bluuebunny/crossref_metadata_2025 huggingface dataset:
Relaxation of guinea‐pig trachea by sodium nitroprusside: cyclic GMP and nitric oxide not involved
Authors: Sadeghi‐Hashjin Gudarz , Folkerts Gert , Henricks Paul A.J. , van de Loo Peet G.F. , Dik Ilse E.M. , Nijkamp Frans P.
Published: July 2012

Sodium nitroprusside (SNP) completely relaxed the guinea‐pig isolated, perfused trachea in a concentration‐dependent manner. Although SNP was less potent by about 2 orders of magnitude, its maximal effect was 25% higher compared to isoprenaline.

SNP (3.2 μm) increased cyclic GMP levels by 300% and relaxed guinea‐pig isolated, perfused trachea by 54%. The SNP‐induced relaxations of the preparations were not affected by the guanylate cyclase inhibitor, methylene blue. Moreover, zaprinast, a cyclic GMP‐specific phosphodiesterase inhibitor which was supposed to enhance SNP‐induced relaxations, decreased the maximal relaxation by 22% (P < 0.001).

In contrast, 8Br‐cyclic GMP (10 μm) increased the cyclic GMP levels by 1100% without inducing a marked relaxation.

SNP (10 μm) and S‐nitroso‐N‐acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 μm), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (< 1 pmol 100 μl−1), but a substantial NO release by SNAP (560 pmol 100 μl−1).

It is concluded that the SNP‐induced tracheal relaxations are probably not mediated by cyclic GMP and NO.
Drug‐induced in vitro inhibition of neutrophil‐endothelial cell adhesion
Authors: Pellegatta Fabio , Lu Yan , Radaelli Antonella , Zocchi Maria Raffaella , Ferrero Elisabetta , Chierchia Sergio , Gaja Gianfranco , Ferrero Maria Elena
Published: July 2012

Leukocyte‐endothelial cell interactions play an important role during ischaemia‐reperfusion events. Adhesion molecules are specifically implicated in this interaction process.

Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia‐reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation.

In basal conditions, defibrotide (1000 μg ml−1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% ± 3.6 (P < 0.05), and after endothelial cell stimulation (TNF‐α, 500 u ml−1) or after leukocyte stimulation (fMLP, 10−7 m), it inhibited leukocyte adhesion by 26.5% ± 3.4 and 32.4% ± 1.8, respectively (P < 0.05).

In adhesion blockage experiments, the use of the monoclonal anntibody anti‐CD31 (5 μg ml−1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti‐LFA‐1 (5 μg ml−1) significantly interfered with the effect of defibrotide.

This result was confirmed in NIH/3T3‐ICAM‐1 transfected cells.

We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM‐1/LFA‐1 adhesion system is involved in the defibrotide mechanism of action.
The mechanism of acidic pH‐induced contraction in aortae from SHR and WKY rats enhanced by increasing blood pressure
Authors: Furukawa Ken‐Ichi , Komaba Junji , Sakai Hiroyuki , Ohizumi Yasushi
Published: July 2012

Effect of pH on vascular smooth muscle contraction was analyzed by use of biochemical and pharmacological techniques.

In the aorta isolated from spontaneously hypertensive rats (SHR) decreasing extracellular pH (pHo) caused a rapid acidification of intracellular pH accompanied by a pHo‐dependent increase in tension. The contraction of the SHR aorta was remarkable compared with that of the Wistar Kyoto rat (WKY) aorta.

Removal of NH4Cl caused a transient decrease in intracellular pH followed by a marked increase in tension.

Both contraction and intracellular Ca2+ mobilization induced by acidic pHo were markedly inhibited by removal of extracellular Ca2+, verapamil and adenosine, whereas these were not affected by tetrodotoxin or Gd3+, a stretch‐activated cation channel blocker. Furthermore, cromakalim (a K+ channel opener) inhibited acidic pHo‐induced contraction (APIC).

Acidic pHo induced a depolarization of cultured smooth muscle cells from SHR aorta.

Blood pressure elevated with increasing age of WKY and SHR accompanied by an increase in APIC. Feeding WKY with NG‐nitro‐L‐arginine, an inhibitor of nitric oxide synthases caused a marked elevation of their blood pressure followed by an increase in APIC.

These results suggest that APIC is caused by Ca2+ influx mediated through the activation of voltage‐sensitive Ca2+ channels mainly due to acidic pHo‐induced depolarization of the plasma membrane of smooth muscle cells. It is also suggested that APIC is strengthened by the elevation of blood pressure.
Activation of potassium currents by inhibitors of calcium‐activated chloride conductance in rabbit portal vein smooth muscle cells
Authors: Toma C. , Greenwood I.A. , Helliwell R.M. , Large W.A.
Published: July 2012

The conventional whole‐cell recording technique was used to study the effects of the chloride channel inhibitors ethacrynic acid, anthracene‐9‐carboxylic acid (A‐9‐C) and indanyloxyacetic acid (IAA) on membrane currents in rabbit portal vein smooth muscle cells at a holding potential of 0 mV.

Using a pipette solution that contained 1 × 10−4 m 1, 2‐bis (2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) and a normal bathing solution the addition of ethacrynic acid (2 × 10−4 m to 1 × 10−3 m) inhibited spontaneous transient outward currents (STOCs) and evoked a concentration‐dependent current at a holding potential of 0 mV. A similar current was activated by IAA (5 × 10−4 m to 1 × 10−3 m) but not by A‐9‐C (1–5 × 10−3 m) at a holding potential of 0 mV.

