Aging-related endothelial dysfunction in the aorta from female senescence-accelerated mice is associated with decreased nitric oxide synthase expression

The present study investigated the time-course for aging-associated effects on contractile and relaxing vascular responses and nitric oxide (NO) production in the aorta from female senescence-accelerated resistant (SAMR1) and prone (SAMP8) mice. Both SAMR1 and SAMP8 were studied at three different ages: 3 (young), 6 (middle age) and 10 (old) months. Concentration–response curves to phenylephrine (10^− 8 to 10^− 5 M) or acetylcholine (10^− 9 to 10^− 5 M) were performed in the aortic rings in the absence or in the presence of NO synthase (NOS) inhibitor L-NAME (10^− 4 M). Protein and gene expression for endothelial NOS (eNOS) was determined by immunofluorescence, Western blot and real-time PCR. Although we have not seen any difference in vascular responses when comparing both strains at 3 months old, we found a significant aging-associated impairment of vascular reactivity that follows a distinct time-course in SAMR1 and SAMP8. In SAMR1, increases in phenylephrine contraction and decreases in acetylcholine relaxation were only seen at 10 months old, while SAMP8 displays altered responses at 6 months that are further impaired at 10 months old. L-NAME treatment enhanced phenylephrine contractions and completely inhibited acetylcholine relaxations in all age groups of SAMR1 and SAMP8. However, the magnitude of increase in phenylephrine contraction by L-NAME was markedly reduced by aging and followed a faster pace in SAMP8. Similar pattern of responses was observed in the time course for changes of eNOS expression, suggesting an earlier and more pronounced aging-associated decrease of NO production and eNOS expression in SAMP8. These results reveal that aging enhances contractile responses to phenylephrine and decreases endothelium-dependent relaxation to acetylcholine in the aorta from female mice by a mechanism that involves a decrease of NO production. This process occurs earlier in the aorta from SAMP8 mice, establishing these mice as suitable model to study cardiovascular aging in a convenient and standard time course.     

Western-type diet induces senescence,modifies vascular function in non-senescence mice and triggers adaptive mechanisms in senescent ones

The effects of high-fat diet ingestion on senescence-induced modulation of contractile responses to phenylephrine (Phe) were determined in aortas of senescence-accelerated (SAMP8) and non-senescent (SAMR1) mice fed (8 weeks) a Western-type high-fat diet (WD). Increased levels of senescence-associated β-galactosidase staining were found in aortas of SAMP8 and SAMR1 with WD. In SAMR1, WD did not modify Phe contraction in spite of inducing major changes in the mechanisms of regulation of contractile responses. Although WD increased NAD(P)H-oxidase-derived O₂⁻ and augmented peroxynitrite formation, we found an increase of inducible NOS (iNOS)-derived NO production which may contribute to maintain Phe contraction in SAMR1 WD. On SAMP8, WD significantly decreased Phe-induced contractions when compared with SAMP8 under normal chow. This response was not dependent on changes of NOS expression, but rather as consequence of increased antioxidant capacity by superoxide dismutase (SOD1). A similar constrictor influence from cyclooxygenase (COX) pathway on Phe responses was found in SAMR1 and SAMP8 ND. However, WD removed that influence on SAMR1, and produced a switch in the balance from a vasoconstrictor to a vasodilator component in SAMP8. These results were associated to the increased COX-2 expression, suggesting that a COX-2-derived vasodilator prostaglandin may contribute to the vascular adaptations after WD intake. Taken together, our data suggest that WD plays a detrimental role in the vasculature of non-senescent mice by increasing pro-inflammatory (iNOS) and pro-oxidative signaling pathways and may contribute to increase vascular senescence. In senescent vessels, however, WD triggers different intrinsic compensatory alterations which include increase of antioxidant activity by SOD1 and vasodilator prostaglandin production via COX-2.     

Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion

Aims/hypothesis

Endogenous NO inhibits insulin release in isolated beta cells and insulin-degrading enzyme activity in hepatocytes, while NO release from endothelial cells has been suggested to enhance insulin action. We assessed the overall effect of systemic inhibition of endogenous NO synthesis on glucose homeostasis in humans.

Methods

Twenty-four non-diabetic volunteers underwent two hyperglycaemic (+7 mmol/l) clamps with either saline or L-NG-nitroarginine methyl ester (l-NAME, at rates of 2.5, 5, 10 and 20 μg?min^?1?kg^?1) infusion. Another five volunteers underwent an OGTT with either saline or l-NAME (20 μg?min^?1?kg^?1) infusion. Blood pressure and heart rate were measured to monitor NO blockade; during the OGTT, endothelial function was assessed by peripheral arterial tonometry and insulin secretion by C-peptide deconvolution and insulin secretion modelling.

Results

Compared with saline, l-NAME at the highest dose raised mean blood pressure (+20?±?2 mmHg), depressed heart rate (?12?±?2 bpm) and increased insulin clearance (+50%). First-phase insulin secretion was impaired, but insulin sensitivity (M/I index) was unchanged. During the OGTT, l-NAME raised 2 h plasma glucose by 1.8 mmol/l (p?<?0.01), doubled insulin clearance and impaired beta cell glucose sensitivity while depressing endothelial function.

Conclusions/interpretation

In humans, systemic NO blockade titrated to increase blood pressure and induce endothelial dysfunction does not affect insulin action but significantly impairs glucose tolerance by increasing plasma insulin clearance and depressing insulin secretion, namely first-phase and beta cell glucose sensitivity.     

Inhibition of atrial fibrillation by low-level vagus nerve stimulation: the role of the nitric oxide signaling pathway

Purpose

We examined the role of the phosphatidylinositol-3 kinase (PI3K)/nitric oxide (NO) signaling pathway in low-level vagus nerve stimulation (LLVNS)-mediated inhibition of atrial fibrillation (AF).

Methods

In 17 pentobarbital anesthetized dogs, bilateral thoracotomies allowed the attachment of electrode catheters to the superior and inferior pulmonary veins and atrial appendages. Rapid atrial pacing (RAP) was maintained for 6 h. Each hour, programmed stimulation was used to determine the window of vulnerability (WOV), a measure of AF inducibility, at all sites. During the last 3 h, RAP was overlapped with right LLVNS (50 % below that which slows the sinus rate). In group 1 (n?=?7), LLVNS was the only intervention, whereas in groups 2 (n?=?6) and 3 (n?=?4), the NO synthase inhibitor N ^G-nitro-l-arginine methyl ester (l-NAME) and the PI3K inhibitor wortmannin, respectively, were injected in the right-sided ganglionated plexi (GP) during the last 3 h. The duration of acetylcholine-induced AF was determined at baseline and at 6 h. Voltage–sinus rate curves were constructed to assess GP function.

Results

LLVNS significantly decreased the acetylcholine-induced AF duration by 8.2?±?0.9 min (p?<?0.0001). Both l-NAME and wortmannin abrogated this effect. The cumulative WOV (the sum of the individual WOVs) decreased toward baseline with LLVNS (p?<?0.0001). l-NAME and wortmannin blunted this effect during the fifth (l-NAME only, p?<?0.05) and the sixth hour (l-NAME and wortmannin, p?<?0.05). LLVNS suppressed the ability of GP stimulation to slow the sinus rate, whereas l-NAME and wortmannin abolished this effect.

Conclusion

The anti-arrhythmic effects of LLVNS involve the PI3K/NO signaling pathway.     

