Methylxanthines Research Articles

Sperm storage in turtles: A male perspective.

The storage and behavior of sperm collected from the epididymis of two emyiid turtles were examined. In Chrysemys picta, the weight of the epididymis does not change significantly throughout the year as does the testis. However, in this species, as well as in Trachemys scripta, the epididymis contains sperm throughout the entire year. Sperm from both species have a relatively low motility and velocity of movement. In C. picta, equally low motilities are observed both in the autumn, shortly after spermiation, and in spring. Motility could be augmented by the addition of isobutyl methyl xanthine (IBMX, 0.5 mmol l(-1)). Epididymal sperm remained viable in excess of 40 days in vitro when stored in F-10 buffer, during which time motility and swimming velocity could be augmented with IBMX. The longevity and low motility of turtle sperm facilitates its storage by either males or females, and creates conditions that promote the wide dissemination, over time, of gametes produced in a narrow time window.

ATM as a target for novel radiosensitizers

DNA damage checkpoints are complex signal transduction pathways that are critical for normal cellular recovery following potentially lethal genotoxic insults. The ataxia-telangiectasia mutated (ATM) protein kinase is a critical component in these pathways and integrates the cellular response to damage by phosphorylating key proteins involved in cell cycle regulation and DNA repair. Lack of normal ATM function in the inherited ataxia-telangiectasia (A-T) syndrome results in a pleiotropic clinical syndrome characterized by a marked increased risk of cancer and profound hypersensitivity to ionizing radiation. Cells derived from patients with A-T share some of these attributes with genomic instability, loss of normal cell cycle arrest pathways, defects in DNA repair and increased radiation sensitivity. The radiosensitivity of A-T cells suggests that pharmacological inhibitors of the ATM kinase should be effective radiosensitizing agents. In fact, caffeine inhibits ATM kinase activity at concentrations that result in an A-T-like phenotype with loss of cell cycle checkpoints and hypersensitivity to ionizing radiation. Although the clinical use of caffeine as a radiosensitizer is limited by potentially lethal systemic toxicities, more potent methyl xanthines may selectively inhibit the ATM pathway at clinically achievable levels. Interestingly, caffeine and other methyl xanthines preferentially radiosensitize cells that lack normal p53 function. Because p53 is commonly inactivated in epithelial malignancies, this suggests that small molecule inhibitors of ATM might selectively sensitize the majority of tumors to the lethal effects of ionizing radiation while sparing normal tissues.

Effects of angiotensin II on adipose conversion and expression of genes of the renin-angiotensin system in human preadipocytes.

A complete functional renin-angiotensin system exists in human adipose tissue, but its regulation and the effects of angiotensin II on cells from this tissue are only beginning to be understood. In this study, we examined the effects of angiotensin II on changes in lipid accumulation, specific glycerol-3-phosphate dehydrogenase activity, and the expression of five genes of the renin-angiotensin system during the adipose conversion of human primary cultured preadipocytes. Angiotensin II leads to a distinct reduction in insulin-induced differentiation, but only has a marginal effect on the adipose conversion of cells stimulated with insulin, cortisol, and isobutyl methyl xanthine. During differentiation, angiotensinogen mRNA levels rise, renin mRNA levels decline, whereas renin-binding protein and angiotensin-converting enzyme levels are unaffected. Angiotensin II downregulates angiotensinogen and renin gene expression, but it does not affect renin-binding protein and angiotensin-converting enzyme levels. Angiotensin II thus prevents the development of adipocytes in contact with high insulin levels, while not inhibiting differentiation, which is further stimulated. Therefore, angiotensin II could be a protective factor against uncontrolled expansion of adipose tissue. Further studies are needed to find out whether the effects of angiotensin II on the renin-angiotensin system are direct feedback loops or secondary to changes in the differentiation program.

Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells.

Therapeutic differentiation of tumor-derived insulin-producing cells selected for resistance to diabetogenic drugs.

Differentiation therapy has been proposed as a new approach to selectively engage the process of tumor cell differentiation during chemotherapy of cancer. Our recent in vitro study suggests that such an approach can be extended and utilized for the selection of tumor-derived insulin-producing cells for transplantation. Repeated treatment with streptozotocin selected toxin resistant subpopulation of insulin producing tumor RINmS cells, characterized increased level of insulin content and secretion. In the present study RINmS cells were found to have higher glucose sensitivity and insulin response compared with parental RINm cells. In addition, compounds known to induce elevated level of cAMP beta-cells, such as isobutyl methyl xanthine, and forskolin, potentiated glucose-induced insulin secretion of RINmS, but had no effect on the naive parental RINm cells. These experiments suggest that differentiation therapy can be utilized for engineering insulin producing cells with improved defense and secretory mechanisms.

