bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of verapamil on intimal thickening and vascular reactivity in the collared carotid artery of the rabbit
Authors: Üstünes Levent , Yasa Mukadder , Kerry Zeliha , Özdemir Necmettin , Berkan Tayfun , Erhan Yildiz , Özer Asli
Published: July 2012

. Intimal thickening is a common site for atherosclerosis. Therefore, we investigated whether the calcium entry blocker verapamil (10 mg kg−1 body weight day−1, s.c.) can retard intimal thickening and changes in vascular reactivity induced by a non‐occlusive, silicone collar positioned around the left carotid artery of rabbits. The contralateral carotid artery was sham‐operated and served as a control.

. Verapamil and placebo (saline 0.1 ml kg−1 day−1, s.c.) treatments were initiated 7 days before placing the collar and lasted 3 weeks. Thereafter, segments were cut from collared and sham‐treated arteries for histology and isometric tension recording.

. The intima/media (I/M) ratio increased after 14 days of collar treatment, but intimal thickening was not inhibited by verapamil (I/M ratio placebo 0.31 ± 0.07, verapamil 0.32 ± 0.09).

. The collar decreased the capacity to develop force, as indicated by the response to a supramaximal concentration of KC1, decreased the sensitivity (pD2) to acetylcholine (ACh) and phenylephrine (Phe), but increased the sensitivity to 5‐hydroxytryptamine (5‐HT).

. Although verapamil did not affect intimal thickening, it normalized the hypersensitivity to 5‐HT in collared arteries.

. The contraction to the supramaximal concentration of KC1 was not affected by verapamil. Verapamil decreased the Emax of ACh, but this was only seen in collar‐treated arteries. Verapamil also decreased the sensitivity to ACh and Phe, in both sham‐ and collar‐treated arteries.

. We conclude that verapamil, without preventing thickening of the intima, can modify collar‐induced changes in vascular reactivity.
Role of protein kinase C in desensitization of spinal δ‐opioid‐mediated antinociception in the mouse
Authors: Narita Minoru , Mizoguchi Hirokazu , Kampine John P. , Tseng Leon F.
Published: July 2012

. Receptor phosphorylation and down‐regulation by protein kinases may be a key event initiating desensitization. The present studies were designed to investigate the effect of a potent protein kinase C (PKC) activator, phorbol 12,13‐dibutyrate (PDBu), on antinociception induced by intrathecal (i.t.) administration of a selective δ‐opioid receptor agonist [D‐Ala2]deltorphin II in the male ICR mouse and on the specific binding of [3H]‐[D‐Ser2, Leu5]enkephalin‐Thr6 (DSLET), a δ‐opioid receptor ligand, in the crude synaptic membrane of the spinal cord.

. Intrathecal (i.t.) pretreatment with PDBu at low doses, which injected alone did not affect the basal tail‐flick latency, dose‐dependently attenuated the antinociception induced by i.t. administration of [D‐Ala2]deltorphin II. The attenuation of i.t.‐administered [D‐Ala2] deltorphin II‐induced antinociception by PDBu was reversed in a dose‐dependent manner by i.t. concomitant pretreatment with a specific PKC inhibitor, calphostin C.

. In the binding experiment, incubation of the crude synaptic membrane of the spinal cord for 2 h at 25°C with PDBu (0.03 to 10 μm) caused a dose‐dependent inhibition of the [3H]‐DSLET binding. Scatchard analysis of [3H]‐DSLET binding revealed that PDBu at 10 μm displayed a 30.7% reduction in the number of [3H]‐DSLET binding sites with no significant change in affinity, compared with the non‐treatment control, indicating that the activation of membrane‐bound PKC by PDBu causes a decrease in the number of specific δ‐opioid agonist binding sites.

. An i.t. injection of [D‐Ala2]deltorphin II produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D‐Ala2]deltorphin II. Concomitant pretreatment with calphostin C markedly prevented the development of acute tolerance to the i.t.‐administered [D‐Ala2]deltorphin II‐induced antinociception. On the other hand, a highly selective protein kinase A (PKA) inhibitor, KT5720, did not have any effect on the development of acute tolerance to [D‐Ala2]deltorphin II antinociception.

. These findings suggest that a loss of specific δ‐agonist binding by the activation of PKC by PDBu is involved in the PDBu‐induced antinociceptive unresponsiveness to δ‐opioid receptor agonist in the mouse spinal cord. Based on the acute tolerance studies, we propose that PKC, but not PKA, plays an important role in the process of homologous desensitization of the spinal δ‐opioid receptor‐mediated antinociception.
Relevance of p‐glycoprotein for the enteral absorption of cyclosporin A: in vitro‐in vivo correlation
Authors: Fricker G. , Drewe J. , Huwyler J. , Gutmann H. , Beglinger C.
Published: July 2012

. The interaction of cyclosporin A (CyA) with p‐glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco‐2 cells and an intubation study in healthy volunteers.

. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco‐2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 μm. At higher concentrations permeation increased over‐proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 μl min−1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 μm and a Jmax of 6.5 picomol min−1 per filter.

. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomcyin and a non‐immunosuppressive CyA‐derivative. All compounds inhibit p‐glycoprotein‐mediated transport processes. Basolateral to apical permeation of CyA showed a dose‐dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco‐2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A.

. Western blot analysis of Caco‐2 cells with the monoclonal antibody C219 confirmed the presence of p‐glycoprotein in the used cell system.

. When the absorption of CyA in the gastrointestinal (G***I)‐tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in absorption exhibited a marked correlation (r = 0.994) to the expression of mRNA for p‐glycoprotein over the G**I‐tract (stomach < jejunum < colon).

. All data provide evidence that CyA is a substrate of p‐glycoprotein in the G***I‐tract, which might explain the local differences and the high variability in cyclosporin absorption found in vivo.
The cardiovascular and renal functional responses to the 5‐HT1A receptor agonist flesinoxan in two rat models of hypertension
Authors: Chamienia Andrzej L. , Johns Edward J.
Published: July 2012

This study investigated the importance of renal sympathetic nerves in regulating sodium and water excretion from the kidneys of stroke prone spontaneously hypertensive and 2K1C Goldblatt hypertensive rats anaesthetized with chloralose/urethane (17.5/300 mg initially and supplemented at regular intervals), and prepared for measurement of renal function.

In stroke prone spontaneously hypertensive rats, flesinoxan, 30–1000 μg kg−1, i.v., caused graded reductions in blood pressure and heart rate of 74±5 mmHg and 63±9 beats min−1, respectively at the highest dose (P<0.001). Renal blood flow did not change at any dose of drug while glomerular filtration rate fell by some 20% (P<0.001) at the highest dose of drug, absolute and fractional sodium excretions, approximately doubled at 100 μg kg−1, and thereafter fell to below the baseline level at 1000 μg kg−1.

This pattern of excretory response was abolished following acute renal denervation when flesinoxan caused dose‐related reductions in urine flow and sodium excretion, similar to that obtained by a mechanical reduction of renal perfusion pressure.

Flesinoxan administration (30–1000 μg kg−1, i.v.) into 2K1C Goldblatt hypertensive rats caused a maximum decrease in blood pressure and heart rate (both P<0.001) of 34±3 mmHg and 20±6 beats min−1 and while renal blood flow and glomerular filtration rate were autoregulated, from 160 to 125 mmHg, there were dose‐related decreases in urine volume and sodium excretion from the clipped and non‐clipped kidneys of approximately 50–60% at the highest dose.

These findings suggest that in the stroke prone spontaneously hypertensive rat the renal nerves importantly control sodium and water reabsorption at the level of the tubules, whereas in 2K1C Goldblatt hypertensive rats, they play a minor role.
The role of endogenous bradykinin in blood pressure homeostasis in spontaneously hypertensive rats
Authors: Holte Hege Ruge , Bjørnstad‐Østensen Anniken , Berg Torill
Published: July 2012

The role of endogenous bradykinin in mean arterial blood pressure (BP) homeostasis was studied in spontaneously hypertensive (SHR) and normotensive (WKY) rats by the use of a bradykinin B2‐receptor antagonist (BKant; Hoe 140, 11.6 μg kg−1) and converting enzyme (kininase II) inhibitor (captopril, 10 mg). To obtain a response to captopril that was induced through inhibition of kinin‐degradation only and not through inhibition of angiotensin II‐formation, the studies were performed on binephrectomized male rats to eliminate the renin‐angiotensin system.

The role of the nitric oxide (NO) and the adrenergic systems were evaluated by the use of NO‐synthase inhibitor (L‐NAME, 0.3 g kg−1) and phentolamine (2 mg kg−1), respectively.

The rats were anaesthetized and pretreated with two injections of vehicle (PBS) or drugs spaced 5 min apart: PBS + PBS; BKant + PBS; PBS + L‐NAME; BKant + L‐NAME; or phentolamine + L‐NAME. All rats were given captopril 15 min later. Time‐control groups were treated with L‐NAME but not captopril.

In WKY rats, captopril did not significantly alter BP in any of the groups. In the SHR‐PBS + PBS group, on the other hand, captopril induced an immediate fall in BP (ΔBP = −23 ± 4 mmHg, P < 0.0017) which was completely blocked by BKant (ΔBP = 2 ± 2 mmHg) (P < 0.0011). L‐NAME did not significantly alter the immediate hypotensive response to captopril but disclosed a later hypertensive reaction. In L‐NAME + BKant‐treated rats, both the hypotensive response and the late hypertension was abolished. In rats treated with phentolamine + L‐NAME, the immediate fall in BP was not different from the controls whereas the late hypertension was absent.

BKant itself had no effect on basal BP in either WKY or SHR even when a 10 times higher dose was tested in a separate set of experiments. This was true also for conscious, nonnephrectomized SHR rats.