The amplitude of the current evoked by ethacrynic acid and IAA was linearly related to potential between −30 and 0 mV and displayed outward rectification at positive potentials. The current induced by A‐9‐C was evident only at potentials positive to +20 mV.

Glibenclamide (1 × 10−5 m) abolished the current evoked by ethacrynic acid and IAA at potentials negative to +10 mV and partially inhibited the current positive to +10 mV. The glibenclamide‐insensitive current at positive potentials was completely inhibited by 1 × 10−3 m TEA. The A‐9‐C‐evoked current was insensitive to glibenclamide and abolished by 1 × 10−3 m TEA.

The glibenclamide‐sensitive current activated by ethacrynic acid was not sustained and declined to control levels in the continued presence of ethacrynic acid. However, the outwardly rectifying current recorded at +50 mV was well maintained over the same period.

Outwardly rectifying currents evoked by ethacrynic acid and A‐9‐C were observed with a pipette solution containing 1 × 10−2 m BAPTA in cells bathed in Ca‐free extracellular solution containing 5 × 10−4 m BAPTA and 1 × 10−5 m cyclopiazonic acid.

It is concluded that all three chloride‐channel blockers activated an outwardly rectifying, TEA‐sensitive current. Moreover, ethacrynic acid and IAA evoked an additional glibenclamide‐sensitive current which was present at all potentials between −30 and +50 mV.
Endothelins‐induce cyclicAMP formation in the guinea‐pig trachea through an ETA receptor‐ and cyclooxygenase‐dependent mechanism
Authors: EL‐Mowafy Abdalla M. , Abou‐Mohamed Gamal A.
Published: July 2012

The non‐selective endothelin agonist, endothelin‐1 (ET‐1), and the selective ETB receptor agonist, sarafotoxin‐S6c (SRTX‐c), contracted guinea‐pig isolated trachea in a concentration‐dependent manner. The EC50 value for ET‐1 (11 ± 2.1 nM) was significantly higher than that of SRTX‐c (3.2 ± 0.21 nM) and the maximal developed tension due to SRTX‐c was 42.8 ± 2.3% higher than that produced by ET‐1 (P < 0.05).

Pretreatment with the ETA antagonist, BQ‐610, appreciably enhanced the developed tension due to ET‐1 but not SRTX‐c. Likewise, the cyclo‐oxygenase inhibitor, indomethacin, markedly potentiated the contractile responses to ET‐1, but not to SRTX‐c. Combining BQ‐610 with indomethacin was not more effective than either of them in augmenting ET‐1‐evoked tension.

ET‐1 significantly increased cyclic AMP formation in the trachea in concentration‐ and time‐dependent manners. A t1/2 value of 4.3 min, an EC50 value of 20 ± 3 nM and a maximal cyclic AMP increment of 124% above the basal level, were obtained for ET‐1. Similarly but less effectively, ET‐3 (0.1 μm) increased cyclic AMP level (35 ± 3.7% compared to 94 ± 7.8% for the same concentration of ET‐1). By contrast, SRTX‐c did not alter the cyclicAMP level when applied in concentrations up to 1 μm.

Pre‐incubation of the trachea with BQ‐610 (1 μm) or indomethacin (1 μm) prevented cyclicAMP formation by either ET‐1 or ET‐3.

The results of the present study indicate a negative regulatory role mediated by the ETA receptor on the ETB‐triggered mechanical response. This effect is likely to be mediated by activation of adenylate cyclase through a cyclo‐oxygenase‐dependent mechanism.
Evidence for two distinct P2‐purinoceptors subserving contraction of the rat anococcygeus smooth muscle
Authors: Najbar Alicja T. , Li C.G. , Rand Michael J.
Published: July 2012

The effects of the P2‐purinoceptor agonists, adenosine 5′‐triphosphate (ATP), α, β‐methylene ATP (α, β‐MeATP), β, γ‐methylene ATP (β, γ‐MeATP), L‐β, γ‐methylene ATP (L‐β, γ‐MeATP), adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), and 2‐methylthio ATP (2‐MeSATP) were investigated on the isometric tension of the rat anococcygeus muscle.

Non‐cumulative additions of ATP (100–1500 μm), α, β‐MeATP (1–300 μm), β, γ‐MeATP (10–300 μm), L‐β, γ‐MeATP (3–100 μm) and ADPβS (1–100 μm) produced concentration‐dependent contractions, whereas 2‐MeSATP (1–100 μm) had no effect. The rank order of potency was α, β‐MeATP > L‐β, γ‐MeATP ≥ ADPβS > β, γ‐MeATP > > ATP > 2‐MeSATP.

Contractions to cumulative additions of ATP, α, β‐MeATP, β, γ‐MeATP and L‐β, γ‐MeATP were subject to desensitization whilst those to ADPβS were unaffected.

Contractions to ATP, α, β‐MeATP, β, γ‐MeATP and ADPβS were abolished by the non‐selective P2X/P2Y‐purinoceptor antagonist, suramin (100 μm). In contrast, contractions to ATP, α, β‐MeATP and β, γ‐MeATP were not affected by the non‐selective P1‐purinoceptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8SPT, 30 μm). Blockade of P2X‐purinoceptors with the selective P2X‐purinoceptor antagonist pyridoxal‐phosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS, 10 μm) or desensitization with L‐β, γ‐MeATP (10 μm) abolished contractions to α, β‐MeATP, but enhanced those to ADPβS. The P2Y‐purinoceptor antagonist, reactive blue 2 (RB2, 100 μm) enhanced contractions to ATP and α, β‐MeATP but abolished those to ADPβS.

