Irreversible inhibition of forskolin interactions with type I adenylyl cyclase by a 6-isothiocyanate derivative of forskolin.

Forskolin (Fsk) has been demonstrated to interact directly with the enzyme adenylyl cyclase (EC 4.6.1.1) in diverse tissues. However, the ability of Fsk to bind to and activate adenylyl cyclase varies depending on the tissue being studied. Different adenylyl cyclase subtypes have been cloned and expressed in a recombinant Sf9 expression system. This provides an opportunity to study the effects of chemically reactive derivatives of Fsk on individual adenylyl cyclase subtypes in the absence of Gs alpha. Reaction of type I adenylyl cyclase with an isothiocyanate derivative of Fsk (6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin) causes irreversible inhibition of Fsk binding with an IC50 of 300 nM and irreversible inhibition of Fsk activation with an IC50 of 10 microM, suggesting that there are two sites of 6-[[N-(2-isothiocyanatoethyl)amino]carbonyl]forskolin interaction. These studies establish the usefulness of the isothiocyanate derivative of Fsk in localizing the site(s) of Fsk interaction with type I adenylyl cyclase.

Forskolin carbamates: binding and activation studies with type I adenylyl cyclase.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

Interaction of an estramustine photoaffinity analogue with cytoskeletal proteins in prostate carcinoma cells.

To identify specific drug targets of the antimitotic drug estramustine, a photoaffinity analogue, 17-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]estradiol-3-N-bis(2- chloroethyl)carbamate, was synthesized and reacted in competition assays with cytoskeletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties of the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estramustine-resistant cell line (E4), the major microtubule-associated protein (MAP) present was MAP4. In both cytoskeletal fractions and reconstituted microtubules, 17-O-[[2-[3-(4-azido-3-[125I]iodophenyl)propionamido] ethyl]carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamate bound to both MAP4 and tubulin. From competition assays, the apparent binding constant for MAP4 from DU 145 cells was 15 microM. Similar calculations for tubulin gave values of 13 microM (bovine brain), 19 microM (DU 145 wild-type cells), and 25 microM (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direct explanation for the antimicrotubule and antimitotic effects of estramustine.

Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs alpha.

Several forms of adenylyl cyclase (types I, II, V, and VI) have been expressed using the recombinant baculovirus expression system in Sf9 cells. The activation of type I adenylyl cyclase by forskolin and Gs alpha was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by Gs alpha and forskolin. Gs alpha potentiated the effect of forskolin on type II adenylyl cyclase to the greatest extent. Type I and II adenylyl cyclases were photolabeled specifically by an iodinated photoaffinity derivative of forskolin ([125I]-6-AIPP-Fsk). Type I adenylyl cyclase was photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha only slightly increased the labeling efficiency. In contrast, type II adenylyl cyclase was not photolabeled efficiently in the absence of Gs alpha, and the addition of Gs alpha greatly enhanced the labeling efficiency. Photolabeling of type V and VI adenylyl cyclases was detected only in the presence of Gs alpha. Neither calcium/calmodulin nor G protein beta gamma subunits modulated the photolabeling of type I or II adenylyl cyclases. Another iodinated derivative of forskolin, [125I]-6-IHPP-fsk, bound to Sf9 cell membranes expressing type I adenylyl cyclase with high affinity in a filtration binding assay, and the specific binding was not enhanced by the addition of Gs alpha. In contrast, specific binding of [125I]-6-IHPP-Fsk to membranes expressing type II adenylyl cyclase was detected only in the presence of Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

[125I]-labeled forskolin analogs which discriminate adenylyl cyclase and a glucose transporter: pharmacological characterization and localization of binding sites in rat brain by in vitro receptor autoradiography.