Increase in tetrahydrobiopterin concentration with aging in the cerebral cortex of the senescence-accelerated mouse prone 10 strain caused by abnormal regulation of tetrahydrobiopterin biosynthesis

6R-l-Erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH) activity and is a risk factor for cognitive decline and brain atrophy. Previous studies have shown that the decline in TH activity in the cerebral cortex of senescence-accelerated mouse prone 10 (SAMP10) mice is caused, at least in part, by a decrease in Fe, ferritin, and TH phosphorylation. We determined the concentrations of BH4 and the enzymes GTP cyclohydrolase-1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (SPR) in the de novo pathway of BH4 biosynthesis. Dihydrofolate reductase (DHFR), which converts BH2 to BH4 in the salvage pathway of BH4 synthesis was also determined in the cerebral cortex of SAM mice at 3 and 12 months of age. The BH4 concentration was measured by HPLC, and the protein levels of enzymes involved in BH4 synthesis were measured by western blot analysis. At 12 months of age, BH4 concentration in the cerebral cortex of SAMP10 mice showed significantly higher values as compared to that of control mice. Further, the protein level of SPR in SAMP10 mice was significantly higher than that in SAMR1 mice at 3 and 12 months of age. In contrast to SPR, the protein level of DHFR in SAMP10 mice was significantly lower than that in SAMR1 mice. These results indicate that abnormal regulation of BH4 metabolism occurs in the cerebral cortex of SAMP10 where the dysfunction of the salvage pathway of BH4 synthesis may cause overproduction of BH4 through the de novo pathway, which is considered characteristic in the cerebral cortex of SAMP10 with aging. Therefore, there is a possibility that the excess amounts of BH4 lead to age-related brain dysfunction in the cerebral cortex of SAMP10.     

Inhibition of calcium-dependent nitric oxide synthase causes ileitis and leukocytosis in guinea pigs

As nitric oxide reduces gut epithelial permeability, we designed a study to determine if chronic nitric oxide synthase inhibition predisposes the gut to inflammation. Nitric oxide synthase (NOS) inhibitors were administered in the drinking waterad libitum, for seven days: aminoguanidine (10 µg/ml), a selective inhibitor of the inducible form of nitric oxide synthase; andN ^G-nitro-l-arginine methyl ester (l-NAME, 1, 10, and 100 µg/ml), which inhibits both the constitutive and inducible forms. Control animals drank tap water only or water withd-NAME, the inactive enantiomer. After one week, circulating leukocyte count and tissue myeloperoxidase activity were measured.l-NAME (100 µg/ml), but notd-NAME or aminoguanidine, caused a twofold increase in a circulating leukocyte numbers. This increase in leukocyte numbers was time- and dose-dependent, but the differential count was unaltered. Tissue myeloperoxidase (MPO) activity as an index of granulocyte infiltration was comparable in all groups in the stomach, jejunum, colon, liver, lung, kidney, heart, and skeletal muscle. However, ileal MPO activity was elevated threefold in thel-NAME- (100 µg/ml) treated group (P<0.05). Results in thed-NAME and aminoguanidine groups were similar to controls.l-NAME administration resulted in a reduction in NOS activity ([¹⁴C]citrulline formation) in the ileum but not jejunum, whereas cGMP levels were elevated in both ileum and jejunum. We conclude that chronic inhibition of the constitutive form of nitric oxide synthase predisposes the ileum to inflammation and leads to a progressive leukocytosis.     

Western-style diet modulates contractile responses to phenylephrine differently in mesenteric arteries from senescence-accelerated prone (SAMP8) and resistant (SAMR1) mice