Simultaneous addition of α, β‐MeATP and ADPβS produced an additive contraction.

The findings suggest that in the rat anococcygeus, smooth muscle cells are endowed with two distinct P2‐purinoceptors which subserve contractions: a P2X‐purinoceptor activated by ATP and its analogues, and another type of P2‐purinoceptor activated by ADPβS.
Exogenous pulmonary surfactant as a drug delivering agent: influence of antibiotics on surfactant activity
Authors: van ‘t Veen Annemarie , Gommers Diederik , Mouton Johan W. , Kluytmans Jan A.J.W. , Krijt Erik Jan , Lachmann Burkhard
Published: July 2012

It has been proposed to use exogenous pulmonary surfactant as a drug delivery system for antibiotics to the alveolar compartment of the lung. Little, however, is known about interactions between pulmonary surfactant and antimicrobial agents. This study investigated the activity of a bovine pulmonary surfactant after mixture with amphotericin B, amoxicillin, ceftazidime, pentamidine or tobramycin.

Surfactant (1 mg ml−1 in vitro and 40 mg ml−1 in vivo) was mixed with 0.375 mg ml−1 amphotericin B, 50 mg ml−1 amoxicillin, 37.5 mg ml−1 ceftazidime, 1 mg ml−1 pentamidine and 2.5 mg ml−1 tobramycin. Minimal surface tension of 50 μl of the mixtures was measured in vitro by use of the Wilhelmy balance. In vivo surfactant activity was evaluated by its capacity to restore gas exchange in an established rat model for surfactant deficiency.

Surfactant deficiency was induced in ventilated rats by repeated lavage of the lung with warm saline until Pao2 dropped below 80 cmH2O with 100% inspired oxygen at standard ventilation settings. Subsequently an antibiotic‐surfactant mixture, saline, air, or surfactant alone was instilled intratracheally (4 ml kg−1 volume, n = 6 per treatment) and blood gas values were measured 5, 30, 60, 90 and 120 min after instillation.

The results showed that minimal surface tensions of the mixtures were comparable to that of surfactant alone. In vivo Pao2 levels in the animals receiving ceftazidime‐surfactant or pentamidine‐surfactant were unchanged when compared to the surfactant group. Pao2 levels in animals receiving amphotericin B‐surfactant, amoxicillin‐surfactant or tobramycin‐surfactant were significantly decreased compared to the surfactant group. For tobramycin it was further found that Pao2 levels were not affected when 0.2 m NaHCO3 (pH = 8.3) buffer was used for suspending surfactant instead of saline.

It is concluded that some antibiotics affect the in vivo activity of a bovine pulmonary surfactant. Therefore, before using surfactant‐antibiotic mixtures in clinical trials, interactions between the two agents should be carefully evaluated.
Selective and functional 5‐hydroxytryptamine4 receptor antagonism by SB 207266
Authors: Wardle K.A. , Bingham S. , Ellis E.S. , Gaster L.M. , Rushant B. , Smith M.I. , Sanger G.J.
Published: July 2012

The pharmacology of a novel 5‐HT4 receptor antagonist, SB 207266 has been evaluated in vitro in the guinea‐pig distal colon longitudinal muscle myenteric plexus (LMMP) and in vivo in the dog Heidenhain pouch.

SB 207266 is a highly potent antagonist of 5‐HT‐evoked, cholinergically‐mediated contractions in the guinea‐pig distal colon. Low concentrations (0.1–10 nM) produced a parallel shift to the right of the concentration‐effect curve (apparent pA2 10.6±0.1) with no significant effect on the maximum response. With higher concentrations of SB 207266 (30 nM and above) the maximum response to 5‐HT was reduced.

The antagonism seen with SB 207266 cannot be attributed to a non‐selective effect since high concentrations (1 μm) had no effect on cholinergically‐mediated contractions evoked by the nicotinic receptor agonist DMPP in the same preparation.

SB 207266 is not an irreversible antagonist since the effects of the compound were reversible upon washing of the tissue.

In the dog Heidenhain pouch, oral (0.1–100 μg kg−1) and intravenous (0.1–100 μg kg−1) administration of SB 207266 produced a dose‐dependent antagonism of the contractions evoked by a bolus intravenous injection of 5‐HT. An ID50 for SB 207266 of 1.3 μg kg−1 was obtained following i.v. administration and 9.6 μg kg−1 following oral administration.

The antagonistic effects of SB 207266 (0.1–100 μg kg−1) in the dog Heidenhain pouch were long lasting since, following oral administration, the response to 5‐HT was reduced for at least 135 min.

SB 207266 is a highly potent, highly selective and orally active 5‐HT4 receptor antagonist. This compound is the first orally active amide to be identified in this class of antagonists and as such is an important new tool in the evaluation of 5‐HT4 receptor function both in vitro and in vivo.
Intraplantar morphine depresses spinal c‐Fos expression induced by carrageenin inflammation but not by noxious heat
Authors: Honoré Prisca , Buritova Jaroslava , Besson Jean‐Marie
Published: July 2012

We have studied the effects of intraplantar administration of the same doses of morphine on intraplantar carrageenin (6 mg 150 μ***l−1 of saline) and noxious heat (52°C for 15 s) induced spinal c‐Fos expression and inflammation.

