Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

Modulation of thapsigargin-induced calcium mobilisation by cyclic AMP-elevating agents in human lymphocytes is insensitive to the action of the protein kinase A inhibitor H-89

Ca2+ mobilisation from internal stores and from the extracellular medium is one of the primary events involved in lymphocyte activation and proliferation. Regulation of these processes by adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) was studied in Fura2-loaded human peripheral blood lymphocytes. Cytosolic Ca2+ concentration ([Ca2+]i) was measured in single cells by the use of a ratio imaging fluorescence microscope and Ca2+ mobilisation was achieved by the use of the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, thapsigargin (Thg). Our results show that both activation and inhibition of PKA, with forskolin (FSK) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89), respectively, inhibited the Thg-induced Ca2+ entry. Furthermore, FSK also reduced the ability of Thg to release Ca2+ from internal stores. This reduction was inhibited by the adenylyl cyclase (AC) inhibitor 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine (SQ22,536), but not by the PKA inhibitor H89, indicating that cAMP but not PKA is responsible for this effect. FSK effect was mimicked by dibutyryl cAMP (dbcAMP) and by inhibition of phosphodiesterases (PDEs) with rolipram (ROL) and milrinone (MIL). We also showed that a very high concentration of H-89 (100 microM) releases Ca2+ from an intracellular pool, although this action is probably independent of PKA inhibition. Neither 10 microM H-89 nor other cAMP/PKA-modulating drugs had any effect on the basal [Ca2+]i of human lymphocytes. We conclude that PKA may act as a fine modulator of capacitative Ca2+ entry, while cAMP has a PKA-independent interaction with the Ca2+ stores of human lymphocytes.     

Type 5 adenylyl cyclase plays a major role in stabilizing heart rate in response to microgravity induced by parabolic flight.

It is well known that autonomic nervous activity is altered under microgravity, leading to disturbed regulation of cardiac function, such as heart rate. Autonomic regulation of the heart is mostly determined by beta-adrenergic receptors/cAMP signal, which is produced by adenylyl cyclase, in cardiac myocytes. To examine a hypothesis that a major cardiac isoform, type 5 adenylyl cyclase (AC5), plays an important role in regulating heart rate during parabolic flights, we used transgenic mouse models with either disrupted (AC5KO) or overexpressed AC5 in the heart (AC5TG) and analyzed heart rate variability. Heart rate had a tendency to decrease gradually in later phases within one parabola in each genotype group, but the magnitude of decrease was smaller in AC5KO than that in the other groups. The inverse of heart rate, i.e., the R-R interval, was much more variable in AC5KO and less variable in AC5TG than that in wild-type controls. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater in microgravity phase in each genotype group, but the magnitude of increase was much greater in AC5KO than that in the other groups, suggesting that heart rate regulation became unstable in the absence of AC5. In all, AC5 plays a major role in stabilizing heat rate under microgravity.     

Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase

Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-β-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF.     

Cyclooxygenase-2 and adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present experiment, we tested whether or not COX-2 and adenylate cyclase/protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Angiogenesis was enhanced by topical injection of human recombinant basic fibroblast growth factor (bFGF). The enhanced angiogenesis by bFGF was inhibited by indomethacin or selective COX-2 inhibitors, NS398, nimesulide, and JTE-522. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis in a dose-dependent manner, as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by bFGF, 8-bromo-cAMP, forskolin, and amrinone. Angiogenesis was inhibited by indometacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. In addition, angiogenesis without topical injections of the above compounds was also suppressed by SQ22,536, an inhibitor for AC. or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. These results suggested that COX-2 and AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis, and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.     

