Ras inhibits Jun-activated human atrial natriuretic peptide gene transcription in cultured ventricular myocytes.

p21ras is a potent regulator of myogenic cell growth and differentiation. It has been implicated as playing a major role in the genesis of cardiac hypertrophy. We examined the effect of Ras overexpression on human atrial natriuretic peptide (hANP) gene expression, a marker of hypertrophy, in neonatal rat ventricular myocytes. Transient transfection of Haras, which expresses an activated form of p21ras, effected a modest stimulation of basal hANP-chloramphenicol acetyl transferase (hANPCAT) expression. Noteworthy, the same construct inhibited both c-Jun- and Jun B-stimulated hANPCAT activity (60% and 80%, respectively). Cotransfection of a dominant-negative Ras mutant reversed this inhibition completely. The inhibitory effect was promoter selective in this system. Of those tested, only the hANP and cardiac troponin T promoters were suppressed by Ras. The inhibitory effect appears to operate through a Ras-mediated increase in c-Fos activity as evidenced by (1) the absence of additivity of the Ha-Ras- and c-Fos-mediated inhibition at higher levels of proto-oncogene expression, (2) Ras-dependent activation of c-fos gene transcription, inferred from the induction of a c-fos chloramphenicol acetyl transferase reporter (3.4-fold), and (3) reversal of Ras inhibition by a c-fos antisense oligonucleotide but not by a scrambled DNA sequence of identical base composition or the complementary sense oligonucleotide. Our findings suggest that p21tas can exert a wide range of effects on the phenotype of the cardiac ventricular myocyte. The direction that these effects take appears largely to be a function of the preexisting activation state of the cell.

Thrombin inhibits atrial natriuretic peptide receptor activity in cultured bovine endothelial cells.

Thrombin and the atrial natriuretic peptide (ANP) possess a number of functionally antagonistic properties in vascular endothelial cells. Thus, regulatory interactions that modulate the activity of one or the other could have important sequelae with regard to cardiovascular homeostasis. Thrombin treatment effected a dose- and time-dependent reduction in ANP receptor activity (maximal 70% to 80% inhibition) in cultured bovine aortic endothelial cells. This resulted from a decrease in total receptor number as well as a modest reduction in the affinity of the receptor for its ligand. The inhibition was largely confined to the type C receptor population, in that thrombin had no effect on maximal type A receptor-linked cGMP accumulation. The protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate effected a similar reduction in binding activity; however, suppression of protein kinase C activity did not reverse the thrombin effect. Pretreatment of endothelial cells with cycloheximide did not completely prevent the thrombin-dependent inhibition, and thrombin did not effect a reduction in type C receptor mRNA levels, findings that argue for a postsynthetic inhibitory locus. The inhibition of receptor activity was effectively irreversible in that suspension of protein synthesis blocked the recovery of receptor density on the cell surface. Reduction in type C receptor density was accompanied by modest increases in the stability of ANP in the culture medium and enhancement of the cellular cGMP response to the peptide, particularly at low ligand concentrations. These findings demonstrate a potentially important interaction between these two agonist systems in regulating endothelial cell function within the vascular wall.

Differential regulation of natriuretic peptide receptor activity in vascular cells.

We studied the regulation of the individual natriuretic peptide receptor subtypes by 12-O-tetradecanoylphorbol 13-acetate (TPA) and forskolin in cultured bovine aortic endothelial and smooth muscle cells. In cultured endothelial cells, 10(-8) mol/L TPA caused a reduction in atrial natriuretic peptide (ANP) receptor binding activity that was seen as early as 2 hours after the treatment and reached a maximum (88 +/- 3% of control) after 24 hours, whereas the inhibition of ANP-stimulated cyclic GMP (cGMP) accumulation peaked at 2 hours (62 +/- 13% of control) and returned to control levels after 12 hours. After 24 hours of forskolin (10(-4) mol/L) treatment, ANP binding activity fell to 47 +/- 6%, and cGMP accumulation was 52 +/- 11% of control. Northern blot analysis revealed that 10(-8) mol/L TPA suppressed natriuretic peptide C receptor transcript levels, and forskolin increased levels modestly after 24 hours of treatment. Natriuretic peptide A receptor transcript levels remained unchanged by either treatment. In cultured smooth muscle cells, 10(-8) mol/L TPA suppressed ANP binding activity and ANP-stimulated cGMP formation in a fashion similar to that seen in endothelial cells. TPA treatment also resulted in an inhibition of C-type natriuretic peptide-stimulated cGMP production (59 +/- 7% of control); however, this response persisted for as long as 24 hours after addition of the agonist. Treatment with 10(-4) mol/L forskolin produced a time-dependent inhibition of ANP binding activity and did not inhibit cGMP production stimulated by either ANP or C-type natriuretic peptide. In contrast to the effects seen with endothelial cells, TPA caused a dose-dependent stimulation of natriuretic peptide C receptor mRNA, whereas forskolin was inhibitory in smooth muscle cells. These results indicate that the effects of the kinase activators are a function of the individual receptor subtype as well as the cell in which it is expressed and imply a considerable degree of flexibility in the response to regulatory stimuli.

Octreotide therapy in advanced thyroid cancer.

