role of endothelium in the response of the vein wall to magnesium withdrawal

Pflfigers Arch (1992) 420:140-145

Journal of Physiology �9 Springer-Verlag 1992

Role of endothelium in the response of the vein wall to magnesium withdrawal

Csaba Szab6 i, Viktor B6rczi i, Ferenc Schneider 2, Arisztid G.B. Kovdch 1, and Emil Monos i

1Experimental Research Department and 2nd Institute of Physiology, and 2Second Institute of Pathology, Semmelweis University Medical School, UllOi fit 78/a, P.O.B. 448, H-1446 Budapest, Hungary

Received July 26, 1991/Received after revision October 8, 1991/Accepted October 21, 1991

Abstract. Complete absence of magnesium has a two- fold effect on the arterial tone: direct smooth muscle contraction and relaxation via endothelium-derived relaxing factor (EDRF) release. In the present study performed on a systemic vein we investigated (1) which of these effects dominates following reduction of magnesium concentration from 1.2 mM to 0.8 and 0.4 mM and (2) whether the vessel segments asymmetrically respond when the magnesium concentration is reduced on either the intra- or extraluminal side. The effects of reducing magnesium concentration on both the isometric tension of isolated ring preparations and the diameter of isolated, perfused and superfused feline femoral veins were investigated. In noradrenaline-precontracted rings, rapid decreases in the extracellular magnesium concentration from 1.2 mM to 0.8 and 0.4 mM caused relaxation, whereas total omission of magnesium returned the tone to the level of the initial tone induced by noradrenaline. Both in the presence of haemoglobin (5• 10 -6 M), and in vessels without endothelium, lowering the magnesium concentration caused a dose-dependent elevation of the noradrenaline-induced tone. In perfused and superfused noradrenaline-contracted vein segments, each reduction of extraluminal magnesium concentration caused contraction of the vessels, re- gardless of whether the endothelium was intact or not. A decrease in intraluminal magnesium concentration did not alter the diameter of the vessel when the endothelium was intact, but caused contraction when the endothelium was disrupted. The results of the present study demon- strate that both the reduction of magnesium concentration or its complete absence cause an EDRF-mediated relaxation and a directly mediated smooth muscle contraction in the femoral vein of the cat. Within the physiological range of extracellular magnesium concentrations, however, the EDRF-mediated relaxation seems to domi- nate.

Key words: Femoral vein - Smooth muscle tone - Basal release of endothelium-derived relaxing factor

Offprint requests to: C. Szab6

Introduction

A reciprocal relationship between calcium and magnesium ions in the modulation of vascular smooth muscle tone and reactivity has been well established. Reduction of extracellular magnesium concentration enhances calcium influx into the smooth muscle cells leading to increases in tension and in reactivity to vasoconstrictor agents. Since the majority of such measurements have been performed on arterial preparations, much less information is available on the influence of extracellular magnesium on venous smooth muscle tone and reactivity [1, 5, t8, 24].

The endothelium plays an important role in the regulation of vascular tone. Endothelium-derived relaxing factor (EDRF) [6] has been identified as nitric oxide (NO) in arteries as well as in some veins [9, 17]. There are, however, several differences in the endothelium-dependent responsiveness between arteries and veins; veins ex- hibit only moderate endothelium-dependent relaxations. The difference is most likely due to the fact that venous endothelium can produce less EDRF [4, 17, 20].

Extracellular calcium influx is needed for the produc- tion of EDRF [14, 15]. Reduction of the magnesium concentration leads to enhanced calcium influx into endothelial cells and, in turn, to enhanced basal EDRF release which, in turn, causes vessel relaxation. This has been shown for the canine coronary [12], and feline middle cerebral arteries [23]. Gold and co-workers [7] have recently shown that complete removal of magnesium from the extracellular medium enhances EDRF release from bovine intrapulmonary artery and vein and increases the cGMP levels in the vascular smooth muscle.

