bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of 5‐hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes
Authors: Hirst Warren D. , Price Gary W. , Rattray Marcus , Wilkin Graham P. 
Published: August 2006

5‐Hydroxytryptamine (5‐HT) elicited a dose‐dependent stimulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 ± 0.08 (mean ± s.e.mean).
In order to characterize the 5‐HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5‐carboxamidotryptamine (5‐CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5‐CT (pEC50 = 7.81 ±+ 0.09)>5‐methoxytryptamine (5‐MeOT) (pEC50 = 6.86 ±0.36) > 5‐HT (pEC50 = 6.68± 0.08). The following compounds, at concentrations up to 10 μm, did not affect basal cyclic AMP levels 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was meth‐iothepin (p Ki = 7.98 ±0.25)>mesulergine (p Ki = 7.58 ± 0.18)>ritanserin (p Ki = 7.20 ±0.24) >clozapine (p Ki = 7.03±0.19)>mianserin (p Ki= 6.41 ±0.19). The following compounds, at concentrations up to 10 μM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5‐HT7 receptor subtype‐mediated effects.
The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 ± 75% and 572 ± 63% of basal levels for 5‐CT and 5‐HT, respectively) compared to brainstem (274 ± 51% and 318 ± 46%, respectively) and colliculus astrocytes (244 ± 15% and 301±24%, respectively). No significant differences in pEC50 (for either 5‐HT or 5‐CT) values were observed.
Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers specific for the 5‐HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5‐HT6 receptor also amplified a cDNA fragment from the same samples.
From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5‐HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally‐dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5‐HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5‐HT6 and 5‐HT7 receptor messenger RNA.
Furosemide interactions with brain GABAA receptors
Authors: Korpi Esa R , Lüddens Hartmut 
Published: August 2006

The loop diuretic furosemide is known to antagonize the function of γ‐aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits.
Autoradiography with [35S]‐t‐butylbicyclophosphorothionate ([35S]‐TBPS) as a ligand indicated that furosemide (0.1–1 mm) reversed the 5 μm GABA‐induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mm decreased [35S]‐TBPS binding only in a limited number of brain regions, but facilitation of the GABA‐inhibition of the binding was much more widespread.
In well‐washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [35S]‐TBPS binding over basal level in the absence of added GABA. The GABAA antagonist, SR 95531, and the convulsant, Ro 5–4864, blocked this furosemide‐induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [3H]‐SR 95531 and [3H]‐Ro 5–4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA‐antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5–4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed.
In recombinant GABAA receptors, furosemide antagonism of GABA‐inhibition of [35S]‐TBPS binding depended only on the presence of α6 and β2/3 subunits, irrespective of the presence or absence of γ2 or δ subunits.
In α6β3γ2 receptors, clozapine reversed the enhancement of [35S]‐TBPS binding by furosemide in the absence of GABA. However, it failed to affect the GABA‐antagonism of furosemide, suggesting that the enhancement of basal binding and the GABA antagonism might represent two different allosteric actions of furosemide.
In conclusion, the present results indicate that furosemide is a subtype‐selective GABAA antagonist with a mode of action not shared by several other antagonists, which makes furosemide a unique compound for development of potential GABAA receptor subtype‐specific and ‐selective ligands.

British Journal of Pharmacology (1997) 120, 741–748; doi:10.1038/sj.bjp.0700922
Ischaemia‐induced loss or reversal of the effects of the class I antiarrhythmic drugs on vulnerability to fibrillation
Authors: Aupetit Jean‐François , Loufoua‐Moundanga Joseph , Faucon Georges , Timour Quadiri 
Published: August 2006

