bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C in rat brain cortex and hippocampus: effect of repeated administration of fluoxetine and desipramine
Authors: Mann Catherine D. , Vu T. Bich , Hrdina Pavel D.
Published: July 2012

Recent evidence indicates that changes in the activity of cyclic AMP‐dependent protein kinase may be involved in neuroadaptive mechanisms after chronic treatment with antidepressants. The aim of this study was to investigate the effect of repeated administration of fluoxetine (FL) and desipramine (DMI) on the distribution and activity of protein kinase C (PKC) in subcellular fractions of rat cortex (Cx) and hippocampus (Hc) under basal conditions and in response to a single in vivo administration of 5‐HT2A/2C agonist, 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI).

Rats were treated for 21 days with FL (5 mg kg−1 day−1, i.p.) or DMI (10 mg kg−1 day−1, i.p.). DOI was injected to groups of rats receiving repeated doses of antidepressants or to control rats 1 h before ex vivo PKC assay. Distribution of PKC was determined by [3H]‐phorbol‐12,13‐dibutyrate ([3H]‐PDBu) binding and PKC activity by the Amersham enzyme assay system.

Autoradiography of tissue sections revealed decreased [3H]‐PDBu binding in CA1 region of hippocampus (by 18%) and paraventricular thalamic nucleus (by 28%) of rats after repeated administration of FL.

In vitro exposure of brain sections to 50 μm FL resulted in significant decreases (by 23–32%) of [3H]‐PDBu binding in six out of seven regions examined; exposure to 100 μm FL reduced [3H]‐PDBu binding (by 36–52%) in all regions. In contrast, exposure of brain sections to 100 μm DMI failed to alter specific [3H]‐PDBu binding in brain sections.

The activity of PKC in subcellular fractions of Cx and Hc was significantly (by 40–50%) decreased in rats given repeated doses of FL or DMI. A single administration of either drug was without effect.

A single in vivo administration of DOI to control rats resulted in reduced PKC activity (by 30–40%) in the particulate fraction of both Cx and Hc. This response to DOI was similar in DMI‐treated rats but was not seen in rats given repeated doses of FL. A single administration of DOI to animals given repeated doses of FL resulted in PKC activities higher than those seen in rats treated with FL alone.

The results indicate that repeated administration of FL and DMI produced similar changes in basal PKC activity but differentially affected the PKC response to the 5‐HT2A/2C receptor agonist, DOI. The effect on basal PKC activity may result from a post‐receptor action of antidepressants; the alteration of PKC response to DOI after fluoxetine could be due to receptor‐mediated desensitization of the signalling system.
The involvement of ATP‐sensitive potassium channels in β‐adrenoceptor‐mediated vasorelaxation in the rat isolated mesenteric arterial bed
Authors: Randall Michael D. , McCulloch Audrey I.
Published: July 2012

We have used the isolated buffer‐perfused superior mesenteric arterial bed of the rat to assess the involvement of ATP‐sensitive potassium (KAtp) channels in the vasorelaxant responses to β‐adrenoceptor agonists.

The vasorelaxant potencies of the non‐selective β‐adrenoceptor agonist, isoprenaline, the β1 adrenoceptor agonist, dobutamine and the β2‐adrenoceptor agonist, terbutaline were all significantly (P>0.05) reduced (isoprenaline, ED50=265±31 pmol v. 1.05 ± 0.42 nmol; dobutamine, ED50=294± 67 pmol v. 497 ± 115 pmol; terbutaline, ED50= 157 ±26 nmol v. 452 ± 120 nmol) in the presence of the KATP‐channel blocker, glibenclamide.

The presence of glibenclamide only weakly influenced the vasorelaxant properties of salbutamol, a β2‐adrenoceptor agonist, while those of verapamil, a β‐adrenoceptor‐independent vasorelaxant, were unaffected.

In radioligand binding experiments, glibenclamide (1 nM‐100 μm) did not displace any specific [3H]‐dihydroalprenolol binding from rat β‐adrenoceptors. Therefore, glibenclamide does not bind to β‐adrenoceptors at the concentration used in the present investigation.

Vasorelaxant responses to dibutyryl cyclic AMP, the cell permeable analogue of cyclic AMP, were also unaffected by glibenclamide, indicating that the coupling of β‐adrenoceptors to KATP‐channels occurs independently of the elevation of intracellular cyclic AMP.

We have shown that a significant element of the vasorelaxant responses to both β1 and β2 adrenoceptor activation involves the opening of KATP‐channels. In conclusion, KATP‐channels may play a physiological role in β‐adrenoceptor‐mediated vasodilatation.
Polydeoxyribonucleotides and nitric oxide release from guinea‐pig hearts during ischaemia and reperfusion
Authors: Masini E. , Lupini M. , Mugnai L. , Raspanti S. , Mannaioni P.F.
Published: July 2012

Two polydeoxyribonucleotides, produced by the controlled hydrolysis of DNA of mammalian lung (defibrotide and its lower molecular weight fraction, P.O. 085 DV), were studied for their ability to modify the release of nitrite and the coronary flow in perfusates collected from isolated, normally perfused hearts of guinea‐pigs and from hearts subjected to regional ischaemia and reperfusion.

In guinea‐pig normally perfused hearts, both defibrotide (DFT) and its fraction, P.O. 085 DV, increase the amount of nitrite appearing in perfusates in a concentration‐dependent fashion. At the highest concentration studied (10−6 m), P.O. 085 DV was more effective than DFT. A concomitant increase in the coronary flow was observed.

The increase in nitrite in perfusates and the increase in coronary flow induced by both DFT and P.O. 085 DV were significantly reduced by NGmonomethyl‐L‐arginine (l‐NMMA, 10−4 m), an inhibitor of nitric oxide synthase (NOS).

