors: McMurray Gordon , Dass Narinder , Brading Alison F.
Published: January 2005

The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), α,β‐methylene‐ATP (α,β‐MeATP) and 2‐methylthio‐ATP (2‐MeSATP) on longitudinally orientated smooth muscle strips from marmoset urinary bladder were investigated by use of standard organ bath techniques.

After being mounted in superfusion organ baths, 66.7% (n=249) of marmoset detrusor smooth muscle strips developed spontaneous tone, 48.2% of all strips examined developed tone equivalent to greater than 0.1 g mg−1 of tissue and were subsequently utilized in the present investigation.

On exposure to ATP, muscle strips exhibited a biphasic response, a rapid and transient contraction followed by a more prolonged relaxation. Both responses were found to be concentration‐dependent. ADP and 2‐MeSATP elicited a similar response (contraction followed by relaxation), whereas application of α,β‐MeATP only produced a contraction. The potency order for each effect was α,β‐MeATP>>2‐MeSATPATP>ADP (contractile response) and ATP=2‐MeSATPADP>>α,β‐MeATP (relaxational response).

Desensitization with α,β‐MeATP (10 μM) abolished the contractile phase of the response to ATP, but had no effect on the level of relaxation evoked by this agonist. On the other hand, the G‐protein inactivator, GDPβS (100 μM) abolished only the relaxation response to ATP. Suramin (general P2 antagonist, 100 μM) shifted both the contractile and relaxation ATP concentration‐response curves to the right, whereas cibacron blue (P2Y antagonist, 10 μM) only antagonized the relaxation response to ATP. In contrast, the adenosine receptor antagonist, 8‐phenyltheophylline (10 μM), had no effect on the relaxation response curve to ATP.

Incubation with tetrodotoxin (TTX, 3 μM) or depolarization of the muscle strip with 40 mM K+ Krebs failed to abolish the relaxation to ATP. In addition, neither Nω‐nitro‐L‐arginine (L‐NOARG, 10 μM) nor methylene blue (10 μM) had any effect on the relaxation response curve. However, tos‐phe‐chloromethylketone (TPCK, 3 μM), an inhibitor of cyclicAMP‐dependent protein kinase A (PKA), significantly (P<0.01) shifted the curve for the ATP‐induced relaxation to the right.

It is proposed that marmoset detrusor smooth muscle contains two receptors for ATP, a classical P2X‐type receptor mediating smooth muscle contraction, and a P2Y (G‐protein linked) receptor mediating smooth muscle relaxation. The results also indicate that the ATP‐evoked relaxation may occur through the activation of cyclicAMP‐dependent PKA.

British Journal of Pharmacology (1998) 123, 1579–1586; doi:10.1038/sj.bjp.0701774
The effects of nifedipine and other calcium antagonists on the glibenclamide‐sensitive K+ currents in smooth muscle cells from pig urethra
Authors: Teramoto Noriyoshi , Brading Alison F.
Published: January 2005

The effects of nifedipine on both levcromakalim‐induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch‐clamp techniques (conventional whole‐cell configuration and cell‐attached patches).

Nifedipine had a voltage‐dependent inhibitory effect on voltage‐dependent Ba2+ currents at −50 mV (Ki=30.6 nM).

In current‐clamp mode, subsequent application of higher concentrations of nifedipine (30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.

The 100 μM levcromakalim‐induced inward current (symmetrical 140 mM K+ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide‐sensitive current.

When 100 μM nifedipine was included in the pipette solution during conventional whole‐cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.

The levcromakalim‐induced K+ channel openings in cell‐attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.

These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide‐sensitive 43 pS K+ channel activity mainly through extracellular blocking actions on the K+ channel itself.

British Journal of Pharmacology (1998) 123, 1601–1608; doi:10.1038/sj.bjp.0701777
Involvement of calcitonin gene‐related peptide (CGRP) receptors in insulin‐induced vasodilatation in mesenteric resistance blood vessels of rats
Authors: Mimaki Yuichi , Kawasaki Hiromu , Okazaki Masatoshi , Nakatsuma Akira , Araki Hiroaki , Gomita Yutaka
Published: January 2005

The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin gene‐related peptide (CGRP)‐receptor in insulin‐induced vascular responsiveness were investigated in rats.

The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration‐dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94±0.22 and 43.9±5.2, respectively.

This vasodilator response to insulin was unaffected by 100 nM propranolol (β‐adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 μM L‐NG‐nitroarginine (nitric oxide synthase inhibitor), 1 μM ouabain (Na+‐K+ ATPase inhibitor), or 1 μM glibenclamide (ATP sensitive K+‐channel inhibitor).

