Adenylyl Cyclase Activity In Membranes Research Articles

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats.

Low-molecular-weight agonists of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LHCGR), which interact with LHCGR transmembrane allosteric site and, in comparison with gonadotropins, more selectively activate intracellular effectors, are currently being developed. Meanwhile, their effects on testicular steroidogenesis have not been studied. The purpose of this work is to perform a comparative study of the effects of 5-amino-N-tert-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-d] pyrimidine-6-carboxamide (TP4/2), a LHCGR allosteric agonist developed by us, and hCG on adenylyl cyclase activity in rat testicular membranes, testosterone levels, testicular steroidogenesis and spermatogenesis in young (four-month-old), aging (18-month-old) and diabetic male Wistar rats. Type 1 diabetes was caused by a single streptozotocin (50 mg/kg) injection. TP4/2 (20 mg/kg/day) and hCG (20 IU/rat/day) were administered for 5 days. TP4/2 was less effective in adenylyl cyclase stimulation and ability to activate steroidogenesis when administered once into rats. On the 3rd–5th day, TP4/2 and hCG steroidogenic effects in young adult, aging and diabetic rats were comparable. Unlike hCG, TP4/2 did not inhibit LHCGR gene expression and did not hyperstimulate the testicular steroidogenesis system, moderately increasing steroidogenic proteins gene expression and testosterone production. In aging and diabetic testes, TP4/2 improved spermatogenesis. Thus, during five-day administration, TP4/2 steadily stimulates testicular steroidogenesis, and can be used to prevent androgen deficiency in aging and diabetes.

Low-Molecular-Weight Ligands of Luteinizing Hormone Receptor with the Activity of Antagonists

Currently, a search for selective antagonists of luteinizing hormone (LH) receptor, which are required to suppress the steroidogenesis in hormone-dependent tumors and to prevent the ovarian hyperstimulation syndrome, is carried out. One approach to tackle this problem is the development of low-molecular-weight antagonists of the allosteric site of this receptor, which is located in its transmembrane domain. The aim of this work was to develop the heterocyclic compounds, the derivatives of thieno[2,3-d]pyrimidine (TP31), pyrimido[4,5,6-de][1,6]naphthyridine (PP10), and pyrido[3,4-d]pyrimidine (PP17), and to study their ability to affect the functional activity of the LH receptor in the in vitro and in vivo conditions. It was shown that TP31 at micromolar concentrations suppressed stimulating effects of human chorionic gonadotropin (hCG) and TP03, an allosteric agonist of the LH receptor, on the adenylyl cyclase activity in rat testicular membranes; its effect was most pronounced in relation to the stimulating effects of TP03. This was due to a higher selectivity of the TP31 antagonist with respect to the cAMP-dependent signaling cascades, predominantly activated by TP03 and realized through Gs proteins. PP17 inhibited stimulatory effects of hCG and TP03 on the adenylyl cyclase activity to a similar extent but was less active compared to TP31. Upon intratesticular (10 mg/kg) or intraperitoneal (45 mg/kg) administration to male rats, TP31 and PP17 decreased the baseline plasma level of testosterone and inhibited the testosterone production stimulated by hCG (100 IU/rat); the inhibitory effect of TP31 was much more pronounced than that of PP17. PP10 exhibited a weaker antagonistic activity than TP31 and PP17 in the in vitro and in vivo conditions. The data obtained indicated that TP31, the most active functional antagonist among the studied compounds, binded to the allosteric site of the LH receptor, made it less accessible to allosteric agonists and impairs the hormonal signal transduction through the LH receptor. This suggests the prospects of the development of TP31-based inhibitors of LH-dependent pathways and steroidogenesis.