Thus, two opposite effects of magnesium deficiency on vascular tone have been described, i.e. (1) an enhance- ment of the agonist-induced smooth muscle contractions resulting in the elevation of the vascular tone and (2) fa- cilitation of the basal EDRF release resulting in relaxation. Since most previous studies have investigated the effect of complete removal of magnesium on vascular smooth muscle tone [1, 7, 11, 18, 24], one of the goals of the present study was to determine which of the above

141

o p p o s i t e e f fec t s d o m i n a t e s w h e n m a g n e s i u m c o n c e n t r a -

t i o n is r e d u c e d w i t h i n t h e p h y s i o l o g i c a l a n d p a t h o -

p h y s i o l o g i c a l c o n c e n t r a t i o n r a n g e s ( 1 . 2 - 0 . 8 m M a n d

0 . 8 - 0 . 4 m M , r e spec t i ve ly ) [21]. A f u r t h e r g o a l o f t h e p re -

s e n t s t u d y was to i n v e s t i g a t e t h e r e s p o n s e s o f i s o l a t e d , p e r f u s e d a n d s u p e r f u s e d f e l i ne f e m o r a l v e i n s e g m e n t s t o r e d u c i n g t h e m a g n e s i u m c o n c e n t r a t i o n in e i t h e r t h e s u p e r f u s i o n o r p e r f u s i o n s o l u t i o n a n d t o s t u d y t h e ro le o f

t h e e n d o t h e l i u m in t h i s r e s p o n s e .

Materials and methods

General Cats of both sexes, weighing 1.9-2.8 kg and anaesthetized with pentobarbitone sodium (30 mg/kg i.p.) were used. Segments of the femoral vein, with collaterals tied off near their junctions, were transsected and transferred to a thermostated (37~ chamber filled with Krebs-Henseleit (K-H) solution of the following composition (in raM): NaC1 119.0, KC1 4.6, CaC12 1.5, MgC12 1.2, NaHCO 3 15.0, NaH2PO 4 t.2, glucose 6.0. The K - H solution was aerated with 95% O 2 and 5070 CO 2 and had a pH of 7.4. Special care was taken to avoid overstretching or injuring the vessels. Alterations in magnesium concentration of the solution to 0.8, 0.4 or 0 mM were compensated by appro- priate changes of the NaC1 concentration to maintain isotonicity. Ten vessel segments with intact and seven segments with disrupted endothelium (from five animals) were used for the ring preparations, while eight segments (one per animal) were used in the perfusion-superfusion studies.

Experiments with ring preparations. For measurement of isometric force, a 1- to 3-mm-long femoral vein ring segment was placed on two L-shaped stainless steel wires (0.1 mm diameter), one of which was at- tached to a force transducer (model FT03, Grass, Quincy, Mass, USA) [8]. The position of the other wire could be adjusted precisely by a mi- cromanipulator. The vessel rings were equilibrated in control K - H solution for 40-50 rain at a passive tension of 80-120 mg. Separate experiments have shown that this level of passive tension gives a maximum contractile response to noradrenaline in this vessel preparation. After cumulative dose-response curves for noradrenaline (10-8-10 -s M) and acetylcholine (10 - s - 10 -5 M) had been obtained, the vessel rings were contracted with 5 • 10-7 M noradrenaline. This induced 55.5_+6.8% (n = i0) and 55.7_+7.5070 (n = 7) of the maximum contractions elicited by noradrenaline in endothelium-intact and endothelium- denuded venous ring segments respectively. After the vascular tone stabilized, the K - H solution containing the control magnesium concentration (1.2 mM) was replaced with another containing 0.8 mM magnesium. This latter solution contained the same concentration of noradrenaline and was oxygenated and prewarmed to the same temperature as the control bathing medium. After the new tone stabilized, the 0.8 mM magnesium concentration was reduced in a stepwise fashion to 0.4 mM and 0 raM. Repetitive washouts with the control K - H solution returned the vascular tone to its initial level. Upon reachieving initial tone the ring segments were incubated in the presence of haemoglobin (5 x 10 .6 M) for 15 min. They were then recontracted with 5 x 10 -7 M noradrenaline and the above stepwise reduction of magnesium was repeated. For these measurements, however, the magnesium-deficient solutions contained both noradrenaline and haemoglobin. The dilatory effect of acetylcholine was tested also in the presence of haemoglobin.