In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62±0.21 and 81.0±4.6, respectively, significantly greater than in preparations with intact endothelium.

The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8–37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP‐containing nerves, did not affect insulin‐induced vasodilatation.

These results suggest that insulin induces non‐adrenergic, non‐cholinergic and endothelium‐independent vasodilatation, which is partially mediated by CGRP receptors.

British Journal of Pharmacology (1998) 123, 1684–1690; doi:10.1038/sj.bjp.0701779
Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat
Authors: McCulloch Kirsty M. , Docherty Cheryl , MacLean Margaret R.
Published: January 2005

We examined the endothelin (ET) receptors mediating contractions to ET‐1, ET‐3 and sarafotoxin S6c (SX6c) in rat pulmonary resistance arteries by use of peptide and non‐peptide ET receptor antagonists. Changes induced by pulmonary hypertension were examined in the chronically hypoxic rat. The effect of the mixed ETA/ETB receptor antagonist SB 209670 on endothelin‐mediated contraction was also examined in human pulmonary resistance arteries.

In rat vessels, the order of potency for the endothelin agonists was SX6c=ET‐3>ET‐1 (pEC50 values in control rats: 9.12±0.10, 8.76±0.14 and 8.12±0.04, respectively). Maximum contractions induced by ET‐3 and ET‐1 were increased in vessels from chronically hypoxic rats.

The ETA receptor antagonist FR 139317 (1 μM) had no effect on the potency of ET‐1 in any vessel studied but abolished the increased response to ET‐1 in the chronically hypoxic vessels. The ETA receptor antagonist BMS 182874 (1 μM) increased the potency of ET‐1 in control rat vessels without effecting potency in the pulmonary hypertensive rat vessels.

Bosentan (non‐peptide mixed ETA/ETB receptor antagonist) increased the potency of ET‐1 in control rat vessels but was without effect in the pulmonary hypertensive rat vessels. Bosentan (1 μM) inhibited responses to SX6c in control and chronically hypoxic rat vessels with pKb values of 5.84 and 6.11, respectively. The ETB receptor antagonist BQ‐788 (1 μM) did not inhibit responses to ET‐1 in any vessel tested but did inhibit responses to both SX6c and ET‐3 (pKb values in control and chronically hypoxic rat vessels respectively: SX6c 7.15 and 7.22; ET‐3: 6.68 and 6.89). BQ‐788 (1 μM) added with BMS 182874 (10 μM) did not inhibit responses to ET‐1 in control vessels but caused a significant inhibition of responses to ET‐1 in chronically hypoxic preparations.

SB 209670 inhibited responses to ET‐1 in both control and chronically hypoxic vessels with pKb values of 7.36 and 7.39, respectively. SB 209670 (0.1 and 1 μM) virtually abolished responses to ET‐1 in the human pulmonary resistance artery.

In conclusion, in rat pulmonary resistance arteries, vasoconstrictions induced by ET‐1, SX6c and ET‐3 are mediated predominantly by activation of an ETB–like receptor. However, lack of effect of some antagonists on ET‐1 induced vasoconstriction suggests that ET‐1 stimulates an atypical ETB receptor. The increase in potency of ET‐1 in the presence of some antagonists suggests the presence of an inhibitory ETA‐like receptor. The influence of this is reduced, or absent, in the chronically hypoxic rats. Increased responses to ET‐1 are observed in the chronically hypoxic rat and may be mediated by increased activation of ETA receptors. SB 209670 is unique in its potency against responses to ET‐1 in both control and chronically hypoxic rats, as well as human, isolated pulmonary resistance arteries.

British Journal of Pharmacology (1998) 123, 1621–1630; doi:10.1038/sj.bjp.0701785
Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α1B‐adrenoceptor
Authors: Hirasawa Akira , Awaji Takeo , Sugawara Tatsuo , Tsujimoto Aiko , Tsujimoto Gozoh
Published: January 2005

α1B‐adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti‐N‐terminus‐antibody (1B‐N1‐C, (Hirasawa et al., 1996)) facilitated the quantification of cell surface α1B‐adrenoceptor. Also, the cellular distribution of α1B‐adrenoceptors was visually monitored by immunocytochemical confocal microscopy.

Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of α1B‐adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist‐induced internalization. The two processes were separately examined by using α1B‐adrenoceptor inducible DDT1MF‐2 cells for the sorting process and CHO cells stably expressing α1B‐adrenoceptors for the agonist‐promoted internalization.

