[Dietary flavonoids and human health]. / Flavonoïdes alimentaires et santé humaine.

Due to antioxidant properties linked to their polyphenolic structure, dietary flavonoids are supposed to protect the organism against deleterious effects of environmental oxidants. Indeed prospective epidemiologic studies on cohorts have shown inverse correlations between consumption of some foods or beverages with high flavonoid content, (especially flavanols and anthocyanins), and coronary stroke mortality or prevalence of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. These include red wine, some grape juices, red fruits, tea and cocoa, The hypothesis of cause effect relationship between dietary flavonoid intake and observed protection is further supported by several short term controlled randomised clinical trials. However composition of ingested food or beverage is complex and poorly defined, especially their content in different flavonoids. In addition, knowledge on bioavailability of these compounds and their fate in the organism is still limited. The best documented effect is protection or restoration of the vascular endothelium function, principally involving nitric oxide (*NO). It is not established that ingested flavonoids produce a direct antioxidant effect in vivo. By contrast, at the cell level, some flavonoids can modify protein kinases mediated signal transmission, thereby inducing antioxidant and anti-inflammatory genes expression, and, vice versa, inhibiting oxidant and inflammatory gene expression. Presently available information and the important health challenge justify enhanced research in the field.

Highly enriched, minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture.

A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5'-nucleotidase and ouabain-sensitive (Na+ + K+)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.

Nitric oxide generation from extracellularly applied NG-hydroxy-L-arginine in LPS-activated RAW 264 macrophages.

Lipopolysaccharide (LPS)-activated but not control RAW 264 macrophages produced nitric oxide (NO) from extracellularly-applied NG-hydroxy-L-arginine (L-NOHA) in a concentration-dependent manner, as measured by EPR spin trapping and assays for NO2- and NO3-. This production was inhibited by NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, NO-synthase inhibitors, as well as by L-lysine, a competitor for the y+ amino acid carrier system. No significant differences were found between L-NOHA and L-arginine with respect to the rate of NO production and the effects of inhibitors. These results provide evidence that extracellular L-NOHA can enter LPS-activated RAW 264 macrophages via a cationic amino acid carrier system and be metabolized to NO by NO-synthase. The data also suggest that no alternative pathway exists for NO production from L-NOHA in non-activated RAW 264 macrophages.

Induction of nitric oxide synthase and dual effects of nitric oxide and cyclooxygenase products in regulation of arterial contraction in human septic shock.

BACKGROUND: The role of endogenous nitric oxide (NO) and cyclooxygenase metabolites was investigated in contractile responses of small omental arteries from patients with hyperdynamic septic shock. METHODS AND RESULTS: Expression of inducible NO synthase (immunostaining) and a high but variable level of NO production (NO spin trapping) was detected in arteries from patients with septic shock. In these vessels, ex vivo contractile responses to the thromboxane A2 analogue U46619 and to low concentrations of norepinephrine (NE) (up to 10 micromol/L) were not significantly different from controls. However, higher concentrations of NE caused pronounced fading of contraction in septic but not in nonseptic arteries. Exposure to either the NO synthase inhibitor NG-nitro-L-arginine methyl ester or the cyclooxygenase inhibitor indomethacin had no effect in control vessels. However, both inhibitors increased the response to the contractile effects of the 2 agonists only in patients with septic shock. In contrast to NG-nitro-L-arginine methyl ester, which decreased the threshold concentration of the fading effect of NE, indomethacin abolished this effect in arteries from septic patients. CONCLUSIONS: These results provide direct evidence for the induction of NO synthase in small arteries from patients with septic shock. They suggest that in these arteries, increased production of NO, in conjunction with vasodilatory cyclooxygenase metabolites, contributes to counteract hyperreactivity to agonists and decreases the cyclooxygenase product-mediated pronounced fading of contraction caused by a high concentration of NE.

Nitric Oxide and cGMP in Regulation of Arterial Tone.

