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PubMed1998

Interactions of phosducin with the subunits of G-proteins. Binding to the alpha as well as the betagamma subunits.

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Antibodies↗

PubMed1998

Exon sequence is required for regulated RNA splicing of the human fibroblast growth factor receptor-1 alpha-exon.

Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the alpha-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the alpha-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5'-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for alpha-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for alpha-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the alpha-exon.

Alternative Splicing↗

PubMed2000

Inhibition of protein phosphatase-1 by clavosines A and B. Novel members of the calyculin family of toxins.

Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M. K., Pihko, P. M., and Koskinen, A. M. (1997) J. Biol. Chem. 272, 23312-23316; Gupta, V., Ogawa, A. K., Du, X., Houk, K. N., and Armstrong, R. W. (1997) J. Med. Chem. 40, 3199-3206; Gauss, C. M., Sheppeck, I. J., Nairn, A. C., and Chamberlain, R. (1997) Bioorg. Med. Chem. 5, 1751-1773). A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety.

Animals↗

PubMed2000

A rice functional transcriptional activator, RISBZ1, responsible for endosperm-specific expression of storage protein genes through GCN4 motif.

The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endosperm-specific expression. This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins. Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library. The predicted gene products can be divided into two groups based on their amino acid sequences. Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif. Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation. The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins. RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes. When the RISBZ1 promoter was transcriptionally fused to the beta-glucuronidase reporter gene and the chimeric gene was introduced into rice, the beta-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm. These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes.

Amino Acid Motifs↗

PubMed2001

Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

Angiotensin II↗

PubMed2001

Plasmin-mediated release of the guidance molecule F-spondin from the extracellular matrix.

Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.

Amino Acid Sequence↗

PubMed2005

PHOX2B regulates its own expression by a transcriptional auto-regulatory mechanism.

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomain transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5'-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription.

Base Sequence↗

PubMed2006

Versican V0 and V1 guide migratory neural crest cells.

We previously showed the selective expression of the chondroitin sulfate proteoglycans versican V0 and V1 in barrier tissues that impede the migration of neural crest cells during embryonic trunk development (Landolt, R. M., Vaughan, L., Winterhalter, K. H., and Zimmermann, D. R. (1995) Development 212, 2303-2312). To test for an active involvement of these isoforms in the guidance process, we have now established protocols to isolate intact versican V0 and V1 in quantities sufficient for functional experiments. Using stripe choice assays, we demonstrate that pure preparations of either a mixture of versican V0/V1 or V1 alone strongly inhibit the migration of multipotent Sox10/p75NTR double-positive early neural crest stem cells on fibronectin by interfering with cell-substrate adhesion. We show that this inhibition is largely core glycoprotein-dependent, as the complete removal of the glycosaminoglycan chains has only a minor effect on the inhibitory capacity. Our findings support the notion that versican variants V0 and V1 act, possibly in concert with other inhibitory molecules such as aggrecan and ephrins, in directing the migratory streams of neural crest cells to their appropriate target tissues.

Animals↗

PubMed2006

Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids.

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.

Amino Acid Sequence↗

PubMed2004

Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport.

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.

ADP-Ribosylation Factors↗

PubMed2004

The DHE cell line as a model for studying rat gastro-intestinal mucin expression: effects of dexamethasone.

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three nontumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semiquantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme-linked lectin assay. We showed that mRNA of rMucl and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10(-7) mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.

Animals↗

PubMed2002

Melanopsin: a novel photopigment involved in the photoentrainment of the brain's biological clock?

The brain's biological clock located in the suprachiasmatic nucleus (SCN) generates circadian rhythms of physiology and behaviour of approximately 24 hours. The clock needs, however, like a watch that runs too fast or too slow, daily adjustment and the most important stimulus for this adjustment is the environmental light/dark cycle, a process know as photoentrainment. It is well established that the eye contains a separate anatomical and functional system mediating light information to the clock. Until recently, the photopigment responsible for light entrainment of the circadian system has been elusive but recent studies have provided evidence that melanopsin, a recently identified opsin, could be the circadian photopigment. This conclusion is based on the observation that melanopsin is expressed exclusively in retinal ganglion cells projecting to the SCN, a projection known as the retinohypothalamic tract (RHT) and that these ganglion cells are intrinsically photosensitive. Melanopsin is present in the plasma membrane of soma, dendrites and axons forming an extensive photoreceptive network in the entire retina. Although these findings make melanopsin a strong candidate as a circadian photopigment, a number of functional experiments are needed before the role of melanopsin is finally proven.

Animals↗

PubMed2000

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function.

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

Adenosine Diphosphate↗

PubMed2002

Effects of trientine, a metal chelator, on defective endothelium-dependent relaxation in the mesenteric vasculature of diabetic rats.