In the last decade, a number of clinical observations have questioned the efficacy of certain class I antiarrhythmic drugs against ischaemia‐induced ventricular fibrillation. The effects of three drugs of this class, disopyramide (Ia), lignocaine (Ib) and flecainide (Ic) on the vulnerability to fibrillation during experimental ischaemia were investigated.
The study was carried out in anaesthetized, open‐chest pigs (n = 8 for each of the drugs, in addition to the control group, n = 6). Vulnerability to fibrillation was evaluated by measuring electrical fibrillation threshold (EFT) by means of stepwise increased intensity of wide (100 ms) diastolic impulses applied to the ischaemic tissue at a 180 beats min−1 rate. Monophasic action potential (MAP) duration and conduction time in the ischaemic region were also measured.
EFT determinations were performed before and during periods of ischaemia induced by complete occlusion of the left anterior descending coronary artery near its origin. Ischaemic periods of increasing duration (30, 60, 90, 120, 150 s) were induced to determine the electrophysiological changes, of EFT especially, leading to fibrillation.
In the absence of ischaemia, all three drugs, administered by intravenous route (1 mg kg−1 plus 0.04 mg kg−1 min−1) increased EFT to a similar extent (from approximately 7 to 10 mA), despite a 25% prolongation of conduction time.
During ischaemia, none of the drugs prevented the fall in EFT towards 0 mA, resulting in spontaneous fibrillation. After 30 s of ischaemia, they no longer had any capacity for raising EFT and, after 60, 90 and 120 s of ischaemia, the decrease in EFT was exacerbated. This accelerated reduction in EFT shortened the time to onset of fibrillation (after 120 s of ischaemia, 62.5% of fibrillations with fiecainide instead of 12.5 under control conditions, 75% instead of 25 with lignocaine and 50% instead of 25 with disopyramide). The reduction in MAP duration due to ischaemia was also significantly accelerated (at 60 s, 178 ± 5 ms instead of 192 ± 4 with fiecainide, 175 ± 3 ms instead of 194 ± 5 with lignocaine and 180 ± 5 ms instead of 196 ± 3 with disopyramide) and the slowing of conduction was made worse (prolongation of conduction time by 70% instead of 50).
In conclusion, the antifibrillatory properties normally manifested by these drugs are first suppressed, then inverted by ischaemia, depending on oxygen debt varying with severity and duration of ischaemia.
Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium‐dependent relaxation to bradykinin in human small resistance‐like coronary arteries
Authors: Kemp B K , Cocks T M 
Published: August 2006

The effects of the nitric oxide (NO) synthase inhibitor, NG‐nitro‐l‐arginine (l‐NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K+ upon endothelium‐dependent relaxation to bradykinin were investigated in human isolated small coronary arteries.
Endothelium‐dependent relaxations to bradykinin were compared in vessels contracted to ∼50% of their maximum contraction to 124 mm KCl Krebs solution, regardless of treatments, with the thromboxane A2 mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force.
l‐NOARG (100 μm) caused a small but significant, 12% (P<0.01), decrease in the maximum relaxation (Rmax: 91.5±5.4%) to bradykinin but did not significantly affect the sensitivity (pEC50: 8.08±0.17). Increasing the concentration of l‐NOARG to 300 μm had no further effect on the pEC50 or Rmax to bradykinin. HbO (20 μm) and a combination of HbO (20 μm) and l‐NOARG (100 μm) reduced Rmax to bradykinin by 58% (P<0.05) and 54% (P<0.05), respectively. HbO (20 μm) and l‐NOARG (100 μm, combined but not HbO (20 μm) alone, caused a significant 11 fold (P<0.05) decrease in sensitivitiy to bradykinin. HbO (20 μm) decreased the sensitivity to the endothelium‐independent NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), approximately 17 fold (P<0.05).
Raising the extracellular concentration of K+ isotonically to 30 mm, reduced the Rmax to bradykinin from 96.6±3.1% to 43.9±10.1% (P<0.01) with no significant change in sensitivity. A combination of HbO, l‐NOARG and high K+ (30 mm) abolished the response to bradykinin. High K+ did not change either the sensitivity or maximum relaxation to SNAP.
In conclusion, l‐NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non‐NO ‐dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K+‐sensitive vasodilator mechanism, possibly endothelium‐derived hyperpolarizing factor (EDHF).

British Journal of Pharmacology (1997) 120, 757–762; doi:10.1038/sj.bjp.0700928
Alterations of the benzodiazepine site of rat α6β2γ2‐GABAA receptor by replacement of several divergent amino‐terminal regions with the αl counterparts
Authors: Im Wha Bin , Pregenzer Jeffrey F. , Binder Jay A. , Alberts Glen L. , Im Haesook K. 
Published: August 2006

The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype.
The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents.
Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others.
In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.
Hypoxic dilatation of porcine small coronary arteries: role of endothelium and KATP‐channels
Authors: Liu Qiang , Flavahan Nicholas A. 
Published: August 2006