Octreotide is a long-acting somatostatin analog that inhibits cell growth and hormone secretion. It has been successfully used in the management of a variety of endocrine tumors (i.e., acromegaly, carcinoid tumors, gastrinomas). In vitro, octreotide suppresses adenylate cyclase activity, DNA synthesis, and cell growth in cultured thyroid cell lines. Previous studies examining the use of octreotide in the treatment of medullary thyroid cancers, in vivo, report symptomatic improvement from tumor-related hormonal hypersecretion; however, octreotide's ability to suppress tumor growth was limited. In the present study, we examine the efficacy of long-term octreotide administration in six subjects with metastatic thyroid carcinoma, including Hurthle cell (one subject), medullary (one subject) and papillary or mixed papillary/follicular cancer (four subjects). All of the subjects had documented recurrences of their thyroid tumors despite appropriate therapy, and were considered to be untreatable by conventional therapeutic modalities (i.e., radioiodine or surgery). Subjects were monitored while receiving relatively high doses (4 mg daily) octreotide subcutaneously for up to 12 months. Octreotide therapy was very well tolerated; mild gastrointestinal symptoms persisted throughout treatment in one subject. Octreotide did not significantly decrease tumor markers (e.g., thyroglobulin, calcitonin, carcinoembryonic antigen). The carcinomas progressed during treatment, as evidenced by an increase in the size and/or number of metastatic lesions. In summary, in this small series subcutaneous octreotide administration did not appear to be efficacious in the management of advanced thyroid cancers.

Transcriptional regulation of human corticotropin releasing factor gene expression by cyclic adenosine 3',5'-monophosphate: differential effects at proximal and distal promoter elements.

cAMP participates in the regulation of endogenous hypothalamic and placental CRF by increasing levels of both peptide secretion and mRNA expression. In previous studies we have shown that stimulation of the protein kinase A-dependent pathway by cAMP analogues or forskolin produced a dose-dependent increase in levels of CRF mRNA when the intact hCRF gene was stably transfected and expressed in the mouse corticotroph AtT20 cell line. In the present study, we explored the mechanism of the cAMP-dependent increase in CRF gene expression in the stably transfected AtT20 cell line using pharmacologic, slot-blot, and RNase mapping methodologies. Following incubation with cAMP, there was a rapid increase in CRF mRNA which was completely blocked by pre-treatment with actinomycin D, an inhibitor of transcription. Cycloheximide, an inhibitor of protein synthesis, produced an independent increase in CRF mRNA, but did not change the relative induction of CRF mRNA produced by cAMP. Solution hybridization studies using intron- and exon-specific hCRF probes demonstrated a rapid rise in nuclear CRF hnRNA, which was apparent within 15 min of cAMP incubation and preceded the rise in cytoplasmic CRF mRNA. RNase mapping studies demonstrated that CRF transcription was initiated at discrete promoter sites in CRF-AtT20 cells, and that this pattern of promoter utilization was similar to that observed in mRNA derived from sites of endogenous CRF expression, human placenta and human hepatoma NPLC cell line. Treatment with cAMP selectively increased CRF mRNA transcripts initiated at the proximal promoter site, but had little or no effect on transcripts initiated at the distal promoters. We conclude that cAMP effects on CRF gene expression occur rapidly, do not require new protein synthesis, and are initiated within the nuclear compartment, consistent with a direct effect on CRF gene transcription. This effect is mediated predominantly through the proximal promoter element, while more distal promoters are less sensitive to transcriptional activation by cAMP.

Melioidosis. Forgotten, but not gone!

Melioidosis, infection by the soil bacterium Pseudomonas pseudomallei, has the potential for prolonged latency with recrudescence into an acute, often fulminating, and fatal infection. Although the organism is never found in North America, infection is endemic in areas of southeast Asia, and populations of service personnel exposed during the Vietnam war and southeast Asian immigrants are at risk of severe recrudescent disease. Diagnosis, however, has been missed or delayed because of lack of familiarity with this disease. We present a case of recrudescent melioidosis that illustrates the difficulties encountered in diagnosis and treatment. This case involves a 76-year-old Vietnam veteran who presented with melioidosis of the bone 18 years after exposure to the organism and 10 years after a missed diagnosis of latent pulmonary disease. This case illustrates the protean nature of latent infection and the difficulty of selecting successful antibiotic therapy.

Cholinergic drugs alter ciliary muscle response and receptor content.

Topical echothiophate administration to the cynomolgus monkey eye for 5-6.5 months produced marked subsensitivity of the accommodative response to pilocarpine and a 65% decrease in specific high affinity 3H-QNB binding sites (ostensibly indicating muscarinic receptors) in the ciliary muscle. The decrease in 3H-QNB binding sites was quantitatively similar in surgically untouched, totally iridectomized, and ciliary muscle disinserted eyes. Following a 5-month off treatment recovery period, 3H-QNB binding sites increased to more than twice the number in untreated control eyes. In echothiophate-treated eyes whose contralateral eyes had previously received atropine+echothiophate, 3H-QNB binding sites were three to six times more numerous than in other long-term echothiophate-treated eyes, and one to two times more numerous than in untreated controls. Topical pilocarpine administration for 1 day to 7 months reduced ciliary muscle 3H-QNB binding sites by approximately 25%. Alterations in muscarinic receptors during and following cholinergic drug therapy may in part explain the observed subsensitization and recovery of ciliary muscle physiological responses.

Adrenergic and cholinergic receptors in isolated non-pigmented ciliary epithelial cells.

Methods were developed to isolate non-pigmented ciliary epithelium from bovine eyes (BCBE) free from other cell types in the ciliary process. Once background binding due to pigment was minimized, it was possible to assay specific beta adrenergic receptors in these preparations. The BCBE beta adrenergic receptors showed appropriate stereospecificity and an order of binding affinities compatible with a beta 2 adrenergic selectivity. Interestingly, specific high affinity muscarinic cholinergic receptors were detected in both BCBE and primate non-pigmented ciliary epithelium. These muscarinic receptors showed an appropriate affinity and selectivity for agonists and antagonists. Further evaluations of the receptors and responses of non-pigmented ciliary epithelial cells may prove useful in understanding the effects of adrenergic and cholinergic agents on aqueous humor inflow.