Increase In cAMP Phosphodiesterase Activity Research Articles

Uncoupling of vasopressin signaling in collecting ducts from rats with CBL-induced liver cirrhosis.

Vasopressin (AVP) stimulates collecting duct water reabsorption through cAMP-mediated membrane targeting and increased expression of the aquaporin-2 (AQP2) water channel. Rats with liver cirrhosis induced by common bile duct ligation (CBL) show decreased protein expression of AQP2 despite increased plasma concentrations of AVP. The present study was conducted to investigate possible mechanisms behind this uncoupling of AVP signaling. The rats were examined 4 wk after CBL or sham operation. The CBL rats had increased plasma AVP concentrations (CBL: 3.2 +/- 0.2 vs. sham: 1.4 +/- 0.4 pg/ml, P < 0.05) and reduced AQP2 (0.62 +/- 0.11) and phosphorylated AQP2 (0.50 +/- 0.06) protein expression compared with sham-operated rats. However, examination of subcellular AQP2 localization by immunohistochemistry showed unchanged plasma membrane targeting in CBL rats, indicating a sustained ability of AQP2 short-term regulation. In a separate series of animals, thirsting was found to normalize AQP2 expression, indicating that AVP uncoupling in CBL rats is a physiological compensatory mechanism aimed at avoiding dilutional hyponatremia. Studies on microdissected collecting ducts from CBL rats showed decreased cAMP accumulation in response to AVP stimulation. The presence of the nonspecific phosphodiesterase inhibitor IBMX normalized the cAMP accumulation, indicating that cAMP-phosphodiesterase activity is increased in CBL rats. However, in contrast to this, Western blotting showed a decreased expression of several phosphodiesterase splice variants. We conclude that CBL rats develop an escape from AVP to prevent the formation of dilutional hyponatremia in response to increased plasma AVP concentrations. The mechanism behind AVP escape seems to involve decreased collecting duct sensitivity to AVP as a result of increased cAMP-phosphodiesterase activity.

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8).

Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca2+ transients than NTG mice, with (surprisingly) no modification in L-type Ca2+ channel current (ICa,L) (1). In this study, we examined the cardiac response of AC8TG mice to beta-adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and ICa,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 microM) produced a twofold higher stimulation of ICa,L in AC8TG vs. NTG mice. IBMX (10 microM) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 microM) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP-PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca2+ channels and protects the cardiomyocytes from Ca2+ overload.

Insulin increased cAMP phosphodiesterase activity antagonizing metabolic actions of glucagon in rat hepatocytes cultured with herbimycin A

The baseline activity of cyclic nucleotide phosphodiesterase 4 was markedly lowered by primary culture of rat hepatocytes with herbimycin A for 4 h [Eur. J. Biochem. 260 (1999) 398–408.]. We now report that insulin added to this preparation of hepatocytes, which had been completely freed of herbimycin, increased the thus lowered phosphodiesterase activity, consequently antagonizing glucagon-induced production of cAMP and activation of glycogen phosphorylase. The insulin receptor β-subunits and α-tubulin were tyrosine-phosphorylated upon the addition of insulin. The phosphorylation of α-tubulin afforded conditions unfavorable for microtubule assembly that is responsible for phosphodiesterase inhibition. These effects of insulin observed in herbimycin-pretreated hepatocytes were not inhibited by wortmannin that actually abolished insulin-induced activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) under the same conditions. The physiological significance of the insulin action not mediated by PtdIns 3-kinase in herbimycin-pretreated hepatocytes is discussed.

Albuterol-induced downregulation of Gsalpha accounts for pulmonary beta(2)-adrenoceptor desensitization in vivo.

The aim of the present study was to develop a chronic in vivo model of pulmonary beta(2)-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E(2), but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. beta(2)-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsalpha expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin-induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein-coupled receptor kinase-2, but the extent to which these events contributed to beta(2)-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsalpha representing a primary molecular etiology.

