bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide‐induced nitric oxide synthase activity in RAW 264.7 murine macrophages
Authors: Paul Andrew , Pendreigh Robert H , Plevin Robin
Published: July 2012

In RAW 264.7 macrophages, lipopolysaccharide (LPS) and γ‐interferon (IFNγ) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS.

LPS‐induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum.

LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFNγ did not potentiate LPS‐stimulated PKC activity.

The protein kinase C inhibitor, Ro‐318220, abolished both LPS‐ and IFNγ‐stimulated protein kinase C activity and the induction of NOS activity.

LPS‐ and IFNγ‐induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS‐stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA).

Nicotinamide, an inhibitor of poly‐ADP ribosylation, abolished LPS‐ and IFNγ‐induced NOS activity.

Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP‐ribosylation factor, ARF, reduced LPS‐ and IFNγ‐induced NOS activity by approximately 80%.

These results suggest the involvement of protein kinase C, tyrosine kinase and ploy‐ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFNγ.
Comparisons of the effects of nicorandil, pinacidil, nicardipine and nitroglycerin on coronary vessels in the conscious dog: role of the endothelium
Authors: Ghaleh Bijan , Dubois‐Randé Jean‐Luc , Hittinger Luc , Giudicelli Jean‐François , Berdeaux Alain
Published: July 2012

The vasodilator properties of nicorandil on large and small coronary arteries were compared to those of nicardipine, pinacidil, nitroglycerin and acetylcholine in six conscious dogs.

Intravenous bolus injections of acetylcholine (0.1 μg kg−1), nitroglycerin (0.3–3 μg kg−1), pinacidil (10–100 μg kg−1), nicardipine (3–30 μg kg−1) and nicorandil (10–100 μg kg−1) dose‐dependently increased circumflex coronary artery diameter and decreased coronary vascular resistance, indicating vasodilator effects on both conduit and resistance coronary arteries.

Three days after removal of the endothelium of the circumflex coronary artery (balloon angioplasty), pinacidil‐ and nicardipine‐induced dilatation of large coronary arteries was greatly reduced (both − 76%, P<0.01) whereas that produced by nitroglycerin and nicorandil was decreased only slightly and to a similar extent for both drugs (− 19%, P<0.01 and −28%, P<0.05, respectively).

Thus in conscious dogs, nicardipine‐ and pinacidil‐induced dilatation of large coronary arteries is endothelium‐dependent. In contrast, the vasodilator effects of nitroglycerin and nicorandil on conduit vessels are endothelium‐independent.

Finally, our results demonstrate that nicorandil dilates the large coronary arteries through its nitrate‐like action and that the ATP‐potassium channel opening properties of the drug are not involved in this effect in the conscious dog.
Unexpected prosecretory action component of loperamide at μ–opioid receptors in the guinea‐pig colonic mucosa in vitro
Authors: Kromer Wolfgang
Published: July 2012

In a voltage clamp setting (Ussing chamber), the antidiarrhoeal drug, loperamide (Lop) slightly augmented prostaglandin E1 (PGE1) plus theophylline‐stimulated net chloride secretion above control values at low concentrations (10−10 and 10−9 m) but inhibited it at higher concentrations (10−6 and 10−5 m). The apparently weak prosecretory action component of Lop was turned into a clear cut antisecretory effect by pretreatment with 2 times 10−7 m naloxonazine plus 10−7 m CTOP‐NH2 , two selective μ opioid receptor antagonists. This indicates a prosecretory effect of loperamide at μ opioid receptors. The antisecretory effect of low Lop concentrations, uncovered by μ opioid receptor blockade, was prevented by additional blockade of κ opioid receptors by 5 times 10−9 m nor‐binaltorphmiine (nor‐BNI).

The nonselective opioid antagonist, naloxone, at 10−6 m did not significantly reduce either PGE1 plus theophylline‐stimulated net chloride secretion in Lop‐free controls or the antisecretory action of Lop. By contrast, the partial agonist ethylketocyclazocine (EKC), which activates κ but blocks μ opioid receptors, concentration‐dependently inhibited PGE1 plus theophylline‐stimulated net chloride secretion without any consistent prosecretory action component. Nor‐BNI at 5 times 10−8 m significantly blocked the antisecretory action of EKC.

It is concluded that, in the guinea‐pig colonic mucosa under the present conditions, μ opioid receptors mediate enhancement and κ opioid receptors inhibition of PGE1‐stimulated net chloride secretion by low Lop concentrations. The two opposite actions are largely masked by superimposition. An opioid receptor‐independent mechanism of action contributes to the antisecretory effect of Lop at high concentrations.
Effects of portal hypertension on responsiveness of rat mesenteric artery and aorta
Authors: Cawley Teresa , Geraghty J. , Osborne H. , Docherty J.R.
Published: July 2012

We have examined the effects of pre‐hepatic portal hypertension on the responsiveness of rat small mesenteric arteries and aorta. Rats were made portal hypertensive by creating a calibrated portal vein stenosis, or sham‐operated.

In rat mesenteric arteries, there was no significant difference between portal hypertensive and sham‐operated animals in the contractile potency of noradrenaline (NA), but the maximum contractile responses to NA, U46619 and KCl were significantly increased in vessels from portal hypertensive animals. This altered maximum contractile response was not due to alterations in smooth muscle mass.