Intraplantar carrageenin, in awake rats, induced numerous Fos‐like immunoreactive (Fos‐LI) neurones in the dorsal horn of L4‐L5 lumbar segments of the spinal cord and extensive peripheral oedema. At 1 h 30 min, Fos‐LI neurones were preferentially located in the superficial laminae (74 ± 2%) whereas at 3 h, Fos‐LI neurones were observed both in the superficial (45 ± 2%) and deep (37 ± 1%) laminae of the spinal dorsal horn.

Intraplantar morphine dose‐dependently reduced c‐Fos expression induced 1 h 30 min after carrageenin (r = 0.605, P < 0.02), these effects were completely blocked by intraplantar methiodide naloxone (20 μg) (121 ± 22% of control carrageenin expression). The systemic injection of the highest dose of intraplantar morphine (50 μg) had no significant effect on the number of Fos‐LI neurones (88 ± 9% of control carrageenin expression). None of the drugs influenced unilateral peripheral oedema observed 1 h 30 min after carrageenin.

In the second series of experiments, intraplantar morphine dose‐dependently reduced the number of superficial and deep Fos‐LI neurones induced 3 h after carrageenin (r = 0.794, P < 0.0004 and r = 0.698, P < 0.004, respectively). Furthermore, the effects of the highest dose of intraplantar morphine were completely blocked by co‐administration of intraplantar methiodide naloxone (20 μg).

In addition, intraplantar morphine dose‐dependently reduced the ankle (r = 0.747, P < 0.002) and paw (r = 0.682, P < 0.005) oedema observed 3 h after carrageenin, with the effect of the highest dose of intraplantar morphine being completely blocked by co‐administration of methiodide naloxone (98 ± 4% and 102 ± 8% of control paw and ankle oedema, respectively).

Brief noxious heat stimulation, in urethane anaesthetized rats, induced, 2 h after the stimulation, numerous Fos‐LI neurones in the dorsal horn of L3‐L4 lumbar segments of the spinal cord but no detectable peripheral oedema. Fos‐LI neurones were preferentially located in superficial laminae (94 ± 2%) of the spinal dorsal horn. None of the drugs influenced the noxious heat induced c‐Fos expression.

Such results illustrate that peripheral effects of morphine preferentially occur during inflammatory states and outline the interest of extending clinical investigations of the possible use of local injection of morphine in various inflammatory pain states.
Mechanotransduction through the endothelial cytoskeleton: mediation of flow‐ but not agonist‐induced EDRF release
Authors: Hutcheson Iain R. , Griffith Tudor M.
Published: July 2012

We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of the endothelial microfilament and microtubule cytoskeleton to EDRF release evoked by time‐averaged shear stress and by acetylcholine in rabbit abdominal aorta.

Cytochalasin B (1 μm) and phalloidin (100 nM) were used to depolymerize and stabilize, respectively, F‐actin microfilaments. Colchicine (500 nM) was used to inhibit tubulin dimerization and thus disrupt the microtubule network. Experiments were performed before or 1 h after administration of agents to the donor perfusate or organ bath.

In cascade bioassay studies, time‐averaged shear stress was manipulated with dextran (1–4% w/v, 80,000 MW), to increase perfusate viscosity. EDRF release induced by increased perfusate viscosity was significantly (P < 0.01) attenuated by cytochalasin B, phalloidin and colchicine.

Endothelium‐dependent relaxations to acetylcholine (0.01–30 μm) in cascade bioassay and in isolated aortic ring preparations were unaffected by pretreatment with any of these agents both in terms of their EC50 and maximal responses. Endothelium‐independent relaxations to sodium nitroprusside (0.001–10 μm) were similarly unaffected.

We conclude that the endothelial F‐actin microfilament and microtubule networks are involved in the mechanotransduction pathway for flow‐evoked EDRF release in rabbit abdominal aorta. However, these cytoskeletal elements appear to play no role in acetylcholine‐induced EDRF release in this tissue.
Studies on the pharmacology of the inward transport of L‐DOPA in rat renal tubules
Authors: Pinto‐do‐Ó P.C. , Soares‐da‐Silva P.
Published: July 2012

The accumulation of L‐DOPA in suspensions of renal tubules obtained from male Wistar rats occurred through non‐saturable and saturable mechanisms. The kinetics of the saturable component of L‐DOPA uptake in renal tubules was as follows: Vmax = 46.3 ± 2.8 nmol mg−1 protein h−1 and Km = 114.4 (95% confidence limits; 83.8, 156.2) μm (n = 5). The diffusion constant (in nmol−1) of the non‐saturable component for the accumulation of L‐DOPA was 1.3 (1.1, 1.6; n = 8).

The effect of 2, 4‐dinitrophenol was a marked reduction in the tubular uptake of L‐DOPA, with an IC50 value of 12.1 (4.0, 36.9) μm. Cocaine produced a slight (P = 0.08) decrease (22% reduction at 50 μm) in the tubular uptake of L‐DOPA. Corticosterone produced a considerable inhibitory effect on the uptake of L‐DOPA with an IC50 value of 11.0 (3.6, 33.5) μm. The maximal inhibitory effect of probenecid was a 24% decrease in the uptake of L‐DOPA; however, the more selective organic anion transport inhibitor 4, 4′‐diisothiocyantostilbene‐2, 2′‐disulphonic acid (DIDS) was, found not to affect the tubular uptake of L‐DOPA. The organic cation transport inhibitor, cyanine 863, was found to produce a marked decrease in the uptake of L‐DOPA (IC50 = 2.02 [1.07, 3.79]). The cyanine derivatives 1, 1′‐diethyl‐2, 2′‐cyanine (decynium 22) and 1, 1′‐diethyl‐2, 4′‐cyanine (decynium 24) also potently inhibited L‐DOPA uptake with IC50 values (in μm) of 0.63 (0.39, 1.01) and 0.10 (0.08, 0.13), respectively, both compounds were found to be more potent than cyanine 863.

