Effect of ethanol on the in vivo kinetics of thiamine phosphorylation and dephosphorylation in different organs of rat--II. Acute effects.

The effects of acute ethanol administration on different steps of thiamine (T), thiamine monophosphate (TMP) and pyrosphosphate (TPP) metabolism were determined in vivo in nervous tissues (cerebral cortex, cerebellum, brain stem and sciatic nerve) and in other tissues (small intestinal mucosa, kidney, heart, skeletal muscle and liver) of rats. The radioactivity of T and its phosphoesters in plasma and tissues was determined under steady-state conditions and at fixed time intervals (0.25-24 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in the presence of a constant plasma ethanol concentration (37 mM; 1.75 g.l-1) produced by repeated intragastric administration of ethanol. Control animals received water intragastrically. Ethanol-treated rats and controls were starved, with water ad libitum during the 24 hr study period. Data were evaluated by using appropriate compartmental models, which allowed calculation of fractional rate constants, turnover rates and turnover times. In nervous tissues ethanol enhanced TMP entry (without affecting T entry or T and TMP release), reduced turnover time of total T and TPP, caused an almost general enhancement of TPP dephosphorylation without affecting T pyrophosphorylation, and increased markedly T content in the mixture released by tissues. Overall, ethanol appeared to enhance exchanges of T compounds in nervous tissues. In other tissues, the effects of ethanol were less consistent. Ethanol increased T uptake in kidney and liver and T release in liver and heart, but had no effect on T exchanges in the small intestinal mucosa and in skeletal muscle. In the presence of ethanol, TMP uptake increased in heart and skeletal muscle and decreased in the small intestinal mucosa, while TMP release decreased in heart and remained unchanged in all other organs. Turnover times tended to increase for total T and to decrease for TPP. T pyrophosphorylation was generally reduced, and T phosphates dephosphorylation generally enhanced. T became the most abundant component in the mixture released from all tissues.

Effects of secretory stimulation on the Golgi apparatus and GERL of rat tracheal gland cells.

The morphological and cytochemical changes of the Golgi apparatus and GERL of rat tracheal serous cells were studied after in vivo secretory stimulation with pilocarpine. Discharge of PAS-positive secretory granules had started within 30 min and complete discharge was finished within 2 h after stimulation. Reaccumulation of secretory granules became evident at 6 h and the volume of granules was similar to that of unstimulated control cells at 8 h. At 6 h after pilocarpine administration, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules as compared to uninjected controls and this phenomenon continued 12 h after stimulation. GERL appeared to increase in extent and numerous continuities with immature granules were observed. Thiamine pyrophosphatase (TPPase) activity, normally present in trans Golgi saccules was not altered during the secretory cycle. At 2 h after stimulation, tubular structures of GERL, which also react to AcPase, were seldom observed. At 6 h, these structures were more numerous than those of control cells. These results demonstrate that, in rat tracheal serous cells, pilocarpine induces alteration of only GERL, and reconstitution followed by resynthesis of secretory granules is performed without participation of the Golgi stack.

Enzyme-histochemical demonstration of microglial cells in the adult and postnatal rabbit retina.

Enzyme-histochemical methods for thiamine pyrophosphatase (TPPase) and nucleoside diphosphatase (NDPase) were applied to wholemounted rabbit retinae to demonstrate the shape and distribution of microglial cells in early postnatal and adult animals. At birth, microglial cells were already present in the entire retina. They acquired their adult "resting shape" during the first 3 postnatal weeks. Early postnatally labeled microglial cells were scattered throughout the nerve fiber layer, the inner plexiform layer, and the outer plexiform layer (OPL); at adulthood, they were not detected in the OPL. Nissl-stained retinae revealed that the number of microglial cells continuously increased during postnatal development. The same Nissl-stained preparations were used to evaluate the topography of degenerating cells in the developing postnatal retina of the rabbit. Large numbers of degenerating pyknotic cells were observed throughout the entire retinal ganglion cell layer during the first postnatal week. Later their number decreased, and from the third postnatal week onward degenerating cells were rare. Also discussed is that the emergence of microglial cells during development may be related to cell death, whereas at adulthood the function(s) of microglial cells remains obscure. Evidence for the blood-derived origin of microglia was not obtained in this study. It is argued here that if this mode of development, which has been demonstrated for other species, is also applied to the rabbit retina, then microglia would have to migrate over considerable distances, since, postnatally, the rabbit retina is avascular for more than 1 week.

On the histochemistry of neuronal and glial TPPase.

Thiamine pyrophosphatase (TPPase) activity in the neuronal Golgi apparatus and in glia cells was investigated using different tissue preparation techniques such as fresh frozen and fixed frozen sections, the effect of ATP as an activator and the fine structural localization of the enzyme activity in glia cell processes and the glio-vascular contact point was observed under the electron microscope. TPPase was demonstrated in the glia cell using only a fixed frozen tissue preparation, activated by ATP with the concentration as low as 0.1 mM. On the contrary, enzyme activity was detected in the Golgi apparatus of the neuron regardless of tissue preparation, fresh or fixed frozen, or by the addition of ATP to the incubation medium. The identity of the glia cell type showing a positive reaction for TPPase is thought to be part astrocyte and part microglia. The fine structural observation of TPPase localization at the glia cell process ending, particularly at the glio-vascular and glio-fibral (medullated fiber) contact point suggests a functional role of this enzyme for transport of metabolites through glia cell processes to neurons and/or the blood capillary, and also may play a role in regulation of co-carboxylase.