Modification of Acute Stress Reactions by the Actual Magnesium Status and Synthetic Calcium Antagonists

Copyright 1999 by H.G. Classen, G. Fischer, L. Dobler, C. Guigas, B. Herold, H. Hirneth, R. Jacob, C.T. Rieg, Germany
(Explore Issue: Volume 9, Number 2)

From the work of Seyle⁹ it is well known that non-adapted organisms with normal levels of resistance can react pathologically on the exposure to strong stressors via an overactivation of the hypothalamus-pituitary-adrenal system. Conditioning agents or measures -- which are usually not pathogenic when applied alone -- can sensitize diverse organs rather specifically against acute stress reactions; e.g. fasting enhances the development of gastric stress ulcers and electrolyte disturbances favor the occurrence of cardiac necroses^8,9. Although the pathogenesis of both diseases is still unknown in detail the assumption is justified that disturbed Ca:Mg relations play a central role by regulating vascular tone¹ and cellular energy metabolism². With this in mind two series of experiments were performed to examine whether the development of gastric ulcers and the occurrence of electrolyte shifts in cardiac tissue can be influenced (a) by the actual Mg status or (b) by synthetic Ca antagonists.

Materials and Methods

A total of 195 female Sprague-Dawley rats (Süddeutsche Versuchstierfarm, Tuttlingen/FRG) kept under controlled conditions was used in the experiments; their body weight was 90-125 g at the beginning of the stress period. Weighing about 50 g they received a Mg-deficient diet containing 30 to 60 ppm Mg determined by analysis (Altromin, C 1035, Lage/FRG) and demineralized water ad libitum enriched with 2 mmol/1 Mg as MgCl₂ to avoid convulsions or mortality (= Mg-deficient rats) or with 30 mmol/1 Mg as MgCl₂ (= controls) during 21 days.

After fasting for 24 h with drinking-water ad libitum the animals were immobilized in the prone position during 18 h. Drugs, or corresponding amounts of the solvent, were injected s.c. at the beginning and at 6 and 12 h of the immobilization period. At the end of the stress period, all animals were sacrificed. Heart ventricles were quickly excised, dried, weighed and prepared for electrolyte determinations using the atomic absorption spectrophotometry (AAS) technique. The stomachs were excised and opened along the greater curvature. Macroscopical ulcerations visible within the glandular portion were scored using an arbitrary scale of 0-4; however, the results were greatly subjected to subjective errors and are therefore not reported here in detail. Then the tissues were prepared for histological evaluation at circa 100-fold magnification. After standard fixation, 10 sections of circa 6 µm thickness were taken from each stomach at regular intervals and stained using the trichrome technique: the diameters and depths of visible ulcerations were measured under blind conditions and summed up, thus yielding two parameters per one stomach.

In the first series of experiments hypermagnesemia was induced by s.c. injections of 8, 4 and 4 mmol/kg body weight Mg as Mg aspartate HCl, and Mg deficiency was produced as outlined above. Gastric lesions are related to the respective control groups, total diameters of the ulcerations amounting to 2.296 ± 465 and 3.692 ± 621 µm and total depths to 1,343 ± 273 and 1,108 ±203 µm, respectively (mean ± SE; n 20 or 15) [for details see Ref. 5,6].

The second series includes part A and one repetition (= part B); animals or samples of each part were carefully randomized. Myocardial Ca and Mg concentrations are presented as absolute values and are also related to the respective controls, consisting of Mg-deficient animals.

Drugs: Mg aspartate HCl was kindly donated by Verla-Pharm, Tutzing; nifedipine plus solvent by Bayer AG, Leverkusen, and verapamil HCl by Knoll AG, Ludwigshafen.

 

Results

The rats kept on the Mg-deficient diet developed typical erythema of their ears, noses and paws during days 6-17; however, neither convulsions nor mortality occurred. In control experiments any gastric lesions were detectable macroscopically or microscopically after 21 days on the diet followed by a 24-hour fasting period. Therefore, it can be excluded that marginal Mg deficit plus starvation per se induce gastric stress ulcerations.

First Series of Experiments

The results obtained on quantitative evaluation of gastric lesions produced by starvation plus immobilization in Mg-deficient rats or in hypermagnesemic animals are summarized in Table I. Obviously, the ulcerogenic effect of stress exposure is significantly aggravated by Mg deficiency on the one hand and impeded by hypermagnesemia on the other. Therefore, it was decided to investigate the effects of synthetic Ca antagonists in animals sensitized by Mg deficiency.

 

Table I. Influence of decreased and increased Mg supply on stress-induced gastric lesions: quantitative evaluation in percent of controls. Statistical evaluation by Hotelling's T2 test
GROUP	N	GASTRIC LESIONS, DIAMETER	% OF CONTROLS DEPTH	SIGNIFICANCE IN COMPARISON TO CONTROLS, P
MG-DEFICIENT RATS	21	+116	+100	<0.01
HYPERMAGNESEMIC RATS	14	­61	­53	<0.001

Second Series of Experiments

The different concentrations of nifedipine and verapamil HCl applied s.c. are summarized in Table II. Although both synthetic Ca antagonists seemed to have a weak inhibiting effect on the development of stress ulcerations after macroscopic evaluation (not done under blind conditions!) and also after taking into account the quantitative data, the latter did not reach the level of significance even when comparing the products of diameters and depths (since there was no normal distribution, the Mann-Whitney U-test or Kruskal-Wallis analysis of variance were applied).

