A randomized, open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification.

BACKGROUND: Approximately 5%-10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization. PATIENTS AND METHODS: Patients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m2 administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken. RESULTS: Of 71 patients randomized (AZD4547 n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547 n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression. CONCLUSIONS: AZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.

SELECT-2: a phase II, double-blind, randomized, placebo-controlled study to assess the efficacy of selumetinib plus docetaxel as a second-line treatment of patients with advanced or metastatic non-small-cell lung cancer.

BACKGROUND: Combination of selumetinib plus docetaxel provided clinical benefit in a previous phase II trial for patients with KRAS-mutant advanced non-small-cell lung cancer (NSCLC). The phase II SELECT-2 trial investigated safety and efficacy of selumetinib plus docetaxel for patients with advanced or metastatic NSCLC. PATIENTS AND METHODS: Patients who had disease progression after first-line anti-cancer therapy were randomized (2 : 2 : 1) to selumetinib 75 mg b.i.d. plus docetaxel 60 or 75 mg/m2 (SEL + DOC 60; SEL + DOC 75), or placebo plus docetaxel 75 mg/m2 (PBO + DOC 75). Patients were initially enrolled independently of KRAS mutation status, but the protocol was amended to include only patients with centrally confirmed KRAS wild-type NSCLC. Primary end point was progression-free survival (PFS; RECIST 1.1); statistical analyses compared each selumetinib group with PBO + DOC 75 for KRAS wild-type and overall (KRAS mutant or wild-type) populations. RESULTS: A total of 212 patients were randomized; 69% were KRAS wild-type. There were no statistically significant improvements in PFS or overall survival for overall or KRAS wild-type populations in either selumetinib group compared with PBO + DOC 75. Overall population median PFS for SEL + DOC 60, SEL + DOC 75 compared with PBO + DOC 75 was 3.0, 4.2, and 4.3 months, HRs: 1.12 (90% CI: 0.8, 1.61) and 0.92 (90% CI: 0.65, 1.31), respectively. In the overall population, a higher objective response rate (ORR; investigator assessed) was observed for SEL + DOC 75 (33%) compared with PBO + DOC 75 (14%); odds ratio: 3.26 (90% CI: 1.47, 7.95). Overall the tolerability profile of SEL + DOC was consistent with historical data, without new or unexpected safety concerns identified. CONCLUSION: The primary end point (PFS) was not met. The higher ORR with SEL + DOC 75 did not translate into prolonged PFS for the overall or KRAS wild-type patient populations. No clinical benefit was observed with SEL + DOC in KRAS wild-type patients compared with docetaxel alone. No unexpected safety concerns were reported. TRIAL IDENTIFIER: Clinicaltrials.gov NCT01750281.

FGFR2 amplification has prognostic significance in gastric cancer: results from a large international multicentre study.

BACKGROUND: In preclinical gastric cancer (GC) models, FGFR2 amplification was associated with increased tumour cell proliferation and survival, and drugs targeting this pathway are now in clinical trials. METHODS: FGFR2 FISH was performed on 961 GCs from the United Kingdom, China and Korea, and the relationship with clinicopathological data and overlap with HER2 amplification were analysed. RESULTS: The prevalence of FGFR2 amplification was similar between the three cohorts (UK 7.4%, China 4.6% and Korea 4.2%), and intratumoral heterogeneity was observed in 24% of FGFR2 amplified cases. FGFR2 amplification was associated with lymph node metastases (P<0.0001). FGFR2 amplification and polysomy were associated with poor overall survival (OS) in the Korean (OS: 1.83 vs 6.17 years, P=0.0073) and UK (OS: 0.45 vs 1.9 years, P<0.0001) cohorts, and FGFR2 amplification was an independent marker of poor survival in the UK cohort (P=0.0002). Co-amplification of FGFR2 and HER2 was rare, and when high-level amplifications did co-occur these were detected in distinct areas of the tumour. CONCLUSION: A similar incidence of FGFR2 amplification was found in Asian and UK GCs and was associated with lymphatic invasion and poor prognosis. This study also shows that HER2 and FGFR2 amplifications are mostly exclusive.

