Donald W. DuCharme

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)

Purification of an endogenous digitalislike factor from human plasma for structural analysis.

In previous reports, we described the isolation and characterization of an endogenous digitalislike factor (EDLF). In this report, we describe a unique combination of bioassay and large-scale purification methodology that made possible the purification of sufficient quantities of this inhibitor of Na+,K+-ATPase for structural analysis. Using an initial XAD-2 extraction and preparative high-performance liquid chromatography followed by a batch enzyme affinity extraction and two subsequent semipreparative chromatographic steps, 300 1 of human plasma was processed, yielding 31 /tg (53 nmol) of pure EDLF and representing purification on a dry weight basis in excess of 0.6 billionfold. Four divergent pieces of evidence, including chromatographic, mass spectrometric, immunoreactive, and binding characteristics, suggested that the EDLF purified in the present study was either ouabain or an isomer of ouabain. This material may represent a plasma-borne, naturally occurring, selective, high-affinity ligand for the digitalis binding site that may play a significant role in the modulation of the sodium pump and thereby cellular electrolyte homeostasis in humans. {Hypertension 1991;17:923-929)

Bronchopulmonary and cardiovascular effects of prostaglandin D2 in the dog

The effects of intravenously administered prostaglandin D2 (PGD2) on bronchopulmonary and cardiovascular functions were examined in the dog. PGD2 (0.03-1.0 microng/kg) was shown to be more active than PGF2alpha, a known bronchoconstrictor, in decreasing dynamic lung compliance, tidal volume, and expiratory airflow rate, as well as in elevating lung resistance. PGD2 demonstrated a potency approximately 4-6 times that of PGF2alpha on pulmonary mechanics. Atropine sulfate infusions reduced significantly the resistance and compliance responses to PGF2alpha, but only the resistance responses to PGD2, thereby suggesting that part of the bronchoconstrictor activities of these agents involved a cholinergic component. In another series of anesthetized dogs, PGD2 (0.1-10.0 microng/kg) increased pulmonary arterial pressure (comparable to PGF2alpha) and heart rate (greater than PGF2alpha, but less than PGE2), while concomitantly decreasing systemic arterial pressure in a dose-related manner (1/10 that of PGE2). Qualitatively similar alterations in cardiovascular parameters were obtained for PGD2 in conscious dogs. Therefore, potent biologic activity of PGD2 has been shown in the dog. No physiologic or pathologic role for PGD2 has yet been demonstrated, but nonetheless, since it is a naturally occurring PG derived from arachidonic acid, further studies are warranted.

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)

Rat adrenal cortex is a source of a circulating ouabainlike compound.

To determine if the adrenal gland may be the source of plasma-borne ouabainlike compound (OLC) in rats, we 1) measured immunoreactivity expressed as OLC equivalents in extracts from a wide variety of central and peripheral tissues and, for adrenal cortex and medulla, chromatographed the extracts to determine to what extent immunoreactivity in the adrenal was OLC, and 2) measured OLC in the plasma of adrenalectomized and adrenal demedullectomized rats. The highest levels of immunoreactivity were found in adrenal cortex, adrenal medulla, atria, and the pituitary. Based on high-performance liquid chromatographic retention time, immunoreactivity in the adrenal cortex was almost exclusively immunoreactive OLC. Removal of this rich source of OLC from rats resulted in an approximate 50% decrease in circulating levels of OLC by 6 days after removal. Furthermore, although adrenal demedullectomy also caused a decrease in OLC 3 days after surgery, the decline was sustained only with total adrenalectomy, in that plasma levels of OLC in demedullectomized rats 6 days after surgery had returned to levels equal to those of sham controls. Taken together, these findings strongly suggest that the adrenal cortex is a major contributor to circulating OLC in the rat.

Pathogenesis of cardiovascular alterations in dogs treated with minoxidil.

