Melatonin promotes osteoblast differentiation of bone marrow mesenchymal stem cells in aged rats.

OBJECTIVE: The current study was to explore the effect of melatonin on osteoporosis and relevant mechanisms. MATERIALS AND METHODS: We performed micro-CT to detect bone microstructure and ELISA to detect the contents of osteocalcin (OCN) and bone alkaline phosphatase (BAP) in serum. Double fluorescence labeling of calcein and tetracycline and toluidine blue staining were used to determine morphological indexes of bone tissues. Alizarin red staining and Oil Red O staining were performed to recognize bone cells and adipocytes. RT-PCR was performed to determine the expression of osteoblast differentiation related genes. RESULTS: In the current study, data from micro-CT indicated that melatonin significantly increased the bone mass density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th), and decreased the Structure Model Index (SMI) and trabecular Separation/Spacing (Tb.Sp) in elderly rats. Melatonin reduced calcium and phosphorus losses in urine and increased BAP and OCN levels in serum in elderly rats and increased bone formation rate (BFR) and bone mineralization rate (MAR) in elderly rats. Melatonin increased the number of osteoblasts in bone marrow and reduced the number of adipocytes in elderly rats. Melatonin also promoted the expression of osteogenic differentiation genes and suppressed the expression of adipogenic differentiation genes. CONCLUSIONS: WE suggest that melatonin could alleviate osteoporosis in aged rats' models probably by promoting osteoblast differentiation.

P2Y receptor-induced EDHF vasodilatation is of primary importance for the regulation of perfusion pressure in the peripheral circulation of the rat.

Extracellular nucleotides have been shown to induce vasodilatation of conductance arteries by release of the endothelium-derived hyperpolarizing factor (EDHF). As small resistance arteries are of greater importance for blood pressure regulation, a whole rat mesenteric arterial bed preparation was used in the present study when evaluating the physiological relevance for EDHF in mediating nucleotide dilatation. Dilatory responses were examined after pre-contraction with noradrenaline in the presence of 10 mM indomethacin. Adenosine-5'-O-thiodiphosphate (ADPbetaS), adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced vasodilatation (pEC50=6.5-7 and E(max)=40-70%), while uridine diphosphate (UDP) was ineffective. Endothelium-derived hyperpolarizing factor was studied in the presence of 0.5 mM Nvarpi-nitro-L-arginine (L-NOARG). ADPbetaS and UTP induced strong and potent EDHF-dilatations, while ATP only had a weak effect (E(max)=25%). After P2X1 receptor desensitization with 10 microM alphabeta-methylene-adenosine triphosphate, the ATP response was significantly increased (E(max)=65%). Putatively, this could be because of simultaneous activation of both endothelial P2Y receptors and P2X1 receptors on smooth muscle cells, which resulted in the release of EDHF and subsequent hyperpolarization, and depolarization, respectively. Nitric oxide (NO) was studied in the presence of 60 mM K+. ADPbetaS, ATP and UTP induced weak NO dilatations, suggesting a minor role for NO as compared with EDHF. In conclusion, extracellular nucleotides stimulate dilatation by activating P2Y(1) and P2Y(2)/P2Y(4) receptors, but not P2Y(6) receptors. The dilatory responses are mediated primarily by EDHF in the peripheral vascular bed.

Cytochrome P450 metabolites of arachidonic acid may be important mediators in angiotensin II-induced vasoconstriction in the rat mesentery in vivo.

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar-Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12, 12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 omega-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize (14)C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

Neuropeptide Y and mesenteric sympathetic vasoconstriction in pregnant and non-pregnant Wistar-Kyoto rats.

1. We have investigated the role of neuropeptide Y (NPY) in sympathetic vasoconstriction and its possible contribution to decreased mesenteric vasoconstrictor responses to electrical field stimulation (EFS) in pregnancy in vivo using an in situ bloodperfused mesenteric preparation in 18-20 day pregnant and age-matched non-pregnant Wistar-Kyoto (WKY) rats. 2. Mean blood pressure (BP) was decreased in pregnant compared with non-pregnant rats. Mesenteric basal perfusion pressure (PP) was not significantly reduced in pregnancy. BIBP 3226, a specific NPY Y1 receptor antagonist, did not affect mean BP or mesenteric basal PP in pregnant or non-pregnant animals. 3. Mesenteric vasoconstrictor responses to EFS were blunted in pregnant compared with non-pregnant controls. BIBP 3226 significantly suppressed mesenteric vasoconstrictor responses to EFS in both pregnant and non-pregnant animals. Decreased mesenteric responses to EFS in pregnancy were still evident following NPY Y1 receptor blockade. 4. These results suggest that endogenous NPY plays a role in rat mesenteric sympathetic vasoconstriction in vivo. However, NPY is unlikely to be responsible for the impairment of EFS-induced mesenteric vasoconstriction in pregnant WKY rats.