Nitric Oxide (NO) is an important factor in the control of vascular tone and peripheral resistance. Guanosine 3',5'-monophosphate (cGMP) mediates NO-induced vasorelaxation via multiple mechanisms, including decreased Ca(2+) entry and release, enhanced Ca(2+) extrusion, and inhibition of sensitization of myofilaments to Ca(2+) caused by some agonists such as norepinephrine (but not others such as ATP). This may result in differential effects of NO, depending on the agonist and the smooth muscle phenotype. In blood vessels exposed to inflammatory stimuli (for instance in endotoxemia), enhanced NO production causes loss of vascular reactivity to vasoconstrictor agents. This results from the induction of NO synthase activity in vascular cells, especially in the adventitia. The role of the adventitia may explain differences between large and small resistance arteries, in addition to the phenotype of smooth muscle cells. Protein-bound dinitrosyl non-heme iron complexes with thiols can be generated in arteries subsequent to the induction of NO synthase. Low molecular thiols can displace Fe-NO from these complexes, leading to activation of guanylyl cyclase and vasorelaxation. This may represent a novel mechanism of NO storage and release, enabling prolonged effects of NO in blood vessels and, perhaps, protection of vascular tissue against oxidative injury in sepsis and other inflammatory diseases.

Vascular relaxation and cyclic guanosine monophosphate in a rat model of high output heart failure.

OBJECTIVE: Low output heart failure induces abnormalities of endothelium dependent vasodilation, but the mechanisms responsible for this remain unclear. As blood flow can alter endothelial cell function, in particular nitric oxide (NO) release, the activity of endothelium derived relaxing factor (EDRF) was investigated in a rat model of high output heart failure. METHODS: The thoracic aorta upstream of an aorto-caval fistula in rats was submitted to hormonal changes (similar to those in heart failure) and to high blood flow (opposite to that found in low output heart failure). Functional and biochemical arterial properties were studied in aorto-caval fistula rats and in sham operated rats three months after operation. The vascular responses were studied by exposing aortic segments from fistula and sham operated rats to increasing concentrations of agonists. Aortic cyclic guanosine monophosphate (cGMP) concentration was assessed as an index of NO synthase activity. The effect of NO synthase blockade on functional and biochemical arterial properties was also studied. RESULTS: Plasma atrial natriuretic factor (ANF) was increased in fistula rats compared to sham operated rats. The concentrations of acetylcholine or the calcium ionophore A23187 required to produce 10% and 50% maximum relaxation (EC10 and EC50) were similar in the two groups. Relaxation in response to low concentrations of Sin-1 (an NO donor) was shifted rightwards in fistula rats and EC10 was greater than in the controls. The aortic cGMP concentration was higher in aorto-caval fistula rats than in sham operated rats (p = 0.008). The differences between aorto-caval fistula rats and sham operated rats were probably the result of increased basal EDRF-NO release in the former, since NO synthase blockade abolished the differences in both aortic cGMP and the dose-response curve to Sin-1. CONCLUSIONS: The arterial wall upstream of a chronic aorto-caval fistula has increased cGMP content and hyposensitivity to Sin-1, which may be due to enhanced basal EDRF-NO release. These changes, strikingly different from those found in the low output heart failure, suggest that haemodynamic rather than neuroendocrine factors play a determinant role in the altered vasodilator response in heart failure.

Temporal relationships between levels of circulating NO derivatives, vascular NO production and hyporeactivity to noradrenaline induced by endotoxin in rats.