It was concluded that endogenous production of bradykinin was demonstrable through kininase II‐inhibition in hypertensive but not in normotensive rats. However, this endogenous bradykinin did not play a role in basal BP homeostasis. The captopril‐induced hypotension depended on kinin but, under the present conditions, not on NO as a mediator. The fall in BP induced a compensatory adrenergic hypertensive response which was revealed when the continuous NO‐synthesis was blocked by L‐NAME.
Photolysis of the novel inotropes EMD 57033 and EMD 57439: evidence that Ca2+sensitization and phosphodiesterase inhibition depend upon the same enantiomeric site
Authors: Lee John A. , Palmer Sue , Kentish Jonathan C.
Published: July 2012

We studied the effects of flash photolysis on the novel enantiomeric cardiac inotropes EMD 57033 (a calcium sensitizer) and EMD 57439 (a phosphodiesterase III inhibitor) in rat isolated ventricular trabeculae.

In skinned trabeculae, EMD 57439 had no effect on force production, consistent with lack of an active cyclic AMP system in this preparation. In contrast, EMD 57033 potentiated force at partial and maximal activation. A single flash of near u.v. light given at partial activation (30–70%) reduced force potentiation by 52.4 ± 5.2%. No effect was produced by flashes in the presence of EMD 57439 or in the absence of either drug.

The time course of relaxation induced by EMD 57033 photolysis was indistinguishable from that obtained on deactivating the muscle by rapidly lowering Ca2+using photolysis of the caged chelator of calcium, diazo‐2.

In intact, twitching trabeculae, EMD 57033 increased diastolic and peak force and slowed relaxation. These effects were simultaneously reduced by a light flash. In these muscles EMD 57439 reduced force, without affecting the twitch time course. These effects were also reduced by a light flash.

The u.v. absorbance spectra of EMD 57033 and EMD 57439 were identical. After photolysis optical density decreased substantially and the peak shifted from 320 nm to 280 nm.

The proton n.m.r. spectra of these compounds were identical. The main change post‐photolysis was a decrease in the proton signal associated with the enantiomeric carbon atom.

This novel manipulation of the molecular structure of EMD 57033 and EMD 57439 within an experiment thus provides direct evidence linking calcium sensitization to a particular molecular structure. The three main effects of the sensitizer on the twitch were simultaneously abolished and may be mechanistically linked. Flash photolysis may be a useful tool for further investigations of the actions of these compounds. In particular, flash photolysis of the sensitizer represents a novel method of rapidly deactivating cardiac muscle.
Differential effects of nitric oxide donors on basal and electrically evoked release of acetylcholine from guinea‐pig myenteric neurones
Authors: Hebeiß Katalina , Kilbinger Heinz
Published: July 2012

The effects of the nitric oxide (NO) donors, 3‐morpholino‐sydnonimine (SIN‐1), S‐nitroso‐N‐acetylpenicillamine (SNAP) and sodium nitroprusside on basal and electrically evoked release of [3H]‐acetylcholine were studied in myenteric plexus longitudinal muscle preparations of the guinea‐pig small intestine preincubated with [3H]‐choline.

The NO donors concentration‐dependently increased basal release of [3H]‐acetylcholine. The increase in release was calcium‐dependent and was prevented in the presence of tetrodotoxin. Superoxide dismutase (150 u ml−1) potentiated the effect of SIN‐1. The selective inhibitor of soluble guanylyl cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐α]quinoxalin‐1‐one (ODQ, 0.01‐1 μm), antagonized the facilitatory effect of SNAP. 8‐Bromo cyclic GMP and the cyclic GMP‐specific phosphodiesterase inhibitor, zaprinast (both 0.1‐1 mM), also enhanced basal [3H]‐acetylcholine release. The effect of 10 μm SNAP was significantly enhanced in the presence of zaprinast.

The NO donors concentration‐dependently inhibited the electrically evoked release of [3H]‐acetylcholine, whereas 8‐bromo cyclic GMP and zaprinast enhanced the evoked release. The inhibition of acetylcholine release by SNAP was not affected by ODQ (0.01‐1 μm).

It is concluded that NO stimulates basal acetylcholine release from myenteric neurones through activation of guanylyl cyclase. In addition, NO inhibits the depolarization evoked release of acetylcholine by a presynaptic mechanism unrelated to cyclic GMP. The data imply that NO is not only an inhibitory transmitter to intestinal smooth muscles but also a modulator of cholinergic neurotransmission in the myenteric plexus.
Involvement of prostaglandins in the down‐regulation of allergic plasma leakage observed in rats undergoing pleural eosinophilia
Authors: Bandeira‐Melo Christianne , Singh Yajaira , Cordeiro Renato S.B. , e Silva Patrícia M.R. , Martins Marco A.
Published: July 2012

Recent evidence has implicated eosinophils in the inhibition of allergen‐induced rat pleurisy, but the mechanism of this negative modulation is not completely understood. This study was undertaken in order to define the potential role of prostaglandins in this phenomenon.

Wistar rats were actively sensitized by subcutaneous injection of a mixture of ovalbumin and Al(OH)3 and challenged with an intrapleural (i.pl.) injection of ovalbumin (12 μg/cavity) 14 days later.

Analysis of the pleural fluid effluent revealed a massive mast cell degranulation and plasma protein extravasation 4 h post‐challenge. We confirmed that concurrently with selective pleural fluid eosinophilia caused by platelet‐activating factor (PAF), the pleural cavity became hyporesponsive to allergen‐induced protein exudation and to the parallel reduction in the number of intact mast cells.

