bluuebunny/crossref_metadata_2025 huggingface dataset:
Potentiation of P1075‐induced K+ channel opening by stimulation of adenylate cyclase in rat isolated aorta
Authors: Linde Claudia , Quast Ulrich
Published: July 2012

The effects of analogues and stimulators of cyclic AMP on the 86Rb+efflux‐stimulating and binding properties of P1075, an opener of ATP‐dependent potassium channels, were studied in rat aortic rings. The increase in 86Rb+efflux stimulated by P1075 was taken as a qualitative measure of K+ channel opening.

Forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and dibutyryl‐cyclic AMP (db‐cyclic AMP), a membrane permeant cyclic AMP‐analogue, relaxed rat aortic rings contracted by noradrenaline with EC50 values of 0.06, 2 and 10 μM, respectively.

Forskolin, IBMX and db‐cyclic AMP produced concentration‐dependent increases of the 86Rb+efflux induced by P1075 (50 nM) by up to twofold with EC50 values of about 0.1, 1.7 and 81 μM. At these concentrations the agents had little effect on the basal rate of 86Rb+efflux.

The 86Rb+efflux produced by P1075 in the presence of the cyclic AMP stimulators was inhibited by glibenclamide, a blocker of ATP‐sensitive potassium channels.

IBMX (100 μM) induced a leftward shift of the concentration ‐ 86Rb+efflux curve of P1075 without increasing the maximum. The enhancements of P1075‐stimulated 86Rb+efflux produced by combinations of forskolin and IBMX were either additive or less than additive.

The protein kinase A inhibitor, H‐89, inhibited P1075‐stimulated 86Rb+efflux in the presence of IBMX significantly more than in the absence of IBMX, suggesting that the effect of increased cyclic AMP levels is mediated by protein kinase A.

At high concentrations, forskolin and IBMX slightly increased basal 86Rb+efflux and inhibited the tracer efflux induced by P1075.

Binding of [3H]‐P1075 to rat aortic rings was either unaffected or inhibited by forskolin, IBMX and db‐cyclic AMP.

This study shows that moderate stimulation of the cyclic AMP system potentiates the K+ channel opening effect of P1075 by activation of protein kinase A. The fact that binding of [3H]‐P1075 remains unchanged or is diminished favours the hypothesis that the K+ channel openers activate ATP‐dependent K+ channels by an indirect mechanism.
Effect of platelet‐derived growth factor on voltage‐operated calcium channels in rabbit isolated ear artery cells
Authors: Wijetunge S. , Hughes A.D.
Published: July 2012

Platelet derived growth factor (PDGF), AB and BB isoforms (100 μM) increased calcium channel currents measured by whole cell voltage clamp technique in single vascular smooth muscle cells isolated from rabbit ear arteries.

Tyrphostin‐23 (100 μM) a selective inhibitor of protein tyrosine kinases, reduced calcium channel currents. Pre‐incubation with tyrphostin‐23 prevented PDGF‐AB induced increase in calcium channel currents. However, in these same cells 10 nM (+)‐202791, a dihydropyridine calcium channel agonist, did increase calcium channel currents.

Bistyrphostin (10 μM), a selective inhibitor of epidermal growth factor (EGF)‐kinase also reduced calcium channel currents and inhibited PDGF‐AB‐induced increases in calcium channel currents.

Genistein (100 μM) a selective inhibitor of tyrosine kinases, structurally unrelated to the tryphostins, also inhibited calcium channel currents and pre‐incubation with genistein prevented the PDGF‐AB‐induced rise in calcium channel currents.

These results indicate that PDGF increases calcium channel currents in vascular smooth muscle. This action of PDGF probably involves a tyrosine kinase.
5‐HT3 receptor agonist induced carrier‐mediated release of dopamine in rat striatum in vivo
Authors: Santiago Marti , Machado Alberto , Cano Josefina
Published: July 2012

In vivo microdialysis was used to study the effect of phenylbiguanide (PBG), a 5‐hydroxytryptamine3 receptor agonist, on the extracellular output of dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA) in the corpus striatum.

PBG produced a dose‐related (10‐500 μM) increase in the release of dopamine (280‐2000%). DOPAC and HVA output decreased with the perfusion of PBG. This decrease was similar with 50–500 μM PBG. 5‐HIAA output was not affected by any PBG concentration used.

When nomifensine (500 μM) was included in the Ringer solution, the effect of PBG on the release of dopamine was ameliorated or inhibited. However, the effect of PBG (50‐500 μ) on the extracellular output of DOPAC and HVA was similar in the absence and in the presence of nomifensine (500 μM).

Perfusion of MDL 72222, a 5‐hydroxytryptamine3 receptor antagonist, at doses of 50 and 100 μM produced similar decreases (50% of controls) and increases (120% of controls) in the extracellular output of dopamine and DOPAC, respectively. HVA and 5‐HIAA output levels were not affected by either concentration of MDL 72222. MDL 72222 (10 μm) produced a slight ard transient increase in the release of dopamine and a decrease in the extracellular output of DOPAC. HVA and 5‐HIAA extracellular output was not affected by MDL 72222 (10 μM) perfusion.

Co‐perfusion of MDL 72222 (10 and 100 μ) or tetrodotoxin (1 μM) with PBG (50 μM) did not modify the effect produced by PBG (50 μM) alone on the release of dopamine.