P738The restoration effect of metformin on the beta-adrenergic signaling, amp-activated kinase and akt-kinase in the myocardium of diabetic rats

Abstract Background The type 2 diabetic mellitus (DM2) induces the myocardial dysfunctions, the impairment of beta-adrenergic regulations and the alterations of energy homeostasis and apoptotic processes in the myocardium are the main causes of diabetic cardiomyopathy. Metformin (MF) is a first-line drug for management of DM2. However, MF effects on the adrenergic signaling, the cell metabolism and survival in the cardiomyocytes are still poorly understood. Purpose To study MF effects on the beta-adrenergic signaling in myocardial membranes and on the expression and activity of beta1- and beta3-adrenergic receptors (ARs), AMP-activated kinase (AMPK), Akt-kinase and the pro- (Bax) and anti-apoptotic (Bcl-2) proteins in the myocardium of diabetic rats. Methods In male rats, DM2 was induced by high-fat/high-carbohydrate diet (three months) and by a single treatment with low-dose streptozotocin. Diabetic rats were treated with MF at two doses, 100 and 200 mg/kg/day, during 28 days. The stimulating effects of beta-AR agonists on adenylyl cyclase (AC) activity in the cardiac membranes were studied by radioisotope method. The gene expression was estimated by qPCR, and the protein amount and their activity were studied by Western blotting. Four groups of animals, such as control (C, n=12), DM2 (D, n=12), and DM2 treated with MF at the doses 100 (D100, n=9) and 200 mg/kg/day (D200, n=9) were investigated. Results MF restores the stimulating AC effect of beta1-agonists, the beta1-AR gene expression and the beta1/beta3-ARs ratio, all reduced in DM2, and restoration effect of MF in Groups D100 and D200 was similar. Meanwhile, in Group D200, but not in Group D100, the AC effect of beta3-agonists and the expression of beta3-AR, both increased in DM2, returns to the control values. In diabetic myocardium, MF restores the AMPK and Akt-kinase activities, reduced in DM2, and it is illustrated by the increased phosphorylation of AMPK by Thr172 and Akt-kinase by Ser473 and Thr308. Despite the fact that MF at a dose of 200 mg/kg/day to a greater extent, as compared to a dose of 100 mg/kg/day, increases the “stimulating” Thr172-phosphorylation, but at the same time it also enhances the “inhibiting” Ser491-phosphorylation of AMPK, decreasing the resulting stimulation of AMPK. In Groups D100 and D200 the Bax/Bcl-2 ratio, significantly increased in DM2, is reduced. This data indicates the weakening of apoptosis in cardiomyocytes in DM2. Conclusion The four-week MF treatment improves the myocardial beta-adrenergic signaling, normalizing the expression and activity of beta1- and beta3-ARs, restores the activity of AMPK and Akt-kinase, the effector components of the signaling cascades responsible for cell survival and metabolism, and normalizes the expression of apoptotic factors. Thus, the cardioprotective MF effect is largely due to the improved myocardial signaling, including the beta1-AR, AMPK and Akt-kinase signaling pathways. Acknowledgement/Funding This work was supported by the Russian Science Foundation (No. 17-75-30052) and partially by the Government Assignment (AAAA-A18-118012290427-7).

Thienopyrimidine Derivatives Specifically Activate Testicular Steroidogenesis but Do Not Affect Thyroid Functions

The luteinizing hormone (LH) and human chorionic gonadotropin (hCG) stimulate the testosterone (T) production in Leydig cells via specifical binding to the LH/hCG receptor extracellular domain. The LH/hCG receptor can also be activated by low-molecular-weight (LMW) agonists that bind to the allosteric site located inside the transmembrane receptor channel. Since the allosteric sites in the LH/hCG and thyroid-stimulating hormone (TSH) receptors share a similar structure, LMW agonists of the LH/hCG receptor can affect activity of the TSH receptor and the synthesis of thyroid hormones which depend on this receptor. When designing selective activators of steroidogenesis, it is necessary to rule out this possibility. The aim of this study was to explore the effect of the seven previously synthesized thienopyrimidines (TPs), LMW agonists of the LH/hCG receptor, on the basal and hCG- and TSH-stimulated adenylyl cyclase (AC) activity in plasma membranes isolated from the rat testes and thyroid as well as on the basal and luliberin- and thyroliberin-stimulated levels of T and thyroid hormones following intraperitoneal (i.p.) TP administration to male rats. All seven TPs (10−4 M) increased the AC basal activity in rat testicular membranes, and in the case of combined action of TPs and hCG their stimulatory effects were additive. TPs did not affect the basal and TSH-stimulated AC activity in thyroid membranes. When administered to male rats, TPs (i.p., 25 mg/kg) increased the T level. The effect of TP03, a most active LH/hCG receptor LMW agonist, persisted under luliberin-induced activation of the gonadal axis. The nicotinamide moiety of TP21, in contrast to the structurally similar TP03, contains an additional methoxy group which alters receptor specificity of this compound. Thus, with the exception of TP21, all TPs sharing LH/hCG receptor agonistic activity, both under in vitro and in vivo conditions, do not affect the TSH receptor, indicative of their selectivity toward the gonadal axis.

