Time responses of cancellous and cortical bones to sciatic neurectomy in growing female rats.

Effects of unilateral sciatic neurectomy on the responses of both cancellous and cortical bones were studied in growing female rats at 0, 1, 4, 8, and 12 weeks after operation. Using double-fluorescent labeling techniques, histomorphometric analyses were performed on longitudinal sections of proximal tibial metaphyseal secondary spongiosa (PTM) and on cross sections of tibial shaft (TX). In PTM, sciatic neurectomy not only inhibited the age-related bone gain, but also reduced the trabecular bone mass by 46%, which was accompanied by decreases in trabecular number, thickness, and node to node density, and an increase in trabecular separation and free end to free end density. The bone loss occurred mainly between 1 and 4 weeks after operation. A sharp increase in bone formation indices was observed during the first week after nerve section. However, these endpoints quickly dropped to levels lower than those of sham-operated controls at 4 weeks, and were not different from the control levels at 8 weeks after operation. Eroded surface increased progressively after sciatic neurectomy during the 12 weeks experimental period. In TX, sciatic neurectomy inhibited the age-related increase in total tissue area that maintained it at the basal control level. However, the cortical bone area in neurectomized legs was lower than that in sham-operated controls. Sciatic neurectomy also stimulated the bone formation indices on both periosteal and endocortical surfaces during the first week after operation. These endpoints declined sharply between 1 and 4 weeks and then maintained at control levels between 8 and 12 weeks post surgery. Endocortical eroded surface increased 1 week after neurectomy, reached the peak at 8 weeks, and then decreased thereafter. These findings suggest that (1) sciatic neurectomy not only inhibited age-related bone gain but also induced marked bone loss in cancellous bone site and inhibited age-related bone gain in cortical bone site, which mainly resulted from the decrease in bone formation and the increase in bone resorption; (2) the changes in both cancellous and cortical bones responded to sciatic neurectomy occurred mostly within the first 4 weeks and stabilized between 8 and 12 weeks after surgical intervention. In conclusion, the unilateral sciatic neurectomized rat is a complex model in which to study osteopenia. Despite sciatic neurectomy being a simple operation, the interactions of skeletal responses to postsurgical regional acceleratory phenomenon (RAP) and disuse and adaptation changes cannot be clearly differentiated. Furthermore, the complications from growth and aging should be avoided.

Endogenous natriuretic factors 3: isolation and characterization of human natriuretic factors LLU-alpha, LLU-beta 1, and LLU-gamma.

A low molecular weight endogenous substance believed to be responsible for extracellular fluid homeostasis in mammals has been sought for many years. Our goal is to isolate and structurally characterize this putative "natriuretic hormone." We have developed an assay using the conscious rat to measure prolonged natriuresis (Benaksas et al (1993) Life Sciences, 52, 1045-1054), the activity originally described for this putative substance. Using this assay we have identified a number of natriuretic compounds isolated from human uremic urine. The collected urine is processed by ultrafiltration (< or = 3 kDa), gel filtration chromatography (G-25) and extraction with isopropanol and diethyl ether. The organic soluble material is then subjected to sequential high-performance liquid chromatography. We report here the initial characterization of two pure isolates (LLU-alpha and LLU-gamma) obtained by this method, and the structural elucidation of a third pure compound, LLU-beta 1, a natriuretic and previously unreported metabolite of the drug diltiazem.

Chiral pharmacokinetics of Rac-flurbiprofen and pharmacodynamics of anabolic bone response in the normal rat.

The route of administration of the NSAID, flurbiprofen (sq vs. po) resulted in positive and negative results respectively with regard to enhanced cancellous and cortical bone accumulation in the immature rat. This pharmacokinetic study was an effort to understand the pharmacodynamic difference between the two routes of administration observed when the same dose range of drug, given as single daily doses, had been employed in both studies. Conventional chiral pharmacokinetics were evaluated in young rats. A significant difference was observed in the Tmax of the active (S)-enantiomer by both administration routes (sq 4 h and po 1 h). The bioavailability, as evaluated by AUCs favored the sq route as expected. The plasma concentrations over 18 h, at steady state, for one po dose group (0.5 mg/kg/day) fell well within the therapeutic window described by the 0.1 and 0.5 mg/kg sq doses which had demonstrated anabolic bone activity. Oral dosing had exhibited no significant bone activity. We conclude that the pharmacodynamic difference between routes of administration cannot be simply explained on a pharmacokinetic basis. Consequently, experiments detailing the pharmacodynamics and pharmacokinetics of single and multiple dose administration of aryl-propionic acids in normal and osteopenic states need further pharmacologic study.

