Receptor-adenylate Cyclase Research Articles

5-HT1B receptor-AC-PKA signal pathway in the lateral habenula is involved in the regulation of depressive-like behaviors in 6-hydroxydopamine-induced Parkinson’s rats

ABSTRACT Objective The aim of the present study was to investigate whether serotonin1B (5-HT1B) receptor-adenylate cyclase (AC)-protein kinase A (PKA) signal pathway in the lateral habenula (LHb) is involved in Parkinson’s disease-related depression in sham-lesioned and substantia nigra pars compacta (SNc)-lesioned rats. Methods The sucrose preference and forced swim tests were used to measure depressive-like behaviors. In vivo electrophysiology and microdialysis were performed to observe the firing activity of LHb neurons and GABA and glutamate release in the LHb, respectively. Western blotting was used to analyze protein expression of 5-HT1B receptors, AC and phosphorylated PKA at threonine 197 site (p-PKA-Thr197) in the LHb. Results Unilateral 6-hydroxydopamine lesions of the SNc in rats induced depressive-like behaviors. Intra-LHb injection of 5-HT1B receptor agonist CP93129 produced antidepressant-like effects and the antagonist SB216641 induced depressive-like behaviors in sham-lesioned and SNc-lesioned rats. Further, pretreatment with AC inhibitor SQ22536 and PKA inhibitor KT5720 blocked the behavioral effects of CP93129 in the two groups of rats, respectively. CP93129 decreased the firing rate of LHb neurons and release of GABA and glutamate, but increased the GABA/glutamate ratio, while SB216641 induced the opposite effects. Compared with sham-lesioned rats, effects of CP93129 and SB216641 on the depressive-like behaviors, electrophysiology, and microdialysis were decreased in SNc-lesioned rats, which were associated with decreased expression of 5-HT1B receptors, AC and p-PKA-Thr197 in the LHb. Conclusion 5-HT1B receptor-AC-PKA signal pathway in the LHb is involved in the regulation of depressive-like behaviors, and depletion of DA reduces activity of 5-HT1B receptor-AC-PKA signal pathway.

Enhanced oral absorption of teriparatide with therapeutic potential for management of osteoporosis

In this study, a system for oral delivery of recombinant human parathyroid hormone [rhPTH(1-34); teriparatide (TRP)] was developed to enhance oral absorption and to demonstrate an equivalent therapeutic effect to that of subcutaneous (SC) TRP injection. The solid oral formulation of TRP was prepared by electrostatic complexation with l-lysine-linked deoxycholic acid (LDA) and deoxycholic acid (DA) at a molar ratio of 1:5:7 in the aqueous dispersion of non-ionic n-dodecyl-β-d-maltoside (DM) at a 1:15 weight ratio, followed by freeze-drying the dispersal, yielding TRP(1:5:7)-15. As expected, TRP(1:5:7)-15 showed a 414% increase in permeability across the Caco-2/HT29-MTX-E12 cell monolayer, resulting in a 13.0-fold greater oral bioavailability compared with free TRP. In addition, the intestinal transport mechanisms in the presence of specific inhibitors of clathrin-mediated endocytosis, macropinocytosis, and bile acid transporters revealed 44.4%, 28.7%, and 51.2% decreases in transport, respectively, confirming that these routes play crucial roles in the permeation of TRP in TRP(1:5:7)-15. Notably, this formulation showed similar activation of the release of cyclic adenosine monophosphate (cAMP) compared with TRP, suggesting equivalent efficacy in the parathyroid hormone receptor-adenylate cyclase system of osteosarcoma cells. Furthermore, oral TRP(1:5:7)-15 (equivalent to 0.4mg/kg TRP) demonstrated increases in bone mineral density (36.9%) and trabecular thickness (31.3%) compared with untreated glucocorticoid-induced osteoporotic mice. Moreover, the elevated levels of biomarkers of bone formation, including osteocalcin, were also comparable with those after SC injection of TRP (0.02mg/kg). These findings suggest that TRP(1:5:7)-15 can be used as an effective oral therapy for the management of osteoporosis.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulatesantioxidant gene expression andphosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclizeATP to enhance the cAMP basal level that regulatesantioxidant geneexpression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression wasupregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KDwas found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

How cAMP Homeostasis is Controlled in Leishmania by Receptor Adenylate Cyclases and Acidocalcisomal Pyrophosphatases Aiding its Survival in Phagolysosomal Conditions?

