Printed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form resewed ISSN 00144827

Experimental

EFFECTS

OF PURINE

Cell Research 105 (1977) 337-347

CYCLIC

OF NEONATAL PRIMARY U. ARMATO,

ENRICA

NUCLEOTIDES RAT

ON THE

HEPATOCYTES

TISSUE DRAGHI

GROWTH

IN

CULTURE and PAOLA

Tissue Culture Laboratory, Department Universio of Padova, I-35100

G. ANDREIS

of Human Anatomy, Padova, Italy

SUMMARY 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 Gl/S boundary of their growth-division cycle.

In previous work we found that a 20-24 h exposure to both physiological and higher doses of CAMP or db-CAMP stimulated the de novo synthesis of protein [l], RNA, DNA and the proliferation [2-51 of slow cycling, differentiated [6] young rat hepatocytes set into primary monolayer tissue culture. These results appeared to be in keeping with current lines of evidence suggesting that CAMP positively regulates the proliferation of already mitotically activated rat hepatocytes [7-lo]. However, this interpretation of our data was complicated by

the preliminary finding that even another naturally occurring cyclic nucleotide, i.e. cGMP, at high doses could enhance the DNA synthesis of cultivated rat liver cells [2]. In fact, we could not ignore that cGMP, and not CAMP, was indicated by the results of many studies as the positive stimulator of eukaryote cell proliferation [ 1 l-141 and that cGMP might mediate in mammalian liver some at least of the actions of insulin [ 15, 161, a hormone capable of eliciting proliferogenic effects [ 171. Therefore, the purposes of the present investigations were: (i) Exp

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to study in depth the possible role of cGMP, with respect to CAMP, as a physiological intra-cycle regulator of hepatic cell proliferation; (ii) to explore whether in cultivated rat liver cells CAMP and cGMP might counteract each other’s actions as postulated by the “Yin-Yang or dualism” concept of biological regulation [I I-141.

MATERIALS

AND METHODS

Abbreviations. 5’-AMP, adenosine-5’-phosphate; CAMP, adenosine 3’,5’-cyclic monophosphate; dbCAMP, N6,02’-dibutyryl-adenosine 3’,5’-cyclic monophosphate; 5’-GMP, guanosined’-phosphate; cGMP, guanosine 3’,5’-cyclic monophosphate; db-cGMP, Nz,02’-dibutyryl-guanosine 3’,5’-cyclic monophosnhate; U, 15-3H]uridine. Hanks’ basal-Salt Solution (BSS) and Eagle’s Minimum Essential Medium (MEM). Wellcome Ltd: adult and fetal bovine serum, Flow Laboratories Ltdi trypsin 1 : 250 powder, Difco Spa; collagenase type I (from Ciostridium histolyticum), hyaluronidase type II (from ovine testes), glucagon, insulin, CAMP, db-CAMP, 5’-AMP, cGMP, db-cGMP, S’-GMP, adenosine, guanosine, TdR, uridine, colchicine, Sigma Co.; cephaloridin, Eli Lilly Ltd; streptomycin sulfate, Farmitalia Spa; nystatin, Squibb Spa; [5-3H]uridine (U) (spec. act. 33 Ci/mM); methyl[aH]TdR (spec. act., 6.7 Ci/mM), Radiochemical Centre; Ilford K5 Nuclear Emulsion and Hvname. Ilfotd-Ciba Spa; Dia Direct”, Pan Reversal f&s, Agfa Gevaert Spa;‘ribonuclease, Merck GmbH. Primary liver cultures of dispersed hepatocytes were prepared using the pooled livers taken aseptically from 4-day-old Wistar albino rats (S. Morini), both males and females belonging to the same litter, as previously detailed [3, 5, 61. The cultures were incubated at 35’C in a gas phase of 5% (v/v) CO2 in air and checked every day for viability and cell growth according to Armato et al. [ 1, 31. To estimate the flow into DNA synthesis of the hepatocyte population, groups of liver cultures on their 4th day in vitro were treated for 24 h with the followinn compounds directly dissolved into the growth medium in doses ranging from 1.5~ lO-‘3 to 10m3M: (a) CAMP; (b) cGMP; (c) equimolar associations of CAMP with cGMP. After treatment, the cultures were labelled for 1 h at 35°C with 0.5 &i/ml of metbyl[3H]TdR, fixed and processed for radioautoarauhv 13. 51. Phase S hepatocytes were recognized _ in developed and haematoxylin-eosin stained specimens by optical counts at x 1200 [3, 51. Supplementary radioautographic experiments were done by observing for 24 h at 4 h intervals to find out the timing of the onset of entry into DNA synthesis for the most effective doses of CAMP, cGMP and of CAMP plus equimolar cGMP. To evaluate the flow of hepatocytes into mitosis, groups of 4-day-old liver cultures were treated for 20 h with the following compounds in doses ranging from Exp Cell

