Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K.

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell-attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7-2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole-cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain-sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.

Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone.

Activities of Na channels and Na pumps were studied in the rat cortical collecting tubule (CCT) during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet, diuretics, or administration of exogenous aldosterone. Tubules were isolated and split open to expose the luminal membrane surface. Using the whole-cell patch-clamp technique, activities of the apical Na channels and the basolateral Na pumps were measured in principal cells as the currents inhibited by amiloride (10 microM) and ouabain (1 mM), respectively. Na channel current (INa) was not measurable in CCTs from control animals on a normal diet. INa was approximately 200 pA/cell in CCTs from animals on a low-Na diet or infused with aldosterone using osmotic minipumps. Currents attributable to the Na pump (Ipump) were similar in control animals and animals on a low-Na diet. Maximal currents were approximately 35 pA/cell in both groups, and decreased with hyperpolarization of the cell membrane. In contrast, administration of exogenous aldosterone increased Ipump fourfold. Coinfusion of aldosterone and amiloride in vivo through the minipumps did not affect the induction of INa but reduced the induction of Ipump by 80%. We conclude that the induction of channel activity in this tissue is a direct action of aldosterone, whereas the induction of pump activity may be a consequence of the increased Na traffic through the epithelial cells.

Regulation of Na channels of the rat cortical collecting tubule by aldosterone.

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell-attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1-2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.

AL721, a novel membrane fluidizer.

AL721, which is a novel lipid mixture extracted from egg yolks, is believed to be a therapeutic pharmacologic agent. AL721 interacts with membranes of various types of cells with a common mode of action. AL721 modifies cellular membrane composition and fluidity through passive extraction and/or exchange of cholesterol. Physiologically diminished cell function due to rigidification of its membrane is reversible both in vitro and in vivo by AL721. Fluidization of aged membranes with AL721 has been shown to restore brain serotonin receptor function both in vitro and in vivo. AL721 can also successfully restore deficient immune responsiveness of lymphocytes to mitogen stimulation in aged subjects. Drug tolerance to morphine and ethanol develops upon elevation of the viscosity of neuronal cell membranes in order to counteract the fluidization effect of the drug. Treatment of rigidified cellular membranes with AL721 in vivo can markedly reduce withdrawal symptoms. The virucidal effect of AL721 on the human immunodeficiency virus is believed to operate by lowering of viral membrane cholesterol thus interfering with the binding of the viral antigen to the host cell. Non-toxicity of AL721 is clearly demonstrated in animal and human safety studies.

Prenatal diagnosis/treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia).

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance (phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated. A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.

Effect of finasteride on adrenal steroidogenesis in men.

Finasteride, a 5 alpha-reductase inhibitor, does not bind to the androgen receptor and has no other known hormonal activity. To determine what effect, if any, it has on adrenal steroidogenesis, 10 healthy men received 5 mg finasteride daily for 28 days. Adrenocorticotropic hormone (ACTH) stimulation tests were performed before and after 4 weeks of finasteride administration (5 mg daily). Serum levels of 17-hydroxypregnenolone, 17-hydroxyprogesterone, deoxycorticosterone, corticosterone, aldosterone, cortisol, dehydroepiandrosterone, and androstenedione were measured before and 60 minutes after i.v. ACTH. Finasteride decreased serum dihydrotestosterone levels from 31 +/- 5 to 4.4 +/- 1.2 ng/dl (P < 0.001). There were no significant changes in basal or ACTH-stimulated serum levels of adrenal steroids. There was also no significant decrease in the product to precursor ratio for the seven adrenal enzymes tested. Finasteride increased mean serum androstenedione levels by 17% (P = 0.10) and significantly increased the androstenedione to 17-hydroxyprogesterone ratio (P = 0.02 before ACTH and 0.05 after ACTH). These changes are most likely due to inhibition of androstenedione metabolism by 5 alpha-reductase. In conclusion, finasteride has no detectable effect on adrenal steroidogenesis, other than that which can be explained by inhibition of the 5 alpha-reductase enzyme.

Studies on the use of a novel affinity matrix, sepharose amine-succinyl-amine haloperidol hemisuccinate, ASA-HHS, for purification of canine dopamine (D2) receptor.

Haloperidol Hemisuccinate (HHS) was synthesized specifically as a ligand for an affinity chromatography matrix. The affinity chromatography matrix, ASA-HHS was developed which had high affinity and capacity for dopamine D2 receptors in solubilized canine striatal preparations. ASA-HHS also demonstrated nonspecific interaction with the D2 receptor. Two fractions, which bound 3H-spiroperidol specifically, with similar one dimensional SDS-PAGE patterns could be eluted successfully with 20 microM haloperidol in only 30% of the runs. Both fractions represented 300-400 fold purification. Two dimensional IEF-PAGE analysis of one of the fractions demonstrated coelution of beta and gamma actin, alpha and beta tubulin with the 3H-spiroperidol binding sites. The pattern of the proteins eluted from ASA-HHS and the inconsistent recovery of active D2 receptors are discussed.

