Douglas W. Harris

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

A sodium pump inhibitor has been isolated from human plasma and extensively purified. This material, endogenous digitalislike factor, was examined by a variety of mass spectrometric techniques. A low-resolution fast atom bombardment mass spectrometric analysis of a sample of purified endogenous digitalislike factor revealed a single unique molecular ion in the mass range 100-2,500. The accurate mass was determined to be 585.295 Da in a second highresolution fast atom bombardment mass spectrometric experiment Based on this accurate mass, the elemental composition of endogenous digitalislike factor was determined and found to be identical to the elemental composition of the known cardenolide ouabain. Direct comparison of ouabain and endogenous digitalislike factor by linked scan tandem mass spectrometry, derivatization with acetic anhydride coupled with fast atom bombardment mass spectrometry, and analytical high-performance liquid chromatography failed to reveal any differences. We conclude that the endogenous digitalislike factor isolated from human plasma is ouabain or a closely related isomer. (Hypertension 1991;17:930-935)

Development of an immunoassay for endogenous digitalislike factor.

Recently, attempts to purify and identify a circulating inhibitor of the sodium pump have been successful. Based on the outcome of mass spectral analysis of purified inhibitor, we raised in rabbits antibodies to conjugates of the commercially available cardenolide ouabain and used them in the development of an indirect enzyme-linked immunosorbent assay (ELISA) for endogenous digitalislike factor (EDLF). Antisera obtained were of high antibody titer (l:2xlO6) and showed full cross-reactivity with purified EDLF. The antisera were highly specific for ouabain and structurally related cardenolides but showed no cross-reactivity with numerous endogenous steroids and peptides. At each step in the purification of EDLF, inhibition of the sodium pump and immunologic cross-reactivity were inseparable. The ELISA as developed had a working range of 5-2,000 fimol, with an IC50 of 80 fmol/well. Using solid-phase extraction and the ELISA, we determined the circulating level of EDLF in plasma from normal human volunteers to be 138±43 fmol/ml, whereas patients on total parenteral nutrition for at least 1 week had a circulating level of 108 ±17 fmol/ml, suggesting that the circulating factor was of endogenous origin. The ELISA developed appears to measure a naturally occurring counterpart to the cardenolides that could play a role in modulating the sodium pump and thereby cellular electrolyte homeostasis. (Hypertension 1991;17:936-943)

Effect of norepinephrine uptake blocker on β-adrenergic receptors of the rat cerebral cortex

Abstract The effect of chronic (7 day) intravenous infusion of desmethylimipramine (DMI), amitriptyline, nortriptyline, iprindole, zimelidine, clovoxamine, amphetamine or cocaine on the density of β-adrenergic receptors in the rat cerebral cortex has been investigated in this study. All the drugs except amphetamine and cocaine significantly decreased the density of β-adrenergic receptors in the cerebral cortex. The results have been discussed with respect to the role of norepinephrine in regulation of density of β-adrenergic receptors in the cerebral cortex.

High affinity binding of [3H]ethylketocyclazocine to rat brain homogenate.

Ethylketocyclazocine (Ekc), a potent kappa-receptor agonist, binds to opiate receptor sites in rat brain tissue. The binding was saturable with respect to the concentration of [3H]Ekc. The dissociation constant of Ekc-receptor complex was 1.8 nM and the maximum number of binding sites in the whole brain was 9.6 pmol/g of tissue. Opiate agonists and antagonists have a high affinity for [3H]Ekc binding sites, but results of present investigations failed to differentiate opiates thought to be specific for the mu- and kappa-receptors.

Stimulation of cyclic GMP formation in smooth muscle cells by atriopeptin II.

Addition of synthesized atriopeptin II (AP-2), a 23 amino acid peptide of rat atria, to rat thoracic aorta smooth muscle cells results in the stimulation of cyclic GMP production by the cells. The EC50 for the effect is 81 nM and a 7 fold increase occurs at 10 microM AP-2. Cyclic GMP levels increased within 15 seconds after the addition of AP-2 and were maximal at 5 minutes. Cyclic GMP levels in primary rabbit kidney cells were increased 15 fold by 10 microM AP-2. However, no increase in cyclic GMP was detected in WI-38 fibroblast cells after the addition of 10 microM AP-2. Cyclic AMP levels were not affected by AP-2 in any of these cell systems. The effect upon cyclic GMP accumulation was specific for AP-2; none of the other compounds or peptides tested affected cyclic GMP levels.

Development of a sensitive activity assay for high-volume evaluation of human renin inhibitory peptides in rat serum : results with U-71,038

A sensitive activity assay for high volume evaluation of human renin inhibitory peptides (RIPs) in rat sera (range 2–80 ng/ml) was developed based on the low affinity of RIPs to rat renin and their high affinity to human renin. The utility of this activity assay was tested by measuring concentrations of a human RIP, U-71,038 (BOC-Pro-Phe-N-MeHis-Leu Ψ [CHOHCH2]Val-Ile-Amp), in rat sera, determined by the activity assay, by a sensitive radioimmunoassay (RIA), and by tracking tritiated drug. Rats were given radiolabeled drug as an intravenous bolus, and blood samples were collected at various times after dosing. The serum level of U-71,038 equivalents was determined by the three techniques. Whole blood was also counted for total radioactivity to evaluate the potential for U-71,038 incorporation into red blood cells. Results from the three serum assays indicate good agreement between the calculated U-71,038 equivalents for the 30 min and 1 hr collection times. The 2 and 4 hr collection times show excellent agreement for the activity assay and RIA; [3H]-U-71,038 determinations gave substantially higher values. Serum levels for U-71,038 determined 30 min after dosing averaged less than 300 ng equivalents/ml suggesting that less than 1% of the administered dose was in the systemic circulation at that time. Thus, U-71,038 was rapidly cleared. At the 4 hr collection time, the level of U-71,038 equivalents, as determined by activity assay and RIA, was ten times the in vitro IC50 for the renin inhibitory activity of U-71,038. Analysis of whole blood levels of 3[H]-U-71,038 indicated little or no incorporation of drug related material into red blood cells. In addition to predicting pharmacological response, the activity assay can be used to quantify human RIPs in rat serum when biotransformation is absent.

Use of a cell harvester for the collection of tissue fragments in receptor binding assays.

The use of a commercially available cell harvester in radioreceptor binding assays is described. Scatchard analyses of data acquired using the cell harvester is presented for [3H]clonidine, [3H]prazosin, [3H]quinuclidinyl benzilate (QNB), and [3H]flunitrazepam. Dissociation constants (Kd) and maximum number of binding sites (Bmax) obtained for each ligand are in good agreement with those published by others and there is excellent intra- and interassay agreement. Apparent inhibition constants for various common drugs are also included. Analyses of precision, efficiency, and cost support the use of this cell harvester in binding assays.