The aim of the present study was to determine the cellular mechanims and potential mediators involved in hypoxic dilatation of porcine small coronary arteries.
Small coronary arteries were isolated from a branch of the left anterior descending artery of porcine hearts, cannulated with glass micropipettes and studied in a perfusion myograph system. At a transmural pressure of 40 mmHg, the arteries had an internal diameter of 167.8 ± 6.6 μm (n = 37).
In arteries contracted with acetylcholine (ACh), hypoxia (0% O2, 30 min) caused dilatation (86.9± 6.7% relaxation, n = 6) in vessels with endothelium but constriction in endothelium‐denuded vessels.
Hypoxic vasodilatation occurring in arteries with endothelium was abolished by the KAtp channel inhibitor, glibenclamide (0.44 μM), but was not affected by inhibition of nitric oxide synthase (l‐NAME, 44 μM) or cyclo‐oxygenase (indomethacin, 4.4 μM).
Bradykinin evoked endothelium‐dependent relaxation that was inhibited by l‐NAME (44 μM) but not glibenclamide 0.44 μM). Cromakalim (0.1–0.3μM), a KATP channel opener, caused relaxation that was inhibited by glibenclamide, but was not affected by l‐NAME (44 μm) and/or indomethacin (4.4 μm).
Endothelium‐removal inhibited vasodilatation evoked by cromakalim, but increased vasodilator responses to the NO donor, SIN‐1 (10−8 to 10−5 m).
These results indicate that hypoxia acted directly on vascular smooth muscle of small coronary arteries to cause contraction. However, this effect was overwhelmed by endothelium‐dependent relaxation in response to hypoxia. This relaxation was most likely mediated by release of an endothelium‐derived factor, distinct from nitric oxide or prostacyclin, that activated smooth muscle KATP‐channels.
Pharmacological and biochemical evidence for the simultaneous expression of CCKB/gastrin and CCKA receptors in the pig pancreas
Authors: Philippe C. , Lhoste E.F. , Dufresne M. , Moroder L. , Corring T. , Fourmy D. 
Published: August 2006

In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.
Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.
The binding of [M125I]‐BH‐[Thr, Nle]CCK‐9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.
Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK‐9, gastrin, PD 135158, L‐365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK‐9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L‐365,260.
These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.
Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.
In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.
Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone
Authors: Coceani F. , Kelsey L. , Seidlitz E. , Marks G.S. , McLaughlin B.E. , Vreman H.J. , Stevenson D.K. , Rabinovitch M. , Ackerley C. 
Published: August 2006

We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction controlling the formation of endothelin‐1 (ET‐1). In the present study, we have examined whether CO is formed naturally in the vessel.
The CO‐forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO‐2) and inducible (HO‐1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO‐1 was localized to endothelial and muscle cells, while HO‐2 was found only in muscle cells. Inside the muscle cells, HO‐1 and HO‐2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO‐1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma.
CO was formed enzymatically from added substrate (hemin, 50 μm) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 μM).
ZnPP (10 μM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin‐treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2).
ZnPP (10 μm) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 μM)‐contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged.
We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.
Regulation of citrulline recycling in nitric oxide‐dependent neurotransmission in the murine proximal colon
Authors: Shuttleworth C.W.R. , Conlon S.B. , Sanders K.M. 
Published: August 2006

We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.
Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μm). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).
L‐Nitro‐arginine‐methyl ester (l‐NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L‐NAME were reversed by L‐arginine (0.2–2 mM) but not by D‐arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.
L‐Citrulline (0.2 mM) also reversed the effects of L‐NAME, and this action was maintained during sustained exposures to L‐citrulline (0.2 mM). This may reflect intraneuronal recycling of L‐citrulline to L‐arginine.
Higher concentrations of L‐citrulline (e.g. 2 mM) had time‐dependent effects. Brief exposure (15 min) reversed the effects of L‐NAME, but during longer exposures (30 min) the effects of L‐NAME gradually returned. In the continued presence of L‐citrulline, L‐arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L‐citrulline, the ability to generate arginine from citrulline was reduced.
Aspartate (2 mM) had no effect on i.j.ps, the effects of L‐NAME, or the actions of L‐citrulline in the presence of L‐NAME. L‐Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.
S‐methyl‐L‐thiocitrulline (10 μm), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L‐arginine (2 mM), but not by L‐citrulline (2 mM).
These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L‐citrulline to L‐arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L‐citrulline are elevated.
Involvement of tyrosine phosphorylation in endothelin‐1‐induced calcium‐sensitization in rat small mesenteric arteries
Authors: Ohanian Jacqueline , Ohanian Vasken , Shaw Linda , Bruce Christopher , Heagerty Anthony M. 
Published: August 2006

We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist.
Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al.
In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin Al inhibited the contractile response to endothelin‐1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin‐1 (100 nM) in the absence of extracellular calcium.
In rat α‐toxin permeabilized mesenteric arteries endothelin‐1 caused a concentration‐dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin Al did not affect endothelin‐1‐induced calcium sensitization of force.
We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin‐1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.