Elevated expression of Gsα in intrahepatic cholangiocarcinoma associates with poor prognosis.

BACKGROUND: The stimulatory G protein a subunit (Gsα) plays important roles in diverse cell processes including tumorigenesis. Activating mutations in Gsα gene (GNAS) have been reported to be associated with poor prognosis in various human carcinomas. Furthermore, Gsα signaling is crucial in promoting liver regeneration by interacting with growth factor signaling, indicating that Gsα might play a promoting role in cancer development. However, little is known about the correlation between Gsα levels and clinicopathological parameters in intrahepatic cholangiocarcinoma (ICC). METHODS: We performed immunoblotting to examine the expression levels of Gsα and Ki67 proteins in tumor tissues and the corresponding adjacent tissues. A total of 74 pair of specimens resected from 74 ICC patients were examined. The association between Gsα levels and clinicopathological findings and prognosis of the patients was evaluated. RESULTS: Western blotting demonstrated that the expression of Gsα was significantly higher in ICC tissues compared with that in their corresponding adjacent tissues. Gsα protein was highly expressed in about half of ICC tissues (48.6%, 36/74) while only 28.4% (21/74) of tumor adjacent tissues showed Gsα high expression (P=0.011). High Gsα expression in ICC was significantly associated with the numbers of tumor nodules (P=0.037) and lymph node metastases (P=0.010). Moreover, the level of Gsα was significantly and positively correlated with Ki67 expression (P<0.001). In addition, the recurrence-free survival rate and overall survival rate in the Gsα high group were significantly lower than those in the Gsα low group (P=0.004 and P=0.005, respectively). CONCLUSIONS: High Gsα expression is correlated with poor prognosis in ICC patients. Gsα might serve as a potential prognostic indicator of ICC.

Assembly of a beta2-adrenergic receptor--GluR1 signalling complex for localized cAMP signalling.

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Activation of the progesterone-signaling pathway by methyl-beta-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Galphas.

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-beta-cyclodextrin (MebetaCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-beta-cyclodextrin. Biol. Reprod. 70, 1685-1692.]. Here we report that treatment of oocytes with MebetaCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MebetaCD-stimulated phosphorylation of MAPK, and inhibited MebetaCD-induced oocyte maturation. These observations suggest that MebetaCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MebetaCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Galpha(s) is contained in low-density membrane; and treatment of oocytes with progesterone or MebetaCD reduced immunodetectable levels of Galpha(s) protein in cortices and increased internal levels of 45-kDa Galpha(s) in cortical-free extracts. Dose-dependent increases in internal Galpha(s) after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Galpha(s) after hormone treatment was comparable to the decrease in cortical Galpha(s). These results are consistent with a model in which release of Galpha(s) from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.

PCR-based analysis of differentially methylated regions of GNAS enables convenient diagnostic testing of pseudohypoparathyroidism type Ib.

BACKGROUND: Pseudohypoparathyroidism type Ib (PHPIb) is characterized by parathyroid hormone (PTH) resistance, which can lead to hypocalcemia, hyperphosphatemia, and increased serum PTH. The disorder is caused by mutations in regulatory regions of the GNAS gene (GNAS complex locus) that lead to interferences in the methylation status of alternative GNAS promoters, such as exon A/B, NESP55, and XL alpha-s. PHPIb comprises disorders that show distinctive changes in methylation status but share the same clinical phenotype: (a) loss of methylation only at exon A/B of the GNAS gene and involving no other obvious epigenetic abnormalities [e.g., those caused by heterozygous microdeletions in the STX16 (syntaxin 16) region and found in many patients with autosomal dominant (AD) PHPIb]; (b) methylation abnormalities at several differentially methylated regions (DMRs), which are observed in most patients with sporadic PHPIb and some families with AD PHPIb. METHODS: To permit early and reliable diagnosis of suspected PHPIb, we designed methylation-sensitive restriction enzyme-based and bisulfite deamination-based PCR tests for exon A/B and NESP55 DMRs. RESULTS: Both PCR strategies permit proper methylation testing of GNAS and NESP55 DMRs and elucidate different disease subtypes. We have identified a novel microsatellite repeat polymorphism within GNAS exon A/B, and pedigree analyses have shown its presence to be conclusive evidence for familial disease. CONCLUSIONS: We provide a simple diagnostic test for PHPIb, an imprinting disorder caused by different molecular changes within the GNAS complex locus. PHPIb, a complex and diagnostically challenging clinical phenotype, can be treated successfully by taking steps before the manifestation of symptoms to avoid clinical complications in affected patients or asymptomatic members of affected families who show positive results in genetic tests.

