Guanylyl Imidodiphosphate Research Articles

Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC

The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to α3β3 of F1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5’-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.

The effect of 2-month bromocriptine treatment on the activity of the adenylyl cyclase signaling system in the myocardium and testes of rats with type 2 diabetes

Cardiovascular diseases and dysfunctions of the reproductive system are common complications of type 2 diabetes (T2D), and, therefore, development of new approaches for the alleviation of these pathologies is highly important. Bromocriptine (BC), an agonist of type 2 dopamine receptors, recently became widely used for the treatment of type 2 diabetes; this drug both restores energy metabolism and prevents the development of cardiovascular disease. However, the targets of BC and mechanisms underlying its action are still poorly characterized. The aim of the present work was to investigate the effect of BC treatment on the functional activity of the adenylyl cyclase signaling system (ACSS) in the myocardium and testes of male rats suffering from type 2 diabetes induced by high-fat diet and administration of streptozotocin (25 mg/kg). Oral BC administration (0.6 mg/kg every other day for 60 days) started 90 days after the commencement of highfat diet. Body weight and triglyceride levels were elevated in diabetic rats. Glucose tolerance of the animals was impaired, and they developed insulin resistance. BC treatment enabled the recovery of glucose metabolism parameters and increased the sensitivity of tissues to insulin. Adenylyl cyclase (AC) stimulation by guanylyl imidodiphosphate (GppNHp), relaxin, and the β-adrenergic receptor (β-AR) agonists isoproterenol and norepinephrine in the myocardium of diabetic rats was impaired, while the stimulatory effects of the β3-agonists BRL-37344 and CL-316243 persisted. The inhibitory effect of somatostatin on forskolin-stimulated AC activity was attenuated, while the inhibitory effect of norepinephrine mediated by α2-AR was enhanced. BC treatment normalized adrenergic signaling in the myocardium and partially restored the effects of relaxin and somatostatin on AC. AC activity, both basal and stimulated by GppNHp, forskolin, human chorionic gonadotropin, and pituitary AC-activating polypeptide, decreased in the testis of diabetic rats, and the inhibitory effect of somatostatin was attenuated. BC treatment induced only slight changes in the testicular ACSS. Thus, prolonged BC treatment restored the functional activity of ACSS in the myocardium and testes of diabetic rats; this accounts for the therapeutic effect of BC on the impaired functioning of the cardiovascular and reproductive systems in T2D and should be taken into account upon the selection of treatment strategies for T2D and its complications.

Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1

Small GTPases regulate a large variety of key cellular processes. Plant small Rac/Rop GTPases have recently received broad attention as it is becoming clear that these enzymes regulate various plant cellular processes. OsRac1, a rice Rac/Rop protein, is a key regulator of reactive oxygen species (ROS) production and induces immune responses. Although four structures of plant small GTPases have been reported, all of these were of the inactive form. Here, OsRac1 was purified and co-crystallized with the GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP). The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 1.9 Å resolution.

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.

Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.

Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state

The interaction of the rod GTP binding protein, Transducin (G(t)), with bleached Rhodopsin (R(*)) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound G(t) by reconstituting G(t) containing bound GDP (G(t-)GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate (G(t-)p[NH]ppG) with R* under physiological conditions. Release of GDP and p[NH]ppG from G(t) occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of G(t) without bound nucleotide (G(t-)) were generated. When ROS containing bleached rhodopsin (R(*)) were centrifuged in low ionic strength buffer, G(t-) remained associated with the membrane fraction, whereas G(t-)GDP remained in the soluble fraction. These results suggest that G(t-)GDP and G(t-)p[NH]ppG have similar affinities for R(*). The results also suggest that G(t-), rather than G(t-)GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.

