James H. Ludens

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)

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.

Protection from ischemia-induced cerebral edema in the rat by U-50488H, a kappa opioid receptor agonist

U-50488 is a specific kappa opioid agonist which produces in rats water diuresis resulting in an elevation of plasma osmolarity. Pretreatment with U-50488H (the methanesulfonate salt) in Fisher rats prior to 4 h of bilateral carotid occlusion prevented the development of edema in the forebrain, and the effect was greater than that from pentobarbital anesthesia. An additional injection of an antidiuretic hormone which prevented the plasma hyperosmolarity also significantly reduced the anticerebral edemic effects of U-50488H. The plasma osmotic effect, however, may not completely account for the ischemic protection produced by U-50488H.

Displacement of 3H-EKC binding by opioids in rat kidney: A correlate to diuretic activity

Multiple opioid binding sites have been documented in brain tissue. In this study we report on the presence of binding sites for the opioid ethylketocyclazocine (EKC) in a membrane fraction of rat kidney. Binding appeared to be selective in that opioids varied markedly in their capacities to displace 3H-EKC. Correlating with the capacity of an opioid to displace 3H-EKC was the ability to produce diuresis. Although our studies cannot assign a particular physiological or pharmacological role for the renal EKC binding sites, binding studies of this nature may, nonetheless, be a means by which diuretic activity of opioids can be predicted.

Digitalis-like factor and ouabain-like compound in plasma of volume-expanded dogs

Previous studies demonstrated that ouabain-like compound (OLC) is widely distributed in mammalian species and is found in a variety of different tissues. Although much evidence suggests that OLC is endogenous to mammals, little information is available concerning physiological and/or pathophysiological roles for OLC. In this study, generic endogenous digitalis-like factor (E-DLF) was measured using an enzyme bioassay and the more specific OLC was determined using ouabain antisera in plasma drawn from dogs before and 30 and 120 min after massive volume expansion with isotonic saline. Plasma OLC was not changed by the saline load, whereas Na excretion was significantly elevated at the 30-min blood draw and remained elevated at the 120-min blood draw. Because renal exposure to OLC did not change with saline loading, it is unlikely that any portion of the profuse natriuresis in these animals could be attributed to OLC. In contrast, plasma E-DLF was higher after the saline load than before in each of four dogs at 30 and 120 min after the infusion. What portion of the profuse natriuresis can be attributed to E-DLF is unknown, although it is reasonable to assume that nanomolar levels of pump inhibitor contributed to the natriuresis to some degree.

Role of ADH in ethylketocyclazocine-induced diuresis: studies in the Brattleboro rat.

Kappa opioids produce diuresis presumably through ADH. We investigated further the role of ADH in kappa-induced diuresis by utilizing the Brattleboro rat, a strain lacking endogenous ADH. Ethylketocyclazocine (EKC), a kappa opioid prototype, increased urine formation in Sprague-Dawley, but not in Brattleboro rats. Furthermore, EKC pretreatment abolished the antidiuretic response to ADH administered exogenously to Brattleboro rats. Our study suggests that, in addition to a fall in plasma ADH reported previously, kappa opioids have direct effects on the renal response to ADH.

Renal and vascular effects of chemically distinct ATP-sensitive K+ channel blockers in rats

Summary The ATP-sensitive K+ channel Mocker U-37883A was given intravenously (i.v.) to rats to determine if pharmacologic diversity among ATP-sensitive K+ channels observed in vitro is also apparent in vivo. Vascular K+ channel blockade was quantified as inhibition of the decrease in blood pressure (BP) produced by a standard dose of the K+channel opener pinacidil. Renal natriuretic effects were evaluated by an increase in urinary Na+ excretion. In both instances, effects of U-37883A. a guanidine, were compared with those of the sulfonylurea glyburide. In addition, the ability of these K+ channel blockers to lower plasma glucose levels was compared. U-37883A was nine times more potent than glyburide as a natriuretic and a comparable six times more potent than glyburide in blocking pinacidil. suggesting common features between ATP-sensitive K+ channels in vascular smooth muscle (VSM) and renal tubules. In contrast, a dose of U-37883A that blocked pinacidil and increased Na+ excretion had no effect on plasma glucose, whereas a dose of glyburide that was natriuretic and equally as effective against pinacidil as U-37883 A decreased plasma glucose, suggesting that ATP-sensitive K+ channels in pancreatic β cells and renal tubules have dissimilar binding sites and/or features. U-37883A appeared to be more renal/vascular selective, an observation entirely consistent with previous findings in vitro. Our results represent the first in vivo suggestion of structural differences among the channel and/or accessory proteins from this family of K+-selective channels.

Effect of chronic treatment on the cardiovascular and behavioral responses of 8-OH-DPAT in conscious normotensive rats

Cardiovascular and behavioral responses induced by intravenous administration of the serotonin (5-HT)1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), were studied in conscious normotensive rats either after a single administration, after repeated subcutaneous treatments (1 mg/kg daily for 3 days), or after chronic intravenous infusion (200 micrograms/kg per h for 72 h). In naive rats, a single intravenous treatment with 10, 30 or 100 micrograms/kg 8-OH-DPAT produced a blood pressure reduction of approximately 10% and a heart rate reduction of 15-20%. The duration of blood pressure and heart rate reduction was dose-dependent. Behavioral responses were observed (i.e. reciprocal forepaw treading, flat body posture, hind limb abduction and headweaving), the severity and duration of which were also dose-dependent. Subcutaneous pretreatment with 8-OH-DPAT greatly reduced the behavior responses but did not alter the hypotensive or the heart rate response to a single intravenous administration of 8-OH-DPAT. Blood pressure and behavior were not monitored during the subcutaneous pretreatment period. Intravenous infusion of 8-OH-DPAT attenuated both the cardiovascular and behavioral effects to post-infusion intravenous treatment. The differential tolerance development to these responses suggests that 8-OH-DPAT may exert its blood pressure response and its behavior response through two distinct mechanisms.

K-atp-blocking Diuretic Pnu-37883a Reduces Plasma Renin Activity in Dogs

We determined the effects of the K-adenosine triphosphate (ATP)-blocking diuretic PNU-37883A on plasma renin activity (PRA) in conscious and anesthetized dogs. In conscious dogs, oral PNU-37883A (6-60 mg/kg) was less potent than hydrochlorothiazide (0.15-1.5 mg/kg) and furosemide (FURO; 0.3-3.0 mg/kg) but exhibited high natriuretic efficacy with little kaliuresis. Unlike the standard diuretics, PNU-37883A reduced PRA by 46-76%, and its high dose minimally affected 24-h urinary aldosterone excretion. PNU-37883A, 1 mg/kg i.v., also blunted the hyperreninemia induced by 1 mg/kg i.v. FURO. In cannulated dogs, 10 mg/kg i.v. PNU-37883A maximally increased fractional Na+ clearance 140% and reduced PRA 76%, but these effects were accompanied by a mean 13 mm Hg pressor effect. In anesthetized dogs, renal artery-infused PNU-37883A (3 mg/kg/h i.r.a.) increased Na+ excretion and reduced renal venous PRA independent of hemodynamics, whereas half this dosage selectively reduced renal venous PRA and renin release, independent of hemodynamics and natriuresis. These data demonstrate that the K-ATP blocker diuretic PNU-37883A reduces PRA in dogs after oral, i.v., and i.r.a. administration and could be a useful pharmacologic agent for exploring the role of K-ATP channels in regulating renin release.