These results suggest that the effect of PBG on the release of dopamine is mainly carrier‐mediated.
The effects of formoterol on plasma exudation produced by a localized acute inflammatory response to bradykinin in the tracheal mucosa of rats in vivo
Authors: O'Donnell Stella R. , Anderson Gary P.
Published: July 2012

The effects of formoterol, a β2‐adrenoceptor agonist, on plasma protein exudation and microvascular permeability induced by topical, i.e. applied onto the tracheal mucosal surface, bradykinin (10 nmol; 20 μM, 5 min, 0.1 min‐1) were studied in a perfused segment of trachea prepared in situ in anaesthetized rats.

Bradykinin increased the amount of plasma (fluorimetric assay for protein) in the perfusate (response; 10.98 ± 0.357 μ, n = 69; total increase in plasma over basal during 45 min after start of bradykinin application) and 2 responses at a 90 min interval were reproducible. Carbon labelling was seen in tracheal sections from animals that received i.v. colloidal carbon, indicating that bradykinin caused tracheal microvessels to leak (increase in microvascular permeability).

Five minutes after topical formoterol, 5 or 30 nmol (10 or 60 μ perfused for 5 min), the bradykinin response was significantly reduced. The effects of formoterol were not dose‐related, i.e. were maximal at 5 nmol. The bradykinin response was at control levels 30 min after 5 nmol formoterol. After 30 nmol formoterol, the response was still reduced 120 min later. The bradykinin response was significantly reduced 60 min after systemic formoterol (i.p., 0.029 to 870 nmol kg‐1) and, for 290 nmol kg‐1 i.p. formoterol, this reduction was shown to last at least 150 min.

The bradykinin response was not prevented by supramaximal doses of topical (30 nmol) or i.p. (870 nmol kg‐1) formoterol and carbon‐labelled microvessels were seen in tracheal sections from all animals that received formoterol, although these were less in number and less densely labelled than in the absence of formoterol. There was a correlation between the plasma exudation response (μ1) and the number of carbon‐labelled vessels (Spearman's correlation coefficient 0.415, P < 0.001).

In animals pretreated with propranolol (3 μmol kg−1, i.V.), 29 nmol kg−1 formoterol, i.p., did not reduce the bradykinin response. However, propranolol itself markedly potentiated the bradykinin response which confounded the interpretation of its effects on formoterol.

The study has shown, in a preparation of rat trachea in situ, that supramaximal doses of the β 2‐adrenoceptor agonist, formoterol (a) produced a sustained, but incomplete, inhibition of plasma exudation (induced by topical bradykinin), and (b) did not prevent bradykinin‐induced leaky microvessels. The data support the view that, at least in rodent airways, β‐adrenoceptor agonists attenuate, but do not abolish, the microvascular permeability effects of bradykinin, a putative asthma mediator.
Relaxin‐induced increased coronary flow through stimulation of nitric oxide production
Authors: Bani‐Sacchi Tatiana , Bigazzi Mario , Bani Daniele , Mannaioni Pier Francesco , Masini Emanuela
Published: July 2012

Relaxin (RLX) is a multifunctional hormone which, besides its role in pregnancy and parturition, has also been shown to influence the cardiovascular system. In this study, we investigated the effect of RLX on coronary flow of rat and guinea‐pig hearts, isolated and perfused in a Langendorff apparatus. RLX was either added to the perfusion fluid at a concentration of 5 × 10−9 M for a 20‐min perfusion, or given as a bolus into the aortic cannula at concentrations of 10−9 M, 5 × 10−9 M and 10−8 M dissolved in 1 ml of perfusion fluid.

RLX, given either for a 20‐min perfusion or as a bolus in the aortic cannula to guinea‐pig and rat isolated hearts, increased the coronary flow and the amount of nitrite, a stable end‐product of nitric oxide (NO) metabolism, that appeared in the perfusates in a concentration‐dependent fashion.

The increase in coronary flow and in nitrite in the perfusates induced by RLX was significantly reduced by pretreatment with the nitric oxide synthase (NOS) inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 10−4 M).

The effects of RLX on coronary flow and nitrite amounts in the perfusates were compared with those induced by the endothelium‐dependent vasodilator agent, acetylcholine (ACh, 10−8 − 10−7 M), and by the endothelium‐independent vasodilator agent, sodium nitroprusside (SNP, 10−7‐10−6M). The results obtained show that RLX is more effective than ACh and SNP in increasing coronary flow.

The results of this study show that RLX increases coronary flow through stimulation of NO production; hence this hormone should be regarded as a novel agent capable of improving myocardial perfusion.
Pharmacological selectivity of the cloned human P2U‐purinoceptor: potent activation by diadenosine tetraphosphate
Authors: Lazarowski Eduardo R. , Watt William C. , Stutts M. Jackson , Boucher Richard C. , Harden T. Kendall
Published: July 2012

The human P2U‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis.

High basal levels of inositol phosphates occurred in P2U‐purinoceptor‐expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes.

UTP, ATP and ATPγS were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 μM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y‐, P2X‐, and P2T‐purinoceptor agonists, 2MeSATP, α,β‐MeATP, and 2MeSADP, respectively.

Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U‐purinoceptor with an EC50 (720 nM) in the range of the most potent P2U‐purinoceptor agonists. AP4A may be a physiologically important activator of P2U‐purinoceptors.
Inhibition of muscarinic receptor‐induced inositol phospholipid hydrolysis by caffeine, β‐adrenoceptors and protein kinase C in intestinal smooth muscle
Authors: Prestwich S.A. , Bolton T.B.
Published: July 2012

The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or 1‐oleoyl‐2‐acetylglycerol (OAG), (protein kinase C (PKC) activators), 2‐methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)‐stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea‐pig.

Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 μm) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]‐inositol (1,4,5) trisphosphate ([3H]‐Ins(1,4,5)P3) isomer occurring 10 s following incubation.

The β‐adrenoceptor agonist, isoprenaline (10 μm) and dibutyryl cyclic AMP (10 μm), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]‐inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline‐induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)‐sensitive G‐protein mechanism.

TPA (1 μm) and OAG (100 μm) a 1,2‐diacylglycerol (DAG) analogue both reduced the CCh‐induced increases in [3H]‐inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP.

D600 (10 μm), which blocks voltage‐dependent Ca2+ channels, also reduced the CCh‐stimulated levels of [3H]‐inositol phosphates suggesting that some of the agonist‐induced increases are due to a potentiating effect of Ca2+ entering the cell.

Caffeine (0.5–30 mm) significantly inhibited both the basal and CCh‐induced increases in all the [3H]‐inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mm.

Incubation with thapsigargin (1 μm) and ryanodine (10 μm) had no effect on either basal or CCh‐induced inositol phospholipid hydrolysis or cyclic AMP levels.

The results indicate a reciprocal inhibition by β‐adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR‐induced rises in inositol phosphate levels. Diacylglycerols may exert a negative feed‐back inhibition on inositol phosphate production.
Interaction between methotrexate and indomethacin on a human normal haemopoietic cell line
Authors: Hollingsworth S.J. , Shankley N.P. , Anderson E.M. , Bennett A.
Published: July 2012

The interaction between methotrexate and indomethacin has been examined, at a physiological folate concentration (20 nm), on a human normal lymphoblast‐like cell line (RPMI 1788) in vitro.

Indomethacin (1 μg ml−1) increased the reduction of lymphoblast growth caused by methotrexate (10–80 ng ml−1).

Indomethacin (0.1 and 1 μg ml−1) potentiated the cytotoxicity of methotrexate (20 and 40 ng ml−1) after 4 days in culture.

Indomethacin (0.4 μg ml−1) reduced the accumulation of tritium in lymphoblasts incubated with [3H]‐methotrexate after 30 min; therefore initial drug accumulation was not responsible for the potentiation seen after 4 days.

If indomethacin increases the killing of human cancer cells by methotrexate in vivo, with a smaller potentiation on lymphoblasts, this combination may be beneficial in treating human malignancy.
Differentiation by pyridoxal 5‐phosphate, PPADS and IsoPPADS between responses mediated by UTP and those evoked by α,β‐methylene‐ATP on rat sympathetic ganglia
Authors: Connolly Gerald P.
Published: July 2012

The effect of pyridoxal 5‐phosphate, and the 2′,4′ and 2′,5′‐disulphonic acid isomers of 6‐azophenyl‐pyridoxal 5‐phosphate (PPADS and IsoPPADS respectively) on depolarization of the rat superior cervical ganglion evoked by α,β‐methylene‐adenosine 5′‐triphosphate (α,β‐Me‐ATP) and uridine 5′‐triphosphate (UTP) were determined by a grease‐gap recording technique.

Pyridoxal 5‐phosphate (10–100 μm) and PPADS (10–100 μm) enhanced UTP‐ and depressed α,β‐Me‐ATP‐evoked depolarizations but did not significantly alter depolarizations evoked by potassium or hyperpolarizations evoked by adenosine. IsoPPADS (10 μm) depressed α,β‐Me‐ATP‐evoked depolarizations but did not alter depolarizations evoked by UTP. Depolarizations evoked by muscarine were depressed by IsoPPADS but not by pyridoxal 5‐phosphate.

It is concluded that pyridoxal 5‐phosphate, PPADS and IsoPPADS are antagonists at P2X‐purinoceptors but not at the receptors that mediate UTP‐evoked depolarization of the rat superior cervical ganglion. These observations substantiate the recent proposal that the rat superior cervical ganglia possess distinct receptors for purine and pyrimidine 5′‐nucleotides, i.e. P2X‐purinoceptors and pyrimidinoceptors respectively.
Endothelium‐dependent relaxation and hyperpolarization evoked by bradykinin in canine coronary arteries: enhancement by exercise‐training
Authors: Mombouli Jean‐Vivien , Nakashima Mikio , Hamra Mary , Vanhoutte Paul M.
Published: July 2012

Kinins, which are produced locally in arterial walls, stimulate the release of endothelium‐derived vasodilator substances. Therefore, they may participate in the metabolic adaptation to chronic exercise that occurs in the coronary circulation. Experiments were designed to compare the reactivity to bradykinin in coronary arteries isolated from sedentary and exercised‐trained dogs (for 8–10 weeks).

The organ chambers used in this study were designed for measurement of isometric tension and cell membrane potential with glass microelectrodes. Rings of canine isolated coronary arteries with endothelium were suspended in the organ chambers filled with modified Krebs‐Ringer bicarbonate solution (37°C, gassed with 5% CO2 in 95 O2), and were all treated with indomethacin to prevent interference from prostaglandins.

Bradykinin evoked concentration‐dependent relaxations of the coronary arteries. However, the kinin was significantly less potent in relaxing coronary arteries from the sedentary dogs than those from the trained ones.

