The present study provides evidence that the adult mammalian retina is highly sensitive to the excitotoxic action of NMDA. In particular, we have investigated the effects of a single intravitreal injection of different doses of N-methyl-D-aspartate (NMDA) (2–200 nmoles) on the adult rat retina. Morphological evaluation of transverse sections of retinae demonstrated a dose-dependent loss of cells in the ganglion cell layer (GCL) and a reduction in the thickness of the inner plexiform layer. No obvious alterations were noted in the more distal retinal layers. Quantitative analyses of Nissl-stained whole-mounted retinae revealed that administration of 20 nmoles of NMDA resulted in a 70% loss of cells with a soma diameter greater than 8 μm (presumed retinal ganglion cells); a 20% loss of cells with a soma diameter smaller than 8 μm (presumed displaced amacrine cells) was also observed. In addition, NMDA produced a dose-dependent decrease of retinal choline acetyltransferase (ChAT) activity, suggesting that NMDA affects cholinergic amacrine cells as well. MK-801, a non-competitive NMDA antagonist, completely prevented the NMDA-induced loss of cells in the GCL and blocked, in a dose-dependent manner, the NMDA-induced decrease of ChAT activity. The excitotoxic action of NMDA observed in these experiments is thus likely mediated through the NMDA receptor subtype. This ”in vivo” model may be utilized to identify potential drugs that antagonize or limit the deleterious effects consequent to NMDA receptor overstimulation in the central nervous system.

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-lbut not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis. Moreover, during vascular myogenesis there is a quantitative isoactin change from fl-actin, which is predominant in the fetal/neonatal phase of development, to a-actin isoform pertinent to the adult SMC phenotype [4,5, 81. In vitro, even though a shift in the isoactin content towards the expression of a-actin has been described [9, 101, the morphological and structural changes which cultured aorta SMCs may undergo during the phenotypic transition from a 'synthetic' (immature) to 'contractile' (differentiated) state 11 11 do not promote the achievement of the fully differentiated cell phenotype found in vivo [12] (and S. Sartore, A. M. C. Zanellato and A. C. Borrione, unpublished work).Myosin, an essential component of contractile machinery in the sarcomeric and non-sarcomeric system, is present in multiple isoforms [13, 141 which are distributed in a tissuespecific fashion and are developmentally regulated [15]. Electrophoresis in pyrophosphate gels and two-dimensional gel electrophoresis have demonstrated the existence of an embryonic myosin light chain (LZ3) in the chicken gizzard [16]. More recently, biochemical and immunochemical studies have reported on structural differences in the myosin heavy chain (MHC) composition of developing and adult avian To define more precisely the potential changes of SM-MHC and NM-MHC isoforms in developing mammalian SM we have undertaken a combined electrophoretic and immunochemical analysis on rabbit fetal, neonatal and adult aorta SM using monoclonal isoform-specific anti-myosin antibodies. MATERIALS AND METHODS Preparation of immunogenActomyosin from SM of adult bovine aorta was prepared according to the method of [30] and stored in 50% (by vol.) glycerol at -30°C until use. Crude actomyosin of human platelets was prepared following the procedure of [31] except for the final purification on Sepharose 4B column which has been omitted. Myosin extractsSpecimens of thoracic aorta from 29-day-old fetus, 5-dayold and 12-week-old white New ...

234 SMC that accumulate in arterial intima show morphological features that are distinct from those in the medial SMC, including myofilaments, rough endoplasmic reticulum, free ribosomes, and mitochondria. 456 A differential distribution of synthetic organelles and myofilaments can also be demonstrated in cultured vascular SMC 7 and the intimal thickening that follows endothelial injury. 6 The structural and synthetic profile of developing smooth muscle (SM) tissue has some features in common with proliferating SMC found in the first stages of intimal thickening. 8 In this system, after cessation of cell division, SMC partly resume the adult differentiated morphology.9 According to some authors, SMC showing differentiated characteristics are unable to proliferate, and the term "phenotypic modulation" has been used 510 to indicate the existence

The most effective therapy of human prolactinomas is represented by dopamine D-2 receptor agonists; there is, however, a population of nonresponder patients who require surgical intervention. In the present study, we report that prolactinomas totally resistant to pharmacological therapy have a high potential of both growing in soft agar and forming tumors in nude mice and lack D-2 receptors for dopamine. These tumors express the receptors for nerve growth factor (NGF) and are sensitive to its differentiating activity. After exposure to NGF for 4 days, prolactinoma cells decreased their proliferation rate, lost their capability to form colonies in soft agar, lost their tumorigenic activity in nude mice, and reexpressed the lactotroph-specific D-2 receptor protein inhibiting prolactin release. These effects were permanent after NGF withdrawal and were reproducible in vivo in nude mice transplanted with the tumors. NGF in fact remarkably and lastingly depressed tumor growth and induced expression of D-2 receptors when injected intravenously once a day for 5 days into prolactinoma-bearing nude mice. These data suggest that NGF may induce a long-lasting switch of gene expression in human prolactinomas, modifying their transforming phenotype and reverting them to more differentiated, less malignant, dopamine-sensitive lactotroph-like cells. The possibility thus arises that short-term treatment with NGF may restore the refractory patients to conventional pharmacological therapy with D-2 agonists.Nerve growth factor (NGF) is a neurotrophic protein that promotes the growth, differentiation, and survival of peripheral sympathetic neurons, the majority of neural crestderived sensory nerve cells (1-3), and the ascending cholinergic neurons ofthe basal forebrain (4)(5)(6). NGF also converts the pheochromocytoma cell line PC-12 into a sympathetic neuronal phenotype (7,8) and exerts a differentiating action on cells of the immune system (9, 10). The effects of NGF are directly dependent on initial binding to specific cell-surface receptors. Two protein components are required to form the high-affinity ligand-binding site for NGF: the 140-kDa protein-tyrosine kinase, which is encoded by the trkA protooncogene (gpl40trk) (11-13), and the 75-kDa glycoprotein gp75NGFR (14,15 We report here that bromocriptine-resistant prolactinomas lack D-2 receptors. Since these tumors express NGF receptors, we investigated whether they were sensitive to the differentiating action of the neurotrophic factor. The results show that exposure of prolactinoma cells to NGF resulted in their conversion into a more differentiated lactotroph-like phenotype reexpressing the D-2 receptor protein. MATERIALS AND METHODS Preparation of Prolactinoma Cell Cultures and Treatmentwith NGF. Tumor specimens were obtained from eight patients refractory to bromocriptine and five sensitive subjects who did not tolerate the drug. The five sensitive subjects responded to bromocriptine at 5-10 mg/day with a normalization of plasma PRL levels. Three of them were for...

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