The endothelium‐dependent vasodilator, acetylcholine (ACh), enhances the formation of nitrite and the coronary flow. Both the increase in coronary flow and in the formation of nitrite were significantly reduced by L‐NMMA (10−4 m).

In guinea‐pig hearts subjected to ischaemia and reperfusion, the effect of both compounds in increasing the amount of nitrite in perfusates was more evident and more pronounced with P.O. 085 DV.

Reperfusion‐induced arrhythmias were significantly reduced by both compounds to the extent of complete protection afforded by compound P.O. 085 DV.

The cardioprotective and antiarrhythmic effects of DFT and P.O. 085 DV are discussed.
Mechanism of bradykinin‐induced plasma extravasation in the rat knee joint
Authors: Cambridge H. , Brain S.D.
Published: July 2012

We have investigated the mechanism of bradykinin (BK)‐induced plasma extravasation into the knee joint of the anaesthetized rat. Accumulation of [125I]‐human serum albumin within the synovial cavity was used as a marker of increased vascular permeability.

Perfusion with BK (1 μm) produced significant plasma extravasation into the knee which was inhibited by co‐perfusion of the selective bradykinin B2 receptor antagonist D‐Arg‐[Hyp3, Thi5, D‐Tic7Oic8]‐bradykinin (Hoe 140, 200 nM).

The bradykinin B1 receptor agonist, [des‐Arg9]‐BK (up to 100 mM), did not induce plasma extravasation into the knee joint over this time period.

Chemical sympathectomy by chronically administered 6‐hydroxydopamine (6‐OHDA) did not inhibit bradykinin‐induced plasma extravasation. Acute intra‐articular perfusion with 6‐OHDA (to stimulate transmitter release from sympathetic nerve terminals) at concentrations up to 50 mM did not induce significant plasma extravasation. Intra‐articular perfusion of 100 mM 6‐OHDA induced significant plasma extravasation but produced severe systemic toxicity.

The selective neurokinin1 (NK1) receptor antagonist, RP67580 (230 nmol kg−1), or receptor antagonists for the mast cell products histamine and 5‐hydroxytryptamine did not significantly inhibit BK‐induced plasma extravasation.

Co‐perfusion of the NO synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME) (1 mM) did not significantly inhibit the response to BK. 133Xe clearance from L‐NAME (1 mM)‐injected joints was significantly (P < 0.05) reduced compared to D‐NAME injected joints, suggesting a reduction in blood flow as a result of decreased basal NO production. Systemic administration of L‐NAME at doses sufficient to produce significant and sustained elevation of blood pressure (5 or 30 mg kg−1, i.v. 15 min prior to BK perfusion) also failed to significantly inhibit the BK‐induced response.

We conclude that, in normal joints, BK induces plasma extravasation by acting on bradykinin B2 receptors and that this response is not dependent on secondary release of mediators from sympathetic nerve terminals, sensory nerves, mast cells or on generation of NO.
Effects of mastoparan upon the late stages of the ACTH secretory pathway of AtT‐20 cells
Authors: McFerran Brian W. , Guild Simon B.
Published: July 2012

The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of the effects of mastoparan upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway.

Mastoparan (10−8‐10−5 m), an activator of heterotrimeric guanosine 5′‐triphosphate binding proteins (G‐proteins), stimulated ACTH secretion from electrically‐permeabilized AtT‐20 cells in a concentration‐dependent manner in the effective absence of calcium ions with a threshold of 10−6 m. Guanosine 5′‐0‐(3‐thiotriphosphate) (GTP‐γ‐S) (10−8‐10−4m) also stimulated ACTH secretion from electrically‐permeabilized AtT‐20 cells in a concentration‐dependent manner in the effective absence of calcium ions with a threshold of 10−6 m. This GTP‐γ‐S‐evoked secretion is consistent with previous studies which demonstrated that a G‐protein, termed GE, mediates calcium evoked ACTH secretion from AtT‐20 cells. GTP‐γ‐S‐evoked secretion however was not as great as that obtained in response to mastoparan.

Both mastoparan (10−5 m) and GTP‐γ‐S (10−4m) stimulated ACTH secretion from electrically‐permeabilized AtT20 cells in a time‐dependent manner. A time of 30 min was adopted as the standard incubation period for the study of both mastoparan and GTP‐γ‐S‐stimulated ACTH secretion from permeabilized AtT‐20 cells.

Mastoparan (10−8‐10−5 m) stimulated ACTH secretion from permeabilized AtT‐20 cells to the same extent in the presence and absence of the protein kinase C (PKC) inhibitor, chelerythrine chloride (10−5 m).

Mastoparan (10−8‐10−5 m)‐stimulated ACTH secretion from permeabilized AtT‐20 cells was significantly reduced in the presence of guanosine 5′‐0‐(2‐thiodiphosphate) (GDP‐β‐S, 10−4 m).

The mastoparan analogue, Mas‐7 (10−8‐10−5m) stimulated ACTH secretion from permeabilized AtT‐20 cells to a greater extent than mastoparan (10−8‐10−5m) however, the mastoparan analogue Mas‐17 (10−8‐10−5 m) had no effect upon ACTH secretion from permeabilized AtT‐20 cells.

Mastoparan (10−8‐10−5m) stimulated ACTH secretion from permeabilized AtT‐20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a concentration of 5 mM. Mastoparan (10−8‐10−5 m)‐stimulated ACTH secretion as well as control secretion was reduced when ATP was omitted.

