AMP Synthesis Research Articles

Fatty Acid Ethyl Ester Effects on Interleukin‐2 Production, Cyclic AMP Synthesis, and Calcium Influx in Human Mononuclear Cells

Fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, are produced by the esterification of fatty acids and ethanol. Plasma and serum FAEE correlate linearly with blood ethanol levels and are present in organs most commonly damaged by ethanol abuse. Our previous studies have shown that there is significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol. We studied the effects of FAEE on selected early events of mononuclear cell activation that follow stimulation with phytohemagglutinin (PHA). Fatty acid ethyl esters induced changes in cytosolic free calcium ([Ca2+]) levels, the production and secretion of interleukin-2 (IL-2) by the cells, and intracellular cAMP concentrations. A mononuclear fraction of human white blood cells (WBC) was incubated with physiologic doses of FAEE. For experiments involving IL-2 production and calcium influx, PHA-stimulated cells were incubated with 10, 25, 50, or 100 microM ethyl oleate, a representative FAEE species, for 60 minutes. Interleukin-2 was measured by enzyme immunometeric assay and maximum levels of Ca2+ were monitored by spectrofluorimetry. In other experiments, mononuclear cells were incubated with 10, 25, and 50 microM ethyl oleate for 0.08 to 120 minutes, and then the concentration of cAMP was determined by a cAMP competitive enzyme immunoassay system. Fatty acid ethyl esters inhibited the PHA-induced IL-2 production and secretion in activated human mononuclear cells. Fatty acid ethyl esters also inhibited PHA-induced [Ca2+] influx into cells in a dose-dependent fashion. There was a rapid increase in the intracellular cAMP concentration of mononuclear cells induced by FAEE, with FAEE dose dependence. The cAMP concentration decreased as the incubation time with FAEE was increased. Fatty acid ethyl esters inhibited PHA-stimulated IL-2 production and Ca2+ influx into human mononuclear cells and elevated intracellular cAMP concentration. These changes in mononuclear cell signaling may be associated with the immunosuppression associated with alcohol abuse.

Regulation of vascular smooth muscle cell function by adenylyl cyclase isoforms

Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. Regulation patterns differ between the AC isoforms, however the consequences of activation of any individual AC isoform (e.g., to catalyze the synthesis of cAMP) were thought to be common to all isoforms. To examine this hypothesis we studied the effect of adenoviral gene transfer of representative AC isoforms AC I, II, V and VI on whole cell cAMP activity, regulation of vascular smooth muscle cell proliferation, isoproterenol (ISO)-mediated arborization responses (an index of cytoskeletal re-organization) and protein kinase A (PKA) activity Although all AC isoforms when expressed in smooth muscle cells comparably enhanced forskolin (FSK)-stimulated cAMP synthesis (186–208% of the AC activity in GFP-infected cells), only AC I-infected cells demonstrated an enhancement of FSK-stimulated inhibition of proliferation (19±3% of GFP, p<0.01). Only gene transfer of AC VI enhanced ISO-mediated arborization (155±16% of GFP, p<0.05). To further examine the contribution of AC VI to the arborization response, we utilized a “knockdown” approach based on expression of a truncated-AC VI dominant negative mutant (TM1T). TM1T expression significantly reduced FSK-stimulated AC activation (79±8% of GFP alone) and ISO-mediated arborization (54±6% of GFP alone). This reduction in arborization was recovered by the co-expression of AC isoform VI (84±10%). Lastly, FSK-stimulated PKA activity was significantly reduced (61±5% of GFP) by AC I expression and significantly enhanced (132±8% of GFP) by AC VI expression. In aggregate these data support a previously unappreciated role for specific AC isoforms in directing the effect of elevation of intracellular cAMP in the regulation of vascular function. Supported by CIHR.

EFA (9-β-D-erythrofuranosyladenine) is an effective salvage agent for methylthioadenosine phosphorylase-selective therapy of T-cell acute lymphoblastic leukemia with L-alanosine

AbstractThe deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5′deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-β-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 μM with negligible toxicity even at 100 μM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 μM, but higher concentrations were toxic. EFA at 20 μM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.