Striatally-mediated response of some structurally rigid analogues of dopamine.

The potency of structurally rigid analogues of dopamine (DA) at striatal dopamine receptors was evaluated in rats using three types of assessments: (a) effectiveness in producing rotational and sniffing behaviors by intrastriatal injections (b) inhibition of [3H]-spiroperidol binding and (c) stimulation of adenylate cyclase activity. The compounds included apomorphine (APO) and its analogues, (R)-2,10,11-trihydroxyaporphine (R-THA) and (R)-2-hydroxy-10,11-methylenedioxyaporphine (MDO-APO), 2-amino-6,7-dihydroxyaminotetraline (ADTN) and its analogue, exo-2-amino-6,7-dihydroxybenzonorbornene (exo-amine). (R)-THA produced no stereotypy yet it was a potent inhibitor of [3H]-spiroperidol binding and adenylate cyclase activity. MDO-APO was quite active in inducing stereotypy and stimulating cyclase activity, but it showed low potency in displacing [3H]-spiroperidol. The exo-amine and ADTN were equally potent in enhancing rotation and sniffing intensity, however, the former was completely inactive in biochemical assessments. Except for (R)-THA, all DA analogues studied elicited dopaminomimetic behavioral activities of circling and sniffing. Relationships between the actions of these drugs in the behavioral and biochemical assessments are discussed.

An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma.

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.

Simultaneous determination of SU5416 and its phase I and phase II metabolites in rat and dog plasma by LC/MS/MS.

SU5416, Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a cytostatic substance in development as an anti-angiogenic agent. SU5416 has several phase I and phase II metabolites including SU9838, SU6595, SU6689, 5'-hydroxy glucuronide of SU5416 and 5'-acyl glucuronide of SU5416. In order to support the preclinical studies, a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method for simultaneous determination of SU5416 and its metabolites in rat and dog plasma was developed. This method is fast, simple, sensitive (LOQ=2.0 ng/ml), reproducible and has a wide linear range (2.0-5000 ng/ml for SU5416, 2.0-2000 ng/ml for SU6689 and 2.0-1000 ng/ml for SU9838 and SU6595). This method was applied to rat and dog plasma samples obtained from pharmacokinetic and toxicokinetic studies.

Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416.

The pharmacokinetics and allometric relationships of SU5416, a novel small anti-angiogenesis agent, were studied. The pharmacokinetics of SU5416 were examined in mice, rats, dogs, and cancer patients. The in-vitro intrinsic clearance (CLint) was estimated from the in-vitro metabolism study in mouse, rat, dog, monkey and human liver microsomes. The parameters of interest were correlated across species as a function of bodyweight using an allometric approach. The steady-state volume of distribution (Vd(ss)), plasma clearance (CLs), and CLint of SU5416 were well correlated across species. The exponent of the allometric relationship (b) of the corresponding parameters was 0.92, 0.80 and 0.66, respectively. The elimination half-life (t1/2) was consistent across species and independent of bodyweight. The prediction of CLs, Vd(ss), CLint, and t1/2 in humans using the data from mouse, rat, and dog, and monkey (for CLint) was reasonably good (within 4-fold of the observed values). However, an improved prediction (within 2-fold of the observed values) of the corresponding parameters in humans was obtained when extrapolation from only the rodent data was performed, suggesting that the rodent data are sufficient for the scale-up of SU5416 pharmacokinetic parameters in humans. Using allometry, it was possible to achieve reasonable predictions of the pharmacokinetic parameters of SU5416 in cancer patients with various solid tumours.

Automation of steroid radioimmunoassays using a robotics workstation.

OBJECTIVE: To automate the radioimmunoassay (RIA) of steroids in a diagnostic clinical laboratory. SETTING: The Laboratory of Pediatric Endocrinology, Cornell Medical Center-New York Hospital. PRACTICE DESCRIPTION: A tertiary-care center supporting the diagnosis and treatment of pediatric patients with endocrine disorders. PRODUCT COMPARISON: The Packard MultiPROBE Model 100 Automated Liquid Handling System (Packard Instrument Company, Meriden, CT 06450) was compared with the assays typically used to assist in the diagnosis of steroidogenic disorders in children: extraction of biologic fluids, celite partition chromatography, and RIA. MAIN OUTCOME MEASUREMENT: The comparative efficiency, cost-effectiveness, specificity, and sensitivity of the two methods. RESULT: The automated RIAs correlated (r = 0.963) with the established manual assays for steroids. The specificity and sensitivity of the assays were uniformly maintained after automation. CONCLUSION: The automation of steroid RIAs has made the method a highly efficient and cost-effective one for diagnostic clinical laboratories.

