bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the Na+/K+ pump current in small neurons from adult rat dorsal root ganglia
Authors: Hamada Kanako , Matsuura Hiroshi , Sanada Mitsuru , Toyoda Futoshi , Omatsu‐Kanbe Mariko , Kashiwagi Atsunori , Yasuda Hitoshi
Published: April 2003

The present investigation was undertaken to characterize the Na+/K+ pump current in small (25 μm in soma diameter) dorsal root ganglion (DRG) neurons isolated from lumbar L4‐6 segments of adult rats.

The Na+/K+ pump current was identified as an ouabain‐sensitive current during square voltage steps to membrane potentials between +40 and −120 mV, using the whole‐cell patch‐clamp technique in which Ca2+ and K+ channel currents and Na+/Ca2+ exchange currents were minimized. The Na+/K+ pump current was practically time‐independent over the entire voltage range examined and exhibited a voltage‐dependence; its current – voltage (I–V) relationship displayed a positive slope at potentials between −120 and 0 mV but nearly plateau levels at positive membrane potentials.

The concentration‐dependent block of Na+/K+ pump current (activated by 30 mM pipette Na+) by ouabain at concentrations between 0.1 μM and 5 mM was biphasic and was well described using a two‐binding site model with dissociation constants for high‐ and low‐affinity binding sites of 0.20 and 140.1 μM, respectively. The relative amplitude of the Na+/K+ pump current produced by low‐ and high‐affinity sites (probably α1β1 and α3β1 isozymes, respectively) was estimated to be 13 : 1 in the presence of 30 mM Na+ in the pipette solution.

Additionally, the activation of Na+/K+ pump current by pipette Na+ at concentrations ranging from 5 to 100 mM also exhibited a biphasic concentration dependence which can be reasonably well fitted by assuming the existence of two isozymes having high and low affinities for Na+ (6.7 and 67.6 mM, respectively).

Thus, the present investigation provides functional evidence to suggest that the Na+/K+ ATPase comprises two functionally distinct isozymes as expected for α1β1 and α3β1 in rat small DRG neurons.

British Journal of Pharmacology (2003) 138, 1517–1527. doi:10.1038/sj.bjp.0705170
Growth hormone protects human lymphocytes from irradiation‐induced cell death
Authors: Lempereur Laurence , Brambilla Daria , Maria Scoto Giovanna , D'Alcamo Maria , Goffin Vincent , Crosta Lucia , Palmucci Tullio , Rampello Liborio , Bernardini Renato , Cantarella Giuseppina
Published: April 2003

Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r‐hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro.

r‐hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl‐2.

r‐hGH restored mitogen‐stimulated release of IL‐2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r‐hGH‐dependent IL‐2 release.

These results demonstrate that r‐hGH protects irradiated PBL from death in a specific, receptor‐mediated manner. Such effect of r‐hGH on PBL involves activation of the antiapoptotic gene bcl‐2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer.

British Journal of Pharmacology (2003) 138, 1411–1416. doi:10.1038/sj.bjp.0705173
Extracellular ATP‐induced calcium channel inhibition mediated by P1/P2Y purinoceptors in hamster submandibular ganglion neurons
Authors: Abe Mitsuhiro , Endoh Takayuki , Suzuki Takashi
Published: April 2003

The presence and profile of purinoceptors in neurons of the hamster submandibular ganglion (SMG) have been studied using the whole‐cell configuration of the patch‐clamp technique.

Extracellular application of adenosine 5′‐triphosphate (ATP) reversibly inhibited voltage‐dependent Ca2+ channel (VDCC) currents (ICa) via Gi/o‐protein in a voltage‐dependent manner.

Extracellular application of uridine 5′‐triphosphate (UTP), 2‐methylthioATP (2‐MeSATP), α,β‐methylene ATP (α,β‐MeATP) and adenosine 5′‐diphosphate (ADP) also inhibited ICa. The rank order of potency was ATP=UTP>ADP>2‐MeSATP=α,β‐MeATP.

The P2 purinoceptor antagonists, suramin and pyridoxal‐5‐phosphate‐6‐azophenyl‐2′, 4′‐disulfonic acid (PPADS), partially antagonized the ATP‐induced inhibition of ICa, while coapplication of suramin and the P1 purinoceptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), virtually abolished ICa inhibition. DPCPX alone partially antagonized ICa inhibition.

Suramin antagonized the UTP‐induced inhibition of ICa, while DPCPX had no effect.

Extracellular application of adenosine (ADO) also inhibited ICa in a voltage‐dependent manner via Gi/o‐protein activation.

Mainly N‐ and P/Q‐type VDCCs were inhibited by both ATP and ADO via Gi/o‐protein βγ subunits in seemingly convergence pathways.

British Journal of Pharmacology (2003) 138, 1535–1543. doi:10.1038/sj.bjp.0705174
Ethanol tachyphylaxis in spinal cord motorneurons: role of metabotropic glutamate receptors
Authors: Li Hui‐Fang , Wang Meng‐Ya , Knape Jessica , Kendig Joan J
Published: April 2003

Ethanol (EtOH) tachyphylaxis (acute tolerance), a time‐dependent decrease in apparent potency, is known in vivo and in some neuronal preparations. The present studies characterize EtOH tachyphylaxis in spinal motorneurons and test the hypothesis that metabotropic glutamate receptors (mGluRs) play a role.

