Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.     

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.     

Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.     

Various enzymes of isolated nuclear membranes and cell nuclei of the liver and hepatoma 27 of rats]

A comparative study of glucose-6-phosphatase, alcaline RNase, ATPase, inosine diphosphatase and 5'-nucleotidase activities in isolated rat liver and hepatoma-27 nuclei and nuclear envelopes was performed. The tumor nuclear membranes were shown to be free from G-6-Pase activity in contrast to the liver nuclear membranes. The nuclear RNase activity was strongly inhibited in the hepatoma and could be unmasked in the presence of 3-10(-4) M pCMB. Hepatoma nuclear and nuclear envelopes ATP-ase activity was found to be moderately decreased as compared to those of the normal tissue. The values of inosine diphosphatase activity in hepatoma were similar to those in liver. The role of the nuclear envelope in nuclear-cytoplasmic interactions as well as nuclear location of G-6-Pase are discussed.     

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.     

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G₁/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G₁/S block, which correlates with G₂/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G₁/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G₁/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G₂/M phase of the cell cycle in HL-60 cells.     

Isolation of nuclear envelopes with polyanions

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.     

心肌肥厚大鼠心肌细胞核三磷酸肌醇受体的特征

为了研究细胞核三磷酸肌醇受体在心肌肥厚中的作用,制备了腹主动脉缩窄大鼠心肌肥厚模型、用差速离心和密度梯度离心提纯心肌细胞核,以［3H］IP3为配基,采用放射受体分析心肌细胞核膜IP3R与其配体的最大结合容量(Bmax)和解离常数(Kd)。大鼠心肌细胞核上存在IP3R、CaM和PKC激动剂PMA,能显著抑制该受体与IP3的结合(P<0.05);核外［Ca2+］也能剂量依赖的抑制细胞核IP3R与IP3的结合。腹主动脉缩窄术后4周,大鼠心肌显著肥大,伴有明显的血流动力学异常,其心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1.217和2.149倍(P<0.01)。心肌细胞核上存在IP3R,并受CaM和PMA及核外［Ca     

Action of x-ray irradiation on the nuclear envelope of rat liver cells]

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.     

Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored.Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca(2+)/CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase "particulate" cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 5'AMP generation, could elevate "soluble" cGMP pool by enhancing NO bioavailability through NOX2 down-regulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2, may be interesting therapeutic targets in preventing cardiac hypertrophy.     

Cyclic nucleotide phosphodiesterase activity in human peripheral blood lymphocytes and monocytes.

cAMP and cGMP phosphodiesterase (PDE) activity was assayed in human peripheral blood lymphocytes purified by isopycnic centrifugation as well as in lymphocyte preparations further purified to remove contaminating platelets and monocytes. The 16,000 X G supernatant from sonicates of each of these cell preparations contained two hydrolytic activities for cAMP with apparent Km of 1.1 to 2.5 microM and 33 to 66 microM, and a single hydrolytic activity for cGMP with an apparent Km of 6 to 25 microM. When lymphocytes were disrupted by Dounce homogenization, there was only a single, low Km cAMP PDE activity in the homogenate; however, the 16,000 X G supernatant demonstrated 2 Km similar to that seen in sonicated lymphocytes. Treatment of the Dounce preparations with 0.5% Triton X-100 or 1.0% NP-40 converted these preparations to activities similar to those seen in sonicated preparations. cGMP hydrolytic activity was low or absent in the Dounce preparations and was not altered by centrifugation; however, it was markedly enhanced by detergent extraction. These data indicate that human peripheral blood lymphocytes and monocytes have PDE activities similar to those seen in other tissues.     

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.     

NCS 613, a potent and specific PDE4 inhibitor, displays anti-inflammatory effects on human lung tissues

Chronic inflammation is a hallmark of pulmonary diseases, which leads to lung parenchyma destruction (emphysema) and obstructive bronchiolitis occurring in both chronic obstructive pulmonary disease and asthma. Inflammation is strongly correlated with low intracellular cAMP levels and increase in specific cAMP hydrolyzing activity. The aim of the present study was to investigate the role of the cyclic phosphodiesterase type 4 (PDE4) in human lung and to determine the effects of NCS 613, a new PDE4 inhibitor, on lung inflammation and bronchial hyperresponsiveness. High cAMP-PDE activities were found in the cytosoluble fractions from human lung parenchyma and distal bronchi. PDE4 (rolipram sensitive) represented 40% and 56% of total cAMP-PDE activities in the above-corresponding tissues. Moreover, PDE4A, PDE4B, PDE4C, and PDE4D isoforms were detected in all three subcellular fractions (cytosolic, microsomal, and nuclear) with differential distributions according to specific variants. Pharmacological treatments with NCS 613 significantly decreased PDE4 activity and reduced IκBα degradation in cultured parenchyma, both of which are usually correlated with a lower inflammation status. Moreover, NCS 613 pretreatment potentiated isoproterenol-induced relaxations in human distal bronchi, while reducing TNF-α-induced hyperresponsiveness in cultured bronchi, as assessed in the presence of methacholine, U-46619, or histamine. This reducing effect of NCS 613 on human bronchi hyperresponsiveness triggered by TNF-α was related to a lower expression level of PDE4B and PDE4C, as well as a downregulation of the phosphorylated forms of p38-MAPK, CPI-17, and MYPT-1, which are known to control tone. In conclusion, specific PDE4 inhibitors, such as NCS 613, may represent an alternative and isoform-specific approach toward reducing human lung inflammation and airway overreactivity.     

