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1.
Tadasu Nagaoka Taku Shirakawa Junko Kasuya Thomas W. Balon Vincent C. Manganiello Yoko Fujita-Yamaguchi 《Cell biochemistry and biophysics》1998,29(1-2):49-66
Type 3 cyclic nucleotide phosphodiesterase (PDE-3) isoforms exhibit a high affinity (“lowK m”) for cAMP and are specifically inhibited by cGMP and a number of pharmacological agents, which increase myocardial contractility, inhibit platelet aggregation, and increase smooth muscle relaxation. The PDE-3 family consists of at least two isozymes, PDE-3A (cardiac type) and PDE-3B (adipocyte type), with distinct tissue-specific distributions. PDE-3A mRNA is highly expressed in the cardiovascular system, whereas PDE-3B mRNA is primarily expressed in adipocytes and hepatocytes. Toward understanding potential roles of PDE-3 in diabetes mellitus, we have established a specific and sensitive RNase protection assay (RPA) for quantitating PDE-3A and PDE-3B mRNA in rat diabetic models. In fatty Zucker diabetic (ZDF) rats, PDE-3A mRNA, but not PDE-3B mRNA, was expressed in heart, whereas liver and white and brown fat tissues predominantly expressed PDE-3B mRNA. Unexpectedly, PDE-3B mRNA expression was ≈2.5 times higher than PDE-3A mRNA in aorta from both ZDF and Sprague-Dawley (SD) rats. In contrast, expression levels of PDE-3A mRNA in heart were similar in both species. With this RPA, we were thus able to compare PDE-3A and-3B mRNA levels in different tissues as well as in different rat species. 相似文献
2.
Rui He Narcisse Komas Dag Ekholm Taku Murata Masato Taira Steven Hockman Eva Degerman Vincent C. Manganiello 《Cell biochemistry and biophysics》1998,29(1-2):89-111
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1)
and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal
truncation mutants were expressed inEscherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all
PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one
from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited
a high affinity (<1 μM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were
photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid
sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other
PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic
domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of
sensitivity of PDE-3s to cGMP.
These authors contributed equally to this work. 相似文献
3.
The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels. 相似文献
4.
Jackie D. Corbin Alfreda Beasley Illarion V. Turko Tamara L. Haik Kimberly A. Mangum Jack N. Wells Sharron H. Francis Konjeti R. Sekhar 《Cell biochemistry and biophysics》1998,29(1-2):145-157
The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each
is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed
to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction
of either of these inhibitors or the substrates themselves with the catalytic domain of the enzymes has not been identified.
IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a
single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling
of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photo-affinity labeling
was progressively blocked by cGMP at concentrations higher than 10 μM, whereas cAMP or 5′-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5
catalytic site, including IBMX, clMP, and β-phenyl-1,N
2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 μM, which compared favorably with an IC50 of 5 μM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe
with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in
studies to map the catalytic site of PDE-5. 相似文献
5.
Alison M. Michie Graham Rena Margaret M. Harnett Miles D. Houslay 《Cell biochemistry and biophysics》1998,28(2-3):161-185
We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated
PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following
ligation of the TCR (Michie, A. M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M. D. [1996]Cell. Signalling
8, 97–110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein
tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated
by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover,
the PKC-activating phorobol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to
that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3
antibodies. Both the PMA-and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either
cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP
levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased
by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent
induction of PDE-4 activity following crosslinking of the TCR complex. This identifies “crosstalk” occurring between the PKA
and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase
and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. 相似文献
6.