In rat mesenteric arteries, there were no significant differences between portal hypertensive and sham‐operated animals in endothelium‐dependent relaxations to acetylcholine (ACh). The difference between portal hypertensive and sham‐operated rats in the maximum response to U46619 was maintained following a combination of methylene blue (1 μm) and NG‐monomethyl‐l‐arginine (100 μm), suggesting that any differences in endothelial function do not explain differences in the response to vasoconstrictors.

In rat aorta, there were no significant differences between portal hypertensive and sham‐operated animals in the contractile response to NA or KCl or in the endothelium‐dependent relaxations to ACh.

In pithed rats, there was no difference between portal hypertensive and sham‐operated animals in the pressor potency of NA.

It is concluded that portal hypertension produces an increase in the contractile response to the vasoconstrictors NA, U46619 and KCl in rat mesenteric arteries but not in the aorta. This suggests that the diminished responsiveness to vasoconstrictors reported in portal hypertensive rats in vivo is not due to a diminished responsiveness at the level of the vascular smooth muscle.
The inhibition of CYP enzymes in mouse and human liver by pilocarpine
Authors: Kimonen Tanja , Juvonen Risto O. , Alhava Esko , Pasanen Markku
Published: July 2012

Pilocarpine is a cholinomimetic natural alkaloid. Its interactions with testosterone hydroxylations, coumarin 7‐hydroxylase (COH), dimethylnitrosamine N‐demethylase (DMNA), pentoxyresorufin O‐dealkylase (PROD) and 7‐ethoxyresorufin O‐deethylase (EROD), which are indicative of the activities of cytochrome P4502A5 (CYP2A5) or 6, 2E1, 2B, 1A, were examined in mouse and human liver microsomes.

In mouse liver microsomes the IC50 values of pilocarpine were 6 μm for COH and testosterone 15a‐hydroxylase (T15αOH) activities, 4 μm for PROD, ≅ 100 μm for DMNA and testosterone 60‐hydroxylase (T6βOH) activities and > 1 mm for EROD activity.

In human liver microsomes, the IC50 value for COH was 6 μm and for DMNA 10 μm; T15αOH and PROD activities were not detectable but T6βOH and testosterone 16β/2β‐hydroxylase activities were moderately inhibited (IC50 70 μm).

These results suggest that pilocarpine has (i) a high affinity towards phenobarbitone‐inducible CYP2A4/5 and CYP2B activities in mouse liver, (ii) a high affinity towards CYP2A6 in human liver microsomes and (iii) a moderate affinity towards CYP3A enzyme(s) in both microsomal preparations.

The low IC50 concentrations in vitro indicate potential metabolic interactions between pilocarpine and several P450 enzymes.
Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea‐pigs
Authors: ten Berge Donald E.J. , Santing Ruud E. , Hamstra Jacob Jan , Roffel Ad F. , Zaagsma Johan
Published: July 2012

Using a guinea‐pig model of allergic asthma, in which the animals display early (0–5 h) and late phase (8–23 h after antigen challenge) bronchoconstrictor reactions, the function of prejunctional inhibitory M2 and postjunctional M3 receptors in isolated tracheal preparations have been investigated. In addition, cardiac M2 receptor function in vitro and bronchial responsiveness to histamine in vivo were evaluated.

Sensitivity to inhaled histamine was increased 3.1 fold and 1.6 fold after the early and late allergic reactions (i.e. at 5 h and 23 h after a single ovalbumin challenge), respectively. At 23 h after the last of four allergen challenges, executed on four consecutive days, bronchial hyperresponsiveness to histamine was diminished to 1.3 fold.

After the early response, there was no change in cardiac muscarinic M2 receptor function, since in left atria pD2 (‐log EC50) and Emax values of pilocarpine and pKB values of AQ‐RA 741, a selective M2 receptor antagonist, were not significantly different from controls (unchallenged sensitized animals), and this also applied to methacholine pD2 values for muscarinic M3 receptors in tracheal smooth muscle.

Prejunctional inhibitory muscarinic M2 autoreceptors in airway smooth muscle were markedly dysfunctional after the early allergic response, since potentiation of electrically evoked twitch contractions of tracheal preparations by low concentrations of the M2‐selective muscarinic receptor antagonists, gallamine, methoctramine, AQ‐RA 741 and AF‐DX 116, which is the result of M2 receptor blockade, was clearly and significantly diminished compared to controls. However, after the late response, both in single and repeatedly challenged animals, twitch potentiation was not significantly different from and similar to controls, indicating restoration of M2 receptor function during the late allergic reaction.

It is concluded that dysfunction of muscarinic M2 autoreceptors in the airways of sensitized and challenged guinea‐pigs is already present after the early allergic reaction, and that it has recovered after the late response. Since histamine‐induced bronchoconstriction involves vagal pathways, the present results suggest that bronchial hyperresponsiveness to histamine is partly due to M2 autoreceptor dysfunction, leading to increased release of acetylcholine.
Role of endothelium in the human uterine arteries during normal menstrual cycle
Authors: Azuma Hiroshi , Obayashi Satoshi , Hamasaki Hidehisa , Koyama Takao , Aso Takeshi
Published: July 2012

The present experiments were designed to investigate the role of endothelium in the human uterine arteries during the normal menstrual cycle.

