Endothelial cell membranes: polarity of particles as seen by freeze-fracturing.

Freeze-fracturing shows particles within membranes. In plasma membranes of most cells the particles are more strongly bound to the inner half. In unfixed endothelial cells, this polarity is reversed. Glutaraldehyde fixation results in conventional polarity. The reverse polarity may be related to a mechanism for preferential fusion of pinocytotic vesicles with the plasma membrane.

Development of radioimmunoassays for ovine neurophysins. Correlation of neurophysin release with oxytocin- and vasopressin-related stimuli.

Specific homologous RIAs for the ovine neurophysins (oN-I/II, oN-III) were established; the assays had a sensitivity capable of measuring basal levels of the pituitary proteins in sheep plasma. Reduction in blood volume of ewes caused a 200-fold increase in oN-III with minimal changes in oN-I/II levels. After infusion of either hypertonic saline or nicotine, a similar specific increase in oN-III was observed, occurring at a time when the free water clearance rate decreased. Intramuscular administration of estradiol benzoate caused an increase in jugular plasma oN-I/II concentrations without affecting the oN-III concentrations. In one of six lactating ewes, there was an increase in oN-I/II during suckling. The release of oN-I/II, which was accompanied by small increases in oN-III, exhibited a pulsatile profile similar to that reported for the secretion of oxytocin under similar conditions. During vaginal distension, increases in intramammary pressure were accompanied by elevations in levels of oN-I/II and oN-III. It is concluded that oxytocin-related events are associated predominantly with the release of oN-I/II and that stimuli that induce vasopressin release also cause elevation of oN-III. These correlations are consistent with our previous conclusion from biochemical and amino acid sequence analysis of the oNs.

Oxytocin, oxytocin-associated neurophysin, and prostaglandin F2 alpha concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep.

Oxytocin, oxytocin-associated neurophysin (neurophysin), prostaglandin F2 alpha (PGF2 alpha), and progesterone concentrations were measured in the utero-ovarian vein (UOV) of sheep during the estrous cycle and early pregnancy. On days 13-16 of the cycle, large pulses of PGF2 alpha, oxytocin, and neurophysin were measured in samples collected at hourly intervals from the UOV draining a corpus luteum (UOV/CL). Most of the PGF2 alpha pulses (96.5%) coincided with a pulse of both oxytocin and neurophysin, whereas only 55.6% of oxytocin pulses coincided with a pulse of PGF2 alpha. Therefore, during luteolysis in sheep, uterine PGF2 alpha release is closely associated with ovarian oxytocin release, and oxytocin release is unlikely to be dependent upon a uterine PGF2 alpha stimulus. During frequent sampling, coincident oxytocin pulses were measured in 1) both UOVs when a CL was present in both ovaries and 2) the jugular vein, carotid artery, and UOV/CL, with a significantly higher oxytocin pulse concentration occurring in jugular venous compared with carotid arterial plasma. Pituitary and luteal release of oxytocin may, therefore, occur simultaneously and be controlled by a circulating factor in sheep. Compared to days 13-16 of the cycle, significantly (P less than 0.001) fewer pulses of PGF2 alpha, which were significantly (P less than 0.001) smaller in amplitude, were measured in UOV samples collected frequently during early pregnancy. The frequency of oxytocin pulses observed in the UOV/CL of pregnant sheep was not significantly (P greater than 0.1) different from that observed in cyclic ewes, although most (86.4%) oxytocin pulses occurred in the absence of a PGF2 alpha pulse. In contrast, when a pulse of PGF2 alpha was observed in the UOV/CL of pregnant ewes, it usually coincided with a pulse of oxytocin. The suppression of uterine PGF2 alpha release during early pregnancy is not considered to result from a lack of stimulation by oxytocin.

Long term primary monolayer culture of adult murine magnocellular neurons.

Primary monolayer culture of adult murine hypothalamic cells has been carried out for 2 months. Neuron-like cells as well as fibroblast, glial, and ependymal-like cells demonstrated characteristic morphological features which distinguished them from one another. In addition, immunocytochemical identification of cytoplasmic substances cross-reactive with neurophysin and (8-arginine)vasopressin further characterized specific magnocellular neuronal populations throughout the culture period. This culture system should provide a basis for studying neurosecretion at the cellular and molecular levels.

