Enrica Draghi

Effects of purine cyclic nucleotides on the growth of neonatal rat hepatocytes in primary tissue culture.

Abstract cGMP and db-cGMP administered for 20–24 h to neonatal rat hepatocytes in primary culture stimulated their DNA synthesis and proliferation only at concentrations higher than the physiological one, whereas at concentrations equal to or lower than the physiological concentration they were ineffective or inhibitory for both activities. Induction of DNA synthesis to be effected by cGMP required 15 h of treatment, preceded, however, by inhibition of the same process between the 6th and the 14th hour of exposure. In contrast, cAMP and db-cAMP stimulated the flow of cultivated hepatocytes into the S and M stages of their mitotic cycle when administered at very wide concentration range, including the physiological for cAMP and the sub-physiological for db-cAMP. cAMP was effective after 12–14 h of treatment. Equimolar mixtures of cGMP with cAMP and of db-cGMP with db-cAMP also stimulated the proliferative activity of primary hepatocytes, but only at very low doses, which induced a first peak of DNA synthesis between the 2nd and the 6th hour of treatment and a second peak at about the 18th hour. These actions of the cyclic compounds, employed singly or in equimolar combination, were shown to be specific, since they could not be reproduced by their main metabolites. The present results strengthen the view that cAMP plays a pre-eminent role in the positive regulation of hepatocyte proliferation. Contrary to the postulate of the dualistic doctrine, cGMP by itself is not proliferogenic in the physiological range; in fact, cGMP acts as an ancillary, possibly dispensable, compound whose physiological role may be to help, in cooperation with cAMP, liver cells to cross the G1/S boundary of their growth-division cycle.

Primary tissue culture of human adult adrenocortical cells. Methodology and electron microscopic observations on ACTH-deprived and ACTH-treated cortical cells

SummaryA method of primary tissue culture involving both disaggregation of cells by repeated exposure of small tissue fragments to a solution of trypsin, collagenase and hyaluronidase and explantation of the residual tissue fragments intermingled with isolated cells onto polyethylene discs, has been shown to be adequate for the prolonged maintenance (up to 30 days) in vitro of cells arising from decapsulated adult human adrenocortical tissue. The technique and its critical points are discussed.Adrenocortical cells were organized both as outgrowing columns from microexplants or as variously sized islets of monolayered cells. The ultrastructural features of ACTH-deprived adrenocortical cells (i.e., mitochondria with laminar cristae, endoplasmic reticulum mainly consisting of rough profiles, abundance of lipid droplets and β-glycogen particles) suggest that the cells dedifferentiate and retain practically no steroidogenic activity.After 2 days of ACTH-treatment, cultured parenchymal cells were found to be quite similar to the zona fasciculata elements of the normal human adrenal cortex. They were grouped in islets of about 50–100 cells. Rough endoplasmic reticulum had decreased, but smooth endoplasmic reticulum showed focal proliferation. The pleomorphic mitochondria with laminar cristae, transformed into a homogeneous population of round or ovoid mitochondria containing tubulo-vesicular cristae. Lipid droplets and glycogen particles were decreased in number.After 7 days of daily treatment with ACTH, the cortical elements, whose nucleus and cytoplasm seemed to be enlarged, were arranged in clusters formed by up to 300 monolayered elements, in which dividing cells were consistently observed. Their cytoplasm was filled with a meshwork of smooth reticulum tubules, in which scantly ribosome-studded profiles and occasional small stacks of granular cisternae were embedded. Mitochondria were similar to those of the 2 days ACTH-treated cultures. Lipid droplets and glycogen particles were absent. The functional significance of these structural changes as well as the possible mechanism underlying the differentiative effect of ACTH are discussed.Primary cultures of human adult adrenals are proposed as a new tool for studies into the physiopathology of the adrenocortical cells under carefully controlled experimental conditions.mis|It is a pleasure to acknowledge our thanks to Drs. F. Mantero and C. Eccher for kindly supplying the normal human adrenocortical tissue. Thanks are also due to Mr. G. Gottardo for his excellent technical assistance.mis|This work was partly supported by a contract with the CNR-Italy (C.T. 73.00663.04).

