PARP-1 is required for retrieval of cocaine-associated memory by binding to the promoter of a novel gene encoding a putative transposase inhibitor.

Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and short hairpin RNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin immunoprecipitation sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified among PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment markedly increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse.

Antagonistic action of estrogens, flutamide, and human growth hormone on androgen-induced changes in the activities of some enzymes of hepatic steroid metabolism in the rat.

The dose-dependent effects of daily estrogen (estradiol, ethinyl estradiol, diethylstilbestrol) administration on the activities of three hepatic androgen-dependent microsomal enzymes (3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase) in male rats were examined. Antiestrogens were then tested for their ability to block the feminizing action of 10 micrograms estradiol/day on these enzyme activities; nafoxidine and monohydroxytamoxifen were the most effective. The prevention of 5 alpha-dihydrotestosterone-induced changes in these activities in ovariectomized females was investigated. All three estrogens at a dose of 1 microgram blocked the action of 500 micrograms androgen. A similar androgenic blockade was achieved by daily administration of 5 mg flutamide or constant infusion of human GH (5 micrograms/h). Simultaneous administration of 200 micrograms monohydroxytamoxifen prevented the androgen-antagonizing action of estrogens, but not of flutamide nor of GH. Large doses of estrogens have the same repressive effect as androgens on 5 alpha-reductase activity in female castrates. Using the diethylstilbestrol-treated rat as a model, it is demonstrated that this effect can be prevented by antiestrogen, but not by GH. It is concluded that androgens and low doses of estrogens affect these enzyme activities by acting at different levels of central regulation, whereas large doses of estrogens act directly on the liver via hepatic estrogen receptors. These conclusions are corroborated by studies of hepatic estrogen receptor concentrations.

Action of oestrogens and antioestrogens on oestrogen-inducible cytoplasmic and androgen-inducible microsomal activity of 3 alpha-hydroxysteroid dehydrogenase in male rat kidney.

The influence of steroidal and non-steroidal antioestrogenic compounds on the effect of systemically administered oestradiol (OE2) and diethylstilboestrol (DES) was investigated in adult male rats with intact gonads. In this animal model, oestrogens induced the NADP-dependent cytoplasmic activity and prevented the inductive action of androgens on NADP-dependent microsomal activity of renal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH). Simultaneous administration of tamoxifen (0.5 mg/day) with OE2 (5 microgram/day) or DES (10 microgram/day) for 10 days completely blocked the inductive effect of OE2 on cytoplasmic 3 alpha-HSDH, whereas, in the case of the microsomal enzyme, the repressive effects of OE2 and DES were antagonized only to 28 and 16% respectively. Simultaneous administration of 5 alpha-dihydrotestosterone (DHT; 0.5 mg/day) for 10 days antagonized the inductive effect of OE2 on the cytoplasmic enzyme activity to 86% and completely by-passed the repressive effects of OE2 and DES on the microsomal enzyme activity. It is concluded that oestrogenic induction of renal cytoplasmic 3 alpha-HSDH involves an oestrogen receptor mechanism which, in this animal model, can be antagonized by tamoxifen. In contrast, oestrogenic repression of renal microsomal 3 alpha-HSDH is obviously the consequence of the strong antigonadotrophic activity of oestrogens leading to subsequent repression of testicular androgen secretion by mechanisms which can be only weakly antagonized by tamoxifen. Exogenous DHT, even in the presence of OE2 or DES, completely compensates for this centrally mediated deficit of peripheral androgen.

Enzyme activity in kidney, adrenal and gonadal tissue of rats treated neonatally with androgen or oestrogen.

A single injection of 300 mug oestradiol benzoate (OEB) or 1.25 mg testosterone propionate (TP) on day 1 of life led to significant changes in the activity of enzymes involved in steroid hormone metabolism in kidney, adrenal and gonadal tissues of adult rats. In the kidney, the enzyme activities of male rats reacted to OEB, but not TP, by the development of normal female levels. With one exception the enzyme activities of the kidney of female rats did not respond to either steroid. In the adrenal of both sexes 5alpha-reductase reacted to OEB, but not TP treatment, by a fourfold increase in activity. In the ovary all the enzymes investigated responded both to OEB and TP treatment by a fall in activity; 20alpha-hydroxysteroid dehydrogenase activity fell to undetectable levels. In the testis, OEB and TP treatment led to contrasting effects. With the exception of 5alpha-reductase all the enzymes tested in this organ responded to OEB by a rise in activity. Where TP had any effect, it produced a slight decrease in activity.