In vitro and in vivo studies of functional activity of new low molecular weight agonists of the luteinizing hormone receptor

The use of luteinizing hormone (LH) and its structural and functional homolog human chorionic gonadotropin (hCG) leads to hyperstimulation of LH-dependent signalling pathways and to desensitization of LH receptors. Therefore, the development of low molecular weight agonists of the LH receptor without these disadvantages has been carried out in recent years. These agonists, unlike gonadotropins, are active when administered orally. The greatest prospects are associated with the development of the thienopyrimidine derivatives structurally similar to compound Org 43553. The purpose of this work was the synthesis of new thienopyrimidine derivatives TP03 and TP04, the study of the regulation of LH-sensitive adenylyl cyclase signalling system in rat testes, and the evaluation of the ability of TP03 and TP04 to stimulate testosterone synthesis in male rats at different routes of administration. In the concentration range 10–7–10–3 M, TP03 and TP04 induced an increase of the basal adenylyl cyclase (AC) activity in rat testicular membranes with the EC50 values of 390 and 759 nM, respectively. At a concentration of 10–4 M, AC-stimulating effects of TP03 and TP04 were 213 and 122%, respectively, which indicates a higher activity of TP03 under in vitro conditions. At the same time, the AC-stimulating effect of TP03 was approximately 2.5-fold weaker than that of hCG (10–8 M). Upon simultaneous administration of hCG and thienopyrimidine derivatives, their stimulating effects on the AC activity were additive due to the different localization of their binding sites in the LH receptor. Compound TP03 (25 mg/kg), when administered intraperitoneally to male rats, was more efficient than TP04: the maximal increase in the testosterone level (3 h after administration) was by 60% higher than that induced by TP04. TP03 increased the testosterone level after oral administration (50 mg/kg) as well, but the effect was weaker than in the case of the intraperitoneal injection. Orally administered TP04 exhibited a low activity. The results suggest that 5-amino-N-tert-butyl-2-(methylsulfonyl)-4-(3-(nicotinamide)phenyl) thieno[2,3-d]pyrimidine-6-carboxamide (TP03) can be used for the development of the drugs that stimulate steroidogenesis in Leydig cells.

(284) Persistent electrical activity in primary nociceptors after spinal cord injury is maintained by scaffolded adenylyl cyclase and protein kinase A and is associated with altered adenylyl cyclase regulation

Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. We found recently that persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) makes major contributions to chronic reflex hypersensitivity and to spontaneous pain in a rat model of thoracic spinal cord injury (SCI). The occurrence of SCI-induced SA in a large fraction of DRG neurons at and below the injury level for months after SCI, and the persistence of this SA long after dissociation of the neurons provide a special opportunity to define intrinsic cell signaling mechanisms that chronically drive SA at the first stage in pain pathways. We have found that SCI-induced SA generated in small dissociated DRG neurons requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of adenylyl cyclase (AC) activity. Membrane AC activity stimulated by added Ca2+-calmodulin increased modestly, while the inhibition of G(alpha)s-stimulated AC activity by G(alpha)i showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons.