Distribution of immunoreactive calcitonin in the rat pituitary gland.

Immunohistochemical (immunoperoxidase) studies were performed on 478 sections from 97 rat pituitary glands with rabbit antisera to unconjugated human synthetic calcitonin beta-endorphin, and/or ACTH-(17--39). Calcitonin-positive cells were present in a majority of the anterior lobes studied, whereas they were present in only a minority of the intermediate lobes. Calcitonin-positive cells were also present in chronically thyroidectomized animals. Beta-Endorphin-positive cells were uniformly present in the intermediate lobes as were the ACTH-positive cells. In the anterior pituitary lobes, beta-endorphin-positive cells were more populous than the ACTH-positive cells, and in general, there was a dissociation of the cellular elements containing beta-endorphin, ACTH, and calcitonin. Although it remains possible that there is calcitonin-like immunoreactivity within a precursor molecule that is differentially processed by pituitary cells, these studies are more consistent with the view that immunoreactive calcitonin is present in pituitary cells which are not as yet precisely and consistently related to any identifiable population of hormone-producing cells.

Somatostatin in cytotrophoblast of the immature human placenta: localization by immunoperoxidase cytochemistry.

Using the technique of immunoperoxidase cytochemistry, we have demonstrated the presence of immunoreactive somatostatin in the cytotrophoblast. This is contrasted by the immunocytochemical staining of human placental lactogen in the syncytotrophoblast in the adjacent section of the villus tissue of the immature human placenta. It is considered that the somatostatin may be involved in the secretion of human placental lactogen within the two layers of trophoblastic cells.

beta-Endorphin in the human pancreas.

Using a specific antiserum, beta-endorphin was quantitated in 8 human pancreas obtained at autopsy by radioimmunoassay and localized by immunocytochemistry. The mean (+/- SE) concentration of beta-endorphin in pancreatic extracts of 5 non-diabetic adults was 13.5 +/- 9.8 ng/gm with a range of 2.1 to 52.8 ng/gm of tissue. Pancreatic beta-endorphin concentration in two premature infants were within the range found in adults. In one diabetic pancreas, there was no measurable beta-endorphin. Specific beta-endorphin immunofluorescence is localized within the pancreatic islets. This finding suggests that beta-endorphin may participate in intraislet regulation of pancreatic hormone secretion.

Demonstration by immunoperoxidase histochemistry of calcitonin in the anterior lobe of the rat pituitary.

We have demonstrated by a specific immunoperoxidase procedure the presence of calcitonin-containing cells in the rat pituitary gland. These cells are widely distributed throughout the anterior lobe and seem to constitute the entire population of cells of the intermediate lobe. No such cells were seen in the posterior lobe. The presence of calcitonin-containing cells in the pituitary provides novel implications about the physiological significance of this hormone.

Precautions in the application of immunohistochemical techniques to tissues of the pig hypothalamo-neurohypophysial system.

Immunohistochemical procedures were used to localize neurophysin in the hypothalamo-neurohypophysial axis of the domestic pig. The topographical distribution of neurophysin as revealed by the immunofluorescence "sandwich" technique was similar to that found when either the immunoglobulin-peroxidase bridge method or the peroxidase-labeled gamma-globulin technique was employed. However, application of the peroxidase-anti-peroxidase (PAP) complex procedure resulted in nonspecific staining of the magnocellular structures. This phenomenon was attributed to the action of PAP on the tissue and after screening a number of other vertebrate species was found to be unique to the pig. Minimal nonspecific binding of the PAP could be achieved either by reducing the reaction time of PAP to 5 min or, by the addition of 1% (v/v) normal serum to all reagents and wash solution. That the PAP-binding protein is a component of the hypothalamo-neurohypophysial axons is discussed.

Use of immunocytochemical techniques for the localization of human placental lactogen.

The recent claim by Gau and Chard (Br J Obstet Gynaecol 83:876, 1976) that, on theoretical grounds, it may be impossible to demonstrate the presence of human placental lactogen in placental tissue using the immunoperoxidase technique, has been reinvestigated. Placental tissue fragments fixed in Carnoy's fluid retained their morphologic identity compared with tissue fixed in formalin. Using these nonformalin fixed tissues, human placental lactogen was successfully localized within the cytoplasm of the syncytial layer of the placental villus. It is concluded that placental villi at term do in fact contain sufficient human placental lactogen to be demonstrated using the immunoperoxidase technique in contrast to the observation of Gau and Chard.