Endocrine regulation of sex-dependent hydroxysteroid dehydrogenase activities in rat kidney: NADP-dependent microsomal 3alpha- and 20beta-hydroxysteroid dehydrogenase.

The NADP-dependent microsomal kidney enzymes, 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSDH), which exhibit considerable sex differences in their activities (male:female activity ratios, 16:1 and 30:1 respectively), were investigated after interference with the pituitary-gonad and pituitary-adrenal systems. Prepubertal gonadectomy as well as hypophysectomy of mature male rats led to a decline in HSDH activity to almost that found in the normal female rat, whereas activities in female rats were unaffected. Testosterone induced typical male 3alpha-HSDH activity in both gonadectomized and hypophysectomized rats of either sex. Administration of 5alpha-dihydrotestosterone (5alpha-DHT) or 5alpha-androstane-3alpha, 17beta-diol to hypophysectomized male rats was equally effective in restoring full 3alpha- and 20beta-HSDH activities whereas 5alpha-androstane-3beta, 17beta-diol was less effective and dehydroepiandrosterone was ineffective. Simultaneous administration of cyproterone acetate did not block the inductive action of 5alpha-DHT. Administration of chorionic gonadotrophin, pregnant mare serum gonadotrophin or a combination of luteinizing hormone and follicle-stimulating hormone to hypophysectomized male rats all led to parallel increases in the weight of the seminal vesicles and in both renal enzyme activities; administration of growth hormone, prolactin or thyroid-stimulating hormone was ineffective. Adrenalectomy of gonadectomized, but not of hypophysectomized male rats, caused a further drop in activity to the normal female level. Adrenalectomy of otherwise intact rats did not affect either enzyme activity. The hypophysis was involved in the regulation of the two NADP-dependent renal HSDH activities through its gonadotrophic function in male rats; adrenal secretions were of little physiological significance.

Changes in the activities of microsomal enzymes involved in hepatic steroid metabolism in the rat after administration of androgenic, estrogenic, progestational, anabolic and catatoxic steroids.

Several steroids (5 beta-dihydrotestosterone, 19-nortestosterone, methyltrienolone, norethisterone, medroxyprogesterone acetate, cyproterone acetate, chlormadinone acetate and 16 alpha-cyanopregnenolone) were tested for their ability to influence the activities of three sexually differentiated hepatic microsomal enzymes (3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase) in male and female gonadectomized and intact female rats. Of the steroids tested only 5 beta-dihydrotestosterone was completely ineffective. The other tested steroids elicited varying degrees of "masculinization" with a distinct gradation of effect according to the enzyme activity measured and animal model used. 5 alpha-Reductase was the most sensitive enzyme activity and 3 alpha-hydroxysteroid dehydrogenase the least. Male castrates responded better than female castrates, and these in turn better than intact females. The mechanism of action of three of the steroids (methyltrienolone, medroxyprogesterone acetate and norethisterone) was examined. Both flutamide and estradiol were able to block the action of methyltrienolone and medroxyprogesterone acetate, but not that of norethisterone. It is concluded that methyltrienolone and medroxyprogesterone acetate probably masculinize the enzyme activities by the same mechanisms as androgens, whereas the repression of 5 alpha-reductase activity elicited by norethisterone administration involves a different route.

Antagonism of the estradiol-mediated repression of microsomal 3 beta-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances.

5 alpha-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3 beta-hydroxysteroid dehydrogenase activity of male rat liver. With the exception of 5 alpha-dihydrotestosterone, which induced activity in females, none of these substances affected 3 beta-hydroxysteroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 micrograms/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 micrograms/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5 alpha-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.

The efficacy of a new salbutamol metered-dose powder inhaler in comparison with two other inhaler devices.