OBJECTIVE: Lipopolysaccharide (LPS) induces early (within 1 h) and delayed (after several hours) impairment of vascular reactivity to catecholamines whose mechanisms are different, although they probably both involve nitric oxide (NO). Temporal and quantitative relationships between hyporeactivity to noradrenaline and NO production were investigated in a rat model of endotoxaemia allowing to clearly distinguish the two phases of hyporeactivity. METHODS: Anaesthetised rats were infused with LPS (14 mg kg-1 h-1) for 1 h. Pressure responses to noradrenaline (NA) and circulating NO derivatives (nitrosyl haemoglobin, NO2-, NO3-) were monitored for 5 h after the onset of infusion. Reactivity to NA and tissue cyclic GMP level were also assessed ex vivo, in aortic rings taken at different experimental times. RESULTS: LPS-induced early hyporeactivity to NA was associated with a moderate but significant increase in plasma NO3- level, without any significant change in concentration of the other circulating NO derivatives. Neither reactivity ex vivo nor cyclic GMP content were modified in aortae taken after 1 h of LPS infusion. By contrast, delayed hyporeactivity (5 h after the onset of LPS infusion) was associated with a large increase in all circulating NO derivatives (up to 2.5 fold), enhanced aortic cyclic GMP level and aortic hyporeactivity ex vivo. Pre-treatment of rats with NG-nitro-L-arginine methyl ester (1 mg kg-1 i.v.) entirely prevented early hyporeactivity and rise in NO3- concentration. In addition it attenuated in comparable proportion both delayed hyporeactivity to NA in vivo and circulating levels of NO derivatives. CONCLUSION: The results confirm the involvement of NO in the two phases of hyporeactivity to NA induced by LPS. They strongly support the view that a circulating factor is involved in triggering endothelial NO release during the early phase, whereas the delayed phase is associated with a high production of NO in vascular smooth muscle resulting from the induction of NO synthase.

Possible mechanism of nitric oxide production from N(G)-hydroxy-L-arginine or hydroxylamine by superoxide ion.

It has been speculated the N(G)-hydroxy-L-arginine (OH-L-Arg), which is an intermediate in NO production from L-arginine, may be converted to NO by superoxide ion. However, there is still no direct evidence for this conversion. In the present study this was investigated using superoxide ion generated either in acellular or cellular systems. It was found that OH-L-Arg and hydroxylamine were converted to nitrite and nitrate apparently via NO by superoxide ion in aqueous solution. Arginine remained unaffected. These changes were observed during reaction of chemical substances as well as in a biological system (zymosan-activated macrophages in culture). Superoxide dismutase prevented this transformation. OH-L-Arg was also spontaneously hydrolysed to hydroxylamine and L-citrulline, however this occurred at pH > 9 only. Activated microsomes (containing different isoforms of cytochrome P450) were unable to replace NO-synthase in its ability to produce OH-L-Arg from L-arginine. These data support the hypothesis that a pathway alterative to the well-known synthesis of NO by NO-synthase via OH-L-Arg exists. This pathway may involve the production of OH-L-Arg by NO-synthase and decomposition of OH-L-Arg to NO by the action of superoxide ion. Alternatively, hydrolysis of OH-L-Arg to hydroxylamine may occur followed by its oxidation to NO, again by superoxide ion.

Alterations in calcium stores in aortic myocytes from spontaneously hypertensive rats.

The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca2+ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca2+, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR-compared with those from WKY. In Ca(2)+-free medium, Ca2+ release induced by thapsigargin (10 mumol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 mumol/L) did not modify the rises in intracellular Ca2+ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)-induced Ca2+ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca2+ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired during proliferation. Thus, they suggest that differences in Ca2+(-)induced Ca2+ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.

Phorbol ester inhibition of vasopressin-induced calcium efflux from cultured rat aortic myocytes.

The effect of the protein kinase C activator TPA was investigated on AVP-induced 45Ca release from rat aortic myocytes. In the nanomolar range TPA, but not 4 beta-phorbol, reduced the brief 45Ca efflux produced by AVP in the presence or in the absence of extracellular calcium. The maximal effect of TPA was to abolish the response to a half maximally active concentration of AVP, and to reduce by 50% the maximal response to the hormone. These results suggest that protein kinase C activation can exert a negative control on the early AVP-induced calcium mobilization in vascular smooth muscle.

Cyclic AMP-dependent phosphorylation of a 16 kDa protein in a plasma membrane-enriched fraction of rat aortic myocytes.