Calvin Yu-Chian Chen 《Journal of biomolecular structure & dynamics》2013,31(5):627-640
Abstract Erectile dysfunction (ED) is a sexual disorder mainly caused by decrease in cellular concentration of cyclic guanosine monophosphate (cGMP), which is degraded by phosphodiesterase type-5 (PDE-5). As a potent therapeutic target, inhibitors such as Viagra®, Cialis®, and Levitra® have already been developed to target PDE-5 for treating ED; traditional Chinese medicine, Epimedium sagittatum, also has shown prominent results as well. To developed new PDE-5 inhibitors, we performed a virtual screening of traditional Chinese medicine (TCM) database and docking analyses to identify candidates. Known PDE-5 inhibitors were used to construct a three dimensional quantitative structure-activity relationship (3D QSAR) model by HypoGen program. From docking analyses, isochlorogenic acid b was identified as the most potential inhibitory compound. De novo evolution designed 47 derivatives. Of the 47 derivatives, seven were able to map into the pharmacophore model, and these seven compounds were suggested to be the most promising leads for inhibiting PDE-5. An analysis of the hydrogen bond interactions formed between the docked ligands and PDE-5 identified ASN662, SER663 and GLN817 as the most frequently interacting residues. A total of eight novel leading compounds were identified to have favorable interaction with PDE-5. These compounds all had hydrogen bond interactions with three key residues that could be further investigated for understanding of PDE-5 and ligands interaction. 相似文献
7.
Cantin LD Magnuson S Gunn D Barucci N Breuhaus M Bullock WH Burke J Claus TH Daly M Decarr L Gore-Willse A Hoover-Litty H Kumarasinghe ES Li Y Liang SX Livingston JN Lowinger T Macdougall M Ogutu HO Olague A Ott-Morgan R Schoenleber RW Tersteegen A Wickens P Zhang Z Zhu J Zhu L Sweet LJ 《Bioorganic & medicinal chemistry letters》2007,17(10):2869-2873
Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured. 相似文献
8.
Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase 总被引:3,自引:0,他引:3
T Yamamoto S Yamamoto J C Osborne V C Manganiello M Vaughan H Hidaka 《The Journal of biological chemistry》1983,258(23):14173-14177
We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites. 相似文献
9.
Katherine B. Pryzwansky Sudha Kidao Elizabeth P. Merricks 《Cell biochemistry and biophysics》1998,28(2-3):251-275
The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is
an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent
protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence
microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils,
and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of
PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA
were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting
that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D,
and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated
the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4
at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization. 相似文献
10.
Carolyn J. Smith JingZhi He Sidonnie G. Ricketts Jia-Zhen Ding Richard A. Moggio Thomas H. Hintze 《Cell biochemistry and biophysics》1998,29(1-2):67-88
Phosphodiesterase III (PDE-3) inhibitors are inotropes used to treat congestive heart failure (HF). Previous studies showed PDE-3A mRNA levels were reduced in the left ventricle (LV) in dogs subjected to pacing-induced HF. The present study evaluated a time-course for RV-specific changes in PDE-3A mRNAs and proteins after pacing for 3 wk (n=4) or in HF (4–5 wk;n=4–6). Total RNA from LV/RV tissues was isolated for Northern analyses; cytosolic and microsomal proteins were prepared for PDE-3A immunoblots. PDE-3A mRNAs (7–8 and 10 kb) were normalized against glyceraldehyde-3-phosphodehydrogenase (GAPDH) or ribosomal 18s with similar results. PDE-3A/GAPDH ratios in 3 wk were unchanged in LV, but significantly (p<0.05) reduced by 48% in RV vs unpaced controls (n=8). In contrast, PDE-3A (7–8kb)/GAPDH ratios were significantly reduced in HF by 50–59% in both ventricles. Consistent with mRNA levels, significant reductions in microsomal 135 kDa (93–96%) and cytosolic 120 kDa PDE-3A (57–69%) were seen in both ventricles in HF or in the RV at 3 wk; an LV-specific reduction (50%) in cytosolic 80 kDa PDE-3A in HF was also detected. In summary, RV-specific downregulation of PDE-3A mRNA/protein(s) at 3 wk suggests that hemodynamic rather than humoral mechanisms are responsible, and provides a molecular basis for the limited efficacy of milrinone in the progression of HF. 相似文献
11.
