Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

The effects of delta-9-tetrahydrocannabinol on actin microfilaments

Fluorescence staining with rhodamine phalloidin specific for F-actin was employed to examine the effects of delta-9-tetrahydrocannabinol (THC) on the distribution of microfilaments in kangaroo rat epithelial cells (PtK2) and rabbit aortic endothelial cells (RAE). PtK2 cells were more sensitive to THC treatment than RAE cells. Exposure of PtK2 cells to 10 microM THC for 2 h disrupted the microfilament network. After treatment with 20 microM THC for 2 h there was a loss of cell-to-cell contact between PtK2 cells, and at 30 microM THC, the cells started to detach from the substratum. In contrast, microfilament disorganization but not cell detachment was observed in RAE cells at THC concentrations of 80 and 100 microM. The possible mechanisms which may account for the changes in the microfilament system are discussed.     

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration blocked Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromrecuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ be, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ compete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.     

In vitro study of delta-6- and delta-5-desaturation of linoleic and dihomo-gamma-linolenic acids in WKY rats on a high-sodium diet

Delta 6- and delta 5-desaturation activities of linoleic [1(4)C] and dihomo-gamma-linolenic [2(14)C] acids are increased, in vitro, in liver microsomes of normotensive rats WKY, which receive an hypernatriuric diet (NaCl 3%). These results lead to a best known of delta 6- and delta 5-desaturases, which are involved in fundamental steps of linoleic acid metabolites biosynthesis, implicated in blood pressure regulation.     

Teratologic evaluation of synthetic delta-9-tetrahydrocannabinol in rats.

Delta-9-Tetrahydrocannabinol (THC) was dissolved in propylene glycol and 25, 50, or 100 mg/kg administereed dialy sc to pregnant Charles River Sprague-Dawley rats on days 6-15 of gestation (presence of sperm considered day 1). Maternal weight gain was depressed, but a significant decrease in fetal weight occurred only in the 50 mg/kg group. No malformations were noted, only some abnormalities consisting of several instances of rudimentary 14th rib and soft or spongy spinal cords.