bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ca+‐dependent K‐channels in the membrane potential and contractility of aorta from spontaneously hypertensive rats
Authors: Silva Eneida G. , Frediani‐Neto Eugenio , Ferreira Alice T. , Paiva Antonio CM. , Paiva Therezinha B.
Published: July 2012

Contractile responses to KCl and membrane potentials were determined in aortic rings from spontaneously hypertensive rats (SHR), normotensive Wistar rats (NWR) and Wistar Kyoto rats (WKY) both in the absence and in the presence of the Ca2+‐dependent K‐channel blockers, apamin and tetraethylammonium (TEA).

Compared to NWR, aortic rings from WKY and SHR were less reactive and their Ca2+ uptake after stimulation with K+ was decreased.

Smooth muscle cell membrane potentials were higher in aortae from SHR and WKY than in NWR aortae, whereas SHR had higher K+ and lower Na+ intracellular activities than WKY and NWR, suggesting overactivity of the Na+/K+ pump in the hypertensive animals.

Treatment with apamin caused depolarization of WKY and SHR aortae, and increased their contractile responses to the same level as those of the NWR. Treatment with TEA also caused depolarization of aortae from WKY and SHR, but in the SHR the depolarization induced by TEA was smaller than that produced by apamin and the contractile responses to KCl did not reach the level of those of aortae from NWR.

It is concluded that overactivity of Ca2+‐dependent K‐channels in aortae of WKY and SHR contributes to their higher membrane potentials and lower responsiveness to vasoconstrictor stimuli. In SHR, an overactive Na+/K+ pump is also present, and the contribution of apamin‐sensitive Ca2+‐dependent K‐channels to the membrane potential and reactivity appears to be more relevant than that of TEA‐sensitive channels.
Paired‐pulse depression of the N‐methyl‐D‐aspartate receptor‐mediated synaptic potentials in the amygdala
Authors: Huang Chiung‐Chun , Gean Po‐Wu
Published: July 2012

An in vitro slice preparation of rat amygdala was used to study the paired‐pulse depression of the N‐methyl‐D‐aspartate (NMDA) receptor‐mediated synaptic potential e.p.s.p.NMDA.

The e.p.s.p.NMDA was isolated pharmacologically by applying a solution containing the non‐NMDA receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and the γ‐aminobutyric acidA (GABAA) blocker picrotoxin and increasing the stimulus intensity.

When two stimuli of identical strength were applied in close succession, the second e.p.s.p.NMDA was depressed. This paired‐pulse depression was seen with interstimulus intervals of between 100 ms and 2000 ms; the maximal depression was observed at interval of 200 ms.

Superfusion of phaclofen or 2‐hydroxy‐saclofen inhibited the paired‐pulse depression indicating the involvement of GABAB receptors.

Bath applications of Ba2+ or intracellular injection of Cs+ to block post‐ but not presynaptic GABAB receptors failed to inhibit the paired‐pulse depression (PPD).

Incubation of slices with pertussis toxin prevented the postsynaptic hyperpolarization induced by baclofen. The PPD of e.p.s.p.NMDA, however, was not affected by pertussis toxin treatment.

These results suggest that GABA released by the first stimulus acts on GABAB receptors to suppress the second e.p.s.p.NMDA via mechanisms other than activation of a postsynaptic GABAB receptor‐coupled K+ conductance.
Pharmacological reactivity of human epicardial coronary arteries: characterization of relaxation responses to endothelium‐derived relaxing factor
Authors: Stork Andrew P. , Cocks Thomas M.
Published: July 2012

Human epicardial coronary artery rings, freshly obtained from cardiac transplantation patients, commonly examined for their responses to endothelium‐derived relaxing factor (EDRF)‐releasing agents.

Functional antagonism profoundly influenced relaxation responses in this tissue. Increasing force with concentrations of U46619 above 3 nm (40% of maximum contraction response) resulted in a reduction of the maximum response to four vasorelaxants which relax vascular smooth muscle via different mechanisms: the EDRF‐releasing agents, substance P and bradykinin; the endothelium‐independent nitro‐vasodilator, sodium nitroprusside (SNP); and the β‐adrenoceptor agonist, isoprenaline.

Substance P, histamine, bradykinin and the Ca2+ ionophores ionomycin and A23187 all caused concentration‐ and endothelium‐dependent relaxation in vessels pre‐contracted with the thromboxane A2‐mimetic, U46619 (3 nm) to an active force optimal for relaxation responses. Nifedipine (0.1 μm), added to prevent spontaneous contractions, had no effect on relaxation responses to substance P, bradykinin and histamine.

Substance P was the most potent of the EDRF‐releasing agents examined and all agents except for bradykinin caused near‐maximal relaxation. Bradykinin caused only 46.2% ± 7.3% relaxation. Responses were abolished when the endothelium was removed and, except for histamine, were not significantly affected by indomethacin (3–10 μm, P>0.05). Histamine (0.1–10 μm) caused a concentration‐dependent contraction of arterial rings without endothelium.

The L‐arginine analogues NG‐nitro‐L‐arginine (L‐NOARG, 0.1 μm) and NG‐monomethyl‐L‐arginine (L‐NMMA, 0.1 μm) both caused no further contraction in arteries precontracted with U46619 (3 nm) and were in general, poor inhibitors of responses to EDRF agonists. L‐NMMA, but not L‐NOARG, caused small but significant decreases in the maximum responses to substance P, bradykinin (18.5 ± 6.9% and 27.6 ± 10.9% relaxation with L‐NMMA and L‐NOARG, respectively), histamine and A23187 (P < 0.05). The analogues had no effect on SNP responses.