Experiments with perfused and superfused vein segments. For each ex- periment, a I I - 16 mm long femoral vein was placed in a tissue bath containing 80 ml of K - H solution. Each end of the vessel was mounted on a stainless steel cannula. The vein was extended to its in situ length (196_+7~ of the relaxed in vitro length, n = 8). Each vessel was perfused and superfused with K - H solution at a rate of 0.74ml/min and 5 ml/min respectively. Outflow pressure was set to 5.0 mmHg by adjust- ing the height of the outflow tube. Inflow pressure varied between 6.0 and 8.2 mmHg. Vascular diameter and axial force were measured by cantilever strain gauge transducers [19]. Axial force was constant during the experiments and averaged 7.1 + 1.4 g (n = 8).

After a 30-min equilibration period, the extraluminal magnesium concentration was lowered in a stepwise manner by changing the magnesium concentration in the superfusate. Following several washouts with normal K - H solution, noradrenaline (5 • 10 -6 M) was added to the superfusate. After stable diameter had been achieved the stepwise reduction of magnesium concentration in the superfusate was repeated in the presence of 5 • 10-6M noradrenaline. The same protocol was repeated intraluminally with the only difference that the perfusate did not contain noradrenaline; this was always applied extraluminally. In half of the experiments intraluminal stepwise reduction of magnesium concentration was performed first. The above magnesium concentration reduction protocol was also repeated after endothelium removal.

Endothelium removal In the experiments with the ring preparation, the endothelium was removed mechanically by gently rubbing the intimal surface of the vein segment with a stainless steel wire. The effectiveness of this technique was verified in each ring by the absence of relaxation in response to acetylcholine [4, 6].

In the experiments with cylindrical mounted, perfused and superfused vessel segments, the endothelium was removed by luminal perfusion with 0.05% Triton X-100 solution (7.4 ml/min for 1 min) [25]. The dis- ruption of the endothelial layer was confirmed morphologically. After the experiments, the veins were cut into pieces fixed in 4% glutar- aldehyde for 24 h and then washed with buffer solution (cacodylate buffer, pH = 7.2). After dehydration, the vein segments were embedded in Durcupan ACM (Fluka, Buchs, Switzerland). The l%tm-thick sections were stained with toluidine blue and examined by light microscopy. In the control preparations the endothelial layer was intact, whereas the veins perfused with Triton X-100 had no endothelial cells. Occasionally moderate smooth muscle cell degeneration of the innermost layer also occurred. This appears not to have had deleterious functional conse- quences for the smooth muscle since there were no significant differences between the noradrenaline-induced contractions before and after the Triton X-100 perfusion (see Results section).

Drugs. The following drugs were used: acetylcholine chloride (Sigma, St. Louis, Mo., USA), noradrenaline (Gedeon Richter Ltd., Budapest, Hungary), Triton X-100 (Sigma).

Haemoglobin was prepared from carefully oxygenated cat arterial blood as previously described [23]. Blood was collected from the femoral artery of an anaesthetized cat into heparin-containing tubes via a polyethylene cannula. After centrifugation at 2000 g and washing several times with K - H solution, the red blood cells were haemolysed with distilled water and diluted in K - H solution. The oxyhaemoglobin concentration of the stock solutions was measured spectrophotometrically. Acetylcholine was dissolved in saline, noradrenaline in distilled water containing ascorbic acid (1 mM), and Triton X-100 in K - H solution.

Calculations and statistics. The concentration of the agonist inducing half-maximum contraction or relaxation (ECs0) and the maximum active tension developed (Emax) were assessed graphically for each dose- response curve. Student's t-test for paired and unpaired samples was used for statistical analysis of the data, and a difference was considered to be significant if P<0.05. Data are shown as means+standard error of the mean (SEM).