The influence of two known cardiovascular risk factors, aging and consumption of a high-fat diet, on vascular mesenteric artery reactivity was examined in a mouse model of accelerated senescence (SAM). Five-month-old SAM prone (SAMP8) and resistant (SAMR1) female mice were fed a Western-type high-fat diet (WD; 8 weeks). Mesenteric arteries were dissected, and vascular reactivity, protein and messenger RNA expression, superoxide anion (O₂^·−) and hydrogen peroxide formation were evaluated by wire myography, immunofluorescence, RT-qPCR, ethidium fluorescence and ferric-xylenol orange, respectively. Contraction to KCl and relaxation to acetylcholine remained unchanged irrespective of senescence and diet. Although similar contractions to phenylephrine were observed in SAMR1 and SAMP8, accelerated senescence was associated with decreased eNOS and nNOS and increased O₂^·− synthesis. Senescence-related alterations were compensated, at least partly, by the contribution of NO derived from iNOS and the enhanced endogenous antioxidant capacity of superoxide dismutase 1 to maintain vasoconstriction. Administration of a WD induced qualitatively different alterations in phenylephrine contractions of mesenteric arteries from SAMR1 and SAMP8. SAMR1 showed increased contractions partly as a result of decreased NO availability generated by decreased eNOS and nNOS and enhanced O₂^·− formation. In contrast, WD feeding in SAMP8 resulted in reduced contractions due to, at least in part, the increased functional participation of iNOS-derived NO. In conclusion, senescence-dependent intrinsic alterations during early stages of vascular senescence may promote vascular adaptation and predispose to further changes in response to high-fat intake, which may lead to the progression of aging-related cardiovascular disease, whereas young subjects lack the capacity for this adaptation.     

Gathering of aging and estrogen withdrawal in vascular dysfunction of senescent accelerated mice

The aim of this work was to characterize a mouse model of experimental menopause and cardiovascular aging that closely reflects menopause in women. Senescence accelerated mouse (SAM)-Resistant type 1 (SAMR1, n = 30) and SAM-Prone type 8 (SAMP8, n = 30) were separated at 5 months of age into three groups: 1) sham-operated (Sham); 2) ovariectomized (Ovx); and 3) ovariectomized chronically-treated with estrogen (Ovx + E2). Contractile responses to KCl (60 mM) and thromboxane A₂ were greater in aorta from SAMP8 mice compared with SAMR1 in all groups. Neither ovariectomy nor estrogen replacement modified the contractile responses from SAMR1 mice. Conversely, in Ovx SAMP8 the increased maximal contractions were reversed by estrogen treatment. Rings with endothelium from all SAMR1 groups showed a greater relaxation to acetylcholine than SAMP8 groups. In SAMR1, endothelium-dependent relaxation was not altered in Ovx or Ovx + E2 groups. Rings from Ovx SAMP8 showed a decreased maximal response to acetylcholine compared to Sham SAMP8. Estrogen replacement restored the response to acetylcholine altered by ovariectomy. Nitric oxide inhibition by L-NAME markedly reduced acetylcholine responses in all groups, but this effect was less pronounced in SAMP8 and Ovx groups (determined by area under the curve reduction). These results indicate that SAMP8 exhibit a significant decreased endothelium-dependent and NO-mediated relaxation and increased vasoconstrictor responses that are potentiated by the lack of estrogen. Because these responses are closely in agreement with vascular dysfunction observed in menopausal women, we propose SAMP8 Ovx as a new model to concomitantly study the effects of aging and menopause in female mice.     

Depression-like behavior is dependent on age in male SAMP8 mice

Aging is associated with an increased risk of depression in humans. To elucidate the underlying mechanisms of depression and its dependence on aging, here we study signs of depression in male SAMP8 mice. For this purpose, we used the forced swimming test (FST). The total floating time in the FST was greater in SAMP8 than in SAMR1 mice at 9 months of age; however, this difference was not observed in 12-month-old mice, when both strains are considered elderly. Of the two strains, only the SAMP8 animals responded to imipramine treatment. We also applied the dexamethasone suppression test (DST) and studied changes in the dopamine and serotonin (5-HT) uptake systems, the 5-HT_2a/2c receptor density in the cortex, and levels of TPH2. The DST showed a significant difference between SAMR1 and SAMP8 mice at old age. SAMP8 exhibits an increase in 5-HT transporter density, with slight changes in 5-HT_2a/2c receptor density. In conclusion, SAMP8 mice presented depression-like behavior that is dependent on senescence process, because it differs from SAMR1, senescence resistant strain.     

Effects of nesfatin-1 on atrial contractility and thoracic aorta reactivity in male rats

Background: This study aimed to examine the effects of nesfatin-1 on thoracic aorta vasoreactivity and to investigate the inotropic and chronotropic effects of nesfatin-1 on the spontaneous contractions of the isolated rat atria.

