Anne von Euler

The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

Profilin is associated with transcriptionally active genes.

We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.

Glycogen Synthase Kinase (GSK) 3β Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β−/− mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation.

Dopamine D2 receptors attenuate phosphatidylinositol 4,5-bisphosphate in synaptosomal membranes from rat neostriatum.

Abstract: The effects of dopamine on [32P]ATP‐labelled phosphatidylinositol 4‐phosphate, phosphatidylinositol 4,5‐bisphosphate, and phosphatidic acid were analyzed by TLC in synaptosomal membranes of the rat neostriatum. The incorporation of 32P into these compounds was found to be stable within 1 min and was maintained during the 30 min of incubation. Dopamine (0.1–10μM) was found to attenuate the levels of phosphatidylinositol 4,5‐bisphosphate without affecting the levels of phosphatidylinositol 4‐phosphate or phosphatidic acid. The maximal decrease (−35 ± 4%) was reached at 10 μM of dopamine after 30 min of incubation. The dopamine (0.1 μM)‐induced decrease was blocked by the D2 selective antagonist raclopride (1 μM), but not by the D1 selective antagonist SCH 23390 (1 μM). These findings indicate the existence of an intramembrane coupling of dopamine D2 receptors to phosphoinositide turnover and may underlie some of the physiological effects of D2 receptor stimulation.

An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.

Effects of Cystic Fibrosis Serum and Fibroblast Culture Medium on Ion Distribution in Rat Submandibular Gland

The effects of cystic fibrosis (CF) serum and culture medium from CF fibroblasts on ion distribution in rat submandibular gland cells were investigated by X-ray microanalysis. These effects were compared to the effects of normal serum and culture medium from normal fibroblasts, of cholinergic and adrenergic agonists, and of the uncoupler 2,4-dinitrophenol. Incubation of gland tissue with CF serum or normal serum caused a significant decrease in potassium and calcium concentrations and an increase in sodium in mucous acinar and serous granular duct cells. CF serum gave a significantly larger decrease of the potassium level than normal serum. Culture medium from CF fibroblasts altered the cellular ion content in a way similar to CF serum. Exposure to medium from cultured normal fibroblasts did not affect the elemental composition of the gland cells significantly, compared to incubation with fresh medium or buffer. Hence, fibroblast culture medium is more suitable than serum to test specific effects of CF-associated factors. The changes in elemental composition of gland cells caused by CF serum or CF fibroblast culture medium mimic some of the effects of the agonist carbachol. They could, however, also in part result from nonspecific changes in membrane permeability.

Effect of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells.

The effect of chronic treatment with cystic fibrosis (CF) fibroblast medium on rat submandibular gland and pancreas was investigated. Rats were injected for 8 days with conditioned medium from normal or CF fibroblasts. The elemental content of the acinar cells was measured by X-ray microanalysis of cryosections. A significant increase in cellular calcium, and a decrease in cellular sodium concentrations were found after treatment with CF medium. The ultrastructure of the submandibular acinar cells was not affected by the conditioned CF fibroblast culture medium. No effect of treatment with CF medium on ultrastructure and elemental content of pancreatic acinar cells could be demonstrated. The response to alpha-adrenergic, beta-adrenergic, cholinergic, and peptidergic stimulation in submandibular gland acinar cells of rats injected with normal or CF medium was investigated in vitro. With regard to changes in elemental composition after stimulation, no significant differences in response between the two groups could be found. Apparently, a factor in conditioned medium from cultured CF fibroblasts induces a net increase in calcium content of rat submandibular gland acinar cells. Possibly, this factor acts in a similar way in CF patients and may cause elevated calcium levels in CF cells.

Microprobe Analysis in Studies and Diagnosis of Cystic Fibrosisa

Cystic fibrosis (CF) is a generalized exocrinopathy with diverse clinical symptoms, of which chronic obstructive lung disease is the most serious. With an incidence of 1 :2,000 to 1 :4,000 newborns, C F is the most common lethal, congenital, genetic disease among Caucasians. Despite considerable progress in the treatment of the symptoms, still about twenty percent of the patients do not survive beyond the age of twenty.’-3 Several exocrine secretions of C F patients have an abnormal ionic composition: elevated Na, CI, and K in sweat,24 elevated Ca, Na, and C1 in submandibular ~ a l i v a , ~ . ~ and elevated Ca in parotid ~ a l i v a . ~ There is also excessive salivary protein ~ e c r e t i o n . ~ . ~ The secretion of a viscous fluid of abnormal ionic composition by exocrine glands could well be the underlying factor in many of the clinical symptoms of CF, such as blocking of the airways by viscous mucus associated with recurrent and persistent infections, exocrine pancreatic insufficiency, meconium ileus, and infertility in males. The abnormal composition of sweat is routinely used in diagnosis of CF.4 The molecular basis of the disease is not known. Considerable attention has, of course, been given to possible abnormalities in the regulation of ion transport in CF. Decreased ductal reabsorption of Na+,8 possibly caused by increased primary secretion of Ca2+, and decreased ductal reabsorption of chloride ions,’ are among the suggested mechanisms. Also of interest is the activity of a “humoral C F factor” (or, possibly, several “CF factors”), present in serum, saliva, and urine from C F patients. Originally of interest because of its ciliostatic properties,” the C F factor may also be responsible for increased secretion of proteins’’ and Kf12 and decreased ductal reabsorption of N a + ” in salivary glands in experimental animal systems. The nature of this hypothetical C F factor, or its relationship to the molecular basis or the clinical symptoms of the disease, is, however, not known at present. The relevance of abnormal ion transport for a t least some of the clinical symptoms of CF, combined with the possibility of studying abnormalities in ion distribution at the