In the presence of NG‐nitro‐L‐arginine (an inhibitor of nitric oxide synthases), concentration‐relaxation curves to bradykinin were shifted to the right in both types of preparations. Nonetheless, the peptide was still significantly more potent in arteries from exercise‐trained animals.

In the electrophysiological experiments, concentration‐hyperpolarization curves to bradykinin obtained in arteries from sedentary dogs were also significantly to the right of those in vessels from exercise‐trained animals. Thus, in arteries from exercised animals, bradykinin more potently evoked the release of both nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF).

The angiotensin converting enzyme (ACE)‐inhibitor, perindoprilat, shifted to the left the concentration‐relaxation curves to bradykinin obtained under control conditions and in the presence of NG‐nitro‐L‐arginine. The concentration‐hyperpolarization curves to bradykinin were also shifted to the left by perindoprilat. The shift induced by the ACE‐inhibitor in either type of preparation was not significantly different.

These findings demonstrate that exercise‐training augments the sensitivity of the coronary artery of the dog to the endothelium‐dependent effects of bradykinin. This sensitization to bradykinin may reflect an increased role of both NO and EDHF, and is not the consequence of differences in ACE activity in the receptor compartment.
Modulation of agonist‐induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP‐dependent mechanisms
Authors: Hoiting B.H. , Meurs H. , Schuiling M. , Kuipers R. , Elzinga C.R.S. , Zaagsma J.
Published: July 2012

The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8‐bromoadenosine 3′:5′‐cyclic monophosphate (8‐Br‐cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells.

Isoprenaline (pD2 (‐log10 EC50) = 6.32±0.24) and forskolin (pD2 = 5.6±0.05) enhanced cyclic AMP levels in a concentration‐dependent fashion in these cells, while methacholine (pD2 = 5.64±0.12) and histamine (pD2 = 4.90±0.04) caused a concentration‐related increase in [3H]‐inositol phosphates (IP) accumulation in the presence of 10 mM LiCl.

Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0±0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 μm.

Both methacholine and histamine induced a fast (max. in 0.5‐2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+.

Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 μm) significantly attenuated both the Ca2+‐transient and the sustained phase generated at equipotent IP producing concentrations of 1 μm methacholine and 100 μm histamine (approx. 40% of maximal methacholine‐induced IP response), but did not affect changes in [Ca2+]i induced by 100 μm methacholine (95.2±3.5% of maximal methacholine‐induced IP response).

Significant correlations were found between the isoprenaline‐induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist.

These data indicate that (a) cyclic AMP‐dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in β‐adrenoceptor‐mediated relaxation of methacholine‐ and histamine‐induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist‐induced contraction to β‐adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.
Inhibitory effect of noradrenaline uptake inhibitors on contractions of rat aortic smooth muscle
Authors: Huang Yu
Published: July 2012

The effects of noradrenaline (NA) uptake inhibitors on contractions induced by NA, high K+, and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in rat isolated aorta were investigated.

Protriptyline (0.3 μm) and amitriptyline (0.3 μm) produced an approximately parallel shift to the right in the dose‐response curve to NA. Protriptyline (>0.3 μm), amitriptyline (>0.3 μm) and xylamine (0.01‐1 μm) significantly reduced the maximal contractile response to NA. The IC50 values for inhibition of the contractile response to 3 μm NA were 1.58 μm for xylamine, 1.70 μm for amitriptyline and 2.57 μm for protriptyline.

Protriptyline and amitriptyline dose‐dependently inhibited the high K+ (60 mM)‐induced contraction (IC50 = 0.69 μm for protriptyline and IC50 = 3.15 μm for amitriptyline). In contrast, xylamine did not affect the high K(+‐induced contraction.

Protriptyline and amitriptyline dose‐dependently inhibited TPA (1 μm)‐induced contraction in calcium‐free solution; xylamine (up to 30 μm) was without effect. Staurosporine (10 nM) completely inhibited the TPA‐ and NA‐induced contraction.

Protriptyline (3 μm) and amitriptyline (3 μm) caused about 54% and 60% inhibition, respectively, of aortic contractions caused by endothelin‐1 (10 nM) in the absence of endothelium. Xylamine (10 μm) was without effect.

Inhibitory effects of NA uptake inhibitors on contractions were independent of the presence of endothelium and were unaffected by the K+ channel blockers, tetraethylammonium ions (up to 3 mM) and glibenclamide (up to 30 μm).

These results indicate that tricyclic antidepressant drugs such as protriptyline and amitriptyline could act as both postsynaptic adrenoceptor antagonists and direct inhibitors of muscle contraction; whereas, xylamine, a structurally distinct NA uptake blocker might principally exert its action only at α‐adrenoceptors on rat aortic smooth muscle.
The effects of anticonvulsants on 4‐aminopyridine‐induced bursting: in vitro studies on rat peripheral nerve and dorsal roots
Authors: Lees G.
Published: July 2012

Aminopyridines have been used as beneficial symptomatic treatments in a variety of neurological conditions including multiple sclerosis but have been associated with considerable toxicity in the form of abdominal pain, paraesthesias and (rarely) convulsions.

Extracellular and intracellular recording was used to characterize action potentials in rat sciatic nerves and dorsal roots and the effects of 4‐aminopyridine (4‐AP).

