Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells.

The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed.

Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization.

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.

Dopamine enters lactotrophs and reaches their secretory granules.

The presence of dopamine in the lactotroph cell, as well as in isolated prolactin secretory granules, was demonstrated by means of an histochemical reaction for electron microscopy. Biochemical assays further confirmed the presence of dopamine in the secretory granules. Autoradiographic preparations examined by light microscopy showed dopamine internalization in dispersed anterior pituitary cells. Isolated anterior pituitary lactotroph cells incorporated more [3H]dopamine than a fraction containing other anterior pituitary cells.

Dispersion and culture of magnocellular neurons from the supraoptic nucleus of the adult rat.

A simple and efficient procedure for the dispersion and culture of magnocellular neurons of the adult rat hypothalamus was developed. The enzymatically and mechanically dispersed cells were highly viable and showed neurite outgrowth after 3 days of culture. The neurons could be maintained for more than 4 weeks without any sign of deterioration. Light-optic immunocytochemistry of the cultured cells revealed the presence of many oxytocin- and vasopressin-immunoreactive neurons. The results indicate that the method may be suitable for many experimental purposes.

Hormone uptake and morphological changes in cultured pituicytes exposed to oxytocin and vasopressin.

Enzymatically dispersed cells, isolated from adult female rat neural lobes, were cultured for 7 days. Routine cultures showed pituicytes with compact, sometimes ovoid, cell bodies. The cytoplasmic processes of these cells exhibited several varicosities and made contact with neighboring cells forming networks. The cultured pituicytes were immunocytochemically characterized using antisera to glial fibrillary acidic protein and to S-100. Most pituicytes, when exposed during culture to oxytocin (OXY) and vasopressin (VP; 1 microM each), were devoid of their characteristic processes. Immunocytochemical staining for OXY or VP revealed that the pituicytes were capable of incorporating these hormones during culture. In cultures without added hormones, no significant staining reaction for OXY or VP could be detected. The lack of projections in pituicytes exposed to the hormones during culture is in agreement with the morphological changes observed by other authors in situ after acute hormone release. The uptake of OXY and VP may be indicative for a regulatory mechanism, by which the pituicytes control the amount of hormones present in the intercellular space.

Uterine NK cells in murine pregnancy.

Murine uterine natural killer (uNK) cells are transient, short-lived, terminally differentiated lymphocytes found in decidualized endometrium. Cells expressing natural killer cell surface markers are present in uteri of infant mice. Terminal uNK cell differentiation coincides with mesometrial decidual development subsequent to blastocyst implantation and begins about gestation day 5. uNK cells proliferate rapidly and, within 3 days, senescent uNK cells appear in normal implantation sites. Mid-gestation, senescent cells become dominant and uNK cell numbers decline until term when remaining cells are shed with the placenta. Transplantable uNK cell progenitors occur outside the uterus, suggesting that blood cell homing augments any in-utero progenitors. Early in healthy pregnancies, uNK cells produce cytokines and angiogenic molecules. Their lytic capacity in normal gestation and in pregnancy failure is incompletely defined. A significant shift recently occurred in thinking about major uNK cell functions. Activated uNK cells are now considered critical for appropriate endometrial angiogenesis in early implantation site development and in non-gestational endometrium. Because analogous cells appear in the endometria of women during each menstrual cycle and become abundant in early pregnancy, studies involving experimental pregnancy termination in genetically manipulated mice continue to have great importance for understanding regulation at the human maternal-fetal interface.

Evidence in support of a chemiosmotic mechanism for stimulated prolactin release.

In order to assess the involvement of a chemiosmotic mechanism in exocytotic prolactin (PRL) release, dispersed anterior pituitary cells from female rats were used. After primary culture they were incubated in media containing bicarbonate or isethionate ions, probenecid and carbonylcyanide p-trifluoromethoxyphenyl-hydrazone (CCTP). Cells incubated without these additives or stimulated with dibutyryladenosine 3':5'-cyclic monophosphate served as controls. Further, a crude secretory granule suspension was used to study the effects of elevated osmotic pressures. The inhibition of anion transport with probenecid always significantly reduced PRL secretion, whereas bicarbonate ions had the opposite effect. The proton ionophore CCTP only inhibited stimulated release, and the substitution of chloride ions by isethionate was ineffective. Finally, it could be observed that elevated osmotic pressures reduced the lysis of isolated secretory granules when bicarbonate ions were present in the incubation medium. The results indicate that a mechanism requiring protons and bicarbonate ions is involved in stimulated PRL release and that a different mechanism, independent from proton gradients, should be considered for basal PRL secretion.

Adenosinetriphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells.

In Paramecium cells a synchronized discharge of trichocysts (which involves only the final exocytosis steps of membrane fusion, content discharge and membrane resealing) was achieved with ATPase-blockers, Ca2+-ionophores, lipid solvents (including lysolecithin), polyethyleneglycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). Only Dibucain--and to some extent cationic detergents--can trigger exocytosis independently of extracellular Ca2+, possibly by mobilizing intracellular Ca2+. The internal free [Ca2+] necessary for exocytosis can be estimated to be greater than 10(-6) to 10(-4) M. Membrane-free trichocyst contents were isolated by density gradient centrifugation; they are converted from the contracted to the expanded state by Dibucain, CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couple the discharge (stretching) process from membrane-related phenomena. Since only the latter are inhibited by low temperature (0 degrees C), membrane lipids probably have to be in a fluid state for exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion is initiated, an independent matrix-bound system is activated for the synchronized stretching process. The energy requirement for one discharge event is estimated to be about 14 X 10(6) ATP molecules.

