New insights on P2X purinoceptors

Significant advances in understanding of P_2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P_2X receptor heterogeneity, from data with agonists, has recently been reported.A number of new antagonists at P_2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P_2X purinoceptor.Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P_2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P_2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P_2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P_2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P_2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.     

Propofol Increases Contractility during α1a-Adrenoreceptor Activation in Adult Rat Cardiomyocytes

Background: The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca2+ sensitivity, and contraction of individual cardiomyocytes during activation of [alpha]1a adrenoreceptors and to determine the cellular mechanism of action.

Methods: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine ([alpha]1b-adrenoreceptor antagonist) and BMY 7378 ([alpha]1d-adrenoreceptor antagonist). Data are reported as mean +/- SD.

Results: Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKC[alpha], PKC[delta], PKC[varepsilon], and PKC[zeta] reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+-H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001).     

Role of Doppler ultrasound flowmetry in the diagnosis of breast lumps

One hundred and five patients with discrete breast lumps were examined with a 10 MHz Doppler ultrasonic flowmeter. Doppler flow signals were analysed on an Angioscan spectrum analyser. Recordings from the opposite normal breast were taken as controls and signals from the two sides compared. In 23 patients signals from the normal breast could not be recorded and therefore results of the remaining 82 patients are reported. These included 39 patients with carcinoma, 20 with fibroadenoma, 12 with cyclical nodularity and 11 with cysts. Malignant lumps exhibited significantly higher peak systolic (S) and minimum diastolic frequencies (D) in comparison to the control breast. Fibroadenoma also had a higher S and D than those of the opposite normal breast. Signals over cysts and cyclical nodularity showed no significant difference from the recordings over the control side. Despite significantly higher systolic and diastolic frequencies in the cancer group in comparison to benign lumps and normal breast, considerable overlap in the values was seen between cancer and other groups. Therefore the patterns on 10 MHz Doppler sonography are not sufficiently specific to discriminate benign from malignant breast lumps.     

Personal exposure to house dust mite allergen in bed: nasal air sampling and reservoir allergen levels

BACKGROUND: Assessment of personal exposure to dust mite allergen has relied on proxy measures. Only recently has a means to directly measure inhaled allergen particle number become available (the intra-nasal air sampler). OBJECTIVE: To quantify inspired dust mite group 1 and group 2 allergen-bearing particles in bed in undisturbed conditions prior to sleep by nasal air sampling and to investigate the relationship between inhaled particles and reservoir allergen levels. METHODS: Twelve volunteers wore nasal samplers in bed for 6 evenings, nose-breathing in undisturbed conditions. Allergen-bearing particles ('halos') were detected by immunostaining for Der p 1, Der p 2, or Der p 1 and Der p 2 together, and counted by light microscopy. Count data were square root transformed for analysis of variance. Mattress dust samples were assayed for Der p 1 and Der p 2 concentrations. RESULTS: Square root detransformed mean particle counts per 30-min sample were: Der p 1, 4.22; Der p 2, 5.9; Der p 1 + Der p 2, 4.87; and for all samples, 5.01, with no difference between the groups. With replicate samples, halo number correlated significantly with mattress allergen concentrations (Der p 1 r = 0.80, P < 0.01; Der p 2 r = 0.68, P < 0.02). CONCLUSION: Nasal air sampling can be used to quantify nocturnal Der p exposure in undisturbed conditions in an area with moderate exposure to mite allergen and can provide a direct measure of inhaled mite allergen. The choice of either Der p 1 or Der p 2 is appropriate for this purpose.     

Motivation and Maternal Presence during Induction of Anesthesia

Background: The authors developed a measure to determine whether maternal motivation to be present during induction (Motivation for Parental Presence during Induction of Anesthesia [MPPIA]) is related to children's anxiety during the induction process.

Methods: Mothers and children (aged 2-12 yr) undergoing outpatient, elective surgery and general anesthesia were enrolled in this study (n = 289 dyads). Items to assess motivation for parental presence during induction were selected by experts in anesthesiology, psychology, and child development; mothers completed the resulting 14-item measure as well as assessments of anxiety and coping style. Children's anxiety and compliance was assessed during induction of anesthesia. Factor analysis was performed, and maternal motivation was then examined against children's anxiety during induction of anesthesia.