Phosphorylation induced by cAMP-dependent protein kinase was examined in a plasma membrane-enriched fraction from control and beta-adrenergic-stimulated rat aortic myocytes. Phosphorylation of a 16 kDa protein which copurified with the plasma membrane marker (Na+ + K+)-ATPase was most prominent. It was decreased by pretreatment of the myocytes with isoproterenol and the effect of isoproterenol was inhibited by propranolol. Both phosphorylation induced by cAMP-dependent protein kinase and its inhibition by isoproterenol pretreatment declined in preparations exposed to endogenous phosphatase. These results provide strong evidence that beta-adrenergic stimulation of aortic myocytes induces in situ phosphorylation of a 16 kDa plasma membrane protein.

Exogenous NG-hydroxyl-L-arginine causes nitrite production in vascular smooth muscle cells in the absence of nitric oxide synthase activity.

Nitric oxide (NO) production from exogenous NG-hydroxy-L-arginine (OH-L-Arg) was investigated in rat aortic smooth muscle cells in culture by measuring nitrite accumulation in the culture medium. As well, the interaction between OH-L-Arg and L-arginine uptake via the y+ cationic amino acid transporter was studied. In cells without NO-synthase activity, OH-L-Arg (1-1000 microM) induced a dose-dependent nitrite production with a half-maximal effective concentration (EC50) of 18.0 +/- 1.5 microM (n = 4-7). This nitrite accumulation was not inhibited by the NO-synthase inhibitor NG-nitro-L-arginine methyl ester, L-NAME (300 microM). In contrast, it was abolished by miconazole (100 microM), an inhibitor of cytochrome P450. Incubation of vascular smooth muscle cells with LPS (10 micrograms/ml) induced an L-NAME inhibited nitrite accumulation, but did not enhance the OH-L-Arg induced nitrite production. OH-L-Arg and other cationic amino acids, L-lysine and L-ornithine, competitively inhibited [3H]-L-arginine uptake in rat aortic smooth muscle cells, with inhibition constants of 195 +/- 23 microM (n = 12), 260 +/- 40 microM (n = 5) and 330 +/- 10 microM (n = 5), respectively. These results show that OH-L-Arg is recognized by the cationic L-amino acid carrier present in vascular smooth muscle cells can be oxidized to NO and nitrite in these cells in the absence of NO-synthase, probably by cytochrome P450 or by a reaction involving a cytochrome P450 by-product.

Age-related differences in cyclic AMP metabolism and their consequences on relaxation induced by isoproterenol and phosphodiesterase inhibitors in rat isolated aorta.

Experiments were designed to investigate the respective roles of adenylate cyclase and cyclic nucleotide phosphodiesterase in the alterations with age of cyclic AMP metabolism-dependent relaxation in rat aorta. Neither basal nor stimulated aortic adenylate cyclase activities from 7- and 18-week-old rats differed significantly. The maximal cyclic AMP accumulation induced by isoproterenol in the aorta decreased with the age of the rat as did the maximal relaxation produced by the beta-adrenergic agonist in pre-contracted aorta strips. The age-related difference in cyclic AMP accumulation was abolished when the experiment was performed in the presence of a high concentration (500 microM) of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). In addition, four phosphodiesterase inhibitors, IBMX, rolipram, Ro 20-1724 and zaprinast relaxed aorta strips pre-contracted by serotonin with concentration-effect curves which were displaced to the right in the older rats, the maximal effects being unchanged. The results suggest that the previously reported increased phosphodiesterase activity could be responsible for (1) the diminution with age of isoproterenol-induced cyclic AMP accumulation and relaxation in rat aorta; (2) the age-related decrease in potency of phosphodiesterase inhibitors as relaxing agents.

Inhibition of cyclic adenosine-3',5'-monophosphate phosphodiesterase from vascular smooth muscle by rolipram analogues.

