Stimulatory G-protein Research Articles

Sensory receptors—design principles revisited

Sensory receptors—design principles revisited

Control of CYP11B2/CYP11B1 Expression Ratio and Consequences for the Zonation of the Adrenal Cortex

Access of corticotropin to glucocorticoid synthesis in adrenocortical cells is provided by the expression of the ACTH receptor (MC2R). Activation of the MC2R increases stimulatory G-protein, adenylyl cyclase, and protein kinase A (PKA) activities. Furthermore, PKA phosphorylates transcription factors that have a stimulating effect on glucocorticoid synthesis. Sensitivity of adrenocortical cells to renin/angiotensin-2 is conferred by the expression of the inhibitory G-protein-linked angiotensin-2 type 1 receptor (AT1R) that additionally associates to the phospholipase C-activating G-protein q. The AT1R is connected to the adrenal potassium sensory system and regulates calcium influx as well as phospholipase C-β (PLC-β) and thus calmodulin kinase-dependent transcription of steroidogenic enzymes. While AT1R signaling suppresses the influence of corticotropin on the generation of cyclic adenosine monophosphate, the expression of the AT1R and its associated enzyme activities are under the control of glucocorticoids. Thus, dominance of one of the two signaling pathways is dependent on two factors: the extracellular concentration of their ligands and the products of their signaling pathways. These findings are in favor of the hypothesis that the centripetal blood flow through the adrenal gland builds up a glucocorticoid gradient creating a morphogenetic field along which adrenal cortical cells adopt different functional states, leading to the typical zonation of the adrenal cortex.

Beta-adrenergic adaptation in idiopathic dilated cardiomyopathy: differences between children and adults

This editorial refers to ‘Beta-adrenergic adaptation in paediatric idiopathic dilated cardiomyopathy’, by S.D. Miyamoto et al. , doi:10.1093/eurheartj/ehs229 The study of Miyamoto et al .1 found differences in beta-adrenergic adaptation to heart failure (HF) in explanted heart tissue between children and adults with symptomatic dilated cardiomyopathy (DCM). Differences included: (i) down-regulation of beta-1- and beta-2-adrenergic receptors in children but maintained beta-2-adrenergic receptor expression in adults; and (ii), in children, uncoupling of the beta-1-adrenergic receptors and stimulatory G-proteins, among others.1 An important family of cell surface protein receptors, the G-protein-coupled receptors (GPCRs), enable cells to sense and respond to outside signals. For children with cardiomyopathy and heart failure, we need a better understanding of the structure–function relationships and the detailed regulation of GCPRs, as well as the drugs that stimulate or inhibit these relationships. Understanding beta-adrenergic adaptation in children with HF should indicate how cardiomyocytes interact with their extracellular environment and adapt to new situations, such as when epinephrine acts on cells to increase blood pressure and heart rate. Miyamoto and colleagues observed that the GCPR signalling system has become the target of prescription drugs, including beta-blockers, to relieve HF.1 In fact, about half of all medications act on these receptors, so learning about them is key to developing better drugs. The GPCR initiates a cascade of signals into the cell, prompting it to respond appropriately, as well as a secondary cascade in which a feedback signal leads to desensitization. A molecule called beta-arrestin binds to the GPCR and reduces or temporarily stops GPCR's sensitivity. Beta-arrestin, in addition to receptor desensitization, is involved in receptor endocytosis and activating extracellular signal-regulated kinase (ERK) and other …

High Levels of Circulating Epinephrine Trigger Apical Cardiodepression in a β 2 -Adrenergic Receptor/G i –Dependent Manner