In conclusion, EDRF release in human isolated coronary artery is only poorly antagonized by the nitric oxide synthase inhibitors L‐NOARG and L‐NMMA. These results indicate that either the nitric oxide transduction pathway present in human coronary artery is different from that in other tissues or that another factor(s) (e.g. endothelium‐derived hyperpolarizing factor) is also released in response to EDRF‐releasing agents and augments the relaxation to nitric oxide.
Antianginal effects of FK409, a new spontaneous NO releaser
Authors: Kita Yasuhiro , Ozaki Reiko , Sakai Shigeru , Sugimoto Toshiko , Hirasawa Yoshimi , Ohtsuka Minoru , Senoh Hachiro , Yoshida Keizo , Maeda Kazuhiro
Published: July 2012

The aim of this study was to compare antianginal effects of (±)‐(E)‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexeneamide (FK409), a new spontaneous nitric oxide releaser, with those of isosorbide dinitrate (ISDN). We used two types of rat angina model; methacholine‐ and arginine vasopressin (AVP)‐induced coronary vasospasm models.

In the in vitro study, FK409 showed 80 times more potent vasorelaxant effect in dog isolated coronary artery than ISDN (EC50= 16.7 ± 4.8 and 1340 ± 320 nm, respectively).

In the rat methacholine‐induced coronary vasospasm model, FK409 suppressed the elevation of ST segment dose‐dependently and significantly at 0.1 mg kg−1, i.d. On the other hand, ISDN suppressed it significantly at 3.2 mg kg−1, i.d. In addition, the efficacy of 3.2 mg kg−1 ISDN in the model was almost the same as that of 0.1 mg kg−1 FK409.

In the above experiments, FK409 and ISDN decreased mean blood pressure significantly at the maximum dose tested (1.0 mg kg−1, i.d. and 3.2 mg kg−1, i.d., respectively) but did not change heart rate at these doses. Therefore, the hypotensive effect of FK409 was 10 times weaker than the antianginal effect of the compound, while those of ISDN were almost the same.

In the rat AVP‐induced coronary vasospasm model, 32mg kg−1 FK409 significantly inhibited the depression of ST segment 60 min after oral administration. On the other hand, 32 mg kg−1 ISDN did not inhibit it at 60 and 120 min after oral administration.

In conclusion, FK409 inhibits coronary vasospasm more potently in two types of rat angina models than ISDN. In addition, FK409 shows an antianginal effect more selectively that a hypotensive effect, compared with ISDN.
Evidence for reduction of bradykinin‐induced bronchoconstriction in guinea‐pigs by release of nitric oxide
Authors: Ricciardolo Fabio L.M. , Nadel Jay A. , Yoishihara Shigemi , Geppetti Pierangelo
Published: July 2012

In this study the influence of nitric oxide (NO) on the bronchoconstriction induced by bradykinin in anaesthetized and artifically ventilated guinea‐pigs pretreated with atropine was investigated.

Aerosol administration of bradykinin (0.1 −1 mm, 40 breaths) caused a dose‐dependent increase in lung resistance (R1): maximum increase in R1 was 2.5 fold the baseline value. Pretreatment with aerosolized NG‐nitro‐1‐arginine methyl ester (1‐NAME) or NG‐monomethyl‐1‐arginine (1‐NMMA) (1 mm, 10 breaths every 5min for 30min), NO synthase inhibitors, markedly increased the bron‐choconstrictor response to bradykinin. 1‐Arginine, but not D‐arginine, (3 mm, 10 breaths every 5 min for 30 min) reversed the hyperresponsiveness to aerosolized bradykinin caused by 1‐NAME and 1‐NMMA.

1‐NAME (1 mm, 10 breaths every 5 min for 30 min) increased the bronchoconstriction induced by intravenous bradykinin (1‐10nmol kg1). 1‐Arginine, but not D‐arginine, (10 breaths every 5 min for 30 min) reversed the hyperresponsiveness to intravenous bradykinin caused by 1‐NAME.

The increase in RL induced by capsaicin, either aerosol (10 μm, 10 breaths) or i.v. (20 nmol kg−1) was not affected by 1‐NAME (1 mm, 10 breaths every 5 min for 30 min). Acute resection of the vagi did not affect the bronchoconstriction evoked by bradykinin in guinea‐pigs, either in the absence or presence of 1‐NAME (1 mm, 10 breaths every 5 min for 30 min).

These results suggest that, irrespective of the route of administration, bradykinin releases NO or a related molecule which exerts a bronchodilator action that opposes the bronchoconstrictor mechanisms activated by bradykinin itself.
The role of myoendothelial cell contact in non‐nitric oxide‐, non‐prostanoid‐mediated endothelium‐dependent relaxation of porcine coronary artery
Authors: Kühberger Elisabeth , Groschner Klaus , Kukovetz Walter R. , Brunner Friedrich
Published: July 2012

Experiments were designed to analyse the requirement of myoendothelial junctions by bradykinin‐induced endothelium‐dependent relaxations resistant to NG‐nitro‐L‐arginine (L‐NOARG) and indo‐methacin in porcine coronary arteries.

Rings of porcine coronary arteries were contracted with the thromboxane receptor agonist, U46619 and relaxations to bradykinin recorded isometrically. All experiments were performed in the presence of indomethacin. Nitric oxide (NO)‐mediated effects were blocked by the NO synthase inhibitor L‐NOARG (250 μm) and myoendothelial contacts inhibited by treatment with hypertonic solution containing D‐mannitol or sucrose (each 180 μm) or the gap junctional uncoupling agent 1‐heptanol (2 μm). High [K+] solutions (40 μm) were used to probe a possible contribution of endothelium‐derived hyperpolarizing factor (EDHF).

In the presence of endothelium, bradykinin induced concentration‐dependent relaxations with a mean EC50 of 3.2 nm and a maximum response of 95 ± 1% of papaverine‐induced relaxation (control curve).

In the absence of endothelium, bradykinin failed to induce relaxations. Addition of cultured porcine aortic endothelial cells to the organ bath resulted in some relaxation and restored in part the relaxant effect of bradykinin. This endothelial cell‐mediated relaxant effect was completely abolished in the presence of 250 μm L‐NOARG.

