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L S Weinstein

Publications and source records attributed to L S Weinstein.

At least 19 recordsLinked to original sources
PubMed2001

The role of tissue-specific imprinting as a source of phenotypic heterogeneity in human disease.

Genomic imprinting is an epigenetic phenomenon affecting a small number of genes that leads to expression from only one parental allele. Several imprinted genes are important for neurologic development and function and several neurobehavioral disorders are caused by genetic defects involving imprinted genes. For some genes, the imprinting is tissue specific, leading to biallelic expression in some tissues and monoallelic expression in other tissues. Defects involving these genes may produce one restricted phenotype due to loss of expression of the gene product in tissues where the gene is imprinted and, in some instances, a second phenotype due to haploinsufficiency of the gene product in tissues where it is biallelically expressed.

Alleles↗
PubMed2001

Increased insulin sensitivity in Gsalpha knockout mice.

The stimulatory guanine nucleotide-binding protein (G(s)) is required for hormone-stimulated cAMP generation. Gnas, the gene encoding the G(s) alpha-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m-/+) or paternal (+/p-) allele is mutated. Notably, m-/+ mice become obese, whereas +/p- mice are thinner than normal. In this study we show that despite these opposite changes in energy metabolism, both m-/+ and +/p- mice have greater sensitivity to insulin, with low to normal fasting glucose levels, low fasting insulin levels, improved glucose tolerance, and exaggerated hypoglycemic response to administered insulin. The combination of increased insulin sensitivity with obesity in m-/+ mice is unusual, because obesity is typically associated with insulin resistance. In skeletal muscles isolated from both m-/+ and +/p- mice, the basal rate of 2-deoxyglucose uptake was normal, whereas the rate of 2-deoxyglucose uptake in response to maximal insulin stimulation was significantly increased. The similar changes in muscle sensitivity to insulin in m-/+ and +/p- mice may reflect the fact that muscle G(s)alpha expression is reduced by approximately 50% in both groups of mice. GLUT4 expression is unaffected in muscles from +/p- mice. Increased responsiveness to insulin is therefore the result of altered insulin signaling and/or GLUT4 translocation. This is the first direct demonstration in a genetically altered in vivo model that G(s)-coupled pathways negatively regulate insulin signaling.

Animals↗
PubMed2001

Evaluation of operative cholangiography in 2043 patients undergoing laparoscopic cholecystectomy: a case for the selective operative cholangiogram.

BACKGROUND: There is still some controversy over whether to use laparoscopic operative cholangiograms routinely (RLOC) or selectively (SLOC). Due to their high cost as well as other issues, in March 1997 we converted from RLOC to SLOC. The purpose of this study was to validate that decision. METHODS: The results of 2043 laparoscopic cholecystectomies (LC) were compiled and analyzed. The results of RLOC in 1556 patients undergoing LC from March 1990 through February 1997 were compared to the results of SLOC in 16 patients undergoing LC from March 1989 through February 1990 and 471 patients undergoing LC from March 1997 through December 1998. The literature was reviewed and data were compiled. Reasons that are typically given for operative cholangiograms (OC) were collected and scrutinized. Finally, cost surveys for RLOC and related procedures were obtained. RESULTS: Overall, laparoscopic operative cholangiogram (LOC) was attempted in 1661 patients and was successful in 1656 cases (99.7%). Bile duct stones (BDS) were evident in 166 patients. Laparoscopic bile duct exploration (LBDE) was attempted in all cases. None were referred for preoperative endoscopic retrograde cholangiopancreatography (ERCP). In the RLOC group, evidence of BDS was observed in 136 patients (9%). Forty-two were unsuspected (2.8%) and five were false positive (0.3%). In a collection of other RLOC studies, the average rate of unsuspected BDS was 2.9%, while the average rate of false positive cholangiograms was 1.6%. In the SLOC group, LOC was indicated in 139 of 487 patients (28.6%). None of the patients who did not have a LOC developed symptomatic residual BDS in < or = 11 years of follow-up. In a large collection of other SLOC studies, the rate of symptomatic residual BDS was 0.3%. A cost survey in February 1997 revealed that the average minimum global charge (MGC) for an OC was $1283.21; for a transcystic duct LBDE it was $1055.10, and for a transcholedochal LBDE it was $3263.61 [corrected]. The MGC for an ERCP with papillotomy was $4303.00. Thus, to avoid one patient with symptomatic residual BDS, 354 unnecessary procedures (333 RLOC, 18 LBDE, and three postoperative ERCP) costing $473,927.52 would be performed. There were no false negatives, bile duct injuries, or other complications attributable to RLOC or SLOC. CONCLUSIONS: The increased morbidity and cost of RLOC to avoid symptomatic residual BDS is not justified. All other reasons given for RLOC are either flawed or indicate that the procedure can be safely employed selectively. SLOC is an effective method of verifying suspected BDS and is safer and less expensive than RLOC.

