Adipocytokine, progranulin, augments acetylcholine-induced nitric oxide-mediated relaxation through the increases of cGMP production in rat isolated mesenteric artery.

AIM: Progranulin (PGRN) is a novel adipocytokine with anti-inflammatory effects in vascular cells. The aim of this study was to clarify the effects of PGRN on reactivity of isolated blood vessel. METHODS: Isometric contraction of rat isolated superior mesenteric artery was measured. RESULTS: Pre-treatment with PGRN (10-100 ng mL-1 , 30 min) had no effect on noradrenaline- or 5-hydroxytriptamine-induced contraction. On the other hand, pre-treatment with PGRN (100 ng mL-1 ) augmented acetylcholine (ACh; 30 nm)-induced endothelium-dependent relaxation. Pre-treatment with PGRN (100 ng mL-1 ) augmented ACh (10 µm)-induced nitric oxide (NO)-mediated relaxation in the presence of indomethacin (10 µm), a cyclooxygenase inhibitor, and tetraethyl ammonium (10 mm), a non-selective potassium channel blocker. In contrast, pre-treatment with PGRN (100 ng mL-1 ) had no effect on ACh-induced endothelium-derived hyperpolarizing factor-mediated relaxation. Pre-treatment with PGRN (100 ng mL-1 ) had no effect on ACh (10 µm, 1 min)-induced endothelial NO synthase phosphorylation (at Ser1177) as determined by Western blotting. Pre-treatment with PGRN (100 ng mL-1 ) augmented an NO donor, sodium nitroprusside (SNP; 30 nm-1 µm)- but not a membrane-permeable cGMP analogue, 8-bromo-cGMP-induced relaxation. In the presence of 3-isobutyl-1-methylxanthine (100 µm), a phosphodiesterase inhibitor, pre-treatment with PGRN (100 ng mL-1 ) increased SNP (30 nm, 5 min)-induced cGMP production as determined by enzyme immunoassay. CONCLUSION: We for the first time demonstrate that PGRN augments ACh-induced NO-mediated relaxation through the increases of cGMP production in smooth muscle. These results indicate PGRN as a possible pharmacotherapeutic target against cardiovascular diseases including obesity-related hypertension.

Improvement of meal-related symptoms and epigastric pain in patients with functional dyspepsia treated with acotiamide was associated with acylated ghrelin levels in Japan.

BACKGROUND: The aim of this study is to clarify whether acotiamide and rabeprazole combination therapy can improve clinical symptoms, gastric emptying, and satisfaction with treatment in functional dyspepsia (FD) patients more effectively than acotiamide or rabeprazole monotherapy alone. We also aimed to determine whether acotiamide affects these changes via its effect on gastric emptying and appetite-related hormones such as ghrelin. METHODS: We used Rome III criteria to evaluate upper abdominal symptoms and anxiety by the State-Trait Anxiety Inventory (STAI). Gastric motility was evaluated by the (13) C-acetate breath test. Eighty-one FD patients were treated with acotiamide (300 mg/day) (n = 35), acotiamide (300 mg/day) and rabeprazole (10 mg/day) (n = 28), or rabeprazole (10 mg/day) (n = 18) for a period of 4 weeks and followed after 4 weeks of no treatment. Adenocorticotropic hormone (ACTH), cortisol, leptin and ghrelin levels were measured in all FD patients. KEY RESULTS: Acotiamide and rabeprazole combination therapy significantly improved postprandial distress syndrome (PDS)-like symptoms (p = 0.018, p = 0.04 and p = 0.041, respectively) and epigastric pain (p = 0.024) as wells as STAI-state scores (p = 0.04) compared to rabeprazole monotherapy. Both acotiamide monotherapy, and acotiamide taken in combination with rabeprazole, significantly (p = 0.001 and p = 0.02, respectively) improved satisfaction with treatment, compared to rabeprazole monotherapy. Acotiamide and rabeprazole combination therapy had no significant effect on ACTH and cortisol levels in FD patients. Of interest, acotiamide monotherapy, and acotiamide and rabeprazole combination therapy, significantly (p < 0.0001 and p = 0.018, respectively) increased acylated ghrelin/total ghrelin ratios and significantly (p = 0.04) improved impaired gastric emptying compared to rabeprazole monotherapy. CONCLUSIONS & INFERENCES: Further studies are warranted to clarify how acotiamide treatment improves clinical symptoms in FD patients.