The results of the present study demonstrate that mastoparan stimulated ACTH secretion from permeabilized AtT‐20 cells and displayed characteristics consistent with calcium ion‐ and GTP‐γ‐S‐stimulated ACTH secretion from permeabilized AtT‐20 cells. This suggests that in permeabilized AtT‐20 cells, mastoparan directly activates GE and that this G‐protein may be a heterotrimeric G‐protein. This study also suggests mastoparan may be a useful alternative to GTP‐γ‐S as a means of directly activating GE.
Increase of noradrenaline release in the hypothalamus of freely moving rat by postsynaptic 5‐hydroxytryptamine1A receptor activation
Authors: Suzuki M. , Matsuda T. , Asano S. , Somboonthum P. , Takuma K. , Baba A.
Published: July 2012

5‐Hydroxytryptamine (5‐HT) plays a role in the regulation of noradrenergic neurones in the brain, but the precise mechanism of regulation of noradrenaline (NA) release by 5‐HT1A receptors has not been defined. The present study describes the effect of a highly potent and selective 5‐HT1A receptor agonist, 5‐{3‐[[(2S)‐1,4‐benzodioxan‐2‐ylmethyl]amino]propoxy}‐1,3‐benzodioxole HC1 (MKC‐242), on NA release in the hypothalamus using microdialysis in the freely moving rat.

Subcutaneous injection of MKC‐242 (0.5mg kg_1) increased extracellular levels of NA and its metabolite, 3‐methoxy‐4‐hydroxyphenylglycol, in the hypothalamus and hippocampus.

The 5‐HT1a receptor agonists, 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) (0.2 mg kg−1) and buspirone (3 mg kg−1) mimicked the effect of MKC‐242 in increasing NA release in the hypothalamus.

The effects of MKC‐242 and 8‐OH‐DPAT in the hypothalamus were antagonized by pretreatment with WAY100135 (10 mg kg−1), a silent 5‐HT1A receptor antagonist.

Local administration of 8‐OH‐DPAT (10–100 μm), citalopram (1 μm), a 5‐HT reuptake inhibitor, and MDL72222 (10 μm), a 5‐HT3 receptor antagonist, into the hypothalamus, had no effect on NA release.

Intracerebroventricular injection with 5,7‐dihydroxytryptamine caused a marked reduction in brain 5‐HT content, but the treatment affected neither basal NA levels nor the MKC‐242‐induced increase in NA release.

The effect of MKC‐242 in increasing NA release was not attenuated by repeated treatment with the drug (0.5 mg kg−1, once a day for 2 weeks).

The present results suggest that activation of postsynaptic 5‐HT1A receptors increases NA release in the hypothalamus.
Evidence that [3H]‐α,β‐methylene ATP may label an endothelial‐derived cell line 5′‐nucleotidase with high affinity
Authors: Michel A.D. , Chau N.‐M. , Fan T.‐P.D. , Frost E.E. , Humphrey P.P.A.
Published: July 2012

In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [3H]‐α,β‐methylene ATP ([3H]‐α,β‐meATP) labelled high (pKd = 9.5; Bmax = 3.75 pmol mg−1 protein) and low (pKd = 7.2; Bmax = 23.3 pmol mg−1 protein) affinity binding sites. The high affinity [3H]‐α,β‐meATP binding sites in the WEC cell membranes could be selectively labelled with a low concentration of the radioligand (1 nM). In competition studies performed at a radioligand concentration of 1 nM, 88.6% of the sites possessed high affinity (pIC50 = 8.26) for α,β‐meATP.

The high affinity [3H]‐α,β‐meATP binding sites appeared heterogeneous since in competition studies a number of nucleotide analogues (α,β‐meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the sites labelled by 1 nM [3H]‐α,β‐meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69°.

Approximately 60–69% of the binding sites labelled with 1 nM [3H]‐α,β‐meATP possessed high affinity for α,β‐meADP (pIC50 = 8.87), AMP (pIC50 = 7.12), GMP (pIC50 = 7.34), UTP (pIC50 = 6.12), GTP (pIC50 = 7.59), GppNHp (plC50 = 735) and adenosine (pIC50 = 5.45). The sites at which these compounds possessed high affinity were probably the same, since, in the presence of GMP at a concentration (10 μm) sufficient to inhibit selectively the binding of [3H]‐α,β‐meATP, the [3H]‐α,β‐meATP binding sites with high affinity for AMP, UTP, α,β‐meADP, GTP, GppNHp and adenosine were also occluded.

WEC cell membranes were able to metabolize a trace concentration (6 nM) of [3H]‐AMP to [3H]‐adenosine under the conditions of the binding assay. The pIC50 values of adenosine (5.99), GMP (7.55) and the substrate AMP (7.19) for inhibiting this [3H]‐AMPase activity were almost identical to their high affinity pIC50 estimates obtained in the binding assay. Although α,β‐meADP, α,β‐meATP, β,γ‐meATP, ATP, ADP and GppNHp identified heterogeneity in the [3H]‐AMPase activity of the WEC cells, their pIC50 values for inhibiting the major portion of the [3H]‐AMPase activity were similar to their respective high affinity pIC50 values in the binding assay. It thus seems likely that WEC cells express a form of 5′‐nucleotidase that possesses high affinity for both α,β‐meADP and α,β‐meATP and that this enzyme can be labelled by [3H]‐α,β‐meATP.