Influence of relaxin and insulin on the activity of adenylate cyclase in the myometrium of pregnant women with type 1 diabetes mellitus

The study of the influence of relaxin and insulin on the activity of adenylate clase (AC) in the myometrium of pregnant women showed that human relaxin 2 (10-9-10-8 M) causes a stronger stimulating effect on the enzyme activity than insulin. The activating effect of relaxin and insulin was potentiated by a non-hydrolyzable GTP analogue, guanylylimidodiphosphate (GIDP), in the myometrium of pregnant women, which indicates the participation of the stimulating GTP-binding protein (Gs-protein) in the realization of the effect of peptides. In the myometrium of pregnant women with type 1 diabetes mellitus under the action of relaxin, insulin and isoproterenol, a decrease in the sensitivity of AC activity to these hormones was found in comparison with these indicators in the myometrium of pregnant women. With the combined effect of peptides and GIDP in the myometrium of pregnant women with type 1 diabetes mellitus, a weakening of the potentiation of the GIDP effect of hormones on AC activity was observed, which may indicate a decrease in the coupling between receptors, Gs protein, and AC. The studies carried out allow us to conclude that in pregnant women with type 1 diabetes mellitus, the decrease in AC stimulation under the influence of these hormones is associated with a weakening of the Gs-protein function.

Alterations of adenylyl cyclase and G proteins in aortocaval shunt-induced heart failure.

Unlike most other experimental models of congestive heart failure, the volume overload model induced by aortocaval shunt (AVS) in rats was found to exhibit enhanced beta-adrenoceptor (beta-AR) signaling. To study whether the adenylyl cyclase (AC)-G protein system is involved in such a change, we examined cardiac AC activity and protein content as well as G(s)alpha and G(i)alpha activities, protein contents, and mRNA levels in both left (LV) and right (RV) ventricles at the failing stage (16 wk after surgery). Basal and forskolin-stimulated AC activities were significantly increased in both LV and RV from the failing hearts; this change was associated with an upregulation of type V/VI AC protein. In contrast to 5'-guanylyl imidodiphosphate and NaF, the stimulatory effect of isoproterenol on AC was increased in the failing heart. Although G(s)alpha and G(i)alpha protein contents in the failing hearts were not altered, the mRNA level for G(s)alpha was decreased by 20% and that for G(i)alpha was increased by 20%. In addition, the activity of G(s)alpha, but not G(i)alpha, as assessed by toxin-catalyzed ADP ribosylation, was significantly decreased in the failing heart. Losartan and imidapril treatments improved cardiac function and attenuated alterations in mRNA levels for G(s)alpha and G(i)alpha proteins, as well as G(s)alpha activity, without affecting changes in AC protein content or activities in heart failure due to volume overload. These data suggest that increased AC activity may contribute to the enhanced beta-AR signaling in the AVS model of heart failure, whereas alterations in gene expression for G proteins may be of an adaptive nature at this stage of heart failure.

Solubilization of adenylyl cyclase from human myometrium in a alphas-coupled form.

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.

Impairment of adenylyl cyclase and of spatial memory function after microsphere embolism in rats.

The purpose of the present study was to characterize alterations in the adenylyl cyclase (AC), cyclic adenosine 3',5'-monophosphate (cAMP), and spatial memory function after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres (48 microm in diameter) into the right (ipsilateral) hemisphere of rats. Alterations in the AC and cAMP in the cerebral cortex and hippocampus were examined up to 7 days after the embolism. A decrease in the cAMP content was seen in the ipsilateral hemisphere throughout the experiment. Microsphere embolism (ME) decreased the activity of Ca(2+)/calmodulin (CaM)-sensitive AC in the ipsilateral hemisphere throughout the experiment, whereas the basal and 5'-guanylyl imidodiphosphate (Gpp(NH)p)-sensitive AC activities were not altered. Immunoblotting analysis of AC subtypes with specific antibodies showed a decrease in the immunoreactivity of AC-I in the ipsilateral hemisphere during these periods. No significant differences in the immunoreactivity of AC-V/VI and AC-VIII were observed after ME. The levels of GTP-binding proteins Galpha(s), Galpha(i), and Gbetawere unchanged. Furthermore, microsphere-embolized rats showed prolongation of the escape latency in the water maze task determined on the seventh to ninth day after the operation. These results suggest that sustained cerebral ischemia may induce the impairment of the AC, particularly a selective reduction in the AC-I level and activity, coupled with the decrease in cAMP content. This reduction may play an appreciable role in the disturbance in cAMP-mediated signal transduction system, possibly leading to learning and memory dysfunction.