Visceral adipose tissue-derived serine protease inhibitor augments acetylcholine-induced relaxation via the inhibition of acetylcholine esterase activity in rat isolated mesenteric artery.

AIM: Visceral adipose tissue-derived serine protease inhibitor (vaspin) is an adipocytokine with insulin-sensitizing activity originally identified in visceral adipose tissues of obesity-related type II diabetic rats. We previously showed that vaspin inhibits vascular cell migration and apoptosis as well as inflammatory responses, which are crucial for the development of hypertension. However, little is known about the effects of vaspin on vascular reactivity. The aim of this study was thus to explore the effects of vaspin on contraction and relaxation of isolated blood vessel. METHODS: After mesenteric arteries were isolated from male Wistar rats, the effects of pretreatment with vaspin (3 ng mL(-1) , 30 min) on concentration-contraction and concentration-relaxation relationships for each agent were examined. The effects of vaspin on acetylcholine (ACh)-induced phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and ACh esterase (AChE) activity were also examined using Western blotting and colorimetric method respectively. RESULTS: Vaspin did not affect noradrenaline- or 5-hydroxytryptamine-induced contraction. In contrast, vaspin augmented ACh- but not histamine-, A23187- or carbachol-induced NO-mediated relaxation. Vaspin significantly increased ACh-induced eNOS phosphorylation and inhibited AChE activity. CONCLUSION: We for the first time demonstrate that vaspin augments the ACh-induced NO-mediated endothelium-dependent relaxation via the inhibition of AChE activity.

Vaspin prevents elevation of blood pressure through inhibition of peripheral vascular remodelling in spontaneously hypertensive rats.

AIM: Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a relatively novel adipocytokine with protective effects on metabolic diseases including obesity and type II diabetes. We have previously demonstrated that vaspin exerts anti-inflammatory and antimigratory roles through antioxidative effects in vascular smooth muscle cells. As inflammatory responses and migration of smooth muscle in peripheral vascular wall are key mechanisms for the pathogenesis of hypertension, we hypothesized that vaspin could prevent the development of hypertension in in vivo hypertensive animal model. METHODS: Vaspin (1 µg kg(-1)  day(-1) ) was administered intraperitoneally to 5-week-old male spontaneously hypertensive rats (SHR) for 4 weeks. Superior mesenteric artery was isolated and used for measurement of isometric contraction and histological analysis. RESULTS: Long-term vaspin treatment significantly prevented an elevation of systolic blood pressure (SBP) at 8 weeks of age. Vaspin had no effect on reactivity of isolated mesenteric artery from SHR. In contrast, vaspin significantly inhibited mesenteric arterial wall hypertrophy in SHR. Moreover, vaspin significantly inhibited an increase of tumour necrosis factor-α expression and a production of reactive oxygen species in isolated mesenteric artery from SHR. CONCLUSION: This study for the first time demonstrates that vaspin prevents the increase of SBP in SHR through inhibiting peripheral vascular hypertrophy possibly via antioxidative and anti-inflammatory mechanisms.

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells.

AIM: Eukaryotic elongation factor 2 kinase (eEF2K), also known as calmodulin (CaM)-dependent protein kinase (CaMK) III, is a unique member of CaMK family protein. We have recently found that expression of eEF2K protein increased in mesenteric artery from spontaneously hypertensive rats. As pathogenesis of hypertension is in part regulated by vascular structural remodelling via proliferation and migration of vascular smooth muscle cells (SMCs), we tested the hypothesis that eEF2K controls SMCs proliferation and migration. METHODSAND RESULTS: In rat mesenteric arterial SMCs, an eEF2K inhibitor, A-484954 (10 µm), significantly inhibited platelet-derived growth factor (PDGF)-BB (10 ng mL(-1) )-induced SMCs proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay. PDGF-BB (10 ng mL(-1) )-induced SMCs migration was significantly inhibited by A-484954 (10 µm) as determined by a Boyden chamber assay. A-484954 (10 µm) significantly inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K, extracellular signal-regulated kinase (ERK), Akt, p38 and heat-shock protein (HSP) 27 as determined by Western blotting. It was confirmed that a CaM inhibitor, W-7 (50 µm), inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K. In an ex vivo mesenteric arterial ring assay, 10% foetal bovine serum-induced SMCs outgrowth was significantly inhibited by A-484954 (10 µm). CONCLUSION: We for the first time revealed that eEF2K mediates PDGF-BB-induced SMCs proliferation and migration through activating ERK, Akt, p38 and HSP27 signals in a CaM-dependent manner. Our results suggest eEF2K as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Brain-derived neurotrophic factor promotes angiogenic tube formation through generation of oxidative stress in human vascular endothelial cells.