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l.5x10-‘3 to IO+ M: (a) CAMP; (b) db-CAMP; (c) cGMP; (d) db-cGMP; (e) equimolar mixtures of CAMP and cGMP; v) equimolar associations of db-CAMP with db-cGMP; (s) glucagon; (h) insulin; (i) equimolar mixtures of glucagon and insulin (for f$), (h) and (i) the dose range was limited between lo-‘* and lo-’ M). After treatment, the cultures were incubated for 4 h with a medium devoid of cyclic compounds or hormones and containing colchicine (0.12 mM) and ethanol (1.0X 10e4% v/v). In fixed and Feulgen-stained cultures the percentage of hepatocyte population stopped in the so-called C-metaphase was determined as previously described [3,5]. Similar ancillary experiments were aimed at establishing the degree of specificity for the actions brought about by the least efticient dose of the cyclic nucleotides as compared to the effects of their main metabolites at the same concentrations. The morphometric estimation of the apparent surface area of hepatocyte main subcellular compartments and the radioautographic quantitation of their new RNA synthesis were-carried out according to Armato et al. [3, 51. Five different 4-day-old liver cultures were treated for 24 h with either cGMP or equimolar mixtures of cGMP with CAMP in doses ranging from 1.5X 1Om8to 10e3 M, then labelled for 1 h at 35°C with U (0.5 &i/ml of growth medium) and fixed and prepared for radioautography [3,5]. The specificity for RNA labellinn bv U was tested bv the use of ribonuclease [3, 51. - . In any experiment groups of untreated cultures set from the same animal source and fed with normal growth medium were run in parallel as controls. Student’s t-test was used for the evaluation of the results: the difference between two means was taken as significant only if P was found to be less than 0.05.

RESULTS Primary cultures of neonatal rat liver cells

Primary cultures set up from neonatal rat liver cells dispersed by means of a mixture of trypsin with collagenase and hyaluronidase contain not only hepatocytes but even different mesenchymal cell types [ 1, 3, 61. However, the hepatocyte is the cell type essentially considered in this work. Hepatocytes are usually arranged in monolayered Fig. 1. An &day-old primary rat liver culture fixed for 20 min at -30°C in absolute acetone, incubated for I h at 37°C with rabbit anti-adult rat total serum antiserum and then stained for 1 h with fluorescein-conjugated sheep anti-rabbit globulin. Hepatocytes, whose cytoplasm fluoresces brightly, are arranged as an irregularly shaped islet, surrounded by negative mesenchymal cells. x200.

Cyclic nucleotides

2. The same specimen as in fig. I observed by phase contrast microscopy. The fluorescein-positive cells correspond to hepatocytes endowed with a vesicular nucleus and with a dark cytoplasm rich in organelles (arrows). The surrounding mesenchymal cells are flatter than parenchymal cells, thus appearing slightly out of focus, and possess a clear cytoplasm.

Fig.

x200. Fig. 3.

An islet of living primary rat hepatocytes on the 5th day in vitro, surrounded by closely packed mesenchymal cells. The brilliant refractility of numerous, small (thereby physiological) lipid droplets, although masking other cytoplasmic organelles, makes the grouped parenchymal cells easily recognizable. Phase contrast microscopy. X350.

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4. An islet of primary rat henatocvtes Vf) on the 5th day in vitro. Cells attached-to the polyethylene disc were fixed in glutaraldehvde (2.5% w/v) and stained with uranyl acetate. The cytcplasm of parenchymal cells, although appearing like honeycomb because of the lipid extracted during the histological processing, is still much darker. due to its richness in organell&, than that of neighbouring fibroblast-like (FJ or “clear” (C) cells, Phase contrast microscoov. I I Fig.

x350. Fig.

5. A radioautographic image of primary rat liver parenchymal cells incubated with methyl[3H]TdR as detailed in the text. A phase S hepatocyte (arrow) is close to the centre of the field. Hematoxylin-eosin. X1000.