Demonstration of the existence of nitric oxide-independent as well as nitric oxide-dependent vasodilator mechanisms in the in situ renal circulation in near term pregnant rats.

1. We have investigated the role of endogenous nitric oxide on renal vascular reactivity in late pregnancy in in situ blood perfused kidneys of alpha-chloralose anaesthetized Wistar-Kyoto rats. Nitric oxide synthesis inhibition was achieved by intravenous administration of NG-nitro-L-arginine or NG-nitro-L-arginine methyl ester. 2. Intra-arterial mean blood pressure was lower in pregnancy compared with nonpregnant controls. Following nitric oxide synthesis inhibition mean blood pressure increased in both pregnant and nonpregnant groups, but remained lower in pregnant animals. 3. Basal renal perfusion pressure was similar in pregnant and nonpregnant groups. Intravenous administration of Ng-nitro-L-arginine resulted in dose-dependent increases in renal perfusion pressure but responses were substantially depressed in pregnancy. 4. Renal vasoconstrictor responses to regional angiotensin II (AII) were decreased in pregnancy, whereas those to noradrenaline (NA) did not differ from nonpregnant controls. NG-nitro-L-arginine (5 mg kg-1) potentiated renal responses to regional AII and NA in both groups, but AII responses remained lower in pregnancy. Blunted renal AII responses in pregnancy were still evident following large doses of NG-nitro-L-arginine methyl ester (100 mg kg-1). 5. The results demonstrate that nitric oxide synthesis inhibition increases renal perfusion pressure to a lesser extent in pregnant compared with nonpregnant rats, and that reduced renal pressor responses to AII are still evident in pregnancy after nitric oxide synthesis inhibition. 6. These results suggest that although endogenous nitric oxide synthesis modulates renal vasoconstrictor responses in both pregnant and nonpregnant animals, this mechanism does not fully account for the blunted renal vasconstrictor responses to regional AII or nitric oxide inhibitors in near term pregnant rats. The nature of this important physiological vasodilator mechanism in pregnancy remains to be elucidated.

Effects of 17 beta-oestradiol on vascular responses in the in situ blood-perfused mesentery of Wistar-Kyoto rats.

1. To determine whether endogenous oestrogen plays a role in pregnancy induced decreased vascular reactivity we have examined the effects of 17 beta-oestradiol on vasoconstrictor responses to various stimuli using an in situ blood-perfused mesenteric vascular preparation in Wistar-Kyoto (WKY) rats. 2. Daily administration of 17 beta-oestradiol (500 micrograms/kg, s.c.) for 15 days significantly enhanced mesenteric vasoconstrictor responses to noradrenaline (NA), without affecting responses to the electrical stimulation of sympathetic nerves (ES) and angiotensin II (AngII). 3. Nitric oxide (NO) synthesis inhibition by nitro-L-arginine methyl ester (L-NAME; 100 mg/kg, i.v.) significantly potentiated mesenteric vasoconstrictor responses to all stimuli in both 17 beta-oestradiol-treated and control animals. The difference in NA responses between groups was diminished following NO synthesis inhibition. 4. These findings do not support the hypothesis that increased endogenous oestrogen plays a role in decreased mesenteric vascular reactivity in pregnancy. However, responses to oestrogen may be dose-dependent and enhancement of vasoconstrictor responses to NA may be relevant to oral contraceptive-induced hypertension.

The role of angiotensin converting enzyme and nitric oxide in the enhanced systemic depressor responses to bradykinin in pregnant rats.

1. In the present study we have examined the effects of angiotensin I converting enzyme (ACE) inhibition and nitric oxide (NO) synthesis inhibition on bradykinin (BK) depressor responses in pregnancy in both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. Ramipril (10 mg/kg, i.v.) significantly reduced mean blood pressure (MBP) in all animals. Further administration of L-nitro-arginine (L-NOARG, 20 mg/kg, i.v.) increased MBP to a less extent in pregnant WKY and to a similar level in pregnant SHR compared with their non-pregnant controls (controls), respectively. 3. Systemic depressor responses to BK were increased in pregnant WKY and were unchanged in pregnant SHR as compared with their controls. Ramipril (10 mg/kg, i.v.) potentiated BK responses in all groups, and abolished the differences between pregnant and non-pregnant WKY. Further administration of L-NOARG (20 mg/kg, i.v.) did not further influence BK responses in all groups. 4. The results suggest that systemic depressor responses to BK are enhanced in pregnant WKY and unchanged in pregnant SHR. Decreased ACE activity may contribute to enhanced systemic depressor responses to BK in pregnant WKY.