Diabetes mellitus compromises endothelium-dependent relaxation of blood vessels. This has been linked to the generation of reactive oxygen species (ROS), which neutralise nitric oxide (NO) and interfere with vasodilator function. Experiments using chelators have emphasised the importance of ROS produced by transition metal catalysed reactions. However, particularly for the small arteries and arterioles that control microcirculatory blood flow, NO is not the only endothelium-derived mediator; endothelium-derived hyperpolarizing factor (EDHF) has a major role. EDHF-mediated vasodilation is severely curtailed by diabetes; however, little information exists on the underlying pathophysiology. Deficits in the EDHF system, alone or in combination with the NO system, are crucial for the development of diabetic microvascular complications. To further elucidate the mechanisms involved, the aim was to examine the effects of diabetes and preventive and intervention chelator therapy with trientine on a preparation that has well-defined NO and EDHF-mediated responses, the rat mesenteric vascular bed. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine was reduced by 35 and 44% after 4 and 8 weeks of streptozotocin-induced diabetes, respectively. Trientine treatment over the first 4 weeks gave 72% protection; intervention therapy over the final 4 weeks prevented deterioration and corrected the initial deficit by 68%. These responses depend on both NO and EDHF. When the latter mechanism was isolated by NO synthase inhibition, diabetic deficits of 53.4 (4 weeks) and 65.4% (8 weeks) were revealed, that were 65% prevented and 50% corrected by trientine treatment. Neither diabetes nor trientine altered vascular smooth muscle responses to the NO donor, sodium nitroprusside (SNP). Thus, the data suggest that metal catalysed ROS production makes a substantial contribution to defects in both the EDHF and NO endothelial mechanisms in diabetes, which has therapeutic implications for microvascular complications.

Acetylcholine↗

PubMedDate not supplied

Recanalization of the collecting lymphatics in rabbit hind leg.

OBJECTIVE: This study was designed to examine whether mature collecting lymphatics can regenerate in the adult tissue or not. MATERIALS AND METHODS: The X-ray lymphograms were used to detect network of the collecting lymphatics in rabbit hind leg. Regeneration of the lymphatics was observed after surgical removal of the popliteal lymph node or a part of the popliteal afferent lymphatic. Structure and mechanical properties of the lymphatics were also examined by light and electron microscopes and in vitro functional experiments. RESULTS: One week after removal of the lymph node, only an afferent lymphatic and a deposit of the contrast medium at the popliteal region were observed. Four weeks after the removal, the connection of the afferent and efferent lymphatics at the popliteal region, and collateral lymphatics were present in the leg. Further, 4 weeks after 1-mm excisions of a part of the lymphatic, recanalization was observed between the central and peripheral cut ends of the lymphatic but not after 3- and 10-mm excisions. Endothelial cells and smooth muscle cells could be observed by electron microscope, and contractile proteins, and alpha-smooth muscle actin SM1 and SM2 were immunofluorescently detected in both intact and the regenerated lymphatic walls. In both lymphatics, norepinephrine and acetylcholine induced dose-dependent constriction and dilation of the vessels, respectively. CONCLUSION: The present study demonstrated that mature collecting lymphatics are able to regenerate in the adult tissues.

Animals↗

PubMed2002

Cyclooxygenase products do not contribute to the gestational renal vasodilation in the nitric oxide synthase inhibited pregnant rat.

OBJECTIVE: To determine whether vasodilatory cyclooxygenase (COX) products are activated in kidney during chronic nitric oxide synthase inhibition (NOSI) in pregnant rats. METHODS: Chronically catheterized pregnant and nonpregnant Sprague-Dawley female rats were treated with daily NOSI ( L-NAME; 100 mg/L drinking water) for 14 days. Renal function experiments were conducted twice at 7-9 days of chronic NOSI (equivalent to midterm in pregnant rats) and after 14 days (equivalent to late pregnancy). Measurements were made of arterial blood pressure (BP) and renal hemodynamics, specifically glomerular filtration rate (GFR), renal plasma flow (RPF), and renal vascular resistance (RVR), in control and then during acute COX inhibition with indomethacin (2 mg/kg i.v.). RESULTS: Chronic NOSI increased BP and RVR in both virgin and pregnant rats. The normal midterm renal vasodilation was prevented and there was no BP or renal hemodynamic response to acute COX inhibition. By late pregnancy, RPF had fallen further and again, acute COX inhibition had no impact on renal hemodynamics, although a small reduction was seen in GFR in LV. CONCLUSIONS: Vasodilatory COX products cannot compensate for nitric oxide as the renal vasodilatory factor of pregnancy in the rat.

Animals↗

PubMed1997

The first 35 amino acids and fatty acylation sites determine the molecular targeting of endothelial nitric oxide synthase into the Golgi region of cells: a green fluorescent protein study.

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS-green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS-GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS-GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.

3T3 Cells↗

PubMed1984

Induction of muscle genes in neural cells.

The regulation of skeletal muscle genes was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to four different rat neural cell lines. Highly enriched populations of heterokaryons isolated using irreversible biochemical inhibitors were labeled with [35S]methionine and analyzed on two-dimensional gels. Rat skeletal myosin light chains were induced in three of the four cell combinations. The one exception, the S-20 cholinergic cell line, not only failed to synthesize rat muscle proteins but also suppressed chick myogenic functions. Experiments with heterokaryons between chick myocytes and cells from whole embryonic rat brain cultures demonstrated that rat skeletal myosin light chains are inducible in normal diploid neural cells as well as in established neural cell lines. In contrast, dividing cell hybrids between rat myoblasts and rat glial cells were nonmyogenic. These results demonstrate that although neural cells may contain factors that prevent the decision to differentiate along myogenic lines in cell hybrids, most neural cell lines do not dominantly suppress the expression of muscle structural genes in heterokaryons. Furthermore, the skeletal myosin light chain genes in most neural cell lines are regulated by a mechanism that permits them to respond to putative chick skeletal myocyte-inducing factors. The "open" state of these myogenic genes may explain many of the reports of apparent "transdifferentiation" to muscle in neural cultures and neural tumors.

Animals↗