Protein Gsα Research Articles

L-Carnitine Ameliorates the Decrease of Aquaporin 2 Levels in Rats with Cisplatin-Induced Kidney Injury

Background: It has been found that L-carnitine ameliorated cisplatin-induced acute kidney injury (AKI) in rats. However, the detailed role of L-carnitine in improving the renal urinary concentration function in cisplatin-induced AKI is not fully understood. Methods: In this study, 30 Sprague-Dawley rats were divided randomly into 5 groups: control, cisplatin (CIS), L-carnitine (CAR), L-carnitine plus cisplatin (CAR + CIS), and cisplatin plus L-carnitine (CIS + CAR) groups. Cisplatin (7 mg/kg) and L-carnitine (300 mg/kg) were injected intraperitoneally. Urine (24 h) and blood samples were collected to analyze renal urinary concentrating function. Immunoblotting, confocal laser microscopy, and enzyme-linked immunosorbent assays were used to assess the level and localization of the water channel aquaporin (AQP) 2, and levels of stimulatory G protein α subunit (GSα protein), arginine vasopressin (AVP) receptor 2, adenylyl cyclase and serum AVP. Results: Renal urinary concentrating function was improved by L-carnitine in rats with cisplatin-induced AKI. AQP2 expression, which decreased after cisplatin treatment, was improved by L-carnitine in different regions of the kidney. Moreover, our data indicated that L-carnitine could increase AQP2 accumulation at the apical plasma membranes of the renal-collecting ducts. Finally, intervention with L-carnitine effectively improved the expression of AQP2 upstream signaling proteins, such as GSα protein, adenylyl cyclase, and serum AVP levels in rats with cisplatin-induced AKI. Conclusion: L-carnitine resolves the cisplatin-induced urinary concentration defect, which may occur by increasing AVP/cyclic adenosine monophosphate/AQP2 levels, indicating the potential use of L-carnitine to ameliorate the renal urinary concentration effect in cancer patients treated with cisplatin.

Pseudohypoparathyroidism: one gene, several syndromes.

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several sites. GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. Heterozygous inactivating mutations involving the maternal GNAS exons 1-13 cause PHP type Ia (PHP1A). Because of much reduced paternal Gsα expression in certain tissues, such as the proximal renal tubules, thyroid, and pituitary, there is little or no Gsα protein in the presence of maternal GNAS mutations, thus leading to PTH-resistant hypocalcemia and hyperphosphatemia. When located on the paternal allele, the same or similar GNAS mutations are the cause of PPHP. Besides biochemical abnormalities, patients affected by PHP1A show developmental abnormalities, referred to as Albrights hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss-of-methylation (LOM) at exon A/B alone or at all maternally methylated GNAS exons. LOM at exon A/B and the resulting biallelic expression of A/B transcripts reduces Gsα expression, thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, the most frequent disease variant, which remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

Genetic and epigenetic alterations in the GNAS locus and clinical consequences in Pseudohypoparathyroidism: Italian common healthcare pathways adoption.

BackgroundGenetic and epigenetic alterations in the GNAS locus are responsible for the Gsα protein dysfunctions causing Pseudohypoparathyroidism (PHP) type Ia/c and Ib, respectively. For these heterogeneous diseases characterized by multiple hormone resistances and Albright’s Hereditary Osteodystrophy (AHO) the current classification results inadequate because of the clinical overlap between molecular subtypes and a standard clinical approach is still missing.In the present paper several members of the Study Group Endocrine diseases due to altered function of Gsα protein of the Italian Society of Pediatric Endocrinology and Diabetology (ISPED) have reviewed and updated the clinical-molecular data of the largest case series of (epi)/genetically characterized AHO/PHP patients; they then produced a common healthcare pathway for patients with these disorders.MethodsThe molecular analysis of the GNAS gene and locus identified the causal alteration in 74 subjects (46 genetic and 28 epigenetic mutations). The clinical data at the diagnosis and their evolution during up to 15 years follow-up were collected using two different cards.ResultsIn patients with genetic mutations the growth impairment worsen during the time, while obesity prevalence decreases; subcutaneous ossifications seem specific for this group. Brachydactyly has been detected in half of the subjects with epigenetic alterations, in which the disease overts later in life, often with symptomatic hypocalcaemia, and also early TSH and GHRH resistances have been recorded.ConclusionsA dedicated healthcare pathway addressing all these aspects in a systematic way would improve the clinical management, allowing an earlier recognition of some PHP features, the optimization of their medical treatment and a better clinical-oriented molecular analysis. Furthermore, standardized follow-up data would provide new insight into less known aspects.