These hyporesponsive animals presented a significant augmentation in the pleural effluent level of prostaglandin E2 (PGE2), which increased with increasing numbers of eosinophils in the pleural cavity. Furthermore, pretreatment with either indomethacin or aspirin failed to modify allergen‐induced exudation but reversed the exudatory hyporesponsiveness associated with eosinophil recruitment.

Allergic exudation was clearly down‐regulated by the following pretreatments: (i) PGE2 (10 μg/cavity, i.pl.) plus rolipram (40 μg/cavity, i.pl.), (ii) misoprostol (200 μg kg−1, p.o.) or (iii) dibutyryl cyclic AMP (80 μg/cavity, i.pl.).

We conclude that prostaglandins may be implicated in the eosinophil‐mediated inhibition of allergic pleurisy, probably acting via cyclic AMP signalling pathway activation.
Characterization of the P2Y‐purinoceptor involved in the ATP‐induced rise in cytosolic Ca2+ concentration in rat ileal myocytes
Authors: Pacaud Pierre , Feolde Erick , Frelin Christian , Loirand Gervaise
Published: July 2012

The P2‐purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca2+ concentration ([Ca2+]i) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques.

The P2‐purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P2Y1‐receptor.

Short applications of the purinoceptor agonists induced a transient rise in [Ca2+]i in an all‐or‐nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2‐methylthioATP = 2‐chloro‐ATP > ADP > ATP, with concentrations giving [Ca2+]i response in 50% of cells ranging between 3 nM and 0.6 μm. The concentration‐response curves to ADP and ATP were shifted to the right by 10 μm pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS).

Although the rise in [Ca2+]i induced by stimulation of the ileal P2Y‐purinoceptor was inhibited by heparin (5 mg ml−1), we were not able to detect stimulation of phospholipase C under conditions (37°C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)‐induced increase in InsP accumulation was suppressed when the agonist was applied at 20°C while a CCh‐induced [Ca2+]i rise similar to that obtained in response to the P2‐purinoceptor agonist was still observed.

Our results indicate that the rat ileal myocytes express a PPADS‐sensitive P2‐purinoceptor similar to the P2Y1‐receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca2+]i by activation of the inositol 1,4,5‐trisphosphate receptor‐channel of the intracellular Ca2+ store, indicating that they couple to phospholipase C.
Effect of selective blockade of endothelin ETB receptors on the liver dysfunction and injury caused by endotoxaemia in the rat
Authors: Ruetten Hartmut , Thiemermann Christoph
Published: July 2012

We investigated the effects of the selective endothelin (ET)A receptor antagonist BQ‐485 and the selective ETB receptor antagonist BQ‐788 on circulatory failure, multiple organ dysfunction syndrome (MODS) and the alterations in acid base balance caused by endotoxaemia in the anaesthetized rat.

Male Wistar rats were anaesthetized (thiopentone sodium; 120 mg kg−1, i.p.) and received a continuous infusion of vehicle (saline, 0.6 ml kg−1h−1, i.v.), BQ‐485 (10 nmol kg−1 min−1, i.v.) or BQ‐788 (10 nmol kg−1 min−1, i.v.). Fifteen min later, animals received a bolus injection of either saline (0.9% NaCl, 1 ml kg−1, i.v.) or E. coli lipopolysaccharide (LPS, 10 mg kg−1, i.v.).

Injection of LPS resulted in a fall in blood pressure from 115 ± 4 mmHg (time 0) to 82 ± 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the pressor responses to noradrenaline (NA, 1 μm kg−1, i.v.). Infusion of BQ‐788 attenuated the delayed hypotension (at 360 min: 100 ± 4 mmHg, n = 7; P < 0.05) and significantly enhanced the pressor responses elicited by NA (at 60 to 240 min). In contrast, treatment of LPS‐rats with BQ‐485 augmented the hypotension (at 360 min), but did not affect the vascular hyporeactivity elicited by endotoxaemia.

Endotoxaemia for 360 min resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), glutamate‐oxalate‐transferase (GOT) and glutamate‐pyruvate‐transferase (GPT) (indicators of hepatocellular injury), and bilirubin and γ‐glutamyl transferase (γGT) (indicators of liver failure) as well as nitrite (indicator of the induction of nitric oxide synthase; iNOS). Treatment of LPS‐rats with BQ‐788, but not with BQ‐485, attenuated the degree of liver injury and failure, while neither BQ‐788 nor BQ‐485 affected the acute renal failure or the induction of iNOS caused by endotoxin.

Endotoxaemia also caused (within 15 min) an acute metabolic acidosis (falls in pH, HCO3− and base excess) which was compensated by hyperventilation (fall in Paco2). Treatment of LPS‐rats with BQ‐788 or BQ‐485 did not affect the metabolic acidosis caused by LPS.