Effects of Hyaluronic Acid on Ca&lt;sup&gt;2+&lt;/sup&gt; Influx, Lactate Dehydrogenase Activity, and Cyclic AMP Synthesis in Canine Ejaculated Sperm during &lt;i&gt;In Vitro&lt;/i&gt; Capacitation

The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca(2+) influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 microg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca(2+) indicator (Ca(2+)-labeled sperm) were evaluated to assess Ca(2+) influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca(2+)-labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). The percentages of HA-sperm correlated with those of Ca(2+)-labeled sperm (r(2)=0.810). The results indicate that hyaluronic acid increases Ca(2+) influx, LDH activity, and cAMP synthesis in canine ejaculated sperm during capacitation in vitro.

Accelerated Bone Turnover in Pregnant Women with McCune-Albright Syndrome

Bone turnover in pregnant women with McCune-Albright syndrome may be affected by both the syndrome and pregnancy. This study evaluated changes in biochemical bone turnover markers in pregnant women with the syndrome. Serum calcium, phosphorus, 1,25-dihydroxyvitamin D (1,25-(OH)₂D), intact osteocalcin (I-OC) and alkaline phosphatase (ALP), and urinary pyridinoline (Pyr), deoxypyridinoline (D-Pyr) and hydroxyproline (HPR) were measured during pregnancy and postpartum in 2 women with McCune-Albright syndrome. Serum calcitonin (CT), and plasma intact parathyroid hormone (I-PTH) and parathyroid hormone-related protein (PTHrP) were also measured in 1 patient. Serum corrected Ca levels were normal or low-normal; phosphorus levels were normal, and 1,25-(OH)₂D levels increased toward term and decreased thereafter, similar to normal pregnant women. Urinary Pyr, D-Pyr and HPR were elevated during pregnancy compared to normal pregnant women, peaked just after delivery, and decreased thereafter. Serum I-OC and ALP levels were high during pregnancy and postpartum. Intact PTH levels were increased during pregnancy and postpartum compared to normal pregnant women, whereas serum CT and PTHrP levels were not elevated. Both bone formation and absorption appear to be more enhanced during pregnancy and postpartum in women with McCune-Albright syndrome than in normal pregnant women. Additional or amplified cyclic AMP synthesis in bone cells through activation of the α subunit of G protein, independent of hormonal control, may explain the high local bone turnover.

Receptor Signaling and Endocytosis Are Differentially Regulated by Somatostatin Analogs

Upon hormone stimulation, the sst2 somatostatin receptor couples to adenylyl cyclase through G(i/o) proteins and undergoes rapid endocytosis via clathrin-coated pits. In this study, we determined the relationship between the ability of ligands to induce sst2 receptor internalization and inhibit adenylyl cyclase. Immunocytochemical studies demonstrated that peptide agonists [such as somatostatin-14, cortistatin-17, octreotide, vapreotide, KE108 (Tyr0-cyclo[d-diaminobutyric acid-Arg-Phe-Phe-d-Trp-Lys-Thr-Phe]), and SOM230 (cyclo[diaminoethylcarbamoyl-hydroxyproline-phenylglycine-d-Trp-Lys-(4-O-benzyl)-l-Tyr-Phe])] and nonpeptide agonists (such as L-779,976), stimulated the rapid endocytosis of sst2 receptors in human embryonic kidney 293 and CHO-K1 cells. In contrast, two antagonists did not induce receptor endocytosis by themselves and completely blocked agonist stimulation. Using a quantitative enzyme-linked immunosorbent assay to measure sst2 receptor sequestration, we found that peptide agonists varied by more than 100-fold in their potencies but exhibited the same efficacy as somatostatin14. In contrast, L-779,976 did not induce maximal receptor internalization. It is interesting that although betaarrestin-2 was recruited to cell surface sst2 receptors after stimulation with either somatostatin14 or L-779,976, the betaarrestin-receptor complex dissociated earlier in the endocytic pathway with the nonpeptide ligand. Although all agonists, including L-779,976, produced the same maximal inhibition of cyclic AMP, the potency ratio for inhibition of cyclic AMP and stimulation of receptor endocytosis varied by 15-fold. In general, native peptides showed similar potencies for cyclic AMP inhibition and receptor endocytosis, whereas short therapeutic analogs were substantially more potent at inhibiting cyclic AMP synthesis. These results demonstrate that the activity of somatostatin analogs to regulate receptor endocytosis and signaling are not tightly linked and provide compelling evidence for the induction of agonist specific states of the sst2 receptor.