D. H. Maurice 《Cell biochemistry and biophysics》1998,29(1-2):35-47
Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents. 相似文献
12.
The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels. 相似文献
13.
Zhu D Birks EK Dawson CA Patel M Falck JR Presberg K Roman RJ Jacobs ER 《American journal of physiology. Heart and circulatory physiology》2000,279(4):H1526-H1533
20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A (CYP4A) metabolite of arachidonic acid (AA) in human and rabbit lung microsomes and is a dilator of isolated human pulmonary arteries (PA). However, little is known regarding the contribution of P-450 metabolites to pulmonary vascular tone. We examined 1) the effect of two mechanistically distinct omega- and omega1-hydroxylase inhibitors on perfusion pressures in isolated rabbit lungs ventilated with normoxic or hypoxic gases, 2) changes in rabbit PA ring tone elicited by 20-HETE or omega- and omega1-hydroxylase inhibitors, and 3) expression of CYP4A protein in lung tissue. A modest increase in perfusion pressure (55 +/- 11% above normoxic conditions) was observed in isolated perfused lungs during ventilation with hypoxic gas (FI(O(2)) = 0.05). Inhibitors of 20-HETE synthesis, 17-oxydecanoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), increased baseline perfusion pressure above that of vehicle and amplified hypoxia-induced increases in perfusion pressures by 92 +/- 11% and 105 +/- 11% over baseline pressures, respectively. 20-HETE relaxed phenylephrine (PE)-constricted PA rings. Treatment with 17-ODYA enhanced PE-induced contraction of PA rings, consistent with inhibition of a product that promotes arterial relaxation, whereas 6-(20-propargyloxyphenyl)hexanoic acid (PPOH), an epoxygenase inhibitor, blunted contraction to PE. Conversion of AA into 20-HETE was blocked by 17-ODYA, DDMS, and hypoxia. CYP4A immunospecific protein confirms expression of CYP4A in male rabbit lung tissue. Our data suggest that endogenously produced 20-HETE could modify rabbit pulmonary vascular tone, particularly under hypoxic conditions. 相似文献
14.
15.
Sayago CM Beierwaltes WH 《American journal of physiology. Regulatory, integrative and comparative physiology》2001,281(4):R1146-R1151
The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney. 相似文献
16.
We have investigated the vasorelaxant effect of atrial natriuretic peptide (ANP) on isolated non-contracted aorta from coarctation hypertensive rats (HR) and the role of endothelium in this vasorelaxant action. After 7-14 days of surgery, mean blood pressure was higher (P < 0.01) in HR compared with sham operated rats (SR), used as the control. ANP (10(-6) mol/l) significantly lowered basal tone in previously unstimulated HR thoracic aortic rings; however, it had no effect in HR abdominal aorta or in SR abdominal and thoracic aorta. Endothelial destruction potentiated the vasorelaxant effect of ANP on basal tone in HR thoracic aorta. A similar potentiation of the ANP-response was observed by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) mol/l) or methylene blue (2 x 10(-5) mol/l) in unrubbed HR thoracic aorta. Treatment with calcium-free Krebs + EGTA (2 x 10(-3) mol/l) + sodium nitroprusside (10(-5) mol/l) or calcium-free Krebs significantly decreased basal tone and abolished ANP-response. These effects were observed only in HR thoracic aorta. Similarly, staurosporine (10(-7) mol/l) and calphostin C (10(-6) mol/l), inhibitors of protein kinase C (PKC), diminished basal tone and abolished the ANP-response in HR thoracic aorta. Acetylcholine (10(-6) mol/l) had a small but significant action on the basal tone of unrubbed HR thoracic aorta. These results demonstrate that ANP has a vasorelaxant effect on aortic basal tone when the vessel is exposed to high blood pressure. Inhibition of ANP effects on basal tone by calcium-free Krebs and PKC antagonists suggests that the HR aorta increases Ca2+-active tone, that modifies the response of vascular smooth muscle to the vasodilating hormone ANP. 相似文献
17.