AIM: Brain-derived neurotrophic factor (BDNF), a major type of neurotrophins, plays a role in the regulation of synaptic function. Recent studies suggest that BDNF promotes angiogenesis through its specific receptor, tropomyosin-related kinase B (TrkB). However, the detailed mechanisms for this still remain to be determined. Reactive oxygen species (ROS) generation contributes to the regulation of angiogenesis. Thus, we investigated the mechanisms by which BDNF regulates angiogenesis with focusing on ROS in cultured human vascular endothelial cells (ECs). METHODS AND RESULTS: In human umbilical vein ECs, BDNF increased ROS generation as measured fluorometrically using 2' 7'-dichlorofluorescein diacetate as well as NADPH oxidase (NOX) activity as measured by lucigenin assay. BDNF-induced ROS generation and NOX activity were inhibited by K252a, a TrkB receptor inhibitor. BDNF induced phosphorylation of p47 phox, a regulatory component of NOX, which was inhibited by K252a as measured by Western blotting. BDNF increased angiogenic tube formation in ECs, which was completely inhibited by K252a or gp91ds-tat, a NOX inhibitor. BDNF caused Akt phosphorylation in ECs, which was inhibited by K252a or gp91ds-tat. CONCLUSION: The present results for the first time demonstrate that BDNF induces NOX-derived ROS generation through activation of p47 phox in a TrkB receptor-dependent manner, which leads to the promotion of angiogenic tube formation possibly via Akt activation.

Vaspin prevents methylglyoxal-induced apoptosis in human vascular endothelial cells by inhibiting reactive oxygen species generation.

AIM: Vaspin (visceral adipose tissue-derived serine protease inhibitor) is a novel adipocytokine found in visceral white adipose tissues of obese type 2 diabetic rats. We have previously shown that vaspin has anti-inflammatory and antimigratory effects in vascular smooth muscle cells. Methylglyoxal (MGO) is an active metabolite of glucose and mediates diabetic vascular complications including endothelial cell (EC) apoptosis. Nonetheless, effects of vaspin on MGO-induced apoptosis of vascular EC remain to be determined. We investigated the effects of vaspin on MGO-induced apoptosis of human umbilical vein ECs (HUVECs). METHODS: Human umbilical vein ECs were treated with MGO (560 µm, 12 h) in the absence or presence of vaspin (1 ng mL(-1), pre-treatment for 2 h). Cell death was evaluated by a cell counting assay. Apoptosis was determined by a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay. Cleaved caspase-3 expression was determined by Western blotting. Reactive oxygen species (ROS) generation was fluorometrically measured using 2', 7'-dichlorodihydrofluorescein diacetate. NADPH oxidase (NOX) activity was determined by a lucigenin assay. RESULTS: Vaspin significantly inhibited MGO-induced HUVEC death. Vaspin significantly attenuated MGO-increased TUNEL-positive ECs. Moreover, vaspin significantly inhibited MGO-induced caspase-3 cleavage. Vaspin significantly inhibited MGO-induced ROS generation as well as NOX activation. CONCLUSIONS: The present results for the first time demonstrate that vaspin inhibits MGO-induced EC apoptosis by preventing caspase-3 activation via the inhibition of NOX-derived ROS generation.

Nizatidine improves clinical symptoms and gastric emptying in patients with functional dyspepsia accompanied by impaired gastric emptying.

