Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E.

We have reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.     

Recovery of cyclic nucleotide regulation in protein-kinase-defective adrenal cells through somatic cell fusion

A mutant cell line (designated Kin-8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth-inhibition by 8-bromoadenosine 3', 5'-monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin-8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP-dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin-8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP-dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin-8 X C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin-8 X C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50 values for 8BrcAMP. In addition, Kin-8 X C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effects of 8BrcAMP on the steroidogenic activity and morphology of Kin-8 X C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP-responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP-dependent protein kinase for Kin-8 cells and led to the recovery of a cAMP-responsive adrenal phenotype. type. These results provide additional evidence in support of an obligatory role for cAMP-dependent protein kinase in the regulation of adrenal steroidogenesis, cell division, and cell shape.     

Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The roles of cAMP and cAMP-dependent protein kinase in the regulation of adrenocortical functions: analysis using DNA-mediated gene transfer

DNA-mediated gene transfer was used to evaluate the cause and effect relationship between mutations in cAMP-dependent protein kinase activity and cellular resistance of adrenocortical tumor cells to ACTH and cAMP. Protein kinase defective, Kin 8 adrenocortical tumor cells were transformed with genomic DNA from an ACTH- and cAMP-responsive adrenocortical cell line and screened for the recovery of morphological responses to the cAMP analog 8-bromo-cAMP (8BrcAMP). 8BrcAMP-responsive transformants were recovered with a frequency of approximately 0.5 per 10(3) transformation-competent cells. These transformants recovered the ability to round up in the presence of ACTH and were able to respond to both ACTH and 8BrcAMP with increased steroidogenesis. They also recovered cAMP-dependent protein kinase activity. The transformants, however, were unstable and concomitantly lost cAMP-dependent protein kinase activity and steroidogenic and morphological responses to ACTH and 8BrcAMP. These observations suggest that a single gene, probably the gene encoding the regulatory subunit of cAMP-dependent protein kinase, is responsible for the resistance of the Kin 8 mutant to ACTH and cAMP.     

Elevated levels of protein kinase C in Y1 cells which express apolipoprotein E decrease basal steroidogenesis by inhibiting expression of P450-cholesterol side chain cleavage mRNA.

We have previously reported that steroidogenesis is dramatically reduced in mouse Y1 adrenocortical cells which express the human apolipoprotein E gene (Y1-E cells). This suppression results in part from inhibition of cAMP-mediated events. In this report we have examined the expression of protein kinase C (PKC) in the Y1-E cell lines. Total cellular PKC activity in vitro is increased 3-5-fold in the Y1-E cell lines. PKC activity in the particulate and cytosolic fractions is increased to the same relative extent. Increased PKC activity reflects increased levels of PKC mRNA, as determined by Northern blot analysis, and PKC protein, as determined by immunoblot analysis. Increased expression of PKC in the Y1-E cell lines is accompanied by a 2-3-fold increase in diacylglycerol, an in vivo activator of PKC. To determine the contribution of elevated PKC expression to the Y1-E cell phenotype, we utilized the PKC inhibitors, staurosporine and calphostin C. Upon treatment with staurosporine or calphostin C, expression of P450-cholesterol side chain cleavage mRNA is increased severalfold to a level equal to, or greater than, basal expression in the Y1-neo control cell line. Treatment with calphostin C also results in recovery of steroidogenesis in the Y1-E cells to a level comparable to the basal level observed in the Y1-neo control cell line. These results indicate that increased expression of PKC in the Y1-E cell lines decreases basal steroidogenesis by suppressing P450-cholesterol side chain cleavage mRNA expression. Inhibition of PKC, however, does not reverse the block in cAMP-stimulated steroidogenesis in Y1-E cells, suggesting that the pleiotropic effects of apoE expression are not mediated entirely through altered PKC expression.     

Ornithine decarboxylase and polyamine levels are reduced in CHO cells deficient in cAMP-dependent protein kinase

A Chinese hamster ovary cell mutant with greatly reduced catalytic activity of cAMP-dependent protein kinase was compared with wild type cells having normal kinase activity for differences in biosynthesis, uptake and conjugation of polyamines. The inducibility of ornithine decarboxylase in response to cAMP, serum, human chorionic gonadotropin, asparagine and phorbol esters was greatly reduced in the mutant cells. Putrescine, spermidine and spermine levels rose 2–6 fold in wild type cells but in kinase mutant cells the basal and stimulated levels were generally lower. The cellular uptake and conjugation of radiolabelled putrescine and spermidine were reduced 5–10 fold in the kinase mutant cells. These results provide further evidence of the positive regulatory control exerted by cAMP-dependent protein kinase on polyamine biosynthesis.     

The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.     

Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.     

Revertants of a trans-dominant S49 mouse lymphoma mutant that affects expression of cAMP-dependent protein kinase

Phenotypic revertants were isolated from an S49 mouse lymphoma tissue culture cell mutant that lacks cAMP-dependent protein kinase (cA-PK) activity (kin-). The mutant phenotype is trans-dominant and results from a lesion that probably lies outside the cA-PK subunit structural genes. The nature of the event that produces the kin- phenotype is unknown. However, the mechanism that is responsible for its behavior is genetically encoded because: spontaneous revertants arise at low frequency; reversion frequency is increased by mutagen treatment; mutagen-specific classes of revertant phenotypes are induced; and some revertants are temperature-sensitive for expression of cA-PK subunit polypeptides. Additional evidence is provided that argues against structural lesions in cA-PK catalytic (C) subunits as explanatory of the kin- phenotype. Kin- cells do not express an immunologically detectable C polypeptide, whereas C expression is restored in revertant cells. Revertants in which phenotype and cA-PK activity levels are only partially restored to that of wild-type cells contain a commensurately reduced amount of C polypeptide. Finally, the structure of C polypeptide in partial revertants is unaltered from that of wild-type C. The evidence supports the hypothesis that the kin- lesion defines a regulatory gene responsible for setting intracellular levels of cA-PK C subunit expression.     

