bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of BRL55834 in rat portal vein and bovine trachea: evidence for the induction of a glibenclamide‐resistant, ATP‐sensitive potassium current
Authors: Edwards G. , Schneider J. , Niederste‐Hollenberg A. , Noack Th. , Weston A.H.
Published: July 2012

The effects of the benzopyran K‐channel opener, BRL55834, on mechanical activity in bovine trachealis and rat portal vein were studied together with membrane currents in freshly‐isolated single cells derived from these tissues.

BRL55834 (3nM‐l μm) produced a concentration‐dependent relaxation of bovine trachealis precontracted with 100 μm histamine and reduced the spontaneous mechanical activity of rat portal veins, effects which were antagonized by glibenclamide (1–10 μm) but were not reversible on washing. In contrast, charybdotoxin (250 nM) did not modify the spasmolytic effect of BRL55834 in bovine trachealis.

BRL55834 (10 nM‐10 μm) did not relax segments of bovine trachealis precontracted with 80 mM KC1.

In some freshly‐isolated single cells from bovine trachealis held at −10 mV, BRL55834 (3 μm) induced a time‐independent outward K‐current which was partially resistant to inhibition by glibenclamide (10 μm). In other cells, a very noisy, outwardly‐rectifying and charybdotoxin‐sensitive current developed in the presence of BRL55834 (3 μm) and in time‐matched control cells.

In freshly‐isolated single cells from rat portal vein held at −10 mV, BRL55834 (3 μm) induced a time‐and calcium‐independent outward K‐current which was partially resistant (approximately 25% inhibition at +40 mV) to subsequent inhibition by glibenclamide (10 um). In contrast, levcromakalim induced a time‐independent outward K‐current which was completely inhibited by glibenclamide 10 μm.

With the non‐hydrolysable ATP analogue, AMP‐PCP (5 mM), in the pipette, the ability of BRL55834 to induce a time‐independent K‐current in portal vein cells was markedly reduced (approximately 80% inhibition at +40 mV) whereas the effects of 10 um levcromakalim were totally inhibited.

The glibenclamide‐resistant current component induced by BRL55834 was totally inhibited by phentolamine (100 μm), a concentration that had no effect on the peak current (IBK(Ca)) induced by NS1619 (33 μm).

Stationary fluctuation analysis of the noise associated with the glibenclamide‐insensitive K‐current induced by BRL55834 in rat portal vein cells indicated that the unitary current flowing through the underlying channels was 0.26 pA at −10 mV, a value inconsistent with the involvement of BKca.

It is concluded that the relaxations of both bovine trachea and rat portal vein produced by BRL55834 are associated with the opening of K‐channels. These are probably identical to the ATP‐sensitive K‐channel opened by levcromakalim, although the involvement of an additional K‐channel cannot be excluded. The reduced sensitivity of the BRL55834‐induced changes to glibenclamide and to AMP‐PCP may result from avid binding of BRL55834 to its site of action.
The effect of neuropeptide Y on sodium, chloride and potassium transport across the rat distal colon
Authors: Strabel D. , Diener M.
Published: July 2012

Neuropeptide Y (NPY; 10−10–10−7 mol l−1) reduced basal short‐circuit current (Isc.) in a concentration‐dependent manner in the rat distal colon but was ineffective in the proximal colon.

The action of NPY was dependent upon the presence of CI− and HC03‐ anions and was blocked by prior treatment of the tissue with a Cl− channel blocker. The decrease in Isc was associated with an increase in mucosa‐to‐serosa fluxes of Na+, Rb+ (K+) and Cl−, whereas the serosa‐to‐muc osa flux of Cl− was decreased.

The size of the inhibitory NPY effect was linearly correlated with the height of the basal Isc i.e. it inhibited 55% of basal secretory Isc.

The action of NPY was unaffected by indomethacin and tetrodotoxin, when given alone, but was abolished, when the basal Isc was decreased to values near zero by a combination of both inhibitors. This inhibition could be overcome by restoring basal Isc with prostaglandin E2, indicating that the effect of NPY is not mediated by nerves or prostaglandins, but that NPY is only effective, when anion secretion is stimulated by the spontaneous release of neurotransmitters and prostaglandins.

NPY inhibited the increase in Isc induced by veratridine and prostaglandin E2, but it had no effect on the Isc induced by direct stimulation of the adenylate cyclase with forskolin, or on Isc induced by stimulation of the Ca2+–pathway with carbachol. Inhibition of the response to veratridine or prostaglandin E2 by NPY showed the same dependence on the height of the Isc just prior to addition of NPY as seen in control conditions, i.e. NPY inhibited 55% of cyclic AMP‐mediated secretion.

These results suggest that the effect of NPY is mediated by an inhibition of cyclic AMP‐stimulated secretion, which is stimulated in the rat distal colon by a continuous release of prostaglandins and neurotransmitters.
Potassium currents in human freshly isolated bronchial smooth muscle cells
Authors: Snetkov V.A. , Hirst S.J. , Twort C.H.C. , Ward J.P.T.
Published: July 2012

K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch‐clamp technique.

In whole‐cell recordings a depolarization‐induced, 4‐aminopyridine (4‐AP)‐sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA.

In the majority of cells depolarization to −40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4‐AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin‐perforated patch technique.

Single channels with a conductance of about 210 pS were recorded in cell‐attached patch, inside‐out patch, outside‐out patch and whole‐cell recording configurations. Channel open state probability in inside‐out patches was 0.5 at a membrane potential of 4±14 mV (mean±s.d., n= 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX.