Interaction of Relaxin-Like Gonad-Stimulating Substance with Ovarian Follicle Cells of the StarfishAsterina pectinifera

Previously, gonad-stimulating substance (GSS), which acts as a gonadotropin, was purified from radial nerves of the starfish Asterina pectinifera and its structure was elucidated. Here, the interaction of GSS with receptors was examined in ovarian follicle cells, a target of GSS. In competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in the microsomal/plasma membrane fraction of follicle cells. GSS scarcely bound in the cytosolic fraction. Scatchard plots showed the numbers of binding sites (NBS) in whole homogenate and the crude membrane to be 1.65 and 3.42 pmoles/mg protein, respectively. Dissociation constant (K (d)) values in these two preparations were almost the same at about 0.6-0.7 nM. Furthermore, it was shown that GSS stimulated adenylyl cyclase activity in follicle cell membranes in a dose-dependent manner that required GTP. Immunoblotting with specific antibodies for G-protein subunits after SDS-PAGE of the membrane preparation showed both stimulatory (Gs) and inhibitory (Gi) regulatory α-subunits for adenylyl cyclase and a β-subunit. The results strongly suggest that GSS interacts with G-protein-coupled receptors (GPCR) located in the follicle cell membrane to stimulate Gs-protein and adenylyl cyclase activity.

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

AKAP79 Interacts with Multiple Adenylyl Cyclase (AC) Isoforms and Scaffolds AC5 and -6 to α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) Receptors

Spatiotemporal specificity of cAMP action is best explained by targeting protein kinase A (PKA) to its substrates by A-kinase-anchoring proteins (AKAPs). At synapses in the brain, AKAP79/150 incorporates PKA and other regulatory enzymes into signal transduction networks that include beta-adrenergic receptors, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and N-methyl-d-aspartic acid receptors. We previously showed that AKAP79/150 clusters PKA with type 5 adenylyl cyclase (AC5) to assemble a negative feedback loop in which the anchored kinase phosphorylates AC5 to dynamically suppress cAMP synthesis. We now show that AKAP79 can associate with multiple AC isoforms. The N-terminal regions of AC5, -6, and -9 mediate this protein-protein interaction. Mapping studies located a reciprocal binding surface between residues 77-108 of AKAP79. Intensity- and lifetime-based fluorescence resonance energy transfer demonstrated that deletion of AKAP79(77-108) region abolished AC5-AKAP79 interaction in living cells. The addition of the AKAP79(77-153) polypeptide fragment uncouples AC5/6 interactions with the anchoring protein and prevents PKA-mediated inhibition of AC activity in membranes. Use of the AKAP79(77-153) polypeptide fragment in brain extracts from wild-type and AKAP150(-/-) mice reveals that loss of the anchoring protein results in decreased AMPA receptor-associated AC activity. Thus, we propose that AKAP79/150 mediates protein-protein interactions that place AC5 in proximity to synaptic AMPA receptors.

Gi-Dependent Cell Signaling Responses of the Human P2Y14Receptor in Model Cell Systems

Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y(14) receptor (P2Y(14)-R). The P2Y(14)-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y(14)-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y(14)-R activation on mitogen-activated protein kinase signaling also was studied in P2Y(14)-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y(14)-HEK293 and P2Y(14)-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y(14)-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y(14)-HEK293 cells. P2Y(14)-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y(14)-R signals through G(i) to inhibit adenylyl cyclase, and P2Y(14)-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y(14)-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y(14)-R-dependent signal transduction.

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D(4) receptor (D(4)R) activation promotes light adaptation and suppresses the light-sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light- and D(4)R-signaling pathways converge on the type 1 Ca(2+)/calmodulin-stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D(4)R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca(2+)/calmodulin-stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D(4)R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D(4)R, cyclic AMP levels in the dark-adapted retina are significantly lower compared to wild-type retina and are unresponsive to light. These changes in Drd4-/- mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in retinal membranes compared with wild-type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild-type mice treated chronically with a D(4)R antagonist, L-745870. Thus, activation of D(4)R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross-talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.

Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

Dopamine D4 receptors regulate intracellular calcium concentration in cultured chicken cone photoreceptor cells: Relationship to dopamine receptor-mediated inhibition of cAMP formation

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca2+ concentrations ([Ca2+]i) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5–7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K+-stimulated uptake of 45Ca2+ and [Ca2+]i, measured with the Ca2+-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K+-stimulated 45Ca2+ influx or [Ca2+]i by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K+, which requires Ca2+ influx through voltage-gated Ca2+ channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca2+/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca2+]i.

