INTRODUCTION

Magnesium (Mg²⁺) is one of the most important and prominent ions in the human organism, such as calcium (Ca²⁺), sodium (Na), and potassium, and is the major cation that is necessary for the function of hundreds of enzyme systems. Mg²⁺ plays a role in the pathogenesis of hypertension, and a correlation is determined between serum Mg²⁺ and the incidence of cardiovascular diseases.¹ Oral Mg²⁺ intake was shown to act as a natural Ca²⁺ channel blocker.² Additionally, many drugs, such as diuretics and proton-pomp inhibitors, can cause Mg²⁺ deficiency³ since both Mg²⁺ and these drugs use the same transport mechanisms in the organism, such as absorption, metabolism, and excretion. Therefore, when a person is taking these drugs, there is a risk of interaction with Mg²⁺ status.⁴ Additionally, Mg²⁺ depletion is frequently observed in patients who are hospitalized. Generally, it is secondary to renal or intestinal Mg²⁺ loss and is particularly seen in patients with diabetes mellitus who have neuromuscular symptoms, hypokalemia, and cardiovascular complications.⁵ Mg²⁺ treatment is becoming an important adjunct in some conditions, such as eclampsia, cardiovascular diseases, diabetes mellitus, asthma, and others.⁶ The rapid onset of Mg²⁺’s effects suggests an extracellular action on Ca²⁺ entry.⁷

L-type Ca²⁺ channel blockers are usually used to treat several clinical situations, mainly cardiovascular disease, and specifically, hypertension. L-type Ca²⁺ channel blockers have been used for many years for cardiovascular disease, thus their side effect profile has been well studied. However, their effects on the contractility of the gastrointestinal tract are less known.

Voltage-gated Na channels are dynamic membrane proteins and are responsible for starting the action potential in all excitable membranes.⁸ Tappenbeck et al.⁹ observed that the contractility-enhancing effects of local anesthetic are based on interaction with smooth muscle membranes, modulated by its molecular structure and lipophilicity.¹⁰ The ion channels and ion transport systems are located in the cell membranes of the smooth muscle¹¹ as in all other excitable membranes. Na fluxes play a small role in smooth muscle contraction of the feline colon;¹² however, they contribute to the production of slow waves in human interstitial cells of Cajal (ICC).¹³ The ICC cells present in the human intestine have a mechanosensitive Na channel current and play a role in the control of intestinal motor function.¹³

This study investigated the effects of extracellular Mg²⁺ concentration on the angiotensin II (ANG)-induced contraction on isolated ileal segments. Additionally, in different extracellular Mg²⁺ perfusion mediums, the effects of the Ca²⁺ channel blocker, diltiazem, and Na channel blocker, prilocaine, were investigated on the amplitude of angiotensin-induced contractions on the isolated rat ileum. As the Na and Ca²⁺ channel blockers are widely used, this study aimed to determine their possible effects in different extracellular Mg²⁺ concentrations on gastrointestinal system motility.

MATERIALS AND METHODS

In these experiments, Healthy adult Wistar rats (n=20) weighing 150–250 g were used. This study received the approval of the Local Ethics Committee of Near East University (date: 02/20/2020, reference number: 2020/105). The animals were lightly anesthetized with pentobarbital (35 mg/kg i.p) and were killed by decapitation and exsanguination. The ileum strips were mounted in an isolated organ bath containing standard Tyrode solution (mM: NaCl of 137, KCl of 2.68, MgCl₂ of 1.05, CaCl₂ of 1.8, NaH₂ PO₄ of 0.42, NaHCO₃ of 11.9, and glucose of 5.5) and were bubbled with 95% O₂ and 5% CO₂ mixture at 370 C at pH 7.4. Four types of Tyrode solutions were used in these experiments: (i) standard, (ii) Mg-free, (iii) 2-fold Mg²⁺, and (iv) 4-fold Mg²⁺. In separate experiments, the bath medium was changed to either Mg²⁺ -free (omitting MgCl₂ and adding equimolar NaCl), or 2-fold Mg²⁺ and 4-fold Mg²⁺ (Omitting NaCl adding equimolar 2-fold, 4-fold MgCl₂) solutions. The control group used standard Tyrode as the perfusion medium. The ileum pieces contracted with ANG in doses of 1.2 × 10–8 M, which was accepted as the maximal ANG-induced contractions in the control group. Local anesthetic prilocaine was used in doses of 2.3 × 10–5 M. After the addition of the prilocaine doses, ANG was added after a few minutes into the medium and the results were recorded. The Ca²⁺ channel blocker diltiazem was used in doses of 5.5 × 10–6 M. The ileal segments were washed several times and left for 60 min before each agent was added into the bath solution. The Ca²⁺ channel blocker was added first then followed by the addition of prilocaine and ANG into the medium and the results were recorded. These processes were performed in standard Tyrode solution as the control group, Mg²⁺-free, 2-fold Mg²⁺, and 4-fold Mg²⁺ groups.