In current‐clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration‐dependent depolarization.

These results suggest that in human bronchial smooth muscle cells, in marked contrast to other species, the majority of the outward current induced by depolarization is not due to a delayed rectifier, but to the activity of a large conductance, ChTX‐sensitive K+ channel. The Ca2+– and voltage‐dependency of this channel may well allow a sufficiently high open state probability for it to play a part in the regulation of the resting membrane potential.
Modulation by prednisolone of calcium handling in skeletal muscle cells
Authors: Metzinger Laurent , Passaquin Anne‐Catherine , Leijendekker Wim J. , Poindron Philippe , Rüegg Urs T.
Published: July 2012

Increased calcium (Ca2+) influx has been incriminated as a potential pathological mechanism in the chronic skeletal muscle degeneration exhibited by Duchenne muscular dystrophy (DMD) patients. We have studied the influence of the glucocorticoid α‐methylprednisolone (PDN), the only drug known to have a beneficial effect on the degenerative course of DMD, on Ca2+ handling in the C2 skeletal muscle cell line.

PDN, when added 3 days (when myoblasts start to fuse into myotubes) after cell seeding, led to a 2 to 4 fold decrease in cellular Ca2+ uptake. This decrease was independent of the extracellular Ca2+ concentration applied to cells. The effect took at least 24 h in order to become established (PDN of 10−5 m) and took longer for lower PDN concentrations (EC50 of ca. 10−6 m at day 5, 10−6 5 m at day 7 and 10−7 5 m at day 9 in culture).

Cellular calcium accumulation was also decreased in PDN‐treated myotubes exposed to 45Ca2+‐containing medium for 1 to 6 days.

No effect of PDN was seen on 45Ca2+ efflux; a decrease in the amount of 45Ca2+released was observed due to the reduction of cellular 45Ca2+ loading.

PDN treatment led to an approximately 2 fold decrease in basal cytosolic Ca2+ concentration. 6 Three antioxidant drugs (lazaroids), previously shown to enhance in vitro skeletal muscle cell differentiation to the same extent as PDN, induced a similar decrease in Ca2+ influx. 7 Our results suggest that long‐term incubation of C2 cells with PDN leads to a decrease of the size of the cellular Ca2+ pools and to reduced resting cytosolic Ca2+ levels. Part of the beneficial effect of PDN in DMD patients could be attributed to a reduction of Ca2+ influx and of the size of Ca2+ pools in dystrophic muscle fibres.
Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium
Authors: Cross M.L. , Jane S.D. , Wild A.E. , Foreman R.C. , Chad J.E.
Published: July 2012

The aim of these experiments was to determine the ability of the muscle‐type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium‐sensitive fluorescent dye Indo‐1 was used.

Application of the nicotinic agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 ± 22 nM (mean ± s.e.mean; n = 40 cells) rising to 600 ± 81 nM on addition of SDC (10 μm; n=15 cells), whereas no increase in [Ca2+]i was seen in non‐transfected control QT6 fibroblasts (before: 128 ± 9 nM, n = 40; after: 113±13 nM, n= 15).

The increase in [Ca2+]i caused by application of SDC was dose‐dependent, with an EC50 value of 12.7 ± 5.9 μm (n=14).

The responses to SDC in QF18 cells were blocked by prior application of α‐bungarotoxin (200 nM), by the addition of Ca2+ (100 μm), by removal of Na+ ions from the extracellular solution, or by the voltage‐sensitive calcium channel blockers nifedipine and ω‐conotoxin, which act with IC50 values of 100 nM and 100 pM respectively.

We conclude that activation of the nicotinic AChRs leads to a Na+‐dependent depolarization and hence activation of endogenous voltage‐sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.
Distribution and effects of pituitary adenylate cyclase activating peptide in cat and human lower oesophageal sphincter
Authors: Ny L. , Larsson B. , Alm P. , Ekström P. , Fahrenkrug J. , Hannibal J. , Anderssno K.‐E.
Published: July 2012

The localization, tissue concentrations, and effects of pituitary adenylate cyclase activating peptide (PACAP) 27 and 38 were investigated in cat and human lower oesophageal sphincter (LOS), and compared with those of vasoactive intestinal peptide (VIP) and helospectin.

PACAP‐immunoreactive nerve structures were found in the cat and human LOS, with an abundance in the circular smooth muscle layer. PACAP 27‐immunoreactivity was often co‐localized with VIP‐immunoreactivity.

In cat tissue, PACAP (PACAP 27 plus PACAP 38) concentrations were 50 fold lower than VIP concentrations; in human tissue they were 10 fold lower.

PACAP 27, PACAP 38, helospectin I, and VIP induced concentration‐dependent relaxations in circular smooth muscle preparations from cat and human LOS. The order of potency was: VIP > helospectin I ≤ PACAP 27 > PACAP 38. NG‐nitro‐L‐arginine, scopolamine, or apamin, did not influence the relaxant effects of PACAP 27 or VIP.

In cat preparations, both cyclic AMP and cyclic GMP levels were increased after exposure to PACAP 27 and helospectin I, whereas exposure to VIP was followed by an increase in cyclic AMP levels only. In human preparations, there was an increase in cyclic AMP levels without any change in cyclic GMP levels.