Different effects of TPA on two skin-derived cell lines: murine (HEL-30) and human (NCTC) epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) caused a rapid activation of protein kinase C in a murine (HEL-30) and in a human (NCTC) epidermal cell line. In HEL-30 cells, protein kinase C activation is followed by ornithine decarboxylase stimulation and cell proliferation, events inhibited by H-7, a specific inhibitor of protein kinase C. TPA in NCTC cells inhibited the basal ornithine decarboxylase activity and cell growth, whereas H-7 did not modify TPA effect. The response of NCTC cells was not due to direct toxicity of TPA. These data confirm that in murine epidermal cells, the proliferation induced by TPA is mediated by protein kinase C, whereas in a human skin-derived cell line these events are not or inversely associated.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome

The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494. The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results suggest that the gene expression of the operon is repressed strongly under standard conditions.     

Functional modulation of brain sodium channels by cAMP-dependent phosphorylation.

Voltage-gated Na+ channels, which are responsible for the generation of action potentials in brain, are phosphorylated by cAMP-dependent protein kinase in vitro and in intact neurons. Phosphorylation by cAMP-dependent protein kinase reduces peak Na+ currents 40%--50% in membrane patches excised from rat brain neurons or from CHO cells expressing type IIA Na+ channels. Inhibition of basal cAMP-dependent protein kinase activity by transfection with a plasmid encoding a dominant negative mutant regulatory subunit increases Na+ channel number and activity, indicating that even the basal level of kinase activity is sufficient to reduce Na+ channel activity significantly. Na+ currents in membrane patches from kinase-deficient cells were reduced up to 80% by phosphorylation by cAMP-dependent protein kinase. These effects could be blocked by a specific peptide inhibitor of cAMP-dependent protein kinase and reversed by phosphoprotein phosphatases. Convergent modulation of brain Na+ channels by neurotransmitters acting through the cAMP and protein kinase C signaling pathways may result in associative regulation of electrical activity by different synaptic inputs.     

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Extracellular signal-regulated kinases (ERK) 1, 2 are required for luteinizing hormone (LH)-induced steroidogenesis in primary Leydig cells and control steroidogenic acute regulatory (StAR) expression

The luteinizing hormone (LH) plays a critical role in steroidogenesis, by stimulating cAMP-dependent protein kinase A (PKA) and phospholipase A2 activity, and by mobilizing calcium and chloride ions. In contrast, whether the ERK 1, 2 mitogen-activated protein (MAP) kinases are involved in LH-induced steroidogenesis is less obvious. Here, we sought to clarify this point in rat primary Leydig cells, naturally bearing the LH receptor (LH-R) in male, and in the mouse tumoral Leydig cell line (MLTC 1). Pre-incubation of both cell types with the mitogen-activated protein kinase kinase (MEK) inhibitors U0126 and PD98059 reduced LH-induced steroidogenesis, and tonically enhanced the expression of the StAR protein. Furthermore, ERK1, 2 were inducibly phosphorylated following LH exposure of MLTC 1 cells. Altogether, our results indicate that in primary as well as in tumoral Leydig cells, inhibiting MEK dampened LH-induced steroidogenesis but enhanced basal as well as LH-induced StAR expression, suggesting that ERK1,2 could be involved in these responses.     

The involvement of cyclic AMP-dependent protein kinase(s) in the induction of ornithine decarboxylase in the regenerating rat liver and in the adrenal gland after unilateral adrenalectomy

We investigated the temporal relationship between the level of cyclic AMP, the activation of cyclic AMP-dependent protein kinase(s), and the induction of ornithine decarboxylase in two important rapid growth systems: the regenerating rat liver and the remaining adrenal gland following unilateral adrenalectomy. There was a biphasic increase in the aactivity of ornithine decarboxylase at 4 and 14 h following partial hepatectomy. The concentration of cyclic AMP increased 2-fold compared to sham-operated animals within 2–3 h, returned to baseline by 8 h, and was elevated again 3-fold by 12 hours. The activation of cyclic AMP-dependent protein kinase(s) occured in a similar biphasic manner. From a control activity ratio (?cAMP/+cAMP) of 0.4, values for total soluble kinase activation reached 0.75 at both 2 and 14 h. After a delay of 2 h following unilateral adrenalectomy, ornithine decarboxylase activity in the remaining adrenal gland increased 15–20-fold of control level by 8 h. Cyclic AMP concentrations in the adrenal were elevated 3.5-fold within 30 min. The protein kinase activation increased from 0.25 to nearly a totally activated state of 1.0 within 1 h, decline to 0.4 by 2 h, and returned to the level of the sham-operated controls at 8 h. In both the rat liver in response to partial hepatectomy and the adrenal gland undergoing hypertrophy, cyclic AMP-dependent protein kinase(s) was markedly activated prior to increases in ornithine decarboxylase activity.