In sciatic nerve trunks, 1 mM 4‐AP produced pronounced after potentials at room temperature secondary to regenerative firing in affected axons (5–10 spikes per stimulus). At physiological temperatures, after potentials (2–3 spikes) were greatly attenuated in peripheral axons.

4‐AP evoked more pronounced and prolonged after discharges in isolated dorsal roots at 37°C (3–5.5 mV and 80–100 ms succeeded by a smaller inhibitory/depolarizing voltage shift) which were used to assess the effects of anticonvulsants.

Phenytoin, carbamazepine and lamotrigine dose‐dependently reduced the area of 4‐AP‐induced after potentials at 100 and 320 μm but the amplitude of compound action potentials (evoked at 0.5 Hz) was depressed in parallel.

The tonic block of sensory action potentials by all three drugs (at 320 μm) was enhanced by high frequency stimulation (5–500 Hz).

The lack of selectivity of these frequency‐dependent Na+ channel blockers for burst firing, compared to low‐frequency spikes, is discussed in contrast to their effects on 4‐AP‐induced seizures and paroxysmal activity in CNS tissue (which is associated with large and sustained depolarizing plateau potentials).

In conclusion, these in vitro results confirm the marked sensitivity of sensory axons to 4‐AP (the presumptive basis for paraesthesias). Burst firing was not preferenitally impaired at relatively high concentrations suggesting that anticonvulsants will not overcome the toxic peripheral actions of 4‐AP in neurological patients.
A comparative study of the reversal by different α2‐adrenoceptor antagonists of the central sympatho‐inhibitory effect of clonidine
Authors: Vayssettes‐Courchay Christine , Bouysset Françoise , Cordi Alex A. , Laubie Michel , Verbeuren Tony J.
Published: July 2012

The recovery of the clonidine‐induced hypotension, bradycardia and sympatho‐inhibition produced by several putative α2‐adrenoceptor antagonists was investigated in pentobarbitone anaesthetized rats. The activity of four substances containing an imidazoline structure: idazoxan, methoxy‐idazoxan, BRL44408 and atipamezole was compared with the effect of fluparoxan, yohimbine and L‐657,743; in addition the effect of the α1‐adrenoceptor antagonist, prazosin, was also studied.

Prazosin (0.03‐1 mg kg−1, i.v.) failed to alter the sympatho‐inhibitory and hypotensive effects of clonidine (10 μg kg−1, i.v.). L‐657,743 (0.01‐1 mg kg−1, i.v.) induced a recovery of blood pressure, heart rate and renal sympathetic nerve activity. Yohimbine (0.03‐3 mg kg−1, i.v.) completely reversed the sympatho‐inhibitory effect of clonidine but did not alter its hypotensive effect.

The four imidazoline drugs: idazoxan (10–300 μg kg−1, i.v.), methoxy‐idazoxan (1–100 μ kg−1, i.v.), BRL44408 (0.1–3 mg kg−1, i.v.) and atipamezole (0.03‐1 mg kg−1, i.v.) and fluparoxan (10–300 μ kg−1, i.v.) reversed the clonidine‐induced hypotension but produced only a partial recovery of the renal sympathetic nerve activity and of the heart rate. After pretreatment with prazosin (0.1 mg kg−1, i.v.), the recovery of the sympathetic nerve activity elicited by these compounds was significantly higher. In hexamethonium (10 mg kg−1, i.v.) pretreated rats, these five drugs induced dose‐related hypertension which was reduced by pretreatment with prazosin (0.1 mg kg−1, i.v.).

Our results indicate that the putative α2‐adrenoceptor antagonists idazoxan, methoxy‐idazoxan, BRL44408, atipamezole and fluparoxan also have a peripheral hypertensive effect which is mediated through activation of vascular α1‐adrenoceptors; this property of the compounds may be partly responsible for the reversal of the hypotensive action of clonidine. Considering the structure and the affinities of the drugs tested, our data indirectly suggest that α2A‐adrenoceptors may be implicated in the central sympatho‐inhibitory effects of clonidine.
Increase by the ORL1 receptor (opioid receptor‐like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones
Authors: Vaughan Christopher W. , Christie MacDonald J.
Published: July 2012

The actions of the endogenous ORL1‐receptor (opioid receptor‐like1) ligand, nociceptin, on the membrane properties of rat dorsal raphe nucleus neurones were examined by use of whole‐cell patch clamp recording in brain slices. Nociceptin produced an outward current in all neurones tested, with an EC50 of 12 ± 2 nM. Dynorphin A (100 nM to 1 μm) produced little outward current. Outward currents reversed polarity near the predicted K+ equilibrium potential in both 2.5 mM (measured/predicted = −105 mV/ −104 mV) and 6.5 mM (measured/predicted = −80 mV/ −77 mV) extracellular K+. The conductance increase was larger between −120 and −130 mV than between −70 and −80 mV, demonstrating that the nociceptin‐induced K current was due to an increased inwardly rectifying K conductance. The outward current produced by nociceptin was similar to, and occluded by, high concentrations of baclofen, demonstrating actions on the same population of K channels. Naloxone (1 μm) failed to inhibit outward currents produced by nociceptin. These results are consistent with the reported high density of ORL1 receptor mRNA in dorsal raphe nucleus and with inhibitory actions of nociceptin in cells expressing ORL1.
Myocardial protection after monophosphoryl lipid A: studies of delayed anti‐ischaemic properties in rabbit heart
Authors: Baxter G. F. , Goodwin R.W. , Wright M.J. , Kerac M. , Heads R. J. , Yellon D.M.
Published: July 2012

Monophosphoryl lipid A (MLA) is a non‐pyrogenic derivative of Salmonella lipopolysaccharide. Administration of this agent at high doses to rats and at low doses to dogs was previously shown to confer marked protection against ischaemia‐reperfusion 24 h later, although the cellular mechanisms of this delayed protection are obscure. We hypothesized that MLA pretreatment causes the induction of the 70 kDa cytoprotective stress protein HSP70i in the myocardium. If this were the case, protection against ischaemia‐reperfusion injury would be observed both in vitro and in vivo.