Immune biomarker evaluation of sequential tyrosine kinase inhibitor and nivolumab monotherapies in renal cell carcinoma: the phase I TRIBE trial.

Background: Predictive biomarkers for immune checkpoint blockade in the second-line treatment of metastatic renal cell carcinoma (mRCC) are lacking. Materials and methods: Patients with histologically confirmed RCC who started nivolumab after at least 4 months of tyrosine kinase inhibitors (TKIs) were recruited for this study. Serial tissue and blood samples were collected for immune biomarker evaluation. The primary endpoint was to determine the association of specific T-cell subsets with clinical outcomes tested using Wilcoxon rank sum for clinical benefit rate (CBR) and log-rank test for progression-free survival (PFS). Results: Twenty patients were included in this trial with a median age of 64 years and followed-up for a median of 12 months. The median PFS for patients who received TKI was 13.8 months, while for those subsequently treated with nivolumab following TKI therapy, the median PFS was 2.6 months. CBR of nivolumab was 20% with two partial responses. Functionally active programmed cell death protein 1+ CD4+ T cells were enriched in non-responders (q = 0.003) and associated with worse PFS on nivolumab (P = 0.04). Responders showed a significant reduction in the effector CD4+T-cell (TEF) fraction compared to non-responders at 3 months on nivolumab (0.40 versus 0.80, P = 0.0005). CD127+CD4+ T cells were enriched in patients who developed immune-related adverse effects (q = 0.003). Using in-house validated multiplex immunohistochemistry for six markers, we measured tumour-associated immune cell densities in tissue samples. Responders to nivolumab showed a significantly higher mean of immune cell densities in tissue samples compared to non-responders (346 versus 87 cells/mm2, P = 0.04). Conclusions: In this small study, analysis of tissue-based and peripheral blood immune cell subsets predicted clinical outcomes of nivolumab. Further studies are warranted with larger populations to validate these observations.

PD-L1 and immune infiltrates are differentially expressed in distinct subgroups of gastric cancer.

This study investigates the association of PD-L1 expression and immune cell infiltrates and their impact on clinical outcome, in addition to their overlap with microsatellite instability (MSI), HER2 and ATM molecular subgroups of gastric cancer (GC). PD-L1 membrane expression on tumour cells (TC) and infiltrating immune cells (IC), CD3 + T-lymphocytes, CD8+ cytotoxic T-cells, ATM and HER2 were assessed by immunohistochemistry (IHC) in the ACRG (Asian Cancer Research Group) GC cohort (N = 380). EBV status was determined using in situ hybridization and MSI status was performed using PCR and MLH1 IHC. The PD-L1 segment was associated with increased T-cell infiltrates, while the MSI-high segment was enriched for PD-L1, CD3, and CD8. Multivariate analysis confirmed PD-L1 positivity, high CD3 and high CD8 as independent prognostic factors for both disease-free survival and overall survival (all p < 0.05). Patients with MSI-high tumours had better overall survival by both univariate and multivariate analysis. The ATM-low and HER2-high subgroups differed markedly in their immune profile; the ATM-low subgroups enriched for MSI, PD-L1 positivity and CD8 + T-cells, while the HER2 segment was enriched for MSS, with no enrichment for immune markers. Hence, we demonstrate a molecular profiling approach that can divide GC into four molecular subgroups, namely ATM-low, HER2-high, PD-L1 positive and MSI-high with differing levels of immune infiltrates and prognostic significance which may help to stratify patients for response to targeted therapies.

Changes in the expression of guanine nucleotide-binding proteins during differentiation of 3T3-F442A cells in a hormonally defined medium.

