bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular platelet‐activating factor regulates eicosanoid generation in guinea‐pig resident peritoneal macrophages
Authors: Stewart Alastair G. , Phillips Wayne A.
Published: July 2012

The role of intracellular platelet‐activating factor (Paf) in arachidonic acid (AA) mobilization from guinea‐pig peritoneal macrophages has been investigated by use of the potent and selective Paf receptor antagonists, WEB 2086 and CV 6209.

Adherent macrophages contained cell‐associated Paf which was increased by exposure to formyl‐methionyl‐leucyl‐phenylalanine (fMLP), endotoxin and the ionophore, A23187. However, only endotoxin and A23187 caused release of detectable amounts of Paf into the extracellular medium.

Exogenous Paf and each of the above stimuli mobilized previously incorporated [14C]‐AA and increased the generation of prostacyclin (PGI2) in resident macrophages.

WEB 2086 (10–100 μm) and CV 6209 (0.1–10 μm) reduced both basal and stimulated PGI2 generation and WEB 2086 inhibited the mobilization of [14C]‐AA. In addition, WEB 2086 (10 μm) inhibited fMLP‐ and Paf‐induced superoxide anion generation. Responses to A23187 were not inhibited by either antagonist.

Activation of macrophages by fMLP caused a short burst of intracellular Paf generation but none was detected in the supernatants. The time‐course of PGI2 synthesis followed closely that of Paf.

These data suggest that intracellular Paf generation is important for subsequent AA mobilization and may have a wider role in signal transduction processes.
Different time courses of the blockade of sodium current by lignocaine and SUN 1165 in single myocytes isolated from guinea‐pig atrium
Authors: Inomata Norio , Ishihara Takafumi , Akaike Norio
Published: July 2012

The time course of the blockade of sodium currents (INa) by the antiarrhythmic agents, lignocaine and SUN 1165, was studied in single myocytes isolated enzymatically from guinea‐pig atrium, by a new concentration‐jump termed as a ‘concentration‐clamp’ technique. This technique combines an intracellular perfusion and a rapid exchange of external solution surrounding the voltage‐clamped myocyte within 2 ms.

Lignocaine (3.7 × 10−5 m to 3.7 × 10−4 M) inhibited the peak amplitude of INa in a concentration‐dependent fashion. It took 2 to 5 s to reach apparent steady‐state inhibition at the concentrations used. Complete recovery from the inhibition also took 2 to 5 s after washing out the agent. In contrast, the inhibitory effect of SUN 1165 (1 × 10−5 M) on the peak INa gradually progressed and reached a steady‐state level about 2 min after the start of drug‐application. The recovery required more than 10 min after washing out of the agent.

In cardiomyocytes treated with scorpion toxin (5 μg ml−1), the inactivation of INa was greatly inhibited, resulting in the relatively persistent Na inward current (persistent INa) during the depolarizing pulse. Lignocaine (1.1 × 10−4 M) applied during the depolarizing pulse, reduced in a single‐exponential fashion the amplitude of the persistent INa in milliseconds. On the other hand, lignocaine applied several tens of milliseconds before the depolarizing pulse induced only a small reduction of the peak amplitude of the first persistent INa. When SUN 1165 (1 × 10−5 M) was applied during the depolarizing pulse, there was only a small instantaneous reduction of the amplitude of the persistent INa, although it did inhibit time‐dependently, the peak INa. Both agents accelerated the decay phase of the persistent INa in a time‐dependent manner.

These results suggest that lignocaine and SUN 1165 may preferentially interact with the open‐state of the sodium channel rather than with the rested one, although SUN 1165 does so much more slowly.
The modulation by chlormethiazole of the GABAA‐receptor complex in rat brain
Authors: Cross Alan J. , Stirling Janet M. , Robinson Timothy N. , Bowen David M. , Francis Paul T. , Green A. Richard
Published: July 2012

The interactions of chlormethiazole with γ‐aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA‐receptor complex and the GABAB‐receptor have been studied in the rat. The GABAA‐receptor was studied using [3H]‐muscimol, [3H]‐flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]‐butylbicyclophosphorothionate ([35S]‐TBPS) to label the chloride channel.

Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]‐baclofen binding to the GABAB‐receptor.

Chlormethiazole (IC50 = 140 μm) and pentobarbitone (IC50 = 95 μm) both inhibited [35S]‐TBPS binding by increasing the rate of [35S]‐TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]‐TBPS binding.

Chlormethiazole enhanced the binding of [3H]‐muscimol but had no effect on [3H]‐flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]‐muscimol and [3H]‐flunitrazepam binding.

It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA‐receptor. However, chlormethiazole does not interact with the GABAA‐receptor complex in an identical manner to the sedative barbiturate pentobarbitone.
Differences in regional vascular sensitivity to endothelin‐1 between spontaneously hypertensive and normotensive Wistar‐Kyoto rats
Authors: Wright Christine E. , Fozard John R.
Published: July 2012

The systemic and regional haemodynamic effects of porcine endothelin‐1 (endothelin) have been measured in anaesthetized spontaneously hypertensive (SH) rats rendered areflexic by ganglion blockade; comparisons were made with age‐matched Wistar‐Kyoto (WKY) control animals.

In both SH and WKY rats endothelin (0.1–1 nmol kg−1 i.v.) elicited an initial, short‐lived (<2 min), fall in blood pressure which was associated with substantial increases in hindquarter and carotid vascular conductances. Both the blood pressure falls and the peripheral vasodilator responses were greater in SH than in WKY rats.

The initial depressor effects of endothelin were followed by marked and sustained increases in blood pressure associated with constriction in carotid, hindquarter, renal and mesenteric vascular beds. Vasoconstrictor responses were quantitatively similar in the two rat strains.