Rabbits were pretreated with MLA 0.035 mg kg−1, i.v. or vehicle solution. For the in vitro study, hearts were isolated 24 h later and Langendorff‐perfused with Krebs‐Henseleit buffer at 37°C. Global ischaemia was induced for 20 min followed by 120 min reperfusion. Recovery of post‐ischaemic left ventricular function and lactate dehydrogenase efflux was similar in MLA and vehicle pretreated hearts and there was no significant difference in the percentage of infarction of the left ventricle determined by triphenyltetrazolium staining (MLA 22.4±5.2%, vehicle 24.8±5.1%).

When 30 min regional ischaemia and 120 min reperfusion was instituted in pentobarbitone‐anaesthetized rabbits 24 h after pretreatment with MLA or vehicle, the percentage infarction within the risk zone was reduced from 42.6±5.7% in vehicle pretreated animals to 19.6±4.4% in MLA pretreated animals (P<0.01).

Determination of myocardial HSP70i content by Western blot analysis showed that MLA treatment did not increase HSP70i immunoreactivity.

We conclude that MLA at this dose confers protection only against ischaemia‐reperfusion injury in vivo and that this protection is not related to induction of HSP70i. Because protection was observed only in vivo it seems possible that the delayed protection conferred by MLA is mediated by effects on humoral or blood‐borne factors.
Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]‐CGS 21680 binding
Authors: Varani Katia , Gessi Stefania , Dalpiaz Alessandro , Borea Pier Andrea
Published: July 2012

The binding properties of human platelet A2a adenosine receptors, assayed with the A2a‐selective agonist, [3H]‐2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680), are masked by a non‐receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation.

The present paper describes the binding characteristics of the selective A2a agonist, [3H]‐CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies.

Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg−1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]‐CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments.

Thermodynamic data indicated that [3H]‐CGS 21680 binding to the purified receptor is totally entropy‐driven in agreement with results obtained in rat striatal A2a adenosine receptors.

It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets.
Protection of the aged substantia nigra of the rat against oxidative damage by (−)−deprenyl
Authors: de la Cruz Cristina P. , Revilla Elisa , Steffen Vera , Rodríguez‐Gómez Jose A. , Cano Josefina , Machado Alberto
Published: July 2012

We have studied the effect of (−)−deprenyl on the oxidative damage that the rat substantia nigra suffers during aging.

(−)−Deprenyl (2 mg kg−1, three times a week) administered for two months, beginning at 22 months of age, produced a significant increase in tyrosine hydroxylase (TH) activity (2.67±0.40 and 3.64±0.38 nmol mg−1 protein h−1 in untreated aged rats and treated aged rats respectively, P<0.05) and in TH amount (0.072±0.012 and 0.128±0.38 absorbance 405 nm in untreated aged and treated aged rats respectively, P<0.05).

The proteins of aged rat substantia nigra showed a significant decrease of carbonyl groups in treated animals compared with saline‐injected control rats (136.2±21.8 and 71.5±13.2 c.p.m. μg−1 protein in untreated aged and treated aged rats respectively, P<0.05).

The carbonyl groups measured in TH enzyme showed a statistically significant decrease (42.3%) after (−)−deprenyl treatment (471.4±73.0 and 271.9±50.00 c.p.m. in untreated aged and treated aged rats respectively, P<0.001).

All these results suggest that oxidative damage produced during aging is prevented by (−)−deprenyl treatment and could explain the effect of this drug in Parkinson's disease (PD) and other degenerative diseases such as Alzheimer's disease.
Endogenous inotropic factor‐induced endothelium‐dependent relaxation of vascular smooth muscle
Authors: Han Chao , Khatter Jagdish C.
Published: July 2012

Possible contractile or relaxation effects of an endogenous inotropic factor (EIF) isolated and purified from porcine heart left ventricle were examined in rat isolated aortic ring preparations.

EIF induced a dose‐dependent relaxation of the rat isolated aortic ring preparation pre‐contracted with 0.4 μm phenylephrine (PE); 200 μl (in 5 ml bath) of EIF caused relaxation of aortic rings by as much as 67.4±4.5%. In another set of experiments, in the presence of 100 μl EIF, the PE concentration‐response contractile curve shifted to the right, the maximal contractile force was reduced by as much as 32.8% and the EC50 of PE increased from 0.2 to 0.3 μm.

The relaxation effect of EIF was demonstrated to be endothelium‐dependent. Additional experiments demonstrated that EIF‐induced relaxation in an isolated aortic ring could be inhibited by 2 μm NG‐nitro‐L‐arginine methyl ester, a nitric oxide synthase inhibitor, suggesting the involvement of nitric oxide in EIF‐induced relaxation of the muscle.

Atropine (0.2 μm) or indomethacin (10 μm) had no significant effect on EIF‐induced relaxation.