Rolipram [(R,S)-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidone] has been shown to inhibit selectively the cAMP phosphodiesterase (PDE) of vascular smooth muscle. In order to further explore the structural requirements for selective PDE inhibition, we synthesized a series of rolipram derivatives differently substituted either at the pyrrolidinone or at the aromatic ring. Among these compounds, rolipram was the most active compound. Semirigid analogues were prepared and used for an evaluation of the active conformation of rolipram. Structural comparison with two other potent and chemically different smooth muscle cAMP-PDE inhibitors, trequinsin and Ro 20-1724, allows us to propose a first topological model of the smooth muscle cAMP-PDE pharmacophore.

Potentiation by neuropeptide Y of vasoconstriction in rat resistance arteries.

1. The effects of neuropeptide Y (NPY) on resistance arteries were investigated on 3rd generation mesenteric arterioles of the rat. 2. Contractions were elicited by noradrenaline (NA), 5-hydroxytryptamine (5-HT), prostaglandin F2 alpha (PGF2 alpha), depolarization (KCl substituted for NaCl) and by the calcium agonist Bay K 8644, in the absence and in the presence of NPY (100 nM), a concentration which by itself did not induce vasoconstriction. 3. NPY produced a leftward shift of the concentration-response curves to the agonists and to KCl, without any alteration of maximal contractions. 4. NPY also potentiated contractions elicited by addition of CaCl2 to KCl-depolarized vessels, but its effect on calcium-induced contractions decreased with increasing KCl concentrations (from 20 to 100 mM). 5. Calcium-induced contractions were inhibited by the calcium channel blocker nitrendipine, both in the presence and absence of NPY (100 nM). NPY increased slightly (but significantly) the sensitivity to nitrendipine (the apparent KB increased from 2.9 x 10(-10) M to 1.6 x 10(-10) M). 6. The KCl concentration necessary for the maximal effect of Bay K 8644 was decreased in the presence of NPY, and the sensitivity to the calcium channel agonist was increased. 7. Elevating the KCl concentration in the bath from 5 to 20 mM (which gives the same displacement to the left of the KCl concentration-effect curve seen in the presence of NPY) induced a parallel leftward shift of NA and 5-HT concentration-response curves. This shift was identical to the one induced by NPY on 5-HT-evoked contractions, but it was significantly smaller (P less than 0.001) than the shift of the NA concentration-response curve observed in the presence of NPY. In the latter case, NPY enhanced more markedly the contractions induced by low NA concentrations (between 10(-9) and 3 x 10(-8 M) than those induced by high concentrations (up to 3 x 10(-7) M), thus giving a shallow concentration-response curve. 8. The results strongly suggest that NPY partially depolarizes the arterioles and induces an increase in calcium entry through voltage-dependent channels, thus enhancing contractions elicited by agonists or by KCl-depolarization. In addition, they support the view that another mechanism also plays a part in the potentiation by NPY of the effects of low concentrations of NA.

Enhancement by neuropeptide Y (NPY) of the dihydropyridine-sensitive component of the response to alpha 1-adrenoceptor stimulation in rat isolated mesenteric arterioles.

