Darrell D. Fanestil

Interference by lipids in the determination of protein using bicinchoninic acid.

Bicinchoninic acid forms the basis of an analytical method for the determination of protein. The reagent produces a purple complex with cuprous ion (Cu+) in an alkaline environment and is the basis for the monitoring of cuprous ions produced in the reactions of proteins with alkaline Cu2+. This method of protein determination was reported to have greater tolerance to many commonly encountered interfering compounds, when compared to the Lowry technique. However, we have found the bicinchoninic acid technique to produce erroneously high values for protein when common membrane phospholipids were included in the assay. Phospholipids in the presence of bicinchoninic acid produced an absorbance peak similar to that produced by protein. This absorbance was linear with concentration, however, the slope varied for individual phospholipids. The combined absorption of phospholipid and protein was not strictly additive. The results indicate that the presence of appreciable quantities of lipid in samples can cause significant error in the analysis of protein by the bicinchoninic acid procedure.

Mineralo- and glucocorticoid effects on renal excretion of electrolytes

The acute effects of mineralo- and glucocorticoids on urinary electrolyte excretion were studied in the conscious, acutely potassium deprived, adrenalectomized rat. Sodium, potassium, and creatinine were measured in the urine excreted from 2.5 to 5.5 h after injection of one or more of the following steroids: aldosterone (Aldo), 9-alpha fluorocortisol (FC), deoxycorticosterone (DOC), dexamethasone (Dex), and spironolactone (Spiro). The hierarchy (a) for increasing creatinine excretion was Dex>FC>Aldo>DOC>Spiro >none, a hierarchy consistent with glucocorticoid potency; and (b) for producing anti-natriuresis was Aldo>DOC≥FC ≥none=Spiro>Dex, a hierarchy consistent with mineralocorticoid potency. In contrast, the kaliuresis produced by mineralo- and glucocorticoids appears different. A “mineralocorticoid” kaliuresis is 1) elicited by anti-natriuretic doses of Aldo and FC, 2) approximately twice control UKV, 3) unrelated to changes in glomerular filtration rate (GFR), and 4) inhibited by Spiro. A “glucocorticoid” kaliuresis is 1) elicited by Dex and high doses of Aldo and FC, 2) about seven to twenty-fold greater than control UKV, 3) possibly dependent, in part, on changes in GFR, and, 4) not inhibited by Spiro. DOC was not kaliuretic at anti-natriuretic doses. The urinary Na/K ratio was an unreliable index of mineralocorticoid action.

Modification of the peripheral-type benzodiazepine receptor by arachidonate, diethylpyrocarbonate and thiol reagents

Peripheral type benzodiazepine receptors are differentially modified by arachidonate, diethylpyrocarbonate and thiol reagents, as evidenced by the finding that binding of a proposed agonist ([3H]R05-4864) and a proposed antagonist ([3H]PK 11195) to rat kidney mitochondrial membranes can be modified separately. (1) Arachidonate significantly lowered the affinity of the peripheral-type benzodiazepine receptor for R05-4864 and diazepam but did not alter the affinity for PK 11195 and only slightly altered for dipyridamole. In contrast, diethylpyrocarbonate inhibited [3H]PK 11195 binding by 43% while reducing [3H]R05-4864 binding only 16%. (2) Diethylpyrocarbonate treatment causes a reduction in affinity for both dipyridamole and PK 11195, did not change the affinities for R05-4864 and diazepam but increased the affinities for diuretics. PK 11195, R05-4864 and dipyridamole totally protected peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. In contrast, diethylpyrocarbonate inactivation of [3H]PK 11195 binding was increased after preincubation of peripheral benzodiazepine receptors with metolazone. Arachidonate both lowers the affinity for [3H]R05-4864 and eliminated the ability of R05-4864 to protect peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. (3) Among different thiol reagents, only dithiothreitol treatment resulted in 26 and 43% inhibition of [3H]PK 11195 and [3H]R05-4864 binding, respectively. These results indicate that the peripheral-type benzodiazepine receptor molecule possesses at least two different conformations or separate mutually exclusive sites for agonists and antagonists. Additionally, the interaction of the thiazide class of diuretics with peripheral-type benzodiazepine receptors seems to require binding sites on the receptor molecule in addition to those for specific agonist and antagonist, whereas dipyridamole appears to bind to the antagonist site or conformation.