Pretreatment with indomethacin (5 mg kg−1 i.p. or i.v.) did not alter the responses to endothelin, 1 nmol kg−1, in SH rats.

The regional haemodynamic effects of intravenously administered acetylcholine (0.01–1 μg kg−1), nitroprusside (0.3–10 μg kg−1) and angiotensin II (0.01–0.1 μg kg−l) were similar in SH and WKY rats.

Endothelin (10−10−3 × 10−8 m) contracted aortic rings from both SH rats and WKY control animals. Removal of the endothelium enhanced significantly the sensitivity of tissues from both WKY and SH rats to endothelin; the increase in sensitivity was greater in tissues from SH than WKY rats.

The results demonstrate qualitative similarities in the complex haemodynamic effects of endothelin in SH rats and WKY control animals. However, the SH rats display substantially greater vasodilator responses to endothelin than WKY. Eicosanoid generation is not the mechanism of the vasodilator action of endothelin in SH rats under the conditions of our experiments.
Microionophoretic study with milacemide, a glycine precursor, on mammalian central nervous system cells
Authors: Godfraind Jean‐Marie
Published: July 2012

The effect of milacemide, a glycine percursor known to increase γ‐aminobutyric acid (GABA) and glycine content in the brain, and to have anticonvulsant properties, was tested by ionophoresis on 247 neurones situated in the cerebral cortex and in deeper structures of cats and rats anaesthetized with urethane.

Virtually all the neurones, either firing spontaneously or exogenously driven by the excitatory amino acids, glutamate, N‐methyl‐d‐aspartate (NMDA), kainate and quisqualate or by acetylcholine, were reversibly depressed in a dose‐dependent fashion. The same depressant effect was observed in animals pretreated with the monoamine oxidase B inhibitor (IMAO‐B) deprenyl which is known to reduce milacemide metabolism into glycinamide and glycine. Intravenous administration of milacemide (10 to 100 mg kg−1) also depressed the firing induced by glutamate, NMDA and acetylcholine.

When compared to GABA, milacemide was a weaker depressant. However, its effect could still be observed in the presence of the reversible GABAA antagonist, SR 95531, and thus milacemide is unlikely to act through GABA receptors. In addition, on cells unaffected by glycine, milacemide also had a depressant effect, and on cells inhibited by glycine, it was still capable of depressing cell firing during reversible blockade by strychnine of the glycine inhibitory action; thus milacemide is unlikely to act through glycine receptors. Simultaneous release of milacemide and GABA or of milacemide and glycine, did not show potentiation of the inhibitory amino acid action. However, the depressant effect of milacemide was additive with that of GABA and glycine.

No consistent depression of glutamate‐induced firing was obtained by ionophoresis of glycinamide, the first metabolite of milacemide.

It is concluded that milacemide by itself is a depressant agent and that its depressant effect does not necessarily require its metabolism into glycine, or its stimulator effect on the production of GABA.
Modulation of cholinergic neurotransmission in guinea‐pig airways by opioids
Authors: Belvisi M.G. , Stretton C.D. , Barnes P.J.
Published: July 2012

Opioid receptors have been localised on sensory fibres in the vagus nerve and opioids have previously been shown to inhibit non‐adrenergic, non‐cholinergic (NANC) neurotransmission in guinea‐pig bronchi in vitro and in vivo. We have now investigated whether an inhibitory effect could be demonstrated on cholinergic neurotransmission.

Electrical field stimulation (EFS) (8 Hz, 0.5 ms, 40 V for 20 s) produced only a rapid, cholinergic response in the upper trachea but in the lower trachea and main bronchi a cholinergic response which was atropine‐sensitive and a longer lasting NANC contraction that was atropine‐insensitive was demonstrated. This slow contraction could be blocked by tetrodotoxin and capsaicin pretreatment.

[d‐Ala2, NMePhe4, Gly‐ol5]enkephalin (DAMGO), a selective μ‐opioid receptor agonist, inhibited the cholinergic response to EFS at 8 Hz in a dose‐dependent manner in main bronchi (IC50 = 113 nm with a maximal inhibition of 35.7 ± 5.6% 10 μm, n = 5). In the lower trachea, DAMGO inhibited the cholinergic response to a similar extent (inhibition of 35.8 ± 3.5% at 10 μm, n = 5). However, DAMGO had no effect on the contractile response to exogenously applied acetylcholine in the main bronchi. By contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea. DAMGO (1 μm) inhibited the cholinergic response to EFS in a frequency‐dependent manner in the main bronchi with greater inhibition at lower frequencies of stimulation.

The δ‐opioid receptor agonist [d‐Pen2, d‐Pen5]enkephalin (DPDPE) significantly inhibited the cholinergic component of the constrictor response to EFS at 8 Hz in the bronchi but at the highest dose used (10 μm). U‐50,488H, a κ‐receptor agonist, had no inhibitory effect on the cholinergic constrictor component in the main bronchi (10 μm).

DAMGO also inhibited the NANC responses to EFS in the main bronchi in a dose‐dependent manner (with an IC50 = 36 nm and a maximal inhibition of 63.4 ± 8.3%, at 1 μm, n = 5). DAMGO had no effect on contractile responses to exogenously applied substance P (SP). DPDPE (10 μm) was less effective in inhibition of the NANC bronchoconstriction with a maximal inhibition of 29.2 ± 4.2% (n = 7), and U‐50,488H (10 μm) had no inhibitory effect.

After capsaicin pretreatment, which depleted sensory nerves of neuropeptides, the inhibitory effect of DAMGO (1 μm) on cholinergic constriction in main bronchi at 8 Hz was only 13.4 ± 1.9% (n = 13) compared with 32.9 ± 4.0% (n = 9) inhibition in vehicle‐treated controls (P < 0.001).

