Effects of the ethanolic extract of Spirulina maxima on endothelium dependent vasomotor responses of rat aortic rings

Journal of Ethnopharmacology 75 (2001) 37 – 44 www.elsevier.com/locate/jethpharm

Effects of the ethanolic extract of Spirulina maxima on endothelium dependent vasomotor responses of rat aortic rings M.C. Paredes-Carbajal a,*, P.V. Torres-Dura´n b, J.C. Dı´az-Zagoya b, D. Mascher a, M.A. Jua´rez-Oropeza b a b

Department of Physiology, School of Medicine, UNAM, P.O. Box 70 -250, Mexico D.F. 04510, Mexico Department of Biochemistry, School of Medicine, UNAM, P.O. Box 70 -159, Mexico D.F. 04510, Mexico

Received 21 November 2000; received in revised form 21 November 2000; accepted 29 November 2000

Abstract Dietary Spirulina decreases, endothelium-dependently, the responses to vasoconstrictor agonists and increases the endotheliumdependent, agonist-induced, vasodilator responses of rat aorta rings. The aim of this study was to analyze, in vitro, the effects of a raw ethanolic extract of Spirulina maxima on the vasomotor responses of rat aortic rings to phenylephrine and to carbachol. On rings with endothelium, the extract produced the following effects: (a) a concentration-dependent (60– 1000 mg/ml) decrease of the contractile response to phenylephrine; (b) a rightward shift and a decrease in maximal developed tension, of the concentration–response curve to phenylephrine; (c) a concentration dependent relaxation of phenylephrine-precontracted rings. These effects were blocked by L-NAME, and not modified by indomethacin. The extract had no effect on the concentration–response curve to carbachol of rings with endothelium. On endothelium-denuded rings the extract caused a significant rightward shift of the concentration response curve to phenylephrine without any effect on maximal tension development. In the presence of the extract, indomethacin induced a marked decrease in the maximal phenylephrine-induced tension of endothelium-denuded rings. These results suggest that the extract increases the basal synthesis/release of NO by the endothelium and, also, the synthesis/release of a cyclooxygenase-dependent vasoconstricting prostanoid by vascular smooth muscle cells. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Spirulina maxima; Aortic rings; Endothelium; Nitric oxide; Vasomotor responses; Indomethacin; L-NAME

1. Introduction Spirulina maxima is a cyanobacteria belonging to the Oscillatoraceae family (Ciferri, 1983), this cyanobacteria usually grows in alkaline waters of Africa, Asia, North and South America (Ciferri, 1983). Spirulina maxima is widely used as a food supplement because of its high protein content (65%), but it is also rich in carotenes and vitamin E (Santilla´n, 1982). Lipids isolated from Spirulina have been shown to contain high levels of polyunsaturated fatty acids, including linolenic acid which is a precursor of arachidonic acid; this cyanobacteria contains, also, several kinds of sterols (Cohen and Voushak, 1991; Kay, 1991). * Corresponding author. E-mail address: [email protected] (M.C. Paredes-Carbajal).

Toxicological as well as teratological studies of several Spirulina species have not revealed any toxic effect, either during or after its acute or chronic oral administration (Yoshino et al., 1980; Krishnakumari et al., 1982; Chamorro et al., 1988, 1996a,b, 1997; Chamorro and Salazar, 1990; Salazar et al., 1996). A Spirulina-enriched diet has been shown to decrease total cholesterol serum levels and to reduce body weight in obese humans (Nakaya et al., 1988; Becker et al., 1986). It was reported that Spirulina reduces lipoprotein lipase activity in hyperlipidemic rats (Iwata et al., 1990), and prevents the induction of fatty liver by a fructose-rich diet (Gonza´lez de Rivera et al., 1993) or by carbon tetrachloride injury (Torres-Dura´n et al., 1998, 1999). On the other hand, Spirulina not only decreases the cholesterol serum levels but also increases the levels of high density lipoproteins, suggesting that Spirulina may have a protective effect on the cardiovas-