Beta-endorphin and somatostatin in the pancreatic D-cell colocalization by immunocytochemistry.

Utilizing highly specific antisera, beta-endorphin and somatostatin immunoreactivity were identified simultaneously in the D-cell of the pancreas of the rat, guinea pig, and man by the fluorescence-immunocytochemical technique. Our observations are consistent with a modulating role of beta-endorphin either within the D-cell or upon A and B cells, thereby regulating the secretion of insulin and glucagon.

Synergism between bovine seminal plasma extract and testosterone propionate in suppressing serum concentrations of gonadotrophins in acutely castrated rats: a role for inhibin.

The rise in concentrations of FSH and LH in serum seen 24 h after castration was suppressed by the administration of an extract of bull seminal plasma or testosterone propionate at the time of castration. Whereas testosterone propionate preferentially suppressed LH, the seminal plasma extract suppressed FSH and LH equally. Small doses of bull seminal plasma extract and testosterone, that had little effect separately, acted synergistically to supress levels of FSH and LH to those found in intact animals, while combinations of larger doses had little further effect. This selective interaction suggests how inhibin and testosterone might together regulate concentrations of FSH and LH in the blood of the male rat.

Uptake of 125-I-labelled human placental lactogen and human placental lactogen by the tissues of normal and lactating rats.

The rate of clearance from the circulation and uptake into tissues of radioactive label was studied after i.v. injection of 125-I-labelled human placental lactogen (HPL) into rats at various stages of pregnancy. The half-life was obtained for the disappearance of the trichloroacetic acid-precipitable material from the plasma. The half-life, t1/2(S), calculated over the first 5 min after injection of the hormone was 5.4 equals or minus 1.1 (S.D.) min, while a half-life, t1/2(L), of 27.9 equals or minus 2.3 min was obtained from the decay period of 15-35 min. In the non-pregnant and pregnant rat the highest ratio of the radioactivity in an organ to that in the blood was 12-14:1 in the kidney. That the kidney is mainly involved in the uptake of exogenous HPL is further confirmed by the application of the histochemical immunoperoxidase technique. Human placental lactogen was localized in the cells of the proximal tubules of the cortex and to a lesser extent in the tubular lumen and the tubules of the medulla region. UPTAKE OF HPL in vivo occurs in the mammary gland tissue of the post-partum rat and reaches a maximum uptake between 15 and 30 min after injection of the hormone. Furthermore, specific uptake of HPL was observed on the alveolar cell membranes after the incubation of paraffin-embedded sections of formalin-fixed mammary gland and subsequent treatment by the peroxidase-labelled antibody method. These findings support the work of others who have demonstrated the presence of specific membrane receptors in the mammary gland for hormones with prolactin-like activity.

Purification and some properties of ovine placental lactogen.

A method has been described for the purification of ovine placental lactogen (oPL) involving the use of freshly obtained sheep foetal cotyledons. Tissue was extracted with 0.1 M-ammonium bicarbonate and the supernatant fraction, adjusted to pH 7, was brought to 60% saturation with ammonium sulphate. The resulting precipitate was then subjected to a sequence of chromatographic steps using columns of Sephadex G-100 and carboxymethylecellulose. During each stage of the purification, the lactogenic activity was monitored with a pregnant rabbit mammary gland radioreceptor assay. The yield of oPL corresponded to 8 mg/kg wet foetal tissue and the oPL possessed lactogenic activity equivalent to 1 mg ovine prolactin/mg protein and GH-like activity equivalent to 0.8 mg human GH/mg protein. The biological activity of oPL was confirmed using a rabbit intraductal mammary gland assay in vivo. After polyacrylamide gel electrophoresis at pH 8.9, oPL was resolved into one major band (isoelectric point 8.2--8.4) and four minor components, which were thought to be deamidation products of oPL. Microimmunoelectrophoresis and immunodiffusion studies confirmed that the preparation of oPL was free from serum protein contaminants.

Effect of ionic environment on the release of human placental lactogen in vitro.

