The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice.

The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matrices of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH(2)) is significantly increased, making it more active on a molar basis than the 120-kDa cell-binding domain of pFn. Arginine is important to this activity because PHSAN and PHSEN are inactive, as is a randomized sequence peptide, Ac-HSPNR-NH(2). One treatment with Ac-PHSRN-NH(2) stimulates reepithelialization and contraction of dermal wounds in healing-impaired, obese diabetic C57BL6/KsJ db/db mice. Wound closure is equally rapid in treated db/db and db/+ mice and may be more rapid than in untreated nondiabetic db/+ littermates. In contrast, treatment with either Ac-HSPNR-NH(2) or normal saline (NS) has no effect. Analysis of sectioned db/db wounds shows that, in contrast to treatment with Ac-HSPNR-NH(2) or NS, a single Ac-PHSRN-NH(2) treatment stimulates keratinocyte and fibroblast migration into wounds, enhances fibroplasia and vascularization in the provisional matrix, and stimulates the formation of prominent fibers that may be associated with wound contraction.

Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma.

Using naturally serum-free SU-ECM basement membranes as invasion substrates showed that plasma fibronectin was necessary to stimulate invasion by DU 145 human and metastatic MATLyLu (MLL) rat prostate carcinoma cells. This activity mapped to the PHSRN sequence, which induced invasion through alpha5beta1 integrin. PHSCN, a competitive inhibitor, blocked both PHSRN- and serum-induced invasion. Acetylated, amidated PHSCN (Ac-PHSCN-NH2) was 30-fold more potent; however, Ac-HSPNC-NH2 was inactive. Rats receiving injections s.c. with 100,000 MLL cells were treated systemically by i.v. injection three times weekly with 1 mg of either Ac-PHSCN-NH2 or Ac-HSPNC-NH2 beginning 24 h later, three times weekly with 1 mg of Ac-PHSCN-NH2 beginning only after surgery to remove large (2 cm) MLL tumors, or were left untreated. MLL tumors grew rapidly in Ac-HSPNC-NH2-treated and in untreated rats. MLL tumor growth in rats treated with Ac-PHSCN-NH2 beginning 1 day after MLL cell injection was reduced by 99.9% during the first 16 days of treatment, although subsequent tumor growth occurred. MLL tumor cryosections immunostained with anti-PECAM-1 showed that Ac-PHSCN-NH2 inhibited neovascularization by 12-fold during this time. Whether initiated after MLL cell injection or only after MLL tumor removal, Ac-PHSCN-NH2 treatment reduced the numbers of MLL lung colonies and micrometastases by 40- to >100-fold, whereas Ac-HSPNC-NH2 was inactive. Thus, Ac-PHSCN-NH2 may be a potent antitumorigenic and antimetastatic agent for postsurgical use prior to extensive metastasis.

Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum.

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.

Fluorometric analysis of endocytosis and lysosomal proteolysis in the rat visceral yolk sac during whole embryo culture.

Using spectrofluorimetry and fluorescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) during whole embryo culture. Rat conceptuses exposed continuously to FITC-albumin had linear increases of both acid-soluble and acid-insoluble FITC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumulated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of fluorescence. During a chase period following a pulse exposure to FITC albumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of proteinase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluorescence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluorescence microscopy of yolk sacs exposed to FITC-albumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and decreased substantially during a chase period. In conceptuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC-fluorescence whose clearance in pulse-chase experiments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125l-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of the effects of embryotoxicants on yolk sac function during whole embryo culture.

Androgen and estrogen concentrating neurons in chemosensory pathways of the male Syrian hamster brain.

The medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), and medial amygdaloid nucleus (Me) are essential for male sexual behavior in the Syrian hamster. These nuclei received chemosensory stimuli and gonadal steroid signals, both of which are required for mating behavior. The objective of this study was to compare the distribution of androgen- and estrogen-concentrating neurons in MPOA, BNST, and Me in the adult male hamster using steroid autoradiography for estradiol (E2), testosterone (T) and dihydrotestosterone (DHT). Adult males (n = 4 per group) received two i.p. injections of tritiated steroid 4-7 days after castration. Six-microns frozen sections through the brain were mounted onto emulsion-coated slides, and exposed for 11-16 months. In MPOA, BNST, and Me, neurons were more abundant and heavily labelled after [3H]E2 treatment than after either [3H]T or [3H]DHT. Tritiated estradiol- and DHT-labeled cells were found throughout the rostrocaudal extent of Me, with a high concentration in posterodorsal Me. Tritiated testosterone treatment labelled cells largely within posterodorsal Me. In MPOA, the majority of E2-, T-, and DHT-labelled neurons were in the medial preoptic nucleus (MPN) and the preoptic continuation of the posteromedial bed nucleus of the stria terminalis (BNSTpm). Few T-labelled cells were present outside these subdivisions. In the BNST, E2- and DHT-labelled neurons were present in all subdivisions, whereas T labelling was confined to the antero- and posteromedial subdivisions of BNST. These results suggest that the distribution of androgen- and estrogen receptor-containing neurons overlap considerably in nuclei which transmit chemosensory signals in the control of mating behavior.

Physiological and immunocytochemical evidence for a new concept of blood flow regulation in the corpus luteum.

To explain the high rate of blood flow in the corpus luteum, we hypothesize that luteal blood vessels offer minimal resistance to flow and are incapable of vasomotion. This hypothesis was tested in rabbits at mid-pseudopregnancy by measuring blood flow in the corpus luteum and ovarian stroma with tracer-labeled microspheres at three levels of arterial blood pressure, which was manipulated by constricting the aorta above the ovarian artery. In addition, the distribution of vascular smooth muscle in the ovary was evaluated with morphological and immunocytochemical techniques. Decreases in arterial pressure were paralleled by reductions in blood flow in the corpus luteum, whereas ovarian stromal blood flow was unchanged. Consistent with our hypothesis, there was no change in the low level of vascular resistance offered by blood vessels in the corpus luteum, supporting the view that they are maximally dilated and incapable of autoregulation. Morphologically, the vessels within the corpus luteum appeared as large sinusoidal capillaries without smooth muscle, providing an anatomical explanation for the lack of vasomotor control demonstrated physiologically. The absence of vascular smooth muscle was confirmed with immunocytochemistry using an antibody against the muscle-specific intermediate filament, desmin. The fluorescein-labeled antibody decorated arteries and arterioles within the ovarian stroma and near the capsule of the corpus luteum, but did not decorate vessels in the corpus luteum of pseudopregnancy, providing additional evidence that the vessels of the corpus luteum lack the smooth muscle investment necessary to change vascular caliber. From these findings, we have proposed a novel scheme to explain intraovarian blood flow regulation. Vascular resistance in the ovarian stroma, as in most tissues, is acutely regulated by dilation or constriction of intratissue arterioles. In contrast, vascular resistance within the corpus luteum is modeled as a relatively invariable parameter, fixed at a low level by the morphological characteristics of the luteal vasculature. Therefore, the corpus luteum operates on a linear (maximally "vasodilated") pressure-flow curve, does not actively regulate intratissue blood flow, and is subject to acute regulation of perfusion only through changes in extra-luteal vessels.

The roles of prolactin and testosterone in the development and function of granulosa lutein tissue in the rat.

The luteotropic roles of prolactin and testosterone (or estradiol formed in luteal tissue) were investigated in hypophysectomized rats with homografts of granulosa lutein tissue. Using this approach, we could determine the effects of prolactin independently of estrogen, since granulosa lutein tissue does not produce estrogen de novo under these conditions. Luteinizing granulosa cells were expressed from the ovaries of immature pregnant mare's serum gonadotropin-primed Fischer 344 rats 6 h after injection of human chorionic gonadotropin. The cells were transplanted beneath the kidney capsule of adult, hypophysectomized, ovariectomized Fischer 344 recipients, which were treated with hormones daily for 12 or 14 days. In rats without treatment (no hormones, n = 3) and in rats treated with only testosterone (Silastic capsule, n = 6), only small amounts of luteal tissue (less than 5 mg/rat) were found and serum progesterone remained at low concentrations (10 ng or less) throughout the experiment. In contrast, in rats treated either with ovine prolactin (300 micrograms/day, n = 10) or with the combination of prolactin and testosterone (n = 12), serum progesterone increased to 43 ng/ml by Day 8. Beyond Day 8, serum progesterone continued to rise in rats treated with the combination of prolactin and testosterone to reach a mean value of 87 ng/ml by Day 14, and mean homograft wet weight was 49 mg/rat; in rats treated with only prolactin, serum progesterone decreased to 25 ng/ml by Day 14 and homograft wet weight was lower (24 mg/rat). Prolactin and testosterone together stimulated more homograft aromatase activity in vivo than did prolactin alone, but the in vitro production of progesterone was not different.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of prodrugs by rat intestinal mucosal cells: mechanism and pharmaceutical implications.

