High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain.

Ontogenetic studies indicate that inositol phosphate accumulation in rodent brain tissue by cholinergic muscarinic agonists as well as expression of high-affinity neurotensin receptor (NTS1) peak at 7 days after birth. Herein, potential participation of this receptor in such effect was investigated. Cerebral cortex prisms of 7-day-old rats were preloaded with [3H]myoinositol and later incubated during 60 or 20 min in the presence of muscarinic agonist carbachol plus neurotensin and SR 48692, a non-peptide NTS1 antagonist. In 60-min incubation experiments, inositol phosphate accumulation by 10(-3) M carbachol was roughly 320%, an effect which remained unaltered plus 10(-6) M to 10(-4) M neurotensin but partially decreased with equimolar SR 48692 concentration. In 20-min incubation experiments, inositol phosphate accumulation by 10(-3) M carbachol was circa 240%, a value which attained 320-360% plus 10(-7) M neurotensin; this effect was totally blocked by 10(-7) M SR 48692. It was concluded that in inositol phosphate accumulation by carbachol, besides the cholinergic muscarinic receptor, the NTS1 receptor is likewise involved; findings at 60 min are attributable to the effect of endogenous neurotensin whereas those at 20 min most likely involve both endogenous and exogenously added peptide.

Proteases with plasminogen activator activity in hamster oviduct.

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.

Developmental pattern of plasminogen activator activity in chick brain hemispheres.

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.

Developmental pattern of plasminogen activator activity in chick optic lobe.

Plasminogen activators are serine proteases which play a key role in morphogenesis and tissue remodelling. Two different molecular types, tissue-type and urokinase-type, were identified and they were postulated to play a role in neural development. The developing chick optic lobe plays a central role in processing visual information. In previous studies we demonstrated the occurrence of high levels of plasminogen activator activity in this model. The aim of the present paper is to study the temporal pattern of expression of this activity and characterize the type of plasminogen activator expressed in the developing optic lobe. Using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions we measured the protease activity with a radial fibrinolytic assay. Employing different inhibitors of fibrinolytic activity and a zymographic assay, we showed that the developing optic lobe expresses only one type of plasminogen activator which corresponds to an urokinase-type of 70 kDa. Our results indicate that peaks of protease activity temporally correlate with massive neuronal migration, neurite outgrowth and synapse formation and maturation. This suggests that a plasminogen activator could play a role in these developmental events. This consistent pattern of variability strongly suggests that it is developmentally regulated and, if so, it could be a reliable parameter to study neural plastic changes induced by modifications in the environmental stimulation.

Temporal pattern of plasminogen activator activity in the developing chick cerebellum.

Plasminogen activators are considered to be involved in several developmental events. The present work aims at characterizing the developmental pattern of expression of plasminogen activators in the chick cerebellum. Soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions were used for determination of the enzyme activity with a radial fibrinolytic assay. By using specific inhibitors and different anti-plasminogen activators antibodies it is shown that only one type of the enzyme, the urokinase-type plasminogen activator, is expressed during the cerebellum ontogeny. Our results show the existence of a bimodal pattern of enzyme activity with two peaks that temporally coincide with the processes of massive neuronal migration, neurite outgrowth and synapse formation and plasticity. It is proposed that plasminogen activator could play a role in these developmental events and that its pattern of variability is developmentally regulated.

Development of the laminated pattern of the chick tectum opticum.

Several ontogenetic studies have been devoted to the structural organization of the developing tectum opticum. They disagree in many respects because they are based on histological preparations performed with differently oriented planes of section. According to our results the differences found in the literature mainly result from the fact that the developmental gradient axis undergoes remarkable positional changes with respect to both optic lobe and neural tube longitudinal anatomical axes during the early stages of development. The present work is a dynamic description of the tectum opticum lamination based on sections coinciding with the developmental gradient. Since this latter displays a curved disposition, several slightly modified planes of section had to be used to obtain a complete picture along the developmental gradient. The development of the tectal architecture proceeds from a relatively simple organization through increasingly complex multilaminated patterns. A dynamic interpretation of successive images of a particular region observed at increasing developmental stages or of images observed at a particular stage along the entire length of the developmental gradient axis, allows us to propose that embryonic laminae are only transient spatial arrangements of cells actively migrating from the sites where they were generated to those where they will definitively reside. These considerations led us to define a nomenclature that establishes clear correlations between the early transient organizations and the definitive one of the fully developed optic tectum. This type of nomenclature could be usefully applied to describe dynamically the development of structures displaying multilaminated patterns such as other cortical zones of the central nervous system.