These results suggest that in the cat and human LOS, PACAP 27 and VIP can occur within the same nerve structures. PACAP 27 has a potent relaxant action, but its functional importance has to be established.
Interaction between the μ‐agonist dermorphin and the δ‐agonist [D‐Ala2, Glu4]deltorphin in supraspinal antinociception and δ‐opioid receptor binding
Authors: Negri L. , Improta G. , Lattanzi R. , Potenza R.L. , Luchetti F. , Melchiorri P.
Published: July 2012

In rats, the interaction between the μ‐opioid agonist dermorphin and the δ‐opioid agonist [D‐Ala2, Glu4]deltorphin was studied in binding experiments to δ‐opioid receptors and in the antinociceptive test to radiant heat.

When injected i.c.v., doses of [D‐Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail‐flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain δ‐opioid receptors and of 17% of μ‐opioid receptors. Naloxone (0.1 mg kg−1, s.c), and naloxonazine (10 mg kg−1, i.v., 24 h before), but not the selective δ‐opioid antagonist naltrindole, antagonized the antinociception.

In vitro competitive inhibition studies in rat brain membranes showed that [D‐Ala2, Glu4]deltorphin displaced [3H]‐naltrindole from two δ‐binding sites of high and low affinity. The addition of 100 μm Gpp[NH]p produced a three fold increase in the [D‐Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the δ‐agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the δ‐agonist increased 15 times.

Co‐administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D‐Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses.

Pretreatment with naloxone or with the non‐equilibrium μ‐antagonists naloxonazine and β∼ funaltrexamine completely abolished the antinociceptive response of the μ‐δ agonist combinations.

Pretreatment with the δ‐opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin‐[D‐Ala2, Glu4]deltorphin combinations to a value near that observed after the μ‐agonist alone. At the dosage used, naltrindole occupied more than 98% of brain δ‐opioid receptors without affecting μ‐opioid‐receptors.

These data suggest that in the rat tail‐flick test to radiant heat, μ‐ and δ‐opioid agonists co‐operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co‐operative antinociception is probably mediated by co‐activation of the δ‐opioid receptors at the cellular level by the μ‐ and δ‐agonist.
Dual mechanisms of GABAa response inhibition by β‐lactam antibiotics in the pyramidal neurones of the rat cerebral cortex
Authors: Fujimoto Mika , Munakata Mitsutoshi , Akaike Norio
Published: July 2012

The effects of β‐lactam antibiotics on the γ‐aminobutyric acid (GABA)‐induced Cl− current were investigated in pyramidal neurones freshly dissociated from the rat frontal cortex by the use of a nystatin‐perforated patch recording mode under voltage‐clamp conditions.

The GABA‐induced inward current increased in a concentration‐dependent manner with an EC50 of 6.7 × 10−6 m at a holding potential of −40 mV. The GABA response was accompanied by an increase in the membrane conductance and reversed at near the Cl− equilibrium potential.

All β‐lactams (penicillin, imipenem, aztreonam and cefotiam) inhibited the 10−5 m GABA‐induced response in a concentration‐dependent manner with an IC50 and Hill coefficient of 1.3 × 10−3 m and 0.64 for penicillin, 9.6 × 10−4 m and 0.83 for imipenem, 2.5 × 10−3 m and 9.99 for aztreonam, and 2.9 × 10−4 m and 1.03 for cefotiam.

Imipenem inhibited the GABA‐response competitively while penicillin inhibited the same response in a noncompetitive fashion.

The inhibitory action of imipenem showed no voltage‐dependency, whereas the effect of penicillin was voltage‐dependent.

It is thus proposed that some classes of β‐lactams, including imipenem, may have a mechanism that is different from penicillin and competitively affects the GABAA receptor.
Neuromuscular blocking profile of the vecuronium analogue, Org‐9487, in the rat isolated hemidiaphragm preparation
Authors: Prior Chris , Tian Lijun , Mallah Ahmed I. El , Young Louise , Ward Joanna M.
Published: July 2012

The neuromuscular effects of the short‐acting aminosteroid muscle relaxant Org‐9487 have been studied in the in vitro rat phrenic nerve/hemidiaphragm preparation by use of twitch tension and electrophysiological recording techniques.

Org‐9487 (5–100 μm) produced a concentration‐dependent decrease in the amplitude of twitches (0.1 Hz) and tetanic contractions (50 Hz) evoked by motor nerve stimulation. The compound produced fade of force during both 50 Hz stimulation and train‐of‐four stimulation at 2 Hz, indicating a prejunctional component of action.

Anticholinesterases only partially reversed the effect of Org‐9487 on twitch responses. This was possibly because, at the concentrations required to block twitches in the rat, Org‐9487 itself was found to possess significant anticholinesterase activity.

Org‐9487 (3 μm) increased the rundown of endplate current amplitudes during a 2 s train of 50 Hz nerve stimulation. This was because Org‐9487 increased the quantal content of the first endplate current in the train without affecting acetylcholine release towards the latter part of the train.

Org‐9487 (10 μm) produced a voltage‐dependent decrease in the time constant of decay of endplate currents at 32°;C and 0.5 Hz, indicative of a block of endplate ion channels. The blocking rate constant increased with membrane hyperpolarization.
Antithrombotic effect of a novel recombinant hirudin analogue, CX‐397, in a rat arterial thrombosis model
Authors: Takiguchi Yoshiharu , Asai Fumitoshi , Wada Kouichiro , Hayashi Hideya , Nakashima Mitsuyoshi
Published: July 2012

The antithrombotic effect of a new specific thrombin inhibitor, CX‐397, was examined in a photochemically‐induced arterial thrombosis model in the rat femoral artery and compared with that of heparin.