Bradykinin‐induced relaxations in endothelium‐preserved rings were only slightly suppressed by L‐NOARG (86% of control). In vessels partially depolarized by high extracellular [K+] (40 μm) relaxation was reduced to 72% of control. In the presence of L‐NOARG, bradykinin failed to relax partially depolarized vessels.

In the presence of 2 mm 1‐heptanol, 180mM mannitol or 180mM sucrose maximum relaxation to bradykinin was reduced to ∼70%, i.e. to the same extent as in the presence of high [K+]. The remaining relaxation was sensitive to blockade by L‐NOARG.

Tissue cyclic GMP content which reflects NO activity, was increased about 4 fold by bradykinin (300 nm). This increase was unaffected by high [K+], heptanol or sucrose but blocked by L‐NOARG.

Our results suggest that non‐nitric oxide‐ and non‐prostanoid‐mediated endothelium‐dependent relaxation of porcine coronary artery requires functionally intact myoendothelial junctions.
Mechanism of the cardiovascular activity of laudanosine: comparison with papaverine and other benzylisoquinolines
Authors: Chuliá Susana , Ivorra M. Dolores , Lugnier Claire , Vila Elisabeth , Noguera M. Antonia , D'Ocon Pilar
Published: July 2012

The activity of (±)‐laudanosine, a benzyltetrahydroisoquinoline alkaloid, was investigated in pithed rats and rat isolated aorta. Its effects on [3H]‐prazosin, [3H]‐(+)‐cis‐diltiazem and [3H]‐nitrendipine binding to rat cerebral cortical membranes, and on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were investigated.

The dose‐response curve to methoxamine (3–300 μg kg−1, i.v.) in normotensive pithed rats was shifted to the right by (±)‐laudanosine, 3 and 6 mg kg−1.

(±)‐Laudanosine inhibited in a concentration‐dependent manner the contractile responses evoked by noradrenaline (NA 1 μm), depolarizing solution (KC1 80 μm) or depolarizing solution plus phentolamine (10 μm) in rat isolated aorta. The alkaloid appeared to be more potent against NA‐induced contractions.

In Ca2+‐free solution, (±)‐laudanosine (100 μm) inhibited the contraction evoked by NA and did not modify the phasic contractile response evoked by caffeine. The alkaloid did not modify the refilling of the intracellular Ca2+‐stores sensitive to NA or caffeine.

(±)‐Laudanosine inhibited [3H]‐prazosin binding to cortical membranes and also inhibited [3H]‐(+)‐cis‐diltiazem but with a lower potency. [3H]‐nitrendipine binding was not affected by laudanosine.

(±)‐Laudanosine does not have a significant effect on the different forms of PDEs isolated from bovine aorta. In contrast, compounds structurally related to this alkaloid such as papaverine and its derivatives, had a non‐selective or more specific inhibitory effect on these PDE forms. These differences can be explained on the basis of their structural features: the planarity of the isoquinoline ring (papaverine) facilitates the interaction with receptor sites, and the different position of the benzyl group does not modify the activity unless this position leads to the presence of a chiral centre (laudanosine).

These results suggest that (±)‐laudanosine has a selective activity as an α1‐adrenoceptor blocker. Its lack of action on different PDE forms provides us with information about a group of benzylisoquinolines that with small structural changes show a different effect on PDE‐forms isolated from vascular smooth muscle.
Differential sensitivity of 3H‐agonist binding to pre‐ and postsynaptic 5‐HT1A receptors in bovine brain
Authors: Iben Lawrence G. , Mahle Cathy D. , Yocca Frank D.
Published: July 2012

The full and weak partial 5‐HT1A agonist ligands [3H]‐8‐OH‐DPAT and [3H]‐BMY‐7378 were used to characterize the binding parameters of pre‐ and postsynaptic 5‐HT1A binding sites in bovine dorsal raphe and hippocampal membranes, respectively. The Kd and Bmax values for the individual radioligands were indistinguisable across the regions tested, as were the Ki values generated by a series of agents acting at 5‐hydroxytryptamine (5‐HT) receptors.

The concentration‐dependent allosteric attenuation of [3H]‐8‐OH‐DPAT and [3H]‐BMY‐7378 binding produced by the nonhydrolyzable guanyl nucleotide, Gpp(NH)p, generated similar IC50 values within a particular region; however, these were significantly different between regions. While the maximal attenuation of [3H]‐8‐OH‐DPAT and [3H]‐BMY‐7378 binding was similar in dorsal raphe, Gpp(NH)p produced a significantly greater attenuation of [3H]‐8‐OH‐DPAT binding in hippocampal membranes when compared to [3H]‐BMY–7378. The maximal attentuation of [3H]‐8‐OH‐DPAT binding by Gpp(NHp) in hippocampus was also significantly greater than that seen with either radioligand in dorsal raphe.

Although exposure to Gpp(NH)p had no effect on the affinity constants of either radioligand in either region, it produced a concentration‐dependent reduction in the maximal number of binding sites for both radioligands in both regions. While the percentage reduction in Bmax values were similar for both radioligands in the dorsal raphe, Gpp(NH)p reduced the Bmax of [3H]‐8‐OH‐DPAT in hippocampus significantly more than that of [3H]‐BMY–7378.

These results suggest that while pre‐ and postsynaptic 5‐HT1A receptors may share similar pharmacological recognition properties, a region‐dependent difference in the coupling of the 5‐HT1A receptor to G‐proteins may exist.
Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids
Authors: Hirokawa K. , O'Shaughnessy K.M. , Ramrakha P. , Wilkins M.R.
Published: July 2012

These studies examine the effect of retinoids on interleukin 1β (IL‐1β)‐induced nitric oxide synthase (NOS) activity in cultured rat aortic vascular smooth muscle (VSM) cells and isolated rat aortic rings.

All‐trans‐retinoic acid (all‐transM‐RA, 0.1–10 μm) and its active analogues produced concentration‐dependent inhibition of IL‐1β (0.1–10 ng ml−1)‐induced nitrite production in cultured VSM cells. In contrast, the inactive retinoid, Ro 14–6113 (0.1–10 μm), had no effect on IL‐1β‐induced nitrite production.