Type 5 adenylyl cyclase disruption leads to enhanced exercise performance

The most important physiological mechanism mediating enhanced exercise performance is increased sympathetic, beta adrenergic receptor (β‐AR), and adenylyl cyclase (AC) activity. This is the first report of decreased AC activity mediating increased exercise performance. We demonstrated that AC5 disruption, that is, knock out (KO) mice, a longevity model, increases exercise performance. Importantly for its relation to longevity, exercise was also improved in old AC5 KO. The mechanism resided in skeletal muscle rather than in the heart, as confirmed by cardiac‐ and skeletal muscle‐specific AC5 KO's, where exercise performance was no longer improved by the cardiac‐specific AC5 KO, but was by the skeletal muscle‐specific AC5 KO, and there was no difference in cardiac output during exercise in AC5 KO vs. WT. Mitochondrial biogenesis was a major mechanism mediating the enhanced exercise. SIRT1, FoxO3a, MEK, and the anti‐oxidant, MnSOD were upregulated in AC5 KO mice. The improved exercise in the AC5 KO was blocked with either a SIRT1 inhibitor, MEK inhibitor, or by mating the AC5 KO with MnSOD hetero KO mice, confirming the role of SIRT1, MEK, and oxidative stress mechanisms. The Caenorhabditis elegans worm AC5 ortholog, acy‐3 by RNAi, also improved fitness, mitochondrial function, antioxidant defense, and lifespan, attesting to the evolutionary conservation of this pathway. Thus, decreasing sympathetic signaling through loss of AC5 is not only a mechanism to improve exercise performance, but is also a mechanism to improve healthful aging, as exercise also protects against diabetes, obesity, and cardiovascular disease, which all limit healthful aging.     

9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogues: specific stable agonist (SQ 26, 538) and antagonist (SQ 26, 536) of the human platelet thromboxane receptor

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

Adenosine A1 receptor-mediated inhibition of myocardial norepinephrine release involves neither phospholipase C nor protein kinase C but does involve adenylyl cyclase

Stimulation of adenosine A1 receptors in the heart exerts cardioprotective effects by inhibiting norepinephrine (NE) release from sympathetic nerve endings. The intraneuronal signal transduction triggered by presynaptic adenosine A1 receptors is still not completely understood. The objective of the present study was to determine whether phospholipase C (PLC), protein kinase C (PKC), and adenylyl cyclase (AC) are involved in the adenosine A1 receptor-mediated inhibition of endogenous (stimulation-induced) NE release in isolated Langendorff-perfused rat hearts as an approach to elucidate their role in the cardiovascular system. Activation of adenosine A1-receptors with 2-chloro-N6-cyclopentyladenosine (CCPA) decreased cardiac NE release by approximately 40%. Inhibition of PLC with 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U 73122) as well as inhibition of PKC with 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF 109203X) slightly but significantly decreased NE release; however, the suppressive effect of CCPA on NE release was not modulated by U 73122 or GF 109203X. Blockade of AC with 9-(tetrahydro-2'-furyl)adenine (SQ 22536) reversed the inhibitory effect of CCPA on sympathetic neurotransmitter release irrespective of whether PKC was pharmacologically activated by phorbol 12-myristate 13-acetate or was not activated, indicating a PKC-independent but AC-dependent mechanism. Direct stimulation of AC with forskolin increased NE release by approximately 20%; an effect that was antagonized by either CCPA or SQ 22536. These data suggest that the adenosine A1 receptor-mediated inhibition of NE release does not involve PLC or PKC but does involve AC.     

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.     

Nitric oxide production from rat adipocytes is modulated by beta3-adrenergic receptor agonists and is involved in a cyclic AMP-dependent lipolysis in adipocytes.