Results

E x p e r i m e n t s with f e m o r a l vein ring preparat ions

N o r a d r e n a l i n e , in c o n c e n t r a t i o n s b e t w e e n 10 -8 a n d 1 0 - S M c a u s e d d o s e - d e p e n d e n t c o n t r a c t i o n s , w i t h ECs0 a n d Ema x v a l u e s o f 1 . 5 + 0 . 4 • 1 0 - 7 M a n d 3 2 7 9 + 7 2 4 m g

r e s p e c t i v e l y (n = 10). A c e t y l c h o l i n e c a u s e d a d o s e - d e p e n d e n t r e l a x a t i o n i n

c o n c e n t r a t i o n s b e t w e e n 10 -8 a n d 5 • (ECs0: 9 . 5 + 6 . 3 x 10 -7 M) . T h e vessels d i d n o t d i l a t e f u r t h e r af-

142

a

NA: 5x10-7 M

w

/'-"q zl / q

b

' ~ I 5rain I

Fig. l a , b. Representative recordings of isometric tension of an intact feline femoral vein ring during reduction of extracellular magnes ium concentration. Magnes ium was lowered in a stepwise fashion to 0.8, 0.4 and 0 m M from the control 1.2 m M in the absence (a) and in the presence (b) o f 5)< 1 0 - 6 M haemoglobin. The vessel was initially precontracted with 5 • 10 -7 M noradrenaline

ter exposure to 10-SM acetylcholine (n = 10). Vascular tone was decreased by 56.1 +_ 6.3 ~ of the noradrenaline- contracted level in response to 5 x 10 -6 M acetylcholine.

After precontraction of the endothelium-intact rings with 5 • 10-TM noradrenaline (1715_+379 mg active tension, n = 10), sequential, stepwise lowering of the magnesium concentration in the organ chamber (to 0.8, 0.4 and 0mM) caused marked changes in the vascular tone (Figs. 1 a, 2a). At magnesium concentrations of 0.8 and 0.4 mM the tone stabilized at approximately 70~ of its initial level. However, complete absence of magnesium caused the tone to return to a level not significantly different from the precontracted tone. It is worthwhile not- ing that rapid changes in the extracellular magnesium concentration caused biphasic responses in tone: a transient relaxation followed by a change to a new, stabilized level (Fig. I a). The transient and the stabilized tones were not significantly different from each other at 0.8 mM magnesium, but the stable tone was significantly higher than the transient tone at both 0.4 and 0 mM magnesium (P<0.05 and 0.001 respectively, n = 10).

In the presence of haemoglobin (5 • 10 -6 M), acetylcholine n o longer relaxed the vessels except at the highest concentration (10 -5 M), where it caused significant relaxation (7.3_+3.4070, P < 0.05, n = 10). The acetylcholine dose-response curves obtained in the absence and in the presence of haemoglobin were significantly different (P<0.01) over the entire concentration range. Haemo- globin by itself caused a weak but significant ( P < 0.05) contraction in the femoral vein ring (35_+21mg or 1.5+0.6~ of the maximum noradrenaline-induced contraction).

In the presence of haemoglobin, stepwise reduction of the magnesium concentration caused a stepwise elevation in the vascular tone (Figs. 1 b, 2b). This response differed significantly from that of the control rings (Fig. 2 a, b). The response was still biphasic, as in the control rings

a

b

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0.8 0.4 0 Mg '~" mM

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Fig. 2 a - e . Effect of a stepwise reduction in the magnes ium concentration on the tone o f feline femoral vein rings. Magnes ium was lowered from 1.2 m M to 0.8, 0.4 and 0 m M after precontraction was produced by 5 x 1 0 - 7 M noradrenaline: a endothelium-intact ring, h endothelium-intact ring in the presence of 5 • 10-6 M haemoglobin, c endothelium-denuded ring. The tension response to 5 • 10-7 M noradrenaline at 1.2 mM was taken as 100%. *, **, *** Significant differences from the noradrenaline-contracted tone ( P < 0.05, P < 0.01 and P < 0.001 respectively). ~-+, +++ Significant differences between the response o f the intact vessel and the response of the intact vessel in the presence of haemoglobin (a, b) or the response of the endothel ium-denuded vessel (a, c) to similar reduction in magnes ium concentration (P<0.01 and P < 0.001, respectively)

(Fig. 1 a, b), but the transient relaxations were extremely weak. The contractile response to 5 • 10 -7 M noradrenaline in the presence of haemoglobin (2006_+578 mg) was not significantly different from the response in its absence.