RESULTS

The effects of extracellular Mg²⁺ concentrations on the ANG-induced contractions on the isolated ileal strip were examined in these experiments. Additionally, the effects of Ca²⁺ channel blocker diltiazem and Na channel blocker prilocaine on ANG-induced contractions in percentage were examined. The ANG-induced percentage contractions in standard Tyrode, Mg²⁺-free, 2-fold Mg²⁺, and 4-fold Mg²⁺ are shown in Figure 1.

In the standard Tyrode solution (control group), when the Ca²⁺ and Na channel blockers were separately used, the responses to ANG were significantly reduced. Additionally, when the channel blockers were used together, the ANG-induced contraction significantly decreased. As shown in Figure 1, an increased response to ANG in the Mg²⁺-free medium was not statistically significant compared to the control group. Further, in the Mg²⁺-free perfusion medium, when Ca²⁺ and Na channel blockers were used either separately or together, the ANG-induced contractions were higher than the control group although the difference was not statistically significant.

When 2-fold and 4-fold Mg²⁺ medium were used, the contractile responses to ANG were significantly decreased in all high Mg²⁺ perfusion mediums compared to the responses obtained in the standard Tyrode and Mg²⁺-free perfusion mediums. If the 2-fold and 4-fold Mg²⁺ perfusion mediums are compared with each other, the ANG-induced contraction decreased but without any significance.

As shown in Figure 1, diltiazem significantly decreased the ANG-induced contractions in all perfusion mediums used. Additionally, prilocaine decreased the ANG-induced contractions in all perfusion mediums.

Statistical analyses were performed with unpaired t-test. Significance values different from the control were accepted as *p <0.05, **p <0.01 ***p <0.001 vs. values were presented as mean ± SEM (standard error of mean).

DISCUSSION

The present study showed that when Mg²⁺ was omitted from the perfusion medium, the ANG-induced ileal contractions increased, but the responses significantly decreased when Mg was added 2-fold and 4-fold into the perfusion medium. Additionally, when Ca²⁺ and Na channel blockers were added to the perfusion medium, the responses significantly decreased in all used mediums.

Our results showed that in the Mg²⁺-free perfusion medium, the responses to ANG increased. Reducing the concentration of Mg²⁺ (When Mg²⁺ is removed) from the normal Tyrode solution enhanced the spontaneous basal activity, whereas the addition of Mg²⁺ gradually abolished this spontaneous activity. Muscle contraction induced by FS or carbachol was enhanced in Mg²⁺-free Tyrode solution. The addition of Mg²⁺ inhibited the response to both forms of stimulation in a dose-dependent manner.¹⁴

Lin et al.¹⁵ showed that Mg²⁺ isoglycyrrhizinate inhibits L-type Ca²⁺ channels and Ca²⁺ transient. The effect of Mg²⁺ is in a dose-dependent manner in myocyte. Also, it was shown that increased internal free Mg²⁺ concentration partly inhibits the activity of Na/Ca exchange, or might limit its ability to trigger Ca release.¹⁶ Our results showed that as the concentration of Mg²⁺ in the extracellular fluid increased, the ANG-induced contraction percentage decreased. Additionally, the presence of Ca²⁺ and Na channel blockers in the perfusion medium further reduced the responses. Our results showed that the contractile responses significantly decreased with the Ca²⁺ channel blocker. The inhibitory effects of Mg²⁺ were potentiated when the Ca²⁺ concentration in the Tyrode solution was reduced to 0.6 mM, whereas increasing the extracellular concentration of Ca²⁺ (5.4 mM) reduced the inhibitory effects of Mg²⁺. Yu et al.¹⁴ demonstrated that the contractile alterations at different concentrations of extracellular Mg²⁺ correlated with similar changes in intracellular free Ca²⁺.¹⁴ Heron¹⁷ postulated that Mg²⁺ and Ca²⁺ are dependent on each other as the disrupted balance of one has an impact on the other.