Patch clamp studies were carried out in motorneurons in rat spinal cord slices. Currents were evoked by pulses of glutamate, alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) or N‐methyl‐D‐aspartic acid (NMDA).

In nine of 15 cells, ethanol depression of glutamate‐evoked currents was time‐dependent. EtOH depressed current area 36.9±3% at 8–10 min, but only 16.8±3% at 20 min. Mean reduction in depression was 20.1±1%, N=9. Tachyphylaxis was less prominent in currents evoked by AMPA or NMDA, appearing in two of 10 AMPA and three of 11 NMDA currents.

The mGluR agonist trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD) increased, the antagonist (±)‐alpha‐methyl‐4‐carboxyphenylglycine (MCPG) decreased the area of glutamate‐evoked currents. ACPD also increased the area of NMDA‐ and AMPA‐evoked currents.

ACPD increased the incidence of tachyphylaxis in glutamate‐evoked currents to 100% (N=9); MCPG markedly reduced tachyphylaxis. ACPD also increased the incidence of tachyphylaxis in currents evoked by NMDA and AMPA to five of eight and four of seven neurons, respectively.

Block of G‐protein pathways by intracellular GDP‐β‐s abolished tachyphylaxis in glutamate‐evoked currents (N=8); however, currents recovered only partially following EtOH washout.

Activation of mGluRs contributes to neuronal tachyphylaxis to EtOH in spinal cord motorneurons, probably via G‐protein pathways.

British Journal of Pharmacology (2003) 138, 1417–1424. doi:10.1038/sj.bjp.0705175
Characterization of a fluorescent conjugate of the rabbit angiotensin AT1 receptor
Authors: Fortin Jean‐Philippe , Bouthillier Johanne , Bastien Luc , Bachvarov Dimcho R , Marceau François
Published: April 2003

The rabbit AT1 receptor (AT1R) for angiotensin II (AII) has been conjugated to the yellow fluorescent protein (YFP) in order to establish the pharmacological profile of such a fusion protein and to facilitate the study of ligand‐induced regulation.

AII bound AT1R–YFP (KD 8.1 nM in transiently transfected cells) and stimulated HEK 293 cells expressing the fusion protein at concentration ranges similar to the ones that stimulate the contraction of the isolated rabbit aorta. Antagonists found to be insurmountable in the latter assay (candesartan and EXP‐3174 being the most extreme cases) were also insurmountable in the phospholipase A2 assay applied to cells expressing AT1R–YFP, whereas losartan appeared to be surmountable in both assays.

Cells expressing AT1R–YFP exhibited a membrane‐associated fluorescence that was partly and reversibly translocated into intracellular structures upon AII stimulation (confocal microscopy); the nonpeptide antagonists were not active in this respect, but prevented the effect of the agonist.

AII treatment increased the quantity of the fusion protein in cells, and phorbol 12‐myristate 13‐acetate (PMA) treatment even more so (immunoblot, confocal microscopy) but, unlike the agonist, the latter drug did not induce receptor endocytosis. A protein kinase C (PKC) inhibitor prevented the effect of either AII or PMA on AT1R–YFP abundance.

The conjugate AT1R–YFP retains the pharmacological properties of the parent rabbit AT1R. Agonist‐induced downregulation was not documented using this system; to the contrary, we have observed a PKC‐mediated increased expression AT1R–YFP likely to be the result of a decreased breakdown rate of the fusion protein.

British Journal of Pharmacology (2003) 138, 1495–1504. doi:10.1038/sj.bjp.0705176
Identification and functional characterization of hemorphins VV‐H‐7 and LVV‐H‐7 as low‐affinity agonists for the orphan bombesin receptor subtype 3
Authors: Lammerich Hans‐Peter , Busmann Annette , Kutzleb Christian , Wendland Martin , Seiler Petra , Berger Claudia , Eickelmann Peter , Meyer Markus , Forssmann Wolf‐Georg , Maronde Erik
Published: April 2003

The human orphan G‐protein coupled receptor bombesin receptor subtype 3 (hBRS‐3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV‐hemorphin‐7 (VV‐H‐7) and LVV‐hemorphin‐7 (LVV‐H‐7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS‐3 compared to other bombesin receptors.

VV‐H‐7 and LVV‐H‐7 induced a dose‐dependent response in hBRS‐3 overexpressing CHO cells, as well as in NCI‐N417 cells expressing the hBRS‐3 endogenously. The affinity of VV‐H‐7 was higher in NCI‐N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV‐H‐7 and 183±60 μM for LVV‐H‐7 in CHO cells, and 19±6 μM for VV‐H‐7 and 38±18 μM for LVV‐H‐7 in NCI‐N417 cells. Other hemorphins had no effect. Gastrin‐releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines.

Structure‐function analysis revealed that both the N‐terminal valine and the C‐terminal phenylalanine residues of VV‐H‐7 are critical for the ligand‐receptor interaction.