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16 -

7. Abscissa: cyclic nucleotide(s) cont. (moles/l of medium); ordinate: % of hepatocyte population entering mitosis between 20 and 24 h after beginning of exposure to cyclic nucleotide(s). Effects of CAMP (O-O), cGMP (A-A) and of equimolar associations of both compounds (a4) on the flow of cultivated hepatocytes into the M stage of their growth division cycle. 0, Controls. The points are means +S.E.M. of the values from six to twelve cultures.

Fig.

oT z‘-13’ !I, ’ ’ ’ ’ ’ I I I I 0 10 10 12 to' rb to’ ll.SX) 6. Abscissa: cyclic nucleotide(s) cont. (moles/l of medium); ordinate: % of hepatocyte population labelled with methyl[3H]TdR between 24 and 2.5 h after beginning of exposure to cyclic nucleotide(s). Effect of CAMP (04), cGMP (A-A) and of equimolar mixtures of both agents (m-m) on the flow of cultivated neonatal rat hepatocytes into the S phase of their mitotic cycle. 0, Controls. The points are means +S.E.M. of values from six to thirtytwo distinct cultures.

Fig.

homogeneous islets of IO-500 elements (figs l-5). The hepatocytic cell type has been shown: (i) to keep producing for up to the 25th day in vitro exportable serum proteins that are antigenically identical to adult rat serum proteins (figs 1,2) [6]; (ii) to increase significantly, if exposed to phenobarbitone, its capacity to survive cytotoxic concentrations of vinblastine sulphate (see [3] for reference); (iii) unlike the coexisting fibroblast-like cells and so called “clear” cells, to take up intensely and specifically [3H]bilirubin from the growth medium [6]; (iv) to have its incorporation of l-leucine4,S3H affected by CAMP in a characteristic way and quite different from that proper of the mesenchymal cells [ 11. Exp Cell

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DNA synthesis and proliferation cultivated hepatocytes

of

As the data in figs 6-8 show, untreated primary hepatocytes on the 5th day of

8. Abscissa: dibutyryl cyclic nucleotide(s) cont. (moles/l of medium); ordinate: % of hepatocyte population entering mitosis between 20 and 24 h after beginning of exposure to dibutyryl cyclic nucleotide(s). The effects of db-CAMP (B-m), db-cGMP (A-A) and of equimolar mixtures of both agents (O-0) on the flow of cultivated hepatocytes into the M phase of their mitotic cycle. El, Controls. The points are means +S.E.M. of values from six to twenty cultures.

Fig.

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9. Abscissa: (n) controls; (b) cGMP (15 PM); (c) 5’-GMP (15 PM); (d) guanosine (15 PM); ordinate: % of hepatocyte population entering mitosis between 20 and 24 h after beginning of treatments. The specificity of the intra-cycle proliferogenic effect of exogenous cGMP on cultivated hepatocytes. Each histogram shows the mean +S.E.M. of four experiments. Fig. 10. Abscissa: (a) controls; (b) cAMP+cGMP (0.15 PM); (c) 5’-AMP+S’-GMP (0.15 PM); (d) adenosine+guanosine (0.15 PM); ordinate: % of hepatocyte population entering mitosis between 20 and 24 h after beginning of treatments. The specificity of the intra-cycle proliferogenic effect of the least effective equimolar association of CAMP with cGMP. Each histogram shows the mean fS.E.M. of four experiments. Fig.

being in vitro flow moderately into the S and M phases of their mitotic cell cycle. CAMP and db-CAMP stimulated the entry of hepatocytes into DNA synthesis and mitosis when employed at a very wide concentration range including the physiological one for CAMP and the subphysiological one for db-CAMP. cGMP and db-cGMP enhanced the same processes only at concentrations higher than the physiological one, being ineffective or inhibitory at other doses. Equimolar associations of CAMP with cGMP and of db-CAMP with db-cGMP stimulated the growth of hepatocytes when given at very low doses (figs 6-8). It has already been shown [5] that the stimulatory effects brought about by the least effective dose of CAMP (15 nM) are specific for this

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compound. The results in figs 9 and 10 demonstrate the specificity of the stimulatory actions elicited by cGMP used alone (15 pm) and in equimolar association with CAMP (0.15 PM). The results in fig. 11 demonstrate that the most effective dose of CAMP (0.15 mM) significantly increases the flow of cultivated hepatocytes into DNA synthesis after 1214 h of continued treatment. As shown in fig. 12, cGMP (15 PM) used alone significantly decreases the flow of hepatocytes into S phase between the 6th and the 14th hour of treatment, prior to increasing it from the 15th hour onwards. The data in fig.