Cholangiography↗
PubMed2001

Reoperation for hyperparathyroidism in multiple endocrine neoplasia type 1.

BACKGROUND: Patients with multiple endocrine neoplasia type 1 and hyperparathyroidism often undergo multiple operations because of inadequate initial surgery, presence of supernumerary and ectopic glands, regrowth of remnant glands, or autograft hyperfunction. Management of this patient population is complex. METHODS: From January 1975 to December 2000 we performed 94 reoperative parathyroidectomies consisting of 79 neck reexplorations, 12 autograft removals, and 3 median sternotomies in 75 patients. Data were gathered by retrospective chart review and follow-up telephone interviews. RESULTS: Excluding autograft excision, reoperative surgery was successful (normocalcemia longer than 6 months) in 91%; autograft removal was successful in only 58%. With a median follow-up of 59 months, 64% of patients are currently free from hypercalcemia, and this outcome was not influenced by the total number of glands resected. The median time to recurrent hypercalcemia was 125 months. Thirty patients received an autograft after reoperation. The complication rate for all reoperations was 12%, including permanent recurrent laryngeal nerve injury in 2 patients (2.1%). CONCLUSIONS: Reoperative parathyroidectomy in patients with multiple endocrine neoplasia type 1 was safe and successful in the majority of patients; however, recurrent hyperparathyroidism is likely to develop in most individuals beyond 10 years of follow-up. The total number of glands accounted for after reoperation is not associated with successful outcome.

Adult↗
PubMed2001

Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting.

The heterotrimeric G protein G(s) couples hormone receptors (as well as other receptors) to the effector enzyme adenylyl cyclase and is therefore required for hormone-stimulated intracellular cAMP generation. Receptors activate G(s) by promoting exchange of GTP for GDP on the G(s) alpha-subunit (G(s)alpha) while an intrinsic GTPase activity of G(s)alpha that hydrolyzes bound GTP to GDP leads to deactivation. Mutations of specific G(s)alpha residues (Arg(201) or Gln(227)) that are critical for the GTPase reaction lead to constitutive activation of G(s)-coupled signaling pathways, and such somatic mutations are found in endocrine tumors, fibrous dysplasia of bone, and the McCune-Albright syndrome. Conversely, heterozygous loss-of-function mutations may lead to Albright hereditary osteodystrophy (AHO), a disease characterized by short stature, obesity, brachydactyly, sc ossifications, and mental deficits. Similar mutations are also associated with progressive osseous heteroplasia. Interestingly, paternal transmission of GNAS1 mutations leads to the AHO phenotype alone (pseudopseudohypoparathyroidism), while maternal transmission leads to AHO plus resistance to several hormones (e.g., PTH, TSH) that activate G(s) in their target tissues (pseudohypoparathyroidism type IA). Studies in G(s)alpha knockout mice demonstrate that G(s)alpha is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in some tissues (e.g., renal proximal tubule, the major site of renal PTH action), while being biallelically expressed in most other tissues. Disrupting mutations in the maternal allele lead to loss of G(s)alpha expression in proximal tubules and therefore loss of PTH action in the kidney, while mutations in the paternal allele have little effect on G(s)alpha expression or PTH action. G(s)alpha has recently been shown to be also imprinted in human pituitary glands. The G(s)alpha gene GNAS1 (as well as its murine ortholog Gnas) has at least four alternative promoters and first exons, leading to the production of alternative gene products including G(s)alpha, XLalphas (a novel G(s)alpha isoform that is expressed only from the paternal allele), and NESP55 (a chromogranin-like protein that is expressed only from the maternal allele). A fourth alternative promoter and first exon (exon 1A) located approximately 2.5 kb upstream of the G(s)alpha promoter is normally methylated on the maternal allele and transcriptionally active on the paternal allele. In patients with isolated renal resistance to PTH (pseudohypoparathyroidism type IB), the exon 1A promoter region has a paternal-specific imprinting pattern on both alleles (unmethylated, transcriptionally active), suggesting that this region is critical for the tissue-specific imprinting of G(s)alpha. The GNAS1 imprinting defect in pseudohypoparathyroidism type IB is predicted to decrease G(s)alpha expression in renal proximal tubules. Studies in G(s)alpha knockout mice also demonstrate that this gene is critical in the regulation of lipid and glucose metabolism.