We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin‐mediated endocytosis) on these processes.

We found that the agonist‐promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery.

The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein‐coupled receptors.
Nitric oxide regulation of monkey myometrial contractility
Authors: Kuenzli Karri A , Buxton Iain L O , Bradley Michael E
Published: January 2005

We evaluated the effect of the nitric oxide (NO) donor CysNO (S‐nitroso‐L‐cysteine) and endogenous NO upon spontaneous contractility in non‐pregnant cynomolgus monkeys. We also assessed the role of intracellular guanosine 3′,5′‐cyclic monophosphate ([cyclic GMP]i) as a second messenger for NO in monkey uterine smooth muscle.

CysNO reduced spontaneous contractility by 84% (P<0.05) at maximal concentrations, and significantly elevated [cyclic GMP]i (P<0.05). However, increases in [cyclic GMP]i were not required for CysNO‐induced relaxations; CysNO inhibited contractile activity despite the complete inhibition of guanylyl cyclase by methylene blue or LY83,583.

Analogues of cyclic GMP had no significant effect upon spontaneous contractile activity. L‐arginine produced a 62% reduction in spontaneous activity (P<0.05) while D‐arginine had no effect. The competitive nitric oxide synthase (NOS) inhibitor Nω‐nitro‐L‐arginine (L‐NOARG) not only blocked L‐arginine‐induced relaxations, but also significantly increased spontaneous contractile activity when added alone (P<0.05); the inactive D‐enantiomer of NOARG had no such effect.

While both endogenous NO and the NO donor CysNO relax monkey myometrium, this effect is not causally related to CysNO‐induced elevations in [cyclic GMP]i. The failure of cyclic GMP analogues to alter monkey uterine smooth muscle tension also argues against a role for [cyclic GMP]i in the regulation of uterine contractility. Not only do these findings argue for the existence of a functionally‐relevant NOS in the monkey uterus, but increases in contractile activity seen in the presence of NOS inhibitors suggest a role for NO in the moment‐to‐moment regulation of contractile activity in this organ.
Carbachol stimulation of gastric acid secretion and its effects on the parietal cell
Authors: Sandvik Arne K , Mårvik Ronald , Dimaline Rod , Waldum Helge L
Published: January 2005

The acid secretagogue effect of gastrin is mainly mediated by the release of enterochromaffin‐like (ECL) cell histamine, but the mechanism of muscarinic stimulation of acid secretion remains unclear. The results of studying aminopyrine uptake in isolated parietal cells, and histamine release in isolated ECL cells suggest that muscarinic agents may act both directly on the parietal cell and indirectly via histamine release from ECL cells.

We examined parietal and ECL cell responses to the muscarinic agent carbamylcholine (carbachol) in conscious rats and in rat isolated vascularly perfused stomachs.

Intravenous carbachol stimulated acid secretion in conscious gastric fistula rats and increased H+K+ ATPase mRNA abundance, indicating activation of parietal cells. In these experiments there was no increase in portal venous histamine, or in oxyntic mucosal histidine decarboxylase (HDC) enzyme activity and HDC mRNA abundance.

In rat isolated stomachs stimulated with carbachol in the dose range 10 nM–1 mM only the 1 μM concentration increased venous histamine significantly.

We concluded that the muscarinic agent carbachol stimulates acid secretion and H+K+ ATPase mRNA in vivo by a direct effect on the parietal cell, that does not depend on the release of ECL cell histamine.
Discovery of novel melanocortin4 receptor selective MSH analogues
Authors: Schiöth Helgi B , Mutulis Felikss , Muceniece Ruta , Prusis Peteris , Wikberg Jarl E S
Published: January 2005

We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors.

We discovered that compounds with 26 membered rings of [Cys4,D‐Nal7,Cys11]α‐MSH(4–11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D‐Nal7Cys11]α‐MSH(4–11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities.

HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]α‐MSH analogues SHU9119 and HS9510.

HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor.

HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is ∼300 fold higher affinity than for α‐MSH), was also demonstrated to antagonize α‐MSH stimulation of cyclic AMP in MC4 receptor transfected cells.

We found that a compound with a 29 membered ring of [Cys3,Nle10,D‐Nal7,Cys11]α‐MSH(3–11) (HS010) had the highest affinity for the MC3 receptor.

This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.
Effects of adrenomedullin and calcitonin gene‐related peptide on contractions of the rat aorta and porcine coronary artery
Authors: Yoshimoto Ryo , Mitsui‐Saito Minori , Ozaki Hiroshi , Karaki Hideaki
Published: January 2005

Effects of adrenomedullin and α‐calcitonin gene‐related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α‐CGRP were also investigated.