Leishmania donovani while infecting macrophages is exposed to phagolysosome conditions (PC) (37 °C and pH 5.5) from the sandfly gut environment of 22 °C and pH 7.4. This shift in temperature and pH led to increased intracellular cAMP and cAMP‐mediated PKA activity in the parasites, which help in intra‐macrophage survival, a pre‐requisite for infectivity and transformation to amastigotes. Though cAMP and cAMP‐triggered signaling pathways seemed to be one of the important factors for parasite survival and infectivity, the mechanism of cAMP production and its stringent control is obscure. In the absence of typical orthologs for G‐proteins and G‐protein coupled receptors, the precise mechanisms for positive modulation of cAMP production during exposure to PC seemed an important aspect to be studied. Amongst various membrane bound isoforms of receptor adenylate cyclase, two promastigote‐stage specific membrane bound receptor adenylate cyclases (LdRAC‐A and LdRAC‐B), LdRAC‐A appeared to function as a major cAMP generator following PC exposure. Following PC exposure, pyrophosphate (PPi), an energy storage compound as well as a by‐product of cAMP biosynthesis by adenylate cyclase, was found to be decreased. This may be due to microtubule and microfilament‐driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV‐H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over‐expression and conditional silencing demonstrated regulatory role of V‐H+PPase on cAMP trigger and consequent induction of resistance to macrophage‐derived pro‐oxidants and parasite killing. Moreover, immunofluorescence analysis indicated towards a possible colocalization of LdV‐H+PPase and LdRAC‐A during PC exposure. The results suggested that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC‐A‐driven cAMP generation through depletion of PPi pool by LdV‐H+PPase. For the first time, one of the molecular mechanisms behind the positive modulation of cAMP by leishmanial adenylate cyclases in absence of Gproteins was deciphered by us.Support or Funding InformationDST‐INSPIRE, Department of Science and Technology (DST), Govt of India, DST‐PURSE, Department of Science and Technology (DST), Govt of India, UGC‐SAP, UGC, Govt of IndiaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Catheter-based Renal Denervation as a Treatment for Pulmonary Hypertension: Hope or Hype?

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance (PVR) that causes right ventricular (RV) failure and premature death. This hypertensive condition leads to precapillary pulmonary hypertension, defined as a mean pulmonary artery pressure (mPAP) 25 mmHg, pulmonary capillary wedge pressure 15 mmHg, and PVR > 3 Wood units at rest. The prevalence of PAH is estimated to range from 15 to 50 patients/million, with a female predominance (men:women, 1:2) and 1-year and 5-year survival rates of 85% and 57%, respectively. During the last 15 years, treatments for PAH have been studied in multiple clinical trials, with approval subsequently granted for 9 distinct drugs belonging to 4 pharmacological families: prostanoids (epoprostenol, iloprost, treprostinil), endothelin receptor antagonists (bosentan, ambrisentan, macitentan), phosphodiesterase-5 inhibitors (sildenafil, tadalafil), and adenylate cyclase stimulators (riociguat). Despite these therapeutic advances, many patients with PAH show disabling symptoms and an ominous prognosis, underscoring the need for new therapeutic strategies to combat PAH. The pathophysiology of PAH involves excessive vasoconstriction and vascular remodeling. The increased PVR is initially reversible and is due to an imbalance between vasodilatory and vasoconstrictive agents, but can become permanent in later stages. The vascular remodeling is characterized by intimal thickening and fibrosis and the proliferation and migration of vascular smooth muscle cells, with hypertrophy and fibrosis of the tunica media, inflammation, and in situ thrombosis, which trigger the formation of plexiform and obstructive lesions (the most characteristic finding of PAH), all of which lead to RV remodeling. Given our poor understanding of the pathophysiology of PAH, the development of new preclinical models of PAH and their evaluation using appropriate imaging techniques are essential to further our understanding of PAH.

CAMP Signaling: An Important Event Facilitating Survival and Infectivity of Leishmania.