Since some of the actions of retinoids are mediated by induction of transforming growth factor beta (TGF‐β), its effect on inducible NOS activity in VSM cells was examined. TGF‐β produced concentration‐dependent (0.1–10 ng ml−1) inhibition of IL‐1β‐induced nitrite production and the maximum effect (approximately 90% inhibition) was significantly greater than that seen with all‐trans‐RA (approximately 70% with 10 μm). However, an anti‐TGF‐β antibody (50 μg ml−1) which blocked the effect of exogenous TGF‐β (5 ng ml−1) did not significantly reverse the inhibitory action of all‐trans‐RA (10 μm).

In addition to inhibiting IL‐1β‐induced nitrite production, all‐trans‐RA (10 μm) reduced substantially inducible NOS mRNA and protein levels in IL‐1β‐induced VSM cells (P < 0.01).

Incubation of isolated rat aortic rings with IL‐1 β (10 ng ml−1) caused a progressive resistance of the rings to the vasoconstrictor action of phenylephrine (10 nm to 10 μm). This effect was abolished by the addition of the nitric oxide synthase inhibitor L‐NG‐monomethyl‐L‐arginine (L‐NMMA, 1 μm). All‐trans‐RA (IO μm) also markedly and significantly reversed this IL‐1β‐induced vascular hyporeactivity (P < 0.01).

These data show that all‐trans‐RA and other active retinoids are able to block cytokine‐stimulated expression of inducible NOS in cultured VSM cells and isolated aortic rings.
Different endothelin receptors involved in endothelin‐1‐ and sarafotoxin S6B‐induced contractions of the human isolated coronary artery
Authors: Bax Willem A. , Aghai Zohara , VAN Tricht Corina L.J. , Wassenaar Claes , Saxena Pramod R.
Published: July 2012

Endothelin receptors, that mediate contraction of the human isolated coronary artery, were characterized by use of a number of agonists and antagonists. Contraction induced by the non‐selective agonists, endothelin (ET)‐1 and sarafotoxin S6b, was compared in endothelium‐intact and endothelium‐denuded ring segments. The effects of ET‐1 and BQ‐123 (an ETA receptor antagonist) were investigated both in ring segments and in spirally cut strips. Lastly, the effect of phosphoramidon was studied on contraction induced by big‐ET‐1.

The order of agonist potency (pD2) in endothelium‐intact coronary artery ring segments was: ET‐1 (8.27) ≅sarafotoxin S6b (8.16)>big‐ET‐1 (<7.1)≅ET‐3 (<6.9). [Ala1,3,11,15]ET‐1 (ETB receptor agonist) caused significant contraction only at 1 μm, whereas 0.3 μm big‐ET‐3 had no effect. Removal of the endothelium in ring segments did not affect the contractile response to ET‐1 or to sarafotoxin S6b.

After a full concentration‐response curve had been obtained to ET‐1 or sarafotoxin S6b, further contractions of the endothelium‐intact coronary artery segments could only be achieved by applying ET‐1 in segments exposed to sarafotoxin S6b, and not the reverse.

BQ‐123 (0.1 μm) antagonized contractions of endothelium‐intact ring segments induced by sarafotoxin S6b (pKB 7.86). Only 10 μm BQ‐123 antagonized contractions induced by ET‐1 (pKB 5.75). FR139317 was also more potent against sarafotoxin S6b (pKB 8.24‐8.47) than against ET‐1 (pKB 6.11). [Ala1,3,11,15]ET‐1 (1 μm) had no effect on the contractile response to ET‐1 or to sarafotoxin S6b.

In strip preparations with intact endothelium, the pD2 of ET‐1 increased to 9.04 ± 0.16 (vs. 8.50 ± 0.07 in rings), and BQ‐123 (1 μm) caused a rightward shift of the ET‐1 induced concentration‐response curve (pKB 6.62 vs. 5.75 in rings).

Contractile responses to big‐ET‐1 of endothelium‐intact coronary artery segments were attenuated in the presence of phosphoramidon (100 μm), indicating conversion of big‐ET‐1 to ET‐1 within the coronary artery segment.

The present study indicates that ET‐1 and sarafotoxin S6b contract the human isolated coronary artery via different receptors, which can probably be best characterized as subtypes of the ETA receptor. Furthermore, it is demonstrated that the type of preparation (ring or strip) may affect the potency of ET‐1 as an agonist and of BQ‐123 as an antagonist.
Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of the GABAB receptor system
Authors: Malcangio Marzia , Bowery Norman G.
Published: July 2012

Monoarthritis was induced in Lewis rats by interdermal injection in the left hind paw of a suspension of Mycobacterium tubercolusis in mineral oil (500 μg 100 μ1−1). Controls were injected with 100 μ1 mineral oil.

Withdrawal latencies to thermal stimuli of the inflamed paw, the contralateral and both paws of control rats were measured at daily intervals after injection by the plantar test.

After detection of the pain threshold, rat spinal cords were removed and horizontal dorsal slices were mounted in a 3‐compartment bath to measure electrically‐evoked release of substance P‐like immunoreactivity (SP‐LI).

The inflamed paw of monoarthritic rats exhibited a lower pain threshold to thermal stimuli than the contralateral paw of the same animals and both paws of control rats. Inflamed paw hyperalgesia was maximal two days after injection, and declined gradually between 7 to 21 days with no evidence of excitability of withdrawal reflexes after 28 days.

During the 28 days study, monoarthritic rats gained less weight than control rats.

Electrical stimulation of the dorsal roots attached to rat isolated spinal cord slices induced a significant increase (174 ± 18% of basal outflow which was 30.3 fmol 8 ml−1 n = 5) in SP‐LI release.

