Modulation of cAMP-mediated vasorelaxation by endothelial nitric oxide and basal cGMP in vascular smooth muscle

Several studies have addressed the question of starvation effects on immune function by means of changes in lymphocyte subsets, cytokine induction or lymphocyte activation. Anorexia nervosa (AN) patients are severely malnourished and contradictory results have been obtained regarding the accompanying immunodeficiency, including its assignation as a part of the primary nervous disorder. In the present work, an extensive immunological function examination was carried out on 40 AN patients who were compared with a control group of 14 healthy girls. The AN patients were also classified according to their nutritional status (by the Body Mass Index: BMI), this being critical for a better understanding of these secondary immunodeficiency bases. Moreover, another immune system study was performed on five patients after refeeding. Lymphocyte subsets and function, cytokine induction and peripheral blood concentrations, and innate as well as humoral immunity were evaluated. Deregulation in the cytokine network, owing to the interaction of the central nervous (CNS) and immune systems, seems to be the initial immune alteration in AN immunodeficiency but it has not been disproved that the immunodeficiency is a direct consequence of the original psychiatric perturbation. Spontaneous high levels of circulating interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) have been observed; this is probably one of the causes of the anomalies found in the T-cell subpopulations (mainly the naive CD4+CD45RA+ reduction and the cytotoxic CD8+ increase) and T-cell activation status (mainly the down-regulation of the CD2 and CD69 activation pathways). This finally leads to an impairment, not only in T-cell function but also in T-cell to B-cell co-operation. The AN specificity of these results is confirmed by the fact that these immune alterations improve after refeeding and when nutritional status becomes less critical, which also suggests that AN immunodeficiency is indeed secondary to malnutrition.     

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.     

Intracellular application of calmidazolium increases Ca2+ current through activation of protein kinase A in cultured vascular smooth muscle cells

This study compared markers of the metabolic processes occurring in male and female adolescent triathletes from two age groups (over 15 years of age [O15] and under 15 years of age [U15]) during a laboratory based duathlon. Participants were tested on three separate occasions; two peak VO2 tests on a treadmill and cycle ergometer, and a third session involved a simulated duathlon (2 km run, 12 km ride and 4 km run for the O15 group or 1 km run, 8 km ride and 2 km run for the U15). Data collection included performance speed, cardiorespiratory responses and blood borne markers of exercise metabolism. The performance speeds selected by the two age groups did not differ. The mean relative percentage of VO2peak at which subjects participated were 79+/-3, 77+/-4%, for the O15 males and females, and 71+/-5 and 82+/-2%, for the U15 males and females, respectively. While the plasma metabolites of ammonia [NH3] and lactate [La] were not different between age groups and sex (p>0.05) there were however, higher concentrations recorded during the cycling phase when compared with the running phases (p < 0.05). The respective mean concentrations for NH3 and La were 80.5+/-5.6 microM, and 4.9+/-0.3 microM for cycling, and 56.3+/-2.7 microM, and 2.7+/-0.2 microM for the combined running phases.     

Eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells

Experiments were designed to determine whether the omega 3-unsaturated fatty acid eicosapentaenoic acid affects the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells. Incubation of cultured rat or human aortic smooth muscle cells with interleukin-1 beta evoked a time- and concentration-dependent release of nitrite, an oxidation product of nitric oxide. The exposure of cells to interleukin-1 beta in combination with eicosapentaenoic acid caused a significantly larger production of nitrite than that evoked by the cytokine alone. The potentiation by eicosapentaenoic acid was concentration-dependent. The production of nitrite evoked by equieffective concentrations of interleukin-1 beta in the presence and absence of eicosapentaenoic acid were inhibited to a similar extent by nitro L-arginine (an inhibitor of nitric oxide synthase), transforming growth factor beta 1, platelet-derived growth factorAB and thrombin. The addition of interleukin-1 beta-activated smooth muscle cells to suspensions of washed and indomethacin-treated platelets inhibited the aggregation caused by thrombin. The inhibitory effect was enhanced when the smooth muscle cells were exposed to the cytokine in the presence of eicosapentaenoic acid prior to the experiment. Smooth muscle cells exposed to interleukin-1 beta and eicosapentaenoic acid did not affect platelet aggregation in the presence of oxyhemoglobin or methylene blue. Untreated cells or cells exposed to the fatty acid alone did not have such effects. These observations suggest that eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in vascular smooth muscle.     

Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.     

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.     

Glucose-induced changes in Na+/H+ antiport activity and gene expression in cultured vascular smooth muscle cells. Role of protein kinase C

Increased Na+/H+ antiport activity has been implicated in the pathogenesis of hypertension and vascular disease in diabetes mellitus. The independent effect of elevated extracellular glucose concentrations on Na+/H+ antiport activity in cultured rat vascular smooth muscle cells (VSMC) was thus examined. Amiloride-sensitive 22Na+ uptake by VSMC significantly increased twofold after 3 and 24 h of exposure to high glucose medium (20 mM) vs. control medium (5 mM). Direct glucose-induced Na+/H+ antiport activation was confirmed by measuring Na(+)-dependent intracellular pH recovery from intracellular acidosis. High glucose significantly increased protein kinase C (PKC) activity in VSMC and inhibition of PKC activation with H-7, staurosporine, or prior PKC downregulation prevented glucose-induced increases in Na+/H+ antiport activity in VSMC. Northern analysis of VSMC poly A+ RNA revealed that high glucose induced a threefold increase in Na+/H+ antiport (NHE-1) mRNA at 24 h. Inhibiting this increase in NHE-1 mRNA with actinomycin D prevented the sustained glucose-induced increase in Na+/H+ antiport activity. In conclusion, elevated glucose concentrations significantly influence vascular Na+/H+ antiport activity via glucose-induced PKC dependent mechanisms, thereby providing a biochemical basis for increased Na+/H+ antiport activity in the vascular tissues of patients with hypertension and diabetes mellitus.     