It is established that the modulation of beta(3)-adrenoceptor function could be associated with impairment of lipolysis in white fat and be responsible for disturbed lipid metabolism. Though two isoforms of nitric oxide synthase (NOS) were reported in adipocytes, the role of nitric oxide (NO) in adipose tissue is still ambiguous. The present work was directed to study the interplay between NO production and beta-adrenoceptor/cyclic AMP (cAMP) pathway on lipid mobilization (glycerol and nonesterified fatty acids, NEFA) in cultures of rat adipocytes isolated from epididymal white adipose tissue. beta-Nonselective (isoprenaline) and beta(3)-selective (BRL-37344) agonists and the postadrenoceptor agents such as dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methylxanthine significantly increased nitrite, glycerol, and NEFA levels with BRL-37344 being the most potent. Conversely, addition of beta-nonselective (propranolol) or beta(3)-selective (bupranolol) antagonist or the adenylyl cyclase inhibitor (SQ 22,536) significantly reduced beta-agonist-induced NO production and lipolysis. For beta-adrenoceptor agonists, antagonists, and their pairs, there was a positive correlation between medium nitrite and glycerol or NEFA with r(2) being 0.90 and 0.84, respectively. The possible relationship between NO and lipolysis was revealed after adipocyte treatment with nonspecific (N(omega)-nitro-l-arginine methyl ester, l-NAME) and specific (aminoguanidine) NOS inhibitors. Both l-NAME and aminoguanidine significantly inhibited the lipolytic effect of BRL-37344. Moreover, NO-donor (S-nitroso-N-acetylpenicillamine) at higher concentration increased basal glycerol and NEFA levels. 8-bromo-cyclic GMP had no effect on adipocyte lipolysis. These data suggest that beta-adrenergic lipolysis, specifically beta(3)-adrenoceptor effect, which is realized via the adenylyl cyclase/cAMP/protein kinase A signaling cascade, involves NO production downstream of beta(3)-adrenoceptor/cAMP pathway.     

Sustained Toll-Like Receptor 9 Activation Promotes Systemic and Cardiac Inflammation,and Aggravates Diastolic Heart Failure in SERCA2a KO Mice

Aim

Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure.

Methods and Results

Diastolic heart failure was established in 8–10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2μg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation.

Conclusion

Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure.     

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26,536, (8(R)9(S)11(R)12(S)-9α-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I₅₀ value of 1.7 μ . SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH₂ and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9α-homo-9-, 11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A₅₀ value of 2.5 μ . SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a K_i value of 3 μ . Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH₂-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Intermedin/adrenomedullin-2 (IMD/AM2) relaxes rat main pulmonary arterial rings via cGMP-dependent pathway: role of nitric oxide and large conductance calcium-activated potassium channels (BK(Ca))

The present study was designed to investigate the effects of rat intermedin/adrenomedullin2 (rIMD), an agonist for calcitonin-like calcitonin receptors (CRLR), on the isolated rat pulmonary arterial rings (PA). When PA were precontracted with 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U-46619), rIMD (10(-11) to 10(-6)M) induced concentration-dependent relaxation. The pulmonary vasorelaxant response (PVR) to rIMD in PA were completely inhibited by endothelium removal, NG-nitro-L-arginine-methyl-ester (L-NAME), l-N5-(1-iminoethyl)-ornithine hydrochloride (l-NIO) or 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The PVR to rIMD were also significantly attenuated by a protein kinase inhibitor, Rp-8-bromo-beta-phenyl-1,N2-ethenoguanosine 3':5'-cyclic monophosphorothioate sodium salt hydrate (Rp-8-Br-PETcGMPs), cholera toxin and abolished by tetraethylammonium chloride (TEA), iberiotoxin and precontraction with KCl. The relaxant effect was not affected by 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy 1H diindolo [1,2,3fg:3',2',1'kl] pyrrolo [3,4-i] [1,6] benzodiazocine-10-carboxylic acid hexyl ester (KT5720), meclofenamate, glybenclamide or apamin. In parallel with SQ22536 and KT5720 results rolipram pretreatment did not alter the rIMD-induced PVR. The PVR to rIMD was potentialized either in the presence of zaprinast or sildenafil. Since the PVR to rIMD was also significantly reduced by rCGRP(8-37) and hADM(22-52) and rIMD(17-47), the present data suggest that rIMD produces PVR by acting in an indiscriminant manner on functional, and possibly different, endothelial CRLR. In conclusion, rIMD stimulates endothelial CRLR are coupled to release of nitric oxide, activation of guanylate cyclases, and promotion of hyperpolarization through large conductance calcium-activated K(+) channels in rat main PA.     