Implications of P16/CDKN2A deletion in pleural mesotheliomas

Homozygous deletion of P16/CDKN2A is found in approximately 75% of mesotheliomas amd may be the most common genetic alteration in this cancer. In terms of diagnostic applications, its high prevalence makes it a useful marker to distinguish malignant mesothelial cells from benign reactive ones in pleural fluid cytologic preparations. In terms of prognosis, P16/CDKN2A loss is associated with more aggressive clinical behavior in mesotheliomas. The homozygous co-deletion of MTAP, encoding the enzyme methylthioadenosine phosphorylase, in approximately 90% of mesotheliomas with P16/CDKN2A loss has potential therapeutic applications because MTAP-deficient tumors may be responsive to inhibitors of de novo AMP synthesis. Finally, global gene expression profiling using Affymetrix U133A chips finds few gene expression correlates of P16/CDKN2A deletion in pleural mesothelioma, consistent with its non-transcriptional mode of direct action through regulation of cell cycle-related kinase signaling.

Cyclic AMP (cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 inEscherichia coli

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for "luxS regulated") operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.

Structural Basis for the Inhibition of Mammalian Membrane Adenylyl Cyclase by 2 ′(3′)-O-(N-Methylanthraniloyl)-guanosine 5 ′-Triphosphate

Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein alpha subunits (Galpha(s)) and by forskolin (FSK). mACs are inhibited with high potency by 2 '(3')-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Galpha(s).GTPgammaS and the catalytic C1 and C2 domains from type V and type II mAC (VC1.IIC2), bound to FSK and either MANT-GTP.Mg(2+) or MANT-GTP.Mn(2+) have been determined. MANT-GTP coordinates two metal ions and occupies the same position in the catalytic site as P-site inhibitors and substrate analogs. However, the orientation of the guanine ring is reversed relative to that of the adenine ring. The MANT fluorophore resides in a hydrophobic pocket at the interface between the VC1 and IIC2 domains and prevents mAC from undergoing the "open" to "closed" domain rearrangement. The K(i) of MANT-GTP for inhibition of VC1.IIC2 is lower in the presence of mAC activators and lower in the presence of Mn(2+) compared with Mg(2+), indicating that the inhibitor binds more tightly to the catalytically most active form of the enzyme. Fluorescence resonance energy transfer-stimulated emission from the MANT fluorophore upon excitation of Trp-1020 in the MANT-binding pocket of IIC2 is also stronger in the presence of FSK. Mutational analysis of two non-conserved amino acids in the MANT-binding pocket suggests that residues outside of the binding site influence isoform selectivity toward MANT-GTP.

Molecular cloning and characterization of a novel muscle adenylosuccinate synthetase, AdSSL1, from human bone marrow stromal cells

Vertebrates have muscle and non-muscle isozymes of adenylosuccinate synthetase (AdSS, EC 6.3.4.4), which catalyzes the first committed step in AMP synthesis. A novel muscle isozyme of adenylosuccinate synthetase, human AdSSL1, is identified from human bone marrow stromal cells. AdSSL1 is 98% identical to mouse muscle type AdSS1 and contains conserved sequence and structural features of adenylosuccinate synthetase. Human AdSSL1 gene is mapped to chromosome 14p32.33. After stimulation, leukemia cells express AdSSL1 in a time-dependent manner different from that of non-muscle adenylosuccinate synthetase. The human AdSSL1 is predominantly expressed in skeletal muscle and cardiac tissue consistent with the potential role for the enzyme in muscle metabolism. Overexpressed AdSSL1 protein in COS-7 cells locates in cytoplasm. Recombinant AdSSL1 protein possesses typical enzymatic activity to catalyze adenylosuccinate formation. The identification of human AdSSL1 with predominant expression in muscle tissue will facilitate future genetic and biochemical analysis of the enzyme in muscle physiology.