Pretreatment with CX‐397 (10, 20 and 40 μg kg−1 min−1, i.v.) from 15 min before the experiment prolonged the time required for thrombotic occlusion of the artery in a dose‐dependent manner. The antithrombotic efficacy of CX‐397 was associated with modest increases in activated partial thromboplastin time (APTT) and template bleeding time.

On the other hand, heparin at a dose of 450 μg kg−1 markedly prolonged APTT and the bleeding time, but did not inhibit thrombo‐occlusion.

CX‐397 selectively inhibited platelet aggregation and concurrent secretion of 5‐hydroxytryptamine (5‐HT) and thromboxane A2 (TXA2) production from platelets in response to thrombin, but not to collagen and ADP, in a dose‐dependent manner (5–100 ng ml−1).

CX‐397 at 10 μg kg−1 combined with vapiprost, a TXA2 receptor antagonist, at O.l mg kg−1 significantly prevented occlusion, whereas, at these doses, neither drug alone had much effect.

These results demonstrate that CX‐397 may prove to be more efficient for preventing platelet‐rich thrombosis than heparin. Thrombin may play an important role in the rat thrombosis model.

The additive antithrombotic effect of the combination of thrombin inhibitor and TXA2 receptor antagonist at low doses suggests that thrombin and TXA2 may work in concert to produce thrombosis.
Direct demonstration of sulphonylurea‐sensitive KATP channels on nerve terminals of the rat motor cortex
Authors: Lee Kevin , Dixon Alistair K. , Rowe Iain C.M. , Ashford Michael L.J. , Richardson Peter J.
Published: July 2012

We examined whether ATP‐sensitive potassium (KATP) channels are present on presynaptic terminals of the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. A novel fused nerve terminal preparation was developed which produced structures amenable to patch clamp methods. In inside‐out recordings a K+channel was observed which possessed all the major features of the Type 1 KATP channel, including sensitivity to ATP and the antidiabetic sulphonylureas.
GABA‐mediated inhibition of the anaphylactic response in the guinea‐pig trachea
Authors: Gentilini Grazia , Franchi‐Micheli Sergio , Mugnai Sabrina , Bindi Daniela , Zilletti Lucilla
Published: July 2012

In sensitized guinea‐pigs, the effects of γ‐aminobutyric acid (GABA) and GABAmimetic drugs have been investigated on tracheal segments contracted by cumulative application of an allergen (ovoalbumin, OA) and on serosal mast cells. The same drugs have also been tested on activation of alveolar macrophages isolated from unsensitized guinea‐pigs.

Superfusion with GABA (1–1000 μM) reduced the contraction intensity of tracheal strips. The effect of GABA (100 μM) was not affected by the carrier blockers, nipecotic acid and β‐alanine (300 μM each). It was mimicked by the GABAB agonist (−)−baclofen (100 μM) but not 3‐aminopropanephosphinic acid (100 μM, 3‐APA). The GABAA agonist, isoguvacine (100 μM) did not exert any effect. GABA (10 μM)‐induced inhibition of tracheal contractions was reduced by the GABAB antagonist, 2‐hydroxysaclofen (100 μM, 2‐HS), but not by the GABAA antagonist, bicuculline (30 μM).

The reduction in contraction intensity induced by GABA (100 μM) was prevented by a 40 min preincubation of tracheal strips with capsaicin (10 μM), but not tetrodotoxin (TTX, 0.3 μM). The effect of GABA (1000 μM) was absent after preincubation with indomethacin (2.8 μM) but unmodified when nordihydroguaiaretic acid (NDGA, 3.3 μM) was used. Finally, removal of the epithelium prevented the GABA effect.

Anaphylactic histamine release from serosal mast cells isolated from sensitized animals was not affected either by GABA (10–1000 μM) or the selective receptor agonists (−)−baclofen (0.1–1000 μM) and isoguvacine (10–1000 μM). The release of platelet‐activating factor (PAF) from alveolar macrophages stimulated by formyl‐Met‐Leu‐Phe (FMLP; 1 μM) was modified neither by GABA (100 μM) nor by (−)−baclofen (100 μM).

In conclusion, these data show that GABA can inhibit allergic phenomena in the guinea‐pig airways through activation of GABAB receptors. An involvement of neuropeptidergic sensory structures is suggested but a role for epithelial cells and arachidonate metabolites is not definitely proved.
Effects of vitamin E deficiency on vasomotor activity and ultrastructural organisation of rat thoracic aorta
Authors: Rubino Annalisa , Loesch Andrzej , Goss‐Sampson Mark A. , Milla Peter , Burnstock Geoffrey
Published: July 2012

The effects of vitamin E deficiency were evaluated in aortic rings isolated from rats maintained on a diet deficient in vitamin E.

Endothelium‐dependent vasodilator responses to acetylcholine (ACh) and calcium ionophore, A23187, were reduced in preparations from treated animals, compared to the age‐matched controls. The maximal vasodilatation to ACh was 66.4±9 (n = 4) and 38.8±7 (n = 4) % in control and 10 month‐treated preparations, respectively.

The endothelium‐independent vasodilator responses to sodium nitroprusside as well as the concentration‐dependent contractile responses to noradrenaline, did not differ between treated and control preparations.

Electron microscopic examination of vascular segments and revealed that, following vitamin E deficiency, normal tissue organisation was disrupted, the endothelial monolayer either not being in contact with the underlying tissue or being absent in most of the areas analysed.