铁对血管收缩活动的影响及其机制 总被引:4,自引:2,他引:2
动脉粥样硬化的发生和铁引起的氧化应激密切相关。铁对血管的直接效应及其对血管收缩功能的影响尚不明确。本文采用血管环灌流装置 ,观察铁对离体SD大鼠去内皮胸主动脉环的直接效应 ,及对去内皮主动脉环KCl和苯肾上腺素 (PE)引发的收缩效应的影响。结果显示 :( 1) 10 0 μmol/L枸橼酸铁 (FAC)引起大鼠血管环发生相位性收缩 ,最大收缩幅度可达KCl诱发的最大收缩的 2 4 0 2± 2 3 7%。当 [Ca2 +]o 增加 1倍时 ,铁所致的血管环收缩幅度明显增加 (P <0 0 1)。阻断L 型钙通道后 ,铁所致的血管环收缩幅度明显降低 (P <0 0 1)。在无钙液中 ,用佛波酯收缩血管环 ,待收缩稳定后给予FAC ,此时收缩幅度增加 49 18± 3 75 %。 ( 2 )铁孵育 3 0min后 ,KCl引起血管环收缩的幅度显著降低 (P <0 0 1)。铁孵育可使PE引起的收缩量 -效曲线右移 (P <0 0 5 )。 ( 3 )二甲基亚砜、过氧化氢酶和谷胱甘肽可明显降低铁对PE血管收缩反应的抑制作用 (P <0 0 5 )。从这些结果可得到以下结论 :铁可引起胸主动脉发生相位性收缩 ,其机制可能与L 型钙通道短暂开放导致钙离子内流 ,及平滑肌对钙的敏感性增加有关 ;较长时间与铁孵育后 ,可对血管收缩功能产生损伤 ,氧自由基的生成增加和细胞内GSH的水平降低可能参与铁对收缩功能的 相似文献
18.
Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver 总被引:1,自引:0,他引:1
T Yamamoto S Yamamoto V C Manganiello M Vaughan 《Archives of biochemistry and biophysics》1984,229(1):81-89
Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP. 相似文献
19.
Peral De Bruno M Vincent PA Romano L Guardia DC Coviello A De Vito E 《American journal of physiology. Heart and circulatory physiology》2003,284(2):H704-H710
The effect of a novel enzyme (PreR-Co) that activates renal prorenin was studied on rabbit aortas with and without endothelium. It was tested 1) in the basal tone of nonstimulated or ANG II-sensitized rings or rings precontracted with norepinephrine (NE), PGF(2alpha), high KCl concentration, and 2) in rings pretreated with enalaprilat, losartan, PD-123319, N(omega)-nitro-l-arginine methyl ester, HOE-140, indomethacin, or serine protease inhibitors (PMSF, aprotinin, or soybean trypsin inhibitor); kallilkrein and bradykinin were also tested in ANG II-sensitized rings. PreR-Co produced a vasorelaxant effect in the basal tone and in the precontracted rabbit aorta. The effect was endothelium independent, potentiated by endothelium removal or nitric oxide (NO) synthase inhibition, and abolished by boiling the enzyme. In addition, the effect improved when basal tone was increased in ANG II-sensitized aortic rings or in precontracted vessels. No activation of the ANG II, bradykinin, prostaglandin, or NO pathway mediating the PreR-Co response could be obtained, suggesting a direct action of the enzyme. This action seems to be dependent on esterasic activity because serine protease inhibitors like PMSF and aprotinin were able to block the vasorelaxant effect of PreR-Co. 相似文献
20.
Natalie Saldou Preston A. Baecker Bin Li Zhengyu Yuan Rena Obernolte James Ratzliff Eric Osen Kurt Jarnagin Earl R. Shelton 《Cell biochemistry and biophysics》1998,28(2-3):187-217
Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order
to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various
functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One
form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography.
The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (>95%) as
estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could
be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for
PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation
in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers
and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4
are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized
physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in
cells. 相似文献