In the presence of 10 μm GMP, the affinity estimates for α,β‐meADP, AMP, GMP, GTP, GppNHp, ADP and adenosine at the high affinity [3H]‐α,β‐meATP binding sites that remained available, were low and similar to their affinity estimates at the high affinity [3H]‐α,β‐meATP binding sites of rat vas deferens. Since the high affinity [3H]‐α,β‐meATP binding sites in rat vas deferens are thought to be P2x purinoceptors it is possible that the high affinity [3H]‐α,β‐meATP binding sites in the WEC which possess low affinity for α,β‐meADP are also P2x purinoceptors.
Noradrenaline contractions of human prostate mediated by α1c‐(α1c‐) adrenoceptor subtype
Authors: Marshall Ian , Burt Richard P. , Chappie Christopher R.
Published: July 2012

The subtype of α1‐adrenoceptor mediating contractions of human prostate to noradrenaline was characterized by use of a range of competitive and non‐competitive antagonists.

Contractions of the prostate to either noradrenaline (pD2 5.5), phenylephrine (pD2 5.1) or methoxamine (pD2 4.4) were unaltered by the presence of neuronal and extraneuronal uptake blockers. Noradrenaline was about 3 and 10 times more potent than phenylephrine and methoxamine respectively. Phenylephrine and methoxamine were partial agonists.

Pretreatment with the alkylating agent, chlorethylclonidine (10−4 m) shifted the noradrenaline concentration‐contraction curve about 3 fold to the right and depressed the maximum response by 31%. This shift is 100 fold less than that previously shown to be produced by chlorethylclonidine under the same conditions on α1B‐adrenoceptor‐mediated contractions.

Cumulative concentration‐contraction curves for noradrenaline were competitively antagonized by WB 4101 (pA2 9.0), 5‐methyl‐urapidil (pA2 8.6), phentolamine (pA2 7.6), benoxathian (pA2 8.5), spiperone (pA2 7.3), indoramin (pA2 8.2) and BMY 7378 (pA2 6.6). These values correlated best with published pKi values for their displacement of [3H]‐prazosin binding on membranes expressing cloned α1c‐adrenoceptors and poorly with values from cloned α1b‐ and α1dadrenoceptors.

The good correlation between the functional data on the prostate and the binding data on the expressed α1c‐subtype clone for the affinities of the competitive antagonists suggests that they are the same subtype. As the expressed α1c‐adrenoceptor clone corresponds to the α1A‐adrenoceptor expressed in tissues, contraction of the human prostate to noradrenaline is therefore mediated by an α1A‐adrenoceptor.
Characterization of neuropeptide Y (NPY) receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226
Authors: Abounader Roger , Villemure Jean‐Guy , Hamel Edith
Published: July 2012

We have characterized pharmacologically the receptor subtype(s) responsible for the neuropeptide Y (NPY)‐induced vasoconstriction in human cerebral arteries. NPY, PYY and several of their derivatives with well defined affinities at the known Y1 and Y2 receptor subtypes were used. Moreover, we tested the ability of the new Y1 receptor antagonist, BIBP 3226, to antagonize the NPY‐induced cerebral vasoconstriction.

NPY, PYY and their agonists with high affinities at the Y1 receptor subtype ([Leu31‐Pro34]‐NPY and [Leu31‐Pro34]‐PYY) elicited strong, long lasting and concentration‐dependent contractions of human cerebral arteries. Compounds with Y2 affinity such as PYY3–36 or NPY13–36 either elicited a submaximal contraction at high concentrations or failed to induce any significant vasomotor response. Also, the application of NPY or the specific Y1 agonist, [Leu31‐Pro34]‐NPY, to human cerebral vessels pretreated with the Y1 agonist, NPY13–36, resulted in contractile responses identical to those obtained when these compounds were tested without prior application of NPY13–36.

The order of agonist potency at the human cerebrovascular receptor was: [Leu31‐Pro34]‐NPY= [Leu31‐Pro34]‐PYY≤NPY>PYY>PYY3–36> > >NPY13–36, which corresponded to that reported previously at the neuronal and vascular Y1 receptors.

Increasing concentrations (10−9‐10−6m) of the Y1 receptor antagonist, BIBP 3226, to human cerebral vessels caused a parallel and rightward shift in the NPY dose‐response curves without any significant change in the maximal contractile response. The calculated pA2 was 8.52±0.13, a value compatible with the reported affinity at the rodent and human Y1 receptor.

We conclude that Y1 receptors exclusively, mediate the NPY‐induced contraction in human cerebral arteries and we show that BIBP 3226 is a potent and competitive antagonist of this Y1‐mediated vasoconstriction.
Effect of interleukin‐4 and interleukin‐10 on leucocyte migration and nitric oxide production in the mouse
Authors: Perretti Mauro , Szabó Csaba , Thiemermann Christoph
Published: July 2012

The effect of systemic treatment of mice with murine recombinant interleukin‐4 (IL‐4) or interleukin‐10 (IL‐10) on neutrophil infiltration into a specific tissue site and nitric oxide (NO) production from peritoneal macrophages was investigated.

Intravenously (i.v.) administered IL‐4 (0.01–10 μg per mouse, approximately 0.3–300 μg kg−1, i.v.) and IL‐10 (0.01‐1 μg per mouse, approximately 0.3–30 μg kg−1, i.v.) dose‐dependently inhibited neutrophil accumulation into a 6‐day‐old murine air‐pouch induced by local application of interleukin‐1β (IL‐1β, 5 ng), with approximate ED50s of 0.35 and 0.90 μg, respectively. Neither IL4 (1 μg, 30 μg kg−1, i.v.) nor IL‐10 (1 μg, 30 μg kg−1, i.v.) prevented leucocyte accumulation in the mouse air‐pouches when interleukin‐8 (IL‐8, 1μg.) was used as chemoattractant. Similarly, neither cytokine had any effect on the in vitro up‐regulation of CD11b antigen on the surface of murine circulating neutrophils.