Background— Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β 2 -adrenergic receptor (β 2 AR) from canonical stimulatory G-protein–activated cardiostimulant to inhibitory G-protein–activated cardiodepressant pathways. Methods and Results— We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G i inactivation by pertussis toxin pretreatment. β 2 AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a β 2 AR-Gi–dependent manner. Preventing epinephrine-G i effects increased mortality in the Takotsubo model, whereas β-blockers that activate β 2 AR-G i exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. Conclusions— We suggest that biased agonism of epinephrine for β 2 AR-G s at low concentrations and for G i at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β 2 ARs explaining the differential regional responses. We suggest this epinephrine-specific β 2 AR-G i signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Basic Science of Cardiac Resynchronization Therapy

Electromechanical dyssynchrony can markedly worsen heart failure (HF) morbidity and mortality, independent of traditional risk factors.1–4 Depending on the metric used, current estimates of the prevalence of dyssynchrony vary from 25–30% in patients with HF (based on QRS widening) up to 60%, based on tissue Doppler or MRI measures of dyssynchronous contraction of the left ventricle (LV).5,6 Cardiac resynchronization therapy (CRT) or biventricular pacing has emerged as a promising option to treat patients with HF and dyssynchronous contraction.7–9 The past few decades have seen the rise of pharmacotherapy, primarily through agents that antagonize the effect of excessive concentrations of circulating neurohormones, yet, HF-related morbidity and mortality remain high.3–6 Biventricular stimulation has been demonstrated to improve contractile performance in patients with mechanical dyssynchrony acutely and chronically while also prolonging long-term survival—something not yet achieved by drug therapy.10 Although the clinical and mechanical effectiveness of CRT are well described, 30% of patients do not benefit from CRT and clinical criteria to identify CRT nonresponders remain elusive8,11,12 Currently, the most widely used predictor of reverse remodeling is the presence of marked mechanical dyssynchrony before CRT, as indexed by the width of the QRS.13 Mechanical dyssynchrony seems important, yet imaging-based measures have not predicted response well14 and even improvement in dyssynchrony after initiation of CRT only weakly predicts chronic response.15 Limited understanding of the molecular mechanisms underlying reverse cardiac remodeling induced by CRT has hampered the selection of potential responders. In this review, we focus on the electrophysiological aspects and molecular networks underlying the benefits of CRT. We will review how CRT homogenizes regional differences in stress kinase signaling and electric remodeling and then review its global effect on myocyte function and its …

Ser/ Thr residues at α3/β5 loop of Gαs are important in morphine‐induced adenylyl cyclase sensitization but not mitogen‐activated protein kinase phosphorylation

The signaling switch of β2-adrenergic and μ(1) -opioid receptors from stimulatory G-protein (G(αs) ) to inhibitory G-protein (G(αi) ) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1/2 activation. Post-translational modifications, including dephosphorylation of G(αs) , enhance opioid receptor coupling to G(αs) . In the present study, we substituted the Ser/Thr residues of G(αs) at the α3/β5 and α4/β6 loops aiming to study the role of G(αs) lacking Ser/Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC(50) = 22.8 ± 3.4 μm) in G(αs) -transfected S49 cyc- cells but not in nontransfected cells. However, there was no significant difference between the G(αs) -wild-type (wt) and mutants. Morphine (10 μm) inhibited AC activity more efficiently in cyc- compared to G(αs) -wt introduced cells (P < 0.05); however, we did not find a notable difference between G(αs) -wt and mutants. Interestingly, G(αs) -wt transfected cells showed more sensitization with respect to AC after chronic morphine compared to nontransfected cells (101 ± 12% versus 34 ± 6%; P < 0.001); μ1-opioid receptor interacted with G(αs) , and both co-immunoprecipitated after chronic morphine exposure. Furthermore, mutation of T270A and S272A (P < 0.01), as well as T270A, S272A and S261A (P < 0.05), in α3/β5, resulted in a higher level of AC supersensitization. ERK1/2 phosphorylation was rapidly induced by isoproterenol (by 9.5 ± 2.4-fold) and morphine (22 ± 2.2-fold) in G(αs) -transfected cells; mutations of α3/β5 and α4/β6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that G(αs) interacts with the μ1-opioid receptor, and the Ser/Thr mutation to Ala at the α3/β5 loop of G(αs) enhances morphine-induced AC sensitization. In addition, G(αs) was required for the rapid phosphorylation of ERK1/2 by isoproterenol but not morphine.

