Chronic alcohol intake reduces retinoic acid concentration and enhances AP-1 (c-Jun and c-Fos) expression in rat liver

OBJECTIVES: In vivo studies of the human coronary resistance circulation cannot control for indirect effects of myocardial metabolism, compression, and neurohumoral influences. This study directly examined the vasodilator responses of the human coronary microcirculation to both receptor-dependent and -independent agonists. METHODS: Atrial arterioles were dissected from human right atrial appendage (103 +/- 2 microns diameter, n = 185 vessels from 145 patients) obtained at the time of cardiopulmonary bypass and left ventricular vessels from explanted human hearts (148 +/- 10 microns diameter, n = 57 vessels from 18 patients). After dissection, vessels were mounted onto pipettes in Kreb's buffer under conditions of zero flow and at a constant distending pressure of 60 mmHg. Drugs were applied extraluminally and steady state changes in diameter measured with videomicroscopy. RESULTS: After contraction by endothelin or spontaneous tone, increasing concentrations of adenosine diphosphate (ADP) produced a similar dose-dependent dilation in vessels from atria (maximum 89 +/- 4%, n = 76) and ventricles (maximum 74 +/- 9%, n = 10). The dilation to ADP was abolished by mechanical removal of the endothelium. Similar dilator responses were found to bradykinin, substance P, arachidonic acid, and the calcium ionophore A23187 in both atria and ventricle. In contrast, acetylcholine (ACh) constricted all atrial vessels (-58 +/- 3%, n = 63) regardless of patient age or underlying disease. This constriction was attenuated by denudation, but not affected by inhibition of nitric oxide synthase or cyclo-oxygenase. Microvessels isolated from human ventricle exhibited a heterogeneous response to ACh with dilation being the predominant response. CONCLUSIONS: We conclude that isolated human coronary arterioles demonstrate endothelium-dependent dilation. However, the response to acetylcholine is unique with vasoconstriction in atrial vessels and dilation in ventricular arterioles.     

Differential role of vasoactive prostanoids in porcine and human isolated pulmonary arteries in response to endothelium-dependent relaxants

The pig is increasingly being used in medical research, both as a model of the human cardiovascular system, and as a possible source of organs for xenotransplantation. However, little is known about the comparative functions of the vascular endothelium between porcine and human arteries. We have therefore compared the effects of two endothelium-dependent vasorelaxants, acetylcholine (ACh) and the Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) on the porcine and human isolated pulmonary artery using isometric tension recording. ACh and CPA produced endothelium-dependent relaxations of both the human and porcine pulmonary arteries. In the porcine pulmonary artery, the cyclo-oxygenase inhibitor, flurbiprofen had no effect on relaxations to ACh (Emax: control 67.8+/-8.8% versus 72.4+/-9.5% (n=11)) or CPA (Emax: control 79.6+/-5.0% versus 94.0+/-10.6% (n=7)). The nitric oxide synthase inhibitor, L-NAME converted relaxations to both ACh and CPA into contractile responses (maximum response: ACh 30.0+/-11.1% (n = 10); CPA 80.4+/-26.2% (n = 8) of U46619-induced tone). These contractile responses in the presence of L-NAME were abolished by flurbiprofen. In the human pulmonary artery, L-NAME and flurbiprofen partly attenuated relaxations to ACh (Emax: control: 45.1+/-12.1%; flurbiprofen: 33.4+/-13.5%; L-NAME: 10.1+/-7.2%) and CPA (Emax: control: 78.1+/-5.5%; flurbiprofen: 69.6+/-7.2%; L-NAME 37.9+/-10.7% of U46619-induced tone). These responses were abolished by the combination of both inhibitors. We have demonstrated that while the release of nitric oxide is important in responses to endothelium-dependent vasorelaxants in both human and porcine pulmonary arteries, in the human arteries, there is an important role for vasorelaxant prostanoids whilst in the porcine arteries, vasoconstrictor prostanoids are released.     