Treatment of mice with lipopolysaccharide (LPS, 0.3 mg kg−1, i.p.) caused an increase in the formation of NO (measured as nitrite accumulation) in the supernatant of peritoneal macrophages ex vivo. Pretreatment of mice with IL4 (0.01‐1 μg i.v., 20 min before LPS), but not with IL‐10 (1 μg i.v., 20 min before LPS), caused a dose‐dependent reduction in this LPS‐stimulated formation of nitrite by peritoneal macrophages ex vivo.

Activation of murine macrophages with LPS (1 μg ml−1 for 24 h) in vitro caused a significant increase in nitrite release in the supernatant of these cells. Pretreatment of either J774.2 or peritoneal macrophages with IL4 (0.1‐1 μg ml−1, 20 min before LPS), but not with IL‐10 (1 μg ml−1, 20 min before LPS) caused a concentration‐related attenuation of this LPS‐stimulated nitrite formation.

Thus, both IL‐4 and IL‐10 inhibit the migration of leucocytes (stimulated by IL‐1β) in vivo; IL‐4 (but not IL‐10) inhibits the induction of NO synthase caused by LPS in murine macrophages in vitro and ex vivo.
Characterization of the functional muscarinic receptors in the rat urinary bladder
Authors: Longhurst Penelope A. , Leggett Robert E. , Briscoe Janice A.K.
Published: July 2012

Muscarinic receptors mediating contraction of the rat urinary bladder were characterized functionally in vitro by use of atropine, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP methiodide), 4‐diphenylacetoxy‐N‐(2‐chloroethyl)‐piperidine hydrochloride (4‐DAMP mustard), hexahydro‐sila‐diphenidol hydrochloride (HHSiD), the p‐fluoro analogue of hexahydro‐sila‐diphenidol hydrochloride (p‐F‐HHSiD), methoctramine, and pirenzepine.

(+)‐cis‐Dioxolane contracted bladder strips in a concentration‐dependent manner with an EC50 of 0.169±0.018 μm and an Emax of 7.84±0.67 g.

Concentration‐effect curves to (+)‐cis‐dioxolane were shifted to the right in the presence of the antagonists in a concentration‐dependent manner. The rank order of antagonist affinities against the (+)‐cis‐dioxolane response was (pA2 values in the parentheses) atropine (9.28)≥4‐DAMP methiodide (9.04)>HHSiD (8.01) > p‐F‐HHSiD (7.28)=pirenzepine (7.12)≥ methoctramine (6.77, 7.25). The profile resembles that associated with the M3 receptor subtype.

Atropine, 4‐DAMP methiodide, pirenzepine, and methoctramine had no effects on the contractile response to 120 mM KCl. However, HHSiD and p‐F‐HHSiD decreased the response to KCl, and 4‐DAMP mustard increased it.

Contractile responses to electrical field stimulation (1–32 Hz, 0.05 ms pulse duration) were biphasic in nature. The tonic response was suppressed more than the phasic response by all antagonists except methoctramine. The suppression was not always concentration‐dependent, and did not seem to be related to antagonism of any one receptor subtype.

Our findings are consistent with the minority M3 receptors mediating the contractile response to muscarinic stimulation by (+)‐cis‐dioxolane in the rat bladder.
SMS 201–995‐induced stimulation of gastric acid secretion via the dorsal vagal complex and inhibition via the hypothalamus in anaesthetized rats
Authors: Yoneda Masashi , Taché Yvette
Published: July 2012

SMS 201–995, a somatostatin analogue which interacts with highest affinities at somatostatin receptor subtypes 5 > 2 ≥ 3, was microinjected into selective brain sites and its influence on pentagastrin (10 μg kg−1 h−1, i.v.) ‐stimulated gastric acid secretion was investigated in rats anaesthetized with urethane. Gastric acid secretion was measured by flushing the stomach with saline through a gastric cannula every 10 min.

SMS 201–995 microinjected into the dorsal vagal complex (DVC, 7, 15, 30 and 60 ng) dose‐dependently increased pentagastrin‐stimulated gastric acid secretion. The peak acid response was reached within 20 min and returned to basal level 50 min post‐injection. SMA 201–995 (30 ng) microinjected into the surrounding area or the central amygdala did not modify pentagastrin‐stimulated acid secretion.

SMS 201–995 injected into the lateral ventricle (i.c.v., 100, 200, or 300 ng), paraventricular nucleus (PVN) or lateral hypothalamus (LH) (7.5, 15, or 30 ng) dose‐dependently inhibited pentagastrin‐stimulated gastric acid secretion. SMS 201–995 (30 ng) microinjected into the area surrounding the PVN or LH did not modify the acid secretion response to pentagastrin.

Vagotomy prevented the effects of SMS 201–995 (30 ng) microinjected into the DVC and LH.

Spinal cord transection abolished the inhibitory action of SMS 201–995 (30 ng) microinjected into the PVN but not the LH.