0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 3 9 0 - 1

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cular system (Kay, 1991). Furthermore, it has been observed that dietary Spirulina decreases, in an endothelium-dependent manner, the responses to vasoconstrictor agonists and increases the endothelium-dependent agonist induced vasodilator responses in aorta rings (Paredes-Carbajal et al., 1997). On the other hand, it was demonstrated that Spirulina maxima prevented the decrease of the endothelium-dependent vasodilator responses induced by a fructose rich diet, in the rat (Paredes-Carbajal et al., 1998). To date, the biochemical nature of the constituents responsible for the Spirulina maxima effects have not been elucidated although some constituents, like C-phycocyanin, from Spirulina have been shown to have an hepatoprotective action on carbon tetrachloride injury (Vadiraja et al., 1998). Therefore, the aim of this study was to analyze, in 6itro, the effects of a raw ethanolic extract of Spirulina maxima (water/ethanol, 9:1, v/v), on the vasomotor responses of rat aortic rings. 2. Material and methods

2.1. Reagents Glucose and organic solvents were from Merck (Mexico), all other chemicals were from Sigma (St Louis, MO). Indomethacin was dissolved in 4% sodium carbonate. L-phenylephrine hydrochloride, carbachol (carbamoylcholine chloride) and N…-nitro-L-arginine methyl ester hydrochloride (L-NAME) were dissolved in deionized water. The spray-dried powder of Spirulina maxima employed in these experiments was a generous gift from Spirulina Mexicana (Mexico). The water – ethanol extract (9:1, v/v, 100 ml) was prepared with 5 g of dry powder of Spirulina at 70°C and vigorously mixed for 1 h. Afterwards, the mixture was filtered and lyophilized (20.91 9 0.56% w/w yielding from starting crude material). The lyophilized extract was solubilized in deionized water and incorporated into the perfusion solutions to give final concentrations of 0.06, 0.12, 0.25, 0.50, and 1.0 mg/ml.

2.2. In 6itro measurements of 6ascular responses The experiments were performed on aorta rings from male rats of the Wistar strain, weighing 250 – 300 g. All animals were kept in individual cages with free access to food and water and exposed to 12-h light-dark cycle. After 12 h fasting, animals were killed by cervical dislocation and the thoracic aorta immediately removed, cleaned of connective and adipose tissue and cut transversely into 2 mm long rings. Special care was taken to avoid damage to the endothelium. In every other ring, the endothelium was removed by gently rubbing the intimal surface.

For each experiment a pair of rings from the central portion of the same aorta (one with intact endothelium, the other without a functional endothelium) was used. Each of these rings was suspended horizontally in the same miniature organ chamber (volume 1 ml) between two stainless steel hooks. One of the hooks was fixed to the chamber wall while the other was attached to an isometric force transducer (Grass, FT 03). The vessels were continuously superfused (3 ml/min) with prewarmed (37°C) aerated (95% O2 and 5% CO2) modified Tyrode solution (composition in mM: NaCl, 137; KCl, 2.7; MgCl2, 0.69; NaHCO3, 11.9; NaH2PO4, 0.4; CaCl2, 1.8 and glucose, 10; pH was adjusted to 7.4). The rings were initially stretched until resting tension reached 2 g and allowed to equilibrate for 1 h; during this period the resting tension was continuously monitored (Grass, Model 79 Polygraph) and, if needed, readjusted to 2 g by further stretching. Before starting an actual experiment, responsiveness of each pair of rings to phenylephrine and to carbachol was tested. This was achieved by switching the superfusing Tyrode solution for 10 min to one containing phenylephrine (10 − 5 M) and, thereafter, to one containing, in addition to phenylephrine, carbachol (10 − 5 M). Carbachol-induced relaxation of the phenylephrine precontracted vessels was taken as evidence for the preservation of an intact endothelium whereas lack of relaxation confirmed the absence of a functional endothelium.

2.3. Experimental protocol To evaluate the effects of the Spirulina extract on the contractile responses and to determinate its effective concentration, in a first series of experiments, the effects of non-cumulatively increasing concentrations of the extract (0.06, 0.125, 0.250, 0.50 and 1.0 mg/ml) on the contractile responses to phenylephrine (10 − 5 M) were analyzed. In a second series of experiments the contractile responses of pairs of aortic rings to cumulatively increasing concentrations (10 − 9 − 10 − 5 M) of the h1-adrenergic agonist phenylephrine (concentration –response curve to phenylephrine) were initially recorded. Once tension development in response to the highest phenylephrine concentration reached its peak value, superfusion was switched to solutions having, in addition to phenylephrine, successively increasing concentrations (10 − 9 − 10 − 5 M) of carbachol (concentration –response curve to carbachol). The results obtained by these procedures were considered as control responses. After a 30-min washout period, the concentration –response curves to phenylephrine were repeated in the presence of the Spirulina extract, (0.50 mg/ml). The latter curves were determined in the absence and in the presence of the