Effects of Hoe 140 on systemic depressor responses to bradykinin and mesenteric vascular reactivity in vivo in pregnant Wistar-Kyoto rats.

1. To examine possible changes in vascular reactivity to exogenous bradykinin (BK) and the possible role of endogenous BK in reduced mesenteric vascular reactivity in pregnant rats. The authors studied the effects of Hoe 140 on systemic depressor responses to BK and on mesenteric vascular reactivity in in situ blood-perfused mesenteric resistance vessels of 18-20 day pregnant and age-matched non-pregnant Wistar-Kyoto rats (WKY). 2. Mean intra-arterial blood pressure (MBP) of pregnant rats was lower than non-pregnant controls. Basal mesenteric perfusion pressure (BPP) was slightly, but not significantly, reduced in the pregnant group. Neither MBP nor BPP was significantly influenced by Hoe 140 (1 mg/kg. s.c.) 3. Systemic depressor responses to BK (1-30 micrograms/kg, i.v.) were significantly increased in pregnant rats at 1 and 3 micrograms/kg. Hoe 140 completely abolished systemic depressor responses to BK in either pregnant or non-pregnant animals. 4. Mesenteric vascular responses to regional administration of noradrenaline, electrical stimulation of sympathetic nerve and angiotensin II were overall decreased in pregnant compared with non-pregnant groups, but those responses were not significantly affected by Hoe 140 in either groups. 5. The results suggested that although systemic depressor responses to exogenous BK were increased, endogenous BK does not contribute to decreased mesenteric vascular reactivity in vivo in pregnant WKY.

Nitric oxide-mediated changes in vascular reactivity in pregnancy in spontaneously hypertensive rats.

1. To examine the mechanisms which may account for pregnancy-induced vasodilatation in spontaneously hypertensive rats (SHR), we have investigated the changes in vascular reactivity and the effects of endothelial nitric oxide (NO) inhibition in the in situ blood-perfused, mesenteric resistance vessels of 18-20 day pregnant SHR. The effects of NG-nitro-L-arginine (L-NOARG) were compared in pregnant and nonpregnant SHR and gestation matched normotensive Wistar-Kyoto (WKY) rats. 2. Intra-arterial mean blood pressures (MBP) were similar in pregnant and nonpregnant SHR. Basal perfusion pressures (BPP) were decreased in pregnant compared with nonpregnant SHR. Pregnant WKY had lower MBP and BPP than either pregnant or nonpregnant SHR. 3. Vasoconstrictor responses to electrical stimulation (ES) and intra-arterial noradrenaline (NA) were decreased in pregnant compared with nonpregnant SHR. These responses were still greater in pregnant SHR when compared with pregnant WKY. Vascular reactivity to angiotensin II (AII) in pregnant SHR was reduced to a similar level to that in pregnant WKY. 4. L-NOARG (5 mg kg-1, i.v.), an inhibitor of nitric oxide synthase, increased MBP and BPP in all groups. After L-NOARG, BPP were equalized between pregnant and nonpregnant SHR. Pregnant WKY still showed lower MBP and BPP than SHR groups. 5. L-NOARG potentiated vascular responses to ES, NA and AII in all groups. The blunted vascular responses to NA and ES were normalized and the reactivity to AII was only partially reversed in pregnant SHR compared with nonpregnant SHR. Pregnant WKY still had much lower vascular responses to ES and NA than either pregnant or nonpregnant SHR. L-NOARG enhanced vascular responses to All to a greater extent in pregnant SHR than in pregnant WKY.6. These results demonstrate that blunted responses to NA and ES were NO-dependent, while diminished reactivity to AII was only partially dependent on NO in the in situ blood perfused mesenteric resistance vessels of pregnant SHR.7. The present results in pregnant SHR differ from our previous finding with pregnant normotensive WKY, in which blunted responses to NA, but not to ES, were equalized by L-NOARG. Pregnancy induced vasodilatation in hypertensive rats appears to be more dependent on endothelial NO than in normotensive WKY. A defect of the endothelial NO generating pathway which promotes vasodilatation in pregnancy may contribute to the predisposition of women with essential hypertension to develop pre-eclampsia.