Effects of chronic vitamin E deficiency on vascular function--a study of sympathetic nerves, smooth muscle and endothelium of the mesenteric arterial bed of the rat

1. Male rats were deprived as weanlings of dietary vitamin E for 2, 4, 6, 10 and 12 months. Mesenteric arterial beds from these rats and from age-matched controls were isolated and perfused with Krebs solution at a constant flow rate (5 ml min-1). The function of perivascular sympathetic nerves, smooth muscle and endothelium was assessed. 2. At 12 months vitamin E deficient rats exhibited the characteristic symptoms of vitamin E deficiency, namely poor coat condition, muscle wasting, kyphoscoliosis and impaired gait. In the isolated mesenteric arterial bed electrical field stimulation (EFS) of perivascular nerves (4-32 Hz, 90 V, 1 ms, for 30 s) elicited frequency-dependent vasoconstrictor responses which were unaffected by vitamin E deficiency except at 12 months, at which age responses were significantly greater than those of the controls at 24 and 32 Hz (P < 0.01). 3. Exogenous noradrenaline (NA; 0.15-500 nmol) elicited dose-dependent vasoconstriction which was similar in vitamin E-deficient and control preparations at all ages. Potassium chloride (0.15 mmol) also produced similar vasoconstrictor responses in vitamin E-deficient and control preparations at each age. 4. Tone of the preparations was raised by continuous perfusion with methoxamine (4-70 microM), producing similar increases in perfusion pressure in vitamin E-deficient and control preparations at each age. Endothelium-dependent dose-dependent vasodilatation to adenosine 5'-triphosphate was significantly impaired in mesenteric arterial beds from 12 month-old vitamin E-deficient rats compared with the controls (P < 0.05). Relaxation to acetylcholine was not significantly different at any age. 5. Endothelium-independent vasodilatation to sodium nitroprusside was similar in vitamin E-deficient rats and age-matched controls. 6. These results suggest that long term (12 months) deprivation of dietary vitamin E may impair endothelial function in mesenteric arteries of the rat. Sympathetic perivascular nerve constrictor function was increased at 12 months. There were no functionally expressed changes in the vascular smooth muscle, which appears to be more resilient to the effects of oxidative stress in vitamin E deficiency.     

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

Arteriolar reactivity in conscious diabetic rats: influence of aminoguanidine treatment

The effect of 6 weeks' streptozotocin (STZ)-induced (70 mg/kg) diabetes and aminoguanidine (AG) treatment (50 mg/kg s.c. or 250-750 mg/l given in drinking water) on arteriolar reactivity to vasoactive substances was investigated in conscious rats. Studies were performed in untreated control rats (n = 13), STZ-induced diabetic rats (n = 11), AG-treated control rats (n = 12), and AG-treated diabetic rats (n = 12). Rats were provided with a dorsal microcirculatory chamber that allowed intravital microscopy of striated muscle arterioles of varying diameter (A1, large; A2, intermediate; and A3, small arterioles) in conscious animals. The mean arterial pressure (MAP) and arteriolar diameter responses to intravenous infusion of the following drugs were examined: the endothelium-dependent vasodilator acetylcholine (ACh; 3, 10, and 30 microg x kg(-1) x min(-1)), the potassium-channel opener levcromakalim (LC; 30 microg/kg), and the vasoconstrictor agents ANG II (0.1 and 0.3 microg x kg(-1) x min(-1)) and norepinephrine (NE; 0.2, 0.6, and 2.0 microg x kg(-1) x min(-1)). Baseline MAP was lower in both diabetic groups versus the nondiabetic groups (P < 0.05). AG treatment had no influence on baseline MAP. The absolute change in MAP after drug infusion tended to be lower in the diabetic rats than in their nondiabetic littermates. Arteriolar vasodilatory responses to ACh and LC were attenuated in the diabetic animals (1 +/- 7 vs. 19 +/- 7% [P < 0.05] and 7 +/- 3 vs. 34 +/- 8% [P < 0.01] in A2, respectively). AG treatment of diabetic animals did not prevent the development of this disturbance. Vasoconstrictor responses were not influenced by the diabetic state. In the intermediate arterioles of AG-treated control rats, a hyperresponse was observed after ANG II infusion (-10 +/- 2 vs. -2 +/- 2%; P < 0.05) and a hyporesponse was observed after ACh and LC infusion (2 +/- 3 and 15 +/- 6%, respectively; P < 0.05 vs. untreated control rats). These data indicate that 6 weeks of experimental diabetes is associated with a decreased endothelium-dependent and -independent vasodilatation. AG treatment had no beneficial effect on this disturbance.     

