Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.     

Epinephrine: A Potential Neurotransmitter in Retina

Abstract: Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) are present in rat retina. DA is the major catecholamine, whereas NE and EPI represent ∼5% of the DA content. DA is contained in a subpopulation of amacrine cells and has been the subject of numerous studies. We investigated the origin and properties of NE and EPI in retina. Following superior cervical ganglionectomy, there was a decrease in NE content, but no decrease in EPI or phenylethanolamine- N -methyltransferase (PNMT) activity. PNMT in retina has many of the substrate-specificity and inhibitor-sensitivity characteristics of other tissues. Enzyme activity is enhanced in newborn rats by treatment with dexamethasone. Exposure to a lighted environment increases retinal EPI in normal and superior cervical ganglionectomized rats. EPI content increased for more than 2 h in a lighted environment. We conclude that most of the NE is contained within the sympathetic neurons that innervate the eye from the superior cervical ganglion, whereas EPI is contained in retinal elements that are responsive to photic stimulation.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood-Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Ethanol Inhibits Basic Fibroblast Growth Factor-Mediated Proliferation of C6 Astrocytoma Cells

Abstract: Early ethanol exposure alters the proliferative activity of glial and neuronal precursors in the developing CNS. The present study tests the hypothesis that ethanol-induced alterations in cell proliferation result from interference with growth factors. An in vitro model of astroglia (C6 astrocytoma cells) was used to study the effects of ethanol on proliferation mediated by basic fibroblast growth factor (bFGF). bFGF stimulated the proliferation of C6 cells. This bFGF-enhanced proliferation was evident by increases in total cell number, DNA synthesis (as measured by [³H]thymidine incorporation), and the number of cells that took up bromodeoxyuridine. A synthetic peptide that specifically blocked the binding of bFGF to its high-affinity receptor completely abolished the proliferation-promoting effect of bFGF. The action of another mitogen for C6 cells, insulin-like growth factor-1, was not affected by this peptide. Therefore, the bFGF-stimulated proliferation was mediated through a specific bFGF receptor. Ethanol inhibited bFGF-mediated proliferation in a concentration-dependent manner. Ethanol concentrations of 100 and 200 mg/dl partially inhibited bFGF-mediated proliferation (by 58 and 74%, respectively), whereas concentrations of ≥400 mg/dl completely abolished the growth-stimulating effect of bFGF. Our data show that ethanol alters proliferative activity of C6 cells by disrupting the action of bFGF. The target of ethanol neurotoxicity is a receptor-mediated activity. bFGF can affect cell proliferation by a non-receptor-mediated intracellular pathway, but ethanol does not have an impact on this pathway.