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time of exposure to CAMP (0.15 mM) (hours); ordinate: (fef) % of hepatocyte population labelled after 1 h of incubation with methyl[3H]TdR; (right) % of hepatocyte population in mitosis. The timing of the onset of the stimulation by exogenous CAMP of the transit of hepatocytes into the S (O-O) and M (W-m) stages of their mitotic cycle. Mitotic activity is the sum of the fractions of cells which were in prophase, metaphase, anaphase and telophase at the time of the exposure of the cultures to methyl[3H]TdR. The points are means +_S.E.M. of the values from four cultures. Fig. 12. Abscissa: time of exposure to cGMP (15 PM) (hours); ordinate: (left) % of hepatocyte population labelled after 1 h of incubation with methyl[3H]TdR; (right) % of hepatocyte population in any stage of mitosis. The time-dependent varying effects of cGMP on the entry of cultivated hepatocytes into the S (O-0) and M (B-B) stages of their mitotic cycle. The points are means +S.E.M. of the values from four cultures, Fig.

11. Abscissa:

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26%; (c) “clear” cells, 35%. Only CAMP alone was (0.15 mM) was found to stimulate the DNA synthesis and proliferation not only of hepatocytes but even of fibroblastlike cells and of “clear” cells (data not shown).

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Fig. 13. Abscissa: time of exposure to equimolar mixtures of CAMP and cGMP (1.5 nM) (hours); ordinate: (left) % of hepatocyte population labelled after 1 h of incubation with methyl[3H]TdR; (right) % of hepatocyte population in any stage of mitosis. The effects of an equimolar mixture of CAMP with cGMP on the flow of cultivated hepatocytes into the S (O-O) and M (Cm) stage of their growth-division cycle. The points are means +S.E.M. of the values from four cultures. Fig. 14. Abscissa: hormone(s) cont. (moles/l of medium); ordinate: % of hepatocyte population entering mitosis between 20 and 24 h after beginning of exposure to hormone(s). The effects of glucagon (A-A), insulin (m-m) and of equimolar mixtures of both hormones (03) on the mitotic activity of cultivated hepatocytes. *, Controls. The points are means +S.E.M. of two experiments, each employing four replicates per point.

13 show that the most effective equimolar combination (1.5 nM) of the two cyclic nucleotides causes a first peak of stimulated entry into DNA synthesis to appear between the 2nd and the 6th hour of treatment and, further, a second peak between the 16th and the 18th hour of exposure. The preliminary results in fig. 14 show that even glucagon and insulin, both when used singly and in equimolar associations, stimulate the growth of cultivated hepatocytes. Finally, the treatments with cyclic nucleotide(s) did not affect the proportions of the principal cell types, which were: (a) hepatocytes, 26 %; (b) fibroblast-like cells, Exp

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30 20 t ':gm

""CleYlUI

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(1.5x)

Fig. IS. Abscissa: cyclic nucleotide(s) cont. (moles/l of medium); ordinate: apparent surface area in pm2. The effect of various concentrations of cGMP (empty symbols) and of equimolar mixtures of cGMP with CAMP (solid symbols) on the morphometric parameters of cultivated hepatocytes. The points are means 5S.E.M. of values of 35 hepatocytes per treatment and per dose from five distinct cultures. If no S.E.M. is shown, it was smaller than the symbol used to represent the point.

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Calcium content of the growth medium of cultivated hepatocytes

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Fig. 16. Abscissa: cyclic nucleotide(s) cont. (moles/l of medium); ordinate: nucleoli/hepatocyte nucleus. The effect of cGMP (0-O) and of equimolar associations of cGMP with CAMP (O-O) on the mean nucleolar numberlhepatocyte nucleus. n , Controls. Data are means +S.E.M. of the values from five distinct cultures.