It is concluded that during vitamin E deficiency both morphological disruption and functional impairment of endothelium occur without observable modification of muscle cell function and morphology.
Activation of rabbit platelets by Ca2+ influx and thromboxane A2 release in an external Ca2+‐dependent manner by zooxanthellatoxin‐A, a novel polyol
Authors: Rho Mun‐Chual , Nakahata Norimichi , Nakamura Hideshi , Murai Akio , Ohizumi Yasushi
Published: July 2012

Zooxanthellatoxin‐A (ZT‐A), a novel polyhydroxylated long chain compound, isolated from a symbiotic marine alga Simbiodinium sp., caused aggregation in rabbit washed platelets in a concentration‐dependent manner (1–4 μM), accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]i).

ZT‐A did not cause platelet aggregation or increase [Ca2+]i in a Ca2+‐free solution, and Cd2+ (0.1‐1 mM), Co2+ (1–10 mM) and Mn2+ (1–10 mM) inhibited ZT‐A‐induced aggregation. SK&F96365 (1–100 μM), a receptor operated Ca2+ channel antagonist, and mefenamic acid (0.1–10 μM), a non‐specific divalent cation channel antagonist, inhibited platelet aggregation and the increase in [Ca2+], induced by ZT‐A.

Indomethacin (0.1–10 μM), a cyclo‐oxygenase inhibitor, and SQ‐29548 (0.1–10 μM), a thromboxane A2 (TXA2) receptor antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT‐A.

Methysergide (0.01‐1 μM), a 5‐HT2 receptor antagonist, inhibited ZT‐A‐induced platelet aggregation but did not affect the increase in [Ca2+]i induced by ZT‐A.

Tetrodotoxin (1 μM), a Na+ channel blocker and chlorpheniramine (1 μM), a H1‐histamine receptor antagonist, neither affected ZT‐A‐induced platelet aggregation nor the increase in [Ca2+]i induced by ZT‐A.

Genistein (1–100 μM), a protein tyrosine kinase inhibitor, and staurosporine (0.01‐1 μM), a protein kinase C inhibitor, also inhibited ZT‐A‐induced platelet aggregation.

The present results suggest that ZT‐A elicits Ca2+‐influx from platelet plasma membranes. The resulting increase in [Ca2+]i subsequently stimulates the secondary release of TXA2 from platelets. Furthermore, the response to ZT‐A may be associated with tyrosine phosphorylation.
The effect of opiates on the terminal nerve impulse and quantal secretion from visualized amphibian nerve terminals
Authors: Lavidis Nickolas A.
Published: July 2012

Secretion of transmitter from amphibian motor nerve terminal release sites is intermittent, spatially non‐uniform and varies considerably throughout the year and during development. The role of opioid receptors in modulating transmitter secretion from amphibian motor nerve terminals is evaluated in this study.

Dynorphin‐A (24 μM) and morphine (500 μM) did not significantly change the shape of the nerve impulse or the consistency with which it was observed, but decreased evoked quantal secretion by more than 50%. These effects of dynorphin‐A and morphine were largely reversed by naloxone (50 μM).

Dynorphin‐A and morphine did not significantly change either the amplitude or the frequency of spontaneous quantal secretions.

There was a uniform decrease in evoked quantal secretion from release sites along terminal branches, irrespective of the quantal content value before drug treatment, indicating no difference in the susceptibility of proximal vs distal release sites to opiates.

Increasing the extracellular calcium concentration (0.3 to 0.4 mM) or trains of conditioning‐test impulses (25 to 100 Hz) resulted in smaller dynorphin‐A or morphine‐induced decreases in evoked quantal secretion.

The decrease in evoked quantal secretion occurs as a result of a uniform decrease in the probability of quantal secretion from release sites without any affect on the propagation of the nerve terminal impulse. Low probability release sites become effectively silent.
Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype
Authors: Dillon Glenn H. , Im Wha Bin , Carter Don B. , McKinley Denise D.
Published: July 2012

We examined how y‐aminobutyric acid (GABA) influences interaction of picrotoxin and tert‐butylbicyclophosphorothionate (TBPS) with recombinant rat α1β2γ2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl− currents measured by the whole‐cell patch clamp technique.

During application of GABA (5 μM) for 15 s, picrotoxin and TBPS dose‐dependently accelerated the decay of inward GABA‐induced currents (a holding potential of −60 mV under a symmetrical C1− gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration‐dependent manner. This indicates their interaction with both GABA‐bound and resting receptors.

The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 μM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 μM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the α1β2γ2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U‐93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono‐ and multiliganded receptors than resting receptors.

The time‐dependent decay and preincubation time‐dependent reduction of initial amplitude of GABA‐induced Cl− currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 μM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.