From pseudohypoparathyroidism to inactivating PTH/PTHrP signalling disorder (iPPSD), a novel classification proposed by the EuroPHP network.

Disorders caused by impairments in the parathyroid hormone (PTH) signalling pathway are historically classified under the term pseudohypoparathyroidism (PHP), which encompasses rare, related and highly heterogeneous diseases with demonstrated (epi)genetic causes. The actual classification is based on the presence or absence of specific clinical and biochemical signs together with an in vivo response to exogenous PTH and the results of an in vitro assay to measure Gsa protein activity. However, this classification disregards other related diseases such as acrodysostosis (ACRDYS) or progressive osseous heteroplasia (POH), as well as recent findings of clinical and genetic/epigenetic background of the different subtypes. Therefore, the EuroPHP network decided to develop a new classification that encompasses all disorders with impairments in PTH and/or PTHrP cAMP-mediated pathway. Extensive review of the literature was performed. Several meetings were organised to discuss about a new, more effective and accurate way to describe disorders caused by abnormalities of the PTH/PTHrP signalling pathway. After determining the major and minor criteria to be considered for the diagnosis of these disorders, we proposed to group them under the term 'inactivating PTH/PTHrP signalling disorder' (iPPSD). This terminology: (i) defines the common mechanism responsible for all diseases; (ii) does not require a confirmed genetic defect; (iii) avoids ambiguous terms like 'pseudo' and (iv) eliminates the clinical or molecular overlap between diseases. We believe that the use of this nomenclature and classification will facilitate the development of rationale and comprehensive international guidelines for the diagnosis and treatment of iPPSDs.

McCune-Albright Syndrome with Hypophosphatemic Rickets

Fibrous dysplasia (FD) is sometimes accompanied by extraskeletal manifestations that can include any combination of cafe-au-lait macules, hyperfunctioning endocrinopathies, such as gonadotropin-independent precocious puberty, hyperthyroidism, growth hormone excess, FGF23-mediated renal phosphate wasting, and/or Cushing’s syndrome, as well as other less common features. The combination of any of these findings, with or without FD, is known as McCune-Albright syndrome (MAS). The broad spectrum of involved tissues and the unpredictable combination of findings is because of a molecular defect due to dominant activating mutations in the widely expressed signalling protein Gsα. These mutations arise sporadically, often early in development, prior to gastrulation and can distribute across many or few tissues. 1,2 We present a case of a 3½ year-old-girl who presented simultaneously with precocious puberty and hypophosphatemic rickets, along with fibrous dysplasia and cafe au lait macules.

Pseudohypoparathyroidism: phenotypic spectrum in kindred

Pseudohypoparathyroidism (PHP) encompasses a heterogeneous group of disorders due to an inactivating mutation in the GNAS gene which encodes the α subunit of Gs proteins (Gsα). Gsα plays a crucial role in intracellular signal transduction of peptides, hormones and neurotransmitter receptors in multiple tissues. Key features of PHP include Albright Hereditary Osteodystrophy (AHO) and biochemical evidence of multiple hormone resistances. There are several conflicting mechanisms for its heterogeneity; a possible explanation is a tissue-specific differential imprinting of Gsα protein. PHP type1a has AHO with multiple hormone resistance and is inherited as maternal imprinting defect. PHP type 1b presents with hormone resistance but no AHO features and is probably due to epigenetic methylation defects. Peudopseudohypoparathyroidism is another subtype with AHO but absence of hormone resistance and is inherited as paternal inactivating mutation. We describe a family with female members having PHP with variable clinical and biochemical features. Two female siblings from non-consanguineous parents, patient A (15 year) & patient B (13 year) presented at birth with raised TSH levels and were diagnosed to have congenital hypothyroidism and treated with thyroxine replacement. Patient A was noted to have infantile obesity with short stature and evolving phenotypic features consistent with AHO. Further history revealed that her sister patient B has similar phenotypic appearance and was confirmed on clinical exam. Biochemical evaluation for Patient A showed a borderline low calcium and normal phosphate level with elevated PTH while Patient B had normal calcium and phosphate levels with high PTH. Both patients entered puberty at 11 years and patient A progressed to breast tanner 4 but has no menarche at 15 years despite pubertal gonadotropin levels (Table ​(Table1),1), advanced bone age (18 years) & mature uterus. Patient B is currently 12.5 years old and has progressed to tanner 3 breasts with no menarche. Mother exhibits features of AHO without hormone resistance. The father and younger brother do not have any features of AHO. Table 1 The two patients with PHP described above had an interesting presentation as congenital hypothyroidism with features of AHO evolving later in life. They exhibit clinical evidence of maternal transmission with end-organ resistance. This family illustrates heterogeneity of presentation of GNAS mutation. Written informed consent was obtained from the patients for publication of this abstract. A copy of the written consent is available for review by the Editor of this journal.