Imprinted Nesp55 influences behavioral reactivity to novel environments.

Genomic imprinting results in parent-of-origin-dependent monoallelic expression of selected genes. Although their importance in development and physiology is recognized, few imprinted genes have been investigated for their effects on brain function. Gnas is a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions. Gnas encodes the stimulatory G-protein subunit Gsalpha and two other imprinted protein-coding transcripts. Of these, the Nesp transcript, expressed exclusively from the maternal allele, codes for neuroendocrine secretory protein 55 (Nesp55), a chromogranin-like polypeptide associated with the constitutive secretory pathway but with an unknown function. Nesp is expressed in restricted brain nuclei, suggesting an involvement in specific behaviors. We have generated a knockout of Nesp55 in mice. Nesp55-deficient mice develop normally, excluding a role of this protein in the severe postnatal effects associated with imprinting of the Gnas cluster. Behavioral analysis of adult Nesp55 mutants revealed, in three separate tasks, abnormal reactivity to novel environments independent of general locomotor activity and anxiety. This phenotype may be related to prominent Nesp55 expression in the noradrenergic locus coeruleus. These results indicate a role of maternally expressed Nesp55 in controlling exploratory behavior and are the first demonstration that imprinted genes affect such a fundamental behavior.

Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level.

Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al.). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate G(s)alpha protein. Expressed in quantitative terms, DIMs contained 30-40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1-3%. The dominant portion (>95%) of FhIPR as well as FhIPR-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient. Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein G(s)alpha from membrane domains; receptor level is unchanged. (ii) Very small fraction (1-3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a "supra-stoichiometric" arrangement of G proteins and corresponding receptors in DIMs.

Cardiac beta-adrenoceptors in chronic uremia: studies in humans and rats.

OBJECTIVES: The purpose of this study was to elucidate whether cardiac beta-adrenergic effects may be blunted in patients on maintenance hemodialysis (HD) and may help to explain autonomic dysfunction. BACKGROUND: Patients on HD often suffer from autonomic dysfunction. METHODS: We investigated the cardiovascular response of five HD patients (age: 46.1+/-7.9 years) and six healthy volunteers (age: 48.2+/-7.5 years) to isoprenaline, pirenzepine and phenylephrine. For analysis of underlying mechanisms of beta-adrenoceptor hyporesponsiveness, six-week-old male Wistar rats were rendered uremic by 5/6-nephrectomy (n = 9; SNX) and were killed for removal of the heart after six to seven weeks. Sham-operated rats (n = 15) served as controls. RESULTS: In the patient study, isoprenaline (3.5, 7, 17, 35 ng/kg/min, i.v.) led to an increase in heart rate, and shortening of the heart rate corrected duration of the electromechanical systole (QS2c), both of which were significantly reduced in HD patients. Baroreflex sensitivity was significantly reduced in HD patients. The response to low parasympathomimetic doses of pirenzepine was unchanged. In the rat study, left ventricular strips were placed in an organ bath, electrically driven and exposed to isoprenaline (10(-11) to 10(-6) mol/liter). While pD2 values were unchanged, maximum effect at the highest concentration was significantly reduced in SNX rats. The response to carbachol was not altered, nor was the M2-cholinoceptor density. There was no difference in beta-adrenoceptor density, or in immunodetectable amount of Gs and Gi protein. Activation of adenylyl cyclase evoked by isoprenaline was significantly reduced in left ventricular membranes of SNX rats, whereas effects of 10 micromol/liter GTP, 10 mmol/liter NaF, 10 micromol/liter forskolin and 10 mmol/liter Mn2+ were not altered. CONCLUSIONS: Cardiac beta-adrenergic responses are blunted in chronic uremia due to reduced isoprenaline-dependent activation of adenylyl cyclase. This might be caused by an "uncoupling" of the receptor or by an inhibition of the receptor by uremic toxins.