Activation of a cGMP‐stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi–endosomal fraction

The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity. Selective effect of cyclic AMP.

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 Autonomic Nervous System and the Immune System in Juvenile Rheumatoid Arthritis

This study demonstrates that juvenile rheumatoid arthritis is associated with a dysregulation of the autonomic nervous system as well as with disturbances in the capacity of the immune system to respond to mediators of the autonomic nervous system. In patients with active disease heart rate at rest is higher than in healthy controls. In addition, 3-hydroxy-4-phenoxyphenylglycol levels in urine are higher in all patients than in the control group. Cardiovascular responses to an orthostatic stress test (tilt up) are reduced in patients with active and nonactive disease. Plasma norepinephrine responses to tilt up are reduced in subjects with active juvenile rheumatoid arthritis. In summary, our data show that patients with juvenile rheumatoid arthritis have an altered function of the autonomic nervous system associated with increased central noradrenergic outflow, presumably leading to increased vasoconstriction, resulting in a decreased response to an orthostatic stressor. The altered function of the autonomic nervous system is associated with changes in the response of leukocytes to mediators of the autonomic nervous system via β2-adrenergic receptors. Leukocytes of patients with active juvenile rheumatoid arthritis have a lower cAMP response to a β2-adrenergic agonist, presumably due to increased cAMP–phosphodiesterase activity in these cells.

Isoprenaline induction of cAMP-phosphodiesterase in guinea-pig macrophages occurs in the presence, but not in the absence, of the phosphodiesterase type IV inhibitor rolipram

The long-term effects of incubating freshly isolated, elicited guinea-pig peritoneal macrophages with the β-adrenoceptor agonist isoprenaline and the selective inhibitor of phosphodiesterase (PDE) IV rolipram, on adenosine-3′,5′-cyclic phosphate (cAMP)-specific PDE IV activity have been investigated. The level of cAMP PDE activity in macrophages was unaffected by long-term exposure of cells to rolipram alone. In contrast, in the presence of isoprenaline (10 μM), a concentration-related (0.05–50 μM) increase in cAMP PDE activity was observed in the cytosolic and paniculate fractions. Incubation with isoprenaline alone did not affect macrophage cAMP PDE. cAMP PDE activities in homogenates of control cells and macrophages treated with isoprenaline (10 μM) and rolipram (5 μM) (3-fold activation) were inhibited by the selective PDE IV inhibitor, rolipram, with similar potencies (IC 50: 2—3 μM). The increase in cAMP PDE activity in response to rolipram and isoprenaline was completely blocked by cyclohexamide (10 μg/ml). Incubating macrophages for 10 min with rolipram increased cAMP accumulation in the presence, but not in the absence, of isoprenaline (10 μM) over the same concentration range that induction of cAMP PDE activity was observed. cAMP levels remained elevated for at least 1 hr. Isoprenaline (10 μM) alone induced a transient elevation in cAMP levels that peaked at 2 min and had returned to basal levels by 10 min. Protein kinase A activity (PKA) was increased almost 10-fold (at 10 min) by exposing cells to rolipram plus isoprenaline and remained elevated for at least 4 hr. Isoprenaline alone induced a small (2-fold) increase in PKA activity and rolipram alone was without effect.

Protein kinase A regulation of cAMP phosphodiesterase expression in rat skeletal myoblasts.