IntroductionLeishmania donovani encounters striking shift in temperature and pH and these act as the key environmental trigger for differentiation. Moreover, the parasite also faces a huge oxidative stress inside macrophages. We showed that differentiation condition‐exposed parasites become resistant to oxidative damage due to increased intracellular cAMP‐mediated responses.ObjectiveFor comprehensive understanding of cAMP signaling, we studied all the enzymes associated with cAMP metabolism, viz. the synthesizing enzyme adenylate cyclase, the degrading enzyme, phosphodiesterase (PDE), the regulatory enzyme pyrophosphatase and the functional enzyme, cAMP‐dependent protein kinase (PKA).ResultsA stage‐specific isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation‐coupled induction of cAMP. The membrane bound acidocalcisomal pyrophosphatase, LdV‐H+PPase, was the major isoform regulating cAMP level in association with LdRACA by modulating the total pyrophosphate pool. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defence. Another cytosolic phosphodiesterase, LdPDED though not differentially expressed, was found to down‐regulate cAMP‐mediated responses by inhibiting catalytic activity of PKA and by increasing its own PDEase activity through phosphorylation. Also, a functional cAMP‐binding effector molecule (a regulatory subunit of PKA, LdPKAR) seemed to modulate metacyclogenesis through induction of autophagy.ConclusionThis study reveals the significance of cAMP signaling in parasite survival and infectivity.

Correlation of serotonin 2A receptor and adenylate cyclase single gene polymorphisms and bipolar disorder

Objective To explore whether there is association between the polymorphisms of serotonin 2A receptor(5-HT2A) rs6311, rs6305 and adenylate cyclase rs2230739 with episode of bipolar disorder. Methods A case-control association study was done in this study, 152 bipolar disorder patients(patients group), and 187 age- and gender-matched controls (control group) were recruited. Ligase detection reaction(LDR) was used to detect the distributive frequency of rs6311, rs6305 and rs2230739 of 152 bipolar disorder patients and 187 normal people. Results The 5-HT2A receptor rs6311 genotypes and alleles frequency distribution between patients group(11, 12, 22 genotypes: 21%, 61%, 18%; 1, 2 alleles: 51%, 49%) and control group(11, 12, 22 genotypes: 44%, 45%, 11%; 1, 2 alleles: 66%, 34%) showed statistically significance (P<0.01). There were no differences of 5-HT2A receptor rs6305 genotypes and alleles between patients group and control group. The frequency of rs2230739 genotypes and alleles distribution between patients group(11, 12, 22 genotypes: 20%, 43%, 37%; 1, 2 alleles: 42%, 58%) and control group(11, 12, 22 genotypes: 17%, 48%, 35%; 1, 2 alleles: 41%, 59%) showed no significant difference(P=0.661, P=0.846). Conclusion The 5-HT2A receptor rs6311 polymorphism may be responsible for an increase in susceptibility to bipolar disorder, indicating that it maybe an marker to bipolar disorder. Key words: Bipolar disorder; 5-HT2A receptor; Adenylate cyclase; Single nucleotide polymorphisms; Biological marker

Hypothesizing repetitive paraphilia behavior of a medication refractive Tourette's syndrome patient having rapid clinical attenuation with KB220Z-nutrigenomic amino-acid therapy (NAAT).

Background and aims Many patients presenting multiple behaviors including drug and food abuse as well as other pathological repetitive unwanted activities such as gambling, self-mutilation and paraphilias may not be appropriately diagnosed. Here we present a case of a male presenting many of these seemingly diverse behaviors and finally diagnosed with reward deficiency syndrome (RDS) by his attending physician. Methods The use of the dopamine agonist, ropinirole after two weeks showed improvement in terms of sexual behavior but tolerance set in and was discontinued especially when an infraction occurred with the patient's insurance. In this article, we carefully explore the potential of ropinirole to downregulate dopamine receptors causing adenylate cyclase receptor supersensitivity and tolerance a feature of neurotransmitter cross-talk. Based on previous scientific evidence showing KB220Znutrigenomic amino-acid therapy (NAAT) to rapidly (post one-hour) activate dopaminergic pathways in both the pre-frontal cortex cingulate gyrus (relapse loci) and ventral tegmental area-caudate-accumbens-putamen (craving and emotion loci) the patient was prescribed NAAT. Results and discussion Within one week of utilization the repetitive paraphilia was eliminated. There were also a number of other positive effects such as enhanced focus that persisted even after the patient stopped using KB220Z suggesting neuroplasticity (e.g. altruistic thoughts). However, these observed profound benefits require more in-depth study, especially in a large cohort against a placebo. While this report focused on a rapid response rather than long-term benefits previously associated with NAAT, it is somewhat encouraging and longer term required follow-up and larger placebo controlled studies are warranted before any definitive conclusions could be gleaned from this case report.