Growing colorectal tumors: minimizing microbial and stromal competition and assessing in vitro selection pressures.

Development of primary colorectal cancer cell lines ishampered by contamination from regional microbes, overgrowthof stromal cells, and purported genetic drift from selectionpressures in vitro. We initiated 32 primaryadenocarcinomas, 3 recurrences and 6 distant metastases incell culture. Twelve cell lines from eleven tumors weregenerated (26.8%) overall. Nine of 32 primary tumorsyielded 10 cell lines, 5 were lost to contamination, 13 wereoverwhelmed by stromal cells, and 5 demonstrated no growth.Addition of isobutyl methyl xanthine (IBMX) to culturelimited fibroblastoid growth. There was no associationbetween tumor location (p = 0.535, mid-P), degree ofdifferentiation (p = 0.850, mid-P) or clinicopathologic stage(p = 0.400, mid-P), and the ability of cells to becomeestablished in culture. The majority of cell lines hadsimilar nuclear DNA content and expression of cell-surfaceantigens compared with their parent tumors. Microbialcontamination and stromal cell overgrowth present thegreatest obstacle to capturing a representative bank ofcolon tumors in vitro.

Functional analysis of desensitization of the beta-adrenoceptor signalling pathway in rat cardiac tissues following chronic isoprenaline infusion.

1. This study examined beta-adrenoceptor signalling in cardiac tissues following infusion of isoprenaline (400 microg kg(-1) h(-1)) or vehicle to rats for 14 days. 2. Isoprenaline infusion caused marked hypertrophy of atria and ventricles and reduced the resting rate of spontaneously beating right atria and the basal force of left atrial contraction. 3. In spontaneously beating right atria, concentration-response curves to isoprenaline and forskolin were shifted 7.9 and 3.2 fold to the right following treatment whereas responses to the cyclic AMP analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphorothioate were unchanged. 4. In electrically driven left atria, concentration-response curves to isoprenaline and forskolin were shifted 4 fold to the right and maximum responses reduced. Responses to dibutyryl cyclic AMP were shifted 3.2 fold to the right but those to Ca2+ were unchanged. 5. Inotropic responses of left and right ventricular papillary muscles to isoprenaline were abolished and markedly reduced respectively by isoprenaline treatment. Responses to forskolin were shifted 5 fold to the right. Responses to dibutyryl cyclic AMP were shifted to the right 3.2 and 2 fold in left and right ventricular papillary muscles. Responses to isobutyl methyl xanthine were shifted to the right 15.8 and 6.3 fold in left and right papillary muscles whereas those to Ca2+ were not significantly altered. 6. This study indicates differences in beta-adrenoceptor desensitization in different regions of the heart following chronic infusion of isoprenaline. Chronotropic responses showed impaired signalling between the receptor and adenylate cyclase whereas inotropic responses exhibited additional desensitization at the level of cyclic AMP dependent protein kinase.

Effects of acute and repeated dose administration of caffeine and pentoxifylline on diazepam-induced mouse behavior in the hole-board test.

The behavioral effects of methyl xanthines and their interactions with benzodiazepines have not been clearly established in animal models of anxiety. The present study extended the previous studies to determine the effects of acute and repeated administration of caffeine, a non-specific phosphodiesterase (PDE) inhibitor and pentoxyfylline, a specific type-4 phosphodiesterase (PDE4) inhibitor on (1) baseline anxiety-like behavior and (2) the response to an acute challenge with diazepam on anxiety-like behavior in the hole-board test. Mice were observed for the number of head-dips they made into the holes of the hole-board apparatus during a 5-min period, starting 30 min after acute (20 mg/kg) and repeated oral dose (20 mg/kg, twice a day for 4 days) administration of caffeine and pentoxifylline. In separate experiments, the response to an acute challenge with graded doses of diazepam (0.375 3 mg/kg, SC) was observed in naive mice or mice on acute and repeated dose regimen with methyl xanthines. Mice on acute but not after repeated dose regimen demonstrated a significantly increased number of hole-dips, indicating an anxiolytic-like effect of methylxanthines. Diazepam at the lower doses (0.375 and 0.75 mg/kg) but not at the highest doses (1.5 and 3 mg/kg) examined produced a significant anxiolytic-like effect. After an acute dose exposure of mice to caffeine and pentoxifylline, a rightward shift in the dose-response curve of diazepam was observed and particularly at 1.5 mg/kg dose, the net effect of diazepam was significantly enhanced which was, however, impaired upon repeated administration, more so with caffeine than with pentoxifylline. It is concluded that the xanthine drugs exert anxiolytic-like activity similar to diazepam in the hole-board test. In addition, they seem to modulate the anxiolytic effects of diazepam after both acute and repeated administration, probably as a result of an endogenous adenosinergic mechanism which may have therapeutic significance.