Diagnosis and Management of Precocious Puberty in Atypical Presentations of McCune-Albright Syndrome: A Case Series Review

Background McCune-Albright syndrome is a rare syndrome, classically defined as the triad of precocious puberty, fibrous dysplasia of bone, and café au lait lesions. Partial or atypical presentations of McCune-Albright syndrome, with only one or two of the classic symptoms, have been described in the literature and remain particularly challenging due to lack of diagnostic phenotype. In these patients, the utility of analysis of mutations in the gene of the α subunit of the stimulatory G-protein is limited and so the diagnosis is often based on clinical judgment. Three atypical cases of suspected McCune-Albright syndrome with gonadotropin-independent precocious puberty are presented. Cases Case #1: A 5-year-old female presented with painlesss vaginal bleeding and was found to have café au lait lesions. She was diagnosed with gonadotropin independent precocious puberty with eventual progression to gonadotropin dependent precocious puberty which was successfully treated with both letrozole and gonadotropin-releasing hormone agonist therapy. Case #2: A 3-year-old female presented with painless vaginal bleeding and was found to have café au lait lesions. She was diagnosed with gonadotropin independent precocious puberty and was successfully treated with letrozole. Case #3: A 5-year-old female presented with fibrous dysplasia and was found to have evidence of uterine and ovarian enlargement on ultrasound. She was diagnosed with gonadotropin-independent precocious puberty and was successfully treated with letrozole. Summary and Conclusion Although different in presentation, all three atypical cases of suspected McCune-Albright syndrome with gonadotropin-independent precocious puberty were successfully treated with aromatase inhibitors. This small case series shows the utility and efficacy of aromatase inhibitors in the setting of atypical cases of suspected MAS with gonadotropin-independent precocious puberty.

Mechanism for XMRV neurotoxicity

Xenotropic murine leukemia virus-related virus (XMRV) has been found in a high percentage of humans with chronic fatigue syndrome (CFS). However, more recent studies have failed to confirm these results, and it now appears likely that the original findings were due to patient sample contamination. Because it initially appeared that XMRV was involved in CFS, we explored potential mechanisms of XMRV neurotoxicity that might underliethe neuromuscular pathology seen in CFS. Indeed, we found that XMRV infection induced apoptosis in SY5Y human neuroblastoma cells. We hypothesized that signaling through the cell-entry receptor for XMRV, the xenotropic and polytropic retrovirus receptor (Xpr1), mediated this toxicity. In support of this hypothesis, SY5Y cells expressing mouse Xpr1, which unlike human Xpr1 does not bind or promote entry of xenotropic retroviruses, were resistant to XMRV toxicity, even though XMRV could still infect these cells. Similarly, SY5Y cells expressing several XMRV binding-defective deletion mutants of human Xpr1 were resistant to XMRV toxicity. These results indicate that Xpr1 mediates the toxicity of XMRV. Xpr1 is related the yeast Syg1 protein, which associates with the β subunit of the yeast G-protein. We found that human Xpr1 is also associated with the human Gβ subunit, and that over expression of mouse or human Xpr1 increased intracellular cAMP, a typical output of stimulatory G-protein signaling. Moreover, increasing the cAMP level in SY5Y cells by direct activation of adenylate cyclase protected the SY5Y cells from the toxic effects of XMRV and polytropic retrovirus infection. These results indicate that Xpr1 is a G-protein-coupled receptor (GPCR), and that xenotropic or polytropic retrovirus binding can disrupt the cAMP-mediated signaling function of Xpr1 leading to apoptosis of infected cells. In addition, we found that this pathway is responsible for the toxicity of the polytropic mink cell focus-forming (MCF) retrovirus in mink cells, the basis for the classic MCF focus assay.Xpr1 orthologs are widely distributed in animals, plants and unicellular organisms, but these proteins show no sequence similarity to known GPCRs. Some proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found no role of Xpr1 in phosphate uptake or its regulation. Lastly, some polytropic retroviruses induce neurologic disease in mice, and we propose that alterations of Xpr1-mediated G-protein signalinglikely are responsible. However, because of recent results indicting that XMRV is not a human retrovirus, a role for XMRV in human disease is unlikely.