Role of nuclear factor-kappaB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappaB (NF-kappaB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-kappaB activation pathway similar to that of IL-1beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1beta and TNF alpha, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1beta, and TNF alpha activated NF-kappaB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-kappaB activation stimulated by TNF alpha, but not by IL-1beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappaB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1beta and TNF alpha, but not neutral SMase, caused a transient decrease in IkappaB-alpha protein levels, whereas IkappaB-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IkappaB-alpha degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappaB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappaB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IkappaB-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).     

Inhibition by the JAK/STAT pathway of IFNgamma- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells

Inducible nitric oxide synthase (iNOS) is induced in many cell types by cytokines and lipopolysaccharide (LPS). Cytokine signal transduction is believed to be mediated primarily through the JAK/STAT pathway. We therefore examined the effects of a JAK2-specific inhibitor, an antisense oligonucleotide to JAK2, and electroporation of neutralizing anti-STAT1 and anti-STAT3 antibodies on IFNgamma- and LPS-stimulated induction of iNOS in vascular smooth muscle cells. Unexpectedly, we found that the JAK/STAT pathway suppresses IFNgamma- and LPS-stimulated iNOS induction in these cells. In contrast, the JAK/STAT pathway appears to have a positive role in iNOS induction in RAW 264.7 macrophages.     

Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells

The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Variability in the proliferative responsiveness of cultured human vascular smooth muscle cells to alpha-thrombin

alpha-Thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for many cell types. In the present study, the responsiveness to alpha-thrombin of cultured human vascular smooth muscle cells (HVSMC) derived from either vein or normal and atherosclerotic arteries was investigated. All HVSMC populations examined responded mitogenically to alpha-thrombin. However, the extent of this response varied between different cell populations. No significant differences were observed between HVSMC derived from vein versus artery or atherosclerotic versus normal tissues. The responsiveness of a specific HVSMC culture to alpha-thrombin was not affected by cell density and remained constant over several passages. Unlike platelet-derived growth factor BB (PDGF-BB), alpha-thrombin did not exhibit any significant chemotactic effects on HVSMC or induce their anchorage independent growth in semi-solid medium. The hypothesis that the observed variability in HVSMC responsiveness to alpha-thrombin is due to the heterogeneity of cultured HVSMC is raised and discussed.     

Advanced glycation endproducts stimulate mitogen-activated protein kinase and proliferation in rabbit vascular smooth muscle cells

Two patients with plasma cell leukemia (PCL) with a t(11;14)(q13;q32) translocation are reported. Case 1 is a 64-year-old woman diagnosed as having primary PCL (IgA/lambda, Stage III) with high serum LDH and beta 2-microglobulin (beta 2MG) levels. She was treated with combination chemotherapy but died of gastrointestinal bleeding on the 45th hospital day. Case 2 is a 52-year-old man, initially diagnosed with multiple myeloma (IgG/kappa, Stage III) in August 1993. Relapse several months after primary chemotherapy was characterized by a rapid increase in plasma cells in peripheral blood, high serum LDH and beta 2MG levels, and resistance to further chemotherapy. Both cases showed complex karyotypic abnormalities including t(11;14), and Northern analysis revealed overexpression of the PRAD1/ cyclin D1 gene. The PRAD1 gene is found on chromosome band 11q13 and encodes cyclin D1. Cyclin D1 plays an important role in control of the cell cycle, and overexpression of PRAD1/cyclin D1 may be involved in disease progression in these cases.     

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.     

Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension

In this study, we examined the regulation of mitogen-activated protein kinase phosphatase (MKP-1) expression by insulin in primary vascular smooth muscle cell cultures. Insulin caused a rapid time- and dose-dependent induction of MKP-1 mRNA and protein expression. Blockade of nitric-oxide synthase (NOS) with NG-monomethyl-L-arginine acetate, and cGMP with RpcGMP, completely inhibited MKP-1 expression. Insulin-mediated MKP-1 expression was preceded by inducible NOS (iNOS) induction and cGMP production. Blockade of phosphatidylinositol 3-kinase (PI3-kinase) signaling with wortmannin inhibited insulin-mediated iNOS protein induction, cGMP production, and MKP-1 expression. To evaluate potential interactions between NOS and the mitogen-activated protein kinase (MAPK) signaling pathways, we employed PD98059 and SB203580, two specific inhibitors of ERKs and p38 MAPK. These inhibitors abolished the effect of insulin on MKP-1 expression. Only PD98059 inhibited insulin-mediated iNOS protein induction. Vascular smooth muscle cells from spontaneous hypertensive rats exhibited a marked decrease in MKP-1 induction due to defects in insulin-induced iNOS expression because of reductions in PI3-kinase activity. Treatment with sodium nitroprusside and 8-bromo-cGMP restored MKP-1 mRNA expression to levels comparable with controls. We conclude that insulin-induced MKP-1 expression is mediated by PI3-kinase-initiated signals, leading to the induction of iNOS and elevated cGMP levels that stimulates MKP-1 expression.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.