Role of cAMP Signaling in the Survival and Infectivity of the Protozoan Parasite,Leishmania donovani

Leishmania donovani, while invading macrophages, encounters striking shift in temperature and pH (from 22°C and pH 7.2 to 37°C and pH 5.5), which act as the key environmental trigger for differentiation, and increases cAMP level and cAMP-mediated responses. For comprehensive understanding of cAMP signaling, we studied the enzymes related to cAMP metabolism. A stage-specific and developmentally regulated isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation-coupled induction of cAMP. The soluble acidocalcisomal pyrophosphatase, Ldvsp1, was the major isoform regulating cAMP level in association with LdRACA. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defense. We identified and cloned a functional cAMP-binding effector molecule from L. donovani (a regulatory subunit of cAMP-dependent protein kinase, LdPKAR) that may modulate metacyclogenesis through induction of autophagy. This study reveals the significance of cAMP signaling in parasite survival and infectivity.

Involvement of Secondary Intracellular Messengers in the Mechanisms of Purinergic Inhibition of Intestinal Smooth Muscles

We studied the role of adenylate cyclase and phospholipase C in the control of ATP-induced relaxation of carbachol-evoked contraction of smooth muscles of the guinea-pig taenia coli. We showed that ATP-induced relaxation of carbachol-caused contraction is completely realized under control conditions via activation of inositol trisphosphate-sensitive (InsP3) receptors of the sarcoplasmic reticulum of smooth muscle cells (SMCs). In the case where phospholipase C was blocked, the relaxing action of ATP on smooth muscles continues to be mediated mostly by activation of InsP3 receptors, but other mechanisms begin to participate in this process. Intracellular processes are also involved in ATP-induced relaxation where the signal is transferred from purine receptors via activation of phospholipase C under conditions of parallel activation of adenylate cyclase by forskolin; these processes also include activation of InsP3 receptors of the sarcoplasmic reticulum of SMCs and some other events. After U73122-induced blocking of phospholipase C and forskolin-induced activation of adenylate cyclase, ATP-caused relaxation can completely be removed by an inhibitor of InsP3 receptors, 2-APB. This indicates that, under the above conditions, such relaxation is realized exclusively via InsP3 receptors of the sarcoplasmic reticulum of SMCs. At the same time, ATP-induced relaxation caused by activation of phospholipase C and inactivation of adenylate cyclase is also nearly completely realized with involvement of InsP3 receptors of the sarcoplasmic reticulum. However, removing the activity of phospholipase C under conditions of blocking of adenylate cyclase and InsP3 receptors of the sarcoplasmic reticulum in SMCs leads to the recovery of ATP-induced relaxation with the participation of the other intracellular processes. Therefore, two intracellular messengers, phospholipase C and adenylate cyclase, are involved in purinergic inhibition of smooth muscles. Upon their action, multiple intracellular signal pathways are triggered. The level of their participation can be influenced by the initial functional state of intestinal SMCs. These changes are always directed toward the maintenance of normal functioning of the respective organs.

Effects of Acetaldehyde on Membrane Potentials and Ionic Currents in Single Cardiac Myocytes

The effect of acetaldehyde on membrane potential and ionic currents in guinea-pig single ventricular myocytes using whole-cell voltage-clamp methods was studied. Exposure to acetaldehde at concentrations between 0.3 and 1 mM increased the overshoot and plateau phase of action potentials in a dose-dependent and reversible manner. The duration of action potentials measured at 90% repolarization was prolonged by acetaldehyde. Potassium currents were not affected by acetaldehyde at concentrations up to 1 mM. Voltage-dependent Ca2+ current (ICa) increased significantly when 0.3 mM acetaldehyde was applied. The effect of acetaldehyde on membrane potential and ionic currents was not modified by the β-adrenergic receptor blocker propranolol. Application of isoproterenol increased ICa in the presence of acetaldehyde. The results suggest that the increased ICa produced by acetaldehyde was not mediated through β1-adrenergic receptor or intracellular adenylate cyclase stimulation.

