David F. Fitzpatrick

Endothelium-dependent vasorelaxation caused by various plant extracts

Summary: In a previous study (Am J Physiol 1993;265: H774–8), we found that certain red wines and other grape products caused endothelium-dependent vasorelaxation. In the present study, aqueous extracts of a variety of vegetables, fruits, teas, nuts, herbs, and spices were tested for their endothelium-dependent relaxing ability in vitro. Rings of rat aorta, with or without an intact endo-thelium, were mounted in tissue baths, contracted with phenylephrine, and then exposed to diluted plant extracts. Many, but not all, extracts exhibited endothelium-dependent relaxations that were reversed by NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, which suggested involvement of nitric oxide, the endo-thelium-derived relaxing factor in the response. Furthermore, extracts that caused relaxation also increased tissue levels of cyclic GMP, the mediator of nitric oxide-induced vascular smooth-muscle relaxation. These results may lend further support to mounting evidence that plant foods contain compounds that, if absorbed intact and in sufficient quantities, could conceivably be beneficial in prevention of cardiovascular disease.

Endothelium-dependent Vascular Effects of Pycnogenol

Pycnogenol (P) is purported to exhibit effects that could be beneficial in terms of prevention of chronic age-related diseases such as atherosclerosis. The most studied of these effects is its antioxidant/free radical-scavenging activity. In this study, we investigated the possibility that this supplement might produce vascular effects by stimulation of nitric oxide (NO) production by vascular endothelial cells. In the in vitro experiments, P (1-10 microg/ml) relaxed epinephrine (E)-, norepinephrine (NE)-, and phenylephrine (PE)-contracted intact rat aortic ring preparations in a concentration-dependent manner. However, when the endothelial lining of the aortic ring was removed, P had no effect, indicating an endothelium-dependent relaxing (EDR) effect. This EDR response was caused by enhanced NO levels, because the NO synthase (NOS) inhibitor N-methyl-L-arginine (NMA) reversed (or prevented) the relaxation, and this response, in turn, was reversed by addition of L-arginine, the normal substrate for NOS. Pycnogenol-induced EDR persisted after exposure of intact rings to high levels of superoxide dismutase (SOD), suggesting that the mechanism of EDR did not involve scavenging of superoxide anion. In addition to causing relaxation, preincubation of aortic rings with P (1-10 microg/ml) inhibited subsequent E- and NE-induced contractions in a concentration-dependent manner. Fractionation of P by Sephadex LH-20 chromatography resulted in three fractions, one of which (fraction 3, oligomeric procyanidins) exhibited potent EDR activity. These results indicate that P, in addition to its antioxidant activity, stimulates constitutive endothelial NOS (eNOS) activity to increase NO levels, which could counteract the vasoconstrictor effects of E and NE. Furthermore, additional protective effects could result from the well-established properties of NO to decrease platelet aggregation and adhesion, as well as to inhibit low-density lipoprotein (LDL) cholesterol oxidation, all of which could protect against atherogenesis and thrombus formation.

Vasodilating procyanidins derived from grape seeds.

Abstract: We have shown in previous work that extracts of grape seeds (GSE) and skins, grape juice, and many red wines exhibit endothelium‐dependent relaxing (EDR) activity in vitro. This EDR activity involves endothelial nitric oxide (NO) release and subsequent increase in cyclic GMP levels in the vascular smooth muscle cells. The NO/cyclic GMP pathway is known to be involved in many cardiovascular‐protective roles. The current study focuses on the isolation and identification of EDR‐active compounds (procyanidins) from GSE. Crushed Concord grape seeds were extracted with methanol and the extract was separated into seven fractions (A‐G) on a Toyopearl TSK‐HW‐40 column. EDR‐active fractions (D‐G) were further separated into 25 individual compound peaks by HPLC, 16 of which were EDR active (threshold for relaxation ranged between less than 0.5 μg/mL and greater than 4 μg/mL). Procyanidin identification was accomplished by electrospray‐ion trap mass spectrometry (ES‐ITMS), MS/MS, and by tannase treatment and acid thiolysis, followed by HPLC and ES‐ITMS of the products. Activity of isolated procyanidins tended to increase with degree of polymerization, epicatechin content, and with galloylation. These EDR‐active compounds (many of which also possess antioxidant activity), individually or in the form of wines, juices, or nutritional supplements, may be useful in preventing or treating cardiovascular diseases.