One‐week after induction of inflammation no differences in the amount of SP‐LI released from the spinal cord of incomplete Freund's adjuvant‐treated rats (IFA) and Freund's adjuvant‐treated rats (CFA) were detected. Two weeks after, CFA spinal cord tended to release more SP‐LI than IFA cords and, 21 days after injection, the spinal cord of CFA rats released significantly more peptide than IFA rats (17.8 ± 2.8 fmol ml−1, n = 12 and 6.9 ± 3.2 fmol ml−1, n = 9, respectively).

Twenty‐one days after treatment, the evoked release from monoarthritic rat spinal cords was increased by 263 ± 42% (n = 3) in the presence of the GABAB receptor antagonist, CGP 36742 (100 μm) which also significantly potentiated monoarthritis‐induced hyperalgesia up to 45min after injection (100mg kg−1, i.p.).

These findings may provide a basis for a novel approach to chronic pain therapy but also an explanation for the lack of analgesia produced by the GABAB agonist, baclofen, in chronic as compared to acute pain.
S14080, a peripheral analgesic acting by release of an endogenous circulating opioid‐like substance
Authors: Ferreira Sérgio H. , Lorenzetti Berenice B. , Devissaguet Michelle , Lesieur Daniel , Tsouderos Yannis
Published: July 2012

The oral administration of a benzothiazolinone derivative (benzoyl‐6 dihydro‐2,3 benzothiazole), S14080, caused dose‐dependent antinociception in the rat paw pressure test, which represents a model of mechanical hyperalgesia. S14080 had no significant effect on the inflammatory oedema induced by carrageenin or on the tail flick test, nor did it possess a notable antipyretic effect.

Post‐treatment with S14080 dose‐dependently antagonized the hyperalgesia induced by prostaglandin E2, bradykinin, dopamine and by the hyperalgesic cytokines reported to be released by carrageenin (tumour necrosis factor α, interleukin‐1 and interleukin‐8).

The blockade of prostaglandin E2‐induced paw hyperalgesia by oral pretreatment of the rats with S14080 was abolished by prior intraplantar administration of either naloxone or NorBNI which are non‐specific and specific κ opioid antagonists, respectively.

Adrenalectomy abolished the oral antinociceptive effect of S14080.

Five consecutive daily injections of S14080 did not produce tolerance such as that seen with the central antinociceptive action of morphine.

As with peripherally acting opiates, the antinociceptive activity of S14080 was abolished by the intraplantar injection of agents which inhibit either arginine synthetase (NG‐monomethyl‐l‐arginine) or the activation of guanylate cyclase (methylene blue).

We conclude that S14080 is a new type of peripheral antinociceptive which, in rats, acts mainly by releasing an endogenous, opioid‐like substance from the adrenal glands.
Angiotensin‐converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco‐2) cells
Authors: Thwaites David T. , Cavet Megan , Hirst Barry H. , Simmons Nicholas L.
Published: July 2012

The role of proton‐linked solute transport in the absorption of the angiotensin‐converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco‐2) cell monolayers.

In Caco‐2 cell monolayers the transepithelial apical‐to‐basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis‐resistant dipeptide, glycylsarcosine (Gly‐Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin‐converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]‐lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mm) was added.

pH‐dependent [14C]‐Gly‐Sar transport was inhibited by the orally‐active ACE inhibitors, enalapril maleate and captopril (both at 20 mm). Lisinopril (20 mm) had a relatively small inhibitory effect on [14C]‐Gly‐Sar transport. pH‐dependent [3H]‐proline transport was not inhibited by captopril, enalapril maleate or lisinopril.

Experiments with BCECF[2′,7′,‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein]‐loaded Caco‐2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (β‐alanyl‐l‐histidine) and the ACE inhibitors, enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco‐2 cell monolayers. However, lisinopril was without effect.

The effects of enalapril maleate and captopril on [14C]‐Gly‐Sar transport and pHi suggest that these two ACE inhibitors share the H+‐coupled mechanism involved in dipeptide transport. The absence of pH‐dependent [14C]‐lisinopril transport, the relatively small inhibitory effect on [14C]‐Gly‐Sar transport, and the absence of lisinopril‐induced pHi changes, all suggest that lisinopril is a poor substrate for the di/tripeptide carrier in Caco‐2 cells. These observations are consistent with the greater oral availability and time‐dependent absorption profile of enalapril maleate and captopril, compared to lisinopril.
Sensitization by calyculin A of brain capillary endothelial cells to endothelin‐1
Authors: Vigne Paul , Breittmayer Jean Philippe , Frelin Christian
Published: July 2012

Cultured brain capillary endothelial cells of the rat respond to endothelin‐1 (ET‐1) by an increased activity of the Na+,K+,2Cl−, cotransporter and a mobilization of intracellular Ca2+ stores.

Calyculin A (1–30 nm), but not okadaic acid, sensitizes up to 100 fold the Na+,K+,2Cl− cotransporter to the action of ET‐1.

Calyculin A (30 nm) does not modify the binding properties of ET‐1 to ETA receptors.

Calyculin A (30 nm) inhibits ET‐1 induced intracellular Ca2+ mobilization.

It is concluded that inhibition of protein phosphatase 1 selectively modifies the repertoire of intracellular actions of ET‐1 and favours actions that are unrelated to the phospholipase C signalling cascade.
Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells
Authors: Zegarra‐Moran Olga , Romeo Giovanni , Galietta Luis J.V.
Published: July 2012

The effect of extracellular nucleotides on the transepithelial ion transport of Madin Darby canine kidney cells (MDCK) was investigated. Cells were grown up to confluency on permeable supports and the short circuit current (Isc) was measured with an Ussing chamber‐like mini‐perfusion system.

Apical ATP stimulated a biphasic Isc increase consisting of a first rapid and transient peak followed by a broader one.

The first peak evoked by ATP was reversibly blocked by basilen blue (BB) in a concentration‐dependent fashion, with an EC50 of 7.5 μm.