The inhibitory effect of decynium 24 (0.2 μm) on the tubular uptake of L‐DOPA was dependent on the pH of the incubation medium; at pH = 6.5 the accumulation of L‐DOPA was reduced up to 37 ± 2% of control values, whereas at pH = 7.4 and pH = 8.2 the accumulation of L‐DOPA was reduced by 56 ± 1% and 60 ± 6%, respectively. Cyanine 863 (2 μm), decynium 22 (1 μm) and decynium 24 (0.2 μm) were found to decrease the Vmax values for the saturable component of L‐DOPA uptake without changes in Km values.

It is concluded that the tubular uptake of L‐DOPA might be promoted through a mechanism which is dependent on the activity of the organic cation transport system.
Endogenous calcium channels in human embryonic kidney (HEK293) cells
Authors: Berjukow Stanislav , Döring Frank , Froschmayr Monika , Grabner Manfred , Glossmann Hartmut , Hering Steffen
Published: July 2012

We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr‐HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr‐HEK were characterized and compared with the properties of a heterologously expressed chimeric L‐type calcium channel construct.

ISr‐HEK activated on depolarization to voltages positive of −40 mV. It had transient current kinetics with a time to peak of 16 ± 1.4 ms (n = 7) and an inactivation times constant of 52 ± 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was −19.0 ± 1.5 mV (n = 7) and the voltage for half maximal steady‐state inactivation was −39.7 ± 2.3 mV (n = 7).

Block of ISr‐HEK by the dihydropyridine isradipine was not stereoselective; 1 μm (+) and (−)−isradipine inhibited the current by 30 ± 4% (n = 3) and 29 ± 2% (n = 4) respectively. (+)‐Isradipine and (−)−isradipine (10 μm) inhibited ISr‐HEK by 89 ± 4% (n = 5) and 88 ± 8% (n = 3) respectively. The 7‐bromo substituted (±)‐isradipine (VO2605, 10 μm) which is almost inactive on L‐type calcium channels also inhibited ISr‐HEK (83 ± 9%, n = 3) as was observed for 10 μm (−)−nimodipine (78 ± 6%, n = 5). Interestingly, 10 μm (±)‐Bay K 8644 (n = 5) had no effect on the current. ISr‐HEK was only slightly inhibited by the cone snail toxins ω‐CTx GVIA (1 μm, inhibition by 17 ± 3%, n = 4) and ω‐CTx MVIIC (1 μm, inhibition by 20 ± 3%, n = 4). The funnel web spider toxin ω‐Aga IVA (200 nM) inhibited ISr‐HEK by 19 ± 2%, n = 4).

In cells expressing ISr‐HEK, maximum inward current densities of 0.24 ± 0.03 pA/pF and 0.39 ± 0.7 pA/pF (at a test potential of −10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 ± 0.18 pA/pF or 1.11 ± 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum‐free medium.

Co‐expression of a chimeric L‐type calcium channel construct revealed that ISr‐HEK and L‐type calcium channel currents could be distinguished by their different voltage‐dependencies and current kinetics. The current density after heterologous expression of the L‐type α1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 ± 0.45 pA/pF) than that of ISr‐HEK under the same conditions.
Desensitization of glucagon‐like peptide 1 receptors in insulininsulinsecreting βTC3 cells: role of PKA‐independent mechanisms
Authors: Gromada Jesper , Dissing Steen , Rorsman Patrik
Published: July 2012

The cellular processes involved in the desensitization of the glucagon‐like peptide 1 receptors were investigated by measurements of the glucagon‐like peptide 1(7–36)amide (GLP‐1(7–36)amide)‐induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin‐secreting βTC3 cells.

In the presence of 11.2 mM glucose, stimulation with GLP‐1(7–36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L‐type Ca2+‐channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP‐1(7–36)amide.

The increase in [Ca2+]i evoked by GLP‐1‐(7–36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4–5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31‐8220.

Short pretreatment of the cells with the phorbol ester, 4‐β‐phorbol‐12‐β‐myristate‐13‐α‐acetate (PMA), an activator of PKC, reduced the GLP‐1(7–36)amide‐evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31‐8220.

The ability of GLP‐1(7–36)amide to increase [Ca2+]i disappeared upon pre‐exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP‐1(7–36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP‐1(7–36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i.

Our data suggest that the GLP‐1(7–36)amide‐evoked increase in [Ca2+]i is initiated by Ca2+‐influx though voltage‐dependent and nifedipine‐sensitive L‐type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP‐1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca2+‐dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP‐1 receptors but that other (PKC‐independent) mechanisms also participate in this process.
Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea‐pig vas deferens: an electrophysiological study
Authors: Brock James A. , Cunnane Thomas C.
Published: July 2012

The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea‐pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro.

Bath application of PGE1 (1–100 nM) or PGE2 (0.1–100 nM) concentration‐dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps.

Local application of PGE1 (10–100 nM) or PGE2 (10–30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20–100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse.

In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence.

The postjunctional electrical activity recorded in the guinea‐pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea‐pig vas deferens.
Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco‐2) cells
Authors: Cavet Megan E. , West Mike , Simmons Nicholas L.
Published: July 2012

Human intestinal epithelial Caco‐2 cells have been used to investigate the transepithelial permeation of the cardiac glycoside, digoxin.

