Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase.

Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not inhibited by antisense-oligonucleotide treatment. Our results demonstrate conclusively that the elimination of ANP-C receptor by antisense oligonucleotide attenuates ANP-induced inhibition of adenylyl cyclase and provide evidence that antisense oligonucleotide of the ANP-C receptor may serve as a useful pharmacological tool to elucidate the physiological functions of the ANP-C receptor.

Angiotensin II enhances the expression of Gialpha in A10 cells (smooth muscle): relationship with adenylyl cyclase activity.

In the present studies, we have investigated the effect of angiotensin II (AII) on guanine nucleotide regulatory protein (G protein) expression and functions in A10 smooth muscle cells. AII treatment of A10 cells enhanced the levels of inhibitory guanine nucleotide regulatory protein (Gi) as well as Gi mRNA and not of stimulatory guanine nucleotide regulatory protein (Gs) in a concentration-dependent manner as determined by immunoblot and Northern blot analysis, respectively. AII-evoked increased expression of Gialpha-2 and Gialpha-3 was inhibited by actinomycin D treatment (RNA synthesis inhibitor). The increased expression of Gialpha-2 and Gialpha-3 by AII was not reflected in functions, because the GTPgammaS-mediated inhibition of forskolin-stimulated adenylyl cyclase and the receptor-mediated inhibition of adenylyl cyclase by AII and C-ANP4-23 [des(Gln18, Ser19, Gln20, Leu21, Gly22) ANP4-23-NH2] were not augmented but attenuated in AII-treated A10 cells. The attenuation was prevented by staurosporine (a protein kinase C inhibitor) treatment. On the other hand, AII treatment did not affect the expression and functions of stimulatory guanine nucleotide regulatory protein (Gs), however, the stimulatory effects of 5'-O-(3-thiotriphosphate), isoproterenol, and N-ethylcarboxamide adenosine (NECA) on adenylyl cyclase activity were inhibited to various degrees by AII treatment. Staurosporine reversed the AII-evoked attenuation of isoproterenol- and NECA-stimulated enzyme activity. From these results, it can be suggested that AII, whose levels are increased in hypertension, may be one of the possible contributing factors responsible for exhibiting an enhanced expression of Gi protein in hypertension.

Angiotensin II modulates ANP-R2/ANP-C receptor-mediated inhibition of adenylyl cyclase in vascular smooth muscle cells: role of protein kinase C.

In the present studies, we have investigated the modulation of atrial natriuretic peptide (ANP) receptor of R2 subtype (ANP-R2/ANP-C) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (angII). C-ANF4-23 [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF4-23-NH2] and AngII inhibited adenylyl cyclase activity in a concentration-dependent manner in vascular smooth muscle cells (VSmc A-10). The maximal inhibitions observed were about 40 and 30%, respectively, with an apparent Ki of about 1 and 10 nm. Pretreatment of the cells with AngII resulted in the attenuation of both C-ANF4-23 and AngII-mediated inhibitions of adenylyl cyclase, without altering [125I]-ANF binding. The levels of Gialpha-2 and Gialpha-3 proteins as determined by immunoblotting were also augmented by AngII treatment. In addition, AngII treatment stimulated the phosphorylation of Gialpha2 but not of Gialpha3 or ANP-C receptor, as revealed by immunoprecipitation of the proteins using specific antibodies after prelabelling the cells with [32P]orthophosphate. Staurosporine and chelerythrine, protein kinase C (PKC) inhibitors at 1 and 100 nm, respectively, prevented the AngII-mediated desensitization of C-ANF 4-23-sensitive adenylyl cyclase. In addition, the AngII-mediated phosphorylation of Gialpha2 protein was also inhibited partially by about 35% by staurosporine treatment. These results suggest that the attenuation of C-ANF4-23-mediated inhibition of adenylyl cyclase activity by AngII may not be attributed to the downregulation of receptors or to the decreased levels of G-proteins, and may involve PKC-dependent mechanisms.

Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggest non-redundant functions for mammalian homologs of Drosophila Groucho.

The Drosophila protein Groucho is involved in the regulation of cell-determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin-like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up-regulated and TLE2 and TLE4 are down-regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up-regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non-redundant roles for individual TLE proteins during both cell-determination and cell-differentiation events.

Differential regulation of G-protein expression by vasoactive peptides.

Vasoactive peptides such as angiotensin II (AII), atrial natriuretic peptide (ANP) and vasopressin play an important role in the regulation of blood pressure. We have recently shown an augmentation of Gi alpha levels in heart and aorta from genetic and experimentally-induced hypertensive rats, which may be attributed to the increased levels of vasoactive peptides. We have therefore investigated the effect of AII and ANP on the expression of G-proteins (Gi alpha and Gs alpha) in cultured vascular smooth muscle cells (VSMC) and their relationship with adenylyl cyclase activity. Exposure of VSMC with AII resulted in the augmentation of the levels of Gi alpha-2 and Gi alpha-3 proteins and Gi alpha-2 and Gi alpha-3 mRNA and not of Gs alpha as determined by immunoblotting and Northern blotting techniques respectively. However, the stimulatory effects of N-ethylcarboxamide adenosine (NECA) and isoproterenol on adenylyl cyclase was diminished by AII treatment, whereas the inhibitory effects of AII and C-ANP4-23 were completely attenuated. On the other hand, pretreatment of the cells with C-ANP4-23 resulted in the reduction of the levels of Gi alpha-2 and Gi alpha-3 and not of Gs alpha. The inhibitory responses of adenylyl cyclase to C-ANP4-23 and AII were also attenuated and the stimulatory effects of GTP gamma S and other agonists were significantly augmented. These data indicate that AII and ANP modulate the expression of Gia protein in a different manner. It may be suggested that the enhanced levels of Gi alpha protein observed in hypertension may be attributed to the augmented levels of AII and not to ANP.

The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of histone H3.

Groucho is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila. We are investigating the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducin-like Enhancer of split (TLE) proteins. We report that Groucho/TLEs are associated with chromatin in live cells and that they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones. This interaction is mediated by the H3 amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing. We also demonstrate that Groucho/TLEs form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3 and that these interactions may be propagated along the chromosome due to the ability of Groucho/TLEs to participate in higher order structures.

Modulation of ANF-R2/ANP-C receptors by angiotensin II in vascular smooth muscle cells.

In the present studies, we have investigated the modulation of atrial natriuretic factor (ANF) receptor of R2 subtype (ANF-R2) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (AII). C-ANF4-23 [C-ANF4-23, [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF4-23-NH2] and AII inhibited adenylyl cyclase activity in control vascular smooth muscle cells (VSMC A-10) by about 40% and 30% respectively. Pretreatment of the cells with AII resulted in the attenuation of both C-ANF4-23- and AII-mediated inhibition of adenylyl cyclase. Losartan treatment of the cells was able to partially block (approximately 50%) the AII- as well as C-ANF4-23-mediated inhibitions of adenylyl cyclase that are completely lost by AII pretreatment. The pretreatment of the cells with AII alone or with losartan did not affect the [125I]-ANF binding to ANF receptors. However, AII treatment resulted in the augmentation of the levels of Gi alpha 2 and Gi alpha 3. On the other hand, staurosporine (a protein kinase C [PKC] inhibitor) treatment of cells before AII treatment was able to prevent the attenuation of both C-ANF4-23 as well as AII-mediated inhibition of adenylyl cyclase elicited by AII. These results indicate that the AII modulation of ANF-R2 receptor-mediated inhibition of adenylyl cyclase is independent of ANF-R2 receptor density or the levels of Gi regulatory protein and may be due to the uncoupling of the ANF-R2 receptor from the Gi protein. This uncoupling may be associated with the phosphorylation of the Gi protein by PKC activated by AII.