Chronic sympathetic activation promotes downregulation of β-adrenoceptor-mediated effects in the guinea pig heart independently of structural remodeling and systolic dysfunction

It is uncertain if downregulation of β-adrenoceptor signaling pathway is promoted by an enhanced adrenergic tone at an early stage of cardiac disease, or it develops secondary to detrimental local myocardial changes in advanced heart failure. We examined the integrity of β-adrenoceptor signaling pathway upon chronic infusion of isoproterenol, a β-adrenoceptor agonist, at a dose producing no structural left ventricular (LV) remodeling and systolic dysfunction. Subcutaneous isoproterenol infusion (400μgkg(-1)h(-1) over 16days) to guinea pigs using osmotic minipumps produced no change in cardiac weights, LV internal dimensions, myocyte cross-sectional area, extent of interstitial fibrosis, and basal contractile function. Isolated, perfused heart preparations from isoproterenol-treated guinea pigs exhibited attenuated responsiveness to acute β-adrenoceptor stimulation, as evidenced by reduced LV developed pressure increase, less shortening of LV epicardial monophasic action potential and effective refractory period, and less myocardial cyclic adenosine monophosphate elevation, in response to isoproterenol exposure, when compared to saline-treated controls. Pharmacological responses to forskolin, an activator of the adenylate cyclase catalytic subunit, were well preserved in isoproterenol-treated hearts. Downregulation of β-adrenoceptor-mediated effects upon chronic isoproterenol infusion was associated with markedly reduced stimulatory G-protein α-subunit (G(sα)) myocardial expression levels. No change in expression levels of β-adrenoceptors, G-protein-coupled receptor kinase 2, inhibitory G-protein α-subunit (G(iα2)), and Ca(v)1.2 and K(v)7.1 ion channels was determined in isoproterenol-treated hearts. We therefore conclude that sustained adrenergic overstimulation may promote downregulation of myocardial β-adrenoceptor-mediated effects independently of structural LV remodeling and systolic failure, an effect attributed to β-adrenoceptor uncoupling from adenylate cyclase due to reduced G(sα)-protein expression.

Current questions of thyroid diseases in childhood

In recent years our knowledge on thyroid diseases in childhood has been increased. Several forms of congenital hypothyroidism (dysgenesis, dyshormongenesis, thyrotropin resistance and some central forms) are consequences of gene mutations. Maternal hypothyroxinemia due to severe iodine deficiency leads to early neurological damage and congenital hypothyroidism. Neonatal screening of congenital hypothyroidism and early treatment with l-thyroxin ensure good prognosis. Differential diagnosis of the various forms of congenital hypothyroidism in newborns is not an easy task. The need for treatment of transient hypothyroxinemia is still controversial. Diagnosis of juvenile lymphocytic thyroiditis can be ascertained by the clinical status, ultrasound examination, detection of anti-peroxydase antibodies, evaluation of thyroid function, and fine needle aspiration cytology. L-thyroxin therapy is recommended in cases of subclinical and manifest hypothyroidism. The transient form of the rare newborn hyperthyroidism is the consequence of maternal Graves-Basedow disease. It can be a sever condition and its permanent form is caused by TSH-receptor gene mutation. In the pathogenesis of autonomic thyroid adenoma mutations of the TSH-receptor and the alpha subunit of the stimulatory G-protein are involved. Treatment of Graves-Basedow disease in childhood is a debated question. The first choice is medical treatment with antithyroid and beta-blocking drugs. However, remission rate is low under this therapy, and the disease is characterised by frequent relapses. For this reason, the necessity of definitive therapy frequently arises. In Europe subtotal thyroidectomy is used as second choice of therapy, but clinical experience in the United States showed that radioiodine treatment is a safe and effective therapy for children and adolescents. Iodine deficient goitre in childhood is a form of iodine deficiency disorder. It is the consequence of adaptation to iodine deficiency. It can be treated by iodine or/and l-thyroxin, and its development can be prevented by iodinated salt. In childhood, thyroid nodule needs for a detailed investigation because of the possibility of thyroid cancer. Medullar thyroid carcinoma indicates genetic screening in the patients and their family, and the presence of disease-causing RET-proto-oncogene mutation confirms the need for total thyroidectomy already in childhood.