The responses of the endothelium-denuded rings were similar to the responses of the endothelium-intact rings in the presence of haemoglobin. Acetylcholine did not relax these rings (n = 7), and reducing the magnesium concentration caused a dose-dependent contraction (Fig. 2b, c). The responses to reduced magnesium were not biphasic,. since at each point the transient relaxation was absent. The magnitudes of the responses of these rings to stepwise reduction in magnesium concentration differed significantly from that of the control veins (Fig. 2a, c), but did not differ from the responses of rings with intact endothelium in the presence of haemoglobin (Fig. 2b). The maximum contractile response of the endothelium-denuded rings to noradrenaline was somewhat smaller than the response of the endothelium-intact vessels, and the ECs0 value was shifted to the right (gmax: 1766_+346 mg, ECs0: 7.8_+3.3x 10-7 M, n = 7). These differences, however, were not significant compared with the responses of the endothelium-intact vessels (P<0.1 in both cases). Noradrenaline (5x 10 -7 M), used for initiating active tone, caused a concentration of 545_+197mg in rings without endothelium; this was significantly (P<0.05) smaller than the responses of the intact rings. This difference and these trends indicate modest damage to the vascular smooth muscle by rubbing. This is not unique in venous preparations after endothelium removal [4].

Experiments with isolated perfused and superfused femoral vein segments

Stepwise lowering of extraluminal magnesium concentration for the control 1.2 mM to 0.8, 0.4 and 0 mM led to a stepwise reduction of the diameter of vein segments

, 0 o 7

L 95- (11 r / ~

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Fig. 3. Effect of

--I--

F / A I / A . / A F / A F / A

2"A

N 0.8

stepwise

0.4

reduction of the magnes ium concentration

0 Mg++ (raM)

from the control 1.2 m M to 0.8, 0.4 and 0 m M on the diameter of the resting and noradrenaline-contracted feline femoral vein. Noradrenaline (5 • 10 -6 M) was applied extraluminally. Absolute values of the diameters with control magnes ium concentration were 1.3-+0.1 m m and I. 1 + 0.1 m m for resting and noradrenaline-contracted state respectively (n = 8). The difference between these absolute values was significant ( P < 0.001). Each of these diameters was taken as 100%. *** Significant reduction of diameter compared to control (P<0.001) . [3, Non-contracted, Mg 2+ , extraluminal; [] noradrenaline-contracted, Mg 2+ , extraluminal

110

1 0 5

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m 9 5 ' 0

9 0 oJ

,x 85'

80

ENDOTHELIUM INTACT (n=8)

++ ++

w *

1'.2 0'.8 0'.4 010

Mg ++ (mM)

143

105 -

o ~ 100 -

'~ 95- E

" 0 90-

~ 85- W

8O

ENDOTHEt.IUM DISRUPTED (n=6)

1'.2 o'.8 o'.4 olo Mg ++ (mM)

Fig. 4. Mean percentage change in diameter of noradrenaline-contracted, perfused feline femoral veins with intact (a) and disrupted (h) endothelium in response to intra- and extraluminal stepwise reduction of the magnes ium concentration from 1.2 m M to 0.8, 0.4 and 0 mM. The diameters of the 5 x 1 0 - 6 M noradrenaline-precontracted vessels in normal magnes ium concentration were taken as 100%. At control magnesium concentration (1.2 mM) the absolute diameters for the two groups with intact endothelium did not differ significantly from the two groups with disrupted endothel ium (1.1_+0.1; 1.1_+0.1; 1.2+_0.2 and 1.2-+0.2 m m respectively). *, **, *** Significant differences between the diameter at 1.2 m M magnes ium (100%) and the diameters at the lowered magnes ium concentrations ( P < 0.05, 0.01 and 0.00! respectively). + +, + + + Significant differences between mean diameters at respective intra- and extraluminal magnes ium concentrations (P<0.01 and P < 0.001 respectively). Mg 2§ extraluminal ( � 9 or intraluminal (A)