Endogenous hBRS‐3 in NCI‐N417 activated by VV‐H‐7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)‐sensitive store followed by a capacitive calcium entry from extracellular space.

VV‐H‐7‐induced hBRS‐3 activation led to phosphorylation of p42/p44‐MAP kinase in NCI‐N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed.

British Journal of Pharmacology (2003) 138, 1431–1440. doi:10.1038/sj.bjp.0705177
Effects of U‐50,488H and U‐50,488H withdrawal on c‐fos expression in the rat paraventricular nucleus. Correlation with c‐fos in brainstem catecholaminergic neurons
Authors: Laorden Maria Luisa , Castells Maria Teresa , Milanés Maria Victoria
Published: April 2003

In the present work, we have studied the expression of Fos during acute and chronic administration of the κ‐opioid receptor agonist U‐50,488H and after U‐50,88H withdrawal in the rat hypothalamic paraventricular nucleus (PVN). Fos production was also studied in brainstem regions that innervate the PVN: the A2 cell group of the nucleus of solitary tract (NTS‐A2) and the A1 cell group of the ventrolateral medulla (VLM‐A1), combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons after acute U‐50,488H administration.

For acute experiments, male rats were treated with saline i.p. for 4 days. On day 5, rats were given saline or U‐50,488H (15 mg kg−1, i.p.). Other groups of rats were rendered tolerant/dependent on U‐50,488H by injecting the drug twice daily (15 mg kg−1, i.p.) for 4 days. Control animals received saline i.p. on the same time schedule. On day 5, rats were treated with vehicle i.p., with U‐50,488H (15 mg kg−1) or with the selective κ opioid‐receptor antagonist nor‐binaltorphimine (Nor‐BNI, 5 mg kg−1, i.p.).

Using immunohistochemical staining of Fos, present results indicate that acute administration of U‐50,488H produced an increase in Fos expression in the PVN and in the noradrenergic A1 and A2 cell groups. Moreover, when double‐label immunohistochemistry was used to identify Fos and catecholaminergic‐positive neurons in the brainstem, it was found that catecholaminergic‐positive neurons in the NTS and VLM showed a significant increase in Fos expression in response to acute U‐50,488H injection. Chronic application of U‐50,488H leads to the development of tolerance towards their effects on Fos expression in the PVN as well as in the NTS and VLM. However, administration of Nor‐BNI to U‐50,488H‐dependent rats did not induce any changes in Fos immunoreactivity in the PVN or in the brainstem.

These findings demonstrate that acute activation of κ‐opioid receptors results in different altered patterns of immediate‐early gene expression in the PVN, which occurs concurrently with an increased activity of their inputs from the brainstem. Interestingly in contrast to morphine withdrawal, present results demonstrate that rats withdrawn from U‐50,488H did show no changes in Fos‐immunoreactivity in the PVN, NTS or VLM, indicating the absence of dependence on the κ‐agonist under the present experimental conditions.

British Journal of Pharmacology (2003) 138, 1544–1552. doi:10.1038/sj.bjp.0705179
Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium‐derived hyperpolarising factor
Authors: Wihlborg Anna‐Karin , Malmsjö Malin , Eyjolfsson Atli , Gustafsson Ronny , Jacobson Kenneth , Erlinge David
Published: April 2003

The present study was aimed at examining P2 receptor‐mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 μM noradrenaline.

Endothelial denudation abolished the dilator responses.

The selective P2Y1 agonist, 2‐MeSADP, induced a potent vasodilatation (pEC50=6.9±0.1). The P2Y1 antagonist of 10 μM, MRS 2216, shifted the 2‐MeSADP concentration‐response curve 1.1 log units to the right. The combined P2Y1 and P2X agonist, 2‐MeSATP, stimulated a dilatation with a potency similar to that of 2‐MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2‐MeSATP and 2‐MeSADP indicating that P2X receptors do not mediate vasodilatation.

Both the P2Y2/4 agonist, UTPγS and the P2Y6 agonist, UDPβS, stimulated potent dilatations (pEC50=7.8±0.4 for UTPγS and 8.4±0.2 for UDPβS).

The 2‐MeSADP‐induced nitric oxide (NO)‐mediated dilatation was studied in the presence of 10 μM indomethacin, 50 nM charybdotoxin and 1 μM apamin. The involvement of the endothelium‐derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L‐NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L‐NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2‐MeSADP dilatation with similar efficacy (Emax=25±5% for NO, 25±6% for EDHF and 27±5% for prostaglandins).

In conclusion, extracellular nucleotides induce endothelium‐derived vasodilatation in human vessels by stimulating P2Y1, P2Y2/4 and P2Y6 receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins

British Journal of Pharmacology (2003) 138, 1451–1458. doi:10.1038/sj.bjp.0705186
Polarized Th1 and Th2 cells are less responsive to negative feedback by receptors coupled to the AC/cAMP system compared to freshly isolated T cells
Authors: Heijink Irene H , Vellenga Edo , Borger Peter , Postma Dirkje S , Monchy Jan G R de , Kauffman Henk F
Published: April 2003

The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T‐cell‐mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and T‐cell clones; yet, effects in differentiated Th1 and Th2 cells are largely unknown.