Beta-2-receptor regulation of immunomodulatory proteins in airway smooth muscle

Airway smooth muscle (ASM) cells have been shown to secrete significant amounts of immunomodulatory factors (IMFs), many of which are typically ascribed to trafficking leukocytes (e.g., GM-CSF, IL-6, IL-13, and eotaxin), and may be indicative of an immunomodulatory role for ASM in control of airway function, as well as in airway diseases states associated with acute and/or chronic inflammation, such as asthma and COPD. Furthermore, epinephrine analogues such as albuterol, which ligate the G-protein coupled beta-2-receptor and have been clinically applied to promote ASM relaxation and bronchodilation in the treatment of asthma and COPD, also have been reported to downregulate IMF release by ASM, both individually and in additive fashion, in combination with corticosteroids. Based on experimental data, an inverse agonist/agonist model is proposed to explain these behaviors modeled on cell stimulatory states and G-protein coupled receptor activation. The ramifications of the model are considered in light of unexplained paradoxical clinical findings, and may provide a model for the understanding of beta-2-receptor agonist modulation of airway inflammation and function.

Identification of a Novel Mutation in a Pseudohypoparathyroidism Family

Pseudohypoparathyroidism type Ia (PHP Ia) is defined as a series of disorders characterized by multihormone resistance in end-organs and Albright hereditary osteodystrophy (AHO) phenotype. PHP Ia is caused by heterozygous inactivating mutations in GNAS, which encodes the stimulatory G-protein alpha subunit (Gsa). A patient with typical clinical manifestations of pseudohypoparathyroidism (PHP) (round face, short stature, centripetal obesity, brachydactyly, and multi-hormone resistance: parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), and gonadotropins) presented at our center. The sequence of the GNAS gene from the patient and her families revealed a novel missense mutation (Y318H) in the proband and her mother. An in vitro Gsa functional study showed that Gsa function was significantly impaired. These results stress the importance of GNAS gene investigation.

The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways.

Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsα subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsα and adenylate cyclase and to lead to the autonomous synthesis of cAMP. Gpa33tm1(GnasR201C)Wtsi/+ mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months but there was a significant increase in adenoma formation when Gpa33tm1(GnasR201C)Wtsi/+ mice were bred onto an ApcMin/+ background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK, and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis.

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a beta-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca(2+) sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased beta myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.

Research Highlights

Research Highlights

Involvement of signaling pathways in bovine sperm motility, and effect of ergot alkaloids