Acetylcholine (ACh) produced a concentration‐dependent relaxation response during the higher level of plasma 17β‐oestradiol (E2) (follicular and luteal phases, E2 = 131.9 ± 15.9 pg ml−1, n = 13; group I). However, the agent did not produce a definite relaxation, but produced a slight contraction during the ovulatory and menstruation phases (E2 = 19.8 ± 2.9 pg mg−1, n = 5; group II). During the follicular and luteal phases (E2 = 181.1 ± 9.0 pg ml−1, n = 6), ACh produced a slight contraction, but not relaxation in 6 cases (group III). Relaxation in response to A23187 in group II was not different from that in group I, while it was significantly (P<0.05 and P <0.005) reduced in group III. Sodium nitroprusside (SNP)‐induced relaxation was similar in the three groups.

Correlation between the maximum response to ACh and the plasma E2 was highly significant (γ = 0.8142, P <0.001) in 18 cases of groups I and II, but not in all 24 cases including group III (γ = 0.1183, NS).

Relaxations in response to ACh in group I or A23187 in all groups were abolished after removal of the endothelium. In group I, ACh‐ and A23187–induced relaxations were greatly inhibited by methylene blue or NG‐nitro‐l‐arginine (l‐NOARG) and partially inhibited by indomethacin. None of these treatments except for methylene blue modified the SNP‐induced relaxation, which was significantly inhibited by methylene blue.

The A23187‐induced relaxation was hardly affected by methylene blue or l‐NOARG in group III, but was partially inhibited by these agents in group II. The effect of indomethacin in inhibiting the A23187 induced‐relaxation was most potent (58.9%) in group III and least (16.9%) in group I.

There were no histological changes in 14 cases out of 18 (groups I and II), but very slight intimal thickening was observed in 4 cases in group I. On the other hand, severe intimal thickening was observed in all 6 cases in group III.

These results indicate that, in human uterine artery strips, ACh and A23187 cause endothelium‐dependent relaxations, which are mediated mainly through EDRF/NO in group I, mainly prostacyclin (PGI2) in group III, or both in group II. It is suggested that lack of the production/release of EDRF/NO and/or of interaction between EDRF/NO and PGI2 might play a role in the formation of intimal thickening in human uterine arteries.
Bradykinin receptors in mouse and rat isolated superior cervical ganglia
Authors: Seabrook G.R. , Bowery B.J. , Hill R.G.
Published: July 2012

The ability of bradykinin and its analogues to depolarize rat and mouse superior cervical ganglia was studied by use of in vitro grease‐gap recording techniques, and the ability of antagonists selective for bradykinin receptor subtypes to block their effects was examined.

Bradykinin (μm) depolarized ganglia from both species, although the magnitude of the maximal response was less in mouse (15 ± 5%, n = 7) than rat tissue (33 ± 6%, n = 7), relative to muscarine (1μm).

Interleukin 1β (30 u ml−1 for 18 h at 37°C) increased the depolarization caused by bradykinin (3μm) in mouse ganglia from 15% to 54% (P<0.001, n=12). Responses to the B1 receptor agonist, [des‐Arg10]‐kallidin (3μm) were similarly potentiated but this was only detected after inhibition of peptidase activity with 10FM captopril (4% to 35%, n = 5).

In ganglia from both species the rank order of agonist potency was bradykinin = [Lys0]‐bradykinin >> [des‐Arg10]‐kallidin. However, like responses to [des‐Arg10]‐kallidin in mouse tissue, both the potency of bradykinin and the maximal depolarization achieved (EC50 = 912 nM; 80%, n = 11) was enhanced following inhibition of angiotensin converting enzyme with 10μm captopril (EC50 = 50 nM; 135%, n = 4).

Responses to bradykinin were selectively antagonized by the B2 receptor antagonist, Hoe 140 but not by the B1 antagonist, [Leu8]‐bradykinin1–8. From Schild analysis the pA2 value for Hoe 140 in mouse tissue was 9.65, although the slope of the regression line was significantly greater than unity, indicating non‐competitive kinetics (slope = 1.88 ± 0.18, n = 9). The depolarization caused by [Lys0]‐bradykinin was also antagonized by Hoe 140 (3 nM).

Thus the predominant bradykinin receptor in mouse superior cervical ganglia is compatible with a B2 subtype. Furthermore the depolarizations caused by B1 and B2 agonists in this tissue can be increased following exposure to interleukin 1β, and by blocking peptide degradation with captopril.
Effects of endothelin receptor antagonism with bosentan on peripheral nerve function in experimental diabetes
Authors: Stevens Elizabeth J. , Tomlinson David R.
Published: July 2012

The effects of the non‐selective endothelin (ET) receptor (ETA/ETB) antagonist, bosentan, on sciatic nerve dysfunction in experimental diabetes were investigated.