Late asthmatic response causes peripheral airway hyperresponsiveness in dogs treated with metopirone

To determine if late asthmatic response (LAR) is associated with hyperresponsiveness of airway smooth muscle itself, we performed antigen challenge in dogs treated with Metopirone. We studied the contractile response to acetylcholine (ACh) in isolated bronchial and bronchiolar segments 8 h after either saline inhalation (the control group) or antigen challenge in dogs demonstrating immediate asthmatic response (IAR) alone and in dogs demonstrating both IAR and LAR. Airway responses to Ascaris suum antigen were assessed by changes in respiratory resistance measured with the forced oscillation technique at 3 Hz. Concentration-response curves of bronchial preparations to ACh did not differ significantly among three groups consisting of the control, IAR and LAR. However, the contractile response of bronchiolar preparations to ACh was significantly greater in the LAR group when compared to the control and IAR groups at the concentrations of ACh ranging from 10(-6) to 3 x 10(-4) M (p < 0.01). SQ 29548, a receptor antagonist of thromboxane A2 and prostaglandin D2 (PGD2), inhibited LAR-induced hyperresponsiveness to ACh in a concentration-dependent fashion. The bronchiolar preparations obtained from dogs showing LAR contained a significantly higher amount of PGD2 than those obtained from dogs showing IAR alone (p < 0.01, n = 6). These results suggest that LAR is associated with hyperresponsiveness of peripheral airway smooth muscle to ACh, and this augmented response to ACh mediates via PGD2 released during LAR.     

Effect of hyperbaric oxygen exposure on pulmonary clearance of 5-hydroxytryptamine

Clearance of 5-hydroxytryptamine (5-HT) by the lungs of normal and vitamin E-deficient rats was evaluated following a 60-min exposure to 100% oxygen (O2) at 4 ATA (HBO). After exposure, lungs were isolated, ventilated, and perfused, with a recirculating system used for measurement of 5-HT clearance. Control lungs were obtained from rats exposed to air at 1 ATA. In control normal rats, fractional clearance of 5-HT was 0.78+/-0.03 (mean+/-SE). Following HBO 5-HT clearance was 0.55+/-0.04 (P less than 0.01). In control vitamin E-deficient rats. 5-HT clearance was 0.85+/-0.05 and was decreased to 0.46+/-0.03 (P less than 0.001) following HBO. To evaluate the effect of recovery time after HBO on 5-HT clearance, separate groups of rats were killed at varying intervals post-HBO. In normal rats, 5-HT clearance had returned to control levels by 3-4 after HBO; in vitamin E-deficient rats, clearance remained unchanged 4 h after HBO and was only 74% (P less than 0.001) of control values 24 h post-HBO. These results indicate that depression of pulmonary 5-HT clearance occurs in rats due to hyperoxia and is potentiated by vitamin E deficiency. This represents a reversible alteration of lung function which requires vitamin E for complete recovery.     

Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia

1. The effect of basal tension (transmural tensions 235 +/- 29 mg wt (low tension: equivalent to approximately 16 mmHg) and 305 +/- 34 mg wt (high tension: equivalent to 35 mmHg)) on rat pulmonary resistance artery responses to endothelin-1 (ET-1) and the selective ET(B)-receptor agonist sarafotoxin S6c (S6c) were studied. The effects of nitric oxide synthase inhibition with N(omega)-nitro-L-arginine methylester (L-NAME, 100 microM) on ET receptor-induced responses, as well as vasodilator responses to acetylcholine (ACh) and S6c, were also investigated. Changes with development of pulmonary hypertension, induced by two weeks of chronic hypoxia, were determined. 2. Control rat preparations showed greatest sensitivity for ET-1 when put under low tension (pEC50: 8.1 +/- 0.1) compared with at the higher tension (pEC50: 7.7 +/- 0.1) and there were significant increases in maximum contractile responses to S6c (approximately 80%) and noradrenaline (approximately 60%) when put under high tension. 3. In control pulmonary resistance arteries, both ET-1 and S6c produced potent vasoconstrictor responses. S6c was 12 fold more potent than ET-1 in vessels set at low tension (S6c pEC50: 9.2 +/- 0.1) and 200 fold more potent than ET-1 when the vessels were set at high tension (S6c pEC50: 9.0 +/- 0.1). Chronic hypoxia did not change the potencies of ET-1 and S6c but did significantly increase the maximum contractile response to ET-1 by 60% (at low tension) and 130% (at high tension). 4. In control rat vessels, L-NAME itself caused small increases in vascular tone (5-8 mg wt tension) in 33-56% of vessels. In the chronic hypoxic rats, in vessels set at high tension, L-NAME-induced tone was evident in 88% of vessels and had increased to 26.9 +/- 6.6 mg wt tension. Vasodilatation to sodium nitroprusside, in non-preconstricted vessels, was small in control rat vessels (2-6 mg wt tension) but increased significantly to 22.5 +/- 8.0 mg wt tension in chronic hypoxic vessels set at the higher tensions. Together, these results indicate an increase in endogenous tone in the vessels from the chronic hypoxic rats which is normally attenuated by nitric oxide production. 5. L-NAME increased the sensitivity to S6c 10 fold (low tension) and 6 fold (high tension) only in chronic hypoxic rat pulmonary resistance arteries. It had no effect on responses to ET-1 in any vessel studied. 6. Vasodilatation of pre-contracted vessels by ACh was markedly greater in the pulmonary resistance arteries from the chronic hypoxic rats (pIC50: 7.12 +/- 0.19, maximum: 72.1 +/- 0.2.0%) compared to their age-matched controls (pIC50: 5.77 +/- 0.15, maximum: 28.2 +/- 2.0%). There was also a 2.5 fold increase in maximum vasodilatation induced by ACh. 7. These results demonstrate that control rat preparations showed greatest sensitivity for ET-1 when set at the lower tension, equivalent to the pressure expected in vivo (approximately 16 mmHg). Pulmonary hypertension due to chronic hypoxia potentiated the maximum response to ET-1. Pulmonary resistance arteries from control animals exhibited little endogenous tone, but exposure to chronic hypoxia increased endogenous inherent tone which is normally attenuated by nitric oxide. Endogenous nitric oxide production may increase in pulmonary resistance arteries from chronic hypoxic rats and attenuate contractile responses to ET(B2) receptor stimulation. Relaxation to ACh was increased in pulmonary resistance arteries from chronic hypoxic rats.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Effects of inhibition of the L-arginine/nitric oxide pathway on vasodilation caused by beta-adrenergic agonists in human forearm

