Richard I. Weiner

Immortalization of hypothalamic GnRH by genetically targeted tumorigenesis

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation.

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen‐activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf‐1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β‐adrenergic receptors. Both VEGF‐ and bFGF‐induced Raf‐1 activity are blocked in the presence of forskolin or 8‐bromo‐cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP‐dependent protein kinase (PKA), since treatment with H‐89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen‐induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen‐induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997.

Signaling Pathways Involved in GnRH Secretion in GT1 Cells

The GT1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the role of the cyclic AMP/protein kinase A, cyclic GMP/protein kinase G and Ca2+/protein kinase C signaling pathways in the regulation of GnRH secretion. Superfusion of GT1-1 cells with the cyclic AMP analog 8-Br-cyclic AMP (0.5 and 2.5 mM) or the adenylate cyclase activator forskolin (1 and 10 microM) for 100 min increased the amplitude of GnRH secretion 2- to 35-fold. The cyclic GMP analog 8-Br-cyclic GMP (2.5 mM) also stimulated the amplitude of GnRH release from superfused GT1-1 cells, although to a much lesser extent (1.5- to 3-fold). The amplitude of GnRH pulses was also stimulated (5- to 50-fold) by the protein kinase C activator TPA (1 microM). Increasing intracellular Ca2+ with an ionophore (ionomycin, 1 microM) or by the Ca2+ channel activator Bay K 8644 (10 microM) also stimulated GnRH release, while secretion was markedly decreased and spontaneous pulsatility abolished by the L-type Ca2+ channel blocker methoxyverapamil (10 microM). These results demonstrate that in GT1 cells the protein kinase A, protein kinase G and protein kinase C pathways are functionally coupled to regulation of GnRH secretion. Furthermore, pulsatile GnRH secretion is coupled to the entry of extracellular Ca2+ via L-type Ca2+ channels.

Differences in the kinetics of dopamine uptake in synaptosome preparations of the median eminence relative to other dopaminergically inervated brain regions.

A comparison of the kinetics of dopamine uptake was made in synaptosomal preparations of the median eminence, striatum and olfactory tubercle. Double reciprocal Lineweaver-Burk plots of the initial velocity versus the concentration of dopamine yielded a single straight line in all three areas. The Michaelis constant (Km) in the median eminence (1.8 +/- 0.9 X 10(-6) M) was significantly higher (p < 0.05) than in the striatum (4.2 +/- 0.8 X 10(-8) M) or olfactory tubercle (6.3 +/- 1.9 X 10(-8) M). Uptake in the median eminence appeared to be predominantly into dopaminergic terminals since preincubation with desipramine did not affect the maximum velocity (Vmax) of dopamine uptake. Observed uptake was predominantly due to transport across the neuronal membrane and not into storage granules, since reserpine only caused a small decrease in uptake. The low affinity of dopamine uptake in median eminence synaptosomes is consistent with the neurosecretory nature of these terminals, whereas, in the striatum and olfactory tubercle, high affinity reuptake is consistent with the role of dopamine as a neurotransmitter.

Immortalization of Neuroendocrine Cells by Targeted Oncogenesis

Publisher Summary This chapter discusses the immortalization of neuroendocrine cells by targeted oncogenesis. By targeting Tag expression to the early developing anterior pituitary with the α-subunit promoter, a developmental precursor of the mature gonadotrope has been immortalized. Although this approach has proved difficult in the neural cells of the brain, targeting with a gene that is very tightly expressed in a small population of neurons prior to the transition to the postmitotic state may be successful. Specific tumors of gonadotropin-releasing hormone (GnRH)-secreting neurons are produced by introduction of a hybrid gene composed of the GnRH promoter coupled to the coding region for Tag into transgenic mice. Clonal cell lines derived from these tumors express GnRH mRNA and secrete GnRH in response to depolarization. Thus, by targeting oncogenesis to a specific, small population of neurons using the regulatory region of a gene that is expressed late in the differentiation of that cell lineage, success is achieved in immortalizing hypothalamic GnRH neurons, which maintain many differentiated functions in culture.

Epoxy Derivatives of Arachidonic Acid Are Potent Stimulators of Prolactin Secretion

Arachidonic acid is metabolized to three distinct classes of metabolites: cyclooxygenase produces prostaglandins, prostacyclins, and thromboxanes; lipoxygenase produces hydroperoxyeicosatetraenoic acids and, epoxygenase, a NADPH-dependent cytochrome P-450 enzyme, produces epoxyeicosatrienoic acids. Addition of 5,6-epoxyeicosatrienoic acid (5,6-EET) to GH3 cells, a rat anterior pituitary cell line, produces a rapid, dose-dependent stimulation of prolactin (PRL) release. Incubation with arachidonic acid (AA) was ineffective at increasing PRL release. The lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), however, increased PRL release from GH3 cells but with a much lower maximal response than 5,6-EET. We examined the role of metabolism inhibitors in 5,6-EET-mediated PRL release. Microsomal and cytosolic epoxide hydrolase (EH) inhibitors do not alter 5,6-EET-induced PRL release, suggesting that EH does not play a significant role in 5,6-EET mediated PRL release from GH3 cells. A chemical analog of 5,6-EET wherein the epoxide oxygen is replaced with a sulfur to afford 5,6-thioepoxyeicosatrienoic acid was also tested and found to stimulate the release of PRL, although not to the same extent as 5,6-EET. Although 5-HETE tends to increase PRL release from GH3 cells, 5,6-EET is significantly more potent at the stimulation of PRL release from GH3 cells.