Methods: Isolated right atria and thoracic aorta were used in organ baths. The reactivity of the thoracic aorta was evaluated by potassium chloride (KCl), phenylephrine (Phe), acetylcholine (ACh), and sodium nitroprusside (SNP). The effects of nesfatin-1 on the spontaneous contractions of the rat atria were also examined.

Results: Nesfatin-1 (0.1–100 ng/ml) produced a concentration-dependent relaxation response in rat thoracic aorta. The relaxant responses to nesfatin-1 were inhibited by the removal of endothelium, NO synthase blocker N-nitro-L-arginine methyl ester (L-NAME, 10^?4 M), and soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10^–5 M). Nesfatin-1 (10 ng/ml, 30 min) increased the relaxation responses to either ACh or SNP, and the contractile response to both Phe and KCl did not significantly change in the arteries that were incubated with nesfatin-1 compared with the controls. The thoracic aorta contractions induced by the stepwise addition of Ca²⁺ to a high KCl solution with no Ca²⁺ were not significantly changed by nesfatin-1. Under calcium-free conditions, the contractions of the thoracic aorta rings incubated with nesfatin-1 in response to Phe were not significantly lower than those of the rings from the control rats. Nesfatin-1 showed positive inotropic and chronotropic effects on rat atria.

Conclusion: Nesfatin-1 significantly changed the vascular responsiveness in rat thoracic aorta and produced positive inotropic and chronotropic effects on rat atria.     

Nitric oxide and prostaglandin as mediators in the pathogenesis of hyperkinetic circulatory state in a model of endotoxemia-induced portal hypertension

Aims

To evaluate the participation of nitric oxide (NO) and prostaglandin (PGI₂) on hyperdynamic state in endotoxemia-induced portal hypertension (EIP) induced by chronic endotoxemia.

Methods

The portal pressure (PP) and mean arterial pressure (MAP) were recorded, in vivo before and after administration of l-NAME (NOS inhibitor) and indomethacin (specific blocker of COX). The vasoactive responses to acetylcholine of thoracic rat aortic rings were studied in vitro before and after nitric oxide and cyclooxygenase blockade using multichannel organ bath. The mRNA expression for isoforms of (cyclooxygenase) COX and nitric oxide synthase (NOS) were analyzed using RT-PCR.

Results

Administration of both l-NAME and indomethacin in EIP rabbits significantly reduced (p < 0.05) the PP and reversed the MAP to normal as compared to sham-operated (SO) rabbits. There was impaired vasodilatory response to acetylcholine in EIP rabbits. l-NAME caused a significant reduction in acetylcholine-induced vasorelaxation in SO rabbits than EIP due to preexisting hyperemia in EIP. Indomethacin partially restored vasoresponsiveness to acetylcholine in EIP group. The mRNA expression of eNOS (endothelial NOS) and COX-1 (constitutive COX) were significantly higher in SO than EIP rabbits. iNOS (inducible NOS) and COX-2 (inducible COX) mRNA expression was seen only in EIP rabbits.

Conclusions

A significant component of acetylcholine-mediated vasorelaxation in EIP model is modulated by eNOS. There was increased production of contractile prostaglandin in EIP rabbits. iNOS and COX-2 play an important role in the hemodynamic abnormalities of PHT. This novel model of PHT produced by chronic splanchnic endotoxemia in rabbit, mimics impaired vasodilation and vasoreactivity akin to other models of PHT.     

Effect of Biliary Cirrhosis on Neurogenic Relaxation of Rat Gastric Fundus and Anococcygeus Muscle: Role of Nitric Oxide Pathway

Background

Cirrhosis, associated with a host of hemodynamic abnormalities, could affect the gastrointestinal (GI) tract motility. On the other hand, the nonadrenergic noncholinergic (NANC) neurotransmission has been shown to play a pivotal role in GI tract motility and has been linked with release of nitric oxide (NO) on electrical stimulation. In this study, we investigated the effect of biliary cirrhosis on the neurogenic relaxation of rat gastric fundus and anococcygeus muscle and also the possible role of nitric oxide system in this manner.