Minoxidil and other potent vasodilators cause coronary arterial injury, right atrial hemorrhagic lesions, and subendocardial necrosis in dogs. This paper discusses the pathogenesis of coronary arterial and right atrial lesions associated with minoxidil in the dog. Acute coronary vascular injury characterized by segmental medial hemorrhage and necrosis and perivascular inflammation occurred only during the first few days of treatment, after which tolerance to further acute injury developed. At 30 d or more of treatment, coronary vascular injury was characterized by perivascular fibrosis rarely attended by medial distortion or hyperplasia and subintimal thickening, changes consistent with responses to previous injury. Right atrial hemorrhagic lesions, unlike coronary vascular injury, often became progressively more extensive with continued treatment. At 3 d, atrial hemorrhage and inflammation were confined to the subepicardium of the right atrium, evidently around affected subepicardial branches of the right coronary artery. At 30 d, fibrovascular proliferative right atrial lesions (granulation tissue with evidence of continual hemorrhage) extended from the epicardium to the myocardium, with eventual replacement of the atrial wall by mature connective tissue at 1 yr of treatment. Minoxidil-induced cardiovascular lesions were not prevented by treatment with a β-blocker (propranolol), or an α-blocker (dibenzylene), or by sympathetic neural activity suppression (surgical sympathectomy or constant carotid sinus nerve stimulation), suggesting that the sympathetic response to the pharmacologic activity of minoxidil was not responsible for the induction of the cardiovascular lesions. Minoxidil-related vascular lesions were confined to the most pharmacologically responsive segment of the arterial system, the coronary arteries, suggesting that medial injury may have been associated with tensile changes in the arterial wall.

The Pharmacologic Basis of the Cardiovascular Toxicity of Minoxidil in the Dog

Minoxidil (MNX), like several other vasoactive drugs, causes cardiovascular toxicity in dogs by undetermined mechanisms. We studied the mechanism of cardiovascular toxicity of MNX [an adenosine triphosphate (ATP)-sensitive potassium channel opener] by blocking its pharmacologic effects with glyburide (an ATP-sensitive potassium channel blocker) in groups of 5 female beagle dogs treated orally for 2 days with 1.0 mg/ kg/day of MNX alone or with glyburide given in 5 or 6 divided doses of 300 mg/kg at 2 hr before and after each dose of MNX and at 3-6-hr intervals thereafter. A third group of 5 dogs received glyburide alone in the same dosing regimen as in the combination group. Mean arterial pressure (MAP), heart rate (HR), the pharmacokinetics of MNX, and gross and microscopic changes in the heart were evaluated. Glyburide did not influence the pharmacokinetics of MNX but prevented or markedly attenuated the MNX-induced cardiovascular lesions (right atrial hemorrhagic lesions, subendocardial necrosis, or coronary arteritis) occurred in dogs whose MNX-induced hemodynamic effects were effectively blocked by glyburide. In conclusion, the cardiovascular toxicity of MNX in dogs is not caused by a direct toxic effect of MNX on the heart but apparently is related to the exaggerated pharmacologic/profound hemodynamic effects it elicits in the dog.

Bronchodilator effects of prostacyclin (PGI2) in dogs and guinea pigs

prostacyclin (PGI2), a recently discovered unstable product in the biosynthetic conversion of prostaglandin endoperoxides, was examined for bronchopulmonary actions. in anesthetized dogs, PGI2 given i.v. (0.3-30.0 microgram/kg) and by aerosol (0.002-0.2%) inhibited significantly PGF2 alpha-induced increases in pulmonary resistance and decreases in dynamic lung compliance in a dose-related fashion. Intrinsically, PGI2 affected resting bronchopulmonary and cardiac functions minimally, but decreased peripheral and pulmonary vascular pressures. PGI2 (0.1-10 mg/kg, i.p.) afforded protection against histamine-induced asphyxial collapse in normal guinea pigs and ovalbumin-induced anaphylaxis in sensitized animals. Cumulative concentrations of PGI2 (1.0 x 10(-9)--3.0 x 10(-4) M) relaxed contractions of the isolated guine pig trachea produced by carbachol. These bronchodilator and hemodynamic effects could not be ascribed to the stable metabolic product of PGI2, because 6-keto-PGF1 alpha was inactive or markedly less active than PGI2 in these test systems. The results of this investigation suggest that PGI2 possesses considerable bronchodilator and vasodilator activity in experimental animal systems.

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs.

Prostaglandin (PG) D3 has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 microgram/kg) of PGD3 and also PGD2 were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD3. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD2, while PGD3 was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 microgram/kg/min) of PGD3 and PGd2 were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD3 infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD2. In contrast, PGD3 was less potent than PGD2 as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD3 and PGD2 were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.

Cardiovascular actions of the hypotensive agent, N, N-Diallylmelamine (U-7720).

SummaryDiallylmelamine is an effective hypotensive agent in hypertensive dogs and rats, having a duration of action exceeding twenty-four hours from a single oral dose. It has limited efficacy in normotensive rats. Hypotensive activity of gradual onset is preceded by a latent period of up to two hours and becomes maximal six hours or more after dosing. This agent does not depress cardiac output or sympathetic vasoconstrictor activity. It is suggested that its hypotensive activity results from a direct effect upon vascular smooth muscle.