Aminoalkylcarbamate derivatives of forskolin have been synthesized at the 6- and 7-hydroxyl positions which have different selectivity for adenylyl cyclase and a glucose transporter, respectively. They were radioiodinated using the Bolton-Hunter reagent to yield [125I]-2-[3-(4-hydroxy-3-iodophenyl)propanamido]-N-ethyl-6- (aminocarbonyl)forskolin ([125I]6-IHPP-Fsk) and [125I]-2-[3-(4-hydroxy-3-iodophenyl)(propanamidol]-N-ethyl-7- (aminocarbonyl)-7-desacetylforskolin ([125I]7-IHPP-Fsk) and tested as autoradiographic probes for adenylyl cyclase and a glucose transporter. In slide-mounted rat brain sections [125I]6-IHPP-Fsk binding was potently inhibited by 1 microM 6-HPP-Fsk (95%) but unaffected by 500 mM D-glucose. In contrast, [125I]7-IHPP-Fsk was only partially inhibited by 1 microM 6-HPP-Fsk (37%), but residual [125I]7-IHPP-Fsk binding was further inhibited 56% by 500 mM D-glucose. These data suggest that while [125I]6-IHPP-Fsk binds exclusively to adenylyl cyclase, a significant fraction of [125I]7-IHPP-Fsk is binding to a glucose transporter in brain. Autoradiographic patterns of [125I]6-IHPP-Fsk and glucose-sensitive [125I]7-IHPP-Fsk binding were different. [125I]6-IHPP-Fsk binding was heterogeneously distributed and resembled [3H] forskolin binding. Highest densities of binding sites were noted in olfactory tubercle, caudate putamen, nucleus accumbens, pyramidal and granule cell layers of hippocampus, molecular layer of cerebellum and substantia nigra. In contrast, of glucose-sensitive [125I]7-IHPP-Fsk, binding appeared more homogeneous and similar to [3H]cytochalasin B, a compound which inhibits glucose transport. Highest densities of binding were noted in caudate putamen, nucleus accumbens, cerebral cortex and molecular layer of cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential identification and localization of adenylyl cyclase and glucose transporter in brain using iodinated derivatives of forskolin.

Two radioiodinated derivatives of forskolin, [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk, were synthesized as specific ligands for adenylyl cyclase and glucose transporter, respectively. [125I]6-IHPP-Fsk bound to bovine brain homogenates with a Kd of 9 nM and binding was inhibited by forskolin but not 1,9-dideoxyforskolin, cytochalasin B, or D-glucose. [125I]7-IHPP-Fsk bound to bovine brain homogenates at two classes of binding sites with Kd's of 56 nM and 4.7 microM; cytochalasin B and D-glucose inhibited 75% of the high affinity binding while having no effect on the low affinity binding. [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk were used to localize adenylyl cyclase and glucose transporter in rat brain by receptor autoradiography. The pattern of binding obtained with [125I]6-IHPP-Fsk was similar to that observed using [3H]forskolin to detect adenylyl cyclase. In contrast, the pattern of binding obtained with [125I]7-IHPP-Fsk was similar to that observed by others using [3H]cytochalasin B to detect glucose transporter. These iodinated ligands are selective for adenylyl cyclase and glucose transporter and require significantly shorter exposure times to yield autoradiographs than tritiated ligands.

Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase.

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

(Aminoalkyl)carbamates of forskolin: intermediates for the synthesis of functionalized derivatives of forskolin with different specificities for adenylyl cyclase and the glucose transporter.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter.

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.

The role of mitochondrial matrix enzymes in the metabolism and toxicity of cysteine conjugates.

The submitochondrial localization and identity of enzymes which metabolize cysteine conjugates were investigated. Glutamine transaminase K was purified from rat kidney mitochondrial soluble fraction and was shown to be a cysteine conjugate beta-lyase. The purified mitochondrial enzyme is similar to the cytosolic glutamine transaminase K whose beta-lyase activity with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is regulated by concurrent transamination (Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) J. Biol. Chem. 261, 15529-15537). However, beta-lyase activity in whole mitochondria is largely independent of regulation by cosubstrates for transamination suggesting that factors present in mitochondria are able to support the beta-lyase activity in the absence of added alpha-keto acid. Fractionation of mitochondria results in a loss of the independent beta-lyase activity. However, the majority of the beta-lyase activity can be recovered in the matrix if it is stimulated by the addition of alpha-keto-gamma-methiolbutyrate. The data suggest that the regulation of beta-elimination by the competing transamination pathway is different for each substrate and that multiple beta-lyases may exist in rat kidney. S-(2-Benzothiazolyl)-L-cysteine (BTC) is a poor substrate for purified glutamine transaminase K from mitochondria and cytosol, but BTC is as active as DCVC in crude mitochondrial matrix suggesting that other enzymes may be present. In contrast to DCVC, with BTC as substrate, the beta-lyase activity of the purified enzyme and enzyme(s) in the mitochondrial matrix is largely alpha-keto acid-independent. The existence of multiple enzymes is also supported by the observation that alpha-keto acids which are not substrates for purified glutamine transaminase K from mitochondria and cytosol do stimulate beta-lyase activity in the mitochondrial matrix fraction. Mitoplasts were found to be sensitive to DCVC toxicity consistent with the matrix localization of beta-lyase activity. The possible role in cysteine conjugate toxicity of matrix enzyme regulation by alpha-keto acids is discussed.

Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals.