Methods

Isolated gastric fundus and anococcygeus strips of sham-operated and biliary cirrhotic (4 weeks after bile duct ligation) rats were mounted under tension in a standard organ bath. Electrical stimulation was applied to obtain NANC-mediated relaxations in precontracted gastric fundus and anococcygeus muscle. The neurogenic relaxations were examined in the presence of different doses of NO synthase inhibitor, N ^w-Nitro-l-Arginine Methyl Ester (l-NAME). The concentration-dependent relaxant responses to the NO donor sodium nitroprusside were also evaluated.

Results

The neurogenic relaxation of both gastric fundus and anococcygeus muscle was significantly (P < 0.001) increased in cirrhotic animals. l-NAME (0.03–1,000 µM) inhibited relaxations in both groups in a dose-dependent manner (P < 0.001), but cirrhotic groups were more resistant to the inhibitory effects of l-NAME (P < 0.01). Sodium nitroprusside-mediated relaxations were similar in two groups.

Conclusions

This study for the first time demonstrated that cirrhosis increases the NO-mediated neurogenic relaxation of both rat gastric fundus and anococcygeus muscle, suggesting a crucial role for the neurogenic NO in the pathophysiology of disturbed GI motility in cirrhosis.     

NO x generation by cultured small intestinal epithelial cells

The effect of cytokines, growth factors, mitogens, and bacterial products on nitric oxide (NO) generation by monolayers of small intestinal epithelial cells-6 (IEC-6) cells was evaluated. Subconfluent IEC-6 cells were maintained in DMEM containing 5% fetal calf serum and after 16–24 hr of incubation, the medium was replaced with fresh medium in the presence or absence of calcium ionophore (CaI),l-NAME,l-NNA, individual growth factors, cytokines, or mitogens. After 72 hr of culture, the media supernatant was collected and NO _x generation was determined. NO synthase activity was determined in sonicated supernatants of IEC-6 cells by [¹⁴C] arginine conversion to citrulline. NO _x generation in subconfluent cultures was greater than in fully confluent cultures, suggesting contact inhibition. NO _x generation by IEC-6 cells was significantly increased by CaI and inhibited byl-NAME andl-NNA. LPS, IL-1β, IL-2, IL-8, IFN-8, TFN-α, EGF, TGF-α, bFGF, and PHA significantly increased NO _x generation. NO synthase activity in IEC-6 cells (4.2±1.7 pmol/min/10⁶ cells) was NADPH dependent. These results suggest that stimulation of NO _x generation by intestinal epithelial cells through cytokine bacterial products and mitogens may be one of the mechanisms responsible for their effects in the intestinal tract.     

The contractile mechanism of beraprost sodium,a stable prostacyclin analog,in the isolated canine femoral vein

Summary The vascular contractile mechanism of prostacyclin (PGI₂) was investigated using beraprost sodium (BPS), a stable PGI₂ analog. Ring strips without endothelium isolated from canine femoral veins and arteries were used. BPS induced a dose-dependent contraction without precontraction and after precontraction with norepinephrine (NE) or 60 mM K⁺ in the veins. In contrast, BPS induced a dose-dependent relaxation after precontraction with U46619, a thromboxane A₂ (TXA₂) analog, or prostaglandin F₂ (PGF₂) in the veins. In the arteries, BPS induced contraction at higher concentrations after precontraction with NE. However, BPS relaxed arteries dose-dependently after precontraction with PGF₂. By pretreatment with 13-azaprostanoic acid (13-APA), a TXA₂/endoperoxide receptor antagonist, the dose-response curve of BPS in the veins was shifted to the right. Schild plot analysis resulted in a linear regression with a slope of 0.86 ± 0.13, which was not significantly different from unity, and the pA₂ value for 13-APA against BPS was 7.10 ± 0.06. By pretreatment with BPS, the dose-response curve of U46619 in the veins was shifted to the right. Kaumann plot analysis resulted in a linear regression with a slope of 0.89 ± 0.09, which was not significantly different from unity, and the pA₂ value for BPS against U46619 was 5.68 ± 0.04. These findings indicate that BPS is a partial agonist for the TXA₂/endoperoxide receptors.     