To obtain differentiated T helper cells, we activated naive T cells for 1 week in the presence of IL‐12 plus α‐IL‐4 to generate Th1‐type cells and in the presence of IL‐4 plus α‐IL‐12 to generate Th2‐type cells.

We demonstrate that, in contrast to freshly isolated T cells, the production of Th1 (IFN‐γ) and Th2 (IL‐4, IL‐5) cytokines in polarized T helper cells is not strictly controlled by the activation of AC/cAMP‐linked β2‐adrenergic and prostaglandin (PG)E2 receptors.

In Th2 cells, PGE2 could still activate the Gs protein‐coupled AC/cAMP system and subsequently induce CREB phosphorylation, whereas PGE2 was unable to activate the cAMP‐dependent pathway in Th1 cells. In both Th1 and Th2 cells, the induction of CREB phosphorylation by β2‐agonist fenoterol was impaired.

The loss of control over cytokine production by cAMP elevating agents in differentiated Th1 and Th2 subsets may have important implications for the regulation of Th1‐ and Th2‐mediated diseases, in particular those associated with the ongoing immune responses.

British Journal of Pharmacology (2003) 138, 1441–1450. doi:10.1038/sj.bjp.0705193
Cannabinoid CB1 receptor‐mediated inhibition of hippocampal acetylcholine release is preserved in aged mice
Authors: Redmer Agnes , Kathmann Markus , Schlicker Eberhard
Published: April 2003

The cannabinoid CB1 receptor inverse agonist/antagonist SR 141716 increases acetylcholine release in rodent hippocampus and improves memory in some experimental paradigms. Since drugs like SR 141716 may represent a novel class of cognition‐enhancing drugs, we wanted to check whether the function of the CB1 receptor is preserved during ageing.

Hippocampal and striatal slices from 2‐ to 3‐ and 24‐ to 28‐month‐old C57BL/6J mice were preincubated with [3H]‐choline or [3H]‐noradrenaline ([3H]‐NA) and superfused.

The cannabinoid receptor agonist WIN 55,212‐2 inhibited, and SR 141716 facilitated, the electrically (3 Hz) evoked tritium overflow in hippocampal slices (preincubated with [3H]‐choline) from young and aged mice to the same extent. The evoked overflow per se was less by 33% in slices from aged animals.

WIN 55,212‐2 and SR 141716 did not affect, but the muscarinic receptor agonist oxotremorine inhibited, the evoked (3 Hz) overflow in striatal slices (preincubated with [3H]‐choline) from young and aged mice to the same extent. The evoked overflow per se tended to be less in slices from aged animals.

The evoked (0.3 Hz) overflow in hippocampal slices (preincubated with [3H]‐NA) was not affected by WIN 55,212‐2 and SR 141716, but was inhibited by histamine (via H3 receptors) in slices from young mice and, to a somewhat less extent, in slices from aged mice. The evoked overflow per se did not differ between age groups.

In conclusion, the function of the CB1 receptor involved in the tonic inhibition of hippocampal acetylcholine release is preserved in aged mice.

British Journal of Pharmacology (2003) 138, 1425–1430. doi:10.1038/sj.bjp.0705194
Effects of flecainide and quinidine on Kv4.2 currents: voltage dependence and role of S6 valines
Authors: Caballero Ricardo , Pourrier Marc , Schram Gernot , Delpón Eva , Tamargo Juan , Nattel Stanley
Published: April 2003

The effects of flecainide and quinidine were studied on wild‐type Kv4.2 channels (Kv4.2WT), channels with deletion of the N‐terminal domain (N‐del) and channels with mutations in the valine residues located at positions 402 and 404 in the presence (V[402,404]I) or in the absence (N‐del/V[402,404]I) of the N‐terminus.

The experiments were performed at 37°C on COS7 cells using the whole‐cell configuration of the patch‐clamp technique.

Flecainide and quinidine inhibited Kv4.2WT currents in a concentration‐dependent manner (IC50=23.6±1.1 and 12.0±1.4 μMat +50 mV, respectively), similar to their potency for the rest of the constructs at the same voltage. In Kv4.2WT channels, flecainide‐ and quinidine‐induced block increased as channel inactivation increased. In addition, the inhibition produced by quinidine, but not by flecainide, increased significantly at positive test potentials. Similar effects were observed in N‐del channels. However, in V[402,404]I and N‐del/V[402,404]I channels, the voltage dependence of block by both quinidine and flecainide was lost, without significant modifications in potency at +50 mV.

These results point to an important role for S6 valines at positions 402 and 404 in mediating voltage‐dependent block by quinidine and flecainide.

British Journal of Pharmacology (2003) 138, 1475–1484. doi:10.1038/sj.bjp.0705199
Benzoquinolines and chloride secretion in murine colonic epithelium
Authors: Cuthbert Alan W
Published: April 2003

In all, 27 compounds related chemically to phenanthrolines were investigated for their ability to stimulate electrogenic chloride secretion in murine colonic epithelia under short circuit current conditions. A total of 11 compounds, not previously examined, were shown to have substantial activity. Cumulative concentration – response relations were obtained in the presence of amiloride to inhibit electrogenic sodium absorption and Ba2+ to prevent potassium secretion.