1. The mechanism by which neuropeptide Y (NPY) potentiates the vasoconstriction induced by alpha 1-adrenoceptor agonists was investigated in 3rd generation mesenteric arterioles of the rat. 2. At a maximally active concentration, nitrendipine (10(-6) M) displaced to the right the concentration-response curves to noradrenaline (pD2 decreased from 6.2 +/- 0.06 to 5.7 +/- 0.03) and phenylephrine (pD2 decreased from 5.6 +/- 0.03 to 5.3 +/- 0.03). Diltiazem (10(-5) M) also shifted to the right the concentration-response curve to phenylephrine (pD2 decreased from 6.0 +/- 0.06 to 5.5 +/- 0.04). In addition, the maximal response to phenylephrine was significantly decreased in the presence of either nitrendipine or diltiazem. 3. In the absence of a calcium channel blocking agent, NPY (100 nM) produced a leftward shift of the concentration-response curves to noradrenaline (pD2 increased from 6.2 +/- 0.06 to 6.5 +/- 0.05) and phenylephrine (pD2 increased from 5.6 +/- 0.03 to 6.0 +/- 0.06 and from 6.0 +/- 0.06 to 6.3 +/- 0.11). In the presence of either nitrendipine (10(-6) M) or diltiazem (10(-5) M), NPY (100 nM) did not alter the concentration-response curves to either noradrenaline or phenylephrine. 4. NPY was added to arterioles brought to the same level of tension (40% of the maximal contraction) either by phenylephrine alone (1.5 x 10(-6) M) or by a higher concentration of phenylephrine (3 x 10(-6) M) followed by the addition of prazosin (1.3 x 10(-9) M; a concentration at which it partially blocks alpha 1-adrenoceptors). In these conditions, the response to phenylephrine was completely abolished by nitrendipine (10-6 M) or by diltiazem (10-5M). Furthermore, NPY (10-1" to 10-7M) increased the arteriolar tension up to the maximal contractile capacity of the vessels with pD2 values of 8.6 + 0.02 and 8.7 + 0.01, in the absence and presence of prazosin, respectively. 5. Prazosin was replaced in the above protocol by other vasodilator agents acting through different mechanisms. Whether in the presence of 2 x 10-7M forskolin, 6 x 10-7M sodium nitroprusside (which stimulate adenylate cyclase or guanylate cyclase, respectively) or 2 x 10- 7M diltiazem (a concentration at which calcium entry is partially blocked), NPY enhanced phenylephrine-induced contraction to the maximum level with an identical potency (pD2 values of the peptide ranged from 8.3 to 8.7). 6. The results show that, in rat mesenteric arterioles, NPY potentiates only the calcium entry blockersensitive component of contraction induced by stimulation of alpha,-adrenoceptors. In addition, they provide evidence that the peptide counteracts with an equal potency the inhibitory effect of partial block of alpha,-adrenoceptors and of relaxing agents acting through different mechanisms. It is suggested that NPY enhances calcium entry induced by stimulation of alpha l-adrenoceptors in this tissue.

Modulation by endothelium of contractile responses in rat aorta in absence and presence of flunarizine.

The possible modulation by endothelium of phenylephrine- and prostaglandin F2 alpha-induced mobilization of calcium for contraction in the rat aorta has been investigated. Contractions elicited by these and other agonists are inhibited in the presence of endothelium. For any single concentration of phenylephrine in the presence of endothelium, the initial phasic components of contractions were significantly greater, the maximal contractions were achieved sooner and were less well maintained as compared to contractions elicited in the absence of endothelium. The kinetic characteristics of contractions stimulated by single concentrations of PGF2 alpha were similar in the presence and absence of endothelium and did not exhibit initial phasic components of contraction. Sub-maximal contractions-elicited by both PGF2 alpha and phenylephrine in the absence of endothelium were inhibited to a greater extent by flunarizine 3 microM than equieffective contractions elicited in the presence of endothelium. Maximal contractions elicited by phenylephrine (1 microM) were inhibited to a similar extent by flunarizine in the presence and absence of endothelium, but maximal contractions elicited by PGF2 alpha (30 microM) were inhibited by flunarizine to a greater extent in the presence than in the absence of endothelium. It is concluded that an endothelium-derived factor, perhaps distinct from endothelium-derived relaxing factor, can modulate the ability of both phenylephrine and PGF2 alpha to mobilize calcium for contraction. This modulatory effect is associated with an enhanced mobilization of intracellular calcium. Thus, submaximal concentrations of both agonists were less dependent on extracellular calcium than on intracellular calcium to elicit contractions in the presence of endothelium, as compared to contractions elicited in the absence of endothelium.

Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery.

