Phosphodiesterase 11A expression in the adrenal cortex, primary pigmented nodular adrenocortical disease, and other corticotropin-independent lesions.

A variety of adrenal tumors and bilateral adrenocortical hyperplasias (BAH) leading to Cushing syndrome (CS) may be caused by aberrant cAMP signaling. We recently identified patients with a micronodular form of BAH that we have called "isolated micronodular adrenocortical disease" (iMAD) in whom CS was associated with inactivating mutations in phosphodiesterase (PDE) 11A ( PDE11A). In the present study, we examined PDE11A expression in normal adrenocortical tissue, sporadic tumors, and hyperplasias without PDE11A mutations, and primary pigmented nodular adrenocortical disease (PPNAD) and adenomas from patients with PRKAR1A and a single tumor with a GNAS mutation. The total number of the tumor samples that we studied was 22. Normal human tissues showed consistent PDE11A expression. There was variable expression of PDE11A in sporadic adrenocortical hyperplasia or adenomas; PPNAD tissues from patients with PRKAR1A mutations expressed consistently high levels of PDE11A in contrast to adenomas caused by GNAS mutations. Phosphorylated CREB was the highest in tissues from patients with iMAD compared to all other forms of BAH and normal adrenal tissue. We conclude that PDE11A is expressed widely in adrenal cortex. Its expression appears to be increased in PPNAD but varies widely among other adrenocortical tumors. PRKAR1A expression appears to be higher in tissues with PDE11A defects. Finally, sequencing defects in PDE11A are associated with a high state of CREB phosphorylation, just like PRKAR1A mutations. These preliminary data suggest that these two molecules are perhaps regulated in a reverse manner in their control of cAMP signaling in adrenocortical tissues.

A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumours: comparison with Carney complex and other PRKAR1A induced lesions.

BACKGROUND: Inactivation of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. METHODS AND RESULTS: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. CONCLUSION: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIalpha as a candidate tumour suppressor gene.

Evidence that barbiturates inhibit antigen-induced responses through interactions with a GTP-binding protein in rat basophilic leukemia (RBL-2H3) cells.

Little is known about the mechanism of action of anesthetics at the biochemical level. The present work, however, gives evidence that barbiturates inhibit inositol phospholipid hydrolysis in both intact and permeabilized rat basophilic leukemia (RBL-2H3) cells by an effect on GTP-binding proteins (G-proteins). Inhibition of hydrolysis was observed when intact cells were stimulated with antigen (DNP24 BSA) or with oligomers of IgE. The inhibition was dependent on the concentration and type of barbiturate used with an order of inhibitory action of secobarbital less than S(-) pentobarbital less than pentobarbital less than R(+) pentobarbital less than phenobarbital. The relatively inactive analogue, (1'RS, 3'SR) 3-hydroxypentobarbital caused little (less than 30% at 1 mM) or no inhibition (at 0.1-0.5 mM). In permeabilized cells, the hydrolysis induced by DNP24 BSA and the nonhydrolyzable analogue of GTP, GTP gamma S (2-100 microM), was also inhibited by pentobarbital. The inhibition of hydrolysis was decreased as pH increased, and was no longer apparent at pH 7.8, a possible indication that the inhibitory effect was due to the unionized form of the drug. In permeabilized cells, the inhibition by pentobarbital occurred in the presence or absence of Ca2+ and was uncompetitive in nature (Km = 7.1 microM for GTP in controls vs. 1.6 microM in the presence of 0.5 mM pentobarbital). Taken together, the data suggest that barbiturates alter the activity of G-proteins independently of Ca2+, and the inhibition may depend on both the hydrophobic properties and the stereospecific and structural features of the molecule.

The demonstration of histamine release in clinical conditions: a review of past and present assay procedures.

Topics related to the measurement of histamine in human plasma and other body fluids are reviewed. These include (1) an overview of the data obtained by the biological, fluorometric and radioenzymatic assays over the past 45 years; (2) the various modifications of the radioenzymatic isotopic assay of histamine and the development of a single extraction step assay; (3) a compilation of values obtained in our laboratory by the radioenzymatic assay of histamine levels in various body fluids in disease states associated with abnormal histamine production or release; and (4) factors that affect histamine levels in plasma and some experimental considerations for monitoring changes in free histamine levels. The last topic includes a discussion of the halflife of histamine in the circulation, its clearance across various vascular beds, and the fact that capillary endothelial cells are one site of inactivation of circulating histamine.