Chromatin-nuclear envelope complex from rat liver: isolation and purification

We describe a method for the isolation of a fraction of nuclear envelope (NE) from rat liver. The method includes mild treatment of pure nuclei with either endonuclease of DNase I under low ionic strength conditions in the presence of magnesium, which allows the nucleomeric organization of the chromatin (Ch) to be preserved. The NEs were purified by centrifugation in sucrose gradients followed by floatation in sucrose. No more than 3% of the Ch present in the purified Ch-NE complexes was due to the non specific adsorption of Ch to the NE. The main components of the complex (Ch and NE) retained their in situ ultrastructure. The complex consisted of 9--10% DNA, 3--4% RNA, about 63% protein and about 24% phospholipids.     

A prenylated flavonol,sophoflavescenol: a potent and selective inhibitor of cGMP phosphodiesterase 5

During the search for naturally occurring cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitors, it was found that the extracts from Sophora flavescens exhibit potent inhibitory activity against cGMP PDE5 prepared from rat diaphragm. Therefore, the inhibitory activities of five flavonoids, kushenol H (1), kushenol K (2), kurarinol (3), sophoflavescenol (4) and kuraridine (5), isolated from S. flavescens were measured against cGMP PDE5 to identify potent cGMP PDE5 inhibitory constituents. Among tested compounds, sophoflavescenol (4), a C-8 prenylated flavonol, showed the most potent inhibitory activity (IC(50)=0.013 microM) against cGMP PDE5 with 31.5- and 196.2-fold selectivity over PDE3 and PDE4, respectively. Kinetic analysis revealed that sophoflavescenol was a mixed inhibitor of PDE5 with a K(i) value of 0.005 microM.     

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity

Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents.     

The cytochrome oxidase activity of the nuclei and nuclear membranes of rat liver cells following partial hepatectomy

The cytochrome oxidase activity of liver cell nuclei determined in the presence of ascorbate and cytochrome c equals, on the average, 1.2 nmol O2/min/mg protein. This activity tentatively named "free" cytochrome oxidase activity is in nuclei 10% and in nuclear envelopes about 30% of the total cytochrome oxidase activity determined in the presence of tetramethyl paraphenylendiamine. Following the partial hepatectomy, the "free" cytochromoxidase activity in the isolated nuclei is 5-6 times that in the control and approaches the total cytochromoxidase activity which remains at the same level. No increase of free cytochrome oxidase activity in the rat liver homogenate and the isolated mitochondria following the partial hepatectomy was observed.     

Selective inhibition of cardiac cyclic nucleotide phosphodiesterases by doxorubicin and daunorubicin

Doxorubicin and daunorubicin, the anthracycline antitumor agents, were evaluated for their in vitro and in vivo effect on phosphodiesterase (PDE) activity in mouse tissues. Doxorubicin at a concentration of 10(-4)M inhibited cardiac c-AMP (adenosine 3',5', monophosphate) PDE activity 50% of the control whereas in lungs and spleen, the activity was inhibited only 20%. On the contrary no effect was seen in kidney and liver. In addition, cardiac c-GMP (guanosine 3',5' monophosphate) PDE appeared less sensitive to doxorubicin than c-AMP PDE though inhibition in heart was more pronounced than in any other tissue. It appears that daunorubicin inhibits c-AMP PDE activity in heart markedly less than doxorubicin. Kinetic studies indicate that both inhibitions of c-AMP and c-GMP PDE by doxorubicin were non-competitive with substrate. Intravenous administration of 20 and 30 mg/kg of free doxorubicin to CDF1 mice resulted in 33 and 39% decreases in cardiac c-AMP PDE activity respectively by 72 hrs. In contrast, similar intravenous injections of same doses of doxorubicin entrapped in cardiolipin liposomes had no effect on c-AMP PDE activity in any tissues. These studies demonstrate the relative selectivity of the cardiac cyclic nucleotide PDE inhibitory effect of doxorubicin suggesting that this inhibition might be one aspect of the mechanism of anthracycline-induced cardiotoxicity.     

Responses of nuclear glucose-6-phosphatase to diabetes and to hydrocortisone administered to normal and diabetic rats differ from those of the microsomal enzyme.

The effect of alloxan-diabetes, and of pharmacological doses of hydrocortisone administered to normal and diabetic rats, on carbamyl phosphate:glucose phosphotransferase and D-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activities of isolated hepatic nuclei and microsomes were studied by assay at pH 7 in the absence and presence of deoxycholate. Hormonally related alterations both in activity levels and in the activation by the detergent (i.e. latency) of activities of the two cellular structural elements differed significantly. Most strikingly, (a) a 3--4-fold increase in the levels of activities of nuclei was seen in response either to diabetes or to hydrocortisone administered to normal rats whether or not detergent was added to preparations prior to assay; (b) the normally low degree of stimulation by detergent of activities of nuclei was unaltered in diabetes, and (c) administration of the glucocorticoid to diabetic rats decreased activity levels and increased their activation by detergent. Directly contrasting responses were noted with isolated microsomal preparations. Fundamental differences in the enzymes in these two organelle preparations are thus demonstrated. It appears that both synthetic and hydrolytic activities of this enzyme of nuclei may be manifest in the presence of requisite substrates, and that activities of this organelle may become increasingly prominent under certain hormonally perturbed conditions.