Preliminary estimates carried out by means of atomic absorption spectrometry have shown that on the 5th day of being in vitro: (i) the calcium content of the growth medium of untreated primary liver cultures was about 1.75 mM; (ii) a 24 h treatment

460 425

Morphometric parameters and RNA synthesis of cultivated hepatocytes

The effects of a 24 h exposure either to cGMP alone or in equimolar combinations with CAMP on the morphometric parameters and mean nucleolar number per nucleus of primary rat hepatocytes are shown in figs 15 and 16. The silver grain background on cultivated hepatocytes from no-tracer exposed specimens was found to range from 0.18 to 0.71 grain/100 pm2. The absolute silver grain counts were related to corresponding apparent surface area values to obtain the U radioactivity concentration values/100 pm2 of each main subcellular compartment of cultivated hepatocytes. In control and cyclic nucleotide(s)treated, U-labelled liver cells incubated with ribonuclease prior to radioautography, the various compartmental U radioactivity concentration values were found not to differ significantly from background values on “cold” hepatocytes, indicating no incorporation of U into DNA. The results in fig. 17 show the effects of cGMP, used singly and in equimolar combinations with CAMP on the de novo RNA synthesis of primary hepatocytes.

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Fig. 17. Abscissa: cyclic nucleotide(s) cont. (moles/l of medium); ordinate: U radioactivity conc.1100 pm* of apparent surface area of the subcellular compartment considered. The effects of cGMP (solid symbols) and of equimolar mixtures of cGMP with CAMP (empty symbols) on the de novo RNA synthesis. X, Controls. The points are means +S.E.M. of the values obtained from five distinct cultures. Exp CellRes

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objected to, because neonatal hepatocytes already cycling in primary culture [3,5] may possess rather high intracellular levels of endogenous cGMP, as other proliferating cell types are reported to have [ 1 l-141 and, hence, they may not respond to the administration of physiological concentrations of exogenous cGMP. However, this objection appears to be ruled out by the finding that extremely low doses of exogenous Fig. 18. Abscissa: cyclic nucleotide(s) cont. (moles/l cGMP slightly, yet significantly, impair the of medium); ordinate: calcium content of the medium DNA synthetic and mitotic activities of the (mg/lOOml). Effects of the treatment with CAMP (A-A); db- hepatocytes. Therefore, we are inclined to CAMP (W-W); cGMP (O-O); and with equimolar consider as more likely another assumption, mixtures of CAMP with cGMP (Q--O) on the coni.e. that cultivated hepatocytes, being withcentration of calcium in the medium of liver cultures. *, Controls. After 4 days of cultivation the cells were of neuro-hormonal exposed to cyclic agent(s).After a 24 h treatmentthe out the bombardment signals normally occurring in vivo, may polyethylene discs with attached cells were stripped off, the growth media underneath removed, centrilevels fuged at 500 g for 15 min and the calcium content of possess very low basal intracellular cGMP and thereby are the supemates determined by atomic absorption spec- of endogenous trometry. Points are means of two experiments, each responsive to minimal exogenous additions employing five replicates per point. of this cyclic nucleotide. Unfortunately, this view lacks direct proof. In conclusion, on the grounds of previous with various doses of CAMP, db-CAMP and [3-51 and present observations we should of equimolar mixtures of CAMP with cGMP assign to CAMP, and not to cGMP, a possignificantly decreased the calcium content sible leading role as the physiological intraof the medium, whereas the treatment cycle accelerator of rat hepatocyte proliwith cGMP alone was much less effective feration. In fact, the total stimulatory ef(fig. 18). fects of CAMP on DNA synthesis and on mitotic activity of cultivated hepatocytes DISCUSSION are not only more intense, but take place The possible major role of CAMP within an even wider range of concentrain the intra-cycle regulation of tions, including physiological, for rat liver. rat hepatocyte proliferation By contrast, cGMP needs to occur in conAlthough cGMP and db-cGMP can easily centrations within the range of pharmacoenter liver cells [ 181, the fact that they have logical action [ 1 I] to produce stimulatory a poor proliferogenic effect on cultivated effects which are at the same time much weaker. Nor should it be overlooked that cycling rat hepatocytes, as disclosed by our studies, does not support the dualistic, bi- extremely low doses of db-CAMP can indirectional contention [ 1l-141 that cGMP is crease the mitotic activity of rat hepatothe physiological positive and CAMP the cytes, whereas db-cGMP is much less effecnegative regulator of cell division, at least tive and only occurs in paraphysiological as far as the mitotically activated rat he- concentrations. Further, our assumption is patocyte is concerned. This view may be strongly supported by several other lines of Exp Cell