precontracted with 5• 10-6M noradrenaline, but not in the absence of noradrenaline (i.e when the vessel was at its resting diameter) (Fig. 3). Furthermore, the response to intra- or extraluminal magnesium reduction was asymmetrical. Reducing the magnesium concentration in the superfusate in the veins with intact endothelium potentiated the noradrenaline-induced contraction. In contrast, reduction of magnesium in the perfusate did not alter the tone significantly. There was a significant difference between these responses (Fig. 4a).

In the absence of an intact endothelium, reduction of either extraluminal or intraluminal magnesium concentration markedly potentiated the noradrenaline-induced tone. There were no significant differences between these responses. (Fig. 4b).

Noradrenaline (5x 10-6M) used for precontraction caused significant contractions reducing the vessel diame-

144

ter to 88+2% of the resting value in the vessels with intact endothelium for extraluminal, and to 87_+ 3 % for intraluminal changes of magnesium, and to 87+3% and 92+ 1% after endothelium removal for similar interven- tions respectively (P<0.01 in all cases). These contractions did not differ significantly from each other.

Discussion

Previous studies on the effects of magnesium withdrawal on isolated blood vessel preparations have demonstrated both endothelium-dependent relaxation [7, 11, 23] and smooth muscle contraction [1]. There are, however, far fewer experimental data on the vascular actions of graded reduction of magnesium concentration in the physiological (approximately 1.2-0.8 mM) or pathophysiological (down to 0.4 mM) ranges [21], since previous studies on arterial or venous preparations evaluated the effect of complete absence of magnesium only [1, 7, 12]. Results of several recent studies from our laboratory indicate that changes in magnesium concentration in the physiological range also have pronounced effects on arterial preparation [5, 23]. Lowering magnesium concentration to 0.8 mM potentiates agonist-induced contractions [5] and enhances basal release of EDRF [23]. The effect of such small alterations in the magnesium concentration on veins has not been investigated to date. The first part of the present study shows that lowering of the extracellular magnesium concentration within the physiological range causes an endothelium-mediated relaxation of noradrenaline-contracted, intact feline femoral vein rings rather than a directly mediated smooth muscle contraction. The latter effect of reducing magnesium concentration over- comes the EDRF-mediated relaxation only if magnesium is completely absent, under which condition the tone re- turns to the initial level induced by noradrenaline. The endothelium-dependent relaxation is probably mediated by EDRF, since it is abolished by the known EDRF-binding substance haemoglobin (16) at a concentration sufficient to inhibit acetylcholine-induced relaxations. In vessels denuded of endothelium, the stepwise reduction of magnesium caused a stepwise elevation of vascular tone. The effect of magnesium reduction was biphasic in the intact vessels. However, the initial, transient relaxation was al- most completely eliminated in the presence of haemoglobin and was absent in the vessels without endothelium. It is therefore probable that the transient relaxation is most- ly due to the endothelium-dependent effect of magnesium reduction, whereas the subsequent sustained tone reflects the sum of the relaxant and contractile effects of low extracellular magnesium. Gold et al. [7] found a similar, transient, endothelium-dependent relaxation of intrapulmonary artery and vein when magnesium was completely absent, followed by an elevation of vascular tone above the initial level of contraction. This latter elevation in the tone was attributed to a direct facilitatory effect of magnesium reduction on vascular smooth muscle contraction.

Both the vasodilator and the vasoconstrictor effects of magnesium reduction result from an enhanced calcium

influx into the endothelial [7, 12, 23] and smooth muscle cells [1, 5, 18, 24], respectively. A recent report from Moncada's laboratory [15] describes an EDRF-mediated relaxation induced by increased extracellular calcium concentrations in the physiological or pathophysiological range. These authors also conclude that calcium exerts its physiological effects by the regulation of EDRF (NO), rather than by a direct action on vascular smooth muscle tone.

