Role of the endothelial reverse mode sodium-calcium exchanger in the dilation of the rat middle cerebral artery during hypoosmotic hyponatremia

doi:10.21203/rs.3.rs-2001332/v1

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Role of the endothelial reverse mode sodium-calcium exchanger in the dilation of the rat middle cerebral artery during hypoosmotic hyponatremia

https://doi.org/10.21203/rs.3.rs-2001332/v1

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The decrease in serum sodium ions concentration below 135 mmol L^− 1 is usually accompanied by a decrease in plasma osmolality (hypoosmotic hyponatremia) and leads to the disorder of intracranial homeostasis mainly due to the cellular swelling. Recently, using in vitro model of hypoosmotic hyponatremia, we have found that a decrease in sodium ions concentration in the perfusate to 121 mmol L^− 1 relaxes the isolated rat middle cerebral artery (MCA). The aim of present study was to explore the mechanism responsible for this relaxation. Isolated, pressurized and perfused MCAs placed in a vessel chamber were subjected to a decrease of sodium ions concentration to 121 mmol L^− 1. The changes in the diameter of the vessels were monitored with a video camera. The removal of the endothelium, inhibition of nitric oxide-dependent signaling or reverse mode sodium-calcium exchanger (NCX) were used to study the mechanism of the dilation of the vessel during hyponatremia. The dilation of the MCA (19 ± 5%, p < 0.005) in low sodium buffer was absent after removal of the endothelium or administration of the inhibitor of the reverse mode of sodium-calcium exchange, and was reversed to constriction after the inhibition of nitric oxide (NO)/cGMP signaling. The dilation of the middle cerebral artery of the rat in 121 mM Na⁺ buffer depends on the endothelium, NO signaling and reverse mode of sodium-calcium exchange. Hyponatremia may constrict large cerebral arteries with impaired NO-dependent signaling and add to vascular spasm such as the one observed in the late phase after subarachnoid hemorrhage.

endothelium

hyponatremia

isolated middle cerebral artery

KB-R7943

nitric oxide

Sodium is the main osmotically active ion of the extracellular fluid. The physiological concentration of Na⁺ in the intracellular space is close to 10 mmol L^− 1 whereas the extracellular concentration is maintained at 144 mmol L^− 1. The difference in Na⁺ concentration across the cell membrane is essential for the maintenance of cell volume, resting membrane potential and sodium-dependent transport systems [1]. Reduction in the extracellular concentration of Na⁺ generates an osmotic gradient across the cell membrane and leads to the inward movement of water and cellular swelling. The cells defend themselves against osmotic swelling by the extrusion ions (K⁺ and Cl⁻) and organic osmolytes (myo-inosytol, phosphocreatine, amino acids) to decrease intracellular osmolarity and volume [2–6]. This response, known as regulatory volume decrease (RVD), is observed in most mammalian cells studied in vitro including vascular endothelial cells [5].

It has been demonstrated by many research groups that, in a variety of cells types, RVD is preceded by an increase in the intracellular concentration of Ca²⁺ [7–10]. The source of these calcium ions is uncertain. The influx of extracellular calcium, the release of calcium from intracellular stores or both have been proposed [8–10].

One of the possible mechanisms introducing Ca²⁺ into the cell under low extracellular sodium conditions could be an Na⁺/Ca²⁺ exchanger (NCX) which is present in the plasma membrane of virtually all cells [11, 12]. The activity and the direction (forward or reverse) of the NCX is controlled mainly by a transplasmalemmal sodium gradient and membrane potential [12, 13]. At a physiological gradient of Na⁺ concentration across the cell membrane, NCX is considered to operate in the forward mode i.e. transports one Ca²⁺ out of the cell in exchange for three Na⁺ entering the cell, thus participating in the intracellular homeostasis of calcium ions (e.g. cardiac muscle, neurons). A reduction in the extracellular concentration of sodium ions decreases the strength of the electrochemical gradient of these ions and may reverse the direction of NCX [14–16]. However, the reverse mode (Ca²⁺ entry) of the NCX has been reported to operate in a variety of cells also under physiological conditions [11, 12]. There is experimental evidence that in blood vessels, Ca²⁺ entry mode of NCX contributes to pressure-induced myogenic constriction [17, 18], as well as to endothelium-dependent vasodilation [19–21].