Studies on the persistence of differentiated functions in rat hepatocytes set into primary tissue culture. II. Production of specific exportable proteins and the effect of purine cyclic nucleotides: an immunofluorescent study.

SummaryImmunofluorescent studies showed that even after 15 days in vitro primary neonatal rat hepatocytes contained in their cytoplasm detectable amounts of different adult rat serum proteins, including fibrinogen and proalbumin. Estimation of the intensity of specific fluorescence revealed that in untreated cultures the hepatocytic content of the various exportable antigens progressively diminished between the 5th and 15th day in vitro. Treatmen with cAMP (10−5 M daily) alone increased in hepatocytic cytoplasm, with respect to parallel controls, the content of total exportable proteins and of proalbumin. Daily administration of an equimolar association (10−5 M) of cAMP with cGMP increased the total protein, proalbumin and fibrinogen content of hepatocytes. Daily treatment with cGMP (10−5 M) alone caused only light and transitory increases in the content of proalbumin and fibrinogen. Rocket immune electrophoresis showed that the hepatocytic secretion of specific proteins into the growth medium persisted up to the 15th day, although progressively diminishing in intensity. The secretion of total exportable proteins and of albumin, but not of fibrinogen, was stimulated by cGMP used alone or coupled with equimolar cAMP.

Neonatal rat hepatocytes in primary tissue culture free from density-dependent regulation of growth

SummaryStudies employing [3H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the degree of cell crowding in vitro, i.e. from 1.45 to 27.55×104 cells per specimen, did not change the rates of entry into DNA synthesis and mitosis of cultivated primary neonatal rat hepatocytes.

Studies on the persistence of differentiated functions in rat hepatocytes set into primary tissue cultures. Specific binding of 3H-bilirubin after eight days of staying in vitro

Preliminary experiments were performed which indicate that even after 8 days of staying in primary in vitro tissue culture rat hepatocytes are still able to take up and bind tritiated, unconjugated bilirubin in a specific fashion and much more intensely than do connective tissue cells present in the same cultures. The data are suggestive of the maintenance in 8-day cultured hepatocytes of at least some of the specific pathways bound to bilirubin metabolism.

Cyclic AMP and cyclic GMP as the respective mediators of the intracycle stimulation of DNA synthesis and mitosis induced by glucagon and insulin in primary neonatal rat hepatocytes.

Abstract Within a wide range of concentrations ( i.e. , from 10 −15 to 10 −8 mole/1), equimolar mixtures of dibutyryl-cyclic AMP and dibutyryl-cyclic GMP or of glucagon and dibutyryl-cyclic GMP or of insulin and dibutyryl-cyclic AMP faithfully mimicked the stimulation of DNA-synthetic and mitotic activities elicited by equimolar associations of glucagon and insulin in 4-to-5-day-old neonatal rat hepatocytes in primary tissue culture. These observations strongly suggest that the intracycle, growth-promoting effects of the two pancreatic hormones are mediated via both purine cyclic nucleotides in the neonatal rat hepatocytes.

Primary Tissue Culture of Normal Adult Human Decapsulated Adrenal Cortex: Radioautographic Studies on the Metabolic Effects of ACTH1-24

ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-3H and 1-leucine-3H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH1-24 (3 X 10(-6) M) the incorporation of the precursors for RNA and DNA synthesis and the cell proliferation were found to be markedly stimulated. The apparent uptake of leucine-3H was instead reduced in 2-day and, less still, in 7-day treated cells. In parallel with ultrastructural studies, these data suggest that ACTH orderly activates the template activity of chromatin while eliciting the differentiation and hypertrophy of human adrenocortical cells.