It was not the aim of the present study to investigate the precise mechanisms by which magnesium exerts its endothelium-mediated effects. However, in addition to an EDRF-mediated action, an effect mediated by the endothelium-derived hyperpolarizing factor (EDHF) may also be involved. Magnesium can alter the synthesis and/or release of these factors, change their half-life or alter the smooth muscle sensitivity to them. On the basis of previous and present results, however, it seems most likely that magnesium reduction primarily modulates EDRF activity, since haemoglobin does not inhibit the EDHF [10]. At least two different EDRFs have been reported in veins. Although the EDRF in canine systemic veins shares some chemical properties with NO, it does not mediate the relaxation via a cGMP pathway but acts rather like sodium nitroprusside [17]. In bovine pulmonary veins, however, EDRF is NO. [9]. Since haemoglobin scavenges both EDRFs [9, 17], the present data cannot distinguish between them. Haemoglobin by itself caused a small, but significant, contraction, suggesting a modest effect of EDRF released under basal conditions in our venous ring preparations.

In the second part of the present study we used perfused vein segments. This method maintains physiological conditions better than do the ring experiments. These conditions include (1) normal intraluminal pressure, (2) maintainance of in situ axial length and the cylindrical geometry of the vessel, (3) application of noradrenaline to the abluminal side in the intrasynaptic concentration range [13], and (4) provision for a more physiological iso- tonic (rather than isometric) contraction or relaxation. Shear stress can induce EDRF release under these con- ditons [9]. In these studies, magnesium thus may exert its modulatory action on this enhanced EDRF release. On the other hand, the effect of intraluminally applied endothelium-dependent relaxants may be less than in the ring preparations, probably due to a continuous washout of EDRF by the perfusion [22].

The results presented here show that lowering the magnesium concentration on the perivascular side potentiates the noradrenaline-induced contractions but does not affect the resting tone of the vessel. Similar behaviour of the rat portal vein has also been reported: magnesium- deficient solutions enhance spontaneous oscillations but do not elevate resting tone [1]. Thus the reactions of veins differ from those of arteries in which magnesium reduction not only facilitates the reaction to constrictors but also elevates basal tone [1]. The exact mechanisms re- sponsible for such differences are not yet known.

The present findings support previous findings that wide variations in magnesium concentration are, them- selves, not likely to cause large changes in the venous tone

145

[24]. However, since sympathetic neural control is a ma- jor mechanism for the in situ regulation of venous tone [3] the observed modulation of active force generation by magnesium suggests an important regulatory role for this ion in neurally regulated venous tone in vivo.

The response to magnesium reduction was asymmetrical, similar to the previously reported asymmetrical responses of perfused arterial and venous preparations to acetylcholine, ATP, thrombin or to aggregating platetets [2, 20]. Vascular endothelium can serve as a barrier to the diffusion of water-soluble drugs from the lumen to the smooth muscle [12]. The extent to which such a role for the endothelium is involved in this response was not examined in the present study but on the basis of the results of the ring studies in which EDRF blockade and endothelium removal were similarly effective, it may be con- cluded that an EDRF-mediated action is more likely than a barrier action in the perfusion studies, too.

In conclusion, the present study demonstrates for the first time that decreases in magnesium concentration in both the physiological and pathophysiological ranges cause an endothelium-dependent relaxation rather than a directly mediated contraction of vascular smooth muscle in veins. This is probably because partial reduction of magnesium concentration causes sufficient activation of the biochemical mechanisms involved in the release of EDRF but only a relatively modest direct activation of the smooth muscle contractile apparatus. The current re- suits emphasize the importance of magnesium in the regulation of venous tone and the protective function of an intact endothelium against the vasoconstriction induced by decreases in magnesium concentration in the physiological and pathophysiological ranges.

Acknowledgements. The authors express their gratitude to E. F~iklya for the technical assistance, and to E Molmir for making the drawings. This work was supported by grant OTKA-I/1314/1988, Hungary.

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