Opioids may reduce the cholinergic neural responses in airways partly via an inhibitory action on excitatory NANC nerves and partly by a direct effect on cholinergic neurotransmission. The opioid receptor involved is of the μ‐opioid receptor subtype.
Evidence that pinacidil may promote the opening of ATP‐sensitive K+ channels yet inhibit the opening of Ca2+‐activated K+ channels in K+ ‐contracted canine mesenteric artery
Authors: Masuzawa Kaoru , Matsuda Tomohiro , Asano Masahisa
Published: July 2012

The effects of cromakalim and pinacidil on contraction and 86Rb efflux were investigated in strips of canine mesenteric artery.

Cromakalim and pinacidil relaxed arterial strips precontracted with 20.9 mm K+ with pD2 values of 6.56 and 5.88, respectively.

High (above 10 μm) concentrations of pinacidil, but not cromakalim, relaxed arterial strips bathed by a medium containing 65.9 mm K+, and inhibited Ca2+‐induced contractions in strips bathed by a medium containing 80 mm K+. These findings suggested that pinacidil may act as an inhibitor of Ca2+ influx.

In arterial strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux.

When the effects of cromakalim and pinacidil on 86Rb efflux were determined in arterial strips contracted with 65.9 mm K+, both drugs increased 86Rb efflux. The increase in 86Rb efflux induced by pinacidil was much smaller than that induced by cromakalim. Under the same conditions, nifedipine decreased 86Rb efflux.

After the addition of nifedipine to arterial strips contracted with 65.9 mm K+, pinacidil produced a greater increase in 86Rb efflux than in the absence of nifedipine, whereas the effects of cromakalim were the same for the two conditions. Therefore, the effects of pinacidil on 86Rb efflux may be the resultant of two opposing effects: an increased 86Rb efflux due to the opening of ATP‐sensitive K+ channels, and a decreased efflux due to the closing of Ca2+‐activated K+ channels.

In causing relaxation, cromakalim was competitively antagonized by glibenclamide with a pA2 value of 7.16. However, glibenclamide antagonism of pinacidil was not of the simple competitive type, suggesting that inhibition of Ca2+ influx may contribute to the relaxant action of pinacidil.

It may be concluded that although the ability of pinacidil to increase 86Rb efflux via ATP‐sensitive K+ channel opening was similar to that of cromakalim, the inhibition of Ca2+ influx by pinacidil may reduce the opening of Ca2+‐activated K+ channels in K+‐contracted arterial strips.
Separate [3H]‐nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla
Authors: Ballesta J.J. , Garcia A.G. , Gutierrez L.M. , Hidalgo M.J. , Palmero M. , Reig J.A. , Viniegra S.
Published: July 2012

Two binding sites for the 1,4‐dihydropyridine (DHP) derivative [3H]‐nitrendipine have been found in the bovine adrenal medulla. The high‐affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg−1 protein) was specifically located in purified plasma membranes. The low‐affinity site (Kd = 252nM and Bmax = 169 pmol mg−1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]‐nitrendipine.

Kinetic analysis of the rates of association and dissociation of [3H]‐nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200‐110 revealed single, homogeneous populations of high‐ and low‐affinity sites in plasma and mitochondrial membranes, respectively.

The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]‐nitrendipine.

In contrast, binding of [3H]‐nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]‐nitrendipine acting in a non‐competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]‐nitrendipine.

These results suggest that both binding sites reflect different receptor entities. The high‐affinity binding site corresponds to the dihydropyridine receptor associated with the L‐type calcium channel. The function of the mitochondrial, low‐affinity binding site is, at present, unknown.
Evidence for prejunctional M2 muscarinic receptors in pulmonary cholinergic nerves in the rat
Authors: Aas Pål , Maclagan Jennifer
Published: July 2012

The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube.

The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists.

In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose‐dependent manner (EC50 0.1 μm) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist.

The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine.

The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve‐induced contraction of tracheal smooth muscle.
Characterization of the dopamine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in vitro
Authors: Evans A.M. , Green K.L.
Published: July 2012

Intracellular recordings have been made of the hyperpolarization of cockroach salivary gland cells induced by nerve stimulation and dopamine.

The relative potency of a number of dopamine antagonists in inhibiting the dopamine‐ and nerve‐mediated hyperpolarization was studied. SCH23390 (10–50 μm), chlorpromazine (0.1–5 μm), haloperidol (10–100 μm) and metoclopramide (1 mm) inhibited the hyperpolarization.

In contrast, domperidone and (±)‐sulpiride potentiated the hyperpolarization induced by both nerve stimulation and dopamine.

Apparent dissociation constants (KDApp) were obtained for the blockade of the dopamine‐induced hyperpolarization. The rank order of potency (KDapp in parentheses) was as follows: chlorpromazine (0.2 μm); haloperidol (3.3 μm); SCH23390 (4.1 μm); metoclopramide (265μm), domperidone and (±)‐sulpiride (inactive).

It is concluded that the receptor subserving the dopamine‐induced hyperpolarization of the salivary gland acinar cells is the same as that mediating the secretory response to dopamine. In addition these data support our findings, which suggested that this receptor is similar to the D1 dopamine receptor, but distinct from the D2 receptor found in mammalian systems.
The effects of neuropeptide Y and its fragments upon basal and electrically stimulated ion secretion in rat jejunum mucosa
Authors: Cox H.M. , Cuthbert A.W.
Published: July 2012

The effects of neuropeptide Y (NPY) and a range of C terminal fragments were investigated both on basal short circuit current (s.c.c.) and electrical field stimulated responses in voltage clamped preparations in rat jejunal mucosa.