Rats with 5–6 weeks untreated streptozotocin‐diabetes exhibited characteristic slowed motor nerve conduction velocity (mean ± s.d., 36.6 ± 3.4 m s−1) and nerve laser Doppler flux (197 ±64 arbitrary units) compared to age‐matched control animals (42.7 ± 2.4 m s−′ and 398 ± 77 arbitrary units, respectively). Preventative treatment of diabetic rats with bosentan at 100 mg kg−1 day−1 p.o. attenuated both these deficits (39.7 ± 3.0 m s−1 and 305 ± 56 arbitrary units, respectively) without affecting mean arterial pressure.

In control and untreated diabetic rats, ET‐1, 1 nmol kg−1 i.v., caused an initial hypotension (duration, 30 ±13 and 26 ± 9 s, respectively; change in mean arterial pressure, −27 ±13 and − 25 ± 7 mmHg, respectively) followed by prolonged hypertension (change in mean arterial pressure, 52 ± 18 and 31 ± 5 mmHg, respectively). Effectiveness of the chronic bosentan treatment was demonstrated by inhibition of the hypotensive response to ET‐1 in treated diabetic rats (duration, 5 ± 2 s; change in mean arterial pressure, − 4 + 2 mmHg) although the hypertension was unaltered (change in mean arterial pressure, 32 + 9 mmHg).

Acute i.v. administration of 10mg kg−1 bosentan caused variable and transient rises in nerve laser Doppler flux in control (78 ± 63 arbitrary units) and untreated diabetic rats (93 ± 77 arbitrary units). Acute bosentan blocked the hypotensive response to subsequent ET‐1 administration and attenuated the later hypertension (change in mean arterial pressure, 21 ±9 mmHg in control, 29 ±10 mmHg in diabetic).

Our results indicate that oral treatment of diabetic rats with an ET receptor antagonist can improve sciatic nerve perfusion and conduction, suggesting that the vasoconstrictor action of endogenous ET may contribute to peripheral nerve dysfunction in experimental diabetes.
Further evidence from functional studies for somatostatin receptor heterogeneity in guinea‐pig isolated ileum, vas deferens and right atrium
Authors: Feniuk W. , Dimech J. , Jarvie E.M. , Humphrey P.P.A.
Published: July 2012

Somatostatin (SRIF) causes a concentration‐dependent inhibition of neurotransmission in guinea‐pig ileum and vas deferens as well as negative inotropy in guinea‐pig isolated right atrium. The SRIF receptors mediating these effects have now been further characterized by use of the peptides BIM‐23027, BIM‐23056 and L‐362855, reported as selective for the recombinant SRIF receptor types, sst2, sst3 and sst5, respectively.

BIM‐23027 was a highly potent agonist at causing an inhibition of neurotransmission in the guinea‐pig ileum (EC50 value 1.9 nM), being about 3 times more potent than SRIF (EC50 value 6.8 nM). In contrast, in both guinea‐pig vas deferens and right atrial preparations, BIM‐23027 was a relatively weak agonist being at least 30–100 times weaker than SRIF. In guinea‐pig atria, BIM‐23027 (3 μu) antagonized the negative inotropic action of SRIF28 (apparent pAb = 5.9±0.1) but had no effect on the negative inotropic action of cyclohexyladenosine.

The inhibitory effect of BIM‐23027 in the guinea‐pig ileum was readily desensitized. Prior exposure to BIM‐23027 (0.3 μm) markedly attenuated the inhibitory effect of SRIF but had no effect on the inhibitory action of clonidine suggesting that BIM‐23027 and SRIF act via a common receptor mechanism.

L‐362855 caused a concentration‐dependent inhibition of neurotransmission in both the guinea‐pig ileum and vas deferens as well as causing negative inotropy in the guinea‐pig atrium but was at least 30–100 times weaker than SRIF. In guinea‐pig isolated atria, L‐362855 (3 μm) did not antagonize the negative inotropic action of SRIF28.

BIM‐23056 in concentrations up to 1 μm was inactive as an agonist in guinea‐pig isolated ileum, vas deferens and atrium and did not antagonize the inhibitory actions of SRIF in any of these preparations.

The results from this study support our previous contention that the sst2 receptor type mediates inhibition of neurotransmission by SRIF in the guinea‐pig ileum. The SRIF receptor type mediating inhibition of neurotransmission in the guinea‐pig vas deferens appears different, but similar, to that mediating negative inotropy in the atrium. However the characteristics of these latter receptors appear different from that of the recombinant sst2, sst3 and sst5 receptors for SRIF described for rat and man.
Glucocorticoid‐induced annexin 1 secretion by monocytes and peritoneal leukocytes
Authors: Coméra Christine , Russo‐Marie Frarçise
Published: July 2012

We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes.

The in vivo treatment of rats with dexamethasone (1.25 mg kg−1) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro, by 10−6m dexamethasone.

Annexin 1 secretion was inhibited in the presence of 20 mM NH4C1 or by conducting the experiments at 18°C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A.

The time necessary for annexin 1 synthesis and secretion was less than 15 min.

These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin‐iβ.
In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs
Authors: Oida Hiroji , Namba Tsunehisa , Sugimoto Yukihiko , Ushikubi Fumitaka , Ohishi Hitoshi , Ichikawa Atushi , Narumiya Shuh
Published: July 2012

Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co‐localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively.

IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes.

IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe.

IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small‐ and large‐sized neurones were labelled but no labelling was found in the glia. Expression of PPT A mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor‐positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor‐positive neurones co‐expressed the EP1, EP3 and EP4 receptor, respectively.

These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.
Effect of chronic morphine treatment on transmitter release from sympathetic varicosities of the mouse vas deferens
Authors: Lavidis Nickolas A.
Published: July 2012

Transmitter release from sympathetic varicosities of mouse vasa deferentia removed from animals which were chronically treated with morphine for 7 to 9 days has been evaluated.

In control preparations increasing the extracellular calcium concentration ([Ca2+]o) from 1 to 2 mM increased transmitter release by 3 fold while increasing [Ca2+]o from 6 to 8 mM increased transmitter release by about 0.9 fold. Introduction of morphine (1.0 μm) produced a uniform decrease in transmitter release, shifting the relationship between transmitter release and [Ca2+]o to the right.

Only sympathetic varicosities with probabilities of transmitter release greater than 0.01 were chosen for this study. In these varicosities the decrease in transmitter release induced by morphine in control preparations (bathed in [Ca2+]o 2.0 mm) was not observed following 7 to 9 days of morphine treatment. When the morphine was acutely withdrawn from these preparations transmitter release was more than 6 times the average level of transmitter release from control preparations.

The morphine induced increase in facilitation of transmitter release while stimulating with short trains of nerve impulses was not observed when the preparations were removed from animals which had been exposed to morphine for 7 to 9 days. When these preparations were acutely withdrawn from morphine there was a further decrease in the level of facilitation and a significant increase in depression of transmitter release when compared to control.

The morphine induced decrease in probability of transmitter release when naive sympathetic varicosities in vitro were bathed with morphine (1 μm) was not observed following chronic morphine treatment of the animals for 7 to 9 days. When the morphine was acutely withdrawn from chronically morphine treated preparations the underlying increase in probabilities of transmitter release of sympathetic varicosities was unmasked.
Calcium compartments in vascular smooth muscle cells as detected by aequorin signal
Authors: Abe Fujio , Mitsui Minori , Karaki Hideaki , Endoh Masao
Published: July 2012

To examine whether cytosolic Ca2+ in smooth muscle cells distributes evenly, cytosolic Ca2+ levels were measured with two different Ca2+ indicators in the ferret isolated portal vein; a fluorescent indicator, fura‐PE3, that shows the average Ca2+ level, and a photoprotein, aequorin, that preferentially shows a high Ca2+ compartment. 2. A noradrenaline (10 μm)‐induced sustained contraction was associated with a sustained increase in the fura‐PE3 signal, or a transient increase followed by small sustained increase in the aequorin signal. A high K+‐induced contraction was associated with a sustained increase in both the fura‐PE3 and aequorin signals. 3. A second application of noradrenaline or high K+ induced reproducible contractions and fura‐PE3 signals. In contrast, the aequorin signal resulting from a second application of noradrenaline or high K+ was much smaller than the first signal. 4. Following a 13 h but not a 3 h resting period, the aequorin signal stimulated by noradrenaline or high K+ recovered, without any change in the contractile response. 5. In Ca2+‐free solution, high K+ was ineffective, whereas noradrenaline induced only a small aequorin signal and contraction compared to those obtained in the presence of external Ca2+. After the addition of Ca2+, the first application of noradrenaline induced a large aequorin signal and a large contraction, although a second application induced a much smaller aequorin signal accompanied by a large contraction. 6. These results suggest that high K+ and noradrenaline increase Ca2+ in at least two cytosolic compartments; a compartment that is coupled to the contractile mechanism (‘contractile’ Ca2+ compartment; major portion of cytoplasm containing contractile elements) and a compartment that is not coupled to contractile mechanisms (‘non‐contractile’ Ca2+ compartment; small sub‐membrane area that does not contain contractile elements). On stimulation, the Ca2+ level in the ‘contractile’ compartment may increase to a level high enough to stimulate myosin light chain kinase but not so high as to consume aequorin rapidly. In contrast, the Ca2+ level in the ‘non‐contractile’ compartment may increase so greatly that aequorin in this compartment is rapidly consumed. These two compartments may be separated by a diffusion barrier and, during a resting period, aequorin may slowly diffuse from the ‘contractile’ compartment to the ‘non‐contractile’ compartment and thus restore the full aequorin signal. An increase in Ca2+ in the ‘non‐contractile’ compartment seems to be dependent mainly on Ca2+ influx and partly on Ca2+ release.
Phosphorylation‐ and voltage‐dependent inhibition of neuronal calcium currents by activation of human D2(short) dopamine receptors
Authors: Brown Nicola A. , Seabrook Guy R.
Published: July 2012

Activation of human D2(8) dopamine receptors with quinpirole (10 nM) inhibits ω‐conotoxin GVIa‐sensitive, high‐threshold calcium currents when expressed in differentiated NG108‐15 cells (55% inhibition at +10 mV). This inhibition was made irreversible following intracellular dialysis with the non‐hydrolysable guanosine triphosphate analogue GTP‐γ‐S (100 μM), and was prevented by pretreatment with pertussis toxin (1 μg ml−1 for 24 h).