Characterization of the thromboxane synthase pathway product 12-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid as a thromboxane A2 receptor antagonist with minimal intrinsic activity

Thromboxane synthase forms thromboxane (TX) A₂ and 12(S)-hydroxyheptadeca-5(Z)-8(E)-10(E)-trienoic acid (HHT) at equimolar amounts. Twelve-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid (Oxo-HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo-HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P-selectin expression using optical aggregometry and flow cytometry. Oxo-HT 0.1 n M to 50 μM did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggregation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619-induced fibrinogen binding and P-selectin expression revealed pA₂ values of 6.1 and 6.6, respectively, which correspond to K_d values of approximately 0.8 μM and 0.3 μM , respectively. Oxo-HT also inhibited U46619 induced shape change (IC₅₀ ? 10 μM ). However, Oxo-HT over a concentration range of 0.1–1 μM enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo-HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo-HT blocked the binding of [³H]SQ 29548 in a concentration-dependent manner, whereas HHT did not displace [³H]SQ 29548. The K_d of Oxo-HT determined from competition binding studies was 7.7 μM , about 10–25-fold higher than the apparent K_d determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA₂ receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude that Oxo-HT is a naturally occurring specific TXA₂ receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulation of TXA₂-induced platelet activation in vivo.     

Calcitonin Gene-Related Peptide: Effect on Contractile Activity and Luminal Cross-Sectional Area in the Isolated,Perfused Porcine Ileum

Because calcitonin gene-related peptide (CGRP) is an abundant peptide in the enteric nervous system we studied the effect of intra-arterial infusions of synthetic human CGRP I in concentrations from 10-¹⁰ to 10 -⁸ mol/1 on contractile activity and luminal cross-sectional area in the isolated perfused porcine ileum, using manometry and impedance planimetry. The frequency of the basal contractile activity was 0.37 ± 0.1 contractions per minute. CGRP induced phasic contractions, which at the highest dose were superimposed on tonic contractions, as determined by measurement of luminal cross-sectional area. The frequency of contractions dose-dependently increased to approximately 10/min at 10 ⁸ mol/1 CGRP. The amplitude of contractions increased from a maximum of 35 cm H₂0 to 51 ± 3 at 5 × 10″⁹ mol/1 CGRP and 52 ± 6 cm H₂0 at 10^?9 mol/1 CGRP. After the termination of CGRP infusion at the highest dose a short phase of up to 5 min with strong tonic contraction was observed. No phasic activity was detected by manometry during this phase. In conclusion, CGRP dose-dependently increased contractile activity in the pig ileum. CGRP may therefore participate in the regulation of small-intestinal motility in the pig.     

Pre- and Post-Synaptic Effects of Spiradoline and U-50488H,Selective Kappa Opioid Receptor Agonists,in Isolated Ileum

In guinea pig ileum, spiradoline (2 × 10^?6M or greater) and U-50488H (3 × 10^?6M or greater) suppressed contractile responses to acetylcholine (ACh), histamine, and BaCl₂. Inhibition by spiradoline (2 × 10^?5 M) of ACh-induced contractions was not antagonized by pretreatment with naloxone (3 × 10^?4 M). Spiradoline (2 × 10^?8 M or greater) and U-50488H (3 × 10^?8 M or greater) caused dose-dependent inhibition of the contractile response of guinea pig ileum to transmural electric stimulation. The inhibitory effect of spiradoline or of U-50488H at low concentrations was reduced by a high concentration of naloxone (3 × 10^?4 M). Spiradoline at low concentrations ranging from 2 × 10^?9 to 2 × 10^?7 M reduced spontaneous contractions in rabbit ileum. Naloxone (3 × 10^?4 g/ml) antagonized the spiradoline-induced inhibition, but marked inhibition by spiradoline at 10^?4 g/ml) was not restored by naloxone. These results suggest that both kappa agonists exert presynaptic inhibitory action on cholinergic nerve endings in the myenteric plexus at a low concentration range of 10^?9 to 10^?7 M and directly inhibit the smooth-muscle motility of the gut at greater concentrations.     