There is evidence that ergot alkaloids can directly interact with mammalian spermatozoa affecting sperm functions. Ergot alkaloids exert their toxic or pharmaceutical effects through membrane receptor-mediated activities. This study investigated the signaling pathways involved in the in vitro inhibitory effects of both ergotamine (ET) and dihydroergotamine (DEHT) on the relative motility of bovine spermatozoa using specific inhibitors. Motile bovine spermatozoa were prepared using a Percoll gradient and incubated with ergot alkaloids with and without signaling pathway inhibitors. Co-incubation of ET or DHET with 100 microM prazosin (alpha 1-adrenergic receptor inhibitor) decreased (p < 0.05) relative motility of spermatozoa when compared with controls. In addition, preincubation of spermatozoa with 10 or 20 microM prazosin and DHET also reduced (p < 0.05) the number of motile spermatozoa. Relative sperm motility (motility of treated spermatozoa normalized to control sperm motility) was increased (p < 0.05) when co-incubations included ET and yohimbine (alpha 2-adrenergic receptor inhibitor); conversely, co-incubation of yohimbine (100 microM) and DHET decreased (p < 0.05) the percentage of motile spermatozoa when compared with controls. Pertussis toxin and cholera toxin (effectors of inhibitory and stimulatory G-proteins, respectively) altered (p < 0.05) relative sperm motility in a concentration dependent manner; however, co-incubation of pertussis or cholera toxin with ergot alkaloids had no interactive (p = 0.83) effects on the relative motility of spermatozoa. Co-incubation of Rp-cAMP (a membrane-permeable cAMP inhibitor) with 50 microM DHET had no effect (p > 0.05) on relative sperm motility; whereas, the co-incubation of 22.4 or 44.8 microM Rp-cAMP with 50 microM ET increased (p < 0.05) the percentage of motile spermatozoa when compared with 0 or 224 microM Rp-cAMP (49%, 65%, 59%, and 54%, respectively, for 0, 22.4, 44.8, and 224 microM of Rp-cAMP. An interaction between BAPTA-AM (a chelator of intracellular calcium) and alkaloids also impacted (p < 0.05) relative sperm motility. Generally, co-incubating spermatozoa with BAPTA-AM and ET increased the percentage of motile spermatozoa; however, co-incubation with DHET decreased relative sperm motility except with 41 microM BAPTA-AM. Collectively, these observations suggest that ET and DHET decreased the percentage of motile bovine spermatozoa via alpha adrenergic receptors. However, the second messenger systems involved with ergot alkaloid inhibition of relative motility of bovine spermatozoa remain to be elucidated.

Epigenetics and Imprinted Genes: Insights from the Imprinted Gnas Locus

The hallmarks of epigenetics – the memory of defining earlier developmental events and the distinction of active and inactive genes – are exemplified by imprinted genes. In this article, I shall consider the imprinted Gnas locus in some detail. Gnas encodes the stimulatory G-protein subunit, Gsα, an essential intermediate between receptor coupling and cyclic adenosine monophosphate generation. It provides an excellent illustration of the pleiotropic effects of imprinted genes, particularly on skeletal growth and metabolism, and is a powerful example of the conflicting effects of imprinted genes with opposing patterns of imprinting. I shall describe the effects of Gsα deficiency in humans and the knowledge gained from genetic manipulation in the mouse. Finally, given the pervasive effects of imprinted genes, I shall discuss the likelihood that epigenetic deregulation, for example of imprinted genes, could contribute to the developmental programming of chronic adult diseases.

G-Protein and Signalling in Pituitary Tumours

The genesis of pituitary tumours is still under debate. Although these neoplasias are monoclonal in origin, mutations of GNAS1, the gene encoding the α subunit of the stimulatory G-protein, Gs, are the only mutational changes unequivocally associated with growth hormone (GH)-secreting adenomas. However, despite the growth advantage that this oncogene has been demonstrated to confer in vitro, patients carrying this mutation have a similar clinical and biochemical phenotype to those who do not carry it. This discrepancy is due to the occurrence of events able to counteract the biological effect of the mutation. Consistent with a potential role of the cyclic adenosine monophosphate pathway in the proliferation of somatotrophs, germline mutations of the gene encoding the type 1α regulatory subunit of protein kinase A (PRKAR1A) have been found in patients with the Carney complex, a syndrome including GH-secreting adenomas, whereas alterations in the expression levels of this subunit are frequently observed in sporadic adenomas.