Stimulation of protein kinase C with the diacylglycerol analogue, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (100 μM), also attenuated the inhibition of the sustained calcium current but did not affect the receptor‐mediated decrease in rate of current activation. Similarly, okadaic acid (100 nM), a protein phosphatase 1/2A inhibitor, selectively occluded the inhibition of the sustained current.

The depression of calcium currents by quinpirole (10 nM) was enhanced following intracellular dialysis with 100 μM cyclic adenosine monophosphate (cyclic AMP, 72.8 ±9.8% depression), but was not mimicked by the membrane permeant cyclic GMP analogue, Sp‐8‐bromoguanosine‐3′, 5′:cyclic monophosphorothioate (100 μM).

Inhibition of calcium currents was only partly attenuated by 100 ms depolarizing prepulses to +100 mV immediately preceding the test pulse. However, following occlusion of the sustained depression with okadaic acid (100 nM) the residual kinetic slowing was reversed in a voltage‐dependent manner (P<0.05).

Thus pertussis toxin‐sensitive G‐proteins liberated upon activation of human D2(short) dopamine receptors inhibited high‐threshold calcium currents in two distinct ways. The decrease in rate of calcium current activation involved a voltage‐dependent pathway, whereas the sustained inhibition of calcium current involved, in part, the voltage‐resistant phosphorylation by cyclic AMP‐dependent protein kinases and subsequent dephosphorylation by protein phosphatases 1/2A.
Induction of a novel form of hippocampal long‐term depression by muscimol: involvement of GABAA but not glutamate receptors
Authors: Akhondzadeh S. , Stone T.W.
Published: July 2012

Unlike long‐term potentiation, long‐term depression (LTD) in the central nervous system remains poorly understood. The present study was undertaken to investigate the role of GABAA receptors in LTD and synaptic plasticity.

Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum (0.01 Hz).

Muscimol induced a time‐ and concentration‐dependent LTD of the amplitude of orthodromic potentials. Increasing the stimulation frequency from 0.01 Hz to 1 Hz for 10 s reversed the LTD induced by muscimol. Muscimol also induced LTD in the absence of electrical stimulation.

Adenosine decreased the spike size in a concentration‐dependent manner, but failed to induce LTD.

Alphaxalone and 5α‐pregnan‐3α‐ol‐20‐one at concentrations that did not have any effect themselves on the population spike (0.5 and 1 μM), potentiated the inhibitory effect of muscimol on the population spike size, including concentrations which were not effective by themselves. Both steroids were able to potentiate the ability of muscimol to induce LTD.

Bicuculline, 5 μM, reversed the LTD induced by muscimol, 10 μM.

The NMDA receptor antagonist (±)‐2‐amino‐5‐phosphonopentanoic acid (2‐AP5), the NMDA/ metabotropic antagonist 2‐AP3 and selective metabotropic antagonist L‐(+)‐2‐amino‐3‐phosphonopro‐pionic acid (L(+)‐AP3) failed to modify the LTD. Similarly, quisqualic acid and (1S, 3R)‐aminocyclopentane dicarboxylic acid (ACPD) a selective agonist at metabotropic receptors did not induce LTD or short‐term depression, whereas kynurenic acid prevented the reversal of the LTD obtained at 1 Hz.

It is concluded that LTD can be induced by the selective activation of GABAA receptors. The lack of involvement of glutamate receptors in our protocol confirms the unique nature of the LTD described here. The phenomenon of GABA‐induced LTD and its reversal by 1 Hz stimulation may represent a novel type of long‐lasting depression by which inhibitory interneurones can modulate pyramidal cell excitability in a frequency‐dependent manner.
A role for Q type Ca2+ channels in neurotransmission in the rat urinary bladder
Authors: Frew Robert , Lundy Paul M.
Published: July 2012

In isolated bladder strips of the rat, a substantial component (46%) of the Ca2+‐dependent contractile response to electrical field stimulation (5 Hz) was resistant to combined block of both N and P type Ca2+ channels by ω‐conotoxin‐GVIA (300 nM) and ω‐agatoxin‐IVA (100 nM) respectively.

The resistant portion (non‐N, non‐P) was sensitive to ω‐conotoxin‐MVIIC (3 μM), which in addition to N and P also blocks Q type channels at this concentration. ω‐Conotoxin‐MVIIC administered alone, inhibited the neurogenic response to the same degree as that observed in the combined presence of ω‐agatoxin‐IVA, ωconotoxin‐GVIA and ω‐conotoxin‐MVIIC.

ω‐Agatoxin‐IVA (100 nM), a concentration that fully inhibits P type channels, had a negligible effect on the neurogenic response. Following blockade of N type Ca2+ channels with ω‐conotoxin‐GVIA (300 μM), ω‐agatoxin‐IVA (3 μM) (a concentration well above that used to block P channels, inhibits Q type channels, but spares N type channels), inhibited the residual response to the same degree as ω‐conotoxin‐MVIIC alone.