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Cyclic nucleotides

evidence, e.g. (a) shortly after birth there occurs a significant peak in rat liver CAMP levels [ 191 that coincides with a period of intense mitotic activity [20]; (b) two waves of intracellular CAMP accumulation, the second of which may be indispensable, precede the onset of DNA synthesis in hepatocytes mitotically activated by partial hepatectomy or by infusion (into female rats) of an empirical mixture of tri-iodothyronine, amino acids, glucagon and heparin (TAGH) [7-10, 21, 221; in this mixture glucagon can be effectively substituted by db-CAMP, but not by cGMP [8]; (c) by contrast, no detectable accumulation of cGMP occurs in the liver stump from 5 min to 21 h after partial hepatectomy [7, lo], nor does any change in total guanylate cyclase activity take place during the first 12 h after surgery [23, 241; (d) CAMP, but not cGMP, markedly enhances the incorporation of U into the newly synthesized RNA of cultivated hepatocytes [3-51; and (e) there occurs, in hepatocytes in vivo as well as in several other types of cells cultivated in vitro at least one increase in intracellular CAMP concentration prior to the onset of DNA synthesis [25-311. It is, therefore, our belief that hormones increasing hepatic intracellular levels of CAMP play an important regulatory role in the trophism and growth of rat liver.

The possible minor role of cGMP in the intra-cycle regulation of rat hepatocyte proliferation

Nevertheless, the present observations show that cGMP itself can have some, even if only a slight influence on the progression of cultivated hepatocytes throughout the various stages of their growth-division cycle. As a matter of fact, it appears conceivable that cGMP might, physiologically

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but not indispensably, regulate the transit of mitotically activated liver cells across some point near the Gl/S boundary, since (a) a transient block of hepatocytes prior to the start of DNA synthesis precedes the appearance of the “stimulation” of this process by cGMP (fig. 12); therefore, the peak of DNA synthesizing cells is likely to be due mostly to a partial synchronization of cycling hepatocytes which have eventually escaped the block; moreover, this synchronization effect may also explain why the proliferogenic action of cGMP is not accompanied by marked increases in the RNA synthetic activity of cultivated hepatocytes; (b) a 24 h exposure to extremely low doses of cGMP also impairs the entry of the hepatocytes into DNA synthesis slightly but significantly; (c) a very early wave of hepatocytes entering DNA synthesis appears after a brief incubation of cultures with an equimolar mixture of CAMP and cGMP at a very low dose (1.5 nM), whereas each nucleotide used singly at the same dosage is ineffective or only slightly inhibitory. Incidentally, such a finding also suggests that in 4 days old liver cultures there may exist a subpopulation of hepatocytes whose progress along the cell cycle is stopped just before DNA synthesis, because of insufficiency or absence of appropriate stimulus(i) (hormone(s)?) or factor(s) (cyclic nucleotides? calcium?). Clearly, the association of the two cyclic nucleotides appears, by unknown mechanisms, to make up quickly for the missing stimulus(i) or factor(s), thus awakening the ability to synthesize new DNA in liver cells stopped at the Gl/S boundary. However, it must be stressed that the indispensability of cGMP as a specific regulator of the transit of mitotically activated hepatocytes across the GllS gate remains open to question, for the assumed need for cGMP at this point Exp Cdl

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can be overcome by prolonged treatment with CAMP. In conclusion, the present results are consistent with the hypothesis that in the presence of CAMP-cGMP-physiologically but not indispensably-facilitates the entry of cycling hepatocytes into DNA synthesis by positively acting at some point near the GUS limit line. Conceivably, another example of an ancillary positive role of cGMP in mitotic cell cycle control may be evinced by the pre-neoplastic BALB/3T3 mouse endothelial cells, in which a single burst of cGMP synthesis is produced between two pre-S waves of CAMP accumulation [lo, 291. The “Yin-Yang” hypothesis of bidirectional control: a model which may not explain the intracycle regulation of rat hepatocyte proliferation

Not only the results of experiments employing each cyclic nucleotide alone, but even those of experiments using equimolar mixtures of both cyclic agents on the whole do not fit the “Yin-Yang” or “dualism” hypothesis of bidirectional regulation [ 1 l141. Indeed, even when the results of experiments employing both cyclic nucleotides in a dose range restricted within 1.5~ IO-’ and 10V4 M apparently agreed with the dualistic concept, yet some at least of the basal metabolic activities (e.g. nucleolar and total cell RNA synthesis) of cultivated hepatocytes were found to be significantly and progressively inhibited: an observation not strictly in accord with the bidirectional doctrine. Furthermore, the synergistic stimulatory effects of hepatocyte RNA, DNA synthesis and proliferation brought about by very low concentrations of equimolarly associated CAMP with cGMP (and db-CAMP with db-cGMP) can in no way be Exp Cell