The P2Y receptor agonist, 2‐methylthioATP (2‐MeSATP) caused a single transient Isc increase that was completely blocked by pretreatment with BB. On the contrary, the P2x agonist, α,β‐methylene ATP (α,β‐meATP) was almost completely ineffective on Isc. UTP essentially induced a monophasic response the time‐course of which resembled that of the second peak stimulated by ATP. The agonist potency order was 2‐MeSATP ≥ ATP >> UTP, α<β‐meATP for the first peak and UTP ≥ ATP > 2‐MeSATP > α,β‐meATP for the second peak.

Monolayer incubation with the membrane permeable calcium chelator [bis‐o‐aminophenoxy)‐ethane‐N,N,N′,N′,‐tetraacetic acid, tetra(acetoximethyl)‐ester] (BAPTA/AM) inhibited the ATP‐evoked first peak.

The non‐hydrolyzable ATP analogue, adenosine‐5′‐O‐(3‐thio)‐trisphosphate (ATP‐γ‐S) elicited a biphasic response similar to that of ATP. The P1 receptor agonist, 2‐chloroadenosine and CGS‐21680, were almost unable to induce an Isc increase. These results rule out the involvement of ATP hydrolysis and P1 receptor activation as responsible for Isc increase.

Inhibition of prostaglandins synthesis by indomethacin abolished the second ATP‐evoked peak.

Chloride replacement with gluconate on both sides of the epithelium completely inhibited the second peak induced by ATP but only reduced the amplitude of the first spike.

The results suggest that ATP stimulates Isc increase by two mechanisms. The first one is mediated by a P2Y receptor and by intracellular calcium increase. The second induces prostaglandin synthesis probably through a P2U receptor activation.
Evidence for the involvement of different receptor subtypes in the pre‐ and postjunctional actions of angiotensin II at rat sympathetic neuroeffector sites
Authors: Cox S.L. , Ben A. , Story D.F. , Ziogas J.
Published: July 2012

The effects of the nonpeptide angiotensin II receptor (AT) antagonists losartan and PD 123319 on actions of angiotensin II in the rat caudal artery and rat vas deferens preparations were investigated.

Angiotensin II (1.0 μm) increased perfusion pressure in isolated segments of the rat caudal artery. This increase in perfusion pressure was prevented by the AT1‐antagonist, losartan (0.1 μm) but was not affected by the AT2‐antagonist, PD 123319 (0.1 μm).

Angiotensin II (0.1 – 3.0 μm) produced a concentration‐dependent enhancement of the stimulation‐induced (S‐I) efflux of [3H]‐noradrenaline from isolated segments of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]‐noradrenaline. The maximum enhancement of S‐I efflux was approximately 60% with 1.0 μm angiotensin II.

Losartan (0.01 and 0.1 μm) reduced the enhancement of S‐I efflux produced by 1.0 μm angiotensin II in the caudal artery.

PD 123319 (0.01 μm) did not affect the enhancement of S‐I efflux produced by angiotensin II (1.0 μm) in the caudal artery. However, in a higher concentration (0.1 μm), PD 123319 reduced the enhancement of S‐I efflux produced by 1.0 μm angiotensin II.

Angiotensin II produced concentration‐dependent enhancement of the purinergic twitch responses (1 pulse/60 s) in the rat vas deferens,

Losartan (0.03 μm) and PD 123319 (0.03 μm) each reduced the angiotensin II‐induced enhancement of the twitch responses in the rat vas deferens.

These findings indicate that the enhancement of sympathetic neuroeffector transmission in both the caudal artery and vas deferens of the rat involves angiotensin receptor subtype(s) sensitive to both losartan and PD 123319. In contrast, the direct vasoconstrictor effect of angiotensin II in the rat caudal artery involves activation of a receptor subtype sensitive only to losartan.
Antiproliferative effects of A02011‐1, an adenylyl cyclase activator, in cultured vascular smooth muscle cells of rat
Authors: Yu Sheu‐Meei , Cheng Zhi‐Jiao , Kuo Sheng‐Chu
Published: July 2012

1 The effects of A02011‐1, a pyrazole derivative, on the proliferation of rat vascular smooth muscle cells (VSMCs) were examined.

2 A02011‐1 (1–100 μm) concentration‐dependently inhibited [3H]‐thymidine incorporation into DNA in rat VSMCs that were synchronized by 48 h serum depletion and then re‐stimulated by addition of foetal calf serum (FCS, 10%), platelet‐derived growth factor (PDGF, 10 ng ml−1), 5‐hydroxytryptamine (10 μm) or ADP (10 μm). The inhibitory effect of A02011‐1 was fully reversible. However, FCS‐induced [3H]‐thymidine incorporation into rat endothelial cells was unaffected by A02011‐1.

3 The concentration of A02011‐1 necessary for inhibition of the FCS‐induced proliferation was similar to that necessary for adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) formation. Adenylyl cyclase activity was increased in A02011‐1‐treated VSMCs, whereas cyclic AMP‐specific phosphodiesterase activity was unchanged.

4 A02011‐1 was equipotent with forskolin but was more potent than 8‐bromo‐cyclic AMP against FCS (10%)‐induced proliferation.

5 The antiproliferative action of A02011‐1 was mimicked by 8‐bromo‐cyclic AMP, a membrane‐permeable cyclic AMP analogue and was antagonized by 2′,5′‐dideoxyadenosine, an adenylyl cyclase inhibitor and by Rp‐cyclic AMPS, a competitive inhibitor of cyclic AMP‐dependent protein kinase (PKA) type I and II. 3‐Isobutyl‐1‐methylxanthine (IBMX) caused significant potentiation of the antiproliferative activity of A02011‐1. However, Rp‐8‐bromo‐cyclic GMPS and staurosporine did not affect the antiproliferative activity of A02011‐1.

6 A02011‐1 still inhibited the FCS‐induced DNA synthesis even when added 10–18 h after re‐stimulation of the serum‐starved VSMCs with 10% FCS. Flow cytometry in synchronized cells revealed an acute blockade of FCS‐inducible cell cycle progression at a point in the G1/S phase in A02011‐1‐treated cells. The inhibition of proliferation by A02011‐1 was shown to be independent of cell damage, as documented by several criteria of cell viability.