Results: Factor analysis resulted in four scales with a total variance of 72.3%: MPPIA-Desire, MPPIA-Hesitancy, MPPIA-Anxiety, and MPPIA-Preparation. Analysis supported the reliability (0.89-0.94) and validity of the MPPIA. The authors found that mothers with high MPPIA-Desire and low MPPIA-Hesitancy had children with significantly higher anxiety (P < 0.0001) during induction of anesthesia, as compared with mothers with low MPPIA-Desire and MPPIA-Hesitancy. The authors also found that highly motivated mothers reported significantly higher levels of anxiety (P = 0.007).     

Flavone acetic acid as a modifier of endothelial cell function.

Flavone acetic acid (FAA) causes significant regression of larger established tumors in murine systems in vivo, but is only slightly toxic in vitro. This in vivo effect is thought to be indirect, or immunological, rather than a direct cytotoxic effect on tumor cells. Using the WHFIB fibrosarcoma, which grows both in vivo and in vitro, and the murine endothelial cell line B10, we have studied the effect of FAA on the survival of tumor and endothelial cells in vitro. The times taken for 1 mg ml-1 FAA to reduce survival to 0.1 surviving fraction were 63 hr for B10 and greater than 85 hr for WHFIB in vitro. WHFIB tumors in vivo were more sensitive than tumor cells in vitro, a single dose of 150 mg kg-1 FAA inducing a tumor growth delay of 10 days at treatment size + 2 mm. As FAA is more toxic to tumor-bearing animals than to those which are non-tumor bearing the effect of tumor conditioned medium on the cytotoxicity of FAA toward B10 cells was studied; no enhanced effect was seen. As FAA is only weakly cytotoxic in vitro to endothelial cells, and even less so to tumor cells, sublethal effects of FAA on endothelial cell function in vitro were studied. The permeability of monolayers of human unbilical vein endothelial cells (HUVEC) in vitro is transiently increased by FAA. Also, procoagulant activity of HUVEC is induced by FAA and this activity is further enhanced in the presence of a factor isolated from Meth-A tumor cells.     

The superoxide anion may mediate short- but not long-term effects of ultraviolet radiation on melanogenesis

Abstract The present study was carried out to examine the role of reactive oxygen species in mediating the melanogenic effects of UVR. B16 mouse melanoma cells responded to a single dose of UVR by showing increases in their melanin content. Although there was a small increase in melanin at 48-72 hours, which was associated with a rise in tyrosinase activity at 48 h, the greatest change occurred at 3 h and this was not associated with an increase in tyrosinase activity. This short-term response, unlike the more delayed melanogenic response, was reduced by superoxide dismut-ase (SOD). Xanthine oxidase (XO), which generates the superoxide anion (O₂). also increased the melanin content of B16 melanoma cells with effects at 3 h and 48 h. As with UVR, the delayed response was accompanied by an increase in tyrosinase activity but no such association was evident at 3 h. In addition, the short-term effect, like that seen with UVR, was reduced with SOD and to a lesser extent with catalase. In contrast to the effects found with XO, glucose oxidase, which generates hydrogen peroxide, had no effect on the melanin content or tyrosinase activity of the B16 cells. These results confirm previous observations that UVR is able to act directly on cells to bring about delayed increases in melanogenesis. They further demonstrate that UVR also stimulates melanogenesis through a more rapid action that is not associated with an activation of tyrosinase. This effect could be mediated by the O₂ which, rather than activating tyrosinase, could act by serving as a substrate for the enzyme.     

Reliability of radiographic measurement of fracture angulation in children with femoral shaft fractures

INTRODUCTION: Clinical decisions are based on x-ray measures of radiographs. However, the reliability of assessing the angular measurement of fractured femurs in children is unknown. The purpose of this study was to determine the intra- and interrater reliability of an x-ray assessment of femoral shaft fracture angulation in children. METHODS: On 2 occasions, 3 raters evaluated 30 radiographs of children aged 4 to 10 years, 3 to 24 months after a femoral fracture. The radiographs were evaluated by an orthopedic surgeon, an orthopedic fellow and a research assistant, using a standardized methodology. Reliability was assessed with the intraclass correlation coefficient (ICC), an index of concordance that ranges from 01, where > 0.75 indicates excellent concordance. RESULTS: The ICC for the intrarater ranged from 0.75 to 0.97. The interrater reliability ICC for anterior or posterior angulation was 0.91 (95% confidence interval [CI] 0.840.95) and for varus or valgus angulation was 0.90 (95% CI 0.790.95). A change in measurement of more than 5 degrees exceeds the variation in measurement and, therefore, can be attributed to a true shift in fracture position. CONCLUSIONS: With specific standardized protocols, radiographic assessment of femoral fractures is reliable, irrespective of the examiner's level of experience.