We conclude that picrotoxin and TBPS interact with both resting and GABA‐bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U‐93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.
Evidence for selective inhibition by lysophosphatidylcholine of acetylcholine‐induced endothelium‐dependent hyperpolarization and relaxation in rat mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira
Published: July 2012

The effects of lysophosphatidylcholine (LPC) on acetylcholine‐induced hyperpolarization and relaxation were examined in rat mesenteric arteries. LPC (3‐10 μM) reversibly inhibited endothelium‐dependent hyperpolarization by acetylcholine in a concentration‐dependent manner. LPC (10 μM) inhibited only partially endothelium‐dependent relaxation by acetylcholine. However, acetylcholine‐induced relaxation obtained in the presence of 100 μM NG‐nitro‐L‐arginine was almost completely eliminated by 10 μ LPC. These results indicate that LPC inhibits hyperpolarization and relaxation due to endothelium‐derived hyperpolarizing factor more specifically than the relaxation due to endothelium‐derived nitric oxide.
Effect of diabetes on relaxations to non‐adrenergic, non‐cholinergic nerve stimulation in longitudinal muscle of the rat gastric fundus
Authors: Jenkinson Karl M. , Reid Julianne J.
Published: July 2012

The effect of 8‐week streptozotocin‐induced diabetes has been examined on relaxations to non‐adrenergic, non‐cholinergic (NANC) nerve stimulation in longitudinal strips of rat gastric fundus.

In the presence of noradrenergic and cholinergic blockade and raised tissue tone, electrical field stimulation (0.5‐4 Hz, 30 s trains) induced frequency‐dependent relaxations that were significantly smaller in gastric fundus strips from diabetic rats than in strips from control rats.

NG‐nitro‐L‐arginine methyl ester (NAME, 100 μM) significantly reduced NANC relaxations in muscle strips from both control and diabetic rats, but the reduction was greater in muscle strips from diabetic rats than in those from control rats at frequencies of 2 and 4 Hz. α‐Chymotrypsin (1 u ml−1) slightly reduced relaxations to nerve stimulation in muscle strips from both control and diabetic rats.

> The duration of NANC nerve relaxations (1‐4 Hz, 30 s trains) was smaller in muscle strips from diabetic rats than in those from control rats. The duration of NANC relaxations was reduced by α‐chymotrypsin (1 u ml−1) in muscle strips from control rats but not in muscle strips from diabetic rats.

Relaxations to both nitric oxide (NO; 1–30 μM) and vasoactive intestinal polypeptide (VIP; 0.1‐30 μM) were concentration‐dependent and did not differ between muscle strips from control and diabetic rats.

The results suggest that streptozotocin‐induced diabetes impairs relaxations to NANC nerve stimulation in the rat gastric fundus, which are largely mediated by NO and to a lesser extent by VIP. The impairment appears to occur at the prejunctional level, as smooth muscle reactivity to NO and VIP is not altered.
Effect of trilinolein on cyclic nucleotide formation in human platelets: relationship with its antiplatelet effect and nitric oxide synthesis
Authors: Shen Y.C. , Hong C.Y.
Published: July 2012

Trilinolein, a triacylglycerol with linoleic acid as the only fatty acid residue in all three esterified positions of glycerol, was recently reported to have an inhibitory effect on adrenaline‐induced platelet aggregation. In the present study, we found that trilinolein at concentrations ranging from 0.01 to 1 μM. increased cyclic GMP formation and decreased cyclic AMP formation in washed human platelets. Both NG‐nitro‐L‐arginine methyl ester (L‐NAME) and methylene blue attenuated the trilinolein‐induced increase in cyclic GMP.

Adrenaline decreased not only the production of cyclic AMP but also that of cyclic GMP. Trilinolein antagonized the inhibitory effect of adrenaline on cyclic GMP formation, but potentiated the inhibitory effect of adrenaline on cyclic AMP accumulation.

Both trilinolein and adrenaline enhanced intracellular calcium but the increment of intracellular calcium induced by them was much less than that produced by thrombin.

We propose that the anti‐platelet effect of trilinolein is mediated through an increase in cyclic GMP, and that the change in cyclic GMP results from stimulation of nitric oxide synthesis in platelets.

We also propose that reduction of both cyclic AMP and cyclic GMP are involved in adrenaline‐induced platelet aggregation.
A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca2+‐activated K+ channels in bovine tracheal smooth muscle cells in culture
Authors: Macmillan S. , Sheridan R.D. , Chilvers E.R. , Patmore L.
Published: July 2012

The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca2+‐activated K+ (BKCa) channels in inside‐out and outside‐out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619.

The presence of BKCa channels on inside‐out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+], and sensitivity to the selective BKca channel blocker, iberio toxin.

NS 004 and ND 1619 (3‐30 μM) induced concentration‐related increases in open state probability of BKca channels when applied to either inside‐out or outside‐out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation.

SCA40 (0.1‐10 μM) had no effect on the activity of BKCa channels when applied to either inside‐out or outside‐out patches which subsequently responded to the application of NS 004 (10‐20 μM).

It is concluded that SCA40 does not have a direct effect on BKca channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.
Peripheral GABAA receptor‐mediated effects of sodium valproate on dural plasma protein extravasation to substance P and trigeminal stimulation
Authors: Lee Won Suk , Limmroth Volker , Ayata Cenk , Cutrer F. Michael , Waeber Christian , Yu Xianjie , Moskowitz Michael A.
Published: July 2012

The GABA transaminase inhibitor and activator of glutamic acid decarboxylase, valproic acid is being used for the treatment of migraine. Its mechanism of action is unknown. We tested the effects of sodium valproate and GABAA‐agonist muscimol on dural plasma protein ([125I]‐bovine serum albumin) extravasation evoked by either unilateral trigeminal ganglion stimulation (0.6 mA, 5 ms, 5 Hz, 5 min) or substance P (SP) administration (1 nmol kg−1, i.v.) in anaesthetized Sprague‐Dawley rats.