Animals↗
PubMed2000

Balloon-tipped cannulas for use in laparoscopic diagnosis and treatment of acute upper GI bleeding.

BACKGROUND: Percutaneous balloon-tipped laparoscopic cannulas designed for preperitoneal hernia repair can be readily used to treat gastric bleeding laparoscopically. METHODS: Between 1995 and 1997, we successfully used balloon-tipped cannulas to visualize, biopsy, and suture acutely bleeding gastric lesions in five patients. These case histories are reviewed for this study. RESULTS: Patients received an average of six units of blood preoperatively (range, 0-15). Operative time averaged 207 min (range, 149-270). At surgery, gastrotomies were made for cannula placement under laparoscopic visualization. Operative findings included: lesser curve gastric ulcer, Mallory-Weiss tear, prepyloric ulcer, duodenal ulcer, and angiosarcoma. Three patients had successful percutaneous suture of bleeding gastric lesions. One patient was converted to open surgery. One patient had local resection of an angiosarcoma. CONCLUSION: The laparoscopic use of balloon-tipped cannulas allows the expeditious diagnosis and treatment of acute gastric hemorrhage.

Adult↗
PubMed2000

Identification of a methylation imprint mark within the mouse Gnas locus.

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.

Animals↗
PubMed2000

Variable imprinting of the heterotrimeric G protein G(s) alpha-subunit within different segments of the nephron.

The heterotrimeric G protein G(s) is required for hormone-stimulated intracellular cAMP generation because it couples hormone receptors to the enzyme adenylyl cyclase. Hormones that activate G(s) in the kidney include parathyroid hormone, glucagon, calcitonin, and vasopressin. Recently, it has been demonstrated that the G(s)alpha gene is imprinted in a tissue-specific manner, leading to preferential expression of G(s)alpha from the maternal allele in some tissues. In the kidney, G(s)alpha is imprinted in the proximal tubule but not in more distal nephron segments, such as the thick ascending limb or collecting duct. This most likely explains why in both humans and mice heterozygous mutations in the maternal allele lead to parathyroid hormone resistance in the proximal tubule whereas mutations in the paternal allele do not. In contrast, heterozygous mutations have little effect on vasopressin action in the collecting ducts. In mice with heterozygous null G(s)alpha mutations (both those with mutations on the maternal or paternal allele), expression of the Na-K-2Cl cotransporter was decreased in the thick ascending limb, suggesting that its expression is regulated by cAMP. The G(s)alpha genes also generate alternative, oppositely imprinted transcripts encoding XLalphas, a G(s)alpha isoform with a long NH(2)-terminal extension, and NESP55, a chromogranin-like neurosecretory protein. The role, if any, of these proteins in renal physiology is unknown.

Animals↗
PubMed2000

A GNAS1 imprinting defect in pseudohypoparathyroidism type IB.

Pseudohypoparathyroidism type IB (PHPIB) is characterized by renal resistance to parathyroid hormone (PTH) and the absence of other endocrine or physical abnormalities. Familial PHPIB has been mapped to 20q13, near GNAS1, which encodes G(s)alpha, the G protein alpha-subunit required for receptor-stimulated cAMP generation. However, G(s)alpha function is normal in blood cells from PHPIB patients, ruling out mutations within the G(s)alpha coding region. In mice G(s)alpha is expressed only from the maternal allele in renal proximal tubules (the site of PTH action) but is biallelically expressed in most other tissues. Studies in patients with Albright hereditary osteodystrophy suggest a similar G(s)alpha imprinting pattern in humans. Here we identify a region upstream of the G(s)alpha promoter that is normally methylated on the maternal allele and unmethylated on the paternal allele, but that is unmethylated on both alleles in all 13 PHPIB patients studied. Within this region is an alternative promoter and first exon (exon 1A), generating transcripts that are normally expressed only from the paternal allele, but that are biallelically expressed in PHPIB patients. Therefore, PHPIB is associated with a paternal-specific imprinting pattern of the exon 1A region on both alleles, which may lead to decreased G(s)alpha expression in renal proximal tubules. We propose that loss of exon 1A imprinting is the cause of PHPIB.