The effects of agonist stimulation and beta(2)-adrenergic receptor level on cellular distribution of gs(alpha) protein.

This study examines the effects of adrenergic ligands, cholera toxin, forskolin, and varying levels of beta(2) adrenergic receptors (beta(2)AR) on the cellular distribution of Gs(alpha) subunits in CHO cells. Localization of Gs(alpha) was evaluated by confocal microscopy and beta(2)AR-mediated signalling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0.2 pmol/mg protein beta(2)ARs (WT18), the localization of Gs(alpha) subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gs(alpha) so that Gs(alpha) appeared as intense spots throughout the plasma membrane as well as the cytoplasm. Exposure to a neutral beta(2)AR antagonist, alprenolol, prevented the ISO-stimulated Gs(alpha) translocation from peripheral to inner cytoplasm. In cells expressing high level of beta(2)ARs (8.2 pmol/mg) (WT4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive correlation between receptor expression and receptor-mediated signalling. Basal Gs(alpha) distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gs(alpha) significantly when tested in WT4 clone. No difference in the cellular level of Gs(alpha) protein between WT18 and WT4 clones was detected. Alprenolol did not affect the distribution of Gs(alpha) in WT4 clone. ICI 118,551, a negative beta(2)AR antagonist, altered Gs(alpha) distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that observed in unstimulated WT18 clone. Taken together, these data suggest that: (1) enhanced beta(2)AR-Gs(alpha) coupling induced by agonist stimulation or by increased expression of beta(2)ARs remodel the cellular distribution of Gs(alpha); (2) the alteration in Gs(alpha) distribution induced by beta(2)AR overexpression provides evidence for agonist-independent interaction of beta(2)AR and Gs(alpha), that can be inhibited by a negative antagonist but not by a neutral antagonist; and (3) forskolin influences the activity state of Gs(alpha) that displays a Gs(alpha) distribution pattern comparable to that observed when Gs(alpha) is activated via beta(2)AR stimulation or directly by cholera toxin.

Expression of Gi-2 alpha and Gs alpha in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction.

OBJECTIVE: Patients surviving large transmural myocardial infarction (MI) are at risk for congestive heart failure with attendant alteration of ventricular geometry and scar remodeling. Altered Gi-2 alpha and Gs alpha protein expression may be involved in cardiac remodeling associated with heart failure, however their expression in scar tissue remains unclear. METHODS: MI was produced in Sprague-Dawley rats by ligation of the left coronary artery. Gi-2 alpha and Gs alpha protein concentration, localization and mRNA abundance were noted in surviving left ventricle remote to the infarct, in border and in scar tissues from 8 week post-MI hearts with moderate heart failure. RESULTS: We observed a 4.5- and 5.0-fold increase in immunoreactive Gi-2 alpha protein concentration occurs in the border and scar regions vs. control values, respectively, in 8-week post-MI rat hearts. Similarly, immunoreactive Gs alpha protein concentration was increased 3.4- and 8.2-fold, respectively, in these tissues vs. controls. Double-fluorescence labeling and phenotyping studies revealed that both Gi-2 alpha and Gs alpha proteins were localized to myofibroblasts in the infarct scar and to viable myocytes bordering the scar. Northern analysis revealed that the Gi-2 alpha/GAPDH ratio was increased in both viable and scar regions (1.24- and 1.85-fold respectively) from experimental hearts when compared to sham-operated control values when compared to noninfarcted left ventricle, the value of this ratio in scar tissue was elevated approximately 1.5 fold. The Gs alpha/GAPDH ratio was significantly increased (1.28-fold) only in the scar region vs. control. CONCLUSION: Our results indicate a marked increase in the expression of Gi-2 alpha and Gs alpha from myofibroblasts of the infarct scar as well as remnant myocytes bordering the scar in 8-week post-MI rat hearts. We suggest that these changes may be associated with ongoing remodeling in the infarct scar in chronic post-MI phase of this experimental model.