Receptors for VIP and PACAP in Guinea Pig Cerebral Cortex: Effects on Cyclic AMP Synthesis and Characterization by &lt;SUP&gt;125&lt;/SUP&gt;I-VIP Binding

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Raf Kinase Activation of Adenylyl Cyclases: Isoform-Selective Regulation

Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins; however, recently we identified an alternate mechanism by which growth factors sensitize adenylyl cyclase activation. We suggested that this mechanism might involve a Raf kinase-mediated serine phosphorylation of adenylyl cyclase. However, the direct involvement of a specific form of Raf kinase is yet to be demonstrated. Furthermore, whether this mechanism is generalized to other isoforms of adenylyl cyclase is unknown. In human embryonic kidney 293 cells, we now demonstrate that in reconstitution studies, c-Raf kinase can mediate phosphorylation of AC VI. Furthermore, AC VI coimmunoprecipitates with c-Raf. Raf kinase-dependent regulation of adenylyl cyclase VI is dependent on the integrity of Ser750 in the fourth intracellular loop of the enzyme and Ser603/Ser608 in the C1b region of the molecule. To examine how generalized this effect is, we studied representative isoforms of the major subfamilies of adenylyl cyclase viz., AC I, AC II, and AC V. Raf kinase-dependent sensitization/ phosphorylation of adenylyl cyclases is common to AC VI, AC V, and AC II isoforms but not AC I. In aggregate, these studies indicate that Raf kinase associates with adenylyl cyclases. Furthermore, Raf kinase regulation of adenylyl cyclase is isoform-selective. These functional interactions (as well as the physical association) between adenylyl cyclases and Raf kinases suggest an important but previously unrecognized interaction between these two key regulatory enzymes.

Dihydrexidine — The First Full Dopamine D1 Receptor Agonist

The functional role of dopamine D(1) receptors is still controversial. One reason for this controversy is that for a long time the only available agonists for in vivo characterization of dopamine D(1) receptors were benzazepines. Among them was the prototype dopamine D(1) receptor partial agonist, SKF 38393. The lack of a selective and fully efficacious dopamine D(1) receptor agonist hampered basic research on dopamine D(1) receptors and left the potential clinical utility of dopamine D(1) receptor agonists elusive. The research situation improved when the first potent full dopamine D(1) receptor agonist dihydrexidine, a phenanthridine, was introduced in the late 1980s. In contrast to SKF 38393, dihydrexidine was shown to stimulate cyclic AMP synthesis just as well or better than dopamine, and potently displaced [(3)H]SCH 23390 from rat and monkey striatal membranes. Also, dihydrexidine was the first dopamine D(1) receptor agonist that had potent antiparkinsonian activity in a primate model of Parkinson's disease. This finding suggested clinical utility for dopamine D(1) receptor agonists in Parkinson's disease and that this utility might be critically dependent on the intrinsic efficacy of the drug. Clinical utility for dopamine D(1) receptor agonists in other central nervous disorders might also be dependent on the intrinsic efficacy of the drug. However, even though studies with dihydrexidine as a pharmacological tool have pointed to the clinical use for dopamine D(1) receptor agonists, dihydrexidine's unfavorable pharmacokinetic profile and various adverse effects are likely to restrict or even preclude its use in humans. This review article provides an updated overview of the pharmacology of dihydrexidine and discusses possible clinical utility of dopamine D(1) receptor agonists in various central nervous system disorders.

Efficacy of a mitochondrial toxin, F16, in a multi-ATP-depleting regimen + chemotherapy on human breast cancer (MDA-MB-468) xenografts