Transepithelial basal to apical [3H]‐digoxin flux exceeds apical to basal flux, a net secretion of [3H]‐digoxin being observed. At 200 μm digoxin, net secretory flux (Jnet) was 10.8 ± 0.6 nmol cm−2 h−1. Maximal secretory flux (Jmax) of vinblastine was 1.3 ± 0.1 nmol cm−2 h−1. Cellular uptake of digoxin was different across apical and basal cell boundaries. It was greatest across the basal surface at 1 μm, whereas at 200 μm, apical uptake exceeded basal uptake.

Net secretion of [3H]‐digoxin was subject to inhibition by digitoxin and bufalin but was not inhibited by ouabain, convallatoxin, and strophanthidin (all 100 μm). Inhibition was due to both a decrease in Jb‐a and an increase in Ja‐b. Uptake of [3H]‐digoxin at the apical surface was increased by digitoxin and bufalin. All cardiac glycosides decreased [3H]‐digoxin uptake at the basal cell surface (except for 100 μm digitoxin).

The competitive P‐glycoprotein inhibitors, verapamil (100 μm), nifedipine (50 μm) and vinblastine (50 μm) all abolished net secretion of [3H]‐digoxin due to both a decrease in Jb‐a and an increase in Ja‐b. Cellular accumulation of [3H]‐digoxin was also increased across both the apical and basal cell surfaces. 1‐Chloro‐2,4,‐dinitrobenzene (10 μm), a substrate for glutathione‐S‐transferase and subsequent ATP‐dependent glutathione‐S‐conjugate secretion, failed to inhibit net secretion of [3H]‐digoxin. The increase in absorptive permeability Pa‐b (= Ja‐b/Ca) and cellular [3H]‐digoxin uptake upon P‐glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP‐dependent extrusion at the apical surface.

A model for [3H]‐digoxin secretion by the intestinal epithelium is likely to involve both diffusional uptake and Na+‐K+ pump‐mediated endocytosis, followed by active extrusion at the apical membrane.
Evidence for a P2‐purinoceptor mediating vasoconstriction by UTP, ATP and related nucleotides in the isolated pulmonary vascular bed of the rat
Authors: Rubino Annalisa , Burnstock Geoffrey
Published: July 2012

The vasoconstrictor effects of uridine 5′‐triphosphate (UTP), uridine 5′‐diphosphate (UDP), uridine 5′‐monophosphate (UMP) and uridine were tested in the isolated pulmonary vascular bed of the rat. Comparison was made with the effects of adenine nucleotides, adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP) and with adenosine. The effect of P2x‐purinoceptor desensitization and blockade was compared on the vascular responses to uracil and adenine nucleotides.

At doses ranging from 10−8 to 10−5 mol, UTP elicited dose‐dependent vasoconstriction. UDP was equiactive to UTP, while UMP and uridine did not show vasomotor activity. Similarly, ATP showed dose‐related vasoconstrictor activity. ADP was less potent than ATP in eliciting vasoconstriction, while AMP was active only at the higher doses tested and adenosine was ineffective.

Vasoconstriction was produced by ATP analogues with the following order of potency: α,β‐methylene ATP > ATPγS > β,γ‐methylene ATP > 2‐methylthio ATP ≥ ATP.

Desensitization of P2x‐purinoceptors by the selective agonist α,β‐methylene ATP did not modify the vasoconstrictor activity of UTP and UDP and only partially reduced vasoconstrictor responses to ATP, while it abolished vascular responses to α,β‐methylene ATP itself.

The antagonists of P2‐purinoceptors, suramin and pyridoxalphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS), did not affect vascular responses to UTP and UDP, but reduced vasoconstriction evoked by β,γ‐methylene ATP and ATP by about 70 and 30%, respectively.

This study demonstrates that uracil nucleotides, UTP and UDP, elicit vasoconstriction in the rat pulmonary vascular bed. In addition to confirming the presence of classical P2x‐purinoceptors, these results also suggest the presence of a distinct purinoceptor subtype which mediates UTP‐ and ATP‐evoked vasoconstriction in the rat pulmonary circulation.
Human brain somatostatin release from isolated cortical nerve endings and its modulation through GABAB receptors
Authors: Bonanno Giambattista , Gemignani Anita , Schmid Giovanna , Severi Paolo , Cavazzani Paolo , Raiteri Maurizio
Published: July 2012

The release of somatostatin‐like immunoreactivity (SRIF‐LI) in the human brain was studied in synaptosomal preparations from fresh neocortical specimens obtained from patients undergoing neurosurgery to remove deeply sited tumours.

The basal outflow of SRIF‐LI from superfused synaptosomes was increased about 3 fold during exposure to a depolarizing medium containing 15 mM KCl. The K+‐evoked overflow of SRIF‐LI was almost totally dependent on the presence of Ca2+ in the superfusion medium.

The GABAB receptor agonist, (—)‐baclofen (0.3–100 μm), inhibited the overflow of SRIF‐LI in a concentration‐dependent manner (EC50 = 1.84 ± 0.20 μm; maximal effect: about 50%). The novel GABAB receptor ligand, 3‐aminopropyl(difluoromethyl)phosphinic acid (CGP 47656) mimicked (—)‐baclofen in inhibiting the SRIF‐LI overflow (EC50 = 3.06 ± 0.52 μm; maximal effect: about 50%), whereas the GABAA receptor agonist, muscimol, was ineffective up to 100 μm.