Using specific antisera, the expression of the G protein alpha subunits, G8, G(i1), G(i2), G(i3) and G0, were determined in 3T3-F442A cells during their differentiation to adipocytes in a hormonally defined medium. Differentiation caused distinct increases in the expression of two Gs isoforms and decreases in the expression of both G(i2) and G(i3). Differentiation also resulted in a 2- to 4-fold increase in forskolin-stimulated adenylyl cyclase activity and a 15-fold increase in the response of cells to a beta-adrenergic agonist. The increase in Gs expression was also observed, to a lesser degree, in cells maintained at confluence under conditions where morphological conversion was negligible and the decreased expression of G(i2) and G(i3) and the increased beta-adrenergic responsiveness did not occur.

Growth hormone induces the tyrosine phosphorylation and nuclear accumulation of components of the ISGF3 transcription factor complex.

Growth hormone induced the accumulation of the stat91 and p84 subunits of the transcription factor complex ISGF3 in nuclear fractions of 3T3-F442A cells. Nuclear levels of p84 and stat91 peaked 30-60 min after addition of growth hormone. Growth hormone also induced the tyrosine phosphorylation of two proteins, with similar sizes to stat91 and p84, in both nuclear and cytosolic fractions. The time course of growth hormone-induced tyrosine phosphorylation of these proteins paralleled the nuclear accumulation of stat91 and p84. Immunoprecipitation with stat91-specific antibodies confirmed that growth hormone induced the tyrosine phosphorylation of stat91 and an associated protein of M(r) = 120 kDa. These findings suggest a mechanism for the modulation of specific gene transcription by growth hormone.

Divergent regulation of rat adipocyte GLUT1 and GLUT4 glucose transporters by GH.

The effect of GH, in vivo, on the glucose transport systems of rat adipocytes has been investigated. Lowering of serum GH levels, by treatment of rats with an antiserum specific for rat GH (anti-rGH), significantly decreased serum levels of both IGF-I and insulin. Treatment with anti-rGH also increased glucose oxidation and the conversion of glucose to lipid by isolated adipocytes. Adipocyte glucose oxidation and lipid synthesis were measured in the presence of a limiting concentration of glucose and therefore reflect changes in glucose transport. Immunoblot analysis of adipocyte subcellular fractions revealed that anti-rGH induced an increase in the amount of the glucose transporters GLUT1 (1.6-fold) and GLUT4 (2.5-fold) present in plasma membranes and a decrease (39%) in the amount of GLUT4 present in low-density microsomal fractions. Lowering of serum GH also increased, by 36%, the amount of GLUT1 present in a total membrane fraction but had no such effect on GLUT4 levels. Replenishment of serum GH, by concurrent administration of ovine GH to rats, prevented all of these effects of anti-rGH. It was concluded that GH in vivo down-regulates the amount of both GLUT1 and GLUT4 present in rat adipocyte plasma membranes. This reflects a decrease in the total cellular levels of GLUT1 and modification of the subcellular distribution of GLUT4 and results in restriction of adipocyte glucose uptake.

Regulation of the GTP-binding protein-based antilipolytic system of sheep adipocytes by growth hormone.

Chronic exposure of sheep adipose tissue to growth hormone (GH) in vitro decreases the ability of the adenosine analogue, N6-phenylisopropyladenosine (PIA), to inhibit isoprenaline-stimulated lipolysis by a mechanism which is dependent on both gene transcription and protein serine/threonine phosphorylation. The inhibition is not due to a change in ligand binding to the adenosine receptor, the amounts of the three isoforms of the inhibitory GTP-binding protein, Gi, or the maximum (forskolin-stimulated) adenylate cyclase activity. The ability of GH to modulate the PIA-activated adenosine receptor to stimulate dissociation of heterotrimeric Gi was assessed by measurement of pertussis toxin-catalysed ADP-ribosylation of Gi; GH does not appear to alter the interaction between the activated receptor and Gi. The ability of GH to alter the ability of activated Gi to inhibit adenylate cyclase activity was assessed by measuring the ability of a GTP analogue, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), to inhibit forskolin-stimulated adenylate cyclase activity; chronic exposure to GH prevented this effect of p[NH]ppG. Thus the attenuation of the inhibition of lipolysis by PIA by chronic exposure of adipocytes to GH appears to be due to an impairment in the interaction between adenylate cyclase and the alpha subunit of one or more isoforms of Gi.