Effect of global transcriptional regulators related to carbohydrate metabolism on organic solvent tolerance in Escherichia coli

The effect of global transcriptional regulators related to carbohydrate metabolism on organic solvent tolerance (OST) in Escherichia coli has been investigated. The OSTs of the E. coli K-12 BW25113 knockout mutants acrA, acrB, cra, crp, cyaA, fnr, and mlc were investigated on the basis of colony forming efficiency on an agar plate overlaid with organic solvents. The knockout mutants of the cyclic AMP receptor protein (Deltacrp) and adenylate cyclase (DeltacyaA) were found to increase their OSTs. However, their OSTs decreased to the level of the wild-type strain when the DeltacyaA cells were grown in the presence of exogenous cyclic AMP. These results indicate that the formation of the cAMP-Crp complex is related to OST. The microbial-adhesion-to-hydrocarbon test on the Deltacrp and DeltacyaA mutants revealed that these mutants bound less abundantly to the organic solvent phase. In the Deltacrp and DeltacyaA mutants, the expression levels of GadB and NuoG increased to the level of the wild type. The OSTs of DeltagadB and DeltanuoG mutants decreased, suggesting that the expressions of these proteins are involved in the increased OST in the Deltacrp and DeltacyaA mutants.

?-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure

To understanding the reversible or irreversible harm to the beta-adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain beta-adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and beta-adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of beta-adrenergic receptor using the ligand [125I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain beta-adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the beta-adrenergic system of the brain is a partly reversible condition.

Effect of the carboxyl terminus of the human VIP2 receptor (VPAC2) on receptor affinity, adenylate cyclase activation and receptor internalization

Effect of the carboxyl terminus of the human VIP2 receptor (VPAC2) on receptor affinity, adenylate cyclase activation and receptor internalization

Effect of the carboxyl terminus of the human VIP2 receptor (VPAC2) on receptor affinity, adenylate cyclase activation and receptor internalization

Effect of the carboxyl terminus of the human VIP2 receptor (VPAC2) on receptor affinity, adenylate cyclase activation and receptor internalization

Adenosine receptor–adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1 receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2 receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2′,5′-dideoxyadenosine. These data suggest that A1 and A2 adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation. Mol. Reprod. Dev. 58:307–317, 2001. © 2001 Wiley-Liss, Inc.

ANTAGONIST AND AGONIST ACTIVITIES OF THE MOUSE AGOUTI PROTEIN FRAGMENT (91–131) AT THE MELANOCORTIN-1 RECEPTOR

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-α-MSH (Ki 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP91–131; the IC50 values for AP91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91–131 with IC50 values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91–131 in the adenylate cyclase and melanin assays. On the other hand, AP91–131 inhibited cell growth similar to α-MSH (IC50 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism.

Subclinical hypothyroidism occurs in a number of children with Down's syndrome (DS). The reason for the mildly elevated plasma thyrotropin (TSH) concentrations is not known. The present study investigated whether decreased TSH bioactivity plays a role in this phenomenon. A retrospective study of plasma specimens from DS children with mildly elevated plasma TSH concentrations and thyroid hormone levels within the reference range, using a TSH receptor-adenylate cyclase mediated bioassay. Strain JP26 Chinese hamster ovary (CHO) cells, stable transfected with the human TSH receptor, were incubated with unfractionated plasma (1/10 diluted in hypotonic incubation medium) of 10 DS children with subclinical hypothyroidism and nine euthyroid children with insulin-dependent diabetes mellitus as controls. cAMP released in the incubation medium was measured by RIA. Mock-transfected CHO cells were used to correct for non-specific CHO response. WHO Second International Reference Preparation of human TSH was dissolved and diluted in pooled normal human plasma and simultaneously bioassayed to match patient and control results. Plasma TSH levels were slightly increased in DS (mean +/- S.D., 6.5+/-1.3 mU/l, reference range 0.4-4.0 mU/l). Plasma TSH levels for controls (1.3+/-0.4 mU/l) were within the reference range. Plasma thyroid hormone levels in patients and controls were normal, plasma TSH binding inhibitory immunoglobulin and thyroid peroxidase antibodies were negative. cAMP levels (corrected for non-specific CHO response) in DS patients (18.4+/-3.9 pmol/well) and in controls (14.3+/-1.3 pmol/well) did not significantly differ from cAMP levels generated by patient-TSH equivalent TSH standards (16.3+/-0.9 pmol/well). The present results demonstrate normal TSH bioactivity in plasma of DS children, indicating that subclinical hypothyroidism in these patients is of primary (thyroidal) origin.

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.

Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.