MANT-substituted guanine nucleotides: a novel class of potent adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G(s). 2'(3')-O-(N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G(i)/G(o)-proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTPgammaS) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on Galpha(s)- and Galpha(i)-mediated signaling. MANT-GTPgammaS and MANT-GppNHp had lower affinities for Galpha(s) and Galpha(i) than GTPgammaS and GppNHp. In contrast to guanosine 5'-[beta-thio]diphosphate, MANT-GTPgammaS noncompetitively inhibited GTPgammaS-stimulated AC in Galpha(s)-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPgammaS and MANT-GppNHp was not due to Galpha(s) inhibition since it was also observed in Galpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked Galpha(i)-mediated AC inhibition by GTPgammaS and GppNHp in S49 cyc(-) membranes but not AC inhibition by MANT-GTPgammaS and MANT-GppNHp. MANT-GTPgammaS and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.     

6-(2-Furanyl)-9H-purin-2-amine derivatives as A2A adenosine antagonists

Structure-activity relationships have been investigated through substitutions at the 9-position of the 2-amino-6-(2-furanyl) purine (5) to identify novel and selective A(2A) adenosine receptor antagonists. Several potent and selective antagonists were identified. In particular, compounds 20, 25, and 26 show very high affinity with excellent selectivity.     

Type 5 adenylyl cyclase disruption increases longevity and protects against stress

Mammalian models of longevity are related primarily to caloric restriction and alterations in metabolism. We examined mice in which type 5 adenylyl cyclase (AC5) is knocked out (AC5 KO) and which are resistant to cardiac stress and have increased median lifespan of approximately 30%. AC5 KO mice are protected from reduced bone density and susceptibility to fractures of aging. Old AC5 KO mice are also protected from aging-induced cardiomyopathy, e.g., hypertrophy, apoptosis, fibrosis, and reduced cardiac function. Using a proteomic-based approach, we demonstrate a significant activation of the Raf/MEK/ERK signaling pathway and upregulation of cell protective molecules, including superoxide dismutase. Fibroblasts isolated from AC5 KO mice exhibited ERK-dependent resistance to oxidative stress. These results suggest that AC is a fundamentally important mechanism regulating lifespan and stress resistance.     

The A-kinase Anchoring Protein Yotiao Facilitates Complex Formation between Adenylyl Cyclase Type 9 and the IKs Potassium Channel in Heart

The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (I(Ks)) and recruits both PKA and PP1 to regulate I(Ks) phosphorylation and gating. Human mutations that disrupt I(Ks)-Yotiao interaction result in reduced PKA-dependent phosphorylation of the I(Ks) subunit KCNQ1 and inhibition of sympathetic stimulation of I(Ks), which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the I(Ks)-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous I(Ks)-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the I(Ks) channel to achieve localized temporal regulation of β-adrenergic stimulation.     

Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases

Identifying novel allosteric inhibitors of G protein-coupled receptor kinases (GRKs) would be of considerable use in limiting both the extent of desensitization of GPCRs as well as downstream positive regulation through GRKs. Several peptides have previously been identified as inhibitors of specific GRKs, but to date there have been few comparisons of the selectivities of these materials on the seven GRKs, modifications to allow cell penetration, or off-target activities. The goal of this study was to determine if a panel of peptides mimicking domains on either GPCRs or GRKs would exhibit selective inhibition of GRKs 2, 5, 6 and 7 phosphorylation of rhodopsin. Peptides included sequences from GRK5; helices 3, 9, and 10 (α3, α9, and α10) in the RH domain, and the N-terminal peptide (N-Ter), as well as the intracellular loop 1 (iL1) of the β2-adrenergic receptor (β2AR), and the G_α transducin C-tail (TCT). While some selectivity for individual GRKs was found, overall selectivity was limited and often not reflective of structural predictions. Off-target effects were probed by determining peptide inhibition of adenylyl cyclase (AC) and PKA, and while peptides had no effect on AC activity, N-Ter, iL1, and α10 were potent inhibitors of PKA. To probe inhibition of GRK activity in intact cells, we synthesized TAT-tagged peptides, and found that TAT-α9-R169A and TAT–TCT inhibited isoproterenol-stimulated GRK phosphorylation of the β2AR; however, the TAT peptides also inhibited isoproterenol and forskolin stimulation of AC activity. Our findings demonstrate potent peptide inhibition of GRK activities in vitro, highlight the differences in the environments of biochemical and cell-based assays, and illustrate the care that must be exercised in interpreting results of either assay alone.