We have been studying cAMP signaling in L6 myoblasts because of its potential role in regulating the differentiation of these cells into multinucleate myotubes. Previous studies have shown that treatment of L6 myoblasts with cAMP analogs causes an increase in cAMP phosphodiesterase activity. To assess the role of protein kinase A in this cAMP-mediated increase in cAMP phosphodiesterase activity, L6 myoblasts were transfected with a plasmid containing the cDNA for a mutant regulatory subunit of protein kinase A, which functions as a dominant negative inhibitor of this enzyme. The cDNA was under control of the metallothionein promoter in the construct. Induction of the mutant regulatory subunit with Zn2+ decreased cAMP-dependent protein kinase activity by 90%. Zn2+ treatment was also able to completely block the cAMP-mediated increase in phosphodiesterase activity, showing that this effect is mediated by protein kinase A. The activity of the cAMP-induced phosphodiesterase was inhibited by low concentrations of RO 20-1724, showing that it was a member of the type IV low Km cAMP phosphodiesterase family of enzymes. We used the polymerase chain reaction and consensus primers designed to amplify phosphodiesterase sequences to show that L6 myoblasts also contain mRNA for a type IV low Km cAMP phosphodiesterase designated PDE3.1. The levels of this mRNA were increased greatly by treatment with dibutyryl cAMP or forskolin in L6 myoblasts and also in differentiated L6 myotubes. Run-off transcription assays showed that this increase in PDE mRNA was regulated, at least in part, by an increase in the rate of transcription of the PDE3 gene. The induction of PDE3 message by cAMP was blocked when the L6 transfectants were treated with Zn2+ to induce protein kinase A inhibition. Therefore, some of the cAMP-mediated increase in phosphodiesterase activity seen in L6 myoblasts is due to a protein kinase A-mediated increase in PDE3 mRNA. This pathway may serve as a feedback mechanism to modulate the inhibitory effects of cAMP on myogenesis.

Atrial Natriuretic Factor Activates Cyclic Adenosine 3′,5′-Monophosphate Phosphodiesterase in Xenopus Laevis Oocytes and Potentiates Progesterone-Induced Maturation via Cyclic Guanosine 5′-Monophosphate Accumulation

Xenopus oocytes were found to express atrial natriuretic factor (ANF) receptors that activate guanylate cyclase and stimulate cyclic guanosine 5'-monophosphate (cGMP) production in a dose- and time-dependent manner. A truncated fragment of ANF, known to bind to mammalian ANF receptors without stimulating cGMP accumulation, did not elicit a cGMP response in oocytes. In addition, preincubation with ANF increased the number of oocytes that underwent progesterone-induced maturation. The maximally effective dose of ANF (1 microM) elevated intracellular and extracellular cGMP accumulation from 50 to 200 and 5 to 800 fmol/oocyte, respectively, and increased the number of maturing oocytes by up to 3-fold. ANF's effects on progesterone-induced maturation were mimicked by nonhydrolyzable analogues of cGMP. ANF and 8-Br-cGMP had no effect on maturation in the absence of progesterone, indicating that elevation of cGMP alone is not sufficient to induce maturation. Dibutyryl-cGMP was as effective as 8-Br-cGMP, whereas 8-Br-guanosine monophosphate, 8-Br-guanosine, and 8-Br-cyclic inosine monophosphate did not potentiate ovum maturation. In Xenopus oocytes, an initial step in progesterone-induced maturation is the reduction of intracellular cAMP levels; both ANF and 8-Br-cGMP lowered cAMP levels and enhanced progesterone's ability to do so. This decrease in cAMP levels was attributable to increased cAMP-phosphodiesterase activity, which was enhanced by both ANF and 8-Br-cGMP. These findings, and the presence of functional ANF receptors on Xenopus oocytes, demonstrate that ANF can participate in ovum development by stimulation of cGMP accumulation and activation of cAMP phosphodiesterase, thereby potentiating progesterone's ability to decrease cAMP levels and promote ovum maturation.

Elevation of cAMP is required for down-regulation, but not agonist-induced desensitization, of endogenous dopamine D1 receptors in opossum kidney cells. Studies in cells that stably express a rat cAMP phosphodiesterase (rPDE3) cDNA

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.