Pheromone Detection in Male Mice Depends on Signaling through the Type 3 Adenylyl Cyclase in the Main Olfactory Epithelium

Terrestrial vertebrates have evolved two anatomically and mechanistically distinct chemosensory structures: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Although it has been generally thought that pheromones are detected through the VNO, whereas other chemicals are sensed by the MOE, recent evidence suggests that some pheromones may be detected through the MOE. Odorant receptors in the MOE are coupled to the type 3 adenylyl cyclase (AC3), an enzyme not expressed in the VNO. Consequently, odorants and pheromones do not elicit electrophysiological responses in the MOE of AC3-/- mice, although VNO function is intact. Here we report that AC3-/- mice cannot detect mouse milk, urine, or mouse pheromones. Inter-male aggressiveness and male sexual behaviors are absent in AC3-/- mice. Furthermore, adenylyl cyclase activity in membranes prepared from the MOE of wild-type mice, but not AC3-/- mice, is stimulated by 2-heptanone, a mouse pheromone. We conclude that signaling through AC3 in the MOE is obligatory for male sexual behavior, male-male aggressiveness, and the detection of some pheromones.

Novel Mutants of the Human β1-Adrenergic Receptor Reveal Amino Acids Relevant for Receptor Activation

Activation of G protein-coupled receptors like the beta(1)-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human beta(1)-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for beta-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at beta(2)- and beta(3)-receptors; however, they act as antagonists at the beta(1)-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the beta(1)-adrenergic receptor for residues that were identical in the beta(2)- and beta(3)-subtypes but different in the beta(1)-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human beta(1)-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation.

Functional expression of metabotropic GABA B receptors in primary cultures of astrocytes from rat cerebral cortex

GABA B receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA B receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA B receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA B receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 μM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA B agonist ( R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 μM GTP. This effect was reversed by the selective GABA B antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA B receptor isoforms (GABA BR1 and GABA BR2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA B receptors are expressed in rat cerebrocortical astrocytes.

Decreased GTP‐stimulated adenylyl cyclase activity in HPRT‐deficient human and mouse fibroblast and rat B103 neuroblastoma cell membranes

Defect of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT), results in Lesch-Nyhan disease (LND). It is unknown how the metabolic defect translates into the severe neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in GTP, UTP and CTP concentrations in HPRT-deficient cells. Moreover, GTP, ITP, XTP, UTP and CTP differentially support Gs-protein-mediated adenylyl cyclase (AC) activation. Based on these findings we hypothesized that abnormal AC regulation may constitute the missing link between HPRT deficiency and the neuropsychiatric symptoms in LND. To test this hypothesis, we studied AC activity in membranes from primary human skin and immortalized mouse skin fibroblasts, mouse Neuro-2a neuroblastoma cells and rat B103 neuroblastoma cells. In B103 control membranes, GTP, ITP, XTP and UTP exhibited profound stimulatory effects on basal AC activity that approached the effects of hydrolysis-resistant nucleotide analogs. In HPRT- membranes, the stimulatory effects of GTP, ITP, XTP and UTP were strongly reduced. Similarly, in human and mouse skin fibroblast membranes we also observed a decrease in GTP-stimulated AC activity in HPRT-deficient cells compared with the respective controls. In mouse Neuro-2a neuroblastoma membranes, AC activity in the presence of GTP was below the detection limit of the assay. We discuss several possibilities to explain the abnormalities in AC regulation in HPRT deficiency that encompass various species and cell types.

A bifunctional Gα i/Gα s modulatory peptide that attenuates adenylyl cyclase activity

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Gα subunits normally bound to GDP and Gβγ. Activated Gα · GTP then regulates effectors such as adenylyl cyclase. Except for Gβγ, no known regulators bind the adenylyl cyclase-stimulatory subunit Gα s in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Gα i. Herein, we report that KB-752 binds Gα s · GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPγS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Gα i and Gα s.

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Level of β-Adrenergic Receptor Kinase 1 Inhibition Determines Degree of Cardiac Dysfunction After Chronic Pressure Overload–Induced Heart Failure

Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.