Thus, the selective ETB receptor antagonist BQ‐788 attenuated (i) the delayed hypotension, (ii) the vascular hyporeactivity to NA as well as (iii) the degree of hepatocellular injury and dysfunction caused by endotoxin in the anaesthetized rat. In contrast, the selective ETA receptor antagonist did neither attenuate the circulatory failure nor the liver or renal dysfunction associated with endotoxaemia. We propose that the prevention of the hepatocellular dysfunction and injury caused BQ‐788 in endotoxaemia is due to an improvement in oxygen delivery to the liver secondary to (i) inhibition of pre‐sinusoidal constriction, (ii) inhibition of sinusoidal constriction, and (iii) improvement in perfusion pressure.
A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea‐pig cardiac ventricular cells
Authors: Watano Tomokazu , Kimura Junko , Morita Tominori , Nakanishi Hironori
Published: July 2012

The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea‐pig with the whole‐cell voltage‐clamp technique.

No. 7943 at 0.1–10 μm suppressed the outward Na+/Ca2+ exchange current in a concentration‐dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 μm.

No. 7943 at 5–50 μm suppressed also the inward Na+/Ca2+ exchange current in a concentration‐dependent manner but with a higher IC50 value of approximately 17 μm.

In a concentration‐response curve, No. 7943 raised the KmCa2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/Ca2+ exchange current.

The voltage‐gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50s of approximately 14, 8 and 7 μm, respectively.

In contrast to No. 7943, 3′,4′‐dichlorobenzamil (DCB) at 3–30 μm suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 μm, but did not affect the outward exchange current at these concentrations.

We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.
In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway
Authors: Fedele Ernesto , Jin Ying , Varnier Giorgia , Raiteri Maurizio
Published: July 2012

Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely‐moving adult rats.

Intracerebellar administration of ODQ (1–100 μm) via the microdialysis probe inhibited, in a concentration‐dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 μm ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ.

The cyclic GMP response evoked in the cerebellum by NMDA (200 μm) or by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA; 100 μm) was largely attenuated by 100 μm ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation.

ODQ (100 μm) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S‐nitroso‐N‐acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 μm) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the ‘NO receptor’ in a reversible and competitive manner.

Infusion of ODQ (10, 100 or 300 μm) into the hippocampus of freely‐moving rats diminished the basal extracellular level of cyclic GMP. The maximal inhibition amounted to 50% and was produced by 100 μm ODQ.

The cyclic GMP response observed when 1 mM SNAP was perfused in the hippocampus, similar in percentage terms to that seen in cerebellum, was dramatically reduced during co‐infusion of 100 μm ODQ.

ODQ appears to act in vivo as a selective, reversible and possibly competitive inhibitor of the soluble guanylyl cyclase targeted by NO. This enzyme may generate most (about 80%) of the cyclic GMP found under basal conditions in the extracellular space of the cerebellum. In the hippocampus, about 50% of the basal cyclic GMP does not seem to originate from the ODQ‐sensitive soluble guanylyl cyclase.
Influence of contractile agonists on the mechanism of endothelium‐dependent relaxation in rat isolated mesenteric artery
Authors: Plane Frances , Garland Christopher J.
Published: July 2012

This study demonstrates directly that the relative contribution of nitric oxide (NO) and an NO synthase‐independent repolarization to acetylcholine‐evoked relaxation in rat isolated mesenteric resistance arteries is determined by the processes which mediate pre‐contraction. Noradrenaline‐induced contractions were reversed by acetylcholine via both NO and NO synthase‐independent smooth muscle repolarization. In contrast, reversal of contractions to the thromboxane‐mimetic, U46619, by acetylcholine was entirely mediated by the actions of NO, independently of a change in membrane potential.
Contrast between effects of aminobisphosphonates and non‐aminobisphosphonates on collagen‐induced arthritis in mice
Authors: Nakamura Masanori , Ando Takanobu , Abe Mastaka , Kumagai Katsuo , Endo Yasuo
Published: July 2012

Bisphosphonates (BPs) are inhibitors of bone resorption, and many derivatives have been developed for the treatment of enhanced bone resorption. Aminobisphosphonates (aminoBPs) are particularly potent in this respect. We have shown previously that aminoBPs, such as 4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonic acid (AHBuBP), induce histidine decarboxylase, the enzyme forming histamine, and increase macrophages, granulocytes and osteoclast numbers. Non‐aminoBPs do not show this activity.

In the present study, an additional aminoBP, cycloheptyl‐aminomethylene bisphosphonate (CHAMBP), was shown to have similar properties to AHBuBP suggesting that these actions are common among aminoBPs.

In experiments carried out to determine if aminoBPs affect immune responses, we found that CHAMBP and AHBuBP each exacerbated the arthritis induced in mice by the co‐injection of type II collagen and an adjuvant, a model for rheumatoid arthritis. In contrast, dichloromethylene bisphosphonate (C12MBP), a typical non‐aminoBP, did suppress the arthritis.

On the basis of these results, and those obtained previously, we propose that the exacerbating effects of CHAMBP and AHBuBP may be related to their ability to stimulate the synthesis of histamine and to increase macrophages and granulocytes. Conversely, we propose that the suppressive effect of C12MBP on arthritis is related to its cytotoxic action on macrophages or granulocytes.
Renal effects of TAPP, a highly selective μ‐opioid agonist
Authors: Gutkowska Jolanta , Schiller Peter W.
Published: July 2012

The effect of i.v. administration of TAPP, a highly selective and exclusively peripherally‐acting μ‐opioid receptor agonist, on urine output, urinary sodium, potassium and cyclic GMP, and on plasma immunoreactive atrial natriuretic factor (IR‐ANF) levels was studied in conscious normally hydrated female rats (200–250 g).

