Previous studies indicated that partial agonists cause less desensitization of the ␤ 2 -adrenergic receptor (␤AR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of ␤AR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the ␤AR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified ␤AR behaved identically to the wild type receptor with regard to agonist K d , coupling efficiency, and desensitization. The coupling efficiencies for ␤AR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0 -30 min) of agonist-induced desensitization of ␤AR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine > fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the ␤AR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, ␤AR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of ␤AR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of ␤AR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 ؎ 0.82) was not appreciably different from ephedrine (5.0 ؎ 0.96) and significantly less than that caused by albuterol (10.4 ؎ 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of ␤ARK phosphorylation of the ␤AR.The adenylyl cyclase-coupled ␤AR 1 system has served as a model system for the study of the phenomenon of desensitization of G-protein-coupled receptors (1-5). Desensitization is functionally defined as an attenuation of hormonal responsiveness upon agonist stimulation. There are four currently known mechanisms of agonist-induced desensitization that appear to have physiological significance: receptor sequestration/internalization (1), ␤AR kinase (␤ARK) phosphorylation of serines and threonines on the ␤AR C terminus (3, 5), cAMP-dependent protein kinase (PKA) phosphorylation of ␤AR ...

1 Partial agonists of the b 2 -adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human b 2 -adrenoceptors with antagonist. 2 We have compared the activation and desensitization of human b 2 -adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing b 2 -adrenoceptor levels of 40 ± 70 fmol mg 71 , and in human embryonic kidney (HEK) 293 cell lines expressing 2 ± 10 pmol mg 71 . The e cacy observed for the stimulation of adenylyl cyclase by salmeterol was only %10% of that observed for adrenaline in BEAS-2B cells expressing low levels of b 2 -adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and b 2 -adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3 The desensitization and internalization of b 2 -adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4 Our data suggest that the reduction in the rapid phase of desensitization of b 2 -adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of b 2 -adrenoceptor kinase (bARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

The properties of a pneumatic nebullzer/spray chamber system as an Interface for high-performance liquid chromatography/lnductively coupled plasma optical emission spectrometry are examined. A comparison Is made of alternate spray chamber locations with respect to the column and plasma, and transport mechanisms of liquid and aerosol are contrasted. Peak broadening and distortion are found to result from extended liquid transport whereas some signal loss Is found with aerosol transport over a comparable distance. Placement of the spray chamber external to the plasma box Is found to result In peak |ielghts largely Independent of the mobile phase flow rate.The inductively coupled plasma (ICP) has considerable potential as a highly selective detector for high-performance liquid chromatography (HPLC) (1-6). Both Cast et al. ( 1 ) and Fraley et al. ( 2) have studied the influence of many system variables on the performance of a coupled HPLC/ICP system. The effects of mobile phase composition and flow rates on peak areas (1) as well as the comparison of detection limits for a number of elements (1,2,5, 6) were described. In such studies, however, no specific attempts have been made to examine the behavior of the interface in terms of analyte transport properties. This paper will describe such a study and show how the location of the ICP spray chamber can profoundly influence ICP response under a variety of experimental conditions. EXPERIMENTAL SECTIONAll experiments were performed on a modular HPLC/ICP system. The HPLC pump was a Laboratory Data Control Constametric IIG-dual-module solvent delivery system. A Rheodyne Valve Model 7120 fitted with a 50-mL sample loop was used for sample injections. Sample volumes of 40,30, 20, and 10 mL were obtained by use of a microliter Hamilton syringe with a 22 gauge needle.

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Continuous-flow fast atom bombardment mass spectrometry was used for the identification of the intermediates and end products of the tryptic digest of polypeptides throughout the time-course of the reactions. Precursor/product relationships for these peptides were determined with the aid of a simple personal computer program. The C-terminal tryptic peptides were identified by performing a tryptic digest in 50% 18O-enriched buffer which resulted in labeling of all non-C-terminal peptides with 18O. This information, along with the precursor/product correlations, was used to create a sequence-ordered tryptic map of the original polypeptide. Ion intensities of intermediate hydrolysis products are compared for enzyme to substrate ratios of 1:100 and 1:1000 (w/w) over the course of the reaction. Intermediates were found to have significantly longer lifetimes when lower levels of trypsin were present.