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cyclooxygenase inhibitor indomethacin (10 − 5 M), as well as in the presence of both indomethacin and the competitive inhibitor of nitric oxide synthase N…-nitroL-arginine methyl ester (L-NAME, 300 mM). To evaluate a possible relaxant action of the Spirulina extract, the effects of cumulatively and non-cumulatively increasing concentrations of the Spirulina extract (0.06, 0.125, 0.250, 0.50 and 1.0 mg/ml) on phenylephrine (10 − 5 M) precontracted aorta rings were analyzed. In order to explore the interaction of this extract with the endothelium dependent relaxation induced by muscarinic receptor activation, concentration –response curves to carbachol (10 − 9 −10 − 5 M) were obtained in the absence and the presence of the Spirulina extract (0.50 mg/ml) in phenylephrine (10 − 5 M) precontracted aorta rings.

2.4. Data analysis The contractile responses induced by phenylephrine are expressed as tension increment in grams above the basal tension (imposed on the vessel throughout the experiment). Spirulina extract and carbachol-induced relaxations are expressed as the percent reduction in tension relative to maximal tension developed in response to phenylephrine (10 − 5 M). PD2 (-Log of the mean molar concentration of agonist producing 50% of the maximal response) was determined with the software package Graph Pad Prism (San Diego, CA, USA). All data are expressed as means9 SE. Comparisons of means were made by One Way Analysis of Variance (ANOVA) and differences among groups were evaluated using Student – Newman –Keuls Method (SigmaStat software; St Louis, MO, USA). A P-value of 0.05 or less was considered significant.

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6.96% of the maximal response observed in its absence. In aortic rings without endothelium the maximal contractile response to phenylephrine in the presence of Spirulina amounted to 76.099 4.78% of that observed in the absence of the extract.

3.2. Cumulati6e concentration–response cur6es to phenylephrine (10 − 9 − 10 − 5 M) in the presence of Spirulina maxima extract (0.5 mg/ml) Aortic rings with endothelium developed less tension in response to phenylephrine in the presence of Spirulina maxima extract than in its absence. Tension developed in response to concentrations of phenylephrine larger than 10 − 7 M differed significantly between the two groups (compare closed and open circles in Fig. 2). Maximal developed tension amounted to 2.3690.08 and 1.4390.15 g, in the absence and the presence of Spirulina, respectively. PD2 amounted to 7.0890.12 and 6.1890.18, respectively. In the presence of Spirulina extract the addition of indomethacin (10 − 6 M) to the superfusing solutions decreased tension development in response to phenylephrine (compare open circle and open diamonds in Fig. 2); the differences were, however, not significant. Maximal developed tension amounted to 1.4390.15 g, in the absence and 0.6790.28 g in the presence of indometacin. PD2 amounted to 6.1890.18 and 6.059 0.03, respectively. The addition of L-NAME (300 mM) to the superfusing solution reverted the effects of the Spirulina extract and indomethacin (compare open circles, open diamonds and open triangles in Fig. 2, see also Table 1). The maximal contractile responses to phenylephrine (10 − 5 M) of rings without endothelium recorded either

3. Results

3.1. Non cumulati6e concentration – response cur6e to the Spirulina maxima extract in the presence of phenylephrine (10 − 5 M) The addition of Spirulina maxima extract (0.06, 0.125, 0.250, 0.50 and 1.0 mg/ml), to the superfusing solution induced a concentration-dependent decrease in the contractile response of the aortic rings to phenylephrine (10 − 5 M). This reduction was much larger in rings having an intact endothelium (compare open and closed circles in Fig. 1). The maximal inhibition was observed with 1 mg/ml concentration. However, since the recovery using this concentration was incomplete, the concentration of 0.5 mg/ml was considered optimal. In rings with endothelium the maximal contractile response to phenylephrine (10 − 5 M) in the presence of the Spirulina extract (1 mg/ml) was 36.119

Fig. 1. Non-cumulative concentration– response curves of aortic rings, with ( ) and without () endothelium, to Spirulina maxima extract added in the presence of phenylephrine (10 − 5 M). The curves represent the percentage of the maximal contractile response to phenylephrine (10 − 5 M) recorded in the absence of Spirulina. Data are presented as means 9SE of five rats.