Intraperitoneal (i.p.) injection of sodium valproate or muscimol, but not baclofen (≤10 mg kg−1, i.p.) dose‐dependently reduced dural plasma protein extravasation caused either by electrical trigeminal stimulation (ED50: 6.6±1.4 mg kg−1, i.p., and 58 + 18 μg kg−1, i.p. for valproate or muscimol, respectively) or by intravenous substance P administration (ED50: 3.2±1.4 mg kg−1, i.p. and 385±190 μg kg−1, i.p. for valproate or muscimol, respectively).

Valproate (6.6 mg kg−1, i.p.) or muscimol (58 μ kg−1, i.p.) had no effect on mean arterial blood pressure or heart rate when measured for 30 min after i.p. administration.

The GABAA‐antagonist bicuculline (0.01 mg kg−1, i.p.) completely reversed the effect of valproate and muscimol on plasma extravasation following electrical stimulation or substance P administration, whereas the GABAB‐receptor antagonist, phaclofen (0.01‐1 mg kg−1, i.p.) did not. Bicuculline or phaclofen, given alone, did not alter the plasma extravasation response after either electrical stimulation or SP administration.

Valproate decreased plasma extravasation following substance P administration in adult animals, neonatally treated with capsaicin by a bicuculline‐reversible mechanism. This suggests that GABAA‐receptors are not found primarily on those afferent neurones or fibres which are sensitive to capsaicin treatment in neonatal rats.

We conclude that sodium valproate blocks plasma extravasation in the meninges through GABAA‐mediated postjunctional receptors probably within the meninges. The dosages required are comparable to those used clinically. Agonists and modulators at the GABAA receptor may become useful for the development of selective therapeutic agents for migraine and cluster headache.
Pharmacological profile of semotiadil fumarate, a novel calcium antagonist, in rat experimental angina model
Authors: Mori Tomohiro , Ishigai Yutaka , Fukuzawa Akiko , Chiba Kiyoshi , Shibano Toshiro
Published: July 2012

The aim of the present study was to determine whether antianginal efficacy of semotiadil fumarate (SD‐3211), a structurally novel calcium antagonist, is distinct from those of diltiazem, nifedipine and nisoldipine.

First, the duration of the inhibitory effects of semotiadil was compared with that of other Ca2+ antagonists in rat experimental angina evoked by vasopressin. Semotiadil (10 mg kg−1, p.o.) was effective for at least 9 h in the anginal model and those effects of semotiadil were longer‐lasting than those of diltiazem (30 mg kg−1, p.o.), nifedipine (10 mg kg−1, p.o.), and nisoldipine (3 mg kg−1, p.o.).

Second, the selectivity of actions of these Ca2+ antagonists for the coronary arteries and myocardium was evaluated in rat isolated perfused hearts. Diltiazem (10−6 M) reduced cardiac contractility without inhibiting the elevation of perfusion pressure evoked by acetylcholine. Semotiadil (10−7 M) significantly suppressed cardiac contractility and inhibited the coronary response to acetylcholine. In contrast, nifedipine (3 × 10−9‐3times 10−8 M) and nisoldipine (3 × 10−10‐10−08 M) did not reduce cardiac contractility at concentrations which significantly inhibited the increase in perfusion pressure to acetylcholine.

The selectivity of semotiadil for coronary artery and myocardium is intermediate between diltiazem and dihydropyridines tested in the present study.

These findings suggest that semotiadil has an advantage of diltiazem, nifedipine, and nisoldipine in the treatment of angina with regard to long‐lasting action and selectivity for coronary artery and myocardium.
Effects of 8‐bromoguanosine 3′:5′‐cyclic monophosphate on phenylephrine‐induced phosphatidylinositol hydrolysis and contraction in rat caudal artery
Authors: Min Suyin A. Lum , Tabrizchi Reza
Published: July 2012

The effects of 8‐bromoguanosine 3′:5′‐cyclic monophosphate (8‐bromo‐cyclic GMP) on phenylephrine‐induced contractions and phosphatidylinositol (PI) hydrolysis were investigated in rat isolated caudal artery. The effects of the nucleotide were compared to those of felodipine, a dihydropyridine Ca2+ channel antagonist and ryanodine, a putative depletor of intracellular Ca2+ stores. The purpose of this investigation was to examine the regulatory effects of cyclic GMP on receptor‐mediated signal transduction in vascular smooth muscle.

Phenylephrine induced a concentration‐dependent increase in PI hydrolysis that reached a maximum at 10 μM phenylephrine. Pre‐incubation with felodipine (10 nM) significantly reduced PI turnover, but did not affect basal hydrolysis. Similarly, removal of extracellular Ca2+ (2 mM ethylene glycol‐bis(β‐amino‐ethyl ether) N, N, N′, N′‐tetraacetic acid (EGTA)) blocked phenylephrine‐induced PI hydrolysis, but did not affect basal turnover. In contrast, 8‐bromo‐cyclic GMP (10 μM) did not affect phenylephrine‐induced PI hydrolysis, nor did it affect basal turnover.

Phenylephrine induced concentration‐dependent contractions that were inhibited by each of 8‐bromo‐cyclic GMP (10 μM), felodipine (1 nM and 10 nM) and ryanodine (3 μM and 10 μM). In addition, removal of Ca2+ from the physiological salt solution (2 mM EGTA) completely abolished contractions elicited by phenylephrine.

Phenylephrine‐induced contractions were not further affected by felodipine and 8‐bromo‐cyclic GMP applied concomitantly than by equivalent concentrations of felodipine alone. However, ryanodine and 8‐bromo‐cyclic GMP applied together significantly inhibited phenylephrine‐induced contractions in comparison to ryanodine alone.