Ca²⁺ is the most important signaling molecule in all cells and is involved in numerous essential functions including cell life and death. Intracellular Ca²⁺ should be increased for the contraction of smooth muscle cells like all other muscle cells. Cytosolic Ca²⁺ increases by opening the Ca²⁺ channels either in the surface cell membrane or organelles membrane. The smooth muscle cell needs much more Ca²⁺ from the extracellular medium.

Smooth muscle cells have many Ca²⁺ channels, such as voltage-gated Ca²⁺ channels and RyRs for increased intracellular Ca²⁺ concentration. Depolarization causes L-type Ca²⁺ channels to open enabling Ca²⁺ to enter down its concentration gradient into cells. The stimulatory signal results in increased cytosolic Ca²⁺ levels, which activates muscle contraction.¹⁸ In smooth muscle, Ca²⁺ enters the cell through many ways, particularly two different types of Ca²⁺ channels, then activates contractile filaments and induces contraction. These Ca²⁺ channels are inhibited by Ca²⁺ channel blockers and/or nitro compounds. Ca²⁺ inhibits both of these Ca²⁺ channels and relaxes the muscle. A portion of the smooth muscle contraction is due to the release of Ca²⁺ from the cellular storage site. Caffeine and norepinephrine release Ca²⁺ from this store to induce a transient contraction. The contraction induced by caffeine is greatly augmented in the absence of Mg²⁺. The other effect of Mg²⁺ deficiency is to inhibit the effects of various vasodilators. Vascular endothelium releases a substance that relaxes vascular smooth muscle and this relaxation is also inhibited by Mg²⁺ deficiency. Thus, Mg²⁺ has multiple sites, and the mechanisms of action in smooth muscle are still controversial.¹⁹

In gastrointestinal smooth muscle, like all in smooth muscles, the upstroke action potential is principally mediated by Ca²⁺ influx through voltage-dependent L-type Ca²⁺ channels and is responsible for the initiation of contraction. Ca²⁺ channel blockers or Ca²⁺ antagonists reduce smooth muscle contraction by inhibiting Ca²⁺ ions fluxes.²⁰ In this study, diltiazem was used as a Ca²⁺ channel blocker and results indicated that the ANG-induced contraction was reduced in all the used perfusion mediums. Diltiazem blocked the L-type Ca²⁺ channels.²⁰

Our study results showed that when Na ions were blocked by prilocaine Na channel blocker, the ANG-induced contractions significantly reduced. Several investigations have reported the effects of local anesthetic on Na channels in particular.^21-23 The role of Na fluxes in smooth muscle contraction is controversial; however, it has been shown to play a role in the generation of slow-wave in human ICC cells.¹³

CONCLUSION

Changing the extracellular Mg²⁺ concentration affects the ANG-induced contraction in isolated ileal segments. In the Mg²⁺-free perfusion medium, the ANG-induced contraction increased. The addition of 2-fold Mg²⁺ and 4-fold Mg²⁺ into the perfusion medium significantly decreased the responses. The Ca²⁺ channel blocker diltiazem and Na channel blocker prilocaine decreased the isolated rat ileal contractility. Diltiazem decreased Ca²⁺ entry into the gastrointestinal smooth muscle as with any other smooth muscle. Prilocaine decreased Na and probably Ca²⁺ entry into the cells. This study showed that changing the concentration of extracellular Mg²⁺ ions affects gastrointestinal contractility. However, further studies are needed to better clarify the role of extracellular Mg²⁺ and it should be further confirmed by human investigations.

Main Points

• Mg²⁺ ions play an important role in many enzyme systems in the organism.

• The angiotensin-induced contractions in the Mg²⁺-free perfusion medium increased in the isolated gastrointestinal segments.

• When Mg²⁺ ions increased in the perfusion medium either 2-fold or 4-fold, the responses to ANG significantly decreased

• Mg²⁺ ions play an important role in gastrointestinal contractility.

ETHICS

Ethics Committee Approval: This study received the approval of the Local Ethics Committee of Near East University (date: 02/20/2020, reference number: 2020/105).

Informed Consent: Animal experiment.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: E.K., A.Ö.; Design: E.K.; Supervision: E.K., A.Ö.; Resource: E.K., A.Ö.;  Materials: E.K., A.Ö.; Data Collection: E.K.; Analysis: E.K.; Literature Search: E.K., A.Ö.; Writing: E.K., A.Ö; Critical Reviews: E.K.

DISCLOSURES

Financial Disclosure: The author declared that this study had received no financial support.

Conflict of Interest: The authors declare no conflict of interest.