Impairment of cardiac beta-adrenoceptor cellular signaling by decreased expression of G(s alpha) in septic rabbits.

Abnormalities in the beta-adrenergic control of cardiac function play a role in the pathogenesis of several disease states. Because circulatory failure in patients with septic shock is known to be less responsive to catecholamines, we investigated whether the beta-adrenoceptor-linked signal transduction mechanisms are altered in the heart of a septic animal model Rabbits were rendered endotoxemic by an intravenous injection of 100 microg/kg Escherichia coli lipopolysaccharide. Three and 6 h later, the myocardial tissues were used for the experiments. The positive inotropic response to isoproterenol was significantly impaired in papillary muscles isolated from septic rabbits compared with those from controls. The impaired inotropic responsiveness to isoproterenol was not prevented by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine, indicating no involvement of nitric oxide overproduction. Adenylate cyclase activity stimulated with isoproterenol and 5'-guanylyl imidodiphosphate was markedly reduced in septic myocardium. The contractile and adenylate cyclase responses to colforsin daropate, a direct adenylate cyclase activator, were unaffected by sepsis. Radioligand binding experiments with (-)[125I]iodocyanopindolol revealed no significant alteration in myocardial beta-adrenoceptor density or affinity in sepsis. Determination of cardiac G(s alpha) level by Western blotting showed a reduction of approximately 50% in sepsis. The relative content of G(s alpha) messenger RNA in septic myocardium also was reduced from the control level by about 50%, as determined by Northern blot analysis. Little change was found in protein and messenger RNA levels of G(s alpha) in septic myocardium. Impairment of myocardial functional responsiveness to beta-adrenoceptor stimulation appears in the early stage of sepsis. The impaired response to beta-adrenoceptor stimulation in the heart in this pathologic state may result in part from a decreased level of G(s alpha) protein which occurs at the level of gene expression.

Alterations in cardiac adrenergic terminal function and β-adrenoceptor density in pacing-induced heart failure

Congestive heart failure is associated with cardiac adrenergic nerve terminal changes and beta-adrenoceptor density downregulation. To study the temporal sequence of these changes, we performed studies in rabbits at 2, 4, and 8 wk of cardiac pacing (360 beats/min) and at 1, 2, and 4 wk after cessation of pacing. Rapid pacing produced left ventricular (LV) dysfunction and an increase in plasma norepinephrine (NE) in 1-2 wk. At week 2, NE uptake activity, NE uptake-1 density, and adenylyl cyclase responses to isoproterenol, 5'-guanylyl imidodiphosphate [Gpp(NH)p], and forskolin reduced. However, immunostained tyrosine hydroxylase profile, beta-adrenoceptor density, and NE histofluorescence did not reduce until 4-8 wk of pacing. After cessation of cardiac pacing, LV function normalized quickly, followed by return of tyrosine hydroxylase and NE profiles in 1 wk and adenylyl cyclase responses to agonists and NE uptake activity in 2 wk. Myocardial beta-adrenoceptor density returned to normal by 4 wk after cessation of pacing. Our results suggest that there is no permanent structural neuronal damage in the myocardium within the first 8 wk of rapid cardiac pacing. Abnormal myocardial NE reuptake mechanism may play an important pathophysiological role in heart failure.

Effect of Digoxin on Ventricular Remodeling and Responsiveness of beta-Adrenoceptors in Chronic Volume Overload.