Single‐ or double‐ring substitutions in 1,10 phenanthroline (EC50=612 μM) produced only modest increases in affinity of between 0.7‐ and 2.1‐fold.

Naphthalenes with either one or two ring nitrogens showed some activity but had very low affinity, yet some (e.g. isoquinoline, EC50=700 μM) were able to produce maximal responses.

Removal of one nitrogen from 1,10‐phenanthroline or from 4,7‐phenanthroline gave, respectively, 7,8‐benzoquinoline and 5,6‐benzoquinoline. These two were the most active compounds found, the EC50 for 5,6‐benzoquinoline being 29 μM.

Most concentration – response curves were steep with Hill slopes of approximately 3. At high concentration, some compounds inhibited the response, producing a characteristic bell shape.

5,6‐ and 7,8‐benzoquinoline may be useful starting points for the synthesis of more potent agents by the addition of ring substituents.

British Journal of Pharmacology (2003) 138, 1528–1534. doi:10.1038/sj.bjp.0705203
AT1 receptor antagonist therapy preferentially ameliorated right ventricular function and phenotype during the early phase of remodeling post‐MI
Authors: Nguyen Quang T , Colombo Federico , Clement Robert , Gosselin Hugues , Rouleau Jean‐Lucien , Calderone Angelino
Published: April 2003

The influence of AII on contractile dysfunction, regulation of the tyrosine kinase‐dependent signaling molecule extracellular signal‐regulated kinase (ERK), and natriuretic peptide gene expression were examined in the noninfarcted left ventricle (NILV) and right ventricle (RV) during the early phase of remodeling post‐myocardial infarct (MI) in the rat. The selective AT1 receptor antagonist irbesartan was administered <10 h following coronary artery ligation, and rats were killed either at 4‐day or 2‐week post‐MI.

At 4 days post‐MI, left ventricular systolic pressure (LVSP: sham=125±12, MI=91±4 mmHg) was decreased, whereas left ventricular end‐diastolic pressure (LVEDP: sham=9±2, MI=17±2 mm Hg), right ventricular systolic (RVSP: sham=26±1, MI=34±2 mm Hg), and end‐diastolic pressures (RVEDP: sham=3±0.5, MI=7±1 mm Hg) were increased. ERK phosphorylation was significantly elevated in the NILV and RV.

Irbesartan (40 mg kg−1/day−1) administration did not improve left ventricular function, or suppress increased ERK phosphorylation in the 4‐day post‐MI rat. By contrast, irbesartan therapy normalized RVSP (MI+irbesartan=25±1 mm Hg), RVEDP (MI+irbesartan=3±0.3 mm Hg), and reduced ERK1 (MI=3.0±0.6, MI+irbesartan=2.0±0.3‐fold increase), and ERK2 (MI=3.8±0.8, MI+irbesartan=2.2±0.5‐fold increase) phosphorylation.

In 2‐week post‐MI rats, biventricular dysfunction was associated with increased prepro‐ANP, and prepro‐BNP mRNA expression. Irbesartan therapy normalized RVSP, attenuated RVEDP, and abrogated natriuretic peptide mRNA expression (prepro‐ANP; MI=9±2, MI+irbesartan=2±1‐fold increase, prepro‐BNP; MI=6±2, MI+irbesartan=1±1‐fold increase), whereas both transcripts remained elevated in the NILV despite the partial attenuation of LVEDP.

These data suggest that the therapeutic benefit of irbesartan treatment during the early phase of remodeling post‐MI was associated with the preferential amelioration of RV contractile function and phenotype.

British Journal of Pharmacology (2003) 138, 1485–1494. doi:10.1038/sj.bjp.0705212
Different modulation by Ca2+‐activated K+ channel blockers and herbimycin of acetylcholine‐ and flow‐evoked vasodilatation in rat mesenteric small arteries
Authors: Thorsgaard Michael , Lopez Vanesa , Buus Niels H , Simonsen Ulf
Published: April 2003

The present study addressed whether endothelium‐dependent vasodilatation evoked by acetylcholine and flow are mediated by the same mechanisms in isolated rat mesenteric small arteries, suspended in a pressure myograph for the measurement of internal diameter.

In pressurized arterial segments contracted with U46619 in the presence of indomethacin, shear stress generated by the flow evoked relaxation. Thus, in endothelium‐intact segments low (5.1±0.6 dyn cm−2) and high (19±2 dyn cm−2) shear stress evoked vasodilatations that were reduced by, respectively, 68±11 and 68±8% (P<0.05, n=7) by endothelial cell removal. Acetylcholine (0.01–1 μM) evoked concentration‐dependent vasodilatation that was abolished by endothelial cell removal.

Incubation with indomethacin alone did not change acetylcholine and shear stress‐evoked vasodilatation, while the combination of indomethacin with the nitric oxide (NO) synthase inhibitor, NG,NG‐asymmetric dimethyl‐L‐arginine (ADMA 1 mM), reduced low and high shear stress‐evoked vasodilatation with, respectively, 52±15 and 58±10% (P<0.05, n=9), but it did not change acetylcholine‐evoked vasodilatation.