These data suggest that EIF, a novel endogenous inotrope from porcine myocardium, also acts as an endothelium‐dependent vasodilator substance mediating relaxation in the rat isolated aorta mainly by release of nitric oxide. The possibility of EIF acting through muscarinic receptor and the involvement of prostacyclin were excluded.
Comparison of desulfatohirudin (REVASC) and heparin as adjuncts to thrombolytic therapy with reteplase in a canine model of coronary thrombosis
Authors: Martin Ulrich , Dörge Liesel , Fischer Stephan
Published: July 2012

We compared the direct thrombin inhibitor, desulfatohirudin (REVASC) and the indirect thrombin inhibitor, heparin, as adjuncts to thrombolytic therapy with reteplase in a canine model of coronary artery thrombosis.

Reteplase (BM 06.022) is a recombinant unglycosylated variant of human tissue‐type plasminogen activator. Thrombus formation in anaesthetized open chest dogs was induced by electrical injury. Left circumflex coronary artery blood flow was monitored for 210 min with an electromagnetic flow probe. Twenty eight dogs were randomized to receive i.v. heparin (120 iu kg−1 bolus plus 80 iu kg−1 per h) or i.v. hirudin (2.0 mg kg−1 bolus plus 2.0 mg kg−1 per h) 10 min before thrombolysis preceded by i.v. acetylsalicyclic acid (20 mg kg−1) 5 min prior to anticoagulation. Every dog received an i.v. double bolus injection of 0.14±0.14 u kg−1 (= 0.24±0.24 mg kg−1) reteplase, 30 min apart, 1 h after thrombus formation.

At comparable reperfusion rates (12 out of 12 vs. 15 out of 16 dogs), hirudin enhanced time to reperfusion (14.3±1.4 vs. 23.2±3.4 min; P<0.05) and completely prevented reocclusion after reperfusion in contrast to heparin (0 out of 11 vs. 7 out of 11 dogs; P<0.05). Coronary blood flow quality was improved by hirudin as shown by a higher maximum blood flow after reperfusion (130±14.3 vs. 83±9.3% of baseline; P<0.05), a higher blood flow level at 20, 30, 40, and 50 min after onset of thrombolysis (P<0.05) and a longer cumulative patency time (195±1.7 vs. 166±12 min; P<0.05). Activated partial thromboplastin time and buccal mucosa bleeding time were prolonged (P<0.05) by either anticoagulant, but did not differ significantly between groups.

The direct thrombin inhibitor, desulfatohirudin, enhanced thrombolysis, prevented reocclusion and increased blood flow as compared with the indirect thrombin inhibitor, heparin, when investigated at one dose level each and used in conjunction with reteplase.
Receptors for kinins in the human isolated umbilical vein
Authors: Gobeil F. , Pheng L.H. , Badini I. , Nguyen‐Le X.K. , Pizard A. , Rizzi A. , Blouin D. , Regoli D.
Published: July 2012

The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK.

The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) ≥BK (pD2 8.60) > >[Aib7]BK (pD2 6.38) > >desArg9BK and LysdesArg9BK (inactive).

Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive.

Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4–6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists.

When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60)>desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive.

The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK.

The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea‐pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N‐terminal Lys.
Regional haemodynamic effects of antagonists of angiotensin II, endothelin and adrenoceptors in conscious, vasopressin‐deficient, genetically hypertensive rats
Authors: Gardiner S.M. , March J.E. , Kemp P.A. , Bennett T.
Published: July 2012

Male, vasopressin‐deficient, normotensive (DI/N) and hypertensive (DI/H) rats were chronically instrumented (all surgery under sodium methohexitone anaesthesia) to allow assessment of resting haemodynamic status and responses to antagonism of AT1‐receptors (Experiment 1), ETA‐ and ETB‐ receptors (Experiment 2) or adrenoceptors (Experiment 3).

Before any treatment, mean arterial blood pressure (MAP) was higher, and hindquarters vascular conductance was consistently lower in all groups of DI/H rats than in DI/N rats.

In Experiment 1, losartan (10 mg kg−1 i.v.), an AT1‐receptor antagonist, was given 5 h after s.c. injection of saline, (DI/N, n = 8; DI/H, n = 8) or hyperoncotic polyethylene glycol, (DI/N, n = 9; DI/H, n = 9) to induce isosmotic hypovolaemia. In the volume‐replete state, losartan caused similar small falls in MAP in the two groups (maximum Δ MAP; DI/N, −9±2; DI/H, −15±5 mmHg), but the mesenteric and hindquarters vasodilatations were greater in DI/N rats. In the volume‐depleted state the effects of losartan were augmented (Δ MAP; DI/N, −32±3; DI/H. −31±3 mmHg), but its vasodilator effects were still greater in DI/N than in DI/H rats.

In Experiment 2, infusion of the ETA‐ETB‐receptor antagonist, SB 209670 (600 μg kg−1 h−1; DI/N, n = 8; DI/H, n = 9), had haemodynamic effects that were not different from those during saline infusion in DI/N (n = 7) and DI/H rats (n = 8).