Results suggest that neurotransmission in rat urinary bladder is supported by both N and Q type Ca2+ channels.
Histamine H1 receptor occupancy in human brains after single oral doses of histamine H1 antagonists measured by positron emission tomography
Authors: Yanai Kazuhiko , Ryu Jong Hoon , Watanabe Takehiko , Iwata Ren , Ido Tatsuo , Sawai Yoneichi , Ito Kazumi , Itoh Masatoshi
Published: July 2012

Histamine H1 receptor occupancy in the human brain was measured in 20 healthy young men by positron emission tomography (PET) using [11C]‐doxepin.

(+)‐Chlorpheniramine, a selective and classical antihistamine, occupied 76.8±4.2% of the averaged values of available histamine H1 receptors in the frontal cortex after its administration in a single oral dose of 2 mg. Intravenous administration of 5 mg (+)‐chlorpheniramine almost completely abolished the binding of [uC]‐doxepin to H1 receptors (H1 receptor occupancy: 98.2±1.2%).

Terfenadine, a nonsedative antihistamine, occupied 17.2±14.2% of the available H1 receptors in the human frontal cortex after its administration in a single oral dose of 60 mg.

There was no correlation between H1 receptor occupancy by terfenadine and the plasma concentration of the active acid metabolite of terfenadine in each subject.

PET data on human brain were essentially compatible with those on H1 receptor occupancy in guinea‐pig brain determined by in vivo binding techniques, although for the same H1 receptor occupancy the dose was less in human subjects than in guinea‐pigs.

The PET studies demonstrated the usefulness of measuring H1 receptor occupancy with classical and second‐generation antihistamines in human brain to estimate their unwanted side effects such as sedation and drowsiness quantitatively.
Effects of divalent cations and La3+ on contractility and ecto‐ATPase activity in the guinea‐pig urinary bladder
Authors: Ziganshin Airat U. , Ziganshina Lilia E. , Hoyle Charles H.V. , Burnstock Geoffrey
Published: July 2012

Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1–1000 μm) were tested in the guinea‐pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, α,β‐methylene ATP (α,β‐meATP), carbachol (CCh), and KCl; (ii) affect ecto‐ATPase activity.

Ba2+ (10–1000 μm) concentration‐dependently potentiated contractile responses evoked by EFS (4–16 Hz), ATP (100 μm), α,β‐meATP (1 μm), CCh (0.5 μm), and KCl (30 mm). Ni2+ at concentrations of 1–100 μm also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration‐dependent. Cu2+ at a concentration of 10 μm and Cd2+ at a concentration of 1 μm potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 μm and Cd2+ at a concentration of 100 μm inhibited contractions evoked by all stimuli, and at a concentration of 1000 μm Cd2+ abolished any contractions. Responses to ATP and α,β‐meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mm.

Cu2+, Ni2+, Zn2+ and La3+ (100–1000 μm) concentration‐dependently inhibited ecto‐ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 μm.

There was no correlation between the extent of ecto‐ATPase inhibition and the effect on contractile activity of any of the cations.

In conclusion, the ability of some divalent cations to inhibit ecto‐ATPase activity and to potentiate or inhibit contractile responses in the guinea‐pig urinary bladder appear to be independent effects.
Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide‐sensitive K‐channel in smooth muscle cells of rat mesenteric artery
Authors: Zhang H. , Bolton T.B.
Published: July 2012

Single‐channel recordings were made from cell‐attached and isolated patches, and whole‐cell currents zwere recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery.

In single voltage‐clamped cells 1 mm uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 μm) a K‐channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 μm). In single cells from which recordings were made by the ‘perforated patch’ (nystatin pipette) technique, metabolic inhibition by 1 mm NaCN and 10 mm 2‐deoxy‐glucose also evoked a similar glibenclamide‐sensitive current.

Single K‐channel activity was observed in cell‐attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide‐sensitive channel activity. If the patch was pulled off the cell to form an isolated inside‐out patch, similar glibenclamide‐sensitive single‐channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell‐attached mode; channel conductance was 20 pS (60:130 K‐gradient) and openings showed no voltage‐dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K‐channel (KNDP) seen previously in rabbit portal vein.

Formation of an isolated inside‐out patch into an ATP‐free solution did not increase the probability of channel opening which declined with time even when some single‐channel activity had occurred in the cell‐attached mode before detachment. However, application of 1 mm UDP or GDP, but not ATP, to inside‐out patches evoked single‐channel activity. Application of ATP‐free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity.

It is concluded that small conductance K‐channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity. Failure to respond to a fall in ATP concentration upon formation of an inside‐out patch could not be due to dephosphorylation of the channel because sometimes it had been active previously during cell‐attached recording. NDPs, instead of ATP, are more important regulators of KNDP channels. It is suggested that the KNDP is the main target K‐channel for KCOs.
Involvement of endogenous nitric oxide in the mechanism of bradykinin‐induced peripheral hyperalgesia
Authors: Nakamura Akihiro , Fujita Manabu , Shiomi Hirohito
Published: July 2012

When NG‐nitro‐L‐arginine methyl ester (L‐NAME, 0.1–10 nmol) or NG‐monomethyl‐L‐arginine (L‐NMMA, 10 nmol‐1 μmol) was intradermally administered with bradykinin (BK, 3 nmol) into the instep of rat hind‐paws, a dose‐related suppression of BK‐induced hyperalgesia, assessed by the paw‐pressure test, was produced.