These results demonstrate that SMS 201–995 acts in the DVC to enhance and in the LH and PVN to inhibit pentagastrin‐stimulated gastric acid secretion. The action is mediated through vagal (DVC, LH) or spinal (PVN) pathways. The site specific pattern of acid responses to SMS 201–995 may be linked to the distribution of receptor subtypes at these sites that convey the different biological actions of somatostatin.
UTP‐ and ATP‐triggered transmitter release from rat sympathetic neurones via separate receptors
Authors: Boehm Stefan , Huck Sigismund , Illes Peter
Published: July 2012

In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca2+‐dependent and tetrodotoxin‐sensitive [3H]‐noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 μmol 1−1, the concentration used in all subsequent experiments. Pre‐exposure of the neurones to 100 μmol 1−1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre‐exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 μmol 1−1 pyridoxal‐5″‐phosphate or 30 μmol 1−1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 μmol 1−1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.
The antinociceptive effect of 1‐(2‐trifluoromethylphenyl) imidazole (TRIM), a potent inhibitor of neuronal nitric oxide synthase in vitro, in the mouse
Authors: Handy Rachel L.C. , Wallace P. , Gaffen Z.A. , Whitehead K.J. , Moore P.K.
Published: July 2012

1‐(2‐trifluoromethylphenyl)imidazole (TRIM) is a potent inhibitor of neuronal (mouse cerebellar) and inducible (lung from endotoxin‐pretreated rats) isoforms of nitric oxide synthase (NOS) with IC50 values of 28.2 μm and 27.0 μm, respectively. In contrast, TRIM is a poor inhibitor of bovine aortic endothelial NOS with an IC50 of 1057.5 μm. TRIM (10–50 mg kg−1) administered i.p. exhibits dose‐related antinociceptive activity in the mouse (assessed as inhibition of late phase formalin‐induced hindpaw licking behaviour) with an ED50 of 85.8 μmol kg−1. In contrast, TRIM (50 mg kg−1, i.p.) failed to influence mean arterial blood pressure in the urethane‐anaesthetized mouse. Thus, TRIM may be of use as an experimental tool with which to investigate the biological roles of nitric oxide (NO) within the central nervous system.
Similar coronary vascular effects in the rat perfused heart of platelet‐activating factor structural analogues with agonist and antagonist properties
Authors: Man Ricky Y.K. , Kinnaird Anne A.A.
Published: July 2012

Selective blockade of platelet‐activating factor (PAF) receptor subtypes by PAF receptor antagonists has been demonstrated. However, selective activation of PAF receptor subtypes by PAF receptor agonists has not been reported.

When structural analogues of PAF that have been shown to possess either agonist or antagonist effects were administered by a bolus injection in the rat perfused heart, they all showed agonist effects. Lower amounts produced vasodilatation while higher amounts produced vasodilatation followed by vasoconstriction. These coronary vascular effects were typical of that observed with PAF. Lyso‐PAF did not show the same typical pattern of coronary vascular effect, confirming that the detergent effect of PAF structural analogues did not play a role in the coronary vascular effects. Other PAF antagonists, CV‐6209 and WEB 2170, also did not produce the PAF‐like response in the rat perfused heart.

The coronary vascular effects of hexanolamine‐PAF (H‐PAF, putative antagonist) and ethanolamine‐PAF (E‐PAF, agonist) were further studied. Pretreatment with FR‐900452 (a PAF receptor antagonist) or MK‐886 (a leukotriene synthesis inhibitor) significantly reduced the vasodilator and vasoconstrictor effects of H‐PAF and E‐PAF.

Pretreatment of rat perfused hearts with low concentrations of H‐PAF and E‐PAF blocked the response to PAF administration in a dose‐ and time‐dependent manner. However, the pretreatment with either H‐PAF or E‐PAF did not result in a coronary vascular effect expected of a PAF receptor agonist. These results were compatible with H‐PAF and E‐PAF behaving as PAF receptor antagonists.

In summary, our results demonstrate that several PAF structural analogues possess agonist action in the rat perfused heart. Like the coronary vascular effects of PAF, the effects of H‐PAF and E‐PAF were blocked by a PAF antagonist (FR‐900452) and a leukotriene synthesis inhibitor (MK‐886). This suggests that both H‐PAF and E‐PAF mediate their effect through activation of PAF receptors with a subsequent release of leukotrienes that produced vasodilatation and vasoconstriction. Furthermore, pretreatment of perfused hearts with these compounds blocked the response to PAF in these hearts. Thus these compounds can also behave like a PAF receptor antagonist. This latter action may be due to a gradual receptor inactivation or desensitization by the pretreatment of H‐PAF and E‐PAF through a PAF receptor agonist effect rather than being a PAF receptor antagonist.
Correlation of cyclic AMP accumulation and relaxant actions of salmeterol and salbutamol in bovine tracheal smooth muscle
Authors: Ellis Karen E. , Mistry Rajendra , Boyle John P. , Challiss R.A. John
Published: July 2012

The ability of salmeterol to stimulate cyclic AMP accumulation and relaxation has been compared with that of salbutamol in bovine tracheal smooth muscle. In addition, the anti‐spasmogenic effects of these agents and their abilities to modulate histamine‐stimulated [3H]‐inositol phosphate accumulation have also been investigated.

In tissue strips, a close temporal correlation was found to exist between salmeterol (0.1 μm)‐induced relaxation of methacholine (500 nM)‐induced tone and cyclic AMP accumulation, both maximal reversal of induced tone (26.2 ± 6.0%) and maximal levels of cyclic AMP accumulation being achieved after 30–40 min. In contrast to salmeterol, salbutamol exerted greater and more rapid effects on both parameters. Maximal reversal of methacholine‐induced tone (79.3 ± 14.0%) and maximal levels of cyclic AMP accumulation were produced within 5 min.

Salmeterol‐induced cyclic AMP accumulation (EC50 = 5.3 [1.8–15.2] nM) and inhibition of histamine (0.1 mM)‐stimulated [3H]‐inositol phosphate accumulation (IC50=1.4 [0.3–6.3] nM) were both more potent than those induced by salbutamol (EC50= 169 [99–290] nM; IC50= 13.8 [7.0–27.4] nM). However, maximal effects exerted by each of these agents were similar in magnitude.