An open cross-over and randomized study was carried out in order to compare the efficacy and safety of inhaled salbutamol delivered from a new 50 microg dose(-1) metered-dose dry powder inhaler Taifun, and a commercially available 50 microg dose(-1) dry powder inhaler Turbuhaler, and a conventional 100 microg dose(-1) pressurized metered-dose inhaler with a spacer (pMDI+S). Twenty-one patients, aged 21-70 years, with stable asthma and with demonstrated reversibility upon inhalation of salbutamol were included in the study. On three separate study days, the patients received a total dose of 400 microg of salbutamol from the dry powder inhalers and a dose of 800 microg from the pMDI+S in a cumulative fashion: 1,1, 2 and 4 doses at 30 min intervals. The percent change in forced expiratory volume in 1 sec (FEV1), was used as the primary efficacy variable. Salbutamol inhaled via the Taifun produced greater bronchodilation than the other devices. The difference in percent change in FEV1 between the Taifun and the other devices was statistically significant at the two first dose levels, but diminished towards the higher doses when the plateau of the dose-response curve was reached. The estimated relative dose potency of the Taifun was approximately 1.9- and 2.8-fold compared to the Turbuhaler and the pMDI+S, respectively. The Taifun caused a slight, but clinically insignificant, decrease in serum potassium concentration. There were no significant changes in the other safety parameters (blood pressure, heart rate and electrocardiogram recordings) with any of the used devices. In conclusion, this study indicates that salbutamol inhaled via the Taifun is more potentthan salbutamol inhaled from the other devices tested. In practise, a smaller total dose of salbutamol from theTaifun is needed to produce a similar bronchodilatory response. All treatments were equally well tolerated.

Dysphagia in a child with aggressive fibromatosis of the esophagus.

A case of a girl who developed severe dysphagia from an aggressive fibromatosis involving the cervical esophagus is reported. A 3 x 4 cm unencapsulated fibrous tumor was completely excised with the involved esophageal wall. The esophageal defect was bridged by an inverted skin tube with good functional and cosmetic results. Eight years later the patient is disease-free. Primary radical excision is the treatment of choice of aggressive fibromatosis.

A simple method for oral administration of drugs in solid form to fully conscious rats.

The design and application of a simple capsule administration tube for miniature capsules are described. Experiments with rats have shown that the tube is capable of depositing capsules at the distal end of the oesophagus. Regardless of the location of the capsule in the oesophagus, provided normal peristaltic action occurs, the capsule will have reached the stomach and discharged its contents within 10 min. After a short training period of 3-4 days the insertion of the tube does not appear to cause the rats undue discomfort, nor does it cause tissue damage. The procedure, which can be performed rapidly by 1 technician, is ideally suited for dispensing solid materials to fully conscious animals.

In situ measurement of spectral changes in the anterior eye following application of ultraviolet-absorbing compounds.

The ocular structures are very sensitive to damage from ultraviolet (UV) radiation, exposure is linked to corneal and conjunctival damage, cataract formation and may also be implicated in the aetiology of age-related macular degeneration. These structures are usually protected by wearing suitable eyeglasses and goggles. An alternative to conventional eyeglasses/goggles is the concept of "liquid sunglasses" which involve the topical application of eye drops that are designed to block harmful UV radiation reaching the sensitive ocular surfaces. The evaluation of such compounds directly applied to the eye surface requires in situ measurements to compare the efficacy of different formulations. A novel ocular spectrometer system has been used to evaluate changes in the transmission of ultraviolet (UV) radiation through the anterior eye following topical application of candidate UV-absorbing formulations. The key feature of the system is the ability to propagate a beam of light tangentially through the anterior eye using a compact, hand-held lens assembly incorporating UV-transmitting optical fibres. A range of formulations containing UV-absorbing compounds were topically applied to ex vivo rabbit eyes. Significant increases in the absorption of the UV spectrum were detected in seven of the eight formulations studied, demonstrating the potential of this measurement technique in the evaluation of formulations developed as potential topical ocular sunscreens.

Mechanisms of physiological and pharmacological sex hormone action on the mammalian liver.

Androgen and oestrogen receptors have been demonstrated in mammalian liver, but since it is generally accepted that they are probably non-functional at endogenous steroid concentrations, it is not apparent how they mediate physiological influences on this organ. Nor is it certain to what extent pharmacological actions of sex hormones reflect overstimulation of physiological routes or whether alternative mechanisms become available once threshold values have been reached. In this presentation an attempt has been made to answer some of these questions using data obtained from a study of the regulation of the activities of microsomal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) and 5 alpha-reductase in rat liver. Androgens exert their primary physiological and pharmacological influences at the level of the hypothalamus. Oestrogens can elicit three different types of effect-physiological, antiandrogenic and pharmacological--of which the first two involve primary effects on the pituitary. Hepatic oestrogen receptors only become activated when oestrogen concentrations reach pharmacological levels. Progestins probably have no physiological influence on the livers of non-pregnant rats. Their pharmacological actions may either be traced back to secondary androgenic (e.g. medroxyprogesterone acetate, levonorgestrel) or oestrogenic (e.g. norethynodrel, lynestrenol) properties, involving the routes described above, or to independent effects on the central nervous system (e.g. cyproterone acetate modulation of 5 alpha-reductase activity).