TAPP treatment produced a significant dose‐dependent increase of urine output and urinary sodium, potassium and cyclic GMP excretion during the first hour. The highest TAPP dose used (2.5 mg kg−1 body weight) elicited a 10 fold elevation of urine output from 0.23 ± 0.06 ml h−1 to 2.5 ± 0.3 ml h−1 (n = 18) accompanied by augmented sodium [from 17.0 ± 4.7 μEq h−1 to 79 ± 12.7 μEq h−1, n = 18 (P < 0.001)], potassium [from 9.5 ± 2.5 μEq h−1 to 39.4 ± 6.6 μEq h−1, n = 18 (P < 0.005)], and cyclic GMP excretion [from 191 ± 21 pmol h−1 to 1340 ± 322 pmol h−1, n = 18 (P < 0.001)]. Plasma IR‐ANF rose from 22 ± 4 pg ml−1 to 508 ± 22 pg ml−1 (n = 18) (P < 0.001) 5 min after administration of TAPP (2500 μg kg−1).

TAPP lowered systemic blood pressure, also in a dose‐related manner, 1–5 min after injection. This decrease in blood pressure was transient and did not last more than 10 min.

Pretreatment with the opioid antagonist naloxone (0.8 mg per rat) abolished the diuretic, natriuretic and kaliuretic effect of TAPP (250 μg kg−1): urine output dropped from 1.16 ± 0.15 ml h−1, n = 12, to the control value of 0.15 ± 0.06 ml h−1, n = 12 (P < 0.001), sodium excretion fell from 57.5 ± 11 μEq h−1, to 21.3 ± 8.5 μEq h−1, n = 12 (P < 0.001), and potassium excretion decreased from 45.4 ± 9.7 μEq h−1, n = 12, to 16.1 ± 7.0 μEq h−1, (P < 0.001).

Pretreatment with anti‐ANF serum (0.4 ml) abolished the diuretic effect of TAPP: urine output diminished significantly from 1.93 ± 0.28 to 0.88 ± 0.29 ml h−1 (P < 0.01) (n = 6). The TAPP‐induced diuretic action, increased sodium/potassium excretion and elevated urinary cyclic GMP levels were also reversed by anti‐ANF antibodies.

Since TAPP is totally unable to cross the blood‐brain barrier, the ensemble of these observations led to the conclusion that the diuretic, natriuretic, kaliuretic and hypotensive effects produced by this μ‐opioid agonist through interaction with peripheral μ‐opioid receptors occur via ANF release.
Modulation of carbachol‐induced [Ca2+]i oscillations by Ca2+ influx in single intestinal smooth muscle cells
Authors: Komori S. , Iwata M. , Unno T. , Ohashi H.
Published: July 2012

Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 μm), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor‐coupled cationic current (Icat) in guinea‐pig ileal smooth muscle cells by the whole cell patch‐clamp technique.

Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh‐induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations.

Application of nifedipine (1–3 μm) or D600 (2–5 μm) to block the voltage‐gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at −20 mV, but it was without effect in cells held at −60 mV.

Displacement of the holding potential of −20 mV to −60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine‐treated cells, the Icat oscillations varied in frequency voltage‐dependently in a reverse and linear way within the range −80 to 40 mV.

Application of thapsigargin (1 or 2 μm), an inhibitor of Ca2+‐ATPase in the membrane of internal Ca2+ stores, caused CCh‐induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined.

The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh‐induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.
Pyrimidinoceptor‐mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages
Authors: Lin W.W. , Lee Y.T.
Published: July 2012

As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2).

The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP >> ATP, ATPγS, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 μm) were significantly lower than that for ATP and all other analogues tested (> 100 μm).

UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations.

UTP‐, but not ATP‐, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short‐term treatment with phorbol 12‐myristate 13‐acetate (PMA) led to a dose‐dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response.

ATP‐induction of these two transmembrane signal pathways was decreased in high Mg2+‐containing medium but potentiated by the removal of extracellular Mg2+.

Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP.

Both UTP and UDP (0.1–100 μm) induced a sustained increase in [Ca2+]i which lasted for more than 10 min.

Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX‐insensitive G protein, whereas that of cPLA2 is via a PTX‐sensitive G protein‐dependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2. RAW 264.7 cells also possess P2Z purinoceptors which mediate ATP4‐‐induced PLC and PLA2 activation.
Characterization of α1D‐adrenoceptor subtype in rat myocardium, aorta and other tissues
Authors: Deng Xing Fei , Chemtob Sylvain , Varma Daya R.
Published: July 2012

This study was done to characterize the functional role of α1D‐adrenoceptors in rat myocardium, aorta, spleen, vas deferens and prostate by use of the selective antagonist BMY 7378.