Most of the NPY fragments tested had direct effects upon the mucosa, reducing baseline s.c.c. with EC50 values of 1 μm or more. NPY was 30 times more effective than any of the fragments tested and the order of potency was: NPY ≫ NPY (11–36) ≥ (12–36) ≥ (13–36) ≥ (14–36). NPY (15–36), (16–36), (20–36) and (22–36) were still less effective and complete concentration‐response curves could not be constructed. NPY (26–36), des amido NPY and the C‐terminal flanking peptide of NPY (CPON) were all inactive and did not significantly alter responses to NPY.

Electrical field stimulation (EFS) of mucosal preparations elicited rapid transient secretory responses in the presence of hexamethonium and atropine. NPY and fragments attenuated these secretory responses and where concentration‐response relationships could be compared at a given time point the following order of potency was obtained: NPY ≫ NPY (11–36) > NPY (13–36). Again NPY (26–36), des amido NPY and CPON were ineffective, while at single concentrations (300 nm) a graded attenuation of EFS responses was obtained with NPY (14–36) ≥ NPY (15–36) > NPY (16–36) ≥ NPY (20–36) > NPY (22–36).

The attenuation of EFS responses by these peptides was not dependent upon the basal secretory state. Pretreatment of tissues with piroxicam reduced s.c.c. and attenuated further reductions in s.c.c. by NPY, but had no effect upon NPY‐mediated inhibition of electrically‐stimulated secretory responses.

NPY fragments attenuated both basal and EFS generated secretion. Since fragments are effective these receptors must, by definition be Y2‐like. NPY (11–36) and NPY (13–36) at 300 nm and 1 μm did not significantly attenuate secretory responses to either carbachol (CCh) or substance P (SP). A 1 μm concentration of either fragment was equivalent in effect to 30 nm NPY upon basal current, but NPY at this concentration significantly reduced both CCh‐ and SP‐induced secretion. The reduced spectrum of fragment activity together with the different order and potency ratios obtained with these three peptides indicates a presynaptic action for NPY and the fragments.
Functional evidence for heterogeneity of peripheral prejunctional α2‐adrenoceptors
Authors: Connaughton Sonia , Docherty James R.
Published: July 2012

We have examined the potencies of a series of α2‐adrenoceptor antagonists in functional studies of prejunctional α2‐adrenoceptors in rat atrium and vas deferens, and compared potencies with affinities for the α2A‐ligand binding site of human platelet and the α2B‐ site of rat kidney.

Antagonist potency in rat atrium was expressed as an EC30 (concentration producing 30% increase in the stimulation‐evoked overflow of tritium in tissues pre‐incubated with [3H]‐noradrenaline). Antagonist potency in rat vas deferens was expressed as a pA2 or KB at antagonizing the inhibition by the α2‐adrenoceptor agonist xylazine of the isometric twitch to a single stimulus, or as an EC30.

In ligand binding studies, Ki values were obtained for the displacement by α‐adrenoceptor antagonists of [3H]‐yohimbine binding to human platelet or rat kidney membranes.

In functional studies, three antagonists (ARC 239, prazosin and chlorpromazine) distinguished between prejunctional α2‐adrenoceptors of rat atrium (EC30) and rat vas deferens (pA2) and showed 49, 12 and 7 times higher potency in rat atrium, respectively. ARC 239 was also 17 times more potent in rat atrium than rat vas deferens when EC30 values were compared.

The correlation of affinity for the α2A‐ site of human platelet was better with prejunctional potency in rat vas deferens than rat atrium.

The correlation of affinity for the α2B‐site of rat kidney was better with prejunctional potency in rat atrium than rat vas deferens.

It is concluded that prejunctional α2‐adrenoceptors of rat vas deferens and rat atrium differ, and these receptors may resemble the α2A‐ and α2B‐ligand binding sites, respectively.
The effects of substance P analogues on the scratching, biting and licking response induced by intrathecal injection of N‐methyl‐d‐aspartate in mice
Authors: Sakurada Tsukasa , Manome Yoichi , Tan‐No Koichi , Sakurada Shinobu , Kisara Kensuke
Published: July 2012

Intrathecal (i.t.) administration of N‐methyl‐d‐aspartate (NMDA) elicited a dose‐dependent behavioural response consisting of licking, biting and scratching in mice.

Repeated i.t. injections of 0.4 nmol NMDA, at 5 min intervals, resulted in the rapid development of desensitization to this NMDA‐induced behavioural phenomenon.

The NMDA‐induced response was dose‐dependently inhibited by the simultaneous injection of a selective NMDA‐receptor antagonist, d‐2‐amino‐5‐phosphonovaleric acid.

The substance P (SP) analogues [d‐Pro2, d‐Trp7,9] SP and [d‐Arg1, d‐Trp7,9, Leu11] SP (spantide) inhibited NMDA‐induced behavioural responses in a dose‐dependent manner. However, [d‐Phe7, d‐His9] SP (6–11), a SP analogue selective for neurokinin1 (NK1) receptors, failed to inhibit NMDA‐induced responses even at a dose of 4.0 nmol.

These results indicate that NMDA‐induced behavioural responses are mainly mediated through NMDA receptors without affecting NK1 receptors in the spinal cord.
Evidence for BK1 bradykinin‐receptor‐mediated prostaglandin formation in osteoblasts and subsequent enhancement of bone resorption
Authors: Ljunggren Östen , Lerner Ulf H.
Published: July 2012

The effects of the BK1 bradykinin (BK)‐receptor agonist des‐Arg9‐BK on bone resorption and prostaglandin formation in osteoblasts have been studied.