Alleles↗
PubMed2000

Paternal versus maternal transmission of a stimulatory G-protein alpha subunit knockout produces opposite effects on energy metabolism.

Heterozygous disruption of Gnas, the gene encoding the stimulatory G-protein alpha subunit (G(s)alpha), leads to distinct phenotypes depending on whether the maternal (m-/+) or paternal (+/p-) allele is disrupted. G(s)alpha is imprinted, with the maternal allele preferentially expressed in adipose tissue. Hence, expression is decreased in m-/+ mice but normal in +/p- mice. M-/+ mice become obese, with increased lipid per cell in white and brown adipose tissue, whereas +/p- mice are thin, with decreased lipid in adipose tissue. These effects are not due to abnormalities in thyroid hormone status, food intake, or leptin secretion. +/p- mice are hypermetabolic at both ambient temperature (21 degrees C) and thermoneutrality (30 degrees C). In contrast, m-/+ mice are hypometabolic at ambient temperature and eumetabolic at thermoneutrality M-/+ and wild-type mice have similar dose-response curves for metabolic response to a beta(3)-adrenergic agonist, CL316243, indicating normal sensitivity of adipose tissue to sympathetic stimulation. Measurement of urinary catecholamines suggests that +/p- and m-/+ mice have increased and decreased activation of the sympathetic nervous system, respectively. This is to our knowledge the first animal model in which a single genetic defect leads to opposite effects on energy metabolism depending on parental inheritance. This probably results from deficiency of maternal- and paternal-specific Gnas gene products, respectively.

Adrenergic beta-Agonists↗
PubMed2000

Comparison of insufflation vs. retractional technique for laparoscopic-assisted intervertebral fusion of the lumbar spine.

Laparoscopic transperitoneal fusion of the L5-S1 spinal interspace has become a common procedure. Retroperitoneal retraction and laparoscopic instrumentation without insufflation also allows visualization of the upper lumbar spaces, but this procedure is much more difficult to accomplish. We review and compare our results using each of these techniques for the treatment of mechanical instability and chronic back pain. A total of 35 selected patients underwent intervertebral fusion between February 1996 and August 1998. Their mean age was 48 years. There were 22 female and 13 male patients. Standard CO2 insufflation was used in 10 patients with L5-S1 fusions. Retractional gasless technique was used in nine patients with fusions at L5-S1, 16 patients at L4-L5, one patient at L3-L4, three patients at L2-3, and one patient at L1-L2. Thus, we performed a total of 40 lumbar fusions in 35 patients. In the 19 patients with the gasless technique, a balloon dissector and retractor facilitated the retroperitoneal exposure. Seven of these 19 patients were converted to open procedures, most commonly due to lacerations of the peritoneal lining that prohibited visualization. None of the L5-S1 patients with insufflation were converted to open. Mean operative time in the insufflated patients was 152 min vs. 181 min for the retractional technique. There were seven complications in the transperitoneal group: one fusion device migration, one postoperative UTI, one intracerebral hemorrhage, one severe postoperative pancreatitis, and three iliac vein lacerations. There were 16 complications in the retroperitoneal group: one deep vein thromboses, one serosal bowel injury, one small tear in the spleen, one cage migration, one postoperative pulmonary atelectasis, one postoperative hydrocele, four postoperative ileus, and six peritoneal tears. The mean postoperative stay was three days for both groups. There were no deaths. The L5-S1 interspace is best approached transperitoneally for anterior fusion. Although the retroperitoneal retractional technique is much more difficult and has a longer and steeper learning curve, it does allow laparoscopic anterior fusion of the upper lumbar spine.

Female↗
PubMed1999

A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity.

