Mechanism of sodium fluoride-induced contraction in the isolated rat aorta

1247 tered intra-arterially into the carotid artery in order to avoid acute cardiotoxicity, and selective alpha-1 agonists (phenylephrine 0.1-100 /tg/kg and methoxamine 1-1000 /~g/kg) were injec~.ed iitto the yugular 15 minutes after nifedipine pretreatment. In each animal one complete dose-response curve was obtained. Nifedipine effectively antagonizes alpha-1 adrenoceptor mediated increases in diastolic pressure dose-dependently. Table 1 Increase in diastolic pressure (mmHg) (n ~- 5-6), * p < 0.005, ** p < 0.01, * ** p < 0.001. Phenylephrine/tg/kg

Control

Nifedipine/tg/kg 100

0.1 1 10 100 Methoxamine/tg/kg 1 10 100 1000

5.85:0.8 16.0 5:2.9 50.2 5:9.5 113.2 5:7.3

2.25:0.5 ** 7.0 + 1.0 * 27.8 5:3.7 95.6 5:6.3

2.7 5:0.3 9.7 5:0.8 56.2 5:5.6 127.7 5:4.7

1.8 5:0.4 3.6 5:0.4 * * * 32.0 5:4.2 * * 128.4 5:6.8

300 2.65:0.3 4.65:0.3 17.8+0.8 68.8+3.4

1000 ** ** ** ***

1.4+0.3 *** 4.4+0.4 ** 15.85=1.6 ** 69.6+4.6 ***

1 ** 3.85:0.4 *** 32.0 + 3.4 * * 112.0 5:5.9

1.45:0.3 * 3.0+0.3 *** 22.6 5:4.8 * * 98.2 + 7.3 * *

In vitro studies of 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) (Malagodi and Chiou, 1974; Chioti and Malagodi, 1975) have shown this agent to reduce Ca 2+ availability in ~mooth muscle by stabilizing Ca 2+ binding to cellullar Ca 2+ stores. We studied the interaction between TMB-8 and alpha-1 pressor responses in the pithed normotensive rats. Pretreatment with TMB-8 (3000 /~g/kg i.v) was carried out 15 minutes before the administration of the alpha-1 adrenoceptor agonists. The increases in diastolic pressure show no difference with control values. TMB-8 neither potentiates nifedipine inhibition. Conclusion:In the pithed normotensive rats the decrease in extracellular calcium influx seems to be more important to modulate alpha-1 pressor responses than the attenuation of calcium release from intracellular bound stores.

References Malagodi, M.H., Chiou, C.Y., 1974, Eur. J. Pharmacol. 27, 25. Chiou, C.Y., Malagodi, M.H., 1975, Br. J. Pharmacol. 53, 279. P.we.088 [

Mechanism of sodium fluoride-induced contraction in the isolated rat aorta Adeagbo, A.S., Forster, A. a n d Triggle, C.R. Faculty of Medicine, Division of Basic Medical Sciences, Memorial University of Newfoundland St. John's, Newfoundland A I B 3V6, Canada Sodium fluoride (NaF) is known to contract vascular smooth muscle and recently it has been suggested (Zeng, et al., 1989) that this results from the stimulation of a Gi a n d / o r Gp protein(s) that are involved in the gating of voltage-operated calcium channels (VOCCs). The purpose of the present study was to determine the effect of NaF in rat aorta, and, since G-protein activation is an early event in receptor-activated excitation-contraction coupfing, we also examined the effect of agents known to interfere with second messenger systems viz calcium and cyclic AMP (PKA), protein kinase C (PKC) and calmodulin-dependent kinases (CAMK). Rings (4 mm long) from thoracic aortae isolated from male Sprague Dawley rats (Canadian Hybrid Farms, 200-300 g) were mounted in 10 ml organ baths containing Krebs solution at 37°C and gassed with 5% CO2 in oxygen. NaF (3-30 mM) induced consistent concentrati°n'dependent contraction of rat aortic rings, - E C 5 o - - 1 0 mM. The contractile responses were not affected by phenoxybenzamine (PBZ, 0.1 #M), r6fedipine (10 ~tM) or bathing with