BACKGROUND: We examined whether vasodilator responses to beta-agonists in human forearm vasculature are mediated in part through the nitric oxide pathway. METHODS AND RESULTS: We measured forearm blood flow responses to brachial artery infusions of beta-adrenergic agonists in healthy men. Salbutamol was more than 100 times as potent as dobutamine. Cumulative doses of salbutamol (0.3 to 3.5 nmol.min-1) did not cause tachyphylaxis to an identical repeated infusion after a 24-minute recovery period. Vasodilators were infused with this sequence during coinfusion of saline and NG-monomethyl-L-arginine (L-NMMA, 4 mumol.min-1), an inhibitor of nitric oxide synthase. L-NMMA coinfusion inhibited responses (area under the dose-response curve) to isoproterenol (0.01 to 0.1 nmol.min-1) by 59 +/- 7% (n = 5) and inhibited those to salbutamol (0.3 to 3.5 nmol.min-1) by 52 +/- 6% (n = 8). L-NMMA had no significant effect on vasodilator responses to nitroprusside (2.7 to 11.0 nmol.min-1, n = 8), verapamil (20 to 80 nmol.min-1, n = 8), or prostacyclin (0.08 to 0.24 nmol.min-1, n = 8). CONCLUSIONS: These results suggest that beta-adrenergic vasodilator responses in human forearm vasculature are mediated predominantly through beta 2-adrenergic receptors and are dependent on nitric oxide synthesis.     

Altered vasodilator response of coronary microvasculature in pacing-induced congestive heart failure

To characterize vasodilator capacity of small coronary arteries (200-350 microm diameter) in the setting of congestive heart failure, we examined relaxation responses to acetylcholine (10(-9)-10(-4) M) and nitroglycerin (10(-9)-10(-4) M), in the absence and presence of the nitric oxide precursor, L-arginine (10(-4) M). Congestive heart failure was reliably induced in dogs by rapid ventricular pacing (250 beats.min(-1) for 4 weeks). Maximum relaxations (means +/- S.E.) to each vasodilator are expressed as a percentage of the relaxation response to papaverine (10(-4) M). Relaxation responses to the endothelium-dependent relaxing agent, acetylcholine, were not altered at heart failure, or in the presence of L-arginine. Contrary to acetylcholine, relaxations to nitroglycerin were significantly enhanced in heart failure compared to control (83 +/- 25% vs. 25 +/- 6%, respectively, P < 0.05). Although L-arginine, alone, did not cause any vasodilator response in coronary microvessels, it was able to potentiate nitroglycerin relaxations at control (no L-arginine: 25 +/- 6% vs. L-arginine: 135 +/- 66%). In contrast, at heart failure, L-arginine diminished nitroglycerin relaxations (no L-arginine: 83 +/- 25%, vs. L-arginine: 48 +/- 15%). These data indicate a unique vasodilator profile in small coronary arteries at heart failure: endothelium-dependent relaxations are unaltered, whereas responses to nitroglycerin are augmented. Addition of the nitric oxide precursor, L-arginine, did not affect acetylcholine relaxation, yet surprisingly had a differential effect in response to nitroglycerin. Moreover, inhibition of nitric oxide synthase with N(omega)-nitro-L-arginine elicited concentration-dependent constriction in heart failure but not control coronary microvessels. In summary, our study suggests an important role for nitric oxide in vasodilator control of coronary microvessels, which may modify nitrovasodilator therapy in congestive heart failure.     