Effect of lactation on insulin signal transduction in sheep adipose tissue and skeletal muscle.

The molecular basis of the insulin resistance of adipocytes and skeletal muscle during lactation has been investigated in sheep. The number of insulin receptors per adipocyte or per unit membrane protein for skeletal muscle is unchanged by lactation. The ability of insulin to stimulate autophosphorylation of its beta-subunit was enhanced in adipocytes but not in skeletal muscle during lactation. This increased autophosphorylation was due, at least in part, to enhanced tyrosine phosphorylation and was found when both solubilised, immunoprecipitated insulin receptors and intact adipocytes were incubated with insulin. The ability of the insulin receptor kinase to phosphorylate other proteins did not appear to be altered by lactation; this was shown with lectin-purified insulin receptors using the artificial substrate, polyglutamyl tyrosine, and in intact adipocytes. Lactation had no effect on the ability of insulin to activate two key downstream kinases, mitogen-activated protein kinase and phosphatidyl inositol-3-kinase in adipocytes. The study thus shows that the insulin resistance of lactation in sheep is due to changes downstream of the receptor in both adipocytes and skeletal muscle.

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

Chronic effects of somatotropin treatment on response of subcutaneous adipose tissue lipolysis to acutely acting factors in vivo and in vitro.

The basis of the chronic lipolytic effect of somatotropin on adipose tissue was investigated in sheep. Lipolytic rate was assessed in subcutaneous adipose tissue both in vivo, by microdialysis, and in vitro. Somatotropin treatment resulted in a small increase in basal (unstimulated) lipolysis and also in the maximum lipolytic rate observed in the presence of catecholamines both in vivo and in vitro. There was a small increase in the number of beta-adrenergic receptors but no change in the amount of the two isoforms of the stimulatory GTP-binding protein, Gs. Treatment with somatotropin decreased the response to antilipolytic agents such as the adenosine analog N6-phenylisopropyladenosine and prostaglandin E1. There was, however, no change in the number of adenosine receptors or amounts of the inhibitory GTP-binding proteins (Gi-1 plus Gi-2). Somatotropin also decreased prostaglandin E2 production by subcutaneous adipose tissue in vivo. Somatotropin treatment thus alters lipolytic regulation in sheep and this is characterized by changes in a number of proteins involved in this process.

PDH kinase inhibitors: a novel therapy for Type II diabetes?

The pyruvate dehydrogenase multienzyme complex catalyses the oxidative decarboxylation of pyruvate, which is an important regulatory step in oxidative metabolism. Phosphorylation of the E1 (pyruvate decarboxylase) subunit on one of three specific serine residues results in loss of enzyme activity. Four dedicated PDHK (pyruvate dehydrogenase kinase) isoenzymes have been identified, each of which display a distinct tissue-specific expression profile, and have differential regulatory properties. Thus PDHK play a key role in controlling the balance between glucose and lipid oxidation according to substrate supply. Increasing glucose oxidation by inhibiting PDHK may be an effective mechanism to increase glucose utilization; additionally, increasing pyruvate oxidation may further contribute to lowering of glucose level by decreasing the supply of gluconeogenic substrates. A number of PDHK inhibitors are now available to enable this mechanism to be evaluated as a therapy for diabetes. The isoenzyme selectivity profile of AZD7545 and related compounds will be described and evidence for their non-ATP-competitive mode of action presented. These compounds increase PDH activity in vivo, and when dosed chronically, improve glycaemic control in Zucker rats. Furthermore, glucose lowering has been demonstrated in the hyperglycaemic Zucker diabetic fatty rat. This result supports the hypothesis that inhibition of PDHK may be an effective therapy for Type II diabetes.