Mechanisms of vasodilatation in pregnancy: studies of the role of prostaglandins and nitric-oxide in changes of vascular reactivity in the in situ blood perfused mesentery of pregnant rats.

1. To examine the possible mechanisms of the vasodilatation and blunted pressor responses in late pregnancy, we have studied vascular reactivity of the in situ blood perfused mesenteric resistance vessels of 18-20 day pregnant Wistar-Kyoto rats (WKY). 2. Intra-arterial mean blood pressure (MBP) was lower in pregnant rats than in nonpregnant controls. There was no significant difference in basal mesenteric perfusion pressure (BPP) between groups. 3. Vascular reactivity to electrical stimulation (ES) or intra-arterial noradrenaline (NA), angiotensin II (AII) and arginine vasopressin (AVP) was decreased in the preparations from pregnant rats compared to that from nonpregnant controls. Noradrenaline spillover into mesenteric venous blood following ES was similar in pregnant and nonpregnant animals. 4. Indomethacin (5 mg kg-1, i.v.), an inhibitor of cyclo-oxygenase, induced significant increases in reactivity to ES in both pregnant and nonpregnant groups while potentiating the responses to NA and AII in nonpregnant animals only and having no effect on AVP-induced contractions in the preparations from either pregnant or nonpregnant animals. 5. NG-nitro-L-arginine (L-NOARG) (5 mg kg-1, i.v.), an inhibitor of nitric-oxide synthase, increased MBP and BPP in both pregnant and nonpregnant animals, but the difference in MBP between groups was still evident. 6. L-NOARG enhanced mesenteric vascular responses to ES, NA and AII in both pregnant and nonpregnant groups. Only the difference in NA responses between groups was abolished after pretreatment with L-NOARG. 7. These data show that vasoconstrictor responses to a variety of agonists are decreased in the in situ blood-perfused mesenteric resistance vessels of pregnant rats. Increase in endothelial-dependent nitric oxide generation could contribute to the vasodilatation seen in pregnancy but other mechanisms might also be involved. Cyclo-oxygenase products are not responsible for any decreased contractile responses in this preparation.

Study of mechanisms of glucocorticoid hypertension in rats: endothelial related changes and their amelioration by dietary fish oils.

1. To investigate possible mechanisms of increased systolic blood pressure after 1 weeks treatment with dexamethasone and its amelioration by fish oil feeding, we have examined the reactivity of aortic rings and perfused mesenteric resistance vessels. 2. Thirty six Sprague-Dawley rats were initially divided into two groups and fed a semisynthetic diet containing either (10% by weight) hydrogenated coconut oil and safflower oil mixture (HCO/S) (24 rats) or fish oil (12 rats) for 5 weeks. From the end of the fourth week, dexamethasone (1.25 mg ml-1) in drinking water, was given to half the rats on hydrogenated coconut oil (HCO/S+Dex) and to the fish oil-fed group (fish oil+Dex). 3. One week of dexamethasone treatment raised systolic blood pressure in the HCO/S+Dex rats but not in the fish oil+Dex group. 4. Endothelium-dependent relaxation to acetylcholine (ACh) was decreased in aortic rings taken from HCO/S+Dex rats compared to rats on HCO/S alone. Relaxant responses to ACh of aortic rings from rats given fish oil+Dex were intermediate between the three groups. Aortic endothelium-independent responses to sodium nitroprusside (SNP) were unchanged between the groups, while aortic contractile responses to noradrenaline were similar in all the groups. 5. In the perfused mesenteric resistance artery, sensitivity to noradrenaline was decreased in rats given fish oil and dexamethasone compared to the other two groups. There were no differences in resistance vessel relaxation to ACh or SNP between groups. 6. Serum corticosterone levels, used as a marker of dexamethasone absorption, were substantially suppressed in dexamethasone-treated rats but levels were higher in rats on fish oil than on HCO/S diets. 7. We suggest that the glucocorticoid-induced rise in systolic blood pressure may be due in part to decreased aortic compliance as a consequence of impaired endothelium-dependent relaxation and perhaps reduced nitric oxide synthesis. Fish oil feeding may ameliorate this rise in blood pressure through (i) changes in dexamethasone absorption, (ii) decrease in reactivity to noradrenaline of perfused mesenteric resistance arteries, (iii) an increase in endothelium-dependent relaxation to ACh or a combination of these three factors.