Anti‐spasmogenic effects were examined by β‐adrenoceptor agonist application to tissue strips prior to construction of spasmogen concentration‐effect curves. Both salmeterol and salbutamol exerted more marked inhibition of the contractile response induced by histamine than that induced by methacholine, salmeterol being the more potent agent, while salbutamol produced a greater maximal inhibitory effect.

The results demonstrate that salmeterol is a more potent agent than salbutamol and have highlighted a close temporal correlation between promotion of cyclic AMP accumulation and tissue relaxation stimulated by each agent when both parameters are measured under identical conditions.
Ginsenosides‐induced nitric oxide‐mediated relaxation of the rabbit corpus cavernosum
Authors: Chen Xiu , Lee Tony J.‐F.
Published: July 2012

Ginsenosides, the active ingredients extracted from Panax ginseng, have been shown to promote nitric oxide (NO) release in bovine aortic endothelial cells. Since the endothelial cells and the perivascular nerves in penile corpus cavernosum contain NO synthase and an NO‐like substance has been shown to be released from these cells which relaxes corpus cavernosum, the possibility that ginsenosides may relax corpus cavernosum by releasing endogenous NO was examined.

With an in vitro tissue superfusion technique, ginsenosides (250, 500 and 750 μg ml−1) relaxed corpus cavernosum, concentration‐dependently.

Using an in vitro tissue bath technique, acetylcholine (ACh)‐induced relaxations were increased in the presence of ginsenosides (250 *μg ml−1).

Ginsenosides at 100 μg ml−1 significantly enhanced the tetrodotoxin (TTX)‐sensitive relaxation of corpus cavernosum elicited by transmural nerve stimulation.

The ginsenosides‐induced, ACh‐induced and ginsenosides‐enhanced transmural nerve stimulation‐elicited relaxations were significantly attenuated by NG‐nitro‐L‐arginine (100 μM) and oxyhaemoglobin (oxyHb; 5‐10jiM), and were enhanced by superoxide dismutase (SOD; 50u ml−1).

The relaxations and their attenuation by NG‐nitro‐L‐arginine and TTX were associated with increase and decrease in tissue cyclic GMP levels, respectively.

It is concluded that ginsenosides may release NO from endothelial cells, and enhance NO release from endothelial cells elicited by other vasoactive substances and from perivascular nitrergic nerves in the corpus cavernosum. These endothelial and neurogenic effects of ginsenosides in inducing relaxation of the corpus cavernosum may account for the aphrodisiac effect of Panax ginseng.
Suppression of eosinophil function by RP 73401, a potent and selective inhibitor of cyclic AMP‐specific phosphodiesterase: comparison with rolipram
Authors: Soilness John E. , Maslen Christopher , Webber Stephen , Foster Martyn , Raeburn David , Palfreyman Malcolm N. , Ashton Michael J. , Karlsson Jan‐Anders
Published: July 2012

We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP‐specific phosphodiesterase (PDE IV), against partially‐purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea‐pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2−), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea‐pig eosinophils have also been studied.

RP 73401 potently inhibited partially‐purified cyclic AMP‐specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea‐pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM).

Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH).

Reverse transcription polymerase chain reaction (RT‐PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA‐D), showed only mRNA for PDE IVD in guinea‐pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells.

RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (±)‐rolipram (Kiapp =1.7 nM) in displacing [3H]‐(±)‐rolipram from guinea‐pig brain membranes.

In intact eosinophils, RP 73401 potentiated isoprenaline‐induced cyclic AMP accumulation (EC50 = 79 nM). RP 73401 also inhibited leukotriene B4‐induced generation of O2− (IC50 = 25 nM), and the release of major basic protein (IC50‐ 115 nM) and eosinophil cationic protein (IC50 = 7nM). Rolipram was 3–14 times less potent than RP 73401.

Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils (in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.
The anti‐emetic effects of CP‐99,994 in the ferret and the dog: role of the NK1 receptor
Authors: Watson J.W. , Gonsalves S.F. , Fossa A.A. , McLean S. , Seeger T. , Obach S. , Andrews P.L.R.
Published: July 2012

The selective NK1 receptor antagonist, CP‐99,994, produced dose‐related (0.1‐1.0 mg kg−1, s.c.) inhibition of vomiting and retching in ferrets challenged with central (loperamide and apomorphine), peripheral (CuSO4) and mixed central and peripheral (ipecac, cisplatin) emetic stimuli.

Parallel studies with the enantiomer, CP‐100,263 (1 mgkg−1, s.c), which is > 1 000 fold less potent as a NK1 antagonist, indicated that it was without significant effect against CuSO4, loperamide, cisplatin and apomorphine‐induced emesis. Against ipecac, it inhibited both retching and vomiting, expressing approximately l/10th the potency of CP‐99,994.

The 5‐HT3 receptor antagonist, tropisetron (lmgkg−1, s.c.) inhibited retching and vomiting to cisplatin and ipecac, but not CuSO4 or loperamide.

CP‐99,994 (lmgkg−1, i.v.) blocked retching induced by electrical stimulation of the ventral abdominal vagus without affecting the cardiovascular response, the apnoeic response to central vagal stimulation or the guarding and hypertensive response to stimulation of the greater splanchnic nerves. CP‐99,994 (1 mg kg−1, i.v.) did not alter baseline cardiovascular and respiratory parameters and it failed to block the characteristic heart rate, blood pressure and respiratory rate/depth changes in response to i.v. 2‐methyl‐5‐HT challenge (von Bezold‐Jarisch reflex).

Using in vitro autoradiography, [3H]‐substance P was shown to bind to several regions of the ferret brainstem with the density of binding in the nucleus tractus solitarius being much greater than in the area postrema. This binding was displaced by CP‐99,994 in a concentration‐related manner.