M.C. Paredes-Carbajal et al. / Journal of Ethnopharmacology 75 (2001) 37–44

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Fig. 2. Effects of Spirulina maxima extract on the concentration– response curves of aortic rings with endothelium to phenylephrine (10 − 9 − 10 − 5 M). The curves were obtained in the absence of Spirulina maxima extract ( ), in the presence of Spirulina maxima (500 mg/ml) (), in the presence of both Spirulina maxima and indomethacin (10 − 6 M) ( ), and in the presence of Spirulina plus indomethacin plus L-NAME (300 mg/ml) (). Data are presented as means9SE of five rats.

Fig. 3. Effects of Spirulina maxima extract on the concentration–response curves of aortic rings without endothelium to phenylephrine (10 − 9 −10 − 5 M). The curves were obtained in the absence of Spirulina maxima extract ( ), in the presence of Spirulina maxima (500 mg/ml) (), in the presence of both Spirulina maxima (500 mg/ml) and indomethacin (10 − 6 M) ( ), and in the presence of Spirulina plus indomethacin plus L-NAME (300 mg/ml) (). Data are presented as means 9SE of five rats.

in the presence or the absence of Spirulina showed no significant differences (2.2090.19 and 2.439 0.18 g, respectively, see Fig. 3). However, in the presence of the Spirulina extract the concentration response curve was significantly shifted rightwards (PB 0.05). PD2 amounted to 7.629 0.10 and 6.639 0.13, respectively. In the presence of Spirulina extract the addition of indomethacin (10-6 M) to the superfusing solutions significantly decreased tension development in response to phenylephrine (compare open circles and open diamonds in Fig. 3). Maximal developed tension amounted to 2.4390.18 g, in the absence and 0.939 0.21 g in the presence of indomethacin, respectively, the corresponding PD2 values were 6.6390.13 and 6.549

0.23, respectively. In the presence of the extract, the concentration response curve to phenylephrine of endothelium-denuded rings, recorded in the presence of both indomethacin and L-NAME (300 mM), was almost identical to that observed in the absence of these two blocking substances (compare open circles and open triangles in Fig. 3, see also Table 1).

3.3. Effects of Spirulina maxima extract on aortic rings precontracted with phenylephrine (10 − 5 M) In phenylephrine (10 − 5 M) precontracted aortic rings, the addition of cumulatively increasing concentrations of Spirulina maxima extract (0.06, 0.125, 0.25,

Table 1 Effects of Spirulina maxima extract on the concentration-response curves to phenylephrine (10−9–10−5 M) on rat aorta rings

Group

With endothelium PD2a Maximal tensionb

Control Spirulina Spirulina(+)Indomethacinc Spirulina(+)Indomethacin(+)L-NAMEd

7.089 0.12 6.1890.18e 6.0590.03e 6.4090.18e

a

2.36 90.08 1.43 9 0.15e 0.67 90.28e 2.10 9 0.09

Without endothelium PD2 Maximal tension 7.62 9 0.10 6.63 9 0.13e 6.54 9 0.23e 6.55 9 0.04e

2.20 90.19 2.43 9 0.18 0.93 9 0.21e 2.28 9 0.09

PD2: −log of mean molar concentration causing 50% of maximal response to phenylephrine (10−5 M). Maximal tension: Maximal tension (g) developed in response to phenylephrine (10−5 M). c (+) Indom: with indomethacin (10−6 M). d (+) L-NAME: with L-NAME (300 mM). e Denotes a significant difference with the control group (PB0.05). Data are presented as mean 9 SE; n =5 for all groups. b

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0.50 and 1.0 mg/ml) to the superfusing solutions produced a relaxant effect. This effect was both concentration- and endothelium- dependent (compare open and closed circles in Fig. 4). The maximal relaxation was observed with the concentration of 1 mg/ml (47.849 2.99%). However, since the recovery of the rings was incomplete with this concentration, the concentration 0.5 mg/ml was considered optimal.