Recently, we have found that lowering the extracellular concentration of Na⁺ to the level diagnosed in symptomatic patients with hyponatremia (121 mmol L^− 1), elicits dilation of the rat middle cerebral artery [22]. In the present study, we sought to identify the possible mechanism responsible for this dilation. Our data demonstrates that the MCA dilates in low sodium environment in an endothelium- and nitric oxide (NO)/cGMP-dependent fashion. The increase in the concentration of Ca²⁺ required to activate endothelial NO synthase (eNOS) seems to result from the activation of the reverse mode of NCX in the endothelium. In addition, when NO/cGMP pathway is inhibited, the constriction of the MCA in response to the lowering of sodium ions concentration is noted. The last observation suggests that the MCA with dysfunctional endothelium will constrict in response to hypoosmotic hyponatremia of similar magnitude.

The procedures described were approved by the IV Local Ethics Committee for the Care and Use of Laboratory Animals for Experimental Procedures, National Medicines Institute in Warsaw.

Male, adult Wistar rats (270-310g, n = 38) were provided by the Animal House of Mossakowski Medical Research Institute, PAS. The animals were anesthetized with 5% halothane in N₂O/O₂ (70%/30%) and decapitated. The brain was removed and immersed in cold (4°C) 3-(N-morpholino)propanesulfonic acid (MOPS)– buffered saline solution with 1% dialyzed bovine serum albumin (BSA) at pH 7.40.

Isolation and mounting of the MCA

Middle cerebral arteries (n = 76) were isolated and mounted in the organ chamber according to the procedure as described [22].Briefly, MCA was dissected and cleaned of connective tissue. A 3 mm long segment was transferred to the organ chamber filled with MOPS-buffered saline solution containing 1% dialyzed BSA, mounted between two glass micropipettes, and fixed. An inlet pipette was connected to a pressure-servo system. The MCA was perfused at a rate 100 µl/min with MOPS–buffered saline solution containing 1% dialyzed BSA at an intraluminal pressure of 80 mm Hg. The extraluminal bath was replaced with a MOPS buffered saline solution without BSA, slowly heated to 37°C and exchanged at a rate of 20 ml/min with the help of a peristaltic pump (Masterflex, Cole Parmer, USA). The chamber was placed on the stage of an inverted light microscope (Olympus CKX41) equipped with a video camera and a monitor. The internal diameter of the vessel was measured directly from the screen and recorded for off-line analysis. During the equilibration period (1 hour) the vessel slowly developed a myogenic tone (constriction by approximately 35% of the initial diameter). After this time, the response to increased potassium ion concentration (20 mmol L^-1) in the extraluminal bath was tested. Vessels which did not develop a myogenic tone or responded with less than a 25% increase in the diameter to 20 mmol L^-1KCl were discarded.