In Experiment 3, sequential administration of the β2‐adrenoceptor antagonist, ICI 118551 (0.2 mg kg−1 bolus, 0.1 mg kg−1 h−1 infusion), the α2‐adrenoceptor antagonist, idazoxan (0.75 mg kg−1 bolus, 1 mg kg−1 h−1 infusion), and losartan (10 mg kg−1 bolus) had only slight haemodynamic effects in DI/N (n = 8) and DI/H (n = 9) rats. Subsequent administration of the α1‐adrenoceptor antagonist, prazosin (0.5 mg kg−1 bolus, 0.8 mg kg−1 h−1 infusion) caused marked hypotension, although MAP was still higher in DI/H (95±4 mmHg) than in DI/N (75±4 mmHg) rats. However, in this circumstance there were no significant differences between renal, or mesenteric, or hindquarters vascular conductances in the two groups.

The results indicate that the hypertension and hindquarters vasoconstriction in DI/H rats is not dependent on All or endothelin. Moreover, the relative elevation in MAP in DI/H persists in the presence of antagonism of β2, α2‐ and α1‐adrenoceptors, in spite of no significant difference in regional vascular conductances.
The mechanism of relaxation induced by atrial natriuretic peptide in the porcine renal artery
Authors: Seguchi Hiroshi , Nishimura Junji , Toyofuku Kazuki , Kobayashi Sei , Kumazawa Joichi , Kanaide Hideo
Published: July 2012

The mechanisms underlying the relaxation of the porcine renal artery induced by atrial natriuretic peptide (ANP) were investigated, using front‐surface fluorimetry with fura‐2 and receptor‐coupled permeabilization by α‐toxin.

ANP decreased the cytosolic Ca2+ concentration ([Ca2+]i) and tension during the contraction induced by a high external K+ solution, in a concentration‐dependent manner. This ANP‐induced decrease in [Ca2+]i during the contraction induced by high K+ solution was composed of two phases, an initial rapid phase, followed by a maintenance phase. The initial rapid decrease in [Ca2+]i, but not the maintained decrease in [Ca2+]i, was inhibited when the tissue was treated with thapsigargin, a selective Ca2+ pump inhibitor of the sarcoplasmic reticulum. When the tissues were treated with thapsigargin and external Ca2+ was replaced by Ba2+, which cannot be transported by the Ca2+ pump, ANP did not induce a decrease in [Ba2+]i, even though the elevation of tension induced by Ba2+ was strongly inhibited.

In the absence of extracellular Ca2+, ANP inhibited the release of Ca2+ from the intracellular store induced by noradrenaline (NA).

The [Ca2+]i (abscissa scale)‐tension (ordinate scale) relationship observed during the contraction induced by various concentrations of high external K+ solution was shifted downwards by the addition of 10−8 m ANP, indicating that, at any given [Ca2+]i, the tension generated by high K+ solution was considerably inhibited by the additon of 10‐8 m ANP. The [Ca2+]i‐tension curve of the contraction obtained by the cumulative application of external Ca2+ (0–3.75 mM) during depolarization with 118 mM K+ solution was shifted to the left by 3 × 10−7 m NA. This NA‐induced [Ca2+]i‐tension relationship was shifted to the right by 10−8 m ANP, indicating that the ANP‐induced reduction of Ca2+‐sensitivity operates during the contraction induced by NA.

In α‐toxin‐permeabilized preparations, ANP induced relaxation of tissues precontracted with a mixture of 3 × 10−7 m Ca2+, 10−5 m guanosine 5′‐triphosphate (GTP) and 10−6 m NA. Thus a component of ANP‐induced relaxation took place by way of a reduction in the Ca2+ sensitivity of the myofilaments, independent of changes in [Ca2+]i.

These results indicate that ANP induces relaxation of the porcine renal artery by: (1) reducing [Ca2+]i mainly via the activation of the Ca2+ pumps located on the sarcoplasmic reticulum and sarcolemma, as well as via inhibition of agoinist‐induced release of Ca2+ from the intracellular store; and (2) decreasing the Ca2+‐sensitivity of the contractile elements.
Actions of long chain alcohols on GABAa and glutamate receptors: relation to in vivo effects
Authors: Dildy‐Mayfield J.E. , Mihic S.J. , Liu Y. , Deitrich R.A. , Harris R.A
Published: July 2012

The effects of n‐alcohols on GABAa and glutamate receptor systems were examined, and in vitro effectiveness was compared with in vivo effects in mice and tadpoles. We expressed GABAa, NMDA, AMPA, or kainate receptors in Xenopus oocytes and examined the actions of n‐alcohols on receptor function using two‐electrode voltage clamp recording.

The function of GABAa receptors composed of α1β1 or α1β1γ21 subunits was potentiated by all of the n‐alcohols studied (butanol‐dodecanol).

In contrast to GABAA receptors, glutamate receptors expressed from mouse cortical mRNA or from cRNAs encoding AMPA (GluR3)‐ or kainate (GluR6)‐selective subunits were much less sensitive to longer chain alcohols. In general, octanol and decanol were either without effect or high concentrations were required to produce inhibition.

In contrast to the lack of behavioural effects by long chain alcohols reported previously, decanol produced loss of righting reflex in short‐ and long‐sleep mice, indicating that the in vivo effects of decanol may be due in part to actions at GABAa receptors. Furthermore, butanol, hexanol, octanol, and decanol produce similar potentiation of GABAa receptor function at concentrations required to cause loss of righting reflex in tadpoles, an in vivo model where alcohol distribution is not a compromising factor.

Thus, the in vivo effects of long chain alcohols are not likely to be due to their actions on NMDA, AMPA, or kainate receptors, but may be due instead to potentiation of GABAA receptor function.