2096 Background: : Chemotherapy is enhanced by multi-ATP-depleting therapy (Cancer Res. 60: 6776, 2000). Malignant tumors generally increase ATP production to increase cell division. In general, tumors make and consume (deplete) more ATP than normal tissues. If ATP production is equally inhibited in normal and tumor tissues, high ATP consumption (depletion) in tumors should deplete ATP to cytocidal levels, whereas the lower ATP consumption in normal cells should reduce ATP to tolerable levels. This rationale requires multiple ATP inhibitors inhibiting multiple biochemical pathways that produce ATP. Methods: Alanosine (AL), inhibitor of AMP synthesis, + 6-methylmercaptopurine riboside (MMPR), inhibitor of purine synthesis, preceded by PALA, a pyrimidine inhibitor; acronym, PALM. F16 blocks ATP synthesis by selectively accumulating in mitochondria of responsive breast cancers (Cancer Cell 2:29, 2002). Results: F16 alone was not effective. Taxotere alone (TXT), at MTD40, affects 100% PR of tumors. The addition of PALM (P100 AL250 M150), or PALM + F16 10, effects the same 100% P.R. at half the TXT dose (TXT20), suggesting clinical benefit at reduced normal tissue toxicities. Follow-up (120 days) demonstrates substantial tumor recurrence (43–50%) in Groups 1 and 2, and minimal recurrence (17%) in Group 3. Conclusions: A multi-ATP-depleting regimen (PALM + F16) + chemotherapy augments tumoricidal capability. Chemotherapy is at markedly lower doses, strongly suggesting lower toxic side-effects in the clinic. No significant financial relationships to disclose.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by 6125I9-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a Kd of 0.70 nM and a Bmax of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [125I]-VIP.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by [ 125I]-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [ 125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [ 125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a K d of 0.70 nM and a B max of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [ 125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [ 125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [ 125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC 50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [ 125I]-VIP.

Crystal Structure of Fully Ligated Adenylosuccinate Synthetase from Plasmodium falciparum

In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg 2+ and the aspartate analogue, hadacidin at 2 Å resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.

Modeling β-Adrenergic Control of Cardiac Myocyte Contractility in Silico

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans.

Frequent interconversion between yeasts, pseudohyphae and true hyphae is a hallmark of Candida albicans growth in mammalian tissues. The requirement for transient CAP1-dependent pulses of cAMP for generating true hyphae, Hwp1 and virulence raises questions about the role of yeast and pseudohyphal forms in the pathogenesis of candidiasis. In this study, hyperfilamentous mutants, limited in their capacity to produce buds, were generated by disrupting the high-affinity phosphodiesterase gene PDE2. Degradation of cAMP by the PDE2 gene product was confirmed by higher basal cAMP levels in the pde2/pde2 mutant and by accumulation of cAMP to levels permitting germ tube formation upon disrupting PDE2 in the cap1/cap1 mutant. Similar phenotypes of the C. albicans and Saccharomyces cerevisiae pde2/pde2 mutants were found, including sensitivity to nutritional starvation and exogenous cAMP and defective entry into stationary phase. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis. Growth in a multiplicity of forms appears to be a virulence attribute that is controlled by tight coupling of cAMP synthesis and degradation. Delayed increases in PDE2 mRNA in cAMP-deficient cap1/cap1 mutants during germ tube-inducing conditions suggested a mechanism of control involving cAMP-dependent induction of PDE2 mRNA.

Synthesis and characterization of the first fluorescent antagonists for human 5-HT4 receptors.

Fluorescent antagonists for human 5-HT(4) receptors were synthesized based on ML10302 1, a potent 5-HT(4) receptor agonist and on piperazine analogue 2. These molecules were derived with three fluorescent moieties, dansyl, naphthalimide, and NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), through alkyl chains. The synthesized molecules were evaluated in binding assays on the recently cloned human 5-HT(4(e)) receptor isoform stably expressed in C6 glial cells with [(3)H]GR113808 as the radioligand. The affinity values depended upon the basal structure together with the alkyl chain length. The derivatives based on ML10302 were more potent ligands than the derivatives based on piperazine analogue. For ML10302-based ligands, dansyl and NBD derivatives attached through a chain length of one carbon atom 17a and 32, respectively, led to affinities close to the affinity of ML10302. The most potent compounds 17a, 28, and 32 produced an inhibition of the 5-HT stimulated cyclic AMP synthesis in the same cellular system with nanomolar K(b) values. Fluorescent properties of 17a, 28, and 32 were more particularly studied. Interactions of the fluorescent ligand 28 with the h5-HT(4(e)) receptor were indicated using h5-HT(4(e)) receptor transfected C6 glial cell membranes and entire cells. Ligand 28 was also used in fluorescence microscopy experiments in order to label h5-HT(4(e)) receptor transfected C6 glial cells, and subcellular localization of these receptors was more precisely determined using confocal microscopy.