Experimental protocol

After the KCl reactivity test and 15 minutes equilibration period in the MOPS-buffered saline, the vessel was studied according to one of the following paradigms (Table 1). Series 0 was a time control study in which pressurized and KCl reactivity tested MCA was maintained in a MOPS buffered physiological saline solution (Na⁺ = 145 mmol L^-1) and continuously monitored for 180 minutes to find out whether the diameter is stable over time which is needed to perform the longest of the experiments listed below. In series I, the intra- and extraluminal concentration of Na⁺was decreasedto 121 mmol L^-1 for 180 minutes. Although 60^th minute was chosen as a reference time for the measurement of MCA diameter, the vessel was left in low sodium buffer up to 180 minutes to test the stability of MCA diameter under this condition. This low sodium buffer had an osmolality of 268 ± 5 mOsm kg^-1. In series II, the intra- and extraluminal concentration of Na⁺was lowered similarly as in series I but the osmolality of the buffer was maintained at 300 ± 3 mOsm kg^-1with N-methyl-D-glucamine (NMDG). In the remaining series of experiments, the MOPS buffered saline solution contained 121 mmol L^-1Na⁺ and was hypoosmotic (268 ± 5 mOsm kg^-1). In series III, the participation of endothelial cells in the response of MCA to lowering of Na⁺ concentration to 121 mmol L^-1 was investigated. Endothelial cells were removed before decreasing Na⁺ concentration by blowing 8 ml of air through the lumen of the vessel for 15 minutes. The effectiveness of endothelium removal was confirmed by the absence of dilation in response to the intraluminal administration of acetylcholine (ACh, 100 µmol L^-1). Next, the response to the lowering of Na⁺ concentration was studied similarly as in series I. In series IV and V, the participation of nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the response of the MCA to 121 mmol L^-1Na⁺ was tested. To inhibit NO production, 30 minutes before the lowering of Na⁺ concentration, a nonselective NO synthase (NOS) inhibitor - N^G-nitro-L-arginine methyl ester (L-NAME, 10 µmol L^-1) was added to the extravascular perfusate. The synthesis of cGMP was inhibited by the administration of the compound 1H-[1,2,4]Oxadiozolo[4,3-a]quinoxalin-1-one (ODQ, 5 µmol L^-1) to the extravascular perfusate 30 minutes before lowering of Na⁺ concentration. Series VI was designed to study participation of the reverse mode of Na⁺/Ca²⁺ exchanger in the response of the MCA to low sodium environment. In this series, (2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl isothiourea mesylate (KB-R7943, 10 µmol L^-1) was administered to the normal sodium MOPS-buffered saline solution prior to the lowering of sodium ions concentration and maintained throughout the observation time. This dose of KB-R7943 has been used to inhibit Ca²⁺-entry mode of Na⁺/Ca²⁺ exchanger in the endothelial cells.^32,33

In series VII, nimodipine (100nmol L^-1) – the antagonist of L-type Ca²⁺ membrane channels of vascular smooth muscle, was administered to the buffer prior to the lowering of sodium concentration. This experiment was designed to test whether relaxation of the wall of the MCA prior to the application of low sodium buffer affects vasodilatory capacity of the vessel. In the experiment with nimodipine care was taken to avoid light inactivation of the drug.

Table 1 List of the experimental series

series	paradigm	MCA diameter
0	145 mmol L^-1 Na⁺ for 180 minutes	no change
I	121 mmol L^-1 Na⁺ for 180 minutes	stable dilation
II	121 mmol L^-1 Na⁺+ NMDG	dilation
III	- Endo + 121 mmol L^-1 Na⁺	no change
IV	L-Name + 121 mmol L^-1 Na⁺	constriction
V	ODQ + 121 mmol L^-1 Na⁺	constriction
VI	121 mmol L^-1 Na⁺ + KB-R7943	no change
VII	121 mmol L^-1 Na⁺ + Nimodipine	dilation

Drugs and solutions

The normonatremic MOPS buffered saline solution contained (in mmol L^-1): 144.0 NaCl, 3.0 KCl, 2.5 CaCl₂, 1.5 MgSO₄, 1.21 NaH₂PO₄, 0.02 EDTA, 2.0 pyruvate, 5.0 glucose, 2.0 MOPS. The concentration of solutes in the hyponatremic MOPS buffer was the same as in the normonatremic one except for NaCl which was lowered to 120 mmol L^-1to obtain Na⁺ concentration in the buffer equal to 121 mmol L^-1. The hyponatremic MOPS buffer was hypoosmotic in all but one series (series II) of the experiments in which the osmotic pressure was maintained at 300 ± 3 mOsm kg^-1 as mentioned above. Acetylcholine chloride, NMDG, L-NAME and nimodipine were purchased from Sigma-Aldrich. ODQ and KB-R7943 mesylate were obtained from Tocris Cookson Ltd. The drugs were dissolved in a MOPS buffered saline solution except for ODQ and KB-R7943 mesylate which were prepared in DMSO and added to the buffer. The final concentration of DMSO in the MOPS buffered saline did not exceed 0,005%. According to the results of a pilot study, this concentration of DMSO does not affect the diameter of MCA.

