A rapid (less than 2h) method is described for the preparation of synaptosomes from rat brain by using a discontinuous Ficoll/sucrose gradient by a flotation technique. These synaptosomes are metabolically active and minimally (less than 5%) contaminated with 'free' mitochondria as judged by marker-enzyme assays and electron microscopy.
The migration and proliferation of vascular smooth muscle cells (SMCs) during neointima formation in atherosclerosis and angioplasty restenosis is mediated by certain growth factors and cytokines, one action of which may be to promote basement-membrane degradation. To test this hypothesis further, the effects of such growth factors and cytokines on the synthesis of two basement-membrane-degrading metalloproteinases, namely the 72 kDa gelatinase (MMP-2, gelatinase A) and the 95 kDa gelatinase (MMP-9, gelatinase B) and three tissue inhibitors of metalloproteinases (TIMPs) was studied in primary cultured rabbit aortic SMCs. Expression of the 95 kDa gelatinase was increased by phorbol myristate acetate, foetal calf serum, thrombin and interleukin-1alpha (IL-1alpha); platelet-derived growth factor (PDGF) BB alone had no effect but acted synergistically with IL-1alpha. A selective protein kinase C inhibitor, Ro 31-8220, abolished induction of the 95 kDa gelatinase. In contrast, none of the agents tested modulated the synthesis of the 72 kDa gelatinase. We conclude that maximal up-regulation of 95 kDa gelatinase expression requires the concerted action of growth factors and inflammatory cytokines mediated, in part, by a protein kinase C-dependent pathway. TIMP-1 and TIMP-2 were highly expressed, and their synthesis was not affected by growth factors or cytokines. Expression of TIMP-3 mRNAs was, however, increased by PDGF and transforming growth factor beta, especially in combination. Divergent regulation of gelatinase and TIMP expression implies that either net synthesis or net degradation of basement membrane can be mediated by appropriate combinations of growth factors and cytokines.
We investigated the influence of two structurally unrelated inhibitors of matrix-degrading metalloproteinases, Ro 31-4724 and Ro 31-7467, on the primary proliferation of smooth-muscle cells from rabbit aortic explants. Both agents inhibited proliferation in a concentration-dependent manner, but did not affect cell viability. Smooth-muscle cells grown out from explants secreted 95 kDa and 72 kDa gelatinase enzymes that were also inhibited in a concentration-dependent manner by Ro 31-4724 and Ro 31-7467. Interstitial collagenase and stromelysin were not detected. We conclude that metalloproteinases are likely to be involved in the initiation of smooth-muscle proliferation.
Key enzymes of ketone body metabolism (3-hydroxybutyrate dehydrogenase, 3-oxo-acid:CoA transferase, acetoacetyl-CoA thiolase) and glucose metabolism (hexokinase, lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase) have been measured in the brains of foetal, neonatal, and adult guinea pigs and compared to those in the brains of neonatal and adult rats. The activities of the guinea pig brain ketone-body-metabolising enzymes remain relatively low in activity throughout the foetal and neonatal periods, with only slight increases occurring at birth. This contrasts with the rat brain, where three- to fourfold increases in activity occur during the suckling period (0-21 days post partum), followed by a corresponding decrease in the adult. The activities of the hexokinase (mitochondrial and cytosolic), pyruvate dehydrogenase, lactate dehydrogenase, and citrate synthase of guinea pig brain show marked increases in the last 10-15 days before birth, so that at birth the guinea pig possesses activities of these enzymes similar to the adult state. This contrasts with the rat brain where these enzymes develop during the late suckling period (10-15 days after birth). The development of the enzymes of aerobic glycolytic metabolism correlate with the onset of neurological competence in the two species, the guinea pig being a "precocial" species born neurologically competent and the rat being a "non-precocial" species born neurologically immature. The results are discussed with respect to the enzymatic activities required for the energy metabolism of a fully developed, neurologically competent mammalian brain and its relative sensitivity to hypoxia.
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