The inhibition by 10 μm (—)‐baclofen of the K+‐evoked SRIF‐LI overflow was concentration‐dependently prevented by two selective GABAB receptor antagonists, 3‐amino‐propyl (diethoxymethyl)‐phosphinic acid (CGP 35348) (IC50 = 24.40 ± 2.52 μm) and [3‐[[(3,4‐dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) (IC50 = 0.06 ± 0.005 μm).

The inhibition of SRIF‐LI overflow caused by 10 μm CGP 47656 was abolished by 1 μm CGP 52432.

When human synaptosomes were labelled with [3H]‐GABA and depolarized in superfusion with 15 mM KCl, the inhibition by 10 μm (—)‐baclofen of the depolarization‐evoked [3H]‐GABA overflow was largely prevented by 10 μm CGP 47656 which therefore behaved as an autoreceptor antagonist.

In conclusion: (a) the characteristics of SRIF‐LI release from synaptosomal preparations of human neocortex are compatible with a neuronal origin; (b) the nerve terminals releasing the neuropeptide possess inhibitory receptors of the GABAB type; (c) these receptors differ pharmacologically from the GABAB autoreceptors present on human neocortex nerve terminals since the latter have been shown to be CGP 35348‐insensitive but can be blocked by CGP 47656.
B1 bradykinin receptors and sensory neurones
Authors: Davis C.L. , Naeem S. , Phagoo S.B. , Campbell E.A. , Urban L. , Burgess G.M.
Published: July 2012

The location of the B1 bradykinin receptors involved in inflammatory hyperalgesia was investigated.

No specific binding of the B1 bradykinin receptor ligand [3H]‐des‐Arg10‐kallidin was detected in primary cultures of rat dorsal root ganglion neurones, even after treatment with interleukin‐1β (100 iu ml−1).

In dorsal root ganglion neurones, activation of B2 bradykinin receptors stimulated polyphosphoinositidase C. In contrast, B1 bradykinin receptor agonists (des‐Arg9‐bradykinin up to 10 μm and des‐Arg10‐kallidin up to 1 μm) failed to activate polyphosphoinositidase C, even in neurones that had been treated with interleukin‐1β (100 iu ml−1), prostaglandin E2 (1 μm) or prostaglandin I2 (1 μm).

Dorsal root ganglion neurones removed from rats (both neonatal and 14 days old) that had been pretreated with inflammatory mediators (Freund's complete adjuvant, or carrageenan) failed to respond to B1 bradykinin receptor selective agonists (des‐Arg9‐bradykinin up to 10 μm and des‐Arg10‐kallidin up to 1 μm).

Bradykinin (25 nM to 300 nM) evoked ventral root responses when applied to peripheral receptive fields or central terminals of primary afferents in the neonatal rat spinal cord and tail preparation. In contrast, des‐Arg9‐bradykinin (50 nM to 500 nM) failed to evoke ventral root depolarizations in either control rats or in animals that developed inflammation following ultraviolet irradiation of the tail skin.

The results of the present study imply that the B1 bradykinin receptors that contribute to hypersensitivity in models of persistent inflammatory hyperalgesia are located on cells other than sensory neurones where they may be responsible for releasing mediators that sensitize or activate the nociceptors.
The involvement of the endothelium in the relaxation of the leopard frog (Rana pipiens) aorta in response to acetylcholine
Authors: Knight Gillian E. , Burnstock Geoffrey
Published: July 2012

The vasodilator response to acetylcholine (ACh) was investigated in the aortic arches of the leopard frog (Rana pipiens).

With adrenaline pre‐constricted preparations, both ACh and sodium nitroprusside (SNP) caused concentration‐dependent relaxations. Damage to the endothelial layer abolished relaxations to ACh, or reduced them greatly, but had no effect on vasodilatation to SNP.

NG‐Nitro‐L‐arginine methyl ester (L‐NAME; 1–100 μm) concentration‐dependently inhibited relaxations in response to ACh, but had no effect on the ability of SNP to induce vasodilatation.

L‐Arginine (L‐Arg; 100–200 times the concentration of L‐NAME) failed to reverse the inhibitory effect of L‐NAME (1–100 μm) apart from one isolated instance.

In summary, this study has shown endothelium‐dependent vasodilatation to ACh in an amphibian blood vessel that appears to be mediated via nitric oxide (NO). The response to ACh differs from many mammalian preparations in that the inhibitory effect of L‐NAME could not be overcome by L‐Arg, in addition to L‐NAME itself having no direct effect upon the tone of the vessel.
Modulation by locally produced luminal angiotensin II of proximal tubular sodium reabsorption via an AT1 receptor
Authors: Hiranyachattada Siriphun , Harris Peter J.
Published: July 2012

The concentration of angiotensin II reported in proximal tubular fluid in anaesthetized rats is considerably higher than in plasma, indicating secretion of this peptide into the tubular lumen. Shrinking split‐drop micropuncture was used to examine the effect of endogenous angiotensin on sodium and water absorption in the proximal convoluted tubule. Addition of losartan, a nonpeptide AT1 receptor blocker, to intratubular fluid increased fluid uptake by 15.7 ± 3.9% (10−5 m) and 24.7 ± 9.4% (10−4 m) whereas the AT2 inhibitor, PD123319 had no effect. We conclude that angiotensin II is secreted into proximal tubular fluid and, in the anaesthetized rat, is maintained at a concentration that inhibits sodium and water transport via AT1 receptors.
Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices
Authors: Nicol Beverley , Lambert David G. , Rowbotham David J. , Smart Darren , McKnight Alexander T.
Published: July 2012

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K+‐stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 ± 14% and was naloxone (10 μm)‐insensitive excluding an activation of μ, δ and κ opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.
The effect of S‐(+)‐boldine on the α1‐adrenoceptor of the guinea‐pig aorta
Authors: Chuliá Susana , Moreau Joëlle , Naline Emmanuel , Noguera M. Antonia , Ivorra M. Dolores , D'Ocón M. Pilar , Advenier Charles
Published: July 2012

The cardiovascular activity of S‐(+)‐boldine, an aporphine alkaloid structurally related to papaverine, was determined. The work includes functional studies on guinea‐pig isolated aorta contracted with noradrenaline, caffeine, KCl or Ca2+, and on guinea‐pig trachea contracted with acetylcholine or histamine.