Effects of the aqueous fraction of the ethanol extract of the leaves of Cissampelos sympodialis Eichl. in human neutrophils.

An aqueous fraction (10-300 micrograms/mL) of the ethanol extract of the leaves of Cissampelos sympodialis Eichl inhibited N-formyl-Met-Leu-Phe (fMLP)-induced release of lysozyme and myeloperoxidase from human neutrophils. Inhibition by the fraction, as well as by dibutyryl-cAMP and prostaglandin E2, was substantially greater when the cells were pretreated with the phosphodiesterase (PDE) inhibitor isobutyl methyl xanthine (IBMX) indicating that the effect may be mediated by cAMP. Measurement of intracellular cAMP levels showed that the fraction (30-100 micrograms/mL) increased the nucleotide levels in IBMX-pretreated neutrophils which was unaffected by propranolol. Cyclic AMP dependent protein kinase A activity was also increased by the fraction (1.5-100 micrograms/mL). Superoxide anion generation induced by fMLP in cytochalasin B-treated cells primed with PAF was not inhibited by the aqueous fraction. The results indicate that the aqueous fraction of Cissampelos sympodialis inhibits neutrophil degranulation by a cAMP-dependent mechanism which may be relevant to the use of the plant as an anti-asthmatic agent in folk medicine.

Upregulation of angiotensin converting enzyme by atrial natriuretic peptide and cyclic GMP in human endothelial cells.

To examine the role of atrial natriuretic peptide (ANP) and cyclic GMP in the regulation of angiotensin converting enzyme (ACE) in cultured human endothelial cells. Cultured endothelial cells from human umbilical veins (HUVEC) were treated with ANP (0.3-30 nM), 8-Br-cGMP (1-100 microM), Rp-8-Br-PET-cGMPS (1 microM), or the phosphodiesterase inhibitors, zaprinast (10-100 microM), dipyridamole (1-10 microM), or isobutyl methyl xanthine (IBMX, 0.1-0.5 mM). ACE amounts were measured by inhibitor binding assay and cellular cGMP levels by radioimmunoassay. ANP caused a dose dependent increase in ACE measured in intact endothelial cell culture. The stimulatory effect of ANP was blocked by Rp-8-Br-PET-cGMPS, a protein kinase G inhibitor. The cyclic GMP analog, 8-Br-cGMP and the cyclic GMP specific phosphodiesterase inhibitor, zaprinast, both increased ACE. Increase of ACE was also caused by nonspecific phosphodiesterase inhibitors, dipyridamole and IBMX. Intracellular cGMP levels were shown to increase by ANP, and phosphodiesterase inhibitors. These data suggest that cGMP is an intracellular mediator regulating ACE and that ANP induced increase of ACE is mediated via a cGMP dependent mechanism.

Histochemical, clinical, and in vitro β cell responses in a neonate with persistent hyperinsulinaemic hypoglycaemia of infancy

When treatment with diazoxide and somatostatin for persistent hyperinsulinaemic hypoglycaemia of infancy failed, subtotal pancreatectomy was performed on a neonate on day 41. The pancreatic tissue was saved and used...

Synthesis of crown containing imidazo[4,5-e] and-[5,4-e][1,4]diazepines

Starting from benzocrown substituted 4(5)-aminoimidazole-5(4)-carboxamides we have, for the first time, prepared benzocrown imidazo[4,5-e]-and-[5,4-e][1,4]diazepines-cyclic homologs of the corresponding xanthines.

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.

1. On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. 2. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. 5. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. 6. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

Do you know your chocolates? Recognition survey among medical staff of various grades

Gifts of chocolates are common on hospital wards. Preregistration house officers have a massive exposure to a wide range and quantity of chocolates—one of the many early and challenging experiences...

CAMP and Ca2+Involvement in the Mitochondrial Response of Cultured Fetal Rat Hepatocytes to Adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both α- and β-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Pentoxifylline inhibits human T-cell adhesion to dermal endothelial cells.

Pentoxifylline (PTX), a methyl xanthine derivative with phosphodiesterase inhibitory activity, has been shown to have antiinflammatory effects. Previous studies have demonstrated that PTX can suppress TNF alpha production and function, and can inhibit the adhesion of neutrophils and monocytes to endothelial cells. In the present study, we sought to determine whether PTX also interferes with the adhesion of human peripheral blood T lymphocytes to cells of the human dermal endothelial cell line HMEC-1. Using a cell adhesion immunoassay, the effect of different doses of PTX (10(-5)-10(-2) M) on the binding of unactivated or PMA-activated T cells to unstimulated or TNF alpha-stimulated endothelial cells was investigated. In addition, blocking experiments with monoclonal antibodies against pairs of adhesion molecules known to be involved in endothelial cell/T-cell adhesion were performed. Unactivated T cells showed minimal adhesion to unstimulated endothelial cells. PMA-activated T cells showed an eightfold increased binding to TNF alpha-stimulated endothelial cells, which was found to be mediated largely by LFA-1/ICAM-1. PTX inhibited the binding of PMA-activated T cells to TNF alpha-stimulated endothelial cells in a dose-dependent manner. This inhibition was only found when PTX was present during the adhesion assay. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methyl xanthine derivative, or by a combination of two cAMP analogues. The results suggest that interference with T-cell/endothelial cell adhesion, which forms an essential step in the migration of T cells from the peripheral blood into sites of inflammation, may be another explanation for the beneficial effect of PTX in several inflammatory dermatoses.

