Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFN gamma and TNF alpha, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1.

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.

Pubertal development in teleost fish is characterized by gonadal growth that is directly stimulated by the pituitary gonadotropins, FSH and LH. We used a quantitative ribonuclease protection assay to provide, for the first time, the developmental profiles of the alpha-, betaFSH-, and betaLH-subunit gene expression in a seasonal breeding fish, the female striped bass (3-yr study, n = 207). Two-year-old females were sexually immature, although a transient rise in all gonadotropin subunit mRNAs was measured in the pituitary. Pubertal ovarian development occurred in 65% of 3-yr-old females, characterized by the appearance of lipid droplets within the oocytes. This reproductive phase, termed pubertal development, was associated with a 34-fold increase in the mRNA levels of betaFSH and a rise in the pituitary concentration of LH. The first sexual maturation took place in 4-yr-old females and coincided with a 218-fold increase in the mRNA levels of betaFSH. During this time period, the mRNA levels of the alpha and betaLH subunits increased by 11- and 8-fold, respectively. At the final stages of vitellogenic growth, mRNA levels of betaFSH declined to basal levels, whereas the mRNA levels of the alpha and betaLH subunits remained elevated. Throughout the study, pituitary LH concentration was positively correlated to the mRNA levels of betaLH, but plasma levels of LH remained low and unchanged (0.4-0.8 ng/ml) despite increasing levels of pituitary LH concentration, suggesting a regulated secretion pathway. Taken together, the data show that the profiles of betaFSH and betaLH mRNAs appear to follow an annual rhythm that is associated with developmental events in the growing oocytes. In particular, increasing levels of betaFSH mRNA appear to underlie the first sexual maturity in the female striped bass.