Kainate and AMPA receptor binding in seizure-prone and seizure-resistant inbred mouse strains.

Glutamate and its receptors represent the major excitatory neurotransmission system in the mammalian brain and are considered important in the pathogenesis of many neurological diseases. The present study describes saturation binding experiments performed to measure the affinity (Kd) and density (Bmax) of kainate and AMPA receptors in striatum, cortex and hippocampus from mature DBA/2J (DBA) and C57BL/6J (C57) mice. Previous studies have documented that these two strains differ significantly in seizure susceptibility, with DBA mice exhibiting greater sensitivity in various convulsant tests compared to C57 mice. Non-linear regression analysis of binding data together with Student's t-test and ANOVA revealed significantly higher densities of kainate receptors in striatum and of AMPA receptors in cortex of DBA mice. C57 mice exhibited higher striatal [3H]AMPA binding. There were no significant differences between the mouse strains in binding sites prepared from hippocampus and no differences in affinity for either receptor in any brain region studied. The results support a role for kainate and AMPA receptors in seizure sensitivity, possibly by influencing glutamate transmission in specific pathways. It is unlikely, however, that these receptors account for the generation of seizures alone but rather cooperate with other glutamatergic and non-glutamatergic neurotransmitter systems.

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging.

In the present work, we have investigated the effects of the aging process on Ca2+-activated K+ channels (KCa2+) of rat skeletal muscle fibres. KCa2+ channels of adult (5-7 months old) and aged (24-26 months old) rats were surveyed by the patch-clamp technique. In aged rats, KCa2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, KCa2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat KCa2+ channel, measured in the range of potentials from -60 mV to +20 mV, was about 1.5-2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca2+ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K+ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of KCa2+ channels increases with advancing age.

Different sulfonylurea and ATP sensitivity characterizes the juvenile and the adult form of KATP channel complex of rat skeletal muscle.

We have described here the changes of the biophysical and pharmacological properties of the sarcolemmal ATP-sensitive K+ channels (KATP) of rat skeletal muscle fibres, occurring from an early postnatal period (5 days) to adulthood (210 days). The age-dependent changes of the mean current of the KATP channel (channel activity) and the effects of the blockers, ATP and glybenclamide, were examined by using the patch-clamp technique. Measurements of the single channel conductance, open probability and channel density were also performed. Excision of cell-attached patches into an ATP-free solution dramatically increased the KATP channel activity; however, the intensity of this activity was age dependent. The relative activity was low at 5-6 days of postnatal life, increased to a plateau at 12-13 days, then declined toward adult values after 37 days. Two distinct types of the KATP channel complex could be distinguished. The early developmental period (5-6 days) was dominated by a KATP channel having a conductance of 66 pS, a high open probability of 0.602, and an IC50 for ATP and glybenclamide of 123.1 microM and 3.97 microM, respectively. This type of channel disappeared with maturation of the muscle to be replaced by the adult form of the KATP channel. The later developmental period (from 56 days) was dominated by a KATP channel having a 71 pS conductance, but a low open probability of 0.222. This adult channel was also 3.2 and 73.5 times more sensitive to ATP and glybenclamide, respectively. We have also observed that the sensitivity of the KATP channel to ATP and glybenclamide develops differently. Indeed, the greater increase in the sensitivity of the channel to ATP was observed between 5 and 12 days of age. Conversely, the greater enhancement of the sensitivity of the channel to glybenclamide occurred between 12 and 37 days. A further increase of this parameter was also observed between 37 and 56 days of age. The differential age-dependent acquisition of the sensitivity of KATP channels to ATP and glybenclamide poses the hypothesis that in rat skeletal muscle the ATP regulatory site and sulfonylurea site are located on different subunits of the KATP channel complex. The intense KATP channel activity recorded between 12 and 37 days of postnatal life sustains the high resting macroscopic K+ conductance characteristic of the early postnatal development.

The effects of repeated morphine exposure on mu opioid receptor number and affinity in C57BL/6J and DBA/2J mice.