The Neuropeptides Orexin a and B Have An Impact on Functional Properties of Human CD34+ Stem and Progenitor Cells.

Orexin receptors play a role in regulation of sleep-wake-rhythm, food intake and energy homeostasis and they were long thought to be exclusively expressed in the nervous system. During the last years orexin receptors are being identified in a growing number of peripheral tissues. We have earlier detected orexin receptor 1 and 2 expression on human CD34+ blood stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seem to be restricted to the central nerve system to this date. The main downstream signaling pathways of the orexin receptors include Ca2+-dependent signaling associated with activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways. In an attempt to investigate if the receptors are functionally active in CD34+ stem and progenitor cells, we used live cell calcium imaging and stimulated purified CD34+ stem and progenitor cells with orexin A and B. Upon stimulation a massive intracellular calcium release was seen which could not been detected using cells preincubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the selective OX1R-Antagonist SB334867 and CD34 negative cells. Additionally, upon stimulation with orexin A and B we found ERK (1/2) activation at a maximum 3 hours following incubation with orexin A whereas no effect was seen after stimulation with orexin B. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. So far, no effects of orexin stimulation on the proliferation and apoptosis of CD34+ cells were apparent. Remarkably, stimulation with orexin A and B led to a significantly higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors BFU-E (burst forming unit erythrocyte) and CFU-E (colony forming unit erythrocyte). A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significantly higher frequency of LTC-IC (long-term-culture initiating cells) indicating also a more immature phenotype of orexin-stimulated cells and a greater repopulating capacity. The selective orexin receptor antagonist SB-334867 abrogated these effects. No differences could be observed regarding the migration towards SDF-1 with and without stimulation with orexin A and B. Still, orexin A and B led to a decrease in the adhesive capacity of CD34+ stem and progenitor cells to fibronectin coated dishes. Since orexin receptors are coupled to inhibitory G-proteins (Gi/q) and stimulatory G-proteins (Gs) dependent on the tissue, we incubated CD34+ cells with the selective inhibitor of Gi – proteins pertussis toxin concurrently to stimulation with orexins and observed no differences in the adhesive capacity of CD34+ cells compared to the unstimulated controls suggesting coupling of the orexin receptor 1 and 2 to Gi – proteins rather than Gs-proteins in CD34+ cells. Given this functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using ELISA we did not find autocrine production of orexin by CD34+ cells whereas orexin could be detected in the serum obtained by bone marrow biopsies and peripheral blood pointing rather towards a humoral delivery of orexins to CD34+ cells. Taken together, our findings indicate a functional role of the orexin system in CD34+ stem and progenitor cells.

Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial β-Adrenergic Signaling

Although beta-adrenergic stimuli are essential for myocardial contractility, beta-blockers have a proven beneficial effect on the treatment of heart failure, but the mechanism is not fully understood. The stimulatory G protein alpha-subunit (Gsalpha) couples the beta-adrenoreceptor to adenylyl cyclase and the intracellular cAMP response. In a mouse model of conditional Gsalpha deficiency in the cardiac muscle (Gsalpha-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I (cTnI-ND) from restricted removal of the cTnI-specific N-terminal extension. To investigate the functional significance of the increase of cTnI-ND in Gsalpha-DF cardiac muscle, we generated double transgenic mice to overexpress cTnI-ND in Gsalpha-DF hearts. The overexpression of cTnI-ND in Gsalpha-DF failing hearts increased relaxation velocity and left ventricular end diastolic volume to produce higher left ventricle maximum pressure and stroke volume. Supporting the hypothesis that up-regulation of cTnI-ND is a compensatory rather than a destructive myocardial response to impaired beta-adrenergic signaling, the aberrant expression of beta-myosin heavy chain in adult Gsalpha-DF but not control mouse hearts was reversed by cTnI overexpression. These data indicate that the up-regulation of cTnI-ND may partially compensate for the cardiac inefficiency in impaired beta-adrenergic signaling.