Endogenous natriuretic factors 1: sodium pump inhibition does not correlate with natriuretic or pressor activities from uremic urine.

It is our purpose to isolate and characterize the putative "Natriuretic Hormone", ostensibly responsible for ECF homeostasis, as well as identify endogenous pressors and compounds that induce prolonged natriuresis; we report here our initial progress in this area. Large volumes of pooled urine collected from uremic patients were fractionated, and the resulting isolates were evaluated for in vivo natriuretic and pressor effects and Na+/K(+)-ATPase inhibitory activity in renal cells. The purification steps involved ultrafiltration to obtain materials of less than 3000 da, gel filtration, and sequential reversed-phase high performance liquid chromatography (HPLC). After each HPLC step, the fractions were evaluated for their ability to elicit significant natriuresis and/or influence mean arterial pressure in the normal conscious female rat. Each fraction was also assayed for its ability to inhibit Na+/K(+)-ATPase as determined by the inhibition of 86Rb+ uptake into MDBK renal cells. While several of the fractions elicited profound natriuresis and/or pressor activity and other fractions inhibited Na+/K(+)-ATPase, there was no correlation among the activities in individual fractions. We have concluded that this plethora of bioactivities is responsible for much of the confusion and multiplicity of crude isolates claimed to be the putative hormone. Presently we are attempting to purify each of these activities to chemical homogeneity for structure determination.

Rac-flurbiprofen is more ulcerogenic than its (S)-enantiomer.

The most common, and sometimes life-threatening, side-effects associated with the human use of the analgesic, nonsteroidal antiinflammatory drugs (NSAIDs) are gastrointestinal. These include gastritis, ulceration, and severe bleeding. The aryl propionic acid class of NSAIDs are among the most widely used of these drugs in the world, including rac-ibuprofen, rac-flurbiprofen, and rac-ketoprofen. Marketed as racemates, it was assumed that the "inactive" (R)-enantiomers, having no cyclooxygenase inhibiting effect, also had no toxic effect. In a 30-day dose response study of (S)-, (R)-, and rac-flurbiprofen given daily over a range of doses the (R)-isomer alone proved to be without apparent gastrointestinal (GI) toxicity. On the other hand the racemate proved to be 2 to 4 times as ulcerogenic in enantiomerically equivalent doses as the (S)-enantiomer. These results have significant clinical implications.

Endogenous natriuretic factors. 2: Characterization of natriuretic and vasopressive substances from human uremic urine.

It is our intention to isolate, purify, and characterize the putative low-molecular-weight "natriuretic hormone" responsible for extracellular fluid (ECF) homeostasis. Toward this end, we are purifying from human uremic urine, and identifying endogenous vasopressor and natriuretic compounds. Bioactive components from large volumes of pooled urine were purified by ultrafiltration (< or = 3 kDa), gel filtration chromatography, and sequential reverse-phase and normal-phase high-performance liquid chromatography (HPLC). After each HPLC step, the fractions were evaluated in vivo, were assayed for inhibition of Na+/K(+)-ATPase-mediated 86Rb+ uptake, and were checked for cross-reactivity with an anti-ouabain antibody. Fractions assayed in vivo were identified that induced natriuresis, altered mean arterial pressure, or increased plasma cyclic-GMP. Also, many fractions inhibited Na+/K(+)-ATPase and/or cross-reacted with anti-ouabain antibody. None of the in vitro assays correlates with natriuretic or pressor effects. This plethora of bioactivities, revealed only with increased sample purity, may account for much of the confusion and multiplicity of crude isolates claimed to be the putative hormone. Presently we are attempting to purify and identify these natriuretic materials. One of these, a 3-substituted indole, has been partially characterized.