Inhibition of Ca2+‐activated K+ channels with a combination of apamin (0.5 μM) and charybdotoxin (ChTX) (0.1 μM) did not change shear stress‐ and acetylcholine‐evoked vasodilatation. In the presence of indomethacin and ADMA, the combination of apamin (0.5 μM) and ChTx (0.1 μM) increased contraction induced by U46619, but these blockers did not change the vasodilatation evoked by shear stress. In contrast, acetylcholine‐evoked vasodilatation was abolished by the combination of apamin and charybdotoxin.

In the presence of indomethacin, the tyrosine kinase inhibitor, herbimycin A (1 μM), inhibited low and high shear stress‐evoked vasodilatation with, respectively, 32±12 and 68±14% (P<0.05, n=8), but it did not change vasodilatation induced by acetylcholine. In the presence of indomethacin and ADMA, herbimycin A neither changed shear stress nor acetylcholine‐evoked vasodilatation.

The present study suggests that Ca2+‐activated K+ channels sensitive for the combination of apamin and ChTx are involved in acetylcholine‐evoked, mainly non‐NO nonprostanoid factor‐mediated, vasodilatation, while an Src tyrosine kinase plays a role for flow‐evoked NO‐mediated vasodilatation in rat mesenteric small arteries.

British Journal of Pharmacology (2003) 138, 1562–1570. doi:10.1038/sj.bjp.0705214
Synergies at the synapse
Authors: Límbírd Lee E
Published: April 2003

British Journal of Pharmacology (2003) 138, 1387–1388. doi:10.1038/sj.bjp.0705222
Crosstalk between presynaptic angiotensin receptors, bradykinin receptors and α2‐autoreceptors in sympathetic neurons: a study in α2‐adrenoceptor‐deficient mice
Authors: Trendelenburg Anne‐Ulrike , Meyer Angelika , Klebroff Werner , Guimarães Serafim , Starke Klaus
Published: April 2003

In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release‐enhancing effect when presynaptic α2‐autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with α2 antagonists. We now studied this operational condition in α2‐adrenoceptor‐deficient mice. Release of 3H‐noradrenaline was elicited by electrical stimulation.

In tissues from wild‐type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz.

In tissues from mice lacking the α2A‐adrenoceptor (α2AKO) or the α2B‐adrenoceptor (α2BKO), the concentration–response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the α2C‐adrenoceptor (α2CKO) or both the α2A‐ and the α2C‐adrenoceptor (α2ACKO), the concentration–response curves were shifted to the same extent downwards.

As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in α2AKO and α2ACKO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz.

Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven‐fold in WT and α2BKO tissues, three‐fold in α2AKO and α2CKO tissues, and two‐fold in α2ACKO tissues.

Results confirm that angiotensin II and bradykinin require ongoing α2‐autoinhibition for the full extent of their release‐enhancing effect. Specifically, they require ongoing α2C‐autoinhibition. The peptide effects that remain in α2C‐autoreceptor‐deficient mice seem to be because of α2B‐autoinhibition. The results hence also suggest that in addition to α2A‐ and α2C‐ mouse postganglionic sympathetic neurons possess α2B‐autoreceptors.

British Journal of Pharmacology (2003) 138, 1389–1402. doi:10.1038/sj.bjp.0705223
Sarcolipin regulates sarco(endo)plasmic reticulum Ca
2+
-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban
Authors: Asahi Michio , Sugita Yuji , Kurzydlowski Kazimierz , De Leon Stella , Tada Michihiko , Toyoshima Chikashi , MacLennan David H.
Published: April 2003

Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca
2+
-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E
2
conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.
Subtask sequencing in the primary visual cortex
Authors: Roelfsema Pieter R. , Khayat Paul S. , Spekreijse Henk
Published: April 2003

Complex visual tasks can usually be decomposed into a number of simpler subtasks. Whether such subtasks are solved serially or in parallel is subject to considerable debate. Here we investigate how subtasks are coordinated in time by recording from the primary visual cortex of macaque monkeys. The animals were trained to perform both a simple and a composite task. In the simple task, they had to mentally trace a target curve while ignoring a distractor curve. Neuronal responses in the primary visual cortex to the target curve were enhanced relative to responses to the distractor curve 130 ms after stimulus appearance. In the composite task, the monkeys searched for a colored marker and traced a curve that was attached to this marker. In an initial phase of the trials, neuronal responses reflected visual search, and the response enhancement due to curve tracing now occurred after 230 ms, 100 ms later than in the simple task. We conclude that subtasks of the composite task are carried out in a structured and sequential manner that can be monitored in the primary visual cortex.
The genetic basis of adaptive melanism in pocket mice
Authors: Nachman Michael W. , Hoekstra Hopi E. , D'Agostino Susan L.
Published: April 2003