L‐Arginine (1 μmol) but not D‐arginine (1 μmol) reversed the suppressive effects of L‐NAME (10 nmol) and L‐NMMA (1 μmol) on BK‐induced hyperalgesia.

Concomitant intradermal administration of BK (3 nmol) with haemoglobin (1 nmol) significantly suppressed BK‐induced hyperalgesia in the paw‐pressure test. The BK‐induced hyperalgesia was abolished by concomitant intradermal administration of either a guanylate cyclase inhibitor, methylene blue (10 nmol), or LY83583 (1 nmol). In addition, KT5823 (1 nmol) or Rp‐8‐bromoguanosine‐3′:5′‐cyclic monophosphothioate (Rp‐8‐Br‐cGMPS; 1 nmol), an inhibitor of cyclic GMP‐dependent protein kinase, also significantly suppressed BK‐induced hyperalgesia.

The carrageenin‐induced hyperalgesia was significantly attenuated by L‐NAME in a dose‐dependent manner.

L‐Arginine (1 μmol), sodium nitroprusside (1 μmol), dibutyryl cyclic GMP (1 μmol) or 8‐bromo cyclic GMP (1 μmol) all failed to produce any significant relieving effect on the nociceptive threshold of rodent hind‐paws. Concomitant administrations of each agent with a sub‐threshold dose (0.1 nmol) of BK induced significant hyperalgesia.

Rp‐adenosine 3′:5′‐cyclic monophosphothioate (Rp‐cAMPS; 1 nmol), an inhibitor of cyclic AMP‐dependent protein kinase, significantly suppressed BK‐induced mechanical hyperalgesia. Concomitant administration of forskolin (1 nmol) with 8‐bromo cyclic GMP (100 nmol) induced significant hyperalgesia.

In the superfusion experiment of a blister base on the instep of rodent hind‐paws, intradermally administered BK (3 nmol) significantly increased the outflow of both cyclic GMP and cyclic AMP from the blister base. Concomitant administrations of L‐NAME (10 nmol) with BK significantly reduced the BK‐induced outflow of cyclic GMP without affecting the cyclic AMP content.

These results suggest that the NO‐cyclic GMP pathway is involved in the mechanism of BK‐induced hyperalgesia, and an activation of both cyclic GMP‐and cyclic AMP‐second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.
Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells
Authors: Gandía Luis , Villarroya Mercedes , Lara Baldomero , Olmos Virginia , Gilabert Juan‐Antonio , LÓPEZ Manuela G. , Martínez‐Sierra Rafael , Borges Ricardo , García Antonio G.
Published: July 2012

Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide‐spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells.

45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 μm. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 μm).

Whole‐cell Ba2+ currents (IBa) through Ca2+ channels of voltage‐clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 μm, very close to that of K+‐evoked 45Ca2+ uptake. Blockade developed in 10–20 s, almost as a single step and was rapidly and almost fully reversible.

Whole‐cell nicotinic AChR‐mediated currents (250 ms pulses of 100 μm DMPP) applied at 30 s intervals were blocked by otilonium in a concentration‐dependent manner, showing an IC50 of 0.36 μm. Blockade was induced in a step‐wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps.

In experiments with recordings in the same cells of whole‐cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 μm otilonium blocked 87% IDMPP, 7% INa and 13% ICa.

Otilonium inhibited the K+‐evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 μm, very close to the IC50 for blockade of K+‐induced 45Ca2+ uptake and IBa.

Otilonium inhibited the secretory responses induced by 10 s pulses of 50 μm DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP‐evoked responses with an IC50 of 29.8 μm, 4,000 fold higher than that of otilonium.

In conclusion, otilonium is a potent blocker of nicotinic AChR‐mediated responses. The drugs also blocked various subtypes of neuronal voltage‐dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.
Antagonism by SR 48692 of mechanical responses to neurotensin in rat intestine
Authors: Mulè F. , Serio R. , Postorino A. , Vetri T. , Bonvissuto F.
Published: July 2012

The effects of SR 48692 on neurotensin (NT)‐induced mechanical responses were investigated in rat duodenum and proximal colon by use of isometric, isovolumic preparations.

SR 48692 inhibited the relaxant responses to NT in duodenal circular and longitudinal muscle. It also antagonized the NT‐induced contractile effects in duodenal circular muscle and in proximal colon (both muscular layers).

From Schild analysis the pA2 value for SR 48692 was 8.2 in tissues where NT induced relaxant effects and 7.5 in tissues where NT induced contractile effects and the slope of the regression line was not significantly different from unity, indicating competitive antagonism.

SR 48692 did not antagonize the duodenal relaxant effect induced by noradrenaline and the contractile response to carbachol or substance P in duodenum and colon.

Our results demonstrate that SR 48692 selectively antagonizes the mechanical actions of NT in rat intestine and confirm the existence of specific NT receptors. Receptors that subserve a relaxant effect seem to be related, but not identical, to those that mediate contractile effects.