Molecular Docking Analysis Identifies Potential Antagonists of Misregulated Gsα Proteins

McCune-Albright Syndrome (MAS) is a genetic disorder caused by GTPase deficient alleles of Gsα. This disease is characterized by hyperendocrinopathies, skin hyperpigmentation, and polyostotic fibrous dysplasia of bone. While the endocrine disorders can be managed with drugs, the bone disease has no pharmacological treatment, only a path of painful and often unsuccessful orthopedic surgeries. A small molecule inhibitor that is a specific antagonist to the mutant Gs isoform would be of great benefit to MAS patients. Previous work in the lab showed mutations at R231, a surface residue near the GTP-binding site, suppressed the constitutive activity of all MAS-associated mutations in Gs (R201H, R201C, R201S) and partially suppressed the constitutive activity of a Q227L mutation in Gs. Molecules were docked in silico to the R231 region, identifying several molecules with potential to bind to the suppressor site. Transiently transfected HEK cells overexpressing Gs-R201H were treated with each of 18 candidate molecules. Significant reduction in basal cAMP was observed in the presence of either 6-oxo-N-{[2-(pyrrolidin-1-yl)pyridin-4-yl]methyl}-1,6-dihydropyridazine-3-carboxamide or N-{[3-(3-methylphenyl)phenyl]methyl}-6-oxo-1,6-dihydropyridazine-3-carboxamide, while cells transfected with wild-type Gs constructs exhibited no decrease in basal cAMP levels when treated with these compounds. Thus, these compounds may serve as leads for the development of drugs for this rare disease. This work was Supported by NIH grant 1R15ED020190-01

A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gsα.

Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging “active-site” and “activation” loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small substrate predicted to fit into the CTA1 active site). Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα.

Different pattern of epigenetic changes of the GNAS gene locus in patients with pseudohypoparathyroidism type Ic confirm the heterogeneity of underlying pathomechanisms in this subgroup of pseudohypoparathyroidism and the demand for a new classification of GNAS-related disorders.

Disorders characterized by PTH resistance are grouped within the term pseudohypoparathyroidism type I (PHPI). Most subtypes of this disease are caused by genetic or epigenetic changes of the GNAS locus leading to deficiency of the α-subunit of stimulatory G proteins (Gsα). Because the in vitro measured Gsα protein activity is normal in pseudohypoparathyroidism Ic (PHPIc), it had previously been postulated that this subtype is caused by impairment of distinct components of the G protein-signaling pathway. However, recently, pathogenic GNAS mutations in a subset of PHPIc patients were found. To clarify the underlying pathogenic mechanism of GNAS exon 1-13 mutation-negative PHPIc cases by investigating the differentially methylated regions of GNAS for epigenetic abnormalities. The methylation pattern of GNAS exons A/B, AS, XL, and NESP from blood-derived leukocytes of 26 PHPIc patients was assessed by pyrosequencing of bisulfite-converted DNA. Six patients presented with three different patterns of epigenetic changes. One patient had an exclusive loss of methylation of exon A/B associated with a STX16 deletion; four patients had an additional loss of methylation in XL and AS and a gain of methylation in NESP; and one patient presented with partial GNAS methylation changes concerning all differentially methylated regions. Our results confirm that PHPIc is a heterogeneous entity caused in part by impaired Gsα function, not only due to mutations, but also due to abnormal imprinting of GNAS. However, in the majority of cases of PHPIc, the underlying etiopathogenesis remains elusive.

A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses.

Guanine Nucleotide-Binding Protein α Subunit Hypofunction in Children with Short Stature and Disproportionate Shortening of the 4th and 5th Metacarpals

Background:GNAS encodes the α subunit of the stimulatory G protein (Gsα). Maternal inherited Gsα mutations cause pseudohypoparathyroidism type Ia (PHP-Ia), associated with shortening of the 4th and 5th metacarpals. Aims: Here we investigated the Gsα pathway in short patients with distinct shortening of the 4th and 5th metacarpals. Methods: In 571 children with short stature and 4 patients with PHP-Ia metacarpal bone lengths were measured. In identified patients we analysed the Gsα protein function in platelets, performed GNAS sequencing, and epigenetic analysis of four significant differentially methylated regions. Results: In 51 patients (8.9%) shortening of the 4th and 5th metacarpals was more pronounced than their height deficit. No GNAS coding mutations were identified in 20 analysed patients, except in 2 PHP-Ia patients. Gsα activity was reduced in all PHP-Ia patients and in 25% of the analysed patients. No significant methylation changes were identified. Conclusions: Our findings suggest that patients with short stature and distinct metacarpal bone shortening could be part of the wide variety of PHP/PPHP, therefore it was worthwhile analysing the Gsα protein function and GNAS gene in these patients in order to further elucidate the phenotype and genotype of Gsα dysfunction.