BACKGROUND/AIMS: In this crossover study, we investigated whether nizatidine, a H(2)-receptor antagonist, can alleviate clinical symptoms and gastric emptying in patients with Rome III-based functional dyspepsia (FD) with or without impaired gastric emptying. METHODS: We enrolled 30 patients presenting with FD symptoms (epigastric pain syndrome, n = 6; postprandial distress syndrome, n = 24). Rome III-based FD patients were treated with nizatidine (300 mg/day) or placebo for 4 weeks in a crossover trial. Gastric motility was mainly evaluated with the T(max) value using the (13)C-acetate breath test. Meal-related symptoms were defined as postprandial fullness and early satiation. Gastroesophageal symptom was defined as a burning feeling rising from the stomach or lower chest up toward the neck. Acylated- and desacylated ghrelin levels were evaluated by the ELISA method. Clinical symptoms, gastric emptying and ghrelin levels were evaluated at three different points during the study (pretreatment, after 4 weeks former treatment and after 4 weeks later treatment). The primary end point of this study was to determine whether nizatidine would improve clinical symptoms and gastric emptying in FD patients with or without impaired gastric emptying via affecting ghrelin levels. RESULTS: Meal-related symptoms of the patients treated with nizatidine improved significantly (21/30; 70%) compared to those treated with placebo (3/30; 10%). In addition, nizatidine treatment also significantly improved gastroesophageal symptoms (16/30; 53%) compared to those treated with placebo (0/30; 0%). Nizatidine treatment in patients with FD accompanied by impaired gastric emptying significantly improved clinical symptoms and T(max) value as a marker of gastric emptying (10/11, 91%; 9/11, 82%) compared to placebo therapy, respectively. There were no significant differences in ghrelin levels between nizatidine treatment and placebo therapy. CONCLUSION: Nizatidine administration significantly improved both gastric emptying and clinical symptoms in FD patients with impaired gastric emptying.

Protonic diffusion in high-pressure ice VII.

Near ambient pressures, molecular diffusion dominates protonic diffusion in ice. Theoretical studies have predicted that protonic diffusion will dominate at high pressures in ice. We measured the protonic diffusion coefficient for the highest temperature molecular phase of ice VII at 400 kelvin over its entire stable pressure region. The values ranged from 10(-17) to 10(-15) square meters per second at pressures of 10 to 63 gigapascals. The diffusion coefficients extrapolated to high temperatures close to the ice VII melting curve were less by a factor of 10(2) to 10(3) than a superionic criterion of approximately 10(-8) square meters per second, at which protons would diffuse freely.

Chronic effect of doxorubicin on vascular endothelium assessed by organ culture study.

We have attempted to determine the chronic effects of doxorubicin, a commonly used anticancer agent, on vascular endothelium using an organ culture system. In rabbit mesenteric arteries treated with 0.3 microM doxorubicin for 7 days, rounding and concentrated nuclei and TUNEL-positive staining were observed in endothelial cells, indicating DNA damage and the induction of apoptosis. However, the endothelium-dependent relaxation induced by substance P and the expression of mRNA encoding endothelial NO synthase (eNOS) did not differ from those in control arteries. In arteries treated with a higher concentration (1 microM) of doxorubicin, apoptosis and damage to nuclei occurred in the endothelial cells at the third day of treatment, and the detachment and excoriation of endothelium from the tunica interna of the vascular wall were also observed. The impairment of endothelium-dependent relaxation was observed at the fifth day of the treatment with 1 microM doxorubicin. Additionally, apoptotic change in the smooth muscle layer was observed at this concentration of doxorubicin. Apoptotic phenomena were further confirmed by DNA fragmentation using isolated bovine aortic endothelial cells (BAECs) and A7r5 vascular smooth muscle cells, and it was revealed that BAECs are more sensitive than A7r5 to the apoptotic effect of doxorubicin. These results suggest that chronic treatment with doxorubicin at therapeutic concentrations induces apoptosis and excoriation of endothelial cells, which diminishes endothelium-dependent relaxation.

Hypoxia impairs endothelium-dependent relaxation in organ cultured pulmonary artery.

In intrapulmonary arteries cultured under hypoxic conditions (5% oxygen) for 7 days, endothelium-dependent relaxation and cGMP accumulation induced by substance P were decreased as compared to those of a normoxic control (20% oxygen). In rabbit mesenteric arteries exposed to chronic hypoxia, however, endothelial dysfunction was not observed. Furthermore, in endothelium-denuded pulmonary arteries exposed to hypoxia, neither relaxation nor cGMP accumulation due to sodium nitroprusside differed from those of the normoxic control. Hypoxia did not change the mRNA expression of endothelial NO synthase (eNOS), the protein expression of eNOS or the eNOS regulatory protein caveolin-1 as assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) or whole-mount immunostaining. Morphological study revealed atrophy of endothelial cells and condensation of the eNOS protein in many cells. These results suggest that chronic hypoxia impaired NO-mediated arterial relaxation without changing either the eNOS protein expression or the NO-sensitivity of smooth muscle cells in pulmonary arteries. Changes in cell structure and organization may be involved in endothelial dysfunction.