Defect in signal transduction at the level of the plasma membrane accounts for inability of insulin to activate pyruvate dehydrogenase in white adipocytes of lactating rats.

1. The mechanism responsible for the failure of insulin to activate pyruvate dehydrogenase (PDH) in white adipose tissue in vivo during lactation was investigated. 2. Insulin failed to increase PDH in isolated adipocytes from lactating rats. 3. Insulin binding to plasma membranes from adipocytes was unchanged by lactation. 4. Incubation of plasma membranes plus permeabilized mitochondria from adipocytes in the presence of insulin resulted in activation of PDH when the plasma membranes were obtained from virgin rats, whereas no activation was observed when plasma membranes from lactating rats were used. 5. The results show that the failure of insulin to activate PDH in adipose tissue from lactating rats is due to a failure of the signal-transduction system in the plasma membrane at steps subsequent to insulin binding to the insulin receptor.

The generation of inositolglycan mediators from rat liver plasma membranes: the role of guanine nucleotide binding proteins.

The guanine nucleotide dependence for the generation of inositolglycan second messengers from rat liver plasma membranes has been investigated. Plasma membranes, when treated with insulin release a soluble mediator substance which activates pyruvate dehydrogenase (PDH). Guanosine 5'-[3-thio]triphosphate (GTP gamma S) was found to be as potent as insulin in stimulating mediator release. The stimulatory effects of GTP gamma S required the presence of magnesium and following preincubation of membranes with guanosine 5'-[2-thio]diphosphate (GDP beta S) the stimulation of mediator release by either insulin or GTP gamma S was blocked. The activation of PDH by mediator fractions produced in response to either insulin or GTP gamma S was abolished following treatment of the fractions with anti-inositolglycan antibodies. The significance of these observations with respect to the possible involvement of a regulatory guanine-nucleotide binding protein (G-protein) in the generation of insulin mediators is discussed.

Catecholamine activation of pyruvate dehydrogenase in white adipose tissue of the rat in vivo.

Intraperitoneal injections of noradrenaline or adrenaline into rats increased the proportion of pyruvate dehydrogenase in the active state in white adipose tissue; this effect of catecholamines was also apparent in streptozotocin-diabetic rats, showing that it was not due to an increase in serum insulin concentration. The catecholamine-induced increase in pyruvate dehydrogenase of white adipose tissue in vivo was completely blocked by prior injection of either the beta-antagonist propranolol or the alpha 1-antagonist prazosin. Cervical dislocation of conscious rats increased pyruvate dehydrogenase activity of white adipose tissue, which was prevented by prior injection of propranolol. Adrenaline (30 nM) activated pyruvate dehydrogenase in white adipocytes in vitro; the maximum effect of adrenaline required activation of both alpha 1- and beta-receptors. The results show that catecholamines activate pyruvate dehydrogenase of white adipose tissue both in vivo and in vitro and that this effect is mediated by a combination of alpha 1- and beta-adrenergic receptors.

Tissue-specific changes in the ability of insulin and noradrenaline to activate pyruvate dehydrogenase in vivo during lactation in the rat.

Changes are described in the total pyruvate dehydrogenase (PDH) activity, the proportion of PDH in the active state and its control by insulin and noradrenaline in vivo, in white adipose tissue, liver, skeletal muscle and mammary gland with pregnancy, lactation and on weaning. Lactation resulted in a decrease in total PDH in white adipose tissue and an increase in the mammary gland, whereas the proportion in the active state decreased in muscle and increased in the mammary gland. The ability of insulin to activate PDH of white adipose tissue was lost during lactation, whereas it was retained by the other tissues. The ability of noradrenaline to activate PDH was decreased in white adipose tissue but increased in liver during lactation. These various adaptations should limit the use of glucose and lactate carbon by adipose tissue and skeletal muscle during lactation and thereby facilitate their preferential utilization by the mammary gland.

Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways.

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.

Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.