Aspectos clínicos en dos casos de seudohipoparatiroidismo (ia y ib) y estudio molecular del locus GNAS

Pseudohypoparathyroidism (PHP) is a rare disorder, characterized by a tissue resistance to parathyroid hormone (PTH). The two main subtypes of PHP, PHPIa and PHPIb, are caused by alterations in the GNAS locus (20q13.3), which encodes the Gsα protein, essential for the action of PTH and other hormones.PHP-Ia is associated with several hormone resistances, Albright hereditary osteodystrophy (AHO), and reduced Gsα activity. It is caused by inactivating mutations in the GNAS gene. PHP-Ib presents with isolated resistance to PTH, without AHO and with normal to low Gsα activity. It is related to imprinting defects in GNAS.Two unrelated cases of PHP-Ia and PHP-Ib are presented here, focusing on their clinical aspects and in the differential diagnosis with similar pathologies.

Failure of hCG/LH receptors to stimulate the transmembrane effector adenylyl cyclase in human endometrium

The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively; n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p tions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC; however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.

Regulation of renin secretion by renal juxtaglomerular cells

A major rate-limiting step in the renin-angiotensin-aldosterone system is the release of active renin from endocrine cells (juxtaglomerular (JG) cells) in the media layer of the afferent glomerular arterioles. The number and distribution of JG cells vary with age and the physiological level of stimulation; fetal life and chronic stimulation by extracellular volume contraction is associated with recruitment of renin-producing cells. Upon stimulation of renin release, labeled renin granules "disappear;" the number of granules decrease; cell membrane surface area increases in single cells, and release is quantal. Together, this indicates exocytosis as the predominant mode of release. JG cells release few percent of total renin content by physiological stimulation, and recruitment of renin cells is preferred to recruitment of granules during prolonged stimulation. Several endocrine and paracrine agonists, neurotransmitters, and cell swelling converge on the stimulatory cyclic AMP (cAMP) pathway. Renin secretion is attenuated in mice deficient in beta-adrenoceptors, prostaglandin E(2)-EP4 receptors, Gsα protein, and adenylyl cyclases 5 and 6. Phosphodiesterases (PDE) 3 and 4 degrade cAMP in JG cells, and PDE3 is inhibited by cyclic GMP (cGMP) and couples the cGMP pathway to the cAMP pathway. Cyclic AMP enhances K(+)-current in JG cells and is permissive for secretion by stabilizing membrane potential far from threshold that activates L-type voltage-gated calcium channels. Intracellular calcium paradoxically inhibits renin secretion likely through attenuated formation and enhanced degradation of cAMP; by activation of chloride currents and interaction with calcineurin. Connexin 40 is necessary for localization of JG cells in the vascular wall and for pressure- and macula densa-dependent suppression of renin release.

Angiotensin-(3–4) counteracts the Angiotensin II inhibitory action on renal Ca2 +-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3−4) [Ang-(3−4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT2R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3−4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT2R. Stimulation by cholera toxin of Gsα protein structurally linked to AT2R − as revealed by their co-immunoprecipitation − mimicked the effect of Ang-(3−4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3−4), whereas the specific PKA inhibitor, PKAi(5–24) peptide, suppressed the counter-regulatory effect of Ang-(3−4) and the AT2R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3−4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3–4) effect was abrogated by the AT2R antagonist PD123319, whereas the AT1R antagonist Losartan had no effect. Ang-(3−4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3−4) could not displace 3H-Ang II from HEK 293T cells expressing AT2R, but 10−10mol/L Ang-(3−4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3−4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT2R/cAMP/PKA pathway, after inducing conformational changes in AT2R that results in generation of higher-affinity sites for Ang II.