Activation of anterior pituitary folliculo-stellate cells in the formation of estrogen-induced prolactin-secreting tumors

The response of folliculo-stellate (FS) cells of the anterior pituitary to estrogen has been studied in two strains of rat which differ in estrogen responsiveness. Fischer 344 (F344) rats are highly estrogen-responsive in comparison to Sprague-Dawley (S-D) rats. Ovariectomized adults were implanted with silastic capsules containing 17 beta-estradiol benzoate. Control and experimental animals were sacrificed 10 and 20 days after implantation of the silastic capsules. Most FS cells of F344 rats revealed dramatic changes, i.e. activation as phagocytes, after 10 and 20 days of estrogen treatment. These activated FS cells separated themselves from adjacent parenchymal cells and frequently contained phagosomes which included portions of granulated cells, mostly somatotrophs and lactotrophs. They also contained a variety of lysosomal dense bodies and dilated cisterns of endoplasmic reticulum. Endfeet processes abutting the parenchymal basal lamina contained large aggregates of dumbbell-shaped granules and tubulo-vesicles. Subjacent to the endfeet processes the basal lamina was often discontinuous, and profiles suggesting uptake of fragments of the basal lamina by FS cells were evident. Most FS cells of estrogen-treated S-D rats either were identical to those of controls or were only minimaly modified. However, a few activated FS cells were present and these contained a variety of lysosomal dense bodies and phagosomes, as well as lipid inclusions and dilated endoplasmic reticulum. Endfeet projections at the parenchymal basal lamina contained very few dumbbell-shaped granules or tubulo-vesicles, and the basal lamina was intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient removal of dopamine potentiates the stimulation of prolactin release by TRH but not VIP: stimulation via Ca2+/protein kinase C pathway

The effectiveness of thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP) to release prolactin (PRL) after a brief interruption of the tonic inhibitory action of dopamine (DA) was investigated in enzymatically dispersed anterior pituitary cells in superfusion. We also studied the involvement of cAMP and Ca2+/protein kinase C second messenger systems in the mediation of the stimulated PRL release. Anterior pituitary cells from lactating or E2-treated rats were superfused for 10 min with secretagogues either during continual dopamine administration or 10-20 min after a 10-min transient interruption of DA (500 nM). Removal of DA for 10 min resulted in a significant increase in PRL release which had returned to basal levels 10 min after the return of DA to the superfusion. During continuous DA exposure, TRH administration (10 nM) did not alter the rate of PRL release from cells from lactating rats; however, TRH caused a 2-fold increase after the transient interruption of DA. The transient escape from DA inhibition also increased the effectiveness of TRH (100 nM) to release PRL from cells from E2-treated rats (from a 4- to a 15-fold stimulation). In contrast, VIP (0.5 or 5 microM) caused a 2-fold stimulation of PRL release in both cells treated with continuous or transiently interrupted DA.(ABSTRACT TRUNCATED AT 250 WORDS)

β-Endorphin concentrations in serum, hypothalamus and central gray of hypophysectomized and mediobasal hypothalamus lesioned rats

The serum and brain concentrations of beta-endorphin immunoreactivity have been studied in intact, mediobasal hypothalamus (MBH) lesioned and hypophysectomized male rats. After hypophysectomy there is a major reduction (90%) of beta-endorphin concentration in the serum but only a partial reduction (20%) in the mediobasal hypothalamus. However, MBH lesions enhance beta-endorphin serum values in previously hypophysectomized rats. Long-term MBH lesions alone lead to an almost complete disappearance of beta-endorphin in the central gray matter with a slight decrease in the serum. These data clearly show that: (1) the pituitary is the major source of beta-endorphin in the serum; (2) the hypothalamus is the major source of beta-endorphin in the central gray matter; and (3) there is clear influence of the pituitary on hypothalamic beta-endorphin.

[16] Culture of dispersed anterior pituitary cells on extracellular matrix

Publisher Summary This chapter describes methods used in the production of extracellular matrix (ECM), dispersion of anterior pituitary tissue, separation of dispersed cells, and development of a serum-free medium for maintenance of cultured cells. The chapter presents a series of cell dispersion techniques for the anterior pituitary in a variety of species. The chapter focuses on obtaining preparations that were acutely functional rather than on obtaining high yields of cells. For this reason, avoid using proteolytic enzymes, since the use of Viokase destroys cell surface receptors for dopamine in bovine anterior pituitary cells. Dispersed anterior pituitary cells are separated from debris and red blood cells by centrifugation through Ficoll-Hypaque as previously described for the isolation of mouse lymphocytes. The Ficoll-Hypaque is the method of choice based on ease of preparation, cell recovery, and loading capacity.