P2Y12 receptor modulation of ADP-evoked intracellular Ca2+ signalling in THP-1 human monocytic cells.

BACKGROUND AND PURPOSE: The Gi -coupled, ADP-activated P2Y12 receptor is well characterized as playing a key role in platelet activation via crosstalk with the P2Y1 receptor in ADP-evoked intracellular Ca2+ responses. However, there is limited knowledge on the role of P2Y12 receptors in ADP-evoked Ca2+ responses in other blood cells. Here, we investigated the role of P2Y12 receptor activation in the modulation of ADP-evoked Ca2+ responses in human THP-1 monocytic cells. EXPERIMENTAL APPROACH: A combination of intracellular Ca2+ measurements, RT-PCR, immunocytochemistry, leukocyte isolation and siRNA-mediated gene knockdown were used to identify the role of P2Y12 receptor activation. KEY RESULTS: ADP-evoked intracellular Ca2+ responses (EC50 2.7 µM) in THP-1 cells were abolished by inhibition of PLC (U73122) or sarco/endoplasmic reticulum Ca2+ -ATPase (thapsigargin). Loss of ADP-evoked Ca2+ responses following treatment with MRS2578 (IC50 200 nM) revealed a major role for P2Y6 receptors in mediating ADP-evoked Ca2+ responses. ADP-evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 receptor inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3 µM; PSB-0739, IC50 5.6 µM). ADP-evoked responses were suppressed following siRNA-mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that P2Y12 receptor activation positively regulates P2Y6 receptor-mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.

Multiple births following in vitro fertilization treatment: redefining success.

The focus of this article is to review the definition of success following in vitro fertilization (IVF) treatment. Pregnancy rates after IVF have been increasing, but the problem of multiple births with its associated morbidity and mortality has been considerable. This has led to rethinking of assisted reproductive technology (ART) success not only in terms of live birth rates, but also in terms of reduction of multiple births to singleton babies. Single embryo transfer using blastocysts and such other measures are being encouraged. Financial factors and patient satisfaction are key issues. IVF success is thus being redefined.

Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle.

This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding K(V)alpha1 subunits as well as K(V)beta1, K(V)alpha2.1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Ca)beta1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding K(V)alpha2.1 but less or similar amounts encoding K(V)beta1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Cabeta)1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant K(V)alpha1 K(+) current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K(+) current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.

A multiple species approach to sequential learning: are you a man or a mouse?

We have developed a method for studying list learning in animals and humans, and we use variants of the task to examine list learning in rats, mice, and humans. This method holds several advantages over other methods. It has been found to be easily learned without lengthy pretraining. The data gathered with this procedure provide a measure of correct response rates, of incorrect responses and the locations of these responses, and of response latency on a trial-by-trial basis. We have examined mouse, rat, and human list acquisition of patterns ranging from 12 to 48 items in length. This procedure has also been used to examine many aspects of list learning, such as the effects of the placement of phrasing cues that are either consistent or inconsistent with the structure of the list in rats and mice, the effects of phrasing cues of differing modalities in mice, the sensitivity of subjects to violations of list structure in rats, subjects' abilities to "chunk" from nonadjacent serial positions in structured lists in rats, and subjects' sensitivity to serial patterns with multiple levels of hierarchical organization. The procedure has also been used to examine the effects of drugs on sequential learning.

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland.

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L-PRLR) and two of the three short forms (S1- and S3-PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2-PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L-PRLR mRNA. By contrast, S1-PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2-PRLR became undetectable during puberty. Stromal expression of S3-PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases.

The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with beta-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.

Suppression of hippocampal slice excitability by 2-, 3-, and 4-methylpyridine.

The present study assessed the effects of 2-, 3-, and 4-methylpyridine on rat hippocampal slice excitability. Tests of excitatory and inhibitory systems in area CA1 of the hippocampal slice were conducted over a period of 3 h postexposure. Following exposures of 100 microM 2-, 3-, or 4-methylpyridine, evoked population excitatory postsynaptic potential and population spike responses recorded in the cell body field of hippocampal area CA1 were slowly suppressed over the course of 3 h, whereas no effects on local inhibitory processes or latency of evoked responses were detected. No significant differences were observed between agents.

Development and validation of a semi-automated method for L-dopa and dopamine in rat plasma using electrospray LC/MS/MS.

A semi-automated alumina-based extraction method for the determination of L-dopa and dopamine in plasma using liquid chromatography mass spectrometry was validated. The method exploited the use of a Tomtec Quadra 96 liquid handing robot to expedite aluminum oxide extraction for sample clean up. Two 96-well sample plates can be processed in less than 2 h and extracts, collected in a 96-well plate format, can be directly injected onto the ESI/LC/MS/MS instrumentation. Chromatographic separation of the analytes was performed on a reverse-phase ODS column (TosoHaas ODS-80) with a mobile phase of acetonitrile/0.1% formic acid (5/95 v/v) at a flow rate of 0.22 ml/min. Analytes were detected by a triple-quadruple mass spectrometer equipped with an electrospray ionization source (ESI). Recoveries were evaluated for a number of pH modifiers and elution solvents. Under optimized conditions, the mean recoveries of L-dopa and dopamine were 56 and 67%, respectively. Intra-run and inter-run precision, calculated as percent relative standard deviation of replicate quality controls, was in the range of 1.45-10.8% for both L-dopa and dopamine. Intra-run and inter-run accuracy, calculated as percent error, was in the range -2.5 to 6.69% for both analytes. The limit of quantitaiton was 2.5 ng/ml for both L-dopa and dopamine when 100 microl of plasma was extracted. The method is simple, rapid, accurate and suitable for the quantification of L-dopa and dopamine in plasma or other biological fluid samples from clinical, preclinical, or pharmacological studies.