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reconciled with the “Yin-Yang” hypothesis. Therefore, we no longer consider the proliferation of hepatocytes as a process bidirectionally, i.e. antagonistically, controlled by cyclic nucleotides, but assume that in its physiological regulation CAMP plays a major and cGMP possibly a minor cooperative role. This “monodirectional” concept seems at least partly supported by our preliminary observation that in a suitable dose range equimolar mixtures of glucagon and insulin, i.e. of hormones known to increase the hepatic content of cyclic nucleotides [ 15, 16, 191, exert a cooperative proliferogenic effect on cultivated cycling hepatocytes. Such a finding is apparently in keeping with the results of work on the regenerating rat liver [32]. However, the pattern of the proliferogenic curve of insulin, as shown by our preliminary data, is somewhat different from that of cGMP. Hence, one may speculate that, as well as cGMP [ 15, 161, there exist some other intracellular mediator(s) in the proliferogenic activity of insulin on liver cells. Besides, mounting evidence suggests that CAMP interacts closely with calcium ions in the positive control of the proliferation of mitotically actived rat hepatocytes ([7, 9, 10, 33-351; see also fig. 18). Thus, further investigations, some of which are already under way, are required for a better understanding of how hormones, cyclic nucleotides, calcium and even other agents, such as prostaglandins and polyamines [34], interplay in controlling growth processes in the rat liver. The authors are indebted to Dr Robert Mitchell for his helpful review of the manuscript. They also wish to thank Mr Enrico Armato for preparing the illustrations

REFERENCES 1. Armato, U, Andreis, P G, Belloni, A S & Draghi, E, Acta anat 88 (1974) 456. 2. Armato, U, Andreis, P G, Draghi, E & Meneghelli, V, In vitro 9 (1974) 357.

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18. Exton, J H, Hardman, J G, Williams, T F, Sutherland, E W & Park, C R, J biol them 246 (1971) 2658. 19. Christoffersen, T, Msrland, J, Osnes, J B & @ye, I. Biochim bionhvs acta 291 (1973) 246. 20. Rohr, H P, W&.:A, Henning, L C, Riede, U N & Bianchi, N, Lab invest 24 (1971) 128. 21. Ebina, Y, Iwai, H, Fukui, N, Ohtsuka, H & Miura, Y, J biochem 77 (1975) 641. 22. Thrower, S & Ord, M G,Biochemj 144(1974)361. 23. Kimura, M & Murad, F, Proc natl acad sci US 72 (1975) 1%5. 24. Goridis. C & Reutter. W. Nature 257 (1975) 698. ’ 25. Boone, C W, Science 188 (1975) 68. 26. Matsumoto, T & Uchida, T, J biochem 78 (1975) 811. 27. Millis, A J T, Forrest, G A & Pious, A D, Exp cell res 83 (1974) 335. 28. Russell, D H & Stambrook, P J, Proc natl acad sci US 72 (1975) 1482. 29. Seifert, W & Rudland, P S, Proc natl acad sci US 71 (1974) 4920. 30. Thorpe, C W, Bond, J S & Collins, J M, Biochim biophys acta 340 (1974) 413. 31. Zeilig, C E, Johnson, R A, Sutherland, E W & Friedman, D L, Fed proc 33 (1974) 1391. 32. Bucher, L R N & Swaftield, M N, Proc natl acad sci US 72 (1975) 1157. 33. Berridge, M J, Advances in cyclic nucleotide research (ed P Greengard & G A Robison) vol. 6, p. 1. Raven Press, New York (1975). 34. Whittield, J F. Boynton, A L, MacManus, J P, Rixon, R H, Walker, P R & Armato, I-l, Cyclic nucleotides in growth control (ed M Abou-Sabe). Halsted Press, New York (1976). In press. 35 Rixon, R H -& Whitfield, J F, J ~cell physiol 87 (1976) 147.

Received June 30, 1976 Accepted November 10, 1976

E.rp Cell

Rrs

IO5 (1977)

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

Printed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form resewed ISSN 00144827 Experimental EFFECTS OF PUR...
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