Des‐Arg9‐BK (1 μm) stimulated the release of 45Ca from prelabelled neonatal mouse calvarial bones and the formation of prostaglandin E2 (PGE2) in calvarial bones. The stimulatory effect on bone resorption and PGE2 formation could be totally inhibited by indomethacin, flurbiprofen and hydrocortisone.

The BK1 receptor antagonist des‐Arg9‐Leu8‐BK (10 μm) inhibited des‐Arg9‐BK (0.01–0.1 μm)‐induced release of 45Ca from prelabelled neonatal mouse calvarial bones, while leaving BK (0.1–1 μm)‐induced 45Ca release unaffected.

In isolated osteoblast‐like cells from neonatal mouse calvarial bones, des‐Arg9‐BK (1 μm) induced a slowly developing increase in PGE2 formation that was significantly different from untreated controls after 24 h. Treatment with BK caused a rapid burst (within minutes) of PGE2 formation.

Des‐Arg9‐Leu8‐BK (10 μm) selectively inhibited des‐Arg9‐BK (1 μm)‐induced PGE2 and prostacyclin formation in isolated osteoblast‐like cells incubated for 72 h. Des‐Arg9‐Leu8‐BK did not affect BK and Lys‐BK (1 μm)‐induced PGE2 and prostacyclin formation in isolated osteoblast‐like cells incubated for 72 h.

These data indicate that osteoblasts are equipped with BK1‐receptors mediating enhanced prostaglandin formation and subsequent bone resorption.
The crucial role of physiological Ca2+ concentrations in the production of endothelial nitric oxide and the control of vascular tone
Authors: Lopez‐Jaramillo P. , Gonzalez M.C. , Palmer R.M.J. , Moncada S.
Published: July 2012

The effect of varying the extracellular Ca2+ concentration on the basal and acetylcholine (ACh)‐induced release of nitric oxide (NO) from the rabbit aorta was investigated by use of a superfusion bioassay system.

Changes between 0.5 and 2.0 mm in the concentration of Ca2+ superfusing the detector bioassay tissues or perfusing endothelium‐denuded donor aortae had no effect on the tone of these tissues.

Increasing the concentration of Ca2+ perfusing endothelium‐containing donor aortae from zero to 1.25 mm caused a transient (24 ± 9 min), concentration‐dependent basal release of NO, which was attenuated at higher concentrations of Ca2+ (1.5–2.0 mm).

The duration of the effect of Ca2+ on the basal release of NO was increased by a concomitant infusion of l‐arginine (100 μm) through the donor aorta.

Changes in the concentration of Ca2 + between 0.5 and 2.0 mm had a similar biphasic effect on the release of NO induced by ACh, which was also maximal at 1.25 mm Ca2+.

When Ca2+ was removed from the Krebs buffer perfusing the donor aorta, the basal release of NO declined within 2 min. In contrast, the release of NO induced by ACh declined progressively over 60 min.

Thus changes in the concentration of Ca2+ around the physiological range modulate the synthesis of NO by the vascular endothelium and consequently, vascular tone. This may account for the effects of dietary Ca2+ supplements on the control of some hypertensive states.
Vasodilatation and inhibition of mediator release represent two distinct mechanisms for prostaglandin modulation of acute mast cell‐dependent inflammation
Authors: Raud J.
Published: July 2012

Intravital microscopy of the hamster cheek pouch was used to examine the influence of vasodilator prostanoids (prostaglandin E2 (PGE2), PGI2), forskolin, and nitroprusside on the microvascular changes during acute inflammation induced by antigen or histamine. The results extend our previous finding that PGE2 modulates allergic inflammation and histamine release in the cheek pouch model.

The microvascular actions of arachidonic acid and different cyclo‐oxygenase products (PGE2, PGD2, PGI2, PGF2α, and the thromboxane A2 (TXA2)‐analogue U‐44069) were first compared with respect to their effects on arteriolar tone. Of the prostaglandins, only PGE2 and PGI2 were potent vasodilators and markedly increased local blood flow. Nitroprusside and forskolin also caused vasodilatation and increased blood flow, but were somewhat less potent than PGE2 and PGI2.

Topically applied PGE2 and PGI2 in vasodilator concentrations suppressed the antigen‐induced plasma leakage. On the other hand, although the antigen response was predominantly mediated by histamine, both prostaglandins enhanced the plasma leakage evoked by exogeneous histamine.

In contrast, the vasodilator nitroprusside, in a dose causing an increase in blood flow equal to that of PGE2 and PGI2, potentiated both the histamine‐induced plasma leakage, as well as the plasma and leukocyte extravasation after antigen challenge, indicating that the anti‐inflammatory actions of the prostaglandins were unrelated to their vasodilator properties per se.

Because forskolin, a specific activator of adenylate cyclase, mimicked the actions of PGE2 and PGI2, i.e. inhibition of the antigen‐induced plasma extravasation and enhancement of the histamine response, it is possible that the observed antiallergic effects of the prostaglandins were related to accumulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP).

Taken together, there appears to be a competition between pro‐ and anti‐inflammatory effects of PGE2 and PGI2 in reactions involving release of endogeneous inflammatory mediators in vivo, i.e. enhancement of inflammatory mediator target action on one hand (‘two mediator synergism’), and suppression of mediator release on the other. Moreover, the observations indicate that vasodilatation and inhibition of mediator release are two distinct actions of PGE2 and PGI2.
Contractile responses of smooth muscle strips from rat and guinea‐pig urinary bladder to transmural stimulation: effects of atropine and α,β‐methylene ATP
Authors: Brading A.F. , Williams J.H.
Published: July 2012

Strength‐duration curves for threshold mechanical responses to single transmural stimuli were identical for rat and guinea‐pig detrusor. In both species atropine had no effect on the curves, but the curves were shifted to the right by nerve blockade with tetrodotoxin (TTX), and by blockade of P2‐purinoceptors with α,β‐methylene ATP (α,β‐MeATP).