It has been reported that substitution of Arg258, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) alpha-subunit (alphas), to alanine (alphas-R258A) results in decreased activation by receptor or aluminum fluoride (AlF4-) and increased basal GDP release. Arg258 interacts with Gln170 in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF4- and increased thermolability. In this study, we mutate Gln170 to alanine (alphas-Q170A) and demonstrate that this mutant, like alphas-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike alphas-R258A, alphas-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of alphas-R258A was 14-fold higher than normal whereas that of alphas-Q170A was unaffected. Examination of the alphas crystal structure suggests that Arg258, through interactions with Glu50, might constrain the position of Arg201, a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.

Adenylyl Cyclases↗
PubMed1999

Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation.

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.

Aluminum Compounds↗
PubMed1999

Results of heterotopic parathyroid autotransplantation: a 13-year experience.

BACKGROUND: The reported success of heterotopic parathyroid autotransplantation (HPA) in patients with primary hyperparathyroidism varies from 20% to 60%. The purpose of this study was to evaluate our results with HPA to help define its role in this patient group. METHODS: Between July 1985 and June 1998, 44 patients underwent 51 HPA procedures at our institution. Twenty to 25 fragments of parathyroid tissue measuring 1 to 3 mm3 each were placed into the forearm musculature. HPA results were scored as nonfunctional (requiring calcium and vitamin D), partially functional (normocalcemia on calcium alone), fully functional (normocalcemia without supplementation), or hyperfunctional (hypercalcemia without supplementation). RESULTS: Follow-up data were available for 39 patients who underwent 46 autografts (20 immediate and 26 cryopreserved). With a median follow-up of 35 months, 19 autografts (41%) were nonfunctional; 9 autografts (20%) were partially functional; 15 autografts (33%) were fully functional, and 3 autografts (7%) were hyperfunctional. Full function was observed in 35% of immediate and 31% of delayed autografts. CONCLUSIONS: One third of parathyroid autografts develop full function, and an additional one fifth develop partial function. Recurrent hyperparathyroidism is uncommon. No benefit was observed from immediate versus delayed HPA, and the modest success rate of HPA suggests that improvements in technique are warranted.

Adenoma↗
PubMed1999

Identification and localization of G protein subunits in human spermatozoa.

Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility.

Adult↗
PubMed1999

Decreased renal Na-K-2Cl cotransporter abundance in mice with heterozygous disruption of the G(s)alpha gene.

Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein G(s). To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the Gnas gene, which codes for the alpha-subunit of G(s). Outer medullary G(s)alpha protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous G(s)alpha knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-D-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on G(s)alpha-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.

Adenylyl Cyclases↗
PubMed1999

Identification of two novel deletion mutations within the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy.

Albright hereditary osteodystrophy (AHO) is a genetic disorder characterized by short stature, skeletal defects, and obesity. Within AHO kindreds, some affected family members have only the somatic features of AHO [pseudopseudohypoparathyroidism (PPHP)], whereas others have these features in association with resistance to multiple hormones that stimulate adenylyl cyclase within their target tissues [pseudohypoparathyroidism type Ia (PHP Ia)]. Affected members of most AHO kindreds (both those with PPHP and those with PHP Ia) have a partial deficiency of Gs alpha, the alpha-subunit of the G protein that couples receptors to adenylyl cyclase stimulation, and in a number of cases heterozygous loss of function mutations within the Gs alpha gene (GNAS1) have been identified. Using PCR with the attachment of a high melting domain (GC-clamp) and temperature gradient gel electrophoresis, two novel heterozygous frameshift mutations within GNAS1 were found in two AHO kindreds. In one kindred all affected members (both PHP Ia and PPHP) had a heterozygous 2-bp deletion in exon 8, whereas in the second kindred a heterozygous 2-bp deletion in exon 4 was identified in all affected members examined. In both cases the frameshift encoded a premature termination codon several codons downstream of the deletion. In the latter kindred affected members were previously shown to have decreased levels of GNAS1 messenger ribonucleic acid expression. These results further underscore the genetic heterogeneity of AHO and provides further evidence that PHP Ia and PPHP are two clinical presentations of a common genetic defect. Serial measurements of thyroid function in members of kindred 1 indicate that TSH resistance progresses with age and becomes more evident after the first year of life.

Amino Acid Sequence↗