Karen P. Kolbasa

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)

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.

Indomethacin-induced gastric antral ulcers in hamsters

Antral ulcers account for about half of gastric ulcers in humans. An animal model was developed to produce such ulcers. Indomethacin given subcutaneously to normally fed hamsters produced antral ulcers within 1-5 h, dose dependently. These ulcers penetrated the muscularis mucosae. With repeated administration of indomethacin and longer duration of treatment, the lesions became more severe and most animals died with perforated antral ulcers after 2-5 days. Like indomethacin, aspirin given orally also produced antral ulcers in hamsters. Indomethacin reduced the formation of prostaglandin E2, prostaglandin F2 alpha, and 6-keto prostaglandin F1 alpha by the antral mucosa, and increased gastric acid output more than twofold. The ulcers were prevented by various antisecretory agents (cimetidine, methscopolamine bromide, and omeprazole), and the antiulcer dose of each of these agents corresponded to the antisecretory dose. By contrast, several prostaglandins prevented the ulcers at very low, nonantisecretory doses. 16,16-Dimethyl prostaglandin E2 prevented the ulcers at a dose nearly 3000 times lower than the gastric antisecretory ED50. The mechanism by which prostaglandins prevent formation of these ulcers is unknown, but the effect is consistent with cytoprotection, i.e., protection of the gastric mucosa by nonantisecretory doses. Indomethacin-induced antral ulcers appear to depend on two factors: a depletion of prostaglandin content of the antrum and gastric hyperacidity.

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.

Ulcer formation and cytoprotection by acetazolamine

Acetazolamide, a carbonic anhydrase inhibitor, was administered orally and subcutaneously to rats. Acetazolamide increased the gastric ulcerogenicity of indomethacin, but inhibited gastric ulcers produced by acidified aspirin. When administered alone to fasted rats, it did not produce gastric ulcers. Acetazolamide was also cytoprotective for the stomach (it reduced dose dependently the number of gastric necrotic lesions caused by absolute ethanol given orally) and for the small intestine (it prevented dose dependently intestinal lesions produced by administration of a high dose of indomethacin). Acetazolamide did not prevent the antiulcer effect of PGE2 (against aspirin-induced ulcers) nor the cytoprotective effect of 16,16-dimethyl PGE2 (against ethanol-induced gastric lesions). The degree of gastric cytoprotection increased with time after a single administration of acetazolamide; the optimal effect occurred 60 and 90 min after oral and subcutaneous administration, respectively. Pretreatment with indomethacin completely prevented the cytoprotective effect of acetazolamide; this suggests that the cytoprotective effect may be mediated by endogenous release of prostaglandins by the stomach. All the effects of acetazolamide reported here were observed after either oral or subcutaneous administration. The mechanism by which acetazolamide influences ulcer formation and is cytoprotective is unknown.