BACKGROUND: Digoxin improves baroreflex function and reduces neurohumoral activation in severe heart failure, but it is uncertain how digoxin affects ventricular remodeling and progression to left ventricular dysfunction. In addition, the effect of digoxin in in vitro beta-adrenoceptor density and function, and contractile reserve in vivo is not well understood. METHODS AND RESULTS: To study this, we compared digoxin with placebo treatment in rats with chronic volume overload induced by aortocaval fistula and in sham-operated control animals. Left ventricular end-diastolic cavity dimensions (LVDd) and wall thickness were measured weekly by in vivo transthoracic echocardiography, and left ventricular mass (LVM) and percent fractional shortening (%FS) were calculated. Six weeks after fistula creation, simultaneous echocardiographic and invasive hemodynamic evaluation at rest and in response to incremental dobutamine (1-10 µg/kg/min intravenously) were measured. Myocardial plasma membrane beta-adrenoceptor density and maximal adenylate cyclase responses (V(max)) to isoproterenol, 5'-guanylylimi dodiphosphate, and forskolin were measured in vitro. Volume overload induced progressive increases in LVDd and LVM over the 6-week study period. Percent fractional shortening at rest, and the change in %FS in response to dobutamine stress were dramatically reduced 6 weeks after fistula creation. Although 6-week fistula animals had unchanged beta-adrenoceptor density (B(max)) and binding affinity (K(d)) as compared with controls, maximal adenylate cyclase responses to stimulation in vitro (V(max)) were markedly reduced. Digoxin treatment prevented this loss of responsiveness of adenylate cyclase but did not affect beta-adrenoceptor density or affinity in vitro. Digoxin had no effect on LVDd, LVM, %FS, or the response to dobutamine infusion in vivo. CONCLUSIONS: Although digoxin prevented beta-adrenoceptor desensitization and improved in vitro myocardial adenylate cyclase response, the cardiac response to adrenergic stimulation in vivo was not significantly improved. These results suggest that the role of beta-adrenoceptor desensitization in the progression from volume overload hypertrophy to left ventricular dysfunction and heart failure may be less important than previously thought. Furthermore, although digoxin treatment did produce modest hemodynamic benefits, it did not prevent progressive remodeling in this model.

Binding interaction between Tet(M) and the ribosome: requirements for binding.

Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5'-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.

Cross-talk between β-adrenergic and metabotropic glutamate receptors in rat C6 glioma cells

Exposure of rat C6 glioma cells to the β-adrenergic receptor agonist isoproterenol potentiates basal and metabotropic glutamate receptor-stimulated phospholipase C activity in rat C6 glioma cells. After treatment of cells for 24 h with 10 μM isoproterenol, metabotropic glutamate receptors and phospholipase C activity were determined in C6 plasma membranes. Isoproterenol treatment caused an increase of 67% in the total number of binding sites ( B max=12.1±1.8 pmol/mg protein versus B max=20.27±0.88 pmol/mg protein) with K d values of the same order ( K d=1250±101 nM versus K d=1401±211 nM), using l-[ 3H]glutamate as radioligand. On the other hand, basal, guanylyl imidodiphosphate (Gpp[NH]p)- and trans-aminocyclopentane-1,3-dicarboxylic acid ( trans-ACPD)-stimulated phospholipase C activities were also significantly increased in membranes from isoproterenol-treated cells compared to control cells, by 337%, 33% and 40% respectively. Moreover, a significant increase of 94% in the steady-state level of phospholipase C β 1 in membranes from isoproterenol-treated cells compared to control was also detected by immunoblot. These results show that metabotropic glutamate receptors and its effector system, phospholipase C, are affected by isoproterenol treatment, showing the existence of cross-talk between these signal transduction pathways.

Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: definition of its binding profile using agonists, antagonists and inverse agonists.

