A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and of glial cells. The biosynthesis of lipids that occurs in the retina may be distinctly regulated in one neuronal type of cells with respect to another. To study the cell-type-specific aspects of lipid metabolism, a method for the separation of different retinal cell populations is needed. Herein, we describe a very simple procedure to isolate preparations highly enriched in specific retinal cell types that are suitable for in vitro biochemical assays. The method consists of selectively obtaining photoreceptors (PRC) and retina ganglion cells (RGC) from lyophilized chicken retinas using Scotch tape to assess, then, the in vitro incorporation of labeled precursors into phospholipid moieties. When their metabolic capability was assayed, it was found that these cell preparations maintain their enzyme activities intact to incorporate (32)P-phosphate into phospholipids in vitro at a similar rate as observed in fresh tissue after 1 h incubation. The highest proportion of labeling was observed in phosphatidylethanolamine (PE), followed by phosphoinositides (PIPs), phosphatidylcholine (PC) and phosphatidic acid (PA). Phosphatidate-phosphohydrolase (PAPase), a key enzyme of glycerolipid metabolism, exhibits similar levels of activity when assessed in fresh or frozen cell preparations, indicating that the lyophilization procedure does not significantly affect this activity. It is concluded that different cell populations obtained by the experimental procedure described herein, are useful to study the cellular metabolism and its regulation.

Identification of an endogenous inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase as apolipoprotein A1.

A previously described inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (GalNAc-T) (Quiroga et al., 1, 2), was purified from chicken blood serum by a new procedure. When subjected to SDS-PAGE, two major polypeptides of 27 and 70 kDa were observed. When tested in vitro, only the 27 kDa polypeptide inhibited the GalNAc-T. When added to chick cerebral embryonic neurons in culture, both polypeptides inhibited neuritogenesis. Both the 27 kDa and the 70 kDa fractions were present in the cells at 3 h following their addition to the cultures; both polypeptides had aneuritogenic activity and both inhibited the incorporation of [3H]-galactose into the cell gangliosides modifying their labeling pattern to a similar extent. Sequencing of the amino terminal end of the polypeptides showed that 18 and 9 amino acids from, respectively, the 27 and the 70 kDa polypeptides, were 100% homologues with the corresponding region of chick apolipoprotein Al (apo Al). After addition to cells in culture, no interconversion between the two polypeptides was detected after up to 20 h in culture. A monoclonal antibody that recognizes only the 70 kDa polypeptide, blocks its aneuritogenic effect without modifying that of the 27 kDa fraction. It is concluded that the endogenous inhibitor of GalNAc-T is apo Al.

Early inhibition and changes in diurnal rhythmicity of the pituitary-thyroid axis after superior cervical ganglionectomy of rats.

The effect of superior cervical ganglionectomy (SCGx) on the pituitary-thyroid axis was examined in rats. SCGx decreased serum thyrotropin (TSH) and thyroxine (T4) levels for up to 4 days after surgery, during and immediately after completion of anterograde degeneration of regional sympathetic terminals. At later times TSH levels in control and SCGx rats did not differ, but a significant increase of serum T4 was found two weeks after SCGx. A diurnal rhythm in serum TSH and T4 levels with maxima at 11.00 h (TSH) and at 14.00 and 22.00 h (T4) was found in sham-operated rats 3 days after surgery. At this time SCGx evoked a general depression of TSH levels as well as a shift of 3 h in their maximum. A similar shift of the afternoon peak and abolition of the nocturnal peak in serum T4 were detectable in SCGx rats. In SCGx animals examined during anterograde nerve degeneration, i.e. 14 h after surgery, injection of the alpha 1-adrenoceptor blocker phenoxybenzamine negated denervation-induced changes of TSH and counteracted partially T4 effects. The beta-adrenergic blocker propranolol did not modify serum TSH levels in SCGx rats but further decreased serum T4 concentration. Treatment with both drugs simultaneously did no affect TSH release compared to SCGx, phenoxybenzamine-treated rats but effectively decreased serum T4. These results further support the involvement of superior cervical ganglion neurons in the control of thyroid function.

Changes in growth hormone and prolactin release after superior cervical ganglionectomy of rats.

Superior cervical ganglionectomy (SCG X) decreased significantly serum growth hormone (GH) levels in rats 14-96 h after surgery, during and immediately after anterograde degeneration of regional sympathetic terminals. At later times (up to 28 days after SCG X) an increase in serum GH was observed. SCG X augments prolactin (PRL) release, but only at the earliest time examined (14 h after surgery). Injection of the alpha-adrenoceptor blocker, phenoxybenzamine, but not of the beta-blocker, propranolol, negated the depression in serum GH found in SCG X rats 14 h after surgery, without affecting PRL release.

Effects of oestradiol benzoate on the pituitary secretion and peripheral kinetics of thyrotrophin in the rat.

Previous works from this laboratory have demonstrated that oestradiol benzoate (EB) in euthyroid male and female rats induced a significant decrease in the pituitary content of TSH while serum levels of this hormone remained normal. The present work studied the effects of EB (25 micrograms/100 g body weight, during 9 days) on the peripheral metabolism of [125I]rTSH and on the pituitary and plasma concentration of TSH in euthyroid and hypothyroid rats. No significant variations were observed in [125I]rTSH kinetics of EB-treated euthyroid rats vs untreated controls: fractional turnover rate 2.8 +/- 0.2 vs 3.0 +/- 0.3%/min, distribution space 6.5 +/- 0.4 vs 6.8 +/- 0.5 ml/100 g body weight, disposal rate 18.4 +/- 2.4 vs 18.1 +/- 1.9 microU/100 g/min and extrapituitary pool 645 +/- 42 vs 614 +/- 43 microU/100 g body weight. Similarly, in hypothyroid rats oestrogens induced no changes in TSH kinetics except for an increase in distribution space (P less than 0.025). However, oestrogens decreased the pituitary pool of TSH (P less than 0.001) in both euthyroid and hypothyroid rats and increased the plasma TSH in hypothyroid animals (P less than 0.01), all vs their respective controls. Neither hypothyroid group had detectable plasma levels of T4 and T3. In summary: 1) the marked decrease of pituitary TSH with normal plasma TSH induced by EB appears unrelated to the peripheral metabolism of TSH, 2) the results from hypothyroid rats suggest that EB stimulates the release of TSH from the pituitary gland.