Identifying the genes underlying adaptation is a major challenge in evolutionary biology. Here, we describe the molecular changes underlying adaptive coat color variation in a natural population of rock pocket mice,Chaetodipus intermedius. Rock pocket mice are generally light-colored and live on light-colored rocks. However, populations of dark (melanic) mice are found on dark lava, and this concealing coloration provides protection from avian and mammalian predators. We conducted association studies by using markers in candidate pigmentation genes and discovered four mutations in the melanocortin-1-receptor gene,Mc1r, that seem to be responsible for adaptive melanism in one population of lava-dwelling pocket mice. Interestingly, another melanic population of these mice on a different lava flow shows no association withMc1rmutations, indicating that adaptive dark color has evolved independently in this species through changes at different genes.
Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: A potential link between genome surveillance and apoptosis
Authors: Screaton Robert A. , Kiessling Stephan , Sansom Owen J. , Millar Catherine B. , Maddison Kathryn , Bird Adrian , Clarke Alan R. , Frisch Steven M.
Published: April 2003

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.
Inactivation of androgen receptor coregulator ARA55 inhibits androgen receptor activity and agonist effect of antiandrogens in prostate cancer cells
Authors: Rahman Mujib M. , Miyamoto Hiroshi , Lardy Henry , Chang Chawnshang
Published: April 2003

Antiandrogens given to antagonize androgen receptor (AR) activity gradually lose their efficacy as antagonists and eventually function as agonists to promote (instead of block) AR-mediated growth of prostate cancer cells. The mechanisms of how antiandrogens acquire this agonist activity during hormonal therapy are largely unknown. Here, we report that expression of a dominant-negative AR-associated protein 55 (dARA55) coregulator, inhibits AR transcriptional activity and reduces the agonist activity of antiandrogens. Inducibly expressed dARA55 inhibits prostate-specific antigen and cell growth in prostate cancer cells. Further dissection of the molecular mechanism shows dARA55 can selectively suppress endogenous AR-associated protein 55 (ARA55) enhanced AR transactivation by means of interruption of dimerization between ARA55 and ARA55. These results were confirmed by using RNA interference-mediated silencing of the ARA55 gene. These results therefore provide evidence that AR function could be suppressed without mutation or change in AR itself. Taken together, these findings not only demonstrate the important roles of the ARA55 coregulator in the AR-mediated growth of prostate cancer, they also may provide a critical target for developing therapeutic agents for the antiandrogen therapy that almost always fails in the treatment of hormone-refractory prostate cancer.
Characterization of cells and gene-targeted mice deficient for the p53-binding kinase homeodomain-interacting protein kinase 1 (HIPK1)
Authors: Kondo Seiji , Lu Ying , Debbas Michael , Lin Athena W. , Sarosi Ildiko , Itie Annick , Wakeham Andrew , Tuan JoAnn , Saris Chris , Elliott Gary , Ma Weili , Benchimol Samuel , Lowe Scott W. , Mak Tak Wah , Thukral Sushil K.
Published: April 2003

The tumor suppressor p53 is regulated in part by binding to cellular proteins. We used p53 as bait in the yeast two-hybrid system and isolated homeodomain-interacting protein kinase 1 (HIPK1) as a p53-binding protein. Deletion analysis showed that amino acids 100–370 of p53 and amino acids 885-1093 of HIPK1 were sufficient for HIPK1–p53 interaction. HIPK1 was capable of autophosphorylation and specific serine phosphorylation of p53. TheHIPK1gene was highly expressed in human breast cancer cell lines and oncogenically transformed mouse embryonic fibroblasts.HIPK1was localized to human chromosome band 1p13, a site frequently altered in cancers. Gene-targetedHIPK1−/− mice were grossly normal but oncogenically transformedHIPK1−/− mouse embryonic fibroblasts exhibited reduced transcription ofMdm2and were more susceptible than transformedHIPK1+/+ cells to apoptosis induced by DNA damage. Carcinogen-treatedHIPK1−/− mice developed fewer and smaller skin tumors thanHIPK1+/+ mice. HIPK1 may thus play a role in tumorigenesis, perhaps by means of the regulation of p53 and/or Mdm2.
Cripto forms a complex with activin and type II activin receptors and can block activin signaling
Authors: Gray Peter C. , Harrison Craig A. , Vale Wylie
Published: April 2003

Activin, nodal, Vg1, and growth and differentiation factor 1 are members of the transforming growth factor β superfamily and signal via the activin type II (ActRII/IIB) and type I (ALK4) serine/threonine kinase receptors. Unlike activins, however, signaling by nodal, Vg1, and growth and differentiation factor 1 requires a coreceptor from the epidermal growth factor-Cripto-FRL1-Cryptic protein family such as Cripto. Cripto has important roles during development and oncogenesis and binds nodal or related ligands and ALK4 to facilitate assembly of type I and type II receptor signaling complexes. Because Cripto mediates signaling via activin receptors and binds directly to ALK4, we tested whether transfection with Cripto would affect the ability of activin to signal and/or interact with its receptors. Here we show that Cripto can form a complex with activin and ActRII/IIB. We were unable to detect activin binding to Cripto in the absence of ActRII/IIB, indicating that unlike nodal, activin requires type II receptors to bind Cripto. If cotransfected with ActRII/IIB and ALK4, Cripto inhibited crosslinking of activin to ALK4 and the association of ALK4 with ActRII/IIB. In addition, Cripto blocked activin signaling when transfected into either HepG2 cells or 293T cells. We have also shown that under conditions in which Cripto facilitates nodal signaling, it antagonizes activin. Inhibition of activin signaling provides an additional example of a Cripto effect on the regulation of signaling by transforming growth factor-β superfamily members. Because activin is a potent inhibitor of cell growth in multiple cell types, these results provide a mechanism that may partially explain the oncogenic action of Cripto.
The riboswitch-mediated control of sulfur metabolism in bacteria
Authors: Epshtein Vitaly , Mironov Alexander S. , Nudler Evgeny
Published: April 2003