Fish oil feeding selectively attenuates contractile responses to noradrenaline and electrical stimulation in the perfused mesenteric resistance vessels of spontaneously hypertensive rats.

1. The mechanism of the antihypertensive effects of n-3 fatty acids were examined in spontaneously hypertensive rats (SHR) by feeding 'Max EPA' fish oil or hydrogenated coconut oil and determining the responses of perfused mesenteric resistance vessels to various contractile agents and peri-arterial nerve stimulation. 2. Fish oil feeding for 4 weeks caused a decrease in the responses to exogenous noradrenaline and electrical nerve stimulations but had no significant effect on vasopressin and KCl (80 mmol/L) induced contractions. 3. These results provide direct evidence for specific attenuation of vascular responses to sympatho-adrenal stimulation in resistance vessels following fish oil feeding and may account for the antihypertensive effects seen in humans and in some forms of hypertension in rats.

Blood pressure and vascular reactivity changes in spontaneously hypertensive rats fed fish oil.

1. To examine possible mechanisms of antihypertensive effects of feeding fish oil rich in n-3 fatty acids, we have studied vascular reactivity of aortic rings and perfused mesenteric resistance vessels of spontaneously hypertensive rats (SHR) given such a diet. 2. In two experiments, rats were fed a semi-synthetic diet containing either 'fish oil' (10 and 20% by weight) or hydrogenated coconut oil (control) (10 and 20%) for 4 weeks. 3. Blood pressure rose significantly less in the fish oil group than in controls in both experiments. 4. Aortic rings from control rats showed endothelium-dependent relaxations to low concentrations of acetylcholine (ACh) but relaxed less at higher concentrations. In contrast, rings from the fish oil group had relaxations which increased through the range of concentrations used. Indomethacin (10 microM) also increased the relaxation responses seen in rings from control rats, suggesting that fish oil inhibits a contractile cyclo-oxygenase product. This contractile substance may be thromboxane A2 (TxA2) or its endoperoxide precursor, prostaglandin H2 (PGH2) as aortic incubates and serum levels of TxB2 (the stable product of TxA2) were greatly reduced in fish oil-fed rats, and the decrease of relaxant responses to high concentrations of ACh were also blocked by a TxA2/PGH2 receptor blocker (SQ 29548). 5. In contrast to aortic rings, perfused preconstricted mesenteric resistance vessels of control rats relaxed to ACh in a similar fashion to tissues from fish oil-fed rats. However, in this preparation, fish oil feeding enhanced relaxations to sodium nitroprusside (SNP) and contractile responses to noradrenaline were less than controls. After removal of endothelium with 0.05% saponin, contractile responses to noradrenaline increased in both groups but responses from fish oil-treated rats were still attenuated. This suggests that fish oil feeding alters reactivity of mesenteric resistance vessels at the level of the smooth muscle. 6. The results indicate that fish oil feeding may reduce blood pressure by decreasing vascular smooth muscle reactivity to noradrenaline in resistance vessels. The effect may be enhanced by inhibition of an endothelium-derived cyclo-oxygenase product, such as TxA2 or PGH2 in conduit vessels.

Effect of fish oil feeding on blood pressure and vascular reactivity in spontaneously hypertensive rats.

1. To examine possible antihypertensive mechanisms of fish oil feeding, we have studied vascular reactivity of aortic rings and blood pressure of spontaneously hypertensive rats (SHR). 2. SHR were fed a synthetic diet supplemented with either (10% by weight) 'Max EPA' fish oil or hydrogenated coconut oil (saturated fat) for 4 weeks. 3. Mean systolic blood pressure of fish oil fed rats was 9 mmHg lower than saturated fat fed controls. 4. Aortic rings of control SHR had a biphasic response to acetylcholine (ACh), relaxing at lower concentrations but contracting at concentrations higher than 3 x 10(-7) mol/L. No such contractions were seen in tissues of fish oil fed rats. The contractions were abolished by indomethacin, suggesting that they were caused by a cyclo-oxygenase product. 5. Tissue analysis showed that both aortic and serum generation of thromboxane B2(TxB2) was approximately three times less in fish oil fed rats than in control tissues. 6. These results indicate that the lowering of blood pressure in fish oil fed SHR could in part be due to decrease in production of thromboxane (TxA2), a potent vasoconstrictor, hence influencing vascular tone and compliance of the aorta.