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels.

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.

Effects of sequential exposure to multiple concentrations of methylmercury in the rat hippocampal slice.

Two experiments explored the effects of sequential exposure to multiple concentrations of methylmercury (MeHg) on rat hippocampal slice synaptic transmission and excitability in area CA1. When hippocampal slices were exposed to 0.1, 1, 10, and 100 microM MeHg chloride in successive 30-min exposures, MeHg produced an increase in excitability over baseline levels throughout the 1 microM exposure and the first 5 min of the 10 microM exposure, followed by profound suppression of excitability at the 100 microM level. When hippocampal slices were exposed to 10, 25, 50, 75, and 100 microM concentrations, MeHg produced an increase in excitability throughout most of the 10 and 25 microM exposures, followed by profound suppression of excitability at the 50 microM level of exposure. In both series of concentrations, MeHg suppressed local inhibitory systems prior to suppressing excitatory systems. In a third experiment, a single exposure of 50 microM MeHg suppressed both presynaptic and postsynaptic responses recorded in stratum radiatum with the same time course, suggesting that the observed suppressive effects of MeHg were not primarily synaptic.

Fliih, the murine homologue of the Drosophila melanogaster flightless I gene: nucleotide sequence, chromosomal mapping and overlap with Llglh.

The Drosophila melanogaster flightless I gene is involved in cellularization processes in early embryogenesis and in the structural organization of indirect flight muscle. The encoded protein contains a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat protein-protein interaction domain. We have cloned Fliih, the corresponding chromosomal gene from the mouse, and determined its nucleotide sequence (15.6 kb). The predicted Fliih protein of 1271 amino acids is 95% identical to the human FLII protein. Like the human gene, Fliih has 29 introns, compared with 13 in C. elegans and 3 in D. melanogaster. Fluorescence in situ hybridization was used to map Fliih to Chromosome 11B. Fliih lies adjacent to Llglh, the mouse homologue of the D. melanogaster tumor suppressor gene lethal(2) giant larvae. The sequence of the genomic DNA in this area, combined with cDNA sequences, establishes that the 3' ends of the Fliih and Llglh transcripts overlap. The overlap region contains polyA signals for both genes and is conserved between human and mouse.

Quantification and rapid metabolite identification in drug discovery using API time-of-flight LC/MS.

Liquid chromatography/mass spectrometry (LC/MS), utilizing a time-of-flight (TOF) mass analyzer, has been evaluated and applied to problems in bioanalysis for pharmacokinetics and drug metabolism. The data obtained by TOF MS differ from those obtained using quadrupole mass spectrometer instruments in that full-scan spectra can be routinely collected with greater sensitivity and speed. Both quantitative and qualitative information, including compound concentration in rat plasma and full-scan atmospheric pressure ionization mass spectra, are concurrently obtained. This approach has been used to characterize the disposition of several drug compounds that have been simultaneously dosed to rats in a cassette format. Quantitation limits in the 5-25 ng/mL range (approximately 20 nM) were obtained from nominal mass chromatograms (0.5 Da resolution). A reference lock mass was used to provide accurate mass measurement to reach third decimal place accuracy in the monoisotopic molecular weight. An improvement in quantitation limits was demonstrated after using accurate mass determinations. Several possible preliminary drug metabolites were confirmed or refuted, based on accurate mass. The trend of metabolite formation and clearance was qualitatively evaluated.

Primary esophageal repair for Boerhaave's syndrome.

BACKGROUND: Boerhaave's syndrome is the most sinister cause of esophageal perforation. The mediastinal contamination with microorganisms, gastric acid, and digestive enzymes results in a mediastinitis that is often fatal if untreated. METHODS: We present a series of 21 patients seen in our unit in the 10 years 1987 to 1996. Esophageal repair was performed in 17 (81%) of them. After the resuscitation of the patient in the intensive care unit, our strategy is primary esophageal repair with a single layer of interrupted absorbable sutures combined with mediastinal toilet, mediastinal drainage, and drainage gastrostomy. The majority of patients (12/21) were referred more than 24 hours after perforation. RESULTS: The mean age of the patients was 60+/-17 years. The mean stay in the intensive care unit was 1.6+/-1.8 days and the median hospital stay, 14 days. There were three deaths, an overall mortality rate of 14.3%. CONCLUSIONS: When combined with mediastinal toilet, mediastinal drainage, and drainage gastrostomy, primary esophageal repair for Boerhaave's syndrome gives an acceptable mortality and should not be reserved for patients seen within 24 hours after spontaneous rupture.

Iatrogenic oesophageal perforations: a clinical review.

Thirty patients with iatrogenically induced perforation of the oesophagus were managed in our unit between January 1986 and December 1996. Thirteen (43%) of these injuries were referred after upper gastrointestinal endoscopy performed by physicians. Ten (33%) cases were referred by ENT surgeons and general surgeons referred 7 (23%) cases. Of these patients, 15 (50%) had no abnormality of the oesophagus found before perforation. Only 18 (60%) of patients were referred within 24 h of injury. The mean duration of care required in the intensive care unit was 1.5 days +/- 2.5 days and the mean inpatient hospital stay 26.5 days +/- 22.1 days. The mortality was 10% (three cases). Oesophageal perforation remains a serious life-threatening injury. The early diagnosis of this uncommon condition requires a high index of suspicion as the symptoms are often non-specific. Identification of the site of perforation is necessary as the management of cervical and thoracic perforations differs considerably. Early referral combined with appropriate therapy would appear to result in a better outcome than previously published data. It is therefore suggested that patients with this relatively rare condition should be referred as soon as possible to a centre with expertise in its management.