Critical self-association of bile lipids studied by infrared spectroscopy and viscometry.

Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy and viscometry were applied to study the micellization of two bile lipids, sodium taurochenodeoxycholate (NaTCDC) and sodium glycocholate (NaGC), in aqueous solutions. The CH2 stretching bands of the bile lipid hydrocarbon region were shifted to higher frequencies suggesting initial critical micellization at 2.5 mM for NaTCDC and 9 mM for NaGC. An abrupt enhancement of the absorption intensity of the CH3 groups of the sterol rings in bile lipids were under conformational strain at 3.5 mM NaTCDC and 9 mM NaGC. Viscometry measurements showed abrupt changes in viscosities in the region of critical micellar concentration (CMC) of both bile lipids. Both infrared and viscometry studies confirmed the onset of conformational strains in tightly packed lipid micelles at their CMC. In addition, FTIR/ATR spectroscopy has defined the specific hydrophobic interactions which bring about critical micellization of bile lipids.

Assay of brain calmodulin levels using high-performance liquid chromatography.

A simple, sensitive, and efficient HPLC method for the determination of calmodulin levels in brain tissue extracts is described. The assay is linear with respect to both calmodulin and protein concentrations. The specificity and validity of this assay for calmodulin is demonstrated by parallel radioimmunoassay determinations which give equivalent results. Determination of calmodulin levels in various brain regions revealed a high concentration of this protein in the hypothalamus, by comparison to other areas examined.

Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor.

Oxytocin receptors were measured in myometrium and intercaruncular endometrium of cows during pregnancy and parturition. Concentrations of estradiol-17 beta, estrone, and progesterone in peripheral blood were also measured. Receptor concentrations in the endometrium rose almost 200-fold from Day 20 to term (p < 0.0001, ANOVA), from 40 +/- 11 to 7300 +/- 1430 fmol/mg protein. Myometrial receptor concentrations increased 10-fold from 180 +/- 36 fmol/mg on Day 20 to 1850 +/- 360 fmol/mg protein at term (p < 0.0001, ANOVA). During labor, endometrial receptors (6600 +/- 1300 fmol/mg) remained at prelabor values, whereas myometrial receptor concentrations had decreased to 1190 +/- 316 fmol/mg (not significant) and declined further postpartum. Plasma concentrations of progesterone declined from 4-5 ng/ml to about 2 ng/ml between Days 250 and 282 and dropped to < 0.2 ng/ml shortly before delivery. Plasma concentrations of estrone and estradiol-17 beta were below 10-20 pg/ml until Day 230. Estrone concentrations were significantly (p < 0.05) increased by Day 250 and estradiol-17 beta by Day 270, and then both rose rapidly. During labor, plasma estrone was 1135 +/- 245 pg/ml and plasma estradiol-17 beta was 226 +/- 131 pg/ml. The molar ratio of estrone and estradiol-17 beta to progesterone rose from less than 0.01 to 4.4 during labor, and was correlated with oxytocin receptor concentrations in endometrium (r = 0.5160, p < 0.001), but not those in myometrium (r = 0.0122). The regulation of oxytocin receptors by ovarian hormones in the two tissues may therefore differ.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation of the anti-angiogenic compound SU5416.

SU5416 [3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1, 3-dihydro-indol-2-one], an inhibitor of VEGF (vascular endothelial growth factor) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2) is in advanced clinical trials for treatment of AIDS-related Kaposi's sarcoma and colorectal and nonsmall cell lung cancers. Since this chemical class has not been studied previously with therapeutic intent, the present study was designed to investigate the in vitro metabolism of SU5416 by mouse, rat, dog, monkey, and human liver microsomes and to identify the major metabolites of SU5416. An HPLC procedure was developed and validated to resolve and quantify SU5416 and its metabolites. To evaluate the in vitro metabolism of SU5416, pooled liver microsomes from mice, rats, dogs, monkeys, and humans were incubated with SU5416 (25 microM) in the presence of an NADPH-generating system. In the presence of NADPH, mouse, rat, dog, monkey, and human liver microsomes converted SU5416 to at least 12, 9, 9, 7, and 6 polar metabolites, respectively. Microsomal metabolism of SU5416 showed marked species differences in the levels of different metabolites formed. The overall rate of SU5416 metabolism by liver microsomes from the species examined followed the rank order: monkey > or = mouse approximately rat > dog > human. Two major metabolites of SU5416 were identified, a hydroxymethyl derivative of SU5416 (M12) and a carboxylic acid derivative of SU5416 (M6), by spectroscopic methods and comparison with authentic compounds. Both of these oxidative metabolites were further metabolized in vivo through glucuronidation. The metabolic fate of SU5416 in microsomes from various species as well as data from in vivo biotransformation in the rat are discussed.