A conformationally constrained vasopressin analog with antidiuretic antagonistic activity.

Application of information derived from a three-dimensional model of vasopressin bound to its antidiuretic receptor resulted in the design and synthesis of a bicyclic vasopressin analog, [5,8-cyclo(1-beta-mercaptopropionic acid,2-phenylalanine,5-aspartic acid,8-lysine)]vasopressin. The analog acts as an antagonist of the antidiuretic activity of vasopressin.

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.

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.

Isolation and characterization of a sodium pump inhibitor from human plasma.

An endogenous sodium pump inhibitor has been purified from human plasma. The purification scheme involved large scale dialysis, extraction of lyophilized dialysate by methanol followed by preparative and semipreparative scale reverse-phase high-performance chromatography. A single peak of biologically active material was obtained enriched by a factor of greater than 10 billion. This material showed high chromatographic polarity, was inactivated by charring, strong acid, or alkali, and was resistant to short-term boiling. The purified material had a molecular weight between 300 and 900 g/mol and was insensitive to type I esterase and a variety of proteolytic enzymes. The purified factor inhibited the ouabain-sensitive uptake of 86Rb by human erythrocytes, binding of [3H]ouabain, and activity of dog kidney Na,K-adenosine triphosphatase (ATPase) with high affinity (less than 0.3 nM) in a time- and dose-dependent manner. Maximally effective concentrations of the digitalislike factor showed no effect on either human red blood cell Mg- or Ca-ATPase, rabbit muscle sarcoplasmic reticulum Ca-ATPase, or guinea pig stomach H,K-ATPase. The purified material is a highly potent selective inhibitor of the ion transport, receptor, and hydrolytic functions of the sodium pump. The characteristic properties of this substance suggest it may be a mammalian endogenous digitalis and may be similar to the sodium transport inhibitor detected in the plasma of volume-sensitive forms of experimental and human hypertension.

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)

1-(2-aminoethyl)-6-aryl-4H- [1,2,4]triazolo[4,3-a][1,4]benzodiazepines with diuretic and natriuretic activity.

A series of 1-(2-amino-1-phenylethyl)-6-phenyl-4H- [1,2,4]triazolo[4,3-a][1,4]benzodiazepines was prepared and evaluated for diuretic activity. These compounds have diuretic and natriuretic activity but no kaliuretic activity when evaluated by oral administration to the conscious rat. The structure requirements for this activity are discussed. In particular it was found that the 2-aminoethyl side chain at C-1 with hydrogen or methyl substituents on the amino group was required for diuretic activity. A substituent at C-8 was also required; soft substituents such as methylthio and iodo at this position favored activity. Compounds with both phenyl and 2-pyridyl substituents at C-6 were active; substituents on the C-6 phenyl, however, reduced or eliminated the activity. Substituents other than phenyl at the 1-position of the 2-aminoethyl side chain were detrimental to activity; phenyl substitution at this position was required for activity when the substituent at C-8 was chloro but not when it was bromo.

Pyrimidinones. 3. N-substituted 6-phenylpyrimidinones and pyrimidinediones with diuretic/hypotensive and antiinflammatory activity.

In an extensive analysis of the antiviral and interferon-induction structure-activity relationship of 6-arylpyrimidinones we found that modifications at positions 1-4 of the pyrimidine ring resulted in a loss of activity. However, we uncovered interesting hypotensive and antiinflammatory activity with a series of N-substituted analogues, the results of which we report herein.

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.

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.

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.

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.

K+ channel blockers: natriuretic potency correlates to vascular ATP-sensitive K+ channel blockade.

Using analogs of known vascular ATP-sensitive K+ channel (KATP) blockers, we identified compounds with a wide range of potencies (over 500-fold) in their capacity to block the hypotensive response of 0.2 mg/kg pinacidil in rats. The most potent of these, U-97025E, belongs to a newly disclosed class of compounds, the cyanoguanidines. U-97025E at 0.04 mg/kg blocked 50% of the depressor response induced by 0.2 mg/kg pinacidil. The maximal natriuresis induced by U-97025E (0.4 mg/kg i.v.) increased Na+ excretion by approximately 60%. This natriuresis is of the same magnitude as that induced by thiazide without any effect on K+ excretion. We found a high degree of correlation between natriuretic potency and the capacity to block the blood pressure lowering effects of pinacidil, both among closely related analogs and dissimilar compounds. These findings imply an obligatory rather than incidental relationship between vascular KATP blockade and natriuresis. The exact molecular link of the vascular and renal effects remains to be determined.