Localization of G protein alpha-subunits in the human fetal adrenal gland.

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.

Targeted disruption of Gnas in embryonic stem cells.

Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (G alpha(s)) are present in subjects with Albright hereditary osteodystrophy, a syndrome of characteristic developmental defects and, in some patients, resistance to multiple hormones that stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the first step in generating a model of Albright hereditary osteodystrophy, the gene encoding G alpha(s) (Gnas) was disrupted in mouse embryonic stem (ES) cells by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G alpha(s) messenger RNA and G alpha(s) protein in targeted ES cells were approximately 50% of levels in untargeted ES cells. In response to 10 microM forskolin and to various concentrations of isoproterenol (0.1-3.0 microM), cAMP accumulation was reduced in the G alpha(s) knockout ES cell lines, relative to wild-type ES cells and to five of six ES cell lines with randomly integrated targeting vector. These results support the role of G alpha(s) haploinsufficiency in reducing the ability of hormones to generate cAMP in subjects with pseudohypoparathyroidism type Ia. The targeted disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G alpha(s) and cAMP coupled signal transduction in differentiation and development.

Increase in G(i alpha) protein accompanies progression of post-infarction remodeling in hypertensive cardiomyopathy.

Hypertensive cardiac hypertrophy and myocardial infarction (MI) are clinically relevant risk factors for heart failure. There is no specific information addressing signaling alterations in the sequence of hypertrophy and post-MI remodeling. To investigate alterations in beta-adrenergic receptor G-protein signaling in ventricular remodeling with pre-existing hypertrophy, MI was induced by coronary artery ligation in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Ten weeks after the induction of MI, the progression of left ventricular dysfunction and increases in plasma atrial natriuretic peptide (ANP) and cardiac ANP mRNA were more pronounced in SHR than WKY. In addition, the impaired contractile response to beta-adrenergic stimulation was observed in the noninfarcted papillary muscle isolated from SHR. Immunochemical G(s alpha) protein and beta-adrenoceptor density were not significantly altered by MI in both strains. However, immunochemical G(i alpha) was increased (1.5-fold) in the noninfarcted left ventricle of the SHR in which infarction had been induced when compared with that in SHR that underwent sham operation. This increase was observed especially in rats with a high plasma ANP level. Furthermore, there was a positive correlation between G(i alpha) and the extent of post-MI remodeling in WKY. A similar correlation between G(i alpha) and the extent of hypertensive hypertrophy was observed in SHR. In conclusion, the vulnerability of hypertrophied hearts to ischemic damage is greater than that of normotensive hearts. An increase in G(i alpha) could be one mechanism involved in the transition from cardiac hypertrophy to cardiac failure when chronic pressure overload and loss of contractile mass from ischemic heart disease coexist.

HTLV-1-associated myelopathy/tropical spastic paraparesis with pseudohypoparathyroidism.

In many short-stature patients with human T-lymphotrophic virus type I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), signs and symptoms were manifested during childhood. Successive investigations revealed 12 of 14 short-stature patients with pseudohypoparathyroidism (PHP) from the findings of short metacarpi, parathyroid hormone infusion test, immunoblotting of erythrocyte membrane, or lymphocytic Northern blotting of Gsalpha. Patients with PHP probably showed HAM/TSP based on their modified immunologic status. Human T-lymphotrophic virus type I infection did not induce PHP, but PHP may be a risk factor for the occurrence of HAM/TSP.

Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids.

Neuroendocrine secretory protein-55 (NESP-55), the latest addition to the chromogranin family, is a product of a genomically imprinted gene transcribed exclusively from the maternal allele. Initial studies have shown it to have a less widespread distribution than that of chromogranin A in normal tissues. It has also been suggested that NESP-55 may be a marker of neuroendocrine tumors differentiating toward the adrenal chromaffin and pancreatic islet cells. Metastatic gastrointestinal and pulmonary carcinoids may occasionally be difficult to distinguish from pancreatic endocrine tumors (PETs) and pheochromocytomas on morphologic grounds alone. We studied neuroendocrine tumors from these sites to see if NESP-55 expression could reliably discriminate pulmonary and gastrointestinal carcinoids from neuroendocrine tumors arising in the pancreas or the adrenal medulla. Sixty-three neuroendocrine tumors positive for one or more immunohistochemical marker of neuroendocrine differentiation (chromogranin A, chromogranin B, synaptophysin, secretogranin II, neuron-specific enolase) were selected for the study and consisted of 34 typical carcinoids (15 pulmonary, 11 ileal, 4 gastric, and 4 rectal), 19 PETs, and 10 pheochromocytomas (4 sporadic, 3 MEN-2, 2 neurofibromatosis type 1, and 1 VHL). All cases were stained for NESP-55 after microwave antigen retrieval using a rabbit polyclonal antibody at a dilution of 1:1000. Sections of normal adrenal medulla were used as positive controls for NESP-55 staining. Negative controls consisted of omission of primary antibody and replacement with normal rabbit serum at an equivalent concentration. NESP-55 immunoreactivity was seen as brown finely granular cytoplasmic staining with prominent perinuclear accentuation. All gastric and ileal carcinoids studied were completely negative for NESP-55. One of four rectal and 1 of 15 pulmonary carcinoids showed focal positivity for it in less than 5% of tumor cells. In contrast, all 10 pheochromocytomas and 14 of 19 PETs showed strong immunohistochemical staining in a variable proportion of tumor cells. Diffuse positivity (>75% of tumor cells) was seen in 6 of 14 PETs and 8 of 10 pheochromocytomas. Our results indicate that, in contrast to the other granins, NESP-55 reactivity is restricted to endocrine tumors of the pancreas and the adrenal medulla. Immunohistochemical expression of NESP-55 may thus be useful in assigning a pancreatic or adrenal origin to metastatic endocrine tumors of unknown origin.

Coupling efficiency of brain beta-adrenergic receptors to Gs protein in suicide, alcoholism and control subjects.

Abnormal beta-adrenergic receptor (betaAR) density in the brains of suicide victims has been reported, although results of studies are inconsistent. Ethanol modifies betaAR-mediated signal transduction. Moreover abnormal betaAR function has been implicated in alcoholism. BetaAR antagonists, which were used as ligands in previous betaAR binding studies, also bind to 5-HT1B/1Dbeta receptors; hence, their estimates of betaAR density are confounded by binding to 5-HT1B/1Dbeta receptors. More importantly, previous studies did not examine betaAR agonist affinity or coupling efficiency to Gs protein. We investigated agonist affinity and coupling efficiency of betaAR to Gs protein in the brains of ten suicide victims, six subjects with alcoholism, and eight controls. There were no differences in betaAR density in either the frontal cortex or hippocampus of suicide victims or alcoholic subjects compared to controls. Preliminary results indicate betaAR supercoupling in suicide victims in both brain regions and uncoupling in alcoholic subjects in the frontal cortex. These results are discussed in view of the existing literature on the role of betaAR in suicide and alcoholism and the mechanism of action of antidepressants.

Expression of G-protein subtypes in cultured cerebral endothelial cells.

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Hypertolerance to morphine in G(z alpha)-deficient mice.