The in vitro intestinal ring uptake of prednisolone (11 beta,17,21-trihydroxypregna-1,4-diene-3,20-dione), prednisolone 21-succinate, and prednisolone 21-phosphate was compared in rings prepared from rat jejunum and colon. An HPLC assay was developed to determine whether the drug or intact prodrug was taken up by the tissue. In jejunum and colon, the uptake of prednisolone was limited by its solubility (0.84 mM, 37 degrees C). The freely soluble succinate and phosphate esters were well absorbed in the jejunum. The only species detected in jejunal tissue after incubation with prednisolone 21-phosphate was prednisolone, indicating hydrolysis prior to absorption. This implication was verified by light microscopy. Incubation of tissue from the jejunum with prednisolone 21-succinate resulted in uptake of a mixture of prodrug and parent drug, with the latter form predominating. Prednisolone 21-succinate was also absorbed well in the colon, where the predominant species taken up by the tissue was the intact ester. The half-life of the succinate ester in the tissue was approximately 1 h postincubation, implicating enzyme mediation. Uptake of the phosphate ester by the colon was less than 20% of that observed in the jejunum, with the species absorbed still being primarily the parent alcohol. Light microscopy techniques confirmed that prednisolone 21-phosphate and hydrocortisone 21-phosphate are good substrates for brush border membrane alkaline phosphatase in the jejunum, and that lack of this enzyme in colon tissue was most likely responsible for poor uptake in the colon.

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture.

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.

Differential lectin binding to cellular membranes in the epidermis of the newborn rat.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.

Molecular markers of differentiation in the epidermis of the newborn rat.

Regulation in epidermal differentiation can best be studied if molecular mechanism can be associated with structural and functional changes. Such recognized associations include the cessation of mitosis through inhibition of DNA replication by a G1-inhibitor present in the suprabasal cells, the biosynthesis of a tonofilament-protein as an early event in keratinization, the biosynthesis of HRP0 (histidine-rich protein) and its polymerization to HRPI during the formation of keratohyalin, the conversion of HRPI to HRPII coincident with the loss of the nucleus from the granular cell, and the aggregation of the stratum corneum basic protein and keratin filaments to form fibers in the cornified cell. To this list can now be added changes in specificity for lectin-binding to the cell surface as the keratinocyte progresses toward the cutaneous surface. This report presents data on a) the conversion of HRPI to HRPII and b) the differential lectin-binding in the epidermis of the newborn rat. HRPI (Mol. Wgt. greater than or equal to 10(6)) and HRPII (Mol. Wgt. 6 X 10(4)) have similar unique amino acid compositions and exhibit extensive-but not complete-homology in primary structures as determined by peptide mapping after exposure to trypsin. When labeled by exposure in vivo to radioactivity histidine, about 75 of the labeled histidine from both HRPI and HRPII appeared in one peptide fraction in the map, HRPI appears to have on histidine-containing fragment which is not present in HRPII. This peptide appears to contain phosphate and to account for the organically-bound phosphate which was found in HRPI but not defected in HRPII. Changes which occur in the lectin-binding specificity of the cell during differentiation may result from either movement or chemical change in carbohydrates at the cell surface. Immunofluorescent studies have shown that an isolectin from Bandieraea simplicifolia with specificity for alpha-D-galactose binds to the surface of basal and lower spinous cells, a lectin from Ulex europaeus with specificity for alpha-L-focus labels spinous cells, and a second lectin from B. simplicifolia with specificity for N-acetyl-D-glucosamine labels cornified cells. The relationship fo these alterations in the carbohydrates of the cell surface in intracellular structural and/or functional changes in unknown.