The natriuretic activity of the K+ channel blocker U-37883A displays an ADH-dependence.

We reported previously that K+ channel blockers induce diuresis and natriuresis in conscious and anesthetized rats. Free-water clearance studies suggested that K+ channel blockers inhibit NaCl reabsorption in the thick ascending limb (TAL) by blocking K+ recycling through a low-conductance, usually open, apical ATP-sensitive K+ channel. In the present study, we measured the effect of U-37883A (15 mg/kg, i.v.) on Na+ reabsorption in rats preconditioned to alter ADH levels. In water-loaded animals with suppressed ADH levels, U-37883A was 50% less natriuretic than in saline-loaded rats. Infusion of ADH to water-loaded rats restored the natriuretic response to a level comparable to saline-loaded rats. Loss of natriuretic efficacy was not secondary to changes in GFR or renal perfusion pressure since GFRs did not vary before or after drug administration in any of the respective groups. Decreases in blood pressure were not significantly different in saline-loaded, water-loaded and water-loaded/ADH rats. The natriuretic response of U-37883A as varied by ADH levels may be the first observation, in vivo, to support the observation that the cotransporter in TAL can exist in two modes as previously observed in vitro by Hebert and colleagues.

Diuretic activity of N'-disubstituted morpholinoguanidine analogs of U-37883A in rats and dogs.

U-37883A is a K+ sparing diuretic which selectively blocks openers of vascular ATP-sensitive K channels. Many N'-disubstituted morpholinoguanidine (N'-DMG) analogs of U-37883A were synthesized and tested for diuretic activity. In conscious rats, 10-100 mg/kg orally of the most active N'-DMGs increased urine volume (V) and Na+ excretion by up to 4-fold with little kaliuresis. The N'-DMGs U-37997A and U-38658A were less potent than standard diuretics, but did not induce the K+ loss seen with hydrochlorothiazide and furosemide or the K+ retention of amiloride and triamterene. In conscious dogs, 10 mg/kg i.v. of the N'-DMGs U-40389A and U-52090 increased V and Na+ excretion by over 7-fold with little kaliuresis. Despite their attractive diuresis, all of the N'-DMGs had narrow margins of safety. Reflecting their direct myocardial depressant action, in isolated rat hearts, bolus intracoronary U-37883A, U-18177A, and U-38658A (0.25-10 mumol) severely reduced the rate (-10 to -100%) and force (-9 to -100%) of contraction. These studies characterize the eukalemic diuretic activity of N'-DMG analogs of U-37883A, and demonstrate the marked cardiac depression characteristic of the morpholinoguanidine diuretic series.

Systemic and renal haemodynamic effects of renin or angiotensin converting enzyme inhibition in non-human primates.

The cardiovascular actions of a renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), and of an angiotensin converting enzyme (ACE) inhibitor, captopril, were determined in conscious sodium-depleted cynomolgus monkeys. Cardiac output was measured with a thermodilution technique. The hypotension induced by U-71038 was associated with a significant reduction in total peripheral resistance without alteration in cardiac output or the heart rate. A similar reduction in total peripheral resistance was observed after captopril at a dose which caused hypotension equivalent to that elicited by U-71038. The latter effects were not accompanied by significant alterations in cardiac output or the heart rate. The glomerular filtration rate was measured by the plasma disappearance of 125I-sodium iothalamate. Renin or ACE inhibition adequate to cause equivalent hypotensive responses did not change the glomerular filtration rate to a significantly different degree. The systemic and renal haemodynamic profiles of U-71038 and captopril appear to be similar, suggesting that renin and ACE inhibition elicit fundamentally similar cardiovascular effects in conscious sodium-depleted cynomolgus monkeys via a decreased formation of angiotensin II (Ang II).