Data analysis

All data is presented as mean ± standard error (S.E.), n denotes the total number of rats used or the number of vessels per group. The differences between the groups were assessed using one way analysis of variance (ANOVA) followed by post hoc Tukey’s comparison (Statistica 10). Single comparisons were made using the Student’s t-test for paired or unpaired data when appropriate. The differences were considered statistically significant at p<0.05.

Stability of the MCA diameter in normonatremic buffer

The results of the time control series 0 demonstrate that during the observation time of 180 minutes the diameter of pressurized and perfused MCA maintained at 37ºC and pH=7.4 (Table 2: normonatremia, time 0 minutes) remains stable (Table 2: normonatremia, time 60, 120, 180 minutes).

Table 2 The stability of the MCA diameter in series 0 and I

Time (min)	MCA diameter (µm)
Time (min)	145 mmol L^-1 Na⁺	121 mmol L^-1 Na⁺
0	139 ± 8	165 ± 3
60	145 ± 13	162 ± 7
120	139 ± 4	167 ± 7
180	145 ± 5	168 ± 10

Response of the MCA to 121 mmol L^-1 Na⁺. Lack of the effect of hypotonicity

Replacement of the normonatremic MOPS buffered saline solution with the one containing 121 mmol L^-1Na⁺, led to the dilation of the MCA beginning at 15 minutes after induction of hyponatremia. At 60 minutes, the diameter of the MCA increased by 19 ± 5% (from 143 ± 8 µm to 169 ± 7 µm, p<0.005) of normonatremic control (Fig. 1a, Table 2: hyponatremia, time 0 vs time 60 minutes) and remained at this level until the end of the observation time (Table 2: hyponatremia, time 60, 120, 180 minutes). The dilation was solely dependent on the decrease of Na⁺ concentration in the buffer and not on the hypotonicity as the normalization of osmolality with N-methyl-D-glucamine (NMDG) did not affect the response (Fig. 1b).

Effects of the removal of endothelium and inhibition of NO/cGMP signaling pathway on the response of the MCA to 121 mmol L^-1 Na⁺

Successful removal of the endothelium was confirmed with 100 µmol L^-1 acetylcholine which did not affect the diameter of the MCA when applied intravascularly, in contrast to a 34 ± 13% dilation (p<0.05) observed in endothelium-intact vessels. The diameter of the MCA without endothelium did not change when a 121 mmol L^-1 Na⁺ MOPS-buffered saline solution was applied intra- and extraluminally (Fig. 2) suggesting that the endothelium-dependent vasodilator was responsible for the dilation of MCA observed in the previous series. Accordingly, in the next series of experiments, the MCA was pretreated with the extraluminal addition of a nonselective NOS inhibitor – L-NAME (10µmol L^-1). This dose of L-NAME induced a 25 ± 6% constriction (from 152 ± 11 µm to 114 ± 13 µm, p<0.05) during normonatremia. During subsequent lowering of Na⁺concentration to 121 mmol L^-1 the MCA constricted by an additional 10 ± 2% (p<0.05). When ODQ (5 µmol L^-1), an inhibitor of the synthesis of cGMP, was administered during normonatremia the MCA constricted by 35 ± 7% (from 145 ± 10 µm to 94 ± 7 µm, p<0.02). The low sodium buffer administered on the background of ODQ induced further constriction of the vessel by 12 ± 5% (p<0.05).

Role of the reverse mode Na⁺/Ca²⁺ exchanger in the response of the MCA to 121 mmol L^-1 Na⁺.