Sex-specific action of antiandrogens on androgen induced changes in hepatic microsomal 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase activity in the rat.

The ability of two antiandrogens, cyproterone acetate and flutamide, to block the induction of microsomal 3 beta-hydroxysteroid dehydrogenase and repression of microsomal 5 alpha-reductase in rat liver following administration of 5 alpha-dihydrotestosterone was investigated in male and female castrated and female gonad-intact rats. Although both antiandrogens blocked the effects of 5 alpha-dihydrotestosterone on the seminal vesicles and uteri of gonadectomized rats at the doses employed, cyproterone acetate showed no antiandrogenic activity against the two enzyme activities. On the contrary when cyproterone acetate was administered alone it elicited a response similar to that seen after androgen administration; when given simultaneously with 5 alpha-dihydrotestosterone, the induction of 3 beta-hydroxysteroid dehydrogenase activity was greater than after either steroid alone. In contrast the non-steroidal antiandrogen, flutamide, which had no intrinsic effect on either enzyme activity, effectively blocked 5 alpha-dihydrotestosterone mediated changes in the enzyme activities of female rats regardless of gonadal status. However the influence of 5 alpha-dihydrotestosterone administration on these enzyme activities in male rats was not prevented.

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration.

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.

An improved method for the determination of cytoplasmic 3 alpha- and 17 beta- and microsomal 3 alpha-hydroxysteroid dehydrogenase activities in rat liver.

This paper described a modified method for the radiometric determination of hydroxysteroid dehydrogenase activities in rat liver. The principle advantages of this method are the improved precision and a radical reduction in the time involved in performing the assay. The procedure comprises the following steps: incubation of 14C-labelled substrate with coenzyme and cell fraction under optimized conditions; termination of the reaction by addition of organic solvent containing a defined amount of 3H-labelled reaction product; removal of precipitated protein and coenzyme by centrifugation; paper chromatographic isolation of the product; direct quantitation of 14C activity in the product zone of the paper chromatogram. The assay systems have been applied to elucidate and quantitate sex and strain differences in the activities of the above enzymes in Chbb/THOM and Sprague-Dawley rats.

The influence of oestradiol on androgen- and oestrogen-dependent enzyme activities of hepatic steroid metabolism in rats.

Five sexually differentiated enzyme activities of hepatic steroid metabolism (cytoplasmic 17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase; microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase) were investigated in intact, gonadectomized and hypophysectomized rats after administration of a single dose of oestradiol valerate. Oestradiol administration caused a partial or complete feminization of these activities in intact male rats. The influence of oestradiol on these activities in gonadectomized rats was determined by the mode of sex hormone-dependent regulation of the individual activity: the most prominent effects were seen in the oestrogen-dependent activities (17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase); no effect was seen in the completely androgen-dependent 3 alpha-hydroxysteroid dehydrogenase because gonadectomy alone was sufficient to cause complete feminization of the activity. Oestradiol administration had no effect on the activities of hypophysectomized rats. The fact that oestrogen administration to intact male rats caused greater changes than prepuberal gonadectomy demonstrates that oestrogen action is more than simple suppression of testicular function.

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat following gonadectomy.

The activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase of rat liver were determined at different time points after gonadectomy on day 75 of life. Following testectomy the activities in male rats assume female values. However this change is relatively slow, 10--14 days being necessary for significant trends in individual activities to develop, and 40--60 days before the final level of activity is reached. The changes in enzyme activities after ovariectomy are only slight. The change in microsomal 5 alpha-reductase activity following gonadectomy of male rats is biphasic, the activity increasing initially to the normal female level before falling to the intermediate "neonatally androgen-imprinted" level. The reaction of 17 beta-hydroxysteroid dehydrogenase activity to testectomy and ovariectomy indicates that in the course of several years, during which we have investigated the behaviour of this enzyme in Chbb/THOM rats, the regulation of its activity has changed from one of oestrogen dependency to one of androgen dependency.