With short duration pulses of 50 V and less, the responses were nerve‐mediated. Increase in either the strength or duration of the stimulus caused direct muscle stimulation, resistant to blockade with atropine, TTX and α‐β‐MeATP.

The shape of the contractile response to a single nerve stimulus varied from tissue to tissue. The responses could be mono‐, bi‐, or multiphasic. Bi‐ or multiphasic responses were normally seen in tissues which were spontaneously active. The multiphasic nature of the response was enhanced by factors which increased the excitability of the cells and was reduced by factors which decreased the excitability.

The frequency‐response curves in the rat are similar to those previously obtained in the guinea‐pig. Atropine suppresses the high frequency response by 25%, with little effect at low frequencies, whereas desensitization of P2‐purinoceptors with α,β‐MeATP suppresses the responses maximally at low frequencies but still by 75% at high frequencies. A combination of both drugs eliminates the nerve‐mediated responses.

It is concluded that the response to a single nerve stimulus is mediated by a non‐cholinergic transmitter, through activation of P2‐purinoceptors. The possibility that simultaneous release of acetylcholine can modify the excitability of cells and thus the configuration of the response to a single stimulus is discussed.
Release of endothelium‐derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester
Authors: Smith J.A. , Lang D.
Published: July 2012

Cultured aortic endothelial cells of the pig respond to the endothelium‐derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by l‐NG‐monomethyl‐arginine (l‐NMMA), and has been used to investigate the effects of phorbol esters on EDRF release.

Pretreatment with phorbol‐12,13‐dibutyrate (PDB) but not the inactive 4α‐phorbol‐12,13,‐didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10−8 m) in a time and concentration‐dependent manner. PDB did not affect basal cyclic GMP levels.

PDB (3 × 10−7 m), but not PDD (3 × 10−7 m), also inhibited ATP (10−5 m)‐induced increases in cyclic GMP, but did not affect those induced by bradykinin (10−7 m).

Increases in cyclic GMP induced by low (10−7 m) but not high (10−6 m) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 × 10−7 m). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 u ml−1) and catalase (CAT, 100 u ml−1).

SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP‐induced increases in cyclic GMP.

Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10−5 m) were unaffected by PDB pretreatment.

The inhibitory effects of PDB are probably a result of an action of protein kinase C on the steps between receptor occupation and phospholipase C activation.
Mechanism of the potentiation of neurally‐induced bronchoconstriction by gallamine in the guinea‐pig
Authors: Del Monte Marco , Omini Claudio , Subissi Alessandro
Published: July 2012

Electrical stimulation of the cervical vagi (15 Hz, 0.2 ms, 3 s, 7–15 V) produced a slight bronchoconstriction in the anaesthetized guinea‐pig. This effect was fully abolished by atropine, while gallamine (0.1–10 μmol kg−1) produced a dose‐dependent increase up to ten fold.

Gallamine‐induced potentiation of neurally‐mediated bronchoconstriction was not inhibited by depletion of sensory neuropeptides with capsaicin or by pretreatment with pyrilamine. In propranolol‐pretreated guinea‐pigs the potentiation induced by gallamine 3 and 10 μmol kg−1 was inhibited by 40 and 46%, respectively.

Physostigmine (0.5 mg kg−1) produced a very slight and slowly developing bronchoconstriction in the anaesthetized guinea‐pig, which was also potentiated dose‐dependently by gallamine (0.1–10 μmol kg−1).

Gallamine (10 μmol kg−1) potentiated the bronchial anaphylactic response induced by aerosol challenge with ovalbumin in actively sensitized guinea‐pigs.

These results suggest that neither sensory neuropeptides nor histamine are involved in the gallamine‐induced potentiation of neurally‐mediated bronchoconstriction, while inhibition of the sympathetic nervous system may play a minor role. They are in general agreement with the hypothesis that gallamine antagonizes acetylcholine selectively at prejunctional muscarinic receptors in the guinea‐pig airways, thus increasing its release from parasympathetic nerve terminals. These autoreceptors appear to be operant during anaphylactic bronchoconstriction.
The influence of 5‐hydroxytryptamine agonists and antagonists on identified sympathetic preganglionic neurones in the rat, in vivo
Authors: Lewis D.I. , Coote J.H.
Published: July 2012

5‐Hydroxytryptamine (5‐HT) was applied by microiontophoresis in the vicinity of identified sympathetic preganglionic neurones in the upper thoracic spinal cord of the rat, in vivo.

Sympathetic preganglionic neurones responded in one of three ways to 5‐HT: by (a) excitation (76%), (b) inhibition (4%) or (c) in a biphasic manner (5%).

The excitatory responses evoked by 5‐HT were mimicked by 5‐carboxamidotryptamine (5‐CT) and α‐methyl‐5‐hydroxytryptamine (α‐Me‐5‐HT). The inhibitory and biphasic responses evoked by 5‐HT were mimicked by 2‐methyl‐5‐hydroxytryptamine (2‐Me‐5‐HT). The observed responses evoked by 5‐HT and selective agonists may be different on the same cell. In several instances a single neurone excited by one agonist was inhibited by another agonist.

The 5‐HT2‐receptor antagonists, ketanserin and LY 53857, failed to abolish selectively the excitatory responses evoked by 5‐HT and α‐Me‐5‐HT, when applied by microiontophoresis. The antagonists non‐selectively reduced the excitatory responses evoked by 5‐HT, 5‐CT, α‐Me‐5‐HT, d,l‐homocysteic acid (DLH) and noradrenaline (NA). A reduction in synaptically evoked activity was also observed.