Effects of L-arginine and N omega-nitro-L-arginine methyl ester on cardiac perfusion and function after 1-day cold preservation of isolated hearts

BACKGROUND: Coronary flow responses to endothelium-dependent (acetylcholine [ACh] or 5-hydroxytryptamine [5-HT]) and endothelium-independent (adenosine [ADE] or nitroprusside [NP]) vasodilators may be altered before and after 1-day hypothermia during the perfusion of arginine vasopressin (AVP), D-arginine (D-ARG), L-arginine (L-ARG), or nitro-L-arginine methyl ester (L-NAME). METHODS AND RESULTS: Four groups of guinea pig hearts (37.5 degrees C [warm]) were perfused for 6 hours with AVP, L-ARG, L-NAME, or nothing (control). Five heart groups (cold) were perfused with AVP, D-ARG, L-ARG, L-NAME, or nothing (control), but after 2 hours they were perfused at low flow for 22 hours at 3.7 degrees C and again for 3 hours at 37.5 degrees C. ADE, butanedione monoxime, and NP were given for cardioprotection before, during, and after hypothermia. In warm groups, L-ARG did not alter basal flow or ADE, ACh, 5-HT, or NP responses, whereas L-NAME and AVP reduced basal flow and the ADE response, abolished ACh and 5-HT responses, and increased the NP response. In cold groups after hypothermia. L-ARG did not alter basal flow, but L-NAME, AVP, D-ARG, and control reduced flow. In the postcold L-ARG group, ACh increased peak flow, but NP did not increase flow in other cold groups. Effluent L-ARG and L-CIT in the cold control group fell from 64 +/- 9 and 9 +/- 1 micrograms/L at 1 hour to 36 +/- 5 and 5 +/- 1 micrograms/L at 25 hours, respectively. Left ventricular pressure and cardiac efficiency improved more in the postcold L-ARG group than in the postcold D-ARG, AVP, and L-NAME groups. CONCLUSIONS: Endogenous effluent levels of L-ARG and L-CIT decrease after 24 hours in isolated hearts, whereas perfusion of L-ARG improves cardiac performance, basal coronary flow, and vasodilator responses. In contrast, L-NAME, L-ARG, and AVP limit flow and performance but maintain a partial vasodilatory response to NP. Sustained release of NO may account for improved performance after L-ARG after hypothermia.     

Estrogen-mediated neuroprotection after experimental stroke in male rats

BACKGROUND AND PURPOSE: We have previously shown that 17beta-estradiol reduces infarction volume in female rats. The present study determined whether single injection or chronic implantation of estrogen confers neuroprotection in male animals with middle cerebral artery occlusion (MCAO) and whether there is an interaction with endogenous testosterone. METHODS: Male Wistar rats were treated with 2 hours of reversible MCAO. In protocol 1, acute versus chronic estrogen administration was examined in groups receiving the following: Premarin (USP) 1 mg/kg IV, immediately before MCAO (Acute, n=13, plasma estradiol=171+/-51 pg/mL); 7 days of 25 microg (E25, n=10, 10+/-3 pg/mL) or 100 microg 17beta-estradiol (E100, n=12, 69+/-20 pg/mL) by subcutaneous implant; or saline (SAL, n=21, 3+/-1 pg/mL). Laser-Doppler flowmetry was used to monitor the ipsilateral parietal cortex throughout the ischemic period and early reperfusion. At 22 hours of reperfusion, infarction volume was determined by 0 2,3,5-triphenyltetrazolium chloride staining and image analysis. In protocol 2, rats were castrated to deplete endogenous testosterone and then treated with estradiol implants: castration only (CAST, n= 13, estradiol=5+/-2 pg/mL), sham-operated (SHAM, n= 10, 4+/-2 pg/mL), estradiol implant 25 microg (CAST+E25, n=16, 7+/-2 pg/mL) or 100 microg (CAST+E100, n=14, 77+/-14 pg/mL). RESULTS: Cortical infarct volumes were reduced in all estrogen-treated groups: Acute (21+/-4% of ipsilateral cortex), E25 (12+/-5%), and E100 (12+/-3%) relative to SAL (38+/-5%). Caudate infarction was similarly decreased: Acute (39+/-7% of ipsilateral striatum), E25 (25+/-7%), and E100 (34+/-6%) relative to SAL (63+/-4%). Castration did not alter ischemic outcome; cortical and caudate infarction (percentage of respective ipsilateral regions) were 37+/-5% and 59+/-5% in CAST and 39+/-7% and 57+/-5% in SHAM, respectively. Estrogen replacement reduced infarction volume in castrated animals in cortex (19+/-4% in CAST+E25 and 12+/-4% in CAST+E100) and in caudate (42+/-6% in CAST+25 and 20+/-7% in CAST + 100). Laser-Doppler flowmetry results during ischemia and reperfusion was not different among groups. CONCLUSIONS: Both acute and chronic 17beta-estradiol treatments protect male brain in experimental stroke. Testosterone availability does not alter estradiol-mediated tissue salvage after MCAO.     

Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes

1 We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E-deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins. 2 Lumen diameter and wall thickness were measured in pressurized arteries (approximately 250 microm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague-Dawley female rats by use of a dimension analysing system. 3 Treatment with a cyclo-oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (NG-methyl-L-arginine) caused a significant increase in basal tone only in the vitamin E-deprived rats (% tone: 6.2+/-1.1 vs 1.2+/-0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E-deprived rats compared to the controls (26.5+/-2.7 vs 16.4+/-3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E-deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (-17.4+/-4.0%; P<0.05) only the arteries from the vitamin E-deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress-strain relationship were not different between the two groups of rats. 4 In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo-oxygenase pathways but did not alter passive characteristics of mesenteric arteries.     

Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs

Coronary resistance arteries isolated from exercise-trained pigs have been shown to exhibit enhanced myogenic reactivity (J. M. Muller, P. R. Myers, and M. Harold Laughlin. J. Appl. Physiol. 75: 2677-2682, 1993). The purpose of this study was to test the hypothesis that exercise training results in enhanced vasoconstrictor responses of these arteries to all vasoconstrictor stimuli [specifically acetylcholine (ACh), endothelin-1 (ET-1), KCl, and the Ca2+ channel-agonist Bay K 8644]. Female Yucatan miniature swine were trained (Trn) on a motor-driven treadmill (n = 16) or remained sedentary (Sed, n = 15) for 16-20 wk. Arteries 50-120 micron in diameter were isolated and cannulated with micropipettes, and intraluminal pressure was set at 60 cmH2O throughout experiments. Vasoreactivity was evaluated by examining constrictor responses to increasing concentrations of ACh (10(-9) to 10(-4) M), ET-1 (10(-10) to 10(-8) M), KCl (bath replacement with isotonic physiological saline solution containing 30 or 80 mM), and Bay K 8644 (10(-9) to 10(-6) M). Constricted diameters are expressed relative to the passive diameter observed after 100 microM SNP. All four constrictors produced similar decreases in diameter in arteries from both groups [ACh: 0.52 +/- 0.07 (Trn) and 0.54 +/- 0,06 (Sed); ET-1: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); KCl: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); Bay K 8644: 0.86 +/- 0.05 (Trn) and 0. 76 +/- 0.05 (Sed)]. Present results combined with previous observations indicate that exercise training does not alter vasoconstrictor responses of porcine coronary resistance arteries but specifically increases myogenic reactivity. Thus the underlying cellular mechanisms for myogenic tone are altered by training but not receptor-mediated mechanisms (ACh and ET-1) nor voltage-gated Ca2+ channels (KCl and Bay K 8644) in coronary resistance arteries.     

