PubMed · 8097079
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C W Wagner. 1993. Monitoring asthma.. https://pubmed.ncbi.nlm.nih.gov/8097079/
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Rapid regulation of G protein-coupled receptors appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization. In this report we show that the beta-adrenergic receptor kinase (betaARK-1 or GRK2) follows a similar pattern of internalization upon agonist activation of beta2-adrenergic receptors (beta2AR) and that betaARK expression levels modulate receptor sequestration. Stable cotransfected cells expressing an epitope-tagged beta2AR and betaARK-1 show an increased rate and extent of beta2AR internalization compared with cells expressing receptor alone. Moreover, subcellular gradient fractionation studies suggest that betaARK colocalizes with the internalized receptors. In fact, double immunofluorescence analysis using confocal microscopy shows extensive colocalization of beta2AR and betaARK in intracellular vesicles upon receptor stimulation. Our results confirm a functional relationship between receptor phosphorylation and sequestration and indicate that betaARK does not only translocates from the cytoplasm to the plasma membrane in response to receptor occupancy, but shares endocytic mechanisms with the beta2AR. These data suggest a direct role for betaARK in the sequestration process and/or the involvement of receptor internalization in the intracellular trafficking of the kinase.
The involvement of K+ channels in regulating secretion of amylase from isolated rat parotid acini was studied in conjunction with beta-adrenoceptor function. It was observed that increasing the concentration of extracellular K- or adding K+ channel blockers enhanced the secretion of amylase. Among several K+ channel blockers, tetraethylammonium, apamin and charybdotoxin were effective to enhance secretion by 48, 69 and 84%, respectively. Glibenclamide was without effect. A low concentration of isoprenaline (10(-7) M) enhanced secretion by 154% and its simultaneous application with tetraethylammonium gave a synergistic effect, producing 371% stimulation. Combination of tetraethylammonium and a low concentration of carbachol (10(-6) M) did not give the synergistic effect. Isoprenaline at the concentration of 10(-6) M enhanced secretion by 313% and this was reduced to 116% by 10(-5) M valinomycin, a K+ ionophore. Valinomycin was without effect on carbachol (10(-5) M)-induced secretion. Somatostatin (10(-5) M) and morphine (10(-4) M) also reduced isoprenaline-induced secretion of amylase. These results suggested the regulation of Ca(2+)-activated K+ channels by isoprenaline in amylase secretory processes in parotid acini.
Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including beta-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of alpha-nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.