The 5‐HT3‐receptor antagonist, ICS 205–930, failed to abolish the inhibitory responses evoked by 5‐HT.

It was concluded that the excitatory responses evoked by 5‐HT are mediated by a receptor that is neither 5‐HT2 or 5‐HT3, but shows similarities to the 5‐HT1‐like receptor profile. The inhibitory actions of 5‐HT are mimicked by 2‐Me‐5‐HT, but the receptor is not 5‐HT3, or 5‐HT1‐like or 5‐HT2.
Actions of cocaine on rat dopaminergic neurones in vitro
Authors: Lacey M.G. , Mercuri N.B. , North R.A.
Published: July 2012

Intracellular recordings were made from neurones in vitro in the rat substantia nigra zona compacta and ventral tegmental area; the neurones had the characteristic properties of dopamine‐containing cells, and fired action potentials spontaneously.

Cocaine (1–10μm) inhibited spontaneous firing, hyperpolarized the membrane and (in neurones voltage‐clamped at −60 mV) caused an outward membrane current; the minimally effective concentration was 1 μm. These effects were blocked by sulpiride (30 nm–1 μm).

Dopamine (3–100 μm) also inhibited firing, hyperpolarized and caused an outward current. These effects of dopamine were potentiated about five fold by cocaine (10 μm).

It is concluded that cocaine (1–10 μm) inhibits the firing and hyperpolarizes substantia nigra zona compacta neurones in vitro by blocking the uptake of dopamine which the cells continuously release.
Effects of 5‐HT3 receptor antagonists on 5‐HT and nicotinic depolarizations in guinea‐pig submucosal neurones
Authors: Vanner S. , Surprenant A.
Published: July 2012

Intracellular recordings were made from neurones of the guinea‐pig submucosal plexus. The effects of several 5‐hydroxytryptamine3 (5‐HT3) receptor antagonists on depolarizations produced by ionophoretic application of 5‐HT and acetylcholine, as well as on fast excitatory postsynaptic potentials (fast e.p.s.ps) produced by nerve stimulation were examined.

ICS 205–930, GR 38032F, MDL 72222, cocaine and curare all inhibited the fast e.p.s.p. as well as the depolarizations in response to 5‐HT and acetylcholine (ACh) ionophoresis in a dose‐dependent fashion.

IC50 values for ICS 205–930, GR 38032F, MDL 72222, cocaine and curare in inhibiting the 5‐HT mediated depolarizations were 12 nm, 100 nm, 3 μm, 3 μm and 20 μm, respectively.

IC50 values for ICS 205–930, GR 38032F, MDL 72222, cocaine and curare in inhibiting the nicotinic depolarizations were 4 μm, 12 μm, 11 μm, 6 μm and 17 μm, respectively. Similar IC50 values were obtained for inhibition of the fast e.p.s.ps by these antagonists.

The nicotinic receptor blocker, hexamethonium, inhibited the nicotinic depolarization and the fast e.p.s.p. with IC50 values of 10 μm. Hexamethonium (10 μm‐5 mm) did not alter the depolarization induced by 5‐HT.

These results demonstrate that the pharmacological profile of 5‐HT3 receptors present on submucosal neurones is identical to that of 5‐HT3 receptors on myenteric neurones and, thus, provide evidence that the enteric neuronal 5‐HT3 receptor forms a receptor subtype distinct from that characterized in other parts of the autonomic nervous system.
Activation of μ‐ and δ‐opioid receptors present on the same nerve terminals depresses transmitter release in the mouse hypogastric ganglion
Authors: Rogers Helen , Henderson Graeme
Published: July 2012

The inhibitory actions of μ‐ and δ‐opioid receptor agonists on the strong, single fibre synaptic input to neurones contained in the mouse hypogastric ganglion have been examined.

The opioid agonists [d‐Ala2,NMePhe4,Gly‐ol5]enkephalin (DAMGO, 10 nm‐10 μm), morphine (10–30 μm), [d‐Ser2,Leu5,Thr6]enkephalin (DSLET, 3 nm‐1 μm), [d‐Pen2,d‐Pen5]enkephalin (DPDPE, 10 nm‐10 μm), all depressed the single fibre, all‐or‐nothing, nicotinic, excitatory synaptic potential (e.p.s.p.) recorded in mouse hypogastric ganglion neurones. U50488H (0.3–1 μm) was without effect.

The effect of DSLET, but not that of DAMGO, was reversed by the δ‐opioid receptor‐selective antagonist, ICI 174864 (0.3 μm). Naloxone (0.3 μm) antagonized the effect of both DSLET and DAMGO.

The site of action of the μ‐ and δ‐receptor agonists was on the presynaptic terminals, since at the concentrations which depressed the e.p.s.p. these drugs did not affect the resting membrane potential or input resistance of the postganglionic neurone body, nor did they depress the postganglionic, nicotinic response to exogenously applied acetylcholine.

Quantal analysis further confirmed the presynaptic site of action; μ‐ and δ‐opioid receptor agonists decreased the mean number of quanta released per stimulus but did not reduce the mean amplitude of the quantal unit.

It was concluded that μ‐ and δ‐opioid receptors were located on the same presynaptic nerve terminals since, in the same neurones, μ‐ and δ‐opioid receptor agonists depressed the same single fibre inputs.