The Vi has proven to be a protective antigen in two double masked, controlled clinical trials in areas with high rates of typhoid fever (approximately 1% per annum). In both studies the protective efficacy of the Vi was approximately 70%. Approximately 75% of subjects in these areas responded with a fourfold or greater rise of serum Vi antibodies. In contrast, the Vi elicited a fourfold or greater rise in 95-100% of young adults in France and the United States. Methods were devised, therefore, to synthesize Vi-protein conjugates in order to both enhance the antibody response and confer T-dependent properties to the Vi (and theoretically increase its protective action in populations at high risk for typhoid fever). We settled on a method that used the heterobifunctional crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), to bind thiol derivatives of the Vi to proteins. This synthetic scheme was reproducible, provided high yields of Vi-protein conjugates, and was applicable to several medically relevant proteins such as diphtheria and tetanus toxoids. The resultant conjugates were more immunogenic in mice and juvenile Rhesus monkeys than the Vi alone. In contrast to the T-independent properties of the Vi, conjugates of this polysaccharide with several medically relevant proteins induced booster responses in mice and in juvenile Rhesus monkeys. Clinical studies with Vi-protein conjugates are planned. This scheme is also applicable to synthesize protein conjugates with other polysaccharides that have carboxyl functions.

Quipazine-metoclopramide inhibition of CB-154-induced prolactin suppression in rats: neurotransmitter-metabolite correlations.

The ability of quipazine and metoclopramide to protect rats from CB-154-induced suppression of serum prolactin concentrations was studied. These drugs affect whole brain concentrations of dopamine and serotonin, and their major metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. Serum prolactin concentrations have been correlated with the concentrations of the neurotransmitters and their respective metabolites. Differences in the metabolite/precursor ratios have been used to compare turnover rates of the neurotransmitters dopamine and serotonin. Increased turnover of dopamine and decreased turnover of serotonin correlate with elevated prolactin concentrations for quipazine and metoclopramide administered together. The combination of quipazine and metoclopramide protects rats against CB-154-induced prolactin suppression better than either of the drugs given alone. This study suggests that a quipazine-metoclopramide regimen may have therapeutic potential for combating ergotlike fescue and other similar toxicities observed in cattle grazing on endophyte-infected pasture grasses.

A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K.

Cysteine conjugate beta-lyase has been purified from rat kidney cytosol. The enzyme is a 100,000-dalton dimer of two 55,000-dalton subunits and has an absorption maximum at 432 nm. The enzyme has phenylalanine alpha-keto-gamma-methiolbutyrate transaminase activity and appears to be identical to rat kidney cytosolic glutamine transaminase K. Metabolism of S-1,2-dichlorovinyl-L-cysteine (DCVC) by the purified enzyme was dependent on the presence of either alpha-keto-gamma-methiolbutyrate or a protein factor which is present in the cytosolic fraction of rat kidney cortex. The protein factor was identified as a flavin containing L-amino acid oxidase which oxidized DCVC to S-(1,2-dichlorovinyl)-3-mercapto-2-oxopropionic acid. S-(1,2-Dichlorovinyl)-3-mercapto-2-oxopropionic acid has not been previously reported as a metabolite of DCVC. The data also show that rat kidney cytosolic glutamine transaminase K catalyzes both a beta-elimination and a transamination reaction with DCVC when alpha-keto-gamma-methiolbutyrate is present and that amino acid oxidase and alpha-keto-gamma-methiolbutyrate stimulate the enzyme activity by providing amino acceptors. When incubations were done with DCVC as substrate in the presence of excess alpha-keto-gamma-methiolbutyrate, the beta-lyase catalyzed beta-elimination and transamination in a ratio of 1:1.3, respectively. Under conditions where most of the alpha-keto-gamma-methiolbutyrate was consumed, the beta-elimination predominated indicating that the S-1,2-dichlorovinyl-3-mercapto-2-oxopropionic acid pool was consumed by transamination after the alpha-keto-gamma-methiolbutyrate had been depleted. The data are discussed with regard to the importance of these pathways as regulators or participants in the toxicity of S-cysteine conjugates.

31P nuclear magnetic resonance spectroscopic observation of the intracellular transformations of oncostatic cyclophosphamide metabolites.

31P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard. These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide, aldophosphamide, and phosphoramide mustard). Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4. Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3. Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected. Spectral data were used to calculate a rate constant of (5.4 +/- 0.3) X 10(-3) min-1 for the intracellular disappearance of 4 at 23 degrees C. The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.

Effect of clonidine, quipazine, and LY 53857 on the prolactin-suppressant action of bromocriptine in rats.