[Regulation of prolactin secretion at the cellular level]. / Regulación de la secreción de prolactina a nivel celular.

The present review compiles and discusses about two decades of research concerning the mechanisms involved in the regulation of the secretory activity of mammalian prolactin (PRL) containing cells. The topics include methodological aspects (such as dispersion, culture and isolation of anterior pituitary cells), the action of catecholamines, second messengers (calcium, cyclic AMP and phosphatidylinositol metabolites), neuroendocrine modulators and peptides, as well as a chapter about the morphology and biochemistry of the PRL secretory granule. Most of the accumulated data--often dispersed or disconnected--do not allow definitive conclusions and it becomes clear that we are still far away from understanding the mechanisms regulating PRL secretion. One exception is the knowledge about the dopaminergic inhibition of PRL release. Many evidences indicate that dopamine acts as a physiological inhibitor on mammalian PRL secreting cells. Some significant advances also were made studying the mechanism of action of the second messengers and, to a lesser extent, of the thyrotropin-releasing hormone. On the other hand appears an open field, which can be called the "new neuroendocrinology", with its enormous and still growing world of peptides interacting with other neuronal and humoral effectors, and which has overrun and changed the whole traditional neuroendocrinology. In this area much more information is needed and we are hopeful that the study of subpopulations and functionally related cell groups with modern cytochemical techniques (multiple immunostaining, in situ hybridization, etc.) and the development of new specific agonists, antagonists and inhibitors for the characterization of receptors and defined cellular events will lead us to a better understanding of the biochemistry and physiology of PRL secreting cells.

Galanin and cholecystokinin in cultured magnocellular neurons isolated from adult rat supraoptic nuclei: a correlative light and scanning electron microscopical study.

Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.

Coexistence of neuropeptides and their possible relation to neuritic regeneration in primary cultures of magnocellular neurons isolated from adult rat supraoptic nuclei.

The coexistence of vasopressin (VP), oxytocin (OXY), galanin (GAL) and cholecystokinin (CCK) and the synthesis of GAL and CCK during neuritic regeneration was investigated in cultured magnocellular neurons, isolated from adult rat supraoptic nuclei. Double-labelling immunofluorescence was performed after 7 days of culture using primary antibodies for VP, OXY, GAL and CCK (paired in all possible combinations) and secondary antibodies labelled with either fluorescein or rhodamine. Confocal laser scanning microscopy revealed the coexistence of the mentioned peptides in all possible combinations, an unexpected result considering that the only combinations observed in tissue sections are VP-GAL and OXY-CCK. Freshly dispersed cells were devoid of any neuritic processes and showed a very poor immunocytochemical staining reaction for GAL and CCK. In contrast, neurons cultured for 7, 12 and 21 days showed many neurites and a strong immunoreactivity for GAL and CCK indicative of an increased synthesis of both peptides in the regenerating neurons. This increased synthetic activity is consistent with transient upregulation of these peptides observed in situ after hypophysectomy by other authors. The results suggest that the upregulation of GAL and CCK is functionally related to the neuronal regeneration processes observed during culture and that the 'uncommon' coexistences as well as the prolonged sythesis of GAL and CCK may be due to the lack of environmental inputs, which normally regulate the expression and up- and downregulation of these peptides in vivo.

Immunohistochemical localization of neurotensin in a subpopulation of noradrenergic chromaffin cells of the adrenal gland of the flat snake (Waglerophis merremii).

The distribution of neurotensin (NT)-like immunoreactivity (LI) in the adrenal gland of the snake Waglerophis merremii has been examined immunohistochemically. Double staining, combining NT with tyrosine hydroxylase (TH) or calcitonin gene-related peptide (CGRP) antibodies and TH with CGRP antibodies, was also carried out. Results were analyzed by conventional and by confocal fluorescence microscopy. Immunostaining revealed a subpopulation of chromaffin cells containing NT-LI within the dorsal noradrenergic ribbon. In addition, there were some NT-immunoreactive (IR) fibers in this region. NT immunoreactivity was not present within adrenergic chromaffin cells or in cortical tissue. Double staining revealed CGRP-IR fibers innervating most of the chromaffin adrenergic cells. Within the dorsal noradrenergic ribbon, two groups of chromaffin TH-IR cells were present, one receiving a dense CGRP-IR innervation and another without contact with CGRP-IR terminals. The latter chromaffin cells displayed NT-LI. These results show, for the first time, the presence of a neuropeptide in chromaffin noradrenergic cells of a reptilian adrenal gland and open up the possibility that other peptides may also be present in these cells.

Hormone uptake and morphological changes in cultured pituicytes exposed to oxytocin and vasopressin

Enzymatically dispersed cells, isolated from adult female rat neural lobes, were cultured for 7 days. Routine cultures showed pituicytes with compact, sometimes ovoid, cell bodies. The cytoplasmic processes of these cells exhibited several varicosities and made contact with neighboring cells forming networks. The cultured pituicytes were immunocytochemically characterized using antisera to glial fibrillary acidic protein and to S-100. Most pituicytes, when exposed during culture to oxytocin (OXY) and vasopressin (VP; 1 microM each), were devoid of their characteristic processes. Immunocytochemical staining for OXY or VP revealed that the pituicytes were capable of incorporating these hormones during culture. In cultures without added hormones, no significant staining reaction for OXY or VP could be detected. The lack of projections in pituicytes exposed to the hormones during culture is in agreement with the morphological changes observed by other authors in situ after acute hormone release. The uptake of OXY and VP may be indicative for a regulatory mechanism, by which the pituicytes control the amount of hormones present in the intercellular space