Opposite regulation of cAMP concentration in the quail oviduct and the mouse uterus by tamoxifen. Correlation with estrogen-antagonist and estrogen-agonist activity

The ability of estradiol and tamoxifen to regulate cAMP levels and cAMP phosphodiesterase activities has been determined in the quail oviduct and in the mouse uterus. In the quail, tamoxifen (1 mg/kg daily for 3 days) had no effect on oviducal growth but significantly increased cAMP concentration (+49%). Injected concurrently with estradiol, tamoxifen completely inhibited oviduct growth as well as the increase of cAMP phosphodiesterase activity induced by the hormone alone and increased cAMP concentration (+229% over estradiol treated group). In the mouse, estradiol and tamoxifen displayed uterotrophic activity and increased cAMP phosphodiesterase activity. In both groups, cAMP concentration was greatly reduced (−76% in estradiol treated group; −86% in tamoxifen treated group). The opposite regulation of cAMP levels in the quail oviduct and the mouse uterus by tamoxifen reflected large differences in the contribution of calmodulin-dependent and -independent forms of phosphodiesterase to the hydrolysis of cAMP in the two models and the fact that tamoxifen stimulated the activity of the calmodulin-independent isoenzyme, while it competitively inhibited the activation of the calmodulin-dependent isoenzyme by calmodulin. Several lines of evidence strongly suggest that the regulation of cAMP levels is involved in growth-inhibiting or growth-promoting activity of tamoxifen.

Characterization of a Liver Low Michaelis-Menten Constant 3′,5′-Cyclic Adenosine Monophosphate Phosphodiesterase Activity Sensitive to Thyroid Status*

These studies were undertaken to assess the subcellular distribution and some biochemical properties of the hepatic cAMP phosphodiesterase(s) whose activity is modulated by the thyroid status in the rat. Thyroidectomy led to a 2-fold increase in low Michaelis-Menten constant (Km) cAMP phosphodiesterase activity in Golgi-endosomal fractions, but little affected this activity in crude particulate fractions. On analytical sucrose density gradients, an increase in cAMP phosphodiesterase activity in particulate elements which equilibrated at densities 1.17-1.22 was also observed. Acute insulin treatment did not further increase cAMP phosphodiesterase activity in Golgi-endosomal fractions of thyroidectomized rats. Up to 75% of the cAMP phosphodiesterase activity associated with Golgi-endosomal fraction of euthyroid and hypothyroid rats was inhibited by cGMP (IC50, 10 microM and 1 microM, respectively). Activity was also potently inhibited by griseolic acid, cilostamide, and cilostazole (IC50, less than 1 microM) but was much less sensitive to R0-20-1724 (IC50, 1 mM). Treatment of Golgi-endosomal fractions by a hypotonic extract of rat liver lysosomes led to the solubilization of 50% of low Km cAMP phosphodiesterase activity. On sucrose density gradients, the solubilized activity migrated as a slightly asymmetrical peak with a sedimentation coefficient of 6 S in euthyroid rats and 6.9 S in hypothyroid rats. On nondenaturing polyacrylamide gel electrophoresis, the activity migrated as two majors peaks with Rf values of 0.23 and 0.50; only the activity associated with the fast-moving peak was increased by thyroidectomy. On diethylaminoethyl-Sephacel chromatography, four peaks of cAMP phosphodiesterase activity, two of which were cGMP-inhibitable, were resolved. Thyroidectomy increased the activity associated with one of the cGMP-inhibitable peaks (eluted at 0.7-0.9 M sodium acetate) and led to the appearance of a new peak of activity (eluted at 0.4 M), which was not sensitive to cGMP. These results show that the low Km phosphodiesterase activity associated with liver Golgi-endosomal fractions, previously shown to be increased in hyperinsulinemic rats, is also increased in hypothyroid animals. They also suggest that, based on pharmacological and physical criteria, the enzyme species affected by the thyroid status belongs to the cGMP-and cilostamide-inhibited subclass of low Km cAMP phosphodiesterases.

CAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

Modulation of aromatase and P450 cholesterol side-chain cleavage enzyme activities of human placental cytotrophoblasts by insulin and insulin-like growth factor I.

The placenta is the primary source of estrogens and progesterone during pregnancy. Because pregnant diabetic women are reported to have lower serum estrogen and higher progesterone concentrations than nondiabetic pregnant women, we studied the roles of insulin and insulin-like growth factor I (IGF-I) in the regulation of human cytotrophoblastic aromatase and P450 side-chain cleavage enzyme (P450 SCC) activities. Incubation of cytotrophoblasts with insulin or IGF-I for 24 h significantly inhibited the conversion of androstenedione to estrogens by approximately 20-40%. Insulin and IGF-I suppressed aromatization at doses as low as 20 and 10 ng/ml, respectively. Insulin's suppressive effect was demonstrable only after 18-22 h of incubation, suggesting an effect of insulin on aromatase protein mass rather than on aromatase activity. Cytotrophoblasts pretreated with insulin for 24 h possessed 23-30% less aromatase activity than control cells, as quantitated directly by the specific release of 3H2O from [3H]androstenedione, indicating that insulin inhibited estrogen synthesis rather than increased estrogen catabolism. Insulin's suppressive effect on aromatase was not due to a toxic effect of insulin, since incubates exposed to insulin for 24 h showed no decrease in cell number, cellular DNA content, or cellular protein content compared to control incubates. Also, insulin's suppression of aromatization was not due to increased cAMP phosphodiesterase activity, since cotreatment with 1 mM (Bu)2cAMP did not alter insulin's suppressive effect. Blockade of the IGF-I receptor of cytotrophoblasts with alpha IR-3, a monoclonal anti-IGF-I receptor antibody, prevented the suppression of aromatase activity by IGF-I, but did not alter insulin's inhibitory effect. This suggests that the two hormones inhibit aromatization via activation of their specific receptors and not by cross-association. Insulin treatment did not affect P450 SCC activity, whereas IGF-I treatment significantly stimulated P450 SCC activity by 19-36%, as measured by the conversion of 25-hydroxycholesterol to progesterone. These studies indicate that insulin exerts a selective inhibitory effect on cytotrophoblastic aromatase activity, whereas IGF-I inhibits aromatase activity but stimulates P450 SCC activity. Since pregnant diabetic women manifest peripheral hyperinsulinemia, and IGF-I levels in fetal cord sera from diabetic pregnancies are elevated, these observations may help explain the lower serum estrogen and elevated progesterone levels associated with diabetic pregnancy.

Cyclic nucleotide phosphodiesterase in developing rat testis identification of somatic and germ-cell forms

Several forms of phosphodiesterase are present in the male gonad, and their relative concentrations vary during testicular development. On the basis of kinetic analysis and chromatography on DEAE cellulose, 3 forms were separated: (a) a high-affinity cGMP phosphodiesterase, regulated by Ca 2+ and calmodulin, similar to a form described in different tissues (peak I); (b) a high-affinity cAMP form insensitive to Ca 2+, which cannot be readily compared with forms described elsewhere (peak II); and (c) a high-affinity cAMP phosphodiesterase, Ca 2+- and calmodulin-insensitive, corresponding to the “hormone-regulated” form described in several systems (peak III). The elution pattern of peak I and calmodulin stimulation were dependent on free calcium concentration during cytosol preparation and chromatography. This datum and rechromatography in the presence or absence of excess calmodulin suggested that the enzyme complexes calmodulin in a Ca 2+-dependent manner and is therefore activated. Moreover, whereas peak I was observed in all cell compartments of the testis, peak II was present in germ cells and peak III was found mainly in somatic cells. During development of the testis, a relevant enhancement in the ratio between cAMP and cGMP hydrolytic activity was observed together with an overall increase of phosphodiesterase activity, thus suggesting that the previously described increase in cAMP phosphodiesterase activity during testis maturation should be ascribed to forms present in both germ cells and somatic cells.

