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.

A non-radioactive iothalamate and p-aminohippuric acid high-performance liquid chromatographic method for simultaneously measuring glomerular filtration rate and renal blood flow in the rat.

A high-performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p-aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, and an aliquot chromatographed on a C18 reversed-phase column. The mobile phase was comprised of 0.1 M sodium phosphate with 1.2 mM tetrabutylammonium phosphate: methanol, 85:15 (v/v), at a flow rate of 1.0 mL/min. The analytical column eluate was monitored with a UV detector at 254 nm with quantitation achieved using peak-height ratios. The precision of the method was 6.6 and 3.6% for iothalamate in serum and urine, and 5.6 and 4.9% for PAH in serum and urine, respectively. The lower limit of quantitation was 0.63 microgram/mL for iothalamate and 1.25 microgram/mL for PAH in serum, and 3.1 microgram/mL for iothalamate and 1.5 microgram/mL for PAH in urine. Recovery of iothalamate from serum and urine was 99.9 and 93.5%, respectively. Recovery of PAH from serum and urine was 99.8 and 92.6%, respectively. The present study demonstrated that non-radioactive iothalamate and PAH can be measured simultaneously using a HPLC assay to measure GFR and ERBF in the male rat.

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.

Identification and characterization of a ouabain-like compound from human plasma.

The plasma membrane sodium-potassium pumps that regulate intracellular sodium in most animal cells have specific, high-affinity receptors for the digitalis glycosides and their aglycones. This has fostered speculation that there is an endogenous ligand. We have purified and structurally identified by mass spectroscopy an endogenous substance from human plasma that binds with high affinity to this receptor and that is indistinguishable from the cardenolide ouabain. This human ouabain-like compound (OLC) displaces [3H]ouabain from its receptor, inhibits Na,K-ATPase and ouabain-sensitive 86Rb+ uptake, and has cardiotonic actions quantitatively similar to commercial ouabain. Immunoreactive OLC was detected in the plasma of many mammals, and high concentrations were found in the adrenals. The circulating OLC may modulate intracellular Na+ and affect numerous Na+ gradient-dependent processes including intracellular Ca2+ and pH homeostasis in many tissues. Furthermore, altered circulating levels of OLC may be associated with the pathogenesis of certain forms of hypertension.

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 l of human plasma was processed, yielding 31 micrograms (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.

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 high-resolution 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.

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 (1:2 x 10(6)) 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 fmol, 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.

Effects of an endogenous ouabainlike compound on heart and aorta.

An endogenous ouabainlike compound (OLC) has been purified from human plasma, and mass spectrometry has shown it to be indistinguishable from plant-derived ouabain. This human OLC was tested for its effects on evoked tension in guinea pig left atria and aortic rings. The tissues were incubated at 37 degrees C in bicarbonate-buffered physiological salt solution gassed with 95% O2-5% CO2. In atria stimulated electrically at 1 Hz, 85 and 170 nM human OLC increased peak active force to 177 +/- 15% and 313 +/- 32% of control, respectively (n = 3), with little effect on the duration of contraction. On washout of the OLC, peak systolic force returned to the control level with a half-time of 4.3 +/- 0.5 minutes. Similar results were obtained with 160 nM plant-derived ouabain: peak systolic force increased to 310 +/- 31% of control (n = 4) and returned to the control level with a half-time of 3.8 +/- 0.2 minutes during washout. In aortic rings, neither 170 nM human OLC nor 160 nM plant ouabain (30-minute treatments) affected resting (unstimulated) tension, but they increased the contractions evoked by histamine (0.2-1.0 microM) to 156 +/- 13% (n = 4) and 143 +/- 6% (n = 4) of control responses, respectively. The mean half-time for washout of the OLC and plant ouabain-induced augmentation of histamine-evoked tension exceeded 35 minutes. These data show that human OLC has cardiotonic and vasotonic actions qualitatively and quantitatively similar to those observed with plant ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Renin inhibitory peptides. Incorporation of polar, hydrophilic end groups into an active renin inhibitory peptide template and their evaluation in a human renin infused rat model and in conscious sodium-depleted monkeys.

We previously reported that Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]-Ile-Amp (1) is a potent and specific inhibitor of human renin in vitro. It was shown to resist degradation by selected proteases and a rat liver homogenate. It was shown to inhibit plasma renin activity and to reduce blood pressure in renin-dependent-animal models both by the intravenous and by the oral routes using dilute citric acid as vehicle. In an effort to discover compounds with improved pharmacological efficacy, we set out to modify the physical characteristics of this highly lipophilic renin inhibitor by incorporation of hydrophilic end groups. We report here a variety of water-solubilizing groups and the resulting structure-activity relationship of these compounds. They all maintain an extremely high level of enzyme inhibitory activity in vitro. Evaluation of these potent renin inhibitors in a human renin infused rat model suggests that some of these compounds exhibit improved pharmacological efficacy in vivo. This observation was further confirmed in the conscious sodium-depleted cynomolgus monkey. Importantly, the oral efficacy was demonstrated in a water vehicle in the absence of citric acid.

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 beta-blocker (propranolol), or an alpha-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.

Pyrimidine and triazine 3-oxide sulfates: a new family of vasodilators.

Di- and triaminopyrimidine 3-oxides (e.g., 2,4-diamino-6-piperidinylpyrimidine 3-oxide and 2,4-diamino-6-(diallylamino)triazine 3-oxide) react with sources of sulfur trioxide, such as sulfur trioxide trimethylamine or chlorosulfuryl chloride, to yield the corresponding heterocyclic O-sulfates. These sulfates are inner salts with unusual physical properties. The structure of the O-sulfate of 2,4-diamino-6-piperidinylpyrimidine 3-oxide was confirmed by X-ray. These O-sulfates are hypotensives. They apparently act by direct vasodilation.

U-54,669F: a novel hypotensive agent.

U-54,669F, a new antihypertensive agent, administered orally was associated with dose-related hypotensive responses in conscious, spontaneously hypertensive, and normotensive rats (0.015-0.5 mg/kg) and in supine conscious monkeys (1-10 mg/kg). No loss of hypotensive efficacy of U-54,669F was observed after 1 wk of daily repetitive treatment. U-54,669F did not alter electrical postganglionic sympathetic nerve activity or postsynaptic sympathetic function. Hypotensive responses to U-54,669F were blunted in spinal cats. U-54,669F was associated with dose-related decreases in norepinephrine (NE) levels in plasma and in cardiac and splenic tissue, whereas brain NE was unaltered. U-54,669F attenuated vascular responses associated with electrical stimulation of sympathetic nerves. However, at hypotensive doses, U-54,669F did not impair the ability of monkeys to withstand orthostatic stress, or contraction of the nictitating membrane secondary to sympathetic stimulation in the cat. U-54,669F appears to alter peripheral sympathetic neurogenic function, but apparently does not enter the central nervous system and does not impair the ability to withstand orthostatic stress at effective hypotensive doses.

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.

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.

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.