Lovastatin maintains nitric oxide--but not EDHF-mediated endothelium-dependent relaxation in the hypercholesterolemic rabbit carotid artery

The endothelium contributes to the regulation of vascular tone by producing nitric oxide (NO) and the endothelium-derived hyperpolarising factor (EDHF). In hypercholesterolemia, endothelium-dependent relaxation is impaired but can be restored by treatment with lovastatin (LOVAS). We investigated the effects of LOVAS on NO and EDHF-mediated relaxation. Rabbits were fed 1% cholesterol diet for 4 weeks and 0.5%) cholesterol for the following 12 weeks (CHOL-group). The LOVAS group additionally received 10 mg of lovastatin over the last 12-week period. Experiments were performed in carotid artery rings. Relaxant responses to acetylcholine (ACh) were recorded in the presence of indomethacin. Nitro-L-arginine (NOARG, 100 microM) and potassium chloride (KCl, 35 mM) were used to differentiate between NO- and EDHF-mediated relaxations. Cholesterol impaired ACh-induced relaxations and this effect was prevented by LOVAS (control 100+/-1%, CHOL 81+/-6%, LOVAS 98+/-1%). In the presence of NOARG, relaxations to ACh were not different between the LOVAS and CHOL groups (control 78+/-4%, CHOL 64+/-6%, LOVAS 64+/-5%). When KCl was used, ACh-induced relaxations were similar in the LOVAS and control group (control 75+/-5%, CHOL 49+/-6%, LOVAS 76+/-2%). In arteries treated with NOARG and KCl together, no relaxations were observed. Relaxations of arteries from the control group were not affected by 18 h preincubation with lovastatin (10 microM). Lovastatin selectively maintains nitric oxide-mediated endothelium-dependent relaxation in hypercholesterolemic rabbit carotid arteries.     

Genetic studies in osteoporosis]

Vitamin K2 is a known vitamin to promote post-translational modification of vitamin K-dependent protein such as osteocalcin and blood coagulation factors. The effects of vitamin K2 on cortical bone mineral density in osteoporosis has been shown in the phase III DBT trial which had been reported several years ago. However, until now there is no available data regarding to the effect of vitamin K2 on vertebral bone mineral density (LBMD) and on fracture prevention. Thus, a two years randomized open trial to examine the effects of vitamin K2 on LBMD and the fracture prevention in a total of 167 osteoporotic patients had been carried out. The LBMD in vitamin K2 treated group was maintained for 2 years while, that in the control group was deceased to -3% during 2 years observation. The vertebral fracture incidence in the control group was 0.212 +/- 0.038 events/year and that in the treated group was 0.098 +/- 0.029 (p = 0.0186). Vitamin K2 treated group showed significantly lower Glu-osteocalcin level suggesting that vitamin K2 contributed to increase in post-translational modification of osteocalcin. When the treated group was divided into two groups: Group 1 showed low serum Glu-osteocalcin level and Group 2 maintained high Glu-osteocalcin level despite vitamin K2 administration. The LBMD in group 1 significantly higher than that in the Group 2. This may indicate that sufficient tissue supply of vitamin K2 is the limiting factor to increase in LBMD. Furthermore, patients with Apo E4 phenotype showed less response in LBMD comparing to that in the patients without Apo E4. In conclusion, vitamin K2 is effective to maintain trabecular BMD in osteoporosis and effectively prevent future fracture. However, some part of the patients didn't respond to vitamin K2 treatment. Therefore, we have to develop the more practical way(s) to predict the effectiveness of vitamin K2 treatment in osteoporosis.     

Forearm nitric oxide balance, vascular relaxation, and glucose metabolism in NIDDM patients

Endothelium-dependent and -independent vascular responses were assessed in 10 NIDDM patients and 6 normal subjects with no evidence of atherosclerotic disease. Changes in forearm blood flow and arteriovenous (AV) serum nitrite/nitrate (NO2-/NO3-) concentrations were measured in response to intra-arterial infusion of acetylcholine (ACh) (7.5, 15, 30 microg/min, endothelium-dependent response) and sodium nitroprusside (SNP) (0.3, 3, 10 microg/min, endothelium-independent response). Insulin sensitivity (determined by minimal model intravenous glucose tolerance test) was lower in NIDDM patients (0.82 +/- 0.20 vs. 2.97 +/- 0.29 10(4) min x microU(-1) x ml(-1); P < 0.01). Baseline forearm blood flow (4.8 +/- 0.3 vs. 4.4 +/- 0.3 ml x 100 ml(-1) tissue x min(-1); NS), mean blood pressure (100 +/- 4 vs. 92 +/- 4 mmHg; NS), and vascular resistance (21 +/- 1 vs. 21 +/- 1 units; NS), as well as their increments during ACh and SNP, infusion were similar in both groups. No difference existed in baseline NO2-/NO3- concentrations (4.09 +/- 0.33 [NIDDM patients] vs. 5.00 +/- 0.48 micromol/l [control subjects]; NS), their forearm net balance (0.31 +/- 0.08 [NIDDM patients] vs. 0.26 +/- 0.08 micromol/l x 100 ml(-1) tissue x min(-1); NS), and baseline forearm glucose uptake. During ACh infusion, both NO2- and NO3- concentrations and net balance significantly increased in both groups, whereas glucose uptake increased only in control subjects. When data from NIDDM and control groups were pooled together, a correlation was found between the forearm AV NO2- and NO3- differences and blood flow (r = 0.494, P = 0.024). On the contrary, no correlation was evident between NO2- and NO3- concentrations or net balance and insulin sensitivity. In summary, 1) no difference existed in basal and ACh-stimulated NO generation and endothelium-dependent relaxation between uncomplicated NIDDM patients and control subjects; 2) in both NIDDM and control groups, forearm NO2- and NO3- net balance following ACh stimulation was related to changes in the forearm blood flow; and 3) ACh-induced increase in forearm blood flow was associated with an increase in glucose uptake only in control subjects but not in NIDDM patients. In conclusion, our results argue against a role of impaired NO generation and blood flow regulation in determining the insulin resistance of uncomplicated NIDDM patients; rather, it supports an independent insulin regulation of hemodynamic and metabolic effects.     