Table II. The inuence of immobilization and synthetic Ca antagonists on cardiac Ca and Mg concentrations (µg/g dry weight) in rats kept on a Mg-decient diet during 21 days and fasted for 24 h
GROUP	N	CA, µG/G	% OF CONTROLS	MG, µG/G	% OF CONTROLS
PART A CONTROL I	16	180±20		821±140
STRESS I	16	275±100	+53	843±100	+3
STRESS I + 3 X 5 MG N	16	237±70	+32	904±100	+10
STRESS I + 3 X 5 MG V	16	216±40	+20	951±80	+16
PART B CONTROL II	15	162±20		778±70
STRESS II	15	208±60	+28	829±100	+7
STRESS II + 3 X 10 MG V	15	206±60	+27	865±70	+11
STRESS II + 3 X 20 MG V	16	187±30	+15	879±70	+13
N = NIFEDIPINE; V = VERAPAMIL HCL

Myocardial Ca and Mg concentrations are summarized in Table II. In comparison with myocardial Ca and Mg concentration s measured earlier¹¹ in normal rats [153 (124-177) or 941 (840-1,036) µg/g dry weight] both controls (part A and B) kept on Mg-deficient diets showed increased Ca and decreased Mg levels (Table II). In both experiments significant Ca overload resulted after exposure to stress; however, this effect was less pronounced in part B than in part A (53% compared to 28%). This might be due to the fact that (on macroscopic examination) the kidneys of the rats used in part A exhibited only occasionally nephrocalcinotic alterations whereas this disturbance was observed in 89% of the animals used in part B. It might be that in the latter a resulting metabolic acidosis was present protecting against massive myocardial Ca overload^2,3. Stress-induced Ca overload was significantly reduced by verapamil HCl in part A (p>0.05); the same tendency can be observed in part B, however, without reaching level of significance (explanation, see above). In both parts of the experiment, a surprising effect of verapamil HCl on myocardial Mg was observed: in part A, Mg concentrations increased by 16% (p<0.005), and in part B analysis of regression (considering 0, 10 and 20 mg/kg body weight verapamil HCl) revealed significance. Thus, evidence is given that verapamil HCl facilitates myocardial Mg uptake in Mg-deficient animals exposed to acute stress.

Discussion

From the data presented it becomes obvious that decreased Mg supply favors the development of gastric stress ulcerations whereas a marked protective effect results after increased supply. Although the pathogenesis of stress ulcers is not yet fully understood, circulatory disturbances are assumed to be of importance. Since Altura and Altura ¹ have shown that vascular smooth muscles generally contract at low and relax at high extracellular Mg concentrations, it may be speculated that this is also true for gastric and intestinal blood vessels. With this in mind it cannot be excluded that the beneficial effect of Mg-containing antacids is partly due to systemic effects in addition to neutralizing excess acid. The synthetic Ca antagonists used had only weak, if any, effects on these alterations.

However, besides reducing stress-induced myocardial Ca overload, nifedipine and the more intensively studied verapamil HCl facilitated Mg uptake into the heart muscle. These findings support the view of Lehr⁴ who -- studying rats after parathyroidectomy -- has discussed a Mg-sparing effect of verapamil HCl "which might be independent of its Ca antagonistic activity or which might even contribute to this very property". Assuming that catecholamines (released under exposure to stressors) open the slow Ca channels and block the Mg-carrying transport system postulated by Späh and Fleckenstein¹⁰, one could speculate that synthetic Ca antagonists not only block the activated Ca channel but also reopen the Mg transport system to a certain degree.

Keeping in mind that Prasad et al.⁷ -- studying the tone and contractility of isolated blood vessels -- have already described synergistic and even potentiating effects of combined application of verapamil HCl and Mg, our data further support the idea to study the effects of a combined therapy in other disease models.

Resources

1. Altura, B.M.; Altura, B.T.: General anesthetics and magnesium ions as calcium antagonists on vascular smooth muscle: in Weiss, New Perspectives on Calcium Antagonists, pp. 131-145 (Am. Physiological Society, Washington 1981).
2. Fleckenstein, A.; Döring, H.J.; Janke, J; Byon, Y.K.; Basic actions of ions and drugs on myocardial high-energy phosphate metabolism and contractility; in Schmier, Eichler, Handbook of Experimental Pharmacology, Vol. 16, pp. 345-405 (Springer, Berlin 1975).
3. Leder, O.; Paschen, K.; Günther, T.: Die Nierenschädigung bei Magnesiummangel, Magnesium Bull. 3:50-53 (1981).
4. Lehr, D.: Magnesium and cardiac necrosis. Magnesium Bull. 3:178-191 (1981).
5. Mangler, B.; Fischer, G,; Classen, H.G.; The influence of magnesium deficiency on the development of gastric ulcers in rats. Magnesium Bull. 4:9-12 (1982).
6. Mesmer, M.; Fischer, G.; Classen, H.G.: Stress-Abschirmung durch Magnesium. Arzneimittel-Forsch. 31:389-391 (1981).
7. Prasad, D.M.V.; Weiner, R.; Altura, B.M.: Interactions of magnesium and verapamil on tone and contractility of vascular smooth muscle. Eur. J. Pharmacol. 74:263-272 (1981).
8. Selye, H.: The Pluricausal Cardiopathies (Thomas, Springfield 1961).
9. Selye, H.: Stress in Health and Disease (Butterworths, London 1976).
10. 1Späh, F.; Fleckenstein, A,: Evidence of a new, preferentially Mg-carrying, transport system besides the fast Na and the slow Ca channels in the excited myocardial sarcolemma membrane. J. Mol. Cell. Cardiol. 11:1109-1127 (1979).
11. Vormann, J.; Fischer, G.; Classen, H.G.; Thömi, H.: Influence of decreased and increased magnesium supply on the cardiotoxic effects of epinephrine in rats. Arzneimittel-Forsch. 33:205-210 (1983)

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