These results suggest that phospholipase C‐activated PI hydrolysis in the rat caudal artery is dependent on extracellular Ca2+, mediated, in part, through dihydropyridine‐sensitive Ca2+ channels. Inhibition of contraction by felodipine may be brought about through indirect inhibition of IP3 production and subsequent attenuation of intracellular Ca2+ release. 8‐Bromo‐cyclic GMP does not inhibit PI hydrolysis; it may regulate vascular smooth muscle contraction by inhibition of Ca2+ release from IP3‐mediated intracellular stores, but it is unlikely that 8‐bromo‐cyclic GMP affects ryanodine‐sensitive stores.
Relationships between kallikrein secretion, kallikrein excretion and β‐adrenoceptors in kidney cortical slices from neurogenic hypertensive dogs
Authors: Damase‐Michel Christine , Bompart Guy , Durrieu Geneviève , Montastruc Jean‐Louis , Montastruc Paul , Girolami Jean‐Pierre
Published: July 2012

Sinoaortic denervation (SAD) in dogs is characterized by an increase in blood pressure and heart rate as well as the development of renal morphological lesions similar to those observed in essential hypertension in human subjects. To assess the effect of SAD on the secretion of kallikrein kinin systems (KKS), we studied the in vitro secretion of kallikrein by renal cortical slices of normal and neurogenic hypertensive dogs (1 and 18 months after SAD). The method using renal cortical slices allowed the study of secretion of kallikrein independently of renal perfusion pressure. The number of renal β‐adrenoceptors was measured by [125I]‐cyanopindolol binding.

SAD was associated with a marked increase in urinary kallikrein excretion at one month and a significant decrease at 18 months when compared with controls. Both changes were statistically significant (P<0.05). Concurrently, a progressive increase in in vitro kalhkrein secretion was observed (+80±10% and +179±48%, 1 and 18 months after SAD, respectively). Moreover, the cortical slices obtained from sinoaortic denervated dogs contained more kalhkrein than the control cortical slices (+ 32 ±16% and +55 ±7%, 1 and 18 months after SAD, respectively).

Renal β‐adrenoceptor number significantly (P<0.05) decreased 18 months after SAD from 18 ±2 to 8 ± 3 fmol mg−1 protein without any change in affinity constant.

Although there was no test of association, because the number of renal β‐adrenoceptors decreased whereas kallikrein secretion increased, the present data could suggest a β‐adrenoceptor‐mediated inhibition of kallikrein secretion. These results show that although the urinary kalhkrein is decreased, the tissue secretory capacities are enhanced. This could suggest a renal compensatory mechanism possibly involved in tissue protection in dogs after SAD, although such a mechanism is not sufficient to reverse hypertension.
An investigation into the mechanism of capsaicin‐induced oedema in rabbit skin
Authors: Newbold P. , Brain S.D.
Published: July 2012

Oedema formation induced by intradermal capsaicin has been studied in rabbit skin. The effect of the anti‐inflammatory steroid dexamethasone and also of a range of known inhibitors of oedema formation have been investigated in order to elucidate mechanisms involved in capsaicin‐induced oedema formation.

Oedema formation, in response to intradermally‐injected test agents, was measured by the local extravascular accumulation of intravenously injected 125I‐labelled albumin. In separate experiments skin blood flow was assessed by the clearance of intradermally‐injected 133xenon.

Oedema formation induced by intradermal histamine (3 nmol) and bradykinin (1 nmol), when in the presence of vasodilator doses of calcitonin gene‐related peptide (CGRP) (3 pmol) or prostaglandin E1, (PGE1) (10 pmol), was significantly inhibited (P < 0.01) in rabbits pretreated with intravenous dexamethasone (3 mg kg−1, −4h). In contrast dexamethasone had no effect on capsaicin (3 μmol)‐induced oedema formation or, on capsaicin (30–100 nmol)‐induced blood flow.

Oedema formation observed in response to intradermal capsaicin (3 μmol) was significantly inhibited (P < 0.01) when the selective capsaicin antagonist, ruthenium red (3 nmol) was co‐injected. This suggests that the mechanism of capsaicin‐induced oedema involves activation of sensory nerves. However, oedema was not inhibited when capsaicin was co‐injected with the neurokinin NK1 receptor antagonist, RP67580 (10 nmol), the NK2 antagonist SR48960 (10 nmol) or the CGRP antagonist CGRP8–37 (300 pmol).

Oedema formation induced by capsaicin was not inhibited when co‐injected with the histamine H1 receptor antagonist, mepyramine (3 nmol), the PAF antagonist, WEB 2086 (100 nmol), the bradykinin B2 receptor antagonist, Hoe140 (1 nmol), or the cyclo‐oxygenase inhibitor, indomethacin (10 nmol), suggesting that these mediators do not play a major role in the capsaicin‐induced response.

Histological analysis of capsaicin‐treated skin sites revealed undamaged, intact microvessels and lack of haemorrhage. Further, co‐injection of capsaicin with the hydrogen peroxide remover, catalase (2,200 u), had no effect on oedema formation. This suggests that capsaicin does not induce oedema formation secondary to free radical‐induced damage.

These results indicate that capsaicin‐induced oedema in rabbit skin involves activation of sensory nerves. However, the oedema is not inhibited by pretreatment with the anti‐inflammatory steroid, dexamethasone. Further the mechanisms which lead to the oedema formation observed after intradermal capsaicin remain unknown.