1248

calcium-free + EGTA Krebs solution, indicating the contraction was neither mediated via neurotransmitter release from noradrenergic nerve terminals, the activation of VOCCs, nor the influx of extracellular calcium via voltage-independent mechanisms. Also, the NaF contraction of aortic rings isolated from rats pretreated with pertussis toxin (PTX, 50 ~g/kg, Lv.) were not different from rings obtained from untreated rats, implying that the NaF effect in r~t aorta is not mediated via a PTX-sensitive G-protein. BAPTA (intracellular calcium chelator, 100/zM), ryanodine (sarcoplasmic reticulum calcium depleter, 10 M) and TMB-8 (intracellular calcium antagonist, 100 /tM) respectively caused 36.0 5: 6.1~, 16.4 5: 6.8~ and 52.3 5: 7.3~; inhibition of the NaF contractile effect in calcium-free media while causing near complete inhibition of noradrenaline (NA) effect. On the other hand, PKC inhibitors, staurosporine and H-7, potently (ICso - 0.02 and 5.5/tM respectively) inhibited NaF induced contractions, whereas pretreatment with W-7 or calmidozolium (calmodulin inhibitors) only resulted in modest inhibition (ICso - 50 and 67/tM respectively). These data suggest that NaF induced contractions in the rat aorta are mediated in part by activation of PKC and, to a lesser extent, by intracellular calcium mob'dization. The occurrence of a NaF induced contraction in the absence of extracellular Ca 2+ the presence of in~xacellular BAFFA, but sensitive to inhibition by staurosporine, indicates that activation of a PTX-insensitive G protein(s).coupled directly or indirectly to PKC activation leads to an increase m the sensitivity of the contractile elements to low ( < 10 -7 M) levels of intracellular Ca 2+. In conclusion, the contraction o~ the rat aorta is associated with the entry of extracellular Ca 2+, the release of intracellular Ca 2+ and the activation of PKC. Supported by the Medical Research Council of Canada. Reference Zeng, Y.Y., Benishin, G. and Pang, P.H.T., 1989, J. Pharmac. Exptl. Therap. 250: 343-351.

I P.we.089 1

Effect of diosmin on the noradrenafine sensitive intraceHular calcium store in isolated saphenous veins Dacquet, C. and Finer, M. Dept. of Pharmacology, Innothera, 10 a~. Paul Vaillant Couturier, 94111 Arcueil, France

Hemisynthetic diosmin (Diovenor R) is a flavone derivative, indicated in chronic venous insufficiency. Its veinotonic property has been attributed to an indirect mechanism, which is an inhibition of the noradrenaline metabolism (Heusser and Osswald, 1977; Araujo and Osswald, 1989). The purpose of this study was to investigate the possibility of a direct action of diosmin, especially on the intracellular calcium store involved in the venous contractions produced by noradrenaline. Isometric contractions of endothelialized saphenous vein rings from rabbit were recorded. Tissues were bathed with a modified Krebs' solution at 37 o C and aerated by a gas mixture of 95 ~ 02 and 5 ~; CO2. The effect of diosmin was studied on noradrenaline (10/zM) induced contraction after different periods of store filling (0.5-2-5-10-20 or 25 min) in 2.1. mM calcium Krebs' solution, followed by a period of 1.30 min in calcium-free-EGTA solution. In these conditions, noradrenaline produces a transient contraction for which the amplitude can be taken as an indirect measurement of the amount of calcium present in the noradrenaline sensitive intracellular calcium store (Dacquet et al., 1987). The degree of store filling depends on the external calcium cor~centration and on the duration of calcium loading. In control conditions, the time required to fill half of the intracellular calcium store mobilized by noradrenaline was equal to 5.5 + 1.0 rnin of incubation in the presence of extracellular calcium. The time of total tilling was equal to 23 + 3 min. In presence of diosmin (3 and 10/zM), half tilling time of noradrenaline sensitive calcium store was significantly reduced respectively to 1.0 5:0.6 min (p < 0.01; n > 5) and 1.5 5:0.7 (p < 0.02; n > 5). In the same