3.4. Effects of Spirulina maxima extract (0.5 mg /ml) on the cumulati6e concentration – response cur6e to carbachol (10 − 9 – 10 − 5 M) The concentration-respose curve to carbachol (10 − 9 −10 − 5 M) of rings with endothelium; was not modified significantly by the addition of Spirulina maxima extract (0.5 mg /ml) to the superfusion solutions (results not illustrated).

4. Discussion One of the approaches that has been extensively used to elucidate the mechanism by which natural products modify the function of the cardiovascular system, has been to explore the effects of crude extracts of these products on the in vitro responsiveness of isolated vessels to well characterized vasoactive agents. Accordingly we analyzed the effects of an ethanolic extract of the cyanobacteria Spirulina maxima on the responsiveness of the rat aorta to the vasoconstrictor phenylephrine and to the vasodilator carbachol. Phenylephrine, a selective h1-adrenergic agonist, was

Fig. 4. Relaxant effects of Spirulina maxima extract on aortic rings precontracted with phenylephrine (10 − 5 M). The figure ilustrates the cumulative concentration-response curves to Spirulina maxima extract in rings with ( ) and without endothelium (). Data are expressed as percent of maximal phenylephrine-induced tension and shown as means9SE of five rats.

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used assuming that it would activate vascular smooth muscle contraction without simultaneously inducing receptor-mediated release of vasoactive substances from endothelial cells. This assumption is based on the presumptive absence of h1-adrenoceptors in the latter cells (Cocks and Angus, 1983). Carbachol was selected because it consistently produces endothelium-dependent, nitric oxide-mediated, relaxation in precontracted aorta rings whithout having any relaxing effect on vessels lacking a functional endothelium (Furchgott and Zawadzki, 1980). Our method of measuring the in 6itro vascular responsiveness offers some advantages relative to that used in most other similar studies, in which the small vessel segment under study is immersed in a rather large organ bath (5–20 ml). In these conditions, the solution is replaced by means of repeated ‘washing’ with the new solution. Our miniature organ bath (1 ml), on the contrary, is continuously superfused (at 3 ml/min) avoiding in this way, the uncontrolled alteration of the extracellular medium (by either accumulation or depletion of molecules) and, on the other hand, insuring — when desired — its fast and homogeneous modification. In addition, the use of a pair of aorta rings (with and without functional endothelium, respectively) excised from the central portion of the same aorta and placed in the same organ bath, allowed us to differentiate, during each individual experimental procedure, between endothelium-dependent and -independent effects. The ethanolic extract of Spirulina maxima decreased, concentration dependently, the tension developed in response to phenylephrine by the aortic rings having an intact endothelium. This decrease occurred, both when the rings were exposed simultaneously to a concentration of phenylephrine (10 − 5 M) producing a maximal contractile response and increasing concentrations of the extract (Fig. 1), or when they were exposed to increasing concentrations of the extract after having been maximally precontracted by phenylephrine (Fig. 4). The attenuation of the phenylephrine-induced tension development by the extract was also evident by comparing the concentration-response curve to phenylephrine (10 − 9 − 10 − 5 M) obtained in the absence of the extract with that obtained in the presence of a submaximal concentration of the extract. The latter curve was significantly deviated to the right and maximal developed tension was markedly decreased. These effects of the extract were not observed in rings without endothelium and they were blocked by the addition of an inhibitor of the nitric oxide synthase (L-NAME, 300 mM). Taken together, these results suggest that the extract either increases the synthesis/release of nitric oxide (NO) by the endothelium or the biological action of NO on vascular smooth muscle cells. The lack of effect of the extract on the carbacholinduced relaxation of phenylephrine-precontracted

