Dynamics of connexin 43 levels and distribution in the mink (Mustela vison) anterior pituitary are associated with seasonal changes in anterior pituitary prolactin content.

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.

Biochemical and morphological diversity among folliculo-stellate cells of the mink (Mustela vison) anterior pituitary.

The folliculo-stellate (FS) cells are agranular cells of the anterior pituitary whose origin and function are still a matter of debate. This study examined the presence, topography, and morphological characteristics of FS cells in the mink anterior pituitary throughout the annual reproductive cycle. The S-100 protein was used as a FS cell marker. Immunoperoxidase labeling on tissue sections demonstrated the presence of two types of S-100 positive cells. Type 1 cells were stellate-shaped cells whose nuclei were localized near the center of pituitary follicles. In this type, S-100 labeling was strong in anterior pituitary sections obtained during spring, a period characterized by high prolactin pituitary content and low gonadotropin pituitary content. Type 2 cells were rounded cells occupying the periphery of the follicles. During periods of low prolactin pituitary content and high gonadotropin anterior content the type 2 S-100 positive cells formed aggregates of several cells. The total number of S-100 positive cells was constant during these two periods of the annual reproductive cycle, suggesting that type 1 and type 2 may reflect different morphological and physiological states of the same cell. Of the two subunits, alpha and beta, that, combined, form three different dimeric S-100 proteins, mink FS cells expressed mostly the beta subunit. FS cells also expressed the glial fibrillary acidic protein (GFAP). In culture, 8 +/- 3% of anterior pituitary cells were S-100 positive. Cultured S-100 cells were elongated, polygonal, or rounded. The S-100 labeling accumulated in the cytoplasm around and within the nucleus, whereas it was weak in pseudopods and large cytoplasmic vacuoles. The presence of pseudopods suggests that cultured FS cells could migrate. The vacuoles may be related to the phagocytic activity ascribed to these cells. Some FS cells presented membrane blebbing and peripheral vesicles that were immunopositive for S-100 and that may indicate a secretory activity. Cultured FS cells possessed actin filaments organized as a peripheral network; a few actin cables were also observed running across the cytoplasm. Pseudopods depicted a highly organized actin network. The microtubules of FS cells expanded throughout the cytoplasm. The intermediate filaments expressed by cultured FS cells were GFAP and vimentin. GFAP labeling was punctate and vimentin was organized as filaments. All cultured S-100 cells were positive for vimentin, suggesting a mesenchymal origin for the cells, and all cultured S-100 positive cells were positive for GFAP, suggesting a neuroectodermal origin. In conclusion, S-100 positive cells are heterogeneous with respect to cell shape and expression of S-100 subunits in the mink anterior pituitary. The presence of morphologically different S-100 positive cells is modified in accordance with the endocrine status of the animal, suggesting that FS cells may be involved in the modulation of the anterior pituitary endocrine activity in the mink.

Intracellular mechanisms involved in dopamine-induced actin cytoskeleton organization and maintenance of a round phenotype in cultured rat lactotrope cells.

The participation of the actin cytoskeleton in the control of PRL secretion by dopamine (DA) is not yet fully understood. Recently, we demonstrated that DA induces cortical actin assembly and stabilization in anterior pituitary PRL-secreting cells (lactotropes) that can be linked to DA-induced inhibition of PRL secretion. Here we show that DA prevents cell flattening and the formation of cytoplasmic actin cables in cultured rat lactotropes. The effects of DA were reversible, mediated by D2 receptors, exclusive to lactotropes, and independent of other anterior pituitary cells present in the cultures. Because cAMP and Ca2+ mediate DA-induced inhibition of PRL secretion and synthesis, we investigated whether morphological responses to DA were dependent on these second messengers. Either inhibition of protein kinase A activity with the specific inhibitor KT5720 or blockade of Ca2+ channels with nifedipine inhibited cell flattening and induced cytoplasmic actin filament breakdown. Nifedipine was as effective as DA, but KT5720 was less effective than DA. Increased intracellular cAMP levels provoked cell flattening, which was blocked by nifedipine and KT5720, but not by DA. The results suggest that Ca2+-dependent pathways control cell shape in most lactotropes; however, in a subpopulation of lactotropes, cAMP-dependent pathways may also contribute to DA morphological responses. Next, we studied the participation of the Rho family of guanosine triphosphatases, which is known to regulate the dynamics of actin filaments. Inactivation of Rho by C3 exoenzyme induced cytoplasmic actin cable disassembly and lactotrope rounding up. No additive effects were observed among Rho-, cAMP-, and Ca2+-dependent pathways. However, C3-induced morphological responses were blocked by increased cAMP levels, suggesting that Rho-dependent steps are upstream cAMP-dependent steps. DA-induced actin cytoskeleton reorganization in lactotropes may involve modifications in the expression and localization of actin-binding proteins. DA increased expression of the actin anchoring proteins talin and alpha-actinin, but not of vinculin. DA enhanced association of talin to cell membranes. Increased talin-membrane interaction may be implicated in DA-induced maintenance of a round phenotype in lactotrope cells.