S‐(+)‐boldine inhibited in a concentration‐dependent manner the contractile reponse evoked by noradrenaline (10 μm) in guinea‐pig aorta (IC50 = 1.4 ± 0.2 μm) while the KCl depolarizing solution (60 mM)‐ or the Ca2+ (1 mM)‐induced contractions were only partially affected by boldine up to 300 μm. In contrast, papaverine relaxed noradrenaline (NA), KCl or Ca2+ induced contractions showing similar IC50 values in all cases. S‐(+)‐boldine had a greater potency on the contraction elicited by NA whereas papaverine acted in a non‐selective manner.

S‐(+)‐boldine was found to be an α1‐adrenoceptor blocking agent in guinea‐pig aorta as revealed by its competitive antagonism of noradrenaline‐induced vasoconstriction (pA2 = 5.64 ± 0.08), and its potency was compared with that of prazosin (pA2 = 8.56 ± 0.24), a known potent α1‐adrenoceptor antagonist. In contrast, papaverine caused rightward shifts of the NA concentration‐response curves with depression of maximal response indicating that it acts as a non‐competitive antagonist.

Contraction of guinea‐pig aorta induced by caffeine (60 mM) in a Ca2+‐containing Krebs solution was not affected by a 60 min incubation period with different doses of S‐(+)‐boldine (1–300 μm). Papaverine inhibited partially this caffeine‐induced contraction at the maximal dose used (100 μm).

Inositol phosphates formation induced by noradrenaline (10 μm) in guinea‐pig thoracic aorta was inhibited by S‐(+)‐boldine (30 μm but not by papaverine (10 μm).

Contractions of guinea‐pig trachea caused by acetylcholine (100 μm) or histamine (10 μm) were not modified by S‐(+)‐boldine (0.1–100 μm).

These results provide evidence that S‐(+)‐boldine, an aporphine alkaloid, has interesting properties as an α1‐adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the α1‐adrenoceptor present in the guinea pig aorta.
Enhancement of homomeric glycine receptor function by longchain alcohols and anaesthetics
Authors: Mascia Maria Paola , Machu Tina K. , Harris R. Adron
Published: July 2012

The effects of n‐alcohols (ethanol to dodecanol) and anaesthetics on strychnine‐sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric α1 or α2 glycine receptor subunits, with the two electrode voltage‐clamp recording technique.

The glycine‐induced chloride conductance of homomeric α glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric α1 and α2 receptors were potentiated similarly by the n‐alcohols, except that low concentrations of ethanol produced greater potentiation with α1, as previously reported.

Increasing the n‐alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to EC50s for producing loss of righting reflex in tadpoles. However, dodecanol was no more potent than decanol, and only modest potentiation (30–60%) was obtained with dodecanol, in contrast to marked (150–200%) potentiation with the other alcohols. Thus, a ‘cut‐off’ occurred at about dodecanol.

Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric α1 glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective.

Two novel cyclobutane compounds were tested; the anaesthetic compound (1‐chloro‐1,2,2‐trifluorocyclobutane) from 0.5 to 5 mM potentiated the action of glycine in a concentration‐dependent manner; however, the non‐anaesthetic analogue (1,2‐dichloro‐hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 μm) predicted to be anaesthetic, based on the lipid solubility of this compound.

These results suggest that the α subunits of strychnine‐sensitive glycine receptors contain sites of action for n‐alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not for ketamine and etomidate. Potentiation of glycine receptor function may contribute to the anaesthetic action of n‐alcohols and volatile agents.
The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells
Authors: Piatt Kathleen P. , Zwartjes Raymond E. , Bristow David R.
Published: July 2012

After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM γ‐aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor al, a6 and β2,3 subunit protein expression and α1 and α6 subunit steady‐state mRNA levels, was examined using Western blotting and reverse transcriptase‐polymerase chain reaction (RT‐PCR), respectively.

GABA exposure for 2 days decreased α1 (35 ± 10%, mean ± s.e.mean), β2,3 (21±9%) and α6 (28 ± 10%) subunit protein expression compared to control levels. The GABA‐mediated reduction in al subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 μm).

GABA exposure for 8 days increased α1 (26 ± 10%, mean ± s.e.mean) and β2,3 (56 ± 23%) subunit protein expression over control levels, whereas α6 subunit protein expression remained below control levels (by 38 ± 10%). However, after 10 days GABA exposure, a6 subunit protein expression was also increased over control levels by 65 ± 29% (mean ± s.e.mean).

GABA exposure did not change the α1 or α6 subunit steady‐state mRNA levels over an 8 day period, nor did it alter the expression of cyclophilin mRNA over 1–8 days.

These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi‐phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post‐transcriptional and/or post‐translational processes.