Chronic vascular toxicity of doxorubicin in an organ-cultured artery.

1. We investigated the chronic effects of doxorubicin (DXR) on morphological and functional changes in the rabbit mesenteric artery using an organ culture system. 2. In arteries cultured with 0.3 microM DXR for 7 days, the contractions induced by noradrenaline, but not those induced by endothelin-1 or high K(+), were strongly inhibited. This reaction was followed by a decrease in the induction of the alpha(1A)-adrenoceptor without any change in the mRNA level. Inhibition of noradrenaline-induced contractions by DXR was attenuated by superoxide dismutase, and alpha(1A)-adrenoceptor protein expression recovered. 3. In the arteries cultured with 1 microM DXR for 7 days, contractions induced by endothelin-1 or high K(+) and absolute force in the permeabilized muscles were also inhibited. Morphological examinations revealed the existence of concentrated nuclei and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive smooth muscle cells, and internucleosomal DNA fragmentation was also detected, indicating the induction of apoptosis. 4. In the arteries cultured with 10 microM DXR for 7 days, nuclear swelling, karyolysis and random DNA fragmentation indicative of necrosis were observed, and muscle contractility was abolished. 5. These results suggest that 0.3 microM DXR selectively down-regulates the alpha(1A)-adrenoceptor protein expression, resulting in a decrease in the noradrenaline-induced contraction. This down-regulation may be at least partly due to the production of a superoxide radical. DXR also caused a decrease in muscle contractility followed by apoptotic changes at 1 microM and necrotic changes at 10 microM. These changes might be responsible for the disturbance of the circulatory system that is often observed during the course of repetitive chemotherapy.

Morphological and functional changes of rabbit mesenteric artery cultured with fetal bovine serum.

The aim of this study was to examine the morphological and functional changes in rabbit mesenteric arterial tissue cultured with fetal bovine serum. In the endothelium-denuded arteries cultured under a serum-free condition for one week (serum-free arteries), morphology of the smooth muscle layer was intact. In the serum-free arteries, high K+ -induced contraction did not change but norepinephrine-induced contraction slightly decreased compared with that in the freshly isolated arteries, whereas the sensitivity to these stimulants was significantly augmented. In the medial layer of the arteries cultured with 10% fetal bovine serum for one week (serum-treated arteries), proliferation, disorientation and death of smooth muscle cells were observed. In the serum-treated arteries, both the amplitude of contractions induced by high K+ and norepinephrine and the sensitivity to these stimulants were significantly reduced compared with those of the serum-free arteries. The reduced norepinephrine-induced contraction in the serum-treated arteries was partially recovered by adding NG-monomethyl-L-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, to the assay medium. In alpha-toxin permeabilized arteries, the amplitude of Ca2+ -induced contraction and the sensitivity of the contractile apparatus to Ca2+ were significantly reduced after serum-treatment. These results suggest that chronic serum-treatment of rabbit mesenteric arteries impairs muscle contractility by the morphological and phenotypic changes in smooth muscle cells. NO production in smooth muscle cells is also responsible for the decreased contractility after the serum-treatment.

Platelet-derived growth factor causes endothelium-independent relaxation of rabbit mesenteric artery via the release of a prostanoid.