1 The effects of two 8-substituted analogues of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were compared with those of forskolin and isoprenaline on [3H]-noradrenaline release and vasoconstriction induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery, in the absence and in the presence of protein kinase inhibitors. 2 8-Bromo-adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP, 10-300 microM), 8-(4-chlorophenyl-thio)-adenosine 3':5' cyclic monophosphate (8-pCPT-cyclic AMP, 3-300 microM), forskolin (0.3-10 microM) and isoprenaline (1 nM-1 microM) all concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. The effect of cyclic AMP analogues was larger (2.5 fold at 300 microM) than those of cyclic AMP elevating drugs (1.6 fold at 10 microM for forskolin and 1.5 fold at 30 nM for isoprenaline). 3 At concentrations active at the prejunctional level, the four drugs had differential effects on stimulation-induced vasoconstriction, which was enhanced by the two cyclic AMP analogues, decreased by forskolin and not significantly altered by isoprenaline. 4 The [3H]-noradrenaline release-enhancing effects of 8-bromo-cyclic AMP, forskolin and isoprenaline were significantly decreased by the cyclic AMP-dependent protein kinase (PKA) inhibitor (N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5- isoquinolinesulphonamide, di-hydrochloride) (H-89; 100 nM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor, 8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 microM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor,8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 MicroM).At the same concentrations the PKA inhibitor attenuated only the nerve-induced vasoconstrictor responses obtained in the presence of 8-bromo-cyclic AMP, whereas the PKG inhibitor did not modify that obtained in the presence of 8-bromo-cycic AMP or forskolin.5. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (1 MicroM) enhanced nerve-evoked [3H]-noradrenaline release, and this effect was decreased by the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)-maleimide (GF 109203X; 100 nM). However, the latter drug did not modify the enhancing effect of 8-bromo-cyclic AMP on [3H]-noradrenaline release.6. It is concluded that activation of cyclic AMP-dependent protein kinase is involved in the enhancing effect of cyclic AMP-elevating compounds on prejunctional release of noradrenaline. In addition the results provide no clear-cut evidence for a vasodilator role of PKA.

Evidence for N-acetylcysteine-sensitive nitric oxide storage as dinitrosyl-iron complexes in lipopolysaccharide-treated rat aorta.

1. The aim of this study was to assess whether or not vasoactive nitric oxide (NO) stores exist within vascular tissue after lipopolysaccharide (LPS)-treatment. 2. Rat thoracic aortic rings (for contraction experiments) or whole thoracic aortae (for electron paramagnetic resonance (e.p.r.) spectroscopy) were incubated for 18 h at 37 degrees C in the absence (control) or in the presence of LPS (10 micrograms ml-1), with or without L-arginine (L-Arg, 1 mM), the substrate of NO synthase (NOS) or N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NOS. 3. Incubation of rat aortic rings with LPS and L-Arg resulted in a significant decrease of the maximum contractile response to noradrenaline (NA, 3 microM). Addition of L-NAME (3 mM) enhanced contraction towards control values. After precontraction with NA and L-NAME, addition of N-acetyl-L-cysteine (NAC, 0.1 to 10 mM) evoked a concentration-dependent relaxation in rings incubated with LPS and L-Arg, but not in control rings, rings incubated with LPS in the absence of L-Arg or rings incubated with LPS in the presence of L-Arg and L-NAME. Removal of the endothelium did not significantly modify the relaxation induced by NAC. Methylene blue (3 microM), an inhibitor of the activation of guanylyl cyclase by NO, completely abolished the relaxing effect of NAC. 4. The presence of protein-bound dinitrosyl non-haem iron complexes (DNIC) was detected by e.p.r. spectroscopy in aortae incubated with LPS and L-Arg, but not in control aortae. Furthermore in LPS-treated aortae, addition of NAC (20 mM) gave rise to the appearance of an e.p.r. signal characteristic of low molecular weight DNIC. 5. These results provide evidence that, within vascular tissue, NO generated from L-Arg by LPS-induced NOS activity can be stored as protein-bound DNIC in non-endothelial cells. Upon addition of NAC, low molecular weight DNIC are released from these storage sites and induce vascular relaxation probably through guanylyl cyclase activation.