Induced melanin reduces mutations and cell killing in mouse melanoma.

When melanin absorbs light energy, it can produce potentially damaging active oxygen species. There is little doubt that constitutive pigment in dark-skinned individuals is photoprotective against skin cancer, but induced pigment-as in tanning-may not be. The first step in cancer induction is mutation in DNA. The most suitable systems for evaluating the role of melanin are those in which pigment can be varied and mutations can be measured. Several cell lines from Cloudman S91 mouse melanoma can be induced to form large quantities of melanin pigment after treatment with a number of different agents enabling comparison of mutant yields in the same cells differing principally in pigment concentration. In these studies, melanin was induced with synthetic alpha-melanocyte-stimulating hormone and with isobutyl methyl xanthine in the cell line S91/mel. The former inducer produced about 50% more pigment than the latter. Survival and mutation induction at the Na+/K(+)-ATPase locus were studied using ethyl methane sulfonate (EMS), a standard mutagen and five UV lamps emitting near monochromatic and polychromatic UV light in the three wave-length ranges of UV. There was greater protection against killing and mutation induction in the more heavily pigmented cells after exposure to EMS and after irradiation with monochromatic UVC and UVB. There was significant protection against killing by polychromatic UVB + UVA (FS20), but the small degree of protection against mutation was not significant. No significant change in killing and mutation using the same protocol was seen in S91/amel, a related cell line that does not respond to these inducers. No mutants were produced by either monochromatic or polychromatic UVA at doses that killed 50% of the cells. Our results show that induced pigment-shown earlier to be eumelanin (K. A. Cieszka et al., Exp. Dermatol. 4, 192-198, 1995)-is photo- and chemoprotective, but it is less effective in protection against mutagenesis by polychromatic UVB + UVA in a spectrum that more nearly approximates the solar spectrum.

C-Rel Is a Target of Pentoxifylline-Mediated Inhibition of T Lymphocyte Activation

The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell–dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-κB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2Rα chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes.

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.

Regulation of somatostatin gene transcription by cyclic adenosine monophosphate

Cyclic adenosine monophosphate (cAMP) stimulates transcription of somatostatin and other target genes with burstattenuation kinetics. The kinetics of protein kinase (PK-A)—dependent cAMP response element binding protein (CREB) phosphorylation closely parallel the changes in transcription of cAMP-responsive genes by run-on assay. Nuclear translocation of PK-A, visualized by microinjection of fluorescently labeled PK-A holoenzyme, appears to represent the rate-limiting step in CREB phosphorylation and transcriptional activation. We and others have recently characterized a CREB-binding protein (CBP), which specifically recognizes sequences within the Ser133 phosphorylated form of CREB. CBP does not regulate the DNA binding, dimerization, or nuclear targeting properties of CREB, but binds selectively to the kinase-inducible 60 amino acid trans-activation domain (KID) of CREB, critical for PK-A—inducible transcription. We developed an antiserum directed against amino acid 634–648 within the CREB-binding domain of CBP. We detected a 265-kd polypeptide by Western blot as predicted from the cDNA, which coincided with the predominant phospho-CREB—binding activity in Hela nuclear extracts by “Far Western” blot assay. An identical phospho-CREB—binding activity was also found in NIH-3T3 cells. This phospho-CREB—binding protein appeared to be specific for Ser133-phosphorylated CREB, because no such band was detected with CREB labeled to the same specific activity at a nonregulatory phosphoacceptor site (Ser156) by casein kinase II (CKII). Following microinjection into nuclei of NIH-3T3 cells, a cAMP response element (CRE)-lacZ reporter was markedly induced by treatment with 8-Br cAMP plus isobutyl methyl xanthine (IBMX). Coinjection of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP-dependent activity in a dose-dependent manner, but control immunoglobulin G (IgG) had no effect on this response. We can now begin reconstituting PK-A—dependent transcription in vitro, using well-characterized proteins such as CREB, TAF 110, and CBP. The assembly of such factors on cAMP-regulated promoters like somatostatin may enable responsiveness to a variety of hormonal stimuli that employ cAMP as their second messenger.