In dogs, CP‐99,994 (40 μg kg−1 bolus and 300 μg kg−1 h−1, i.v.) produced statistically significant reductions in vomiting to CuSO4 and apomorphine as well as retching to CuSO4.

Together, these studies support the hypothesis that the NK1 receptor antagonist properties of CP‐99,994 are responsible for its broad spectrum anti‐emetic effects. They also suggest that CP‐99,994 acts within the brainstem, most probably within the nucleus tractus solitarius although the involvement of the area postrema could not be excluded.
Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the β‐adrenoceptor and adenylate cyclase in rat heart
Authors: Chatelain P. , Meysmans L. , Mattéazzi J.R. , Beaufort Ph. , Clinet M.
Published: July 2012

The effects of SR 33589 and amiodarone on the cardiac β‐adrenoceptor were studied in vitro and after chronic treatment by means of [125I]‐(−)−iodocyanopindolol ([125I]‐(−)−CYP) binding and measurement of adenylate cyclase activity.

Binding of [125I]‐(−)−CYP was inhibited in a dose‐dependent manner by SR 33589 (IC50 = 1.8 ± 0.4 μM, nH = 0.93 ± 0.06) and amiodarone (IC50 = 8.7 ± 2.0 μM, nH = 0.92 ± 0.03). Saturation binding experiments indicated a non‐competitive interaction such that SR 33589 (1 and 3 μm) and amiodarone (5 and 10 μm) reduced the Bmax of [125I]‐(−)−CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]‐(−)−CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 μm) and amiodarone (30 μM).

Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose‐dependent, but non‐competitive manner, by SR 33589 (isoprenaline‐, glucagon‐ and secretin‐stimulated enzyme inhibited 50% at 6.8 ± 0.6 μM, 31 ± 10 μm and 12 ± 3 μM, respectively) while the basal, GTP‐ and Gpp(NH)p‐stimulated enzyme was inhibited by 5–10% and the NaF and forskolin‐stimulated enzyme by 50% at 500 μM. Amiodarone exhibited a similar pattern of inhibition.

After chronic oral treatment (50, 100, 150 mg kg−1 per day, 14 days), both SR 33589 and amiodarone produced a dose‐dependent decrease in Bmax without any effect on KD as determined from [125I]‐(−)−CYP saturation experiments and a decrease of the isoprenaline‐ and glucagon‐stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units.

Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4 and rT3 were unchanged after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and the level of rT3 were dose‐dependently increased by amiodarone treatment.

In vitro, SR 33589 and amiodarone were characterized as non‐competitive β‐adrenoceptor antagonists. Chronic treatment led to a down‐regulation of the β‐adrenoceptor; the down‐regulation cannot be attributed to an indirect effect mediated by the thyroid hormones. To reconcile these opposing observations, we propose that SR 33589 and amiodarone interact with the β‐adrenoceptor at a site close to the intracellular loops which are involved in the coupling with Gs and contain the phosphorylable sites.
YC‐1 inhibited human platelet aggregation through NO‐independent activation of soluble guanylate cyclase
Authors: Wu Chin‐Chung , Ko Feng‐Nien , Kuo Sheng‐Chu , Lee Fang‐Yu , Teng Che‐Ming
Published: July 2012

Our previous study demonstrated that YC‐1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC‐1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets.

In human washed platelets, YC‐1 inhibited platelet aggregation and ATP released induced by U46619 (2 μm), collagen (10 μg ml−1) and thrombin (0.1 u ml−1) in a concentration‐dependent manner with IC50 values of (μm) 2.1 ± 0.3, 11.7 ± 2.1 and 59.3 ± 7.1, respectively.

In a 30,000 g supernatant fraction from human platelet homogenate, YC‐1 (5–100 μm) increased sGC activity in a concentration‐dependent manner. At the same concentration‐range, YC‐1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets.

MY‐5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC‐1, and shifted the concentration‐anti‐aggregation curve of YC‐1 to the left. In contrast, HL‐725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti‐aggregatory effect caused by YC‐1.

Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC‐1, and attenuated markedly the anti‐aggregatory effect of YC‐1. The adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine (DDA) did not affect YC‐1‐induced inhibition of platelet aggregation.

Haemoglobin, which binds NO, prevented the activation of sGC and anti‐aggregatory effect caused by sodium nitroprusside, but did not affect YC‐1 responses.

These results would suggest that YC‐1 activates sGC of human platelets by a NO‐independent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.
Long‐term carbachol treatment‐induced down‐regulation of muscarinic M2‐receptors but not m2 receptor mRNA in a human lung cell line
Authors: Haddad E.‐B. , Rousell J. , Mak J.C.W. , Barnes P.J.
Published: July 2012

The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells).

Saturation studies performed with the non‐selective hydrophilic ([3H]‐N‐methyl‐scopolamine, [3H]‐NMS) and lipophilic ([3H]‐quinuchdinyl benzilate, [3H]‐QNB) muscarinic antagonists revealed a single class of high affinity binding sites.

Carbachol (1 mM) induced a rapid down‐regulation of [3H]‐NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h.

The loss of [3H]‐QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]‐NMS binding sites as a result of receptor sequestration.

Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin‐induced cyclic AMP accumulation was lost following 24 h carbachol stimulation.

The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol treatment over the time course investigated and half‐life studies with actinomycin D suggest that carbachol had no effect on the stability of m2 mRNA.

The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run‐on assay was unaltered by carbachol stimulation.

These results suggest that homologous sequestration, desensitization, and down‐regulation of M2 muscarinic receptors in HEL 299 cells does not involve transcriptional or post‐transcriptional modifications of m2 muscarinic receptor mRNAs.