Recent evidence has indicated that the mitogen platelet-derived growth factor (PDGF) can act acutely to regulate arterial tone. In this study we demonstrate that the BB isoform of PDGF (PDGF-BB) can cause endothelium-independent relaxation of rabbit isolated mesenteric arteries. In endothelium-denuded arteries, PDGF-BB (40 pM - 8.0 nM) caused concentration-dependent relaxation of noradrenaline-induced tone. The relaxation to PDGF-BB was abolished by a cyclo-oxygenase inhibitor, indomethacin (10 microM) and by the PDGF receptor-associated tyrosine kinase inhibitor, tyrphostin AG1295 (50 microM), but not by N:(G)-monomethyl-L-arginine (L-NMMA, 200 microM), an inhibitor of nitric oxide (NO) synthase. PDGF-BB (4.0 nM) significantly increased the release of prostacyclin (PGI(2)) in endothelium-denuded arteries. Exogenously applied iloprost (1 microM), a stable analogue of PGI(2), relaxed the endothelium-denuded artery. PDGF-BB (4.0 nM) significantly increased the cyclic AMP content. In the absence of Ca(2+), PDGF-BB (4.0 nM) failed to relax the artery, and the release of PGI(2) was almost completely suppressed. In addition, the release of PGI(2) by PDGF-BB (4.0 nM) was significantly reduced in the presence of endothelium. The effect of endothelium was eliminated by L-NMMA (200 microM) and PGI(2) release increased. These data indicate that in rabbit mesenteric arteries, PDGF-BB can evoke endothelium-independent relaxation by stimulating the synthesis of PGI(2). The PDGF-BB-induced prostaglandin synthesis is dependent on both Ca(2+) and tyrosine phosphorylation of the PDGF receptor, and is attenuated by endothelium-derived NO.

Impairment of EDR by a long-term PDGF treatment in organ-cultured rabbit mesenteric artery.

Platelet-derived growth factor (PDGF) has been shown to act chronically on blood vessels to regulate not only proliferation but also vascular tone. These effects may be at least partly due to the chronic effect of PDGF on vascular endothelium. To evaluate this possibility, we examined the effects of PDGF on the endothelium-dependent relaxation (EDR) and total RNA for endothelial nitric oxide (NO) synthase (eNOS) using an organ culture system. In rabbit mesenteric arteries cultured in a serum-free medium for 1 wk, amplitude of the substance P-induced EDR did not change, whereas dependency of the EDR on NO (approximately 60.0% vs. 18.9%) and the total amounts of recoverable eNOS mRNA estimated by RT-PCR were increased compared with those in freshly isolated arteries. Culture with PDGF for 1 wk decreased the relaxant effect of substance P and ionomycin (P < 0.01 compared with the arteries without PDGF), NO production estimated by bioassay (P < 0.01), and eNOS mRNA level, whereas the sodium nitroprusside-induced relaxation did not change. These results suggest that PDGF has a chronic effect on vascular endothelium to decrease eNOS mRNA and NO production and to impair NO-dependent EDR.

Impairment of endothelium-dependent relaxation in the arteries cultured with fetal bovine serum.

Effects of chronic treatment with fetal bovine serum on the function of vascular endothelium were examined using an organ culture system. In the rabbit mesenteric arteries cultured with 10% fetal bovine serum for 7 days, the substance P- or ionomycin-induced endothelium-dependent relaxation was significantly attenuated compared to the arteries cultured in the serum-free condition. The effects of the serum were concentration- and time-dependent. By the treatment with the serum, the amounts of nitric oxide (NO) production and total mRNA for endothelial NO synthase were reduced, whereas the sodium nitroprusside-induced relaxation was rather augmented. These results suggest that chronic treatment of rabbit mesenteric artery with fetal bovine serum decreases endothelial NO synthase mRNA, reduces NO production and impairs endothelium-dependent relaxation.

[Long-term effects of growth-activating agents on smooth muscle contraction and endothelial function in organ-cultured rabbit mesenteric artery].

In the endothelium-denuded arteries cultured in the presence of FBS, morphological (i.e. smooth muscle disorientation and increase in collagen fiber) and phenotypic changes in smooth muscle were observed. Correlated with these changes, contractile force induced by high concentration of KCl and norepinephrine was significantly decreased. In addition, Ca-induced contraction in the permeabilized muscle was also significantly reduced. The reduced contractility in the FBS-treated arteries was partially recovered by the treatment with L-NMMA. In the endothelium-intact arteries cultured in the presence of FBS or PDGF, substance P and ionomycin-mediated, endothelium-dependent relaxation (EDR) was significantly decreased compared to the arteries cultured in serum-free condition. In addition, amounts of NO production and total recoverable eNOS mRNA was reduced in the FBS and PDGF-treated arteries. In these arteries, however, cGMP-dependent relaxation in smooth muscle was not impaired. These results suggest that long-term treatment of vascular tissue with growth-activating agents causes morphological or phenotypic changes nad up-regulation of NO production in smooth muscle, resulting in a reduced contractility. Furthermore, longterm treatment with these agents impairs NO-mediated EDR by decreasing eNOS mRNA and NO production.