7 These results indicate that A02011‐1 inhibition of VSMC proliferation was mediated by cyclic AMP and was due to a delay in the progression from the G1 into S phase of the cell cycle. A02011‐1 did not cause cell toxicity and may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.
Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists
Authors: Richards Mary H. , van Giersbergen Paul L.M.
Published: July 2012

1 A comparative study of receptor activation by ten full and partial muscarinic agonists was undertaken on the five subtypes of human muscarinic receptors expressed at similar receptor densities in Chinese hamster ovary (CHO‐K1) cells. In addition, m1, m2 and m3 receptors were expressed in mouse fibroblast A9L cells in order to compare the influences of cell type on agonist activation of these receptors.

2 Receptor‐effector coupling efficiencies were greater in CHO than A9L cells and agonists displayed greater potencies and similar or greater intrinsic activities at CHOm1 and CHOm3 than A9Lm1 and A9Lm3 receptors. Although m2 receptor density was 6 fold higher in A9L than CHO cells, carbachol elicited significantly greater inhibition of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) formation in CHOm2 cells. These data suggest that not only receptor density but receptor‐effector coupling and/or coupling efficiencies play significant roles in agonist‐induced responses.

3 In CHO cells, receptor‐effector coupling efficiencies were m3 = m1 > m5. Although CHOm5 receptors were the least efficiently coupled, some partial agonists displayed higher intrinsic efficacies at m5 than m3 receptors suggesting that, in CHO cells, m5 and m3 receptors may activate different G proteins and/or effectors to stimulate inositol monophosphate (IP1) formation.

4 McN‐A‐343 was a functionally selective m4 agonist. It had little or no agonist activity at m3 receptors expressed in either A9L or CHO cells. The slopes of McN‐A‐343 concentration‐response curves in CHOm2 cells were significantly lower than the slopes obtained with this compound in CHOm4 cells suggesting that the mode of activation by McN‐A‐343 differed between the two muscarinic receptors negatively coupled to adenylyl cyclase.

5 Cloned receptors provide valuable tools for the study of agonist‐receptor interaction and agonist‐receptor activation but caution should be applied in assuming that the results are valid for all cell types or for tissue‐expressed receptors.
Release by ultraviolet B (u.v.B) radiation of nitric oxide (NO) from human keratinocytes: a potential role for nitric oxide in erythema production
Authors: Deliconstantinos George , Villiotou Vassiliki , Stravrides John C.
Published: July 2012

1 The mechanism of human sunburn is poorly understood but its characteristic features include the development of erythema. In this study we attempted to determine whether human keratinocytes possess a nitric oxide (NO) synthase (NOS), if this enzyme could be activated to release NO following exposure to ultraviolet B (u.v.B) and to define whether this photo‐induced response could be involved in the pathogenesis of sunburn erythema.

2 Treatment of human keratinocytes with various doses of u.v.B (290–320 nm) radiation (up to 100 mJ cm−2) resulted in a dose‐dependent release of NO and cyclic GMP production that was reduced by NG‐monomethyl‐l‐arginine (l‐NMMA).

3 u.v.B irradiation of keratinocyte cytosol at varying doses (up to 50 mJ cm−2), resulted in a gradual rise in NO production, with a concomitant increase in soluble guanylate cyclase activity (sGC).

4 NOS isolated from the keratinocyte cytosol was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins.

5 In reconstitution experiments, when purified NOS was added to purified sGC, both isolated from keratinocyte cytosol, a four fold increase in cyclic GMP was observed. The GMP was increased by NO synthesized following u.v.B radiation (up to 20 mJ cm−2) of NOS.

6 In in vivo experiments, guinea‐pigs were subjected to u.v.B light. A Protection Factor (PF) of 8.71 ± 2.85 was calculated when an emulsified cream formulation containing l‐NMMA (2%) was applied to their skin.

7 The present results indicate that u.v.B radiation acts as a potent stimulator of NOS in keratinocytes. NO is lipophilic and may diffuse out of the keratinocytes, activating sGC in endothelial cells and neighbouring smooth muscle cells. This may be a major part of the integrated response of the skin leading to vasodilatation and erythema.
Bronchodilator and pre‐protective effects of urodilatin in bovine bronchi in vitro: comparison with atrial natriuretic peptide
Authors: Nally Jane E. , Docherty Cheryl C. , Clayton Robin A. , Thomson Neil C.
Published: July 2012

1 This study examined the activity and mechanisms of action of urodilatin in bovine bronchi. For comparison, the ability of urodilatin to evoke bronchodilatation or protect against subsequent challenge was compared to that of the closely related peptide α‐human atrial natriuretic peptide (ANP).

2 Urodilatin reversed methacholine‐evoked contraction in a concentration‐dependent manner in bovine bronchi. In the absence of any attempt to prevent degradation by neutral endopeptidases, urodilatin was more potent than ANP in this tissue.

3 The bronchodilator properties of urodilatin were significantly augmented by the neutral endopeptidase inhibitor, phsophoramidon (3.68 × 10−5 M). This provides evidence for at least partial degradation of urodilatin by neutral endopeptidases. With phosphoramidon present, urodilatin and ANP were equipotent.

4 In the presence of phosphoramidon (3.68 × 10−5 m), pre‐incubation with urodilatin (10−6 M) had a protective effect against subsequent methacholine‐induced contraction. This action of urodilatin was quantitatively similar to that of ANP in the presence of this endopeptidase inhibitor.

5 The actions of urodilatin appear to involve ATP‐sensitive K+ channels since tolbutamide (10−6‐10−5m) significantly attenuated the relaxations induced by this peptide.

6 Small conductance Ca2+‐activated K+ channels seem likewise to be implicated in the actions of urodilatin since blockade of these channels with apamin (10−7‐10−6m) resulted in a marked attenuation of urodilatin‐evoked responses.