The addition of the inhibitor of reverse mode of NCX (KB-R7943, 10 µmol L^-1) to the normal sodium buffer prior to the lowering of Na⁺ concentration resulted in the stable dilation of the MCA by on average 19% (p<0.02) which was considered to be a new baseline. In 121 mmol L^-1 Na⁺ MOPS buffer containing KB-R7943 the diameter of the MCA did not change statistically significant from the baseline. In order to test whether elimination of the MCA response to low sodium buffer after administration of KB-R7943 is not an artefact due to a decreased vasodilatory capacity of the vessel, nimodipine test was performed. The administration of nimodipine (100 nmol L^-1) to the normonatremic buffer resulted in the dilation of MCA by on average 26 % (p<0.05) which was comparable to the response of this vessel to KB-R7943. In 121 mmol L^-1 Na⁺ MOPS buffer containing 100 nmol L^-1nimodipine the MCA dilated by 15 ± 8% (p<0.05) which was not statistically different from the response observed in the control series I (Fig. 3).

We have demonstrated that the dilatation of the isolated, pressurized and perfused rat middle cerebral artery in response to the lowering of Na⁺ concentration in the perfusate i) depends on the lowering of extracellular sodium ion concentration and not on the decrease of osmolality, and ii) requires the presence of an intact endothelium and nitric oxide/cGMP signaling. Our results suggest that the concentration of intracellular calcium ions required for the stimulation of the synthesis of nitric oxide by endothelial cells increases in low sodium environment due to the activation of the reverse mode of the sodium/calcium exchanger. To the best of our knowledge this is the first study which demonstrates that normal cerebral blood vessels relax in response to the lowering of extracellular sodium ions concentration through an endothelium- and NO/cGMP-dependent mechanism.

It is well established that extravascular osmolarity affects the tone of blood vessels. In hypoosmotic conditions they constrict whereas in a hyperosmotic environment they relax [23]. Local increase in tissue osmolarity is considered to be one of the factors mediating functional hyperemia. It should be stressed, however, that our study addresses the problem of systemic decrease of sodium ion concentration. Also, in present study the effect of low sodium on the diameter of MCA did not depend on hypoosmolarity as MCA dilated in a normoosmotic low sodium buffer to the same extent as in the hypoosmotic one. The relaxation of the MCA in low sodium environment observed in our study is endothelium- and NO/cGMP-dependent, since removal of the endothelium and inhibition of NO/cGMP signaling abolish this response. In support of our data, it has been demonstrated that endothelial cells behave as vascular salt sensors and release increased amounts of NO when extracellular sodium ion concentration decreases [24]. It is important to stress that when the NO/cGMP system was inhibited, the constriction of the MCA in low sodium buffer was observed in our study instead of the dilation.

NO is the most prominent endothelium-dependent vasodilator, produced with the participation of a specific endothelial synthase eNOS and released from the endothelium to activate soluble guanylate cyclase in the vascular smooth muscle [25]. The essential role of endothelial NO in the physiological regulation of the cerebral vasculature is well recognized [26, 27]. It is also well established that activation of constitutive eNOS depends on the increase in the intracellular Ca²⁺ concentration. The increase in the intracellular concentration of Ca²⁺ in low sodium/hypoosmotic buffer may be related to cell swelling (RVD does not completely restore the volume) and opening of stretch-activated Ca²⁺ membrane channels [7, 9]. Our present data demonstrate that endothelium-dependent dilation of the MCA in low sodium buffer is eliminated after inhibition of the reverse mode of NCX which strongly suggests that reverse mode of endothelial NCX operates during our experimental conditions. The reversal of the direction of NCX during the reduction in the extracellular concentration of sodium ions has been described in the literature [14, 16]. The participation of the reverse mode of NCX in the physiological regulation of blood vessels is just emerging [16–20, 28, 29]. The reverse mode of NCX has been reported to contribute to the adjustment of the diameter of the posterior cerebral artery to perfusion pressure changes [16] and to the myogenic constriction of rat cremaster muscle first order arterioles [17]. According to Zhang [30] who reviewed the literature on the contribution of arterial NCX to the physiological regulation of the myogenic tone, in pressurized arteries with myogenic tone and arteries in vivo, NCX is likely to operate in the “Ca²⁺ influx” mode. The present study shows for the first time that the “Ca²⁺ influx” mode NCX participates in the maintenance of the myogenic tone of isolated middle cerebral artery, since administration of NCX inhibitor - KB-R7943 resulted in the dilation of this vessel under basal conditions. This inhibitor of NCX, at a dose of 10 µmol L^− 1 used in our study, selectively inhibits the Ca²⁺ entry mode of the exchanger [31–34].