Our laboratory has generated a mouse deficient in the alpha (alpha) subunit of the G protein, G(z), (G(z alpha)) gene and we have examined the involvement of G(z alpha) in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50 degrees C or -5 degrees C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52 degrees C or 0.5 degrees C. Tolerance to morphine was induced in wild type and G(z alpha)-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and G(z alpha)-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the G(z alpha)-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and G(z alpha)-deficient brains. No changes in the affinity of morphine for its receptor or in the density of mu and delta opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of G(z alpha) does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of G(z alpha) delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.

Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat.

OBJECTIVE: Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the susceptibility of adult heart to ischemia-reperfusion (I-R) injury. METHODS: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) groups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R(10)) or 25 minutes of ischemia and 3 hours of reperfusion (I-R(25)). RESULTS: Prenatal hypoxia did not change basal left ventricular (LV) function. I-R(10) produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R(25) caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R(25)-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. There was a significant decrease in LV heat shock protein 70 and endothelial nitric oxide synthase levels in hypoxic hearts. Prenatal hypoxia did not change beta(1)-adrenoreceptor levels but significantly increased beta(2)-adrenoreceptor in the left ventricle. In addition, it increased G(s)alpha but decreased G(i)alpha. CONCLUSIONS: Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in beta(2)-adrenoreceptor and the G(s)alpha/G(i)alpha ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.

Expression of Gsalpha, connexin-43, connexin-26, and EP1, 3, and 4 receptors in myometrium of prelabor singleton versus multiple gestations and the effects of mechanical stretch and steroids on Gsalpha.

OBJECTIVE: We tested the hypothesis that multiple pregnancies would be associated with altered expression of the following three groups of proteins that are key regulators of myometrial function: (i) Gsalpha, (ii) connexins-43 and 26, and (iii) prostanoid EP1, EP3, and EP4 receptors. METHODS: An in vitro model was used to determine the effects of mechanical stretch on myometrial cell Gsalpha expression. Then the effects of the steroid hormones beta-estradiol and progesterone were tested on Gsalpha expression in vitro. All in vitro studies were performed using myometrium from nonlaboring women. RESULTS: There were no differences in the expression of Gsalpha, prostaglandin E2 receptors, or gap junction proteins in myometrium of singleton versus multiple pregnancies. Mechanical stretch did not alter Gsalpha expression in vitro, and Gsalpha expression was unaffected by steroid hormones. CONCLUSION: These results suggest that the methods whereby stretch can promote myometrial contraction are complex or require additional factors than those tested here. Alternatively, cases of multiple gestation that do not result in preterm labor perhaps compensate for the increased stretch by preventing aberrant expression of the proteins investigated in this study.

G-proteins in kidneys of spontaneously hypertensive rats.

G(s alpha)-, total G(i alpha)- and G(q/11alpha)-protein concentrations were investigated by quantitative immunoblotting in membranes of total kidney, renal cortex and medulla as well as in cortical tubules and glomeruli of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto rats (WKY), aged 5 weeks, 3 or 8 months. We found that total kidney of 5 week old SHR possess less G(s alpha)-, G(i alpha)- and G(q/11alpha)-proteins than controls. For G(s alpha)-proteins, differences found in total kidney were mirrored both in cortex (tubules and glomeruli) and in medulla. Decreased G(i alpha)-concentrations were accompanied by lower tubular but higher glomerular levels, while medullar levels were also increased. Decreased G(q/11alpha)-concentrations were reflected in decreased glomerular and medullary concentrations. Kidneys of 3 month old SHR and WKY possessed similar concentrations of all G(alpha)-species. In 8 month old SHR similar G(i alpha)-, but decreased G(s alpha)-and G(q/11alpha)-concentrations were observed. The G(s alpha)-decrease was reflected in cortex and medulla, the G(q/11alpha)-decrease in the medulla. We conclude that the main strain-related differences in G(alpha)-concentrations are seen in prehypertensive SHR.