Hormonal control of phosphodiesterase activity in cultured rat sertoli cells

Phosphodiesterase activity was studied in cultures derived from 19-day-old rats and enriched with Sertoli cells. Pretreatment of such cultures with follicle-stimulating hormone or l-isoproterenol increased cAMP-phosphodiesterase activity 5.2 and 2.0 times, respectively. cGMP-phosphodiesterase activity was not affected. Similar effects were observed in freshly isolated cells. The stimulatory effect was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was mimicked by cholera toxin and dbcAMP. Increased activity was observed after a latent period of 1 h. Stimulation was blocked by cycloheximide and actinomycin D. The enzyme had an apparent K m for cAMP of 1.4 μM. Its activity was enhanced by Mg 2+ but not by Ca 2+. It is concluded that phosphodiesterases play an important role in the hormonal control of Sertoli-cell function and may contribute to the refractory state of these cells after stimulation with various agonists.

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.

Cyclic nucleotides in the regulation of expression of differentiated functions in neuroblastoma cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) may be one of the important factors in regulating the expression of many differentiated functions in neuroblastoma cells, but some of these functions can be induced by agents that do not increase the intracellular level of cAMP. An elevation of the intracellular level of guanosine 3',5'-cyclic monophosphate (cGMP) neither induced differentiation nor antagonized the effects of cAMP. Neuroblastoma cells increased the level of cAMP-binding proteins during differentiation, whereas glial cells and L-cells did not. This might have accounted in part for an increase in the intracellular level of cAMP even in the presence of high phosphodiesterase activity in neuroblastoma cells, since the protein-bound with the same proteins, but cAMP had about 10 times higher affinity than did cGMP. cAMP promoted the organization of microtubules and microfilaments necessary for the expression of differentiated phenotypes. The extension of neurites required the synthesis of new protein, but it did not need the synthesis of new RNA. cAMP induced differentiation in neuroblastoma cells by increasing the expression of some genetic information while suppressing the expression of others; e.g., the activities of neural enzymes increased, whereas the synthesis of histone and the phosphorylation of H1-histone markedly decreased in differentiated cells. A hypothesis was offered: An increase in cAMP phosphodiesterase activity as a result of mutation in the regulatory gene for phosphodiesterase in a single, or group of, dividing nerve cell(s) is the primary lesion that leads to malignancy. Based on the concept that selective cytocytoxic drugs should be used with agents that cause differentiation, a new therapeutic approach was suggested for the treatment of neuroblastoma. This involved administration of sodium butyrate followed by L-DOPA or prostaglandin E1 in the presence of cAMP phosphodiesterase inhibitor followed by the less immunosuppressive vincristine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide.

Alterations in adenosine 3′,5′-monophosphate-binding protein in Walker carcinoma cells sensitive or resistant to alkylating agents

The extent of binding of 8-[ 3H]adenosine 3′,5′-monophosphate (cyclic AMP) to specific sites was measured in Walker carcinoma cells of tissue culture lines, having different degrees of resistance to the cytotoxic effects of alkylating agents. When compared with sensitive cells, the resistant lines showed a loss of binding activity with increasing resistance, when measured at either pH 4.0 or pH 6.5. This applied to cell lines made resistant either to 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) or chlorambucil, although the total loss of binding activity was greater in the former cell lines. Scatchard analysis of binding suggested the presence of two sites in all cell lines with K D I ∼ 1–5 × 10 −9M and K D II ∼ 3 × 10 −8M. The decreased binding activity was not due to an increased cAMP phosphodiesterase activity in the resistant cell lines, since the activity of the high affinity form of this enzyme was either the same as (CB 1954-resistant) or less than (chlorambucil-resistant) that found in sensitive cells. Walker cells with acquired resistance to either CB 1954 or chlorambucil showed a degree of cross resistance to the cytostatic effect of cAMP analogues and of other alkylating agents.