It has been also suggested that reverse mode NCX is involved in the activation of endothelial NOS and release of NO by the rat aortic endothelial cells [19], porcine aortic endothelial cells [20] and cardiac microvascular endothelial cells [14]. According to the results obtained by Bondarenko [18], NCX operates in reverse mode in intact endothelial cells following stimulation by acetylcholine. Our present results strongly suggest that, the activation of the reverse mode of the Na⁺/Ca²⁺ exchanger caused by a decrease of the Na⁺ concentration gradient across the cell membrane, also participates in the transport of Ca²⁺ into the endothelium. Although there are data in the literature questioning KB-R7943 selectivity, they do not apply to the endothelial aspect of our study. It has been demonstrated that, in addition to the inhibition of the reverse mode of NCX, KB-R7943 may block the voltage-dependent, L-type Ca²⁺ channel of the cell membrane [35] However, the dilation of the MCA in low sodium buffer in our experiments is entirely endothelium-dependent and L-type voltage-dependent Ca²⁺ channel doesn’t seem to be present in the endothelium of the rat MCA [36]. Another study questioning selectivity of KB-R7943 demonstrated that, in cultured human umbilical vein endothelial cells and in freshly isolated smooth muscle cells of the mouse aorta, KB-R7943 not only inhibited the reverse mode of NCX but also activated large conductance Ca²⁺-activated K⁺ channel (BK_Ca) [37]. This argument also does not apply to our research because these channels are irrelevant in the endothelium of cerebral blood vessels [38].

Our results suggest that reverse mode of NCX can be stimulated in endothelial cells of intact, isolated large cerebral arteries by a 24 mmol L^− 1 decrease in plasma sodium concentration. This, in turn, suggests that endothelial NCX is much more sensitive to the decrease of Na⁺ gradient across the plasma membrane than the one operating in smooth muscle cells. The elucidation of this issue requires further study.

In summary, these results demonstrate for the first time that during hypoosmotic hyponatremia normal cerebral blood vessels dilate through an endothelium- and NO/cGMP-dependent mechanism. They also suggest that stimulation of the NO/cGMP signaling pathway in the middle cerebral artery of the rat during short-lasting lowering of the extracellular sodium concentration is associated with the activation of “Ca²⁺ entry” mode of Na⁺/Ca²⁺ exchanger in the endothelium.

Last but not least, these results demonstrate that when NO-dependent regulation of the middle cerebral artery is blunted/absent, the vessel constricts in response to the lowering of Na⁺ concentration in the perfusate. The last observation strongly suggests that hyponatremia may contribute to the development of post-SAH cerebrovascular vasospasm as the NO-dependent regulation of cerebral vasculature was reported to be profoundly disturbed after subarachnoid bleeding [39–41].

Ethics approval and consent to participate

Not applicable.

Human and Animal Ethics

No human subjects were involved in the study.

All experimental procedures involving animals were approved by the IV Local Ethics Committee for the Care and Use of Laboratory Animals for Experimental Procedures, National Medicines Institute in Warsaw.

Consent for publication

All authors have seen the final version of the manuscript and have given their consent for publication.

Availability of supporting data

All data that support the finding of the current study are available from corresponding author, upon reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by a grant of the Ministry of Science and Higher Education in Poland [N40119032/3924] and by the Mossakowski Medical Research Institute PAS.

Author information

Authors and Affiliations

Laboratory of Experimental and Clinical Neurosurgery, Mossakowski Medical Research Institute