BMY 7378 inhibited [3H]‐prazosin binding to aortic membranes with a potency (pKi 9.8 ± 0.40) approximately 100 fold higher than in right ventricular membranes (pKi 7.47 ± 0.11) and approximately 1,000 fold higher than that in plasma membranes of the prostate (pKi 6.62 ± 0.39), vas deferens (pKi 6.67 ± 0.15), salivary gland (pKi 6.46 ± 0.38) and liver (6.58 ± 0.06).

BMY 7378 antagonized the positive inotropic effects of phenylephrine (in the presence of 1 μm propranolol) on right ventricles (pA2 7.0 ± 0.11), left atria (pKB 7.04 ± 0.18) and papillary muscles (pKB 6.9 ± 0.1) and inhibited phenylephrine‐induced increase in inositol phosphates.

BMY 7378 was approximately 100 fold more potent as an antagonist of phenylephrine on aortic strips (pA2 9.0 ± 0.13) than on vas deferens (pKB 7.17 ± 0.08) and spleen (pKB 7.16 ± 0.21); it was ineffective on the prostate.

Chloroethylclonidine suppressed the maximal effects of phenylephrine on spleen; 5‐methylurapidil antagonized the effects of phenylephrine on aortic strips (pA2 7.98 ± 0.08), vas deferens (pKB 8.89 ± 0.07) and prostate (pKB 8.85 ± 0.21).

BMY 7378 caused a dose (0.1–100 nmol kg−1)‐dependent decrease in mean blood pressure of urethane‐anaesthetized rats and its hypotensive efficacy was equal to that of hexamethonium.

The data suggest that α1D‐adrenoceptors play a significant role in rat aorta, a minor role in the heart, vas deferens and spleen and virtually no role in the prostate.
Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein
Authors: Shuba Lesya M. , Asai Tatsuya , Pelzer Siegried , McDonald Terence F.
Published: July 2012

Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP‐dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, were applied to guinea‐pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl− channels.

Myocytes in the standard whole‐cell configuration were pulsed to various potentials and Cl− current (IC1) measured as the difference from control background current. GST (1–500 μm) activated a current that had similar biophysical properties (time‐ and voltage‐independent; Cl−‐dependent reversal potential and outward rectification) as IC1 activated by 5 μm FSK. The EC50 for activation of Cl− conductance (gC1) by GST was approximately 100 μm, and gC1 activated by GST (500 μm) was as large as gC1 activated by maximally‐effective FSK (5 μm). Daidzein, a GST analogue with little effect on PTK, was at least one order less effective than GST.

GST responses were rapidly and reversibly inhibited by 0.1‐1 mM VO4 treatments that had little effect on FSK‐activated IC1.

Niflumic acid (100–200 μm) reversibly depressed GST (100 μm)‐activated gC1 by 55%.

GST (50 μm) strongly incremented current in myocytes with cyclic AMP‐dependent CFTR IC1 already activated by maximally‐effective FSK 5 μm.

Based on these results, and on evidence of a synergistic interaction between GST and FSK, we conclude that inhibition of tyrosine phosphorylation by GST causes an activation of cardiac CFTR that is not mediated by an elevation of cyclic AMP.
The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization
Authors: Vigne Paul , Pacaud Pierre , Urbach Valérie , Feolde Erick , Breittmayer Jean Philippe , Frelin Christian
Published: July 2012

Brain capillary endothelial cells responded to uridine 5′‐triphosphate (UTP) and adenosine 5′‐triphosphate (ATP) by activation of phospholipase C and by large changes in [Ca2+]i. These cells expressed mRNA sequences identical to the sequence of the P2Y2‐purinoceptor of rat pituitaries.

Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) at 100 μm did not prevent UTP and ATP induced accumulations of total [3H]‐inositol (poly)phosphates. It inhibited UTP and ATP induced intracellular Ca2+ mobilization (IC50 = 30 γm) by non competitive mechanism.

PPADS (100 μm) inhibited endothelin‐1 induced accumulation of total [3H]‐inositol (poly)phosphates by less than 20% and prevented most of endothelin‐1 induced intracellular Ca2+ mobilization (IC50 = 30 μm).

PPADS (100 μm) had no action on ionomycin induced intracellular Ca2+ mobilization.

Microinjection of inositol (1,4,5)trisphosphate (InsP3) into Xenopus oocytes induced large Ca2+ activated Cl− currents that were prevented by heparin and by PPADS.

It is concluded that PPADS does not recognize rat P2Y2‐purinoceptors and prevents UTP and ATP induced intracellular Ca2+ mobilization by a non‐specific mechanism that could involve the inhibition of InsP3 channels.
Evidence for mediation of L‐2‐chloropropionic acid‐induced delayed neuronal cell death by activation of a constitutive nitric oxide synthase
Authors: Widdowson P.S. , Farnworth M. , Moore R.B. , Dunn D. , Wyatt I.
Published: July 2012

Delayed neuronal cell death elicited by excess excitatory amino acid concentrations has been strongly implicated in many neurological disorders including head trauma, stroke, motor neurone disease and Huntington's disease. We have used the neurotoxin, L‐2‐chloropropionic acid (L‐CPA) to model cellular events in vivo leading to delayed neuronal cell loss which is confined to the cerebellar cortex and can be prevented by inhibitors of nitric oxide synthase such as NG‐nitro‐L‐arginine methyl ester.