The novel benzodioxopiperazine, 5-HT1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT1A receptor (Ki = 0.6 nM versus [3H]-8-hydroxy-dipropylamino-tetralin, [3H]-8-OH-DPAT): its affinity at h5-HT1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22 degrees C, [3H]-S 15535 associated and dissociated from h5-HT1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a Kd estimate of 3.6 nM. In saturation binding experiments, [3H]-S 15535 displayed a Bmax value for h5-HT1A receptors (1630 fmol/mg), higher than that obtained with the agonist [3H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 microM) reduced the binding of [3H]-S 15535 by only 25% compared with 79% for [3H]-8-OH-DPAT at h5-HT1A receptors. [3H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (Bmax = 820 fmol/mg versus 647 fmol/mg for [3H]-8-OH-DPAT). For both h5-HT1A and rat 5-HT1A receptors, the Ki values for competition binding of 15 serotonergic ligands with [3H]-S 15535 was highly correlated with that of [3H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [3H]-S 15535 but not with [3H]-8-OH-DPAT at h5-HT1A receptors. Similarly, the 'antagonists', spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [3H]-S 15535 but not [3H]-8-OH-DPAT. When [3H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 microM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-h exanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [3H]-S 15535 is a highly selective 5-HT1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [3H]-agonists and [3H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [3H]-S 15535 being modulated in an opposite manner to those of agonists.

EVIDENCE FOR A LOCAL ACTION OF MELATONIN ON THE RAT PROSTATE

Purpose Melatonin, secreted by the pineal gland at night, inhibits pubertal development in rats and possibly humans. We have recently found functional specific binding sites for 125I-labeled melatonin (sup 125 I-melatonin) in human benign prostate tissue, localized in the microsomal fraction of the glandular epithelium. The aim of the present study was to set up an animal (rodent) model for the growth inhibitory effects of melatonin on the prostate. Materials and Methods Putative melatonin receptors in the rat ventral prostate were explored by means of autoradiography and receptor binding assays and the ability of melatonin to inhibit stimulated prostate growth was tested in vivo. Results In vitro autoradiography and equilibrium binding experiments demonstrated specific binding sites for 125I-labeled melatonin (sup 125 I-melatonin) associated with the microsomal fraction of the rat ventral prostate cells (apparent dissociation constant 0.9 nM). 125I-melatonin binding was inhibited by 2-iodomelatonin>6-hydroxy-melatonin>melatonin=N-(2,4 dinitrophenyl)-5-methoxytryptamine whereas similar concentrations of serotonin, 5-methoxytryptamine, and tryptamine were less potent. The guanine nucleotide analogs, guanosine 5′-0-[3-thiotriphosphate] and guanosine 5′-0-[2-thio-diphosphate], inhibited specific 125I-melatonin binding whereas 5′-guanylyl imido-diphosphate was less potent. Daily injections of testosterone to castrated rats induced regrowth of the prostate and increased the weight of the seminal vesicles. Administration of melatonin to the rats through drinking water prevented the testerone-mediated regrowth of the prostate but had no effect on the seminal vesicles' weight. Conclusions The results demonstrated putative melatonin receptors in the rat prostate and suggest a direct suppression by melatonin of testosterone-dependent prostate growth.

Adenylyl cyclase in the heart: an enzymocytochemical and immunocytochemical approach.

This review provides a discussion of the localization of adenylyl cyclase (AC) in normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes. This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin. With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes. Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction. Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes demonstrated by enzymocytochemistry.

Prostaglandin H synthase: protein synthesis-independent regulation in bovine aortic endothelial cells.

The objective of the present study was to examine whether prostaglandin H synthase (PGHS) can be regulated by pathways independent of de novo synthesis of PGHS. Incubation of bovine aortic endothelial cells (BAEC) for as short as 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity. PGHS activity induced by NaF was unaffected by either 10 microM cycloheximide or 1 microM actinomycin D. Aspirin (25 microM) completely inhibited resting PGHS activity, and NaF did not induce further stimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, was ineffective. Basic fibroblast growth factor (bFGF) induced a significant increase in PGHS activity within 30 min and was insensitive to cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measured by Western blots, were not affected by NaF or bFGF. The tyrosine kinase inhibitor genistein attenuated PGHS activity that was induced by NaF and bFGF, whereas the tyrosine phosphatase inhibitor, sodium orthovanadate, augmented these responses. The G protein activators 5'-guanylyl imidodiphosphate and guanosine 5'-O-(3-thiotriphosphate) inhibited both resting and NaF-induced PGHS activities. These results suggest-that, in BAEC, PGHS-1 activity can be regulated by tyrosine kinase and/or G proteins, independently of de novo protein synthesis.