The alpha 2-adrenergic agonist clonidine hydrochloride, the serotonin agonist quipazine maleate, and the serotonin (5-HT2) antagonist LY 53857 were tested alone and in various combinations for their capabilities to increase mean serum prolactin (MSP) concentrations in rats given the synthetic ergot alkaloid CB-154 (2-bromo-alpha-ergocriptine), a known prolactin suppressor. The LY 53857 and the combination of clonidine, quipazine, and LY 53857 significantly decreased MSP concentrations. Quipazine given alone (10 mg/kg of body weight) was best able to increase MSP concentration and has potential to antagonize prolactin-depressant effects of ergot alkaloids.

Effect of yohimbine hydrochloride on serum prolactin concentration in the rat: possible antagonist for fescue toxicosis.

Yohimbine hydrochloride is an indole alkaloid which blocks alpha 2-adrenergic and dopamine receptors and stimulates serotonergic receptors. Yohimbine was selected for testing as a possible antagonist in fescue toxicosis. Reduced body weight gains in cattle with chronic fescue toxicosis may be due to ergot alkaloids produced by fungi which infect the fescue grass. Ergot alkaloids stimulate dopamine receptors, antagonize serotonin, and lower serum prolactin concentrations. It was hypothesized that yohimbine may reverse or counteract the effects of the toxic fescue. Investigation was made of the treatment effects of multiple doses of yohimbine given in rats by intraperitoneal and oral routes. Given intraperitoneally once a day for 8 days, yohimbine hydrochloride increased serum prolactin concentrations. When given orally in feed for 7 days, the drug decreased the serum prolactin concentration. The effects of yohimbine on prolactin concentrations were dependent on the dosages and routes of administration. The inability of yohimbine, when given orally, to increase serum prolactin levels decreased its potential usefulness for prolonged treatment of fescue toxicosis.

Reexamination of the protective role of the capsular polysaccharide (Vi antigen) of Salmonella typhi.

The role of the Vi antigen, the capsular polysaccharide of Salmonella typhi, in the pathogenesis of and immunity to typhoid fever remains the subject of controversy. Vi-positive S. typhi resist phagocytosis and the action of serum complement, both of which actions are initiated by antibodies to Vi antigen. Both the laboratory potency in mice and the clinical effectiveness of whole-cell vaccines were related to their content of immunogenic Vi antigen. A Vi polysaccharide used for immunizing humans against experimental challenge with S. typhi failed to prevent typhoid fever; experimental conditions used to prepare this ineffective Vi antigen were shown to denature it and to reduce its immunogenicity. Assay of serum antibodies to Vi antigen with purified Vi antigen is a reliable method for diagnosis of typhoid fever and asymptomatic carriage of S. typhi. Vi polysaccharides prepared by modern techniques passed the requirements for meningococcal polysaccharide vaccines and had approximately 13 times the protective activity in the mouse potency assay as did the US Standard 6A whole-cell typhoid vaccine.

Bioavailability in rabbits of formaldehyde from durable-press textiles.

Carbon-14-labeled formaldehyde was used per se, or was used in the synthesis of dimethyloldihydroxyethyleneurea (DMDHEU), which was incorporated into cotton or cotton/polyester blend fabric. Patches of the fabric containing known quantities of radioactive DMDHEU were applied to the backs of New Zealand White rabbits for periods up to 48 h. The rabbits were placed in specially constructed metabolism chambers designed to prevent either inhalation of volatile material emanating from the fabric or interference of any volatile material from the fabric with trapping of expired carbon dioxide. The results of the studies indicate that aqueous formaldehyde covered with a latex barrier is absorbed and retained in the layers of skin in direct contact with the formaldehyde. Approximately 65% of a dose of [14C] formaldehyde was recovered in skin 4 h after application. Skin samples from the backs of rabbits to which durable-press fabric prepared from radiolabeled DMDHEU had been applied were found to have 0.09-2.61% of the total 14C contained in the cloth patches. The levels of radioactivity recovered from the skin varied with degree of occlusion of the cloth, presence or absence of perspiration, type of synthesis used for the preparation of DMDHEU, and whether cotton or cotton/polyester blend fabric was used. Other tissues and organs had only low levels of radioactivity. Injected [14C] formaldehyde was rapidly expired as 14CO2 (28.6% of the dose within 4 h; 37.0% within 48 h). Metabolism and distribution of formaldehyde was found to be dependent on route of administration: i.e., topical application resulted in high skin levels, whereas intravenous injection led to rapid pulmonary and renal excretion and retention of radioactivity in liver, kidney, and blood.