Experiments were performed to determine whether the constitutive nitric oxide synthase (NOS) or inducible form of NOS (iNOS) was responsible for the neuronal cell death. Activation of NOS was confirmed by a 39% increase in cerebellar total nitrate and nitrite concentrations in L‐CPA‐treated brains, as compared to controls (controls = 2.53 ± 0.10; L‐CPA treated = 3.51 ± 0.31 nmol mg−1 protein, P < 0.01 Student's t tests, n = 6, mean ± s.e.mean). Biochemical measurements of total NOS activity were made in homogenates of cerebellum 6 h and 48 h following L‐CPA administration, times at which L‐CPA concentrations are maximal in brain and a time when there is a high proportion of cerebellar granule cell death, respectively. NOS activity as measured by the amount of [3H]‐arginine converted to [3H]‐citrulline, did not reveal any difference between controls (rats dosed with water) and animals dosed with L‐CPA at either 6 or 48 h following dosing. Furthermore the ability of three NOS inhibitors, NG‐nitro‐L‐arginine, 7‐bromo‐3‐nitroindazole and S‐methylisothiourea to block the conversion of [3H]‐citrulline to [3H]‐arginine was identical at 6 and 48 h time points in control and L‐CPA treated rats.

Quantitative autoradiography using [3H]‐NG‐nitro‐L‐arginine was used to measure the relative anatomical distribution and amount of NOS enzyme in the cerebellum of controls and L‐CPA‐treated rats 48 h following dosing. There was no significant alteration in the binding of [3H]‐NG‐nitro‐L‐arginine to granular and molecular layers of the cerebellum of control and L‐CPA‐treated rat brains.

Western blotting using antibodies against the inducible NOS enzyme failed to detect the protein in cerebellums of L‐CPA‐treated rats when measured 48 h after L‐CPA dosing.

In conclusion, the increase in cerebellar nitrate/nitrite concentrations in L‐CPA‐treated rats provides further evidence for activation of NOS in the cerebellum following administration of L‐CPA. The failure to demonstrate an increase in NOS activity at 6 or 48 h in L‐CPA‐treated rats as compared to controls suggests that the source of nitric oxide responsible for the granule cell death must originate from the constitutive NOS enzyme, probably the neuronal form which is highly enriched in the cerebellum. This hypothesis was further substantiated by Western blotting and quantitative autoradiography.
Interactions between nitric oxide and prostanoids in isolated perfused kidneys of the rat
Authors: Ziyyat Abderrahim , Zhang Bei‐Li , Benzoni Daniel
Published: July 2012

The present study was aimed to assess the interaction between nitric oxide (NO) and thromboxane (Tx) A2‐prostaglandin (PG) H2 in single‐pass perfused isolated kidneys of the rat.

Noradrenaline (NA, 63 and 110 nM) dose‐dependently elevated the renal vascular resistance (RVR), the glomerular filtration rate (GFR) and the urinary excretion of sodium (UNaV). Infusion of Nγ‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μm), an inhibitor of NO synthesis, enhanced the effects of NA on RVR and on UNaV, but decreased those on GFR. The TxA2‐PGH2 (TP) receptor blockade by GR32191B (10 μm) attenuated this potentiating effect of L‐NAME.

When renal perfusion pressure was stepwise increased from 90 to 150 mmHg, L‐NAME similarly decreased renal perfusion flow rate and GFR.

The venous excretion of TxB2 and 6‐keto‐PGF1α was increased by L‐NAME in baseline conditions as well as after NA or increasing renal perfusion pressure (RPP).

These results suggest that: (1) TxA2 and PGH2 play an important role in the overall effect of the renal prostanoids, (2) NO strongly interacts with the cyclo‐oxygenase pathway and reduces the prostanoid synthesis in the kidney, and (3) the pressor effect of L‐NAME partly relies upon the vasoconstrictor effect of TxA2 and PGH2.
Regulation of human basophil function by phosphatase inhibitors
Authors: Peirce Matthew J. , Warner Jane A. , Munday Michael R. , Peachell Peter T.
Published: July 2012

Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE‐mediated release of the pre‐formed mediator, histamine from human basophils in a time‐ and dose‐dependent manner. Optimal inhibition (77 ± 4%,P< 0.0001) of histamine release was observed following a 2 h incubation with 1 μmokadaic acid.

Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE‐dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 μmfor okadaic acid and 7.5 μmfor okadaol, whereas okadaone was inactive.

The structurally‐unrelated PP inhibitor, calyculin A, also inhibited IgE‐dependent histamine release from basophils dose‐dependently and was approximately six fold more potent than okadaic acid.

The IgE‐mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC500.3 μm) > okadaol (3 μm) > okadaone (inactive).

Okadaic acid, okadaol and okadaone (all at 3 μm) inhibited the IgE‐mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 ± 9% (P< 0.002), 48 ± 14% (P< 0.05) and 8 ± 7% (P= 0.31), respectively.

Extracts of purified human basophils liberated32P from radiolabeled glycogen phosphorylase and this PP activity was inhibited by 17 ± 3% (P< 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 ± 1% (P< 0.0001) by a higher (5 μm) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 μm) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils.

In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.