C57BL/6J (B6) mice self-administer substantial quantities of morphine compared to DBA/2J (D2) mice, and most of the genetic component of this strain difference has been attributed to a locus on chromosome 10 in the vicinity of the mu opioid receptor gene. To compare binding characteristics of mu opioid receptor populations between the two strains, mice were given single daily injections of a long-acting preparation of morphine sulfate (80 mg/kg, s.c.) or saline for a period of seven days, and euthanatized six hours after the last injection. Brains were removed and dissected into specific regions. Receptor binding studies were performed on frontal cortex and striatum. Data were analyzed using non-linear regression, and Kd and Bmax comparisons made between strains and treatments. Specific [3H]DAMGO binding in striatum indicates that the density of mu opioid receptors in saline-treated B6 mice and saline-treated D2 mice does not differ significantly. After repeated morphine injection, B6 mice exhibited a decrease in striatal [3H]DAMGO binding, indicating a downregulation of receptor density by approximately 45% (p=.0003 vs saline-treated B6), a phenomenon not observed in D2 mice. In frontal cortex, no differences in [3H]DAMGO binding were observed between strains or treatment groups. These results demonstrate a significant difference between mu opioid receptor regulation in B6 and D2 mice, and may underlie well documented strain differences in specific opioid-related behaviors.

High-performance liquid chromatographic assay for dilevalol in human plasma and urine using a PRP-1 column and fluorimetric detection.

A single high-performance liquid chromatographic (HPLC) assay for the quantitative determination of dilevalol, the R,R isomer of labetalol, was developed for both plasma and urine. A significantly improved limit of detection for dilevalol in plasma was accomplished by extensive modification of an HPLC assay originally developed in our laboratory for labetalol. This simplified method is readily adaptable to urine and represents the first reported HPLC assay for the quantitative determination of dilevalol in this biofluid. Drug was recovered from plasma or urine by partition into diethyl ether under mildly alkaline conditions and back-extraction into dilute acid. Reversed-phase separation of dilevalol and the internal standard was accomplished on a 150 X 4.1 mm column commercially packed with a spherical (5 micron) macroporous copolymer (PRP-1). No interferences were observed in extracts obtained from drug-free plasma or urine. Selectivity for dilevalol in the presence of other beta-blockers was established. This method demonstrated a linear detector response to concentrations of unchanged drug typically observed in urine and plasma following once-a-day treatment with dilevalol hydrochloride (100-800 mg). The lowest limit of reliable quantitation was established at 1 ng/ml in plasma. The intra-assay precision (coefficient of variation) remained less than 6% at all concentrations evaluated from 1 to 800 ng/ml. In urine, the lowest limit of quantitation was validated to 20 ng/ml where the intra-assay precision (coefficient of variation) for unchanged drug was less than 4% at all concentrations evaluated up to 400 ng/ml. This method is suitable for routine quantitation of unchanged drug in human plasma and urine following the administration of therapeutically effective doses of dilevalol hydrochloride.

High-performance liquid chromatographic assay for azatadine in human urine.

A high-performance liquid chromatographic assay for the quantitative determination of azatadine and a base (1 M sodium hydroxide) hydrolyzable conjugate of azatadine in human urine has been developed. Reversed-phase separation of azatadine and the internal standard, 8-chloroazatadine, was accomplished on a 300 X 3.9 mm I.D. mu Bondapak CN column. Following liquid-liquid extraction from urine, azatadine was quantitatively determined by UV detection at 214 nm. No interferences were observed in the extracts obtained from drug-free urine. Detector response (peak area ratio) was linear from 10 to 2500 ng/ml. This method has been shown to provide accurate and precise determinations of the unchanged and hydrolyzed drug in human urine, following the twice daily oral administration (1-2 mg) of azatadine maleate.

High-performance liquid chromatographic method for the determination of trace amounts of acetaminophen in plasma.

A method is described for the rapid, sensitive, and precise quantitation of acetaminophen in human plasma. The assay involved a single acetonitrile extraction and high-performance liquid chromatographic analysis using a reverse-phase column, with a mobile phase of methanol and water. The limit of quantitation of acetaminophen by this method was 8 ng/mL; only 0.1 mL of the plasma sample was required for the determination. N-Propionyl-p-aminophenol was used as the internal standard.