Soleus muscles of SAMP8 mice provide an accelerated model of skeletal muscle senescence

Animal models are valuable research tools towards effective prevention of sarcopenia and towards a better understanding of the mechanisms underlying skeletal muscle aging. We investigated whether senescence-accelerated mouse (SAM) strains provide valid models for skeletal muscle aging studies. Male senescence-prone mice SAMP6 and SAMP8 were studied at age 10, 25 and 60 weeks and compared with senescence-resistant strain, SAMR1. Soleus and EDL muscles were tested for in vitro contractile properties, phosphocreatine content, muscle mass and fiber-type distribution. Declined muscle mass and contractility were observed at 60 weeks, the differences being more pronounced in SAMP8 than SAMP6 and more pronounced in soleus than EDL. Likewise, age-related decreases in muscle phosphocreatine content and type-II fiber size were most pronounced in SAMP8 soleus. In conclusion, typical features of muscular senescence occur at relatively young age in SAMP8 and nearly twice as fast as compared with other models. We suggest that soleus muscles of SAMP8 mice provide a cost-effective model for muscular aging studies.     

Pharmacology of the Thromboxane Receptor Antagonist and Thromboxane Synthase Inhibitor BM‐531

BM‐531 (N‐tert‐butyl‐N'‐[(2‐cyclohexylamino‐5‐nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA₂ receptors labeled with [3H]SQ‐29548 (IC₅₀= 0.0078 μM) is higher than sulotroban (IC₅₀= 0.93 μM) and SQ‐29548 (IC₅₀= 0.021 μM). Moreover, BM‐531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet‐rich plasma BM‐531 prevents platelet aggregation induced by arachidonic acid (600 μM) (ED₁₀₀= 0.125 μM), U‐46619, a stable TXA₂ agonist (1 μM) (ED₅₀= 0.482 μM) or collagen (1 μg/mL) (percentage of inhibition: 42.9% at 10 μM) and inhibits the second wave of ADP (2 μM)‐induced aggregation. On the other hand, when BM‐531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA‐100®) is significantly prolonged. In addition, at the concentrations of 10 and 1 μM, BM‐531 totally prevents the production of TXB₂ by human platelets activated by arachidonic acid. Finally, at 10 μM, BM‐531 significantly prevents rat fundus contractions induced by U‐46619 but not by prostacyclin. These results suggest that BM‐531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

Distribution of the activity of the angiotensin-converting enzyme in the rat aorta and changes in the activity with aging and by the action of l-NAME

The activity of the angiotensin-converting enzyme (ACE) of the inner surface (the endothelium surface) of rat aorta sections has been studied depending on their distance from the aortic arch, age of rats, and the duration of treatment of rats with the NO synthase inhibitor, N_ω-nitro-l-arginine (l-NAME). The activity of ACE of aorta sections was determined by measuring the hydrolysis of hippuryl-l-histidyl-l-leucine and was expressed as picomoles of Hip–His–Leu hydrolyzed per minute per square millimeter of the endothelium surface. It was found that the ACE activity considerably varies along the aorta of young rats. This variability decreases with increasing age of rats and by the action of l-NAME. The average ACE activity in the aorta increases with the age of rats and with increasing time of l-NAME treatment. Enalapril normalizes the distribution of the ACE activity along the aorta and decreases the average ACE activity. The changes in the distribution of the ACE activity along the aorta and in the average ACE activity in the aorta with increasing age of the rat and by the action of l-NAME may play a role in the development of atherosclerosis of vessels on aging and the inhibition of formation of nitric oxide.