Inhibitory Effects of GHRH Antagonists on Human GH-Secreting Adenoma Tissue

Experimental data indicate that antagonists of growth hormone-releasing hormone (GHRH) could be used clinically in disorders characterized by excessive GHRH/growth hormone (GH) secretion, but direct evidence for the effectiveness of GHRH antagonists on human pituitary tissue is still lacking. In this study, we investigated the inhibitory effect of our GHRH antagonists MZ-4-71 and JV-1-36 and the somatostatin (SST) analog RC-160 on superfused pituitary cells obtained from a human GH-secreting adenoma. Using Western blot analysis and immunohistochemistry, we demonstrated profuse expression of the GHRH receptor and its major splice variant SV1 and an increase in the expression of Gsa protein in the adenoma tissue. Exposure of the tumor cells to exogenous pulses of GHRH induced definite GH responses, causing a 3- to 5-fold elevation of the basal GH level. The antagonists MZ-4-71 and JV-1-36 did not alter basal GH secretion, indicating that the adenoma cells did not secrete GHRH in an autocrine manner. However, both antagonists prevented the stimulatory effect of exogenous GHRH. Similarly to the GHRH antagonists, neither SST-14 nor the SST analog RC-160 had an effect on the basal GH secretion of the tumor cells, but both peptides inhibited the stimulatory effect of exogenous GHRH, with RC-160 being more potent than SST. Our study provides direct evidence for the effectiveness of potent GHRH antagonists such as MZ-4-71 and JV-1-36 on human pituitary GH-secreting adenoma tissue and strongly suggests that these drugs could be used for therapy of GHRH-associated forms of acromegaly, particularly for those patients in whom surgery fails or is not an option.

Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation

Oxidative stress has been shown to increase the expression of G(i)α proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H(2)O(2), which induces oxidative stress, could also enhance the expression of G(i)α proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H(2)O(2) increased the expression of G(i)α proteins and not of G(s)α protein in a concentration- and time-dependent manner. A maximal increase of ∼40-50% was observed at 100 μM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H(2)O(2) also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H(2)O(2)-induced enhanced expression of G(i)α proteins to control levels. The increased expression of G(i)α proteins was reflected in enhanced G(i) functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas G(s)α-mediated stimulations of AC were significantly decreased. Furthermore, H(2)O(2)-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H(2)O(2) increases the expression of G(i)α proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.

Contribution of G inhibitory protein alpha subunits in paradoxical hyperalgesia elicited by exceedingly low doses of morphine in mice

AimsAlthough morphine, at higher doses, induces analgesia, it may also enhance sensitivity to pain at extremely low doses as shown in studies for testing an animal's sensitivity to pain. We used an antisense approach capable of selectively down-regulating in vivo Gi(G inhibitory protein),Go and Gs members of the Gα sub-family protein subunits in order to establish if these proteins might be implicated in the effects induced by extremely low morphine doses on acute thermonociception. Main methodsMice pretreated with a morphine hyperalgesic dose (1μg/kg) were submitted to hot plate test after pre-treatment with antisense oligodeoxynucleotides (aODNs) targeting Giα, Goα and Gsα regulatory proteins. The association of G-protein (guanine nucleotide-binding regulatory protein) coupled receptors with G protein was investigated using co-immunoprecipitation procedure. Key findingsThe downregulation of the Giα1–3 and Goα1 proteins reversed the licking latency responses induced by 1μg/kg morphine administration toward the basal value whereas downregulation of the Goα2 and Gsα proteins did not significantly modify the hyperalgesic response. SignificanceThese results suggest that G inhibitory proteins play a role in the production of low dose evoked morphine hyperalgesia in mouse. Immunoprecipitation studies revealed that both μ opioid receptor (μOR) and α2 adrenoreceptor (α2 AR) are bound to G inhibitory proteins in hyperalgesic response to morphine extremely low dose. Both μOR and α2 AR appear to be necessary for low morphine dose induced hyperalgesic response through G inhibitory proteins.

Exploring protein lipidation with chemical biology.

Exploring protein lipidation with chemical biology.

Pseudohypoparathyroidism: Diagnosis and Treatment

The term pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of various hormones (primarily PTH) that activate cAMP-dependent pathways via Gsα protein. The two main subtypes of PHP, PHP type Ia, and Ib (PHP-Ia, PHP-Ib) are caused by molecular alterations within or upstream of the imprinted GNAS gene, which encodes Gsα and other translated and untranslated products. A PubMed search was used to identify the available studies (main query terms: pseudohypoparathyroidism; Albright hereditary osteodystrophy; GNAS; GNAS1; progressive osseous heteroplasia). The most relevant studies until February 2011 have been included in the review. Despite the first description of this disorder dates back to 1942, recent findings indicating complex epigenetic alterations beside classical mutations at the GNAS complex gene, pointed out the limitation of the actual classification of the disease, resulting in incorrect genetic counselling and diagnostic procedures, as well as the gap in our actual knowledge of the pathogenesis of these disorders. This review will focus on PHP type I, in particular its diagnosis, classification, treatment, and underlying molecular alterations.