Adrenomedullin and α‐CGRP caused a concentration‐dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and α‐CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG‐monomethyl‐L‐arginine (L‐NMMA, 100 μM), but not by a cyclo‐oxygenase inhibitor, indomethacin (10 μM).

Adrenomedullin and α‐CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle.

An antagonist of the CGRP1 receptor, CGRP (8–37), antagonized the relaxant effects of α‐CGRP and the β‐isoform of CGRP (β‐CGRP) but not those of adrenomedullin in the rat aorta.

In the porcine coronary artery contracted with U46619, adrenomedullin and α‐CGRP caused a concentration‐dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or α‐CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation.

Adrenomedullin and α‐CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery.

CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α‐CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β‐isoform of CGRP (β‐CGRP).

These results suggest that in the rat aorta, adrenomedullin and α‐CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α‐CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α‐CGRP, β‐CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α‐CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β‐CGRP.

British Journal of Pharmacology (1998) 123, 1645–1654; doi:10.1038/sj.bjp.0701805
Effects of calcium and potassium supplements on arterial tone in vitro in spontaneously hypertensive rats
Authors: Tolvanen Jari‐Petteri , Mäkynen Heikki , Wu Xiumin , Hutri‐Kähönen Nina , Rsukoaho Heikki , Karjala Kirsi , Pörsti Ilkka
Published: January 2005

Calcium and potassium intakes inversely correlate with blood pressure in experimental hypertension. Therefore, we examined the effects of calcium and potassium supplements alone and in combination on arterial tone in spontaneously hypertensive rats (SHR). Wistar‐Kyoto (WKY) rats served as normotensive controls. Calcium and potassium contents in the control diet were both 1%, while those in supplemented chows were 3% and 3.5%, respectively. The sodium content of all diets was moderately elevated to 1.1%.

After 12 weeks of the study systolic blood pressures in SHR on high calcium and on high potassium diets were markedly lower (about 53 and 58 mmHg, respectively) than in hypertensive controls, while combined supplementation of these cations reduced blood pressure even further (about 69 mmHg).

Responses of mesenteric arterial rings in vitro were examined at the end of the study. Both high calcium and high potassium diets improved the impaired relaxation to acetylcholine (ACh) in SHR, while the combination of these supplements completely normalized this response. Cyclo‐oxygenase inhibition by diclofenac augmented the relaxation to ACh in hypertensive controls but not in the other groups. Nevertheless, enhanced endothelium‐mediated dilatation was still observed in the presence of diclofenac and the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) in all supplemented groups. Interestingly, additional blockade of Ca2+‐activated K+ channels by tetraethylammonium abolished the improved relaxation to ACh in SHR on high calcium and on high potassium, but distinct responses were still observed in WKY rats and SHR on the combined supplement.

When hyperpolarization of smooth muscle was prevented by precontraction of the preparations with 50 mM KCl, only marginal differences were observed in the diclofenac and L‐NAME‐resistant relaxations to ACh between the study groups. Finally, endothelium‐independent vasorelaxations of noradrenaline‐precontracted rings to nitroprusside, isoprenaline and cromakalim were comparably augmented by all supplements.

In conclusion, the vascular mechanisms underlying the antihypertensive effect of high calcium and high potassium diets during moderately elevated sodium intake in SHR may involve enhanced arterial hyperpolarization, increased smooth muscle sensitivity to nitric oxide and decreased production of vasoconstrictor prostanoids. The administration of these cations in combination was more effective than either of them alone in reducing blood pressure and restoring arterial tone.
Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse
Authors: Rees D D , Monkhouse J E , Cambridge D , Moncada S
Published: January 2005

The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse.

Administration of endotoxin (E. Coli, 026:B6, 12.5 mg kg−1, i.v.) elevated the concentration of tumour necrosis factor‐α (TNF‐α) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin‐6 (IL‐6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study.

Endotoxin (12.5 mg kg−1, i.v.) induced the expression of a Ca2+‐independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period.

The vasopressor effect of noradrenaline (0.5–4 μg kg−1 min−1, i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg−1, i.v).

The NO synthase inhibitor NG‐monomethyl‐L‐arginine HCl (L‐NMMA; 1–10 mg kg−1, i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg−1, i.v.).

In an attempt to maintain a constant blood concentration, L‐NMMA was administered as a continuous infusion (10 mg kg−1 h−1, i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg−1, i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation.

The fall in blood pressure following endotoxin (3 mg kg−1, i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase.

In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.