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rings makes it less plausible that the extract enhances the biological activity of NO or protects its breakdown, rather than increasing its syntesis/release. The endothelium-dependent effects of the extract on the concentration –response curve to phenylephrine suggests that it increases the basal, but tone related, release of NO, rather than the receptor mediated release of NO. Since the tone related NO synthesis/release is mediated by the calcium-dependent eNOS (Adeagbo et al., 1994), it is reasonable to assume that the extract produces, in the endothelial cells, an increase of the intracellular Ca2 + concentration. This increase could arise either from Ca2 + inflow or from intracellular Ca2 + release or from both processes. The composition of the ethanolic extract of Spirulina maxima is currently under investigation, but it should be quite complex (Chamorro et al., 1996a). It contains lipids, including saturated and unsaturated fatty acids, cholesterol, carotenoids as well as alcohols and products of the denaturation products of the abundant (approx. 65%) proteins present in the dried Spirulina powder (Kay, 1991). Further studies are necessary in order to determine which of the constituents of the extract are responsible for the decrease in tension development in response to phenylephrine and whether that constituent(s) cause(s) or not an increase of the intracellular Ca2 + concentration. An increase of the intracellular Ca2 + concentration in endothelial cells is associated with the endothelium-dependent relaxation induced by red wine polyphenolic compounds (Andriambeloson et al., 1997). Similarly, (-)epichatechin which induces endothelium-dependent NO-mediated relaxations of rat isolated mesenteric arteries, increases intracellular Ca2 + levels in cultured human umbilical vein endothelial cells (Huang et al., 1999). We are not aware of any previous study regarding the effects of a Spirulina extract on the in vitro responsiveness of isolated vessels to vasoactive agonists. The present study is, however, in the same line as several reports regarding the vasomotor responses to some plant extracts or to their constituents. One of the first studies of this type, demonstrated that some grape products (including grape juice, grape skin extracts, certain wines as well as the flavonoid tannic acid, a constituent of wine) induce concentration- and endothelium-dependent relaxations, mediated by the NOcGMP pathway, of phenylephrine-precontracted rat aorta rings (Fitzpatrick et al., 1993). Similar results were reported more recently by Flesch et al. (1998) who showed that certain wines and the flavonoids tannic acid and quercetin induced in both human coronary artery and rat aorta rings precontracted with phenylephrine an endothelium dependent, NO mediated relaxation. Several other studies (Cishek et al., 1997; Andriambeloson et al., 1997; Stoclet et al., 1999) have also reported endohelium-dependent, NO-medi-

ated, relaxations of isolated vessel from several species induced by flavonoids contained in red wine. Similarly, flavonoids, as well as procyanidins in the vasoactive extracts of several other plants like tea (Huang et al., 1999), cocoa (Karim et al., 2000), or hawthorn (Kim et al., 2000) have been shown to induce endothelium-dependent relaxation of isolated vessels. On the other hand, different terpenes in the extracts from several plants have, also, been shown to inhibit (mostly endothelium-dependently) the noradrenaline or phenylephrine induced contraction of isolated vessels from several animal species. For instance, the diterpene jatrophone, present in the plant Jatropha elliptica, relaxes concentration- and endothelium-dependently the contraction induced in rat aorta rings by noradrenaline (Duarte et al., 1992), whereas the sesquiterpene polygodial isolated from the plant Drymis winteri was shown to relax concentration- and endothelium-dependently the phenylephrine-induced contractions of rings from rabbit pulmonary artery or thoracic aorta as well as rings from guinea-pig pulmonary artery (Andre´ et al., 1999). The ginsenoside Rg3, a triterpene glycoside isolated from Panax gingseng, also, induces a concentration- and endothelium-dependent, NO-mediated relaxation of phenylephrine precontracted rat aorta rings (Kim et al., 1999). The significant rightward shift of the concentration – response curve to phenylephrine caused by the Spirulina extract in endothelium denuded rings without an associated decrease of the maximal tension development (see Fig. 3 and Table 1) may be explained by the assumption that some constituent of the extract could be a weak competitive antagonist of the h1-adrenoceptors of the aortic vascular smooth muscle. This possibility was, however, not further explored. The addition of indomethacin, (10 − 6 M) in the presence of the extract (Fig. 3 and Table 1) caused a marked (more than 50%) decrease in the maximal tension developed by the endothelium-denuded rings in response to phenylephrine. This indicates that, in the presence of the extract, at least 50% of the tension developed by endothelium-denuded rings in response to phenylephrine is elicited by a cyclooxygenase dependent vasoconstricting prostanoid. Since on rings with an intact endothelium, the addition of indomethacin in the presence of the extract did not decrease significantly the maximal tension development, such a vasoconstricting prostanoid must have its origin in non-endothelial cells; possible candidates are either vascular smooth muscle cells or fibroblasts of the adventitia. It is quite plausible, that the high content of gamma linolenic acid of Spirulina maxima, a precursor of arachidonic acid, plays a role in the synthesis of such a vasoconstricting prostanoid. The results of the present study allow to conclude that the extract increases the basal synthesis/release of

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