Prolactin stimulation of protein kinase C activity in rat aortic smooth muscle

Prolactin (PRL) activated protein kinase C (PKC) in a dose dependent manner in rat aortic smooth muscle. Aortic strips incubated with sub-nanomolar concentrations of ovine PRL for 25 min. at 37 degrees C showed a significant stimulation of PKC activity in both cytosolic and particulate fractions. This activation could be blocked using either anti-PRL antibodies or 1-(5- isoquinolinesulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The results further support the role of PKC in the signal transduction pathway for PRL action and suggest that this activation may be involved in vascular smooth muscle function.

Stimulation of calcium uptake into aortic microsomes by cyclic AMP and cyclic AMP-dependent protein kinase.

SummaryEnhancement of calcium uptake into rabbit aortic microsomes was seen at a cyclic AMP concentration of 10−6M in the presence of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37). Protein kinase alone also increased calcium uptake, but cyclic AMP alone was without effect. The results suggest that stimulation of calcium sequestration may be the mechanism of cyclic AMP involvement in vascular smooth muscle relaxation.

Analysis of the relationship between pharmacological inhibition of cyclic nucleotide phosphodiesterase and relaxation of canine tracheal smooth muscle

Abstract Dose-response curves for muscular relaxation produced by five phosphodiesterase inhibitors were compared to curves for inhibition of phosphodiesterase-catalyzed breakdown of cyclic AMP or cyclic GMP. The rank-order of potency of the agents as muscular relaxants was similar to their order of potency as phosphodiesterase inhibitors. When enzyme activity was measured with 1.5. 38 or 400 μM cyclic AMP or 1.5 μM cyclic GMP as substrate, it was found that two agents, caffeine and SQ 20,009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b) -pyridine-5-car☐ylic acid, ethyl ester, HCl], inhibited activity in concentrations equal to or slightly less than were required for muscular relaxation. The remaining three agents, theophylline, MIX (1-methyl-3-isobutylxanthine), and ICI 58,301 (3-acetamido-6-methyl-8- n -propyl-s-triazolo[4,3-a]pyrazine), were required in 2.4 to 10.3-fold higher concentrations for enzyme inhibition than for muscular relaxation. It was concluded that, although these findings are generally consistent with the hypothesis that phosphodiesterase inhibition is important in the mechanism of action of the drugs tested, it may be necessary to take into account additional, presently unknown factors to explain fully the relaxant effects of these drugs on respiratory smooth muscle.

Effect of Ca++ on triphosphoinositide extraction in fusing myoblasts

Ca++-dependent degradation of triphosphoinositide has been postulated to regulate levels of membrane-bound Ca++ and to generate a 1,2-diacylglycerol fusogen in cell fusion. Triphosphoinositide metabolism was therefore studied during Ca++-induced fusion of cultured chick embryo myoblasts. Using a frequently cited extraction procedure, it was found that apparent Ca++-dependent triphosphoinositide degradation was actually due to inhibition of extraction. A new procedure using the ion-pairing reagent tetrabutylammonium sulfate was developed which was unaffected by Ca++ and gave 2- to 20-fold greater extraction of triphosphoinositide than existing procedures. With this procedure, no changes in triphosphoinositide metabolism were found during myoblast fusion.

Decreased sensitivity of spontaneously hypertensive rat aortic smooth muscle to vasorelaxation by atriopeptin III

The effects of a synthetic form of Atrial Natriuretic Factor (ANF) on spontaneously hypertensive rat aortic smooth muscle were investigated using either an alpha-adrenoceptive agonist (phenylephrine) or an agent which partially depolarized the plasma membrane (20mM KCl) as a contractile agent. The relaxant response was studied under conditions resembling normal physiological calcium ion levels (1.5mM) as well as over a range of calcium ion concentrations (0.1-2.5mM). The results demonstrate a hyporesponsiveness of hypertensive aorta to vasorelaxation induced by synthetic ANF, which is more apparent when the tissue is contracted with KCl. The results also suggest that ANF, which has been shown previously to inhibit intracellular and receptor operated calcium channel mobilization only, may additionally work through a mechanism which is related to the voltage induced calcium flux across the membrane, which also is inhibited less in hypertensive smooth muscle.

Atrial peptides inhibit protein kinase C-mediated contraction in rat aorta

The effects of atriopeptin III on phorbol ester-induced contraction were examined in aorta from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Precontracted SHR aorta was less responsive than WKY aorta to atriopeptin III-induced relaxation. Additionally, SHR aorta had significantly greater basal and phorbol ester-stimulated protein kinase C (PKC) activity than WKY aorta. The altered PKC response as well as hyporesponsiveness to atriopeptin III in SHR aorta may contribute to an altered vascular response and to the pathogenesis of hypertension.