Short-term incubation of human placental tissue in Krebs-Ringer bicarbonate buffered media with various concentrations of K+ and Ca2+ showed a graded response in human placental lactogen (HPL) release at different Ca2+ concentrations, but no effect at increased K+ concentration. Media with high Ca2+ caused an inhibition of release, while Ca2+-free media caused a stimulation in HPL release. High concentrations of Mg2+ inhibited release minimally, while Ba2+ had no effect. There was no change in HPL release of HPL but the significance of this effect is unknown. These results suggest that Ca2+ is not required for HPL secretion from placental tissue. It seems that HPL secretion in vitro does not follow the usual pattern where a physiological stimulus or high K+ concentration causes inward movement of calcium which couples stimulation to secretion.

Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species.

Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivites as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny.

Secretion of neurophysins by the ovary in sheep.

Measurements of venoarterial concentration differences across the ovary in anesthetized sheep have demonstrated that the ovary secretes ovine neurophysin I/II (oNP I/II) and that this process is stimulated by the prostaglandin F2 alpha analogue, cloprostenol. A parallel increase in the secretion of oxytocin (OT) was observed in response to cloprostenol, and the mean molar ratio of oNP I/II to OT secreted was 1.2. There was no detectable ovarian secretion of oNP III. Secretion of oNP I/II and OT was absent after hysterectomy. The data support other evidence indicating that the corpus luteum synthesizes OT, and confirm that the neurophysin associated with OT in the sheep is oNP I/II.

Differential immunostaining of adenohypophysial cells with antisera to ACTH and beta-endorphin.

Three antisera, raised in rabbits against human beta-endorphin were denoted Y-10, Y-18 and R-230 and tested for their ability to immunohistochemically stain cells in teh adenohypophysis of the rat and dog. By radioimmunoassay Y-10 was highly specific towards beta-endorphin with minimal cross-reactivity against beta-LPH while Y-18 and R-230 cross-reacted with both beta-endorphin and beta-LPH on an equimolar basis. In the rat pituitary, Y-18 and R-230 antisera stained cells identified as corticotrophs. With the dog, however, beta-endorphin-staining cells in the anterior pituitary and infundibulum did not necessarily co-stain for ACTH-like material. When the beta-endorphin antiserum, Y-10, was applied to rat anterior pituitary tissues, the subsequent positive immunocytochemical staining was associated not only with corticotrophs but also with somatotrophs. The findings are consistent with (a) a differential processing of the 31K pro-opiocortin and (b) the presence in rat somatotrophs of determinants that cross-react immunologically with some beta-endorphin antisera.

Presence of neurophysin-like material in the pituitary corticotrophs and melanotrophs and cells of the arcuate nucleus of the rat as revealed by immunocytochemistry.

Highly purified porcine neurophysin-II, prepared from pig posterior pituitary lobe tissue was injected into fifteen rabbits in the preparation of anti-neurophysin sera. All antisera, when used in association with immunohistochemical procedures, gave an immunoreaction in structures of the rat hypothalamo-neurohypophysial system. Four antisera, however, also stained cells of the arcuate nucleus, corticotrophs and melanotrophs. Staining of the latter two cell groups also occurred in tissues obtained from Brattleboro rats. Preadsorption of the latter neurophysin antisera with either alpha-MSH, beta-LPH, beta-endorphin, ACTH (1-24), ACTH (17-39), ACTH (1-39), gastrin and CCK, failed to inhibit the staining of the corticotrophs, melanotrophs and cells of the arcuate nucleus. Inhibition of staining was achieved only by preadsorption of the antineurophysin sera with the neurophysin antigen or an homogenate prepared from the anterior pituitary. These results support the observation by others that the biosynthesis of the ACTH-beta-endorphin system in the pituitary and hypothalamus may also be accompanied by the appearance of neurophysin.

Immunohistochemical localization of neurophysin and oxytocin in the sheep corpora lutea.

Antisera raised against neurophysin, oxytocin and vasopressin were used, in conjunction with the immunofluorescence procedure, to localize the neuropeptides in corpora luteal tissue collected from sheep at estrus. Specific immunostaining for neurophysin and oxytocin was observed in all the giant cells of the corpora lutea. In the series of corpora lutea examined staining for vasopressin was not observed.