A novel, nongenomic action of estrogen on the cardiovascular system

To examine the time course and mechanisms of action of single doses of estrogen on the skin microvasculature, two double-blind placebo-controlled cross-over studies were conducted in healthy young men using the noninvasive technique of laser Doppler velocimetry with iontophoretic application of vasodilator substances. Estradiol (2 mg sublingually) produced a significant increase in the response to the endothelial vasodilator acetylcholine (ACh) after 15 min, but not after 20 or 30 min. The mean plasma estradiol concentration increased from 89.4 +/- 9 pmol/L at baseline to 486.6 +/- 218 pmol/L at 15 min. An i.v. bolus of 25 mg conjugated equine estrogens produced significant increases in the responses to ACh at 15 and 20 min but not at 30 min. There was no change in responses to the nonendothelial vasodilators sodium nitroprusside or nicotine, and administration of placebo produced no change in ACh responses at any time point. These experiments show that, at plasma estradiol concentrations within the physiological range for premenopausal women, estrogens act directly on the cutaneous microvasculature through a rapid onset, rapid offset, nongenomic mechanism that is specific to the endothelium; in addition, it supports the view that estrogens can act on the male cardiovascular system in a manner that is potentially clinically beneficial.     

Metabotropic glutamate receptor antagonists, like GABA(B) antagonists, potentiate dorsal root-evoked excitatory synaptic transmission at neonatal rat spinal motoneurons in vitro

Recordings of whole-cell synaptic current responses elicited by electrical stimulation of dorsal roots were made from motoneurons, identified by antidromic invasion, in isolated spinal cord preparations from five- to eight-day-old Wistar rats. Supramaximal electrical stimulation of the dorsal root evoked complex excitatory postsynaptic currents with mean latencies (+/- S.E.M.) of 6.1 +/- 0.26 ms, peak amplitude of -650 +/- 47 pA and duration of 4.30 +/- 0.46 s (n=34). All phases of excitatory postsynaptic currents were potentiated to approximately 20% above control levels in the presence of the metabotropic glutamate receptor antagonists S-2-amino-2-methyl-4-phosphonobutanoate (MAP4; 200 microM; n=15) and 2S, 1'S,2'S-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 200 microM; n=9). A similar level of potentiation was produced by the GABA(B) receptor antagonist 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-p hosphinic acid (CGP55845; 200 nM; n=5). MAP4 (200 microM) produced a six-fold rightward shift in the concentration-effect plot for the depressant action of the metabotropic glutamate receptor agonist S-2-amino-4-phosphonobutanoate (L-AP4), whereas CGP55845 produced no significant change in the potency of L-AP4. MAP4 did not antagonize the depressant actions of baclofen (n=8), 1S,3S-1-aminocyclopentane-1,3-dicarboxylate (n=4) or 2-S,1'S,2'S-2-(carboxycyclopropyl)glycine (n=4). The metabotropic glutamate receptor antagonists produced no change in the holding current of any of the neurons, indicating that they had no significant postsynaptic excitatory actions. These results are the first to indicate a possible physiological role for metabotropic glutamate receptors in the spinal cord. Like GABA(B) receptors, they control glutamatergic synaptic transmission in the segmental spinal pathway to motoneurons. This is likely to be a presynaptic control mechanism.