Many operons in Gram-positive bacteria that are involved in methionine (Met) and cysteine (Cys) biosynthesis possess an evolutionarily conserved regulatory leader sequence (S-box) that positively controls these genes in response to methionine starvation. Here, we demonstrate that a feed-back regulation mechanism utilizes
S
-adenosyl-methionine as an effector.
S
-adenosyl-methionine directly and specifically binds to the nascent S-box RNA, causing an intrinsic terminator to form and interrupt transcription prematurely. The S-box leader RNA thus expands the family of newly discovered riboswitches, i.e., natural regulatory RNA aptamers that seem to sense small molecules ranging from amino acid derivatives to vitamins.
Simulating disorder–order transitions in molecular recognition of unstructured proteins: Where folding meets binding
Authors: Verkhivker Gennady M. , Bouzida Djamal , Gehlhaar Daniel K. , Rejto Paul A. , Freer Stephan T. , Rose Peter W.
Published: April 2003

A microscopic study of functional disorder–order folding transitions coupled to binding is performed for the
p
27 protein, which derives a kinetic advantage from the intrinsically disordered unbound form on binding with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex. Hierarchy of structural loss during
p
27 coupled unfolding and unbinding is simulated by using high-temperature Monte Carlo simulations initiated from the crystal structure of the tertiary complex. Subsequent determination of the transition-state ensemble and the proposed atomic picture of the folding mechanism coupled to binding provide a microscopic rationale that reconciles the initiation recruitment of
p
27 at the cyclin A docking site with the kinetic benefit for a disordered α-helix in the unbound form of
p
27. The emerging structural polarization in the ensemble of unfolding/unbinding trajectories and in the computationally determined transition-state ensemble is not determined by the intrinsic folding preferences of
p
27 but rather is attributed to the topological requirements of the native intermolecular interface to order β-hairpin and β-strand of
p
27 that could be critical for nucleating rapid folding transition coupled to binding. In agreement with the experimental data, the disorder–order folding transition for
p
27 is largely determined by the functional requirement to form a specific intermolecular interface that ultimately dictates the folding mechanism and overwhelms any local folding preferences for creating a stable α-helix in the
p
27 structure before overcoming the major free energy barrier.
Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA
Authors: Iversen T.-G. , Skretting G. , van Deurs B. , Sandvig K.
Published: April 2003

To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3–5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.
Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1)
Authors: Ara Toshiaki , Nakamura Yuri , Egawa Takeshi , Sugiyama Tatsuki , Abe Kuniya , Kishimoto Tadamitsu , Matsui Yasuhisa , Nagasawa Takashi
Published: April 2003

Primordial germ cells (PGCs) are the founders of sperm or oocytes. PGCs migrate through the tissues of the embryos and colonize the gonads during development. However, the cytokines essential for colonization of the gonads by PGCs in mammals remain unclear. Stromal cell-derived factor-1 (SDF-1, also called PBSF and CXCL12) is a member of chemokines, a family of structurally related chemoattractive cytokines. SDF-1 and its primary physiologic receptor CXCR4 have multiple essential functions in development including colonization of bone marrow by hematopoietic cells and neuron localization within cerebellum during embryogenesis as well as B lymphopoiesis and cardiovasculogenesis. Here, we have shown that PGCs have cell-surface expression of CXCR4 and that, in SDF-1
−/−
mice, PGCs undergo directed migration through tissues of embryos, but the numbers of PGCs in the gonads are significantly reduced. The proliferation of PGCs within the gonads seems normal in the mutant mice. These findings reveal the essential role for SDF-1 in murine PGC development likely by controlling colonization of the gonads by PGCs.
BIR-1, a
Caenorhabditis elegans
homologue of Survivin, regulates transcription and development
Authors: Kostrouchova Marta , Kostrouch Zdenek , Saudek Vladimir , Piatigorsky Joram , Rall Joseph Edward
Published: April 2003

bir
-
1
, a
Caenorhabditis elegans
inhibitor-of-apoptosis gene homologous to Survivin is organized in an operon with the transcription cofactor
C. elegans
SKIP (
skp
-
1
). Because genes arranged in operons are frequently linked functionally, we have asked whether BIR-1 also functions in transcription.
bir
-
1
inhibition resulted in multiple developmental defects that overlapped with
C. elegans
SKIP loss-of-function phenotypes: retention of eggs, dumpy, movement defects, and lethality.
bir
-
1
RNA-mediated interference decreased expression of several
gfp
transgenes and the endogenous genes
dpy
-
7
and
hlh
-
1
. Immunoblot analysis revealed decreased phosphoacetylated histones in
bir
-
1
RNA-mediated interference-treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone-regulated transcription and has an additive effect with SKIP. These results show that BIR-1 functions in the regulation of transcription and development.