The cortical actin cytoskeleton of lactotropes as an intracellular target for the control of prolactin secretion.

We investigated the role of cortical actin filaments (F-actin) in the regulation of PRL secretion in cultured normal anterior pituitary cells. F-actin dynamics were evaluated by fluorescence microscopy, and PRL secretion from attached cells was measured by the reverse hemolytic plaque assay. F-actin localized to the periphery of lactotropes. PRL-releasing factors such as TRH, vasoactive intestinal peptide (VIP), and forskolin, or removal of the PRL-inhibiting factor dopamine (DA) from cultures chronically exposed to DA, caused fragmentation, i.e. focal disassembly of cortical F-actin. Basal, VIP-, and DA withdrawal-induced cortical F-actin disassembly were dependent on extracellular Ca2+ whereas TRH- and forskolin-induced disassembly were not. Short-term (5 min) treatment of cells with the F-actin-disrupting agent cytochalasin D (CD) enhanced basal PRL secretion but did not further stimulate TRH- or VIP-induced PRL secretion. The results support the existence of a causal link between F-actin disassembly and increased PRL secretion. On the other hand, exposure of cultures to DA decreased the percentage of cells showing cortical F-actin disassembly within minutes. Longer treatments (2-4 h) caused stabilization of cortical actin filaments as revealed by the protection vis-a-vis the depolymerizing effect of CD. The protective effect was specific for lactotropes and was evident with DA concentrations as low as 50 nM. Chronic exposure of the cells to DA blocked CD- and TRH-evoked actin disassembly and PRL secretion while VIP-induced effects were partially inhibited. Stabilization of F-actin with the marine sponge venom, jasplakinolide, also decreased basal and stimulated PRL secretion. In conclusion, our results suggest that, first, the cortical actin cytoskeleton of lactotropes is an integrator of the multiple factors regulating PRL secretion directly on the lactotrope, and second, the tonic inhibition of PRL secretion is mediated, at least in part, by DA-induced stabilization of cortical F-actin.

Cytogenetic effects of cadmium accumulation on water hyacinth (Eichhornia crassipes).

Cadmium was bioassayed to observe cytogenetic effects in the water hyacinth (Eichhornia crassipes). Plants were exposed for 96 hr to freshwater containing 0.01, 0.05, 0.10, 1.5, and 10 mg/liter of cadmium. Metal concentrations in tissues were determined by atomic absorption spectrophotometry. The highest level was found in roots, thus root-tip cells were used for cytogenetic studies; after 24 hr of exposure, micronuclei, c-mitotic effects, and pycnosis were detected and after 48 hr polyploidy was observed. A linear relationship between frequencies of micronuclei and cadmium concentrations was found: at 1.5, and 10 mg/liter micronuclei numbers were always the lowest. The inhibition of cell proliferation. shown by the low mitotic index, was proportional to the concentration and time of exposure. From the results presented in this paper it may be concluded that water hyacinth is a good sensor, due to its fast rate of metal accumulation, which allows an easy way to determine the presence of potential mutagenic compounds in water.