7 The presence of charybdotoxin (10−9 m‐10−7 m) had no significant effect on subsequent responses to urodilatin suggesting that large conductance Ca2+‐activated K+ channels are not involved in the relaxations evoked by this peptide.

8 In the presence of phosphoramidon (3.68 × 10−5 m), urodilatin (10−6 m) evoked elevation of cyclic GMP levels within bovine bronchial tissue. Levels of cyclic GMP increased significantly within 5–10 s in response to this peptide and preceded the initiation of relaxant responses. Maximum increases in cyclic GMP levels were reached within 5 min; the time required for maximal relaxation evoked by this peptide.

9 In conclusion, urodilatin, like ANP reversed and protected against, subsequent methacholine‐induced bronchoconstriction; an action enhanced by the presence of phosphoramidon (3.68 × 10−5 M). Associated with these actions of urodilatin was a rise in cyclic GMP levels as well as the opening of ATP‐sensitive K+ and small conductance Ca2+‐activated K+ channels.
Increase by NG‐nitro‐L‐arginine methyl ester (L‐NAME) of resistance to venous return in rats
Authors: Wang Yong‐Xiang , Lim Su Lin , Pang Catherine C.Y.
Published: July 2012

1 The effects of the nitric oxide (NO) synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), on mean circulatory filling pressure (MCFP), total peripheral resistance (TPR), cardiac output (CO) and resistance to venous return (Rv) were studied in rats.

2 In conscious, unrestrained rats, L‐NAME (0.5–16 mg kg−1) dose‐dependently increased mean arterial pressure (MAP) but not MCFP, an inverse index of venous compliance, either in the absence or presence of the ganglionic blocker mecamylamine (l0 mgkg−1).

3 In pentobarbitone‐anaesthetized rats, L‐NAME (2, 4, 8 mg kg−1) increased MAP and reduced CO in a dose‐related manner but did not change MCFP. TPR (+ 84, +140 and +192%) as well as Rv (+ 62, + 72, +110%) were dose‐dependently increased by L‐NAME.

4 Our results show that L‐NAME reduces CO by increasing arterial as well as venous resistances. L‐NAME does not affect MCFP.
Blockade of mast cell histamine secretion in response to neurotensin by SR 48692, a nonpeptide antagonist of the neurotensin brain receptor
Authors: Miller Lisa A. , Cochrane David E. , Carraway Robert E. , Feldberg Ross S.
Published: July 2012

1 Pretreatment of rat isolated mast cells with SR 48692, a nonpeptide antagonist of the neurotensin (NT) receptor, prevented histamine secretion in response to NT.

2 This inhibition was rapid in onset (∽1 min) and dependent upon the concentration of SR 48692 (IC50 ∽1–10 nM).

3 SR 48692 (l‐1000 nM) did not inhibit histamine secretion elicited by substance P, bradykinin or compound 48/80, or by anti‐IgE stimulation of sensitized mast cells.

4 When SR 48692 was injected intradermally (5 pmol in 50 μl) into anaesthetized rats, 15 min before the intradermal injection of NT, it reduced the effect of NT on vascular permeability.

5 When injected intravenously, SR 48692 attenuated the effects of NT on haematocrit and blood stasis.

6 These results demonstrate that SR 48692 selectively antagonizes the actions of NT on rat isolated mast cells as well as mast cells in vivo. Given the demonstrated specific interaction of SR 48692 with receptors for NT in brain, our results suggest the presence of specific NT receptors on mast cells.
Different effects of salmeterol, formoterol and salbutamol on cholinergic responses in the ferret trachea
Authors: Bergendal A. , Linden A. , Skoogh B.‐E. , Löfdahl C.‐G. , Lötvall J.
Published: July 2012

1 In the present study, the inhibitory effects of the selective β2‐adrenoceptor agonists, salmeterol, formoterol and salbutamol, have been investigated on contractions of ferret trachea induced both by endogenous and exogenous acetylcholine. The aim of the study was to evaluate quantitative and/or qualitative differences in response which may indicate both pre‐and post‐junctional sites of action. The non‐selective β‐antagonist, sotalol, was used to estimate β‐adrenoceptor involvement.

2 Isometric tension was measured in ferret isolated tracheal strips. The inhibitory effects of the drugs were studied on tonic contractions induced by pre‐junctional activation with electrical field stimulation (EFS) (2 Hz, 700 mA) or postjunctional activation with exogenous acetylcholine (ACh) (0.5 μm, about EC80), giving a similar degree of smooth muscle response.

3 Concentration‐response experiments were performed with formoterol (0.3 nM‐0.3 μm) and salmeterol and salbutamol (10 nM‐10 μm). The experiments ended with the addition of sotalol (10 μm).

4 All three P‐agonists inhibited the contractions in a concentration‐dependent manner. Salbutamol, formoterol and salmeterol inhibited the EFS‐induced contractions by 66(8)%, 105(5)% and 103(8)% (mean(s.e.mean)) respectively. ACh‐induced contractions were inhibited by 37(6)%, 72(11)% and 33(8)%. Theophylline (10nM‐3mM) inhibited the contractions to the same degree.

5 β‐Adrenoceptor blockade by sotalol significantly antagonized the inhibitory effects of salbutamol and formoterol on both EFS‐ and ACh‐induced contractions. The effect of salmeterol on ACh‐induced contraction was also significantly antagonized, whereas the inhibition of EFS‐induced contraction was virtually unaffected.

6 In conclusion, salbutamol, salmeterol and formoterol produced greater inhibitory effects in preparations contracted by EFS than in preparations contracted by exogenously‐added ACh. In the case of formoterol and salbutamol, the effects on both levels are most probably due to β‐adrenoceptor stimulation, whereas for salmeterol the dominant pre‐junctional effect is probably not mediated via β‐adrenoceptors. This non‐β‐mediated effect could represent an additional relaxant mechanism for salmeterol.