The potassium channel blockers barium and quinine, at concentrations known to block opioid‐activated somatic potassium conductances, reduced slightly but did not abolish the μ‐ and δ‐opioid receptor‐mediated inhibition of the e.p.s.p.
Sodium nitroprusside modulates the fibrinolytic system in the rabbit
Authors: Lidbury Paul S. , Korbut Richard , Vane John R.
Published: July 2012

We have investigated the effect of sodium nitroprusside (NP) and glyceryl trinitrate (GTN) on fibrinolysis in anaesthetized rabbits ex vivo and in vitro by measurement of euglobulin clot lysis time (ECLT), plasma levels of tissue plasminogen activator (t‐PA) activity, plasma t‐PA antigen levels and plasminogen activator inhibitor (PAI‐1) activity.

In vivo, NP (30 μg kg−1), GTN (30 μg kg−1) and prostacyclin (3 μg kg−1) caused similar transient decreases in left ventricular systolic pressure. However, while prostacyclin induced near‐maximal inhibition of ex vivo platelet aggregation, NP or GTN had no effect.

Ex vivo, NP caused a significant decrease in ECLT and an increase in plasma t‐PA activity.

Intravenous co‐administration of t‐PA (30 μg kg−1) with NP caused substantial prolongation of plasma t‐PA activity, without affecting t‐PA antigen levels.

In whole blood in vitro, NP (30 μg kg−1) prevented the time‐dependent increase in PAI‐1 activity and inhibited inactivation of added t‐PA (10 ng ml−1).

We propose that NP exhibited fibrinolytic activity through increased t‐PA activity as a result of inhibition of PAI‐1 release from platelets. These results could have important therapeutic consequences when t‐PA and nitrate treatments are combined.
An examination of the 5‐HT3 receptor mediating contraction and evoked [3H]‐acetylcholine release in the guinea‐pig ileum
Authors: Fox Alyson J. , Morton Ian K.M.
Published: July 2012

The relative contributions of two classes of 5‐hydroxytryptamine (5‐HT) receptor (5‐HT2 and 5‐HT3) to the contractile action of 5‐HT, 2‐methyl‐5‐hydroxytryptamine (2‐methyl‐5‐HT) and α‐methyl‐5‐hydroxytryptamine (α‐methyl‐5‐HT) were studied in the guinea‐pig ileum longitudinal muscle‐myenteric plexus strip (LMMP) preparation. Contractility studies were combined with an analysis of the effects of the three agonists on [3H]‐acetylcholine ([3H]‐ACh) release from preparations preincubated with [3H]‐choline.

In contracting the LMMP, 5‐HT was approximately one order of magnitude more active than 2‐methyl‐5‐HT and α‐methyl‐5‐HT, with relative activities for 5‐HT: 2‐methyl‐5‐HT: α‐methyl‐5‐HT of 1.00:0.13:0.10.

Ketanserin (1 μm) was without effect on the concentration‐response curves for contraction to 5‐HT, 2‐methyl‐5‐HT or α‐methyl‐5‐HT, whilst ondansetron (GR38032F; 1 μm) produced a parallel rightward displacement of the upper part of the concentration‐response curves to 5‐HT and α‐methyl‐5‐HT and of the entire curve to 2‐methyl‐5‐HT.

In increasing the spontaneous release of [3H]‐ACh from the LMMP, 5‐HT was again approximately one order of magnitude more active than 2‐methyl‐5‐HT and α‐methyl‐5‐HT with relative activities for 5‐HT: 2‐methyl‐5‐HT: α‐methyl‐5‐HT of 1.00: 0.19: 0.11.

Ondansetron (1 μm) greatly attenuated the increase in spontaneous [3H]‐ACh release evoked by all three agonists. pKB estimates of 7.62 ±0.12 and 7.64 ± 0.09 were obtained for ondansetron antagonism of 5‐HT and 2‐methyl‐5‐HT‐evoked increases respectively.

These data suggest that the contractile action of 5‐HT, 2‐methyl‐5‐HT and α‐methyl‐5‐HT in the guinea‐pig ileum can, under these conditions, be accounted for largely in terms of 5‐HT3 receptor activation. Estimates for pKB obtained with ondansetron are in accordance with those previously obtained from contractility studies in this preparation and these findings are discussed in terms of the postulated existence of subtypes of 5‐HT3 receptors.
A novel series of non‐quaternary oxadiazoles acting as full agonists at muscarinic receptors
Authors: Freedman S.B. , Harley E.A. , Patel S. , Newberry N.R. , Gilbert M.J. , McKnight A.T. , Tang J.K. , Maguire J.J. , Mudunkotuwa N.T. , Baker R. , Street L.J. , MacLeod A.M. , Saunders J. , Iversen L.L.
Published: July 2012

A novel series of non‐quaternary oxadiazole‐based muscarinic agonists demonstrated high affinity for muscarinic receptors.

These agonists possessed high efficacy in the nanomolar range at muscarinic receptors in the superior cervical ganglion, atrium and ileum but did not show selectivity across the tissue preparations.

Two amino oxadiazoles, one from a quinuclidine series (L‐660,863) and one from a 1‐azanorbornane series (L‐670,207) possessed a high ratio of potency for displacing the binding of [3H]‐N‐methylscopolamine ([3H]‐NMS) to potency for displacing the agonist [3H]‐oxotremorine‐M ([3H]‐oxo‐M) (NMS/oxo‐M ratio) predictive of high efficacy in the cortex.

The two azanorbornane derivatives L‐670,548 and L‐670,207 stimulated the turnover of phosphatidylinositol in the cortex with a potency higher than that obtained with any other known muscarinic agonist (ED50 0.26 and 0.18 μm respectively).

The maximum response obtained with L‐670,207 was greater than that observed for carbachol but was comparable to that of the natural ligand acetylcholine.

These oxadiazole muscarinic agonists are among the most potent and efficacious non‐quaternary muscarinic agonists ever described.