Current Triton X-100 treatments do not allow a complete plasminogen activator extraction from developing nervous tissue.

Determinations of plasminogen activator (PA) activity are usually performed in Triton X-100-treated tissue homogenates or crude membrane fractions. Such preparations usually involve a single Triton X-100 treatment. In the present paper we describe the pattern of variability of PA activity measured in different fractions obtained from the developing chick CNS by a repetitive procedure of Triton X-100 treatment and ultracentrifugation. To further characterize this PA activity we have also performed zymographic analyses during the embryonic development and the early postnatal life. Our results show that: a) a single Triton X-100 treatment does not completely extract the enzyme and this lead to an underestimation of the total PA activity; b) the PA activity is associated with the particulate component of the total tissue homogenate requiring its complete solubilization more drastic Triton X-100 treatments; c) better estimations of total and specific activities are obtained by using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions; d) the developing chick optic lobe expresses only one kind of PA molecule along the entire development; e) the level of PA activity vary characteristically during the ontogeny and the early postnatal life indicating the existence of a developmentally regulated mechanism of PA expression.

Urokinase-type plasminogen activator activity increases during the growth of two murine mammary adenocarcinomas with different metastasizing abilities.

Urokinase-type plasminogen activator (u-PA) plays an important role in tumor growth and metastasis. The aim of this work was to study the u-PA production, in vitro and in vivo, in a transplantable murine mammary adenocarcinoma (M3), moderately metastatic to lung, and in a related tumor variant (MM3), highly metastatic to the same organ, during tumor development. At different times post-transplantation, tumors were employed to prepare either primary cell cultures or homogenates. PA activity from conditioned media (CM), cell lysates (CLs) and tumor homogenates (THs) was quantitated by means of a fibrinolytic assay. Immunoneutralization and zymographic assays were performed to identify the PA present in both tumors. PA activity in CM, CLs and THs, that was undetectable at early stages, increased significantly along the growth of M3 adenocarcinoma. Secreted PA activity in MM3 CM was measurable at early stages and consistently increased up to 37 days post-transplantation, but a marked fall of activity was found at 48 days. PA activity in MM3 THs exhibited the same enhancement and late fall found in vitro. A positive correlation was observed between tumor size and THs PA values in both tumors. The PA present in cell cultures and THs was identified as of the u-PA type. These results support the hypothesis that high u-PA levels are important for tumor invasion and that the stage of tumor development is a critical factor in their PA activity.

Correlation between urokinase-type plasminogen activator production and the metastasizing ability of two murine mammary adenocarcinomas.

Plasminogen activator (PA) activity was studied in a transplantable murine mammary adenocarcinoma (M3), moderately metastatic to lungs, and in a highly metastatic variant tumor (MM3) to establish whether a correlation existed between this enzyme and the tumors' metastasizing abilities. The cell-associated and secreted PA activities from primary cultures of both tumors, as well as from solid tumor homogenates, were quantitated by means of a fibrinolytic assay. Immunoneutralization and zymographic assays were done to identify the PA present in both tumors. In culture, the highly metastasizing MM3 cells produced (secreted plus cell-associated activator) 3.3-fold higher PA levels than the M3 cells. This difference was mainly attributed to the enhanced secreting ability of MM3, as these tumor cells secreted 102 times their cell-associated PA activity within 24 hr, while M3 cells secreted only 11 times this activity during the same period. PA activity was also significantly higher in MM3 than in M3 tumor homogenates. Acid-labile inhibitors and non-specific proteases were not detected. In both tumors, PA was characterized as urokinase-type, with a molecular weight of approximately 48 kDa. These results suggest that, in this model, PA plays a role in metastasis formation.

Protein-synthesizing machinery in the growing oocyte of the cyclic mouse. A quantitative electron microscopic study.

A quantitative ultrastructural evaluation of the oocyte ribosomal population was carried out during the oocyte growth, bearing in mind that this period of the mouse oogenesis displays the greatest activity of ribosomal RNA synthesis. At the onset of growth almost 3/4 of the oocyte ribosomes exist as singles, these become polysomal ribosomes as growth progresses. At the same time the number of ribosomes increases. Once the major growth period has elapsed, the number of ribosomes starts to decrease just when lattice-like structures exhibiting a periodic organization begin to accumulate in the oocyte cytoplasmic matrix. Evidence, like the particulate organization of these lattices, the size of their particles, its digestion by RNase, and the time of the lattice appearance, together with data reported by several authors, allows one to suggest that near the end of the oocyte growth a great part of the ribosomes are stored in the lattices to be used during early development.