Inhibitors of peripheral-type benzodiazepine receptors present in human urine and plasma ultrafiltrates

Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.

Licorice, computer-based analyses of dehydrogenase sequences, and the regulation of steroid and prostaglandin action

Licorice, an ingredient in candy, chewing gum and medicines, has provided an important clue in discovering a novel mechanism that regulates the actions of mineralocorticoids in mammals (Stewart et al., 1987). This insight came as a result of an unexpected and initially unexplainable effect of licorice in causing sodium retention in some humans; that is, licorice acted like the major human mineralocorticoid aldosterone. Identification of the active principle in licorice as glycyrrhetinic acid, which has a structure with some resemblance to steroids (Fig. l), raised the possibility that licorice acted by a mechanism that involved the mineralocorticoid receptor. However, neither glycyrrhetinic acid nor its carbohydrate-containing analog, glycyrrhizic acid, binds steroid receptors with sufficient affinity to make it likely that the mineralocorticoid effect was due to an inherent steroid-like activity (Ulmann et al., 1975; Armanini et al., 1983). Soon thereafter, studies on mineralocorticoid receptors revealed another puzzling finding: both endogenous glucocorticoids (e.g. cortisol and corticosterone) and the mineralocorticoid aldosterone had similar affinities for the receptor that regulated sodium and potassium transport in the kidney (Beaumont and Fanestil, 1983; Krozowski

Interactions of lipids with peripheral-type benzodiazepine receptors

Peripheral-type benzodiazepine receptors (PBRs) are present at high densities in the rat kidney distal tubule. [3H]RO 5-4864 binding to PBRs in kidney membranes is inhibited by several unidentified low molecular weight hydrophobic compounds in urine and serum. We tested representative hydrophobic compounds from several lipid classes for ability to inhibit binding to rat kidney PBRs of two high affinity ligands, [3H]RO 5-4864 and [3H]PK 11195. Unsaturated fatty acids and alcohols inhibited [3H]RO 5-4864 binding with half-maximal inhibition occurring at 3 X 10(-6) M to 10(-4) M. Inhibitory potency increased with the degree of unsaturation. Phospholipids inhibited [3H]RO 5-4864 in the same concentration range, with inhibitory potency in this case dependent both upon an unsaturated fatty acid moiety and upon the polar head group. Phosphatidylethanolamine was the most potent phospholipid tested (IC50 = 2 X 10(-6) M), whereas phosphatidylcholine was not inhibitory. Although phospholipids inhibited both [3H]RO 5-4864 and [3H]PK 11195 binding equally, unsaturated fatty acids had a much greater inhibitory effect upon [3H]RO 5-4864 than upon [3H]PK 11195 binding. Similar effects were obtained with digitonin-solubilized PBRs. These data demonstrate that in our experiments PBR binding was inhibited by specific lipids and that binding of proposed agonist (RO 5-4864) and antagonist (PK 11195) ligands was differentially affected by unsaturated fatty acids.

Specific binding of deoxycorticosterone by canine kidney cells in culture

Abstract The Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, have the ability to bind deoxycorticosterone (DOC). Cystosol from MDCK cells exposed to 6nM[H 3 ]-DOC at 4°C bound 0.405 pmol/mg protein. Of this, 0.335 pmol/mg protein was specifically bound, as assessed by displacement by a 100 fold excess of non-radioactive DOC. At 60 nM [H 3 ]-DOC, 5.33 pmol/mg protein was bound; of which 4.01 pmol/mg was specifically bound. This binding could not be attributed to binding by protein in the incubation medium and did not occur in a hamster kidney cell line (BHK). The [H 3 ]-DOC binding was depressed by other steroids with the following order of effectiveness DOC > progesterone ≈ aldosterone > corticosterone > dexamethasone ≈ cortisol ≈ none. By Scatchard analysis, the equilibrium dissociation constant ( K D ), was 10 −7 M with 4–10 × 10 5 cystosol binding sites per cell. Incubation of intact cells at 37°C with [H 3 ]-DOC resulted in the uptake of the steroid into an enriched nuclear fraction such that the amount of [H 3 ]-DOC bound per mg nuclear protein was 3–13 times greater than in the cytosol protein from those cells. Therefore, the DOC binding in MDCK cells resembles in several respects the interaction of a steroid with its specific tissue receptor.

Photoaffinity labeling of peripheral-type benzodiazepine receptors in rat kidney mitochondria with [3H]PK 14105

The peripheral-type benzodiazepine receptor in rat kidney has been identified by photoaffinity labeling. PK 14105, a derivative of the selective peripheral-type ligand PK 11195, was used to covalently label peripheral-type benzodiazepine receptors. In the absence of UV light PK 14105 demonstrated reversible, high affinity (KD = 4.8 nM) binding to rat kidney mitochondrial membranes. Inhibition of the reversible binding of [3H]PK 14105 by various benzodiazepine and other ligands demonstrated that this ligand bound with all the characteristics expected of a ligand interacting specifically with peripheral-type benzodiazepine receptors. A similar order of relative potencies was obtained for inhibition of photolabeling, indicating that reversible binding and photolabeling occurred at the same class of binding sites. Examination of photolabeled binding sites from kidney, heart, brain and adrenal membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the probe is photoincorporated into a single peptide of Mr = 18,500. The results indicate that [3H]PK 14105 identifies the ligand binding domain of the peripheral-type benzodiazepine receptor, which is a peptide with Mr = 18,500, that is of similar size in kidney, heart, brain and adrenals.

Thiazide Diuretic Receptors in Spontaneously Hypertensive Rats and 2-Kidney 1-Clip Hypertensive Rats

Thiazide diuretic receptor density was assessed in kidneys from spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats by measuring hydroflumethiazide-displaceable 3H-metolazone binding to renal membranes in vitro. Renal thiazide receptor density was not significantly different in 4 week old SHR and WKY rats, but was significantly increased by 20%-40% in 14-49 week old SHRs compared to WKY rats. Affinity of receptors for 3H-metolazone did not differ between SHRs and WKY rats at any age. In WKY rats with 2 kidney-1 clip (2K-1C) hypertension, thiazide receptor density was not significantly different in either clipped or unclipped kidneys from sham-operated controls. Thus, increased renal thiazide receptor density occurs in SHRs along with the development of hypertension and does not appear to be secondary to increased renal perfusion pressure. This increase may reflect altered hormonal or ionic input to the distal tubule and may contribute to elevated sodium reabsorption in this segment in the SHR.

Competitive inhibition of dexamethasone binding to the glucocorticoid receptor in HTC cells by tryptophan methyl ester

Abstract We have previously reported that the binding of adrenal and sex steroid hormones to specific receptors is inhibited by a variety of protease inhibitors and substrates, including tryptophan methyl ester (TME). Now, using the method of Scatchard, we report on the effect of D-TME on the binding of [3H]-dexamethasone to the glucocorticoid receptor found in HTC cells. We find that the measured equilibrium dissociation constant of [3H]-dexamethasone for HTC cytosol is raised from 3.10 ± 0.32 × 10−9 M in the absence of TME to 7.3 ± 0.63 × 10−9 M in the presence of 5 mM TME, and 10.1 ± 0.60 × 10−9 M in the presence of 7.5 mM TME. The maximal number of [3H]-dexamethasone binding sites is 5.5 × 10−13 moles per mg protein and is unaltered by d -TME. Therefore, we conclude that TME, a competitive inhibitor of chymotrypsin is also a competitive inhibitor of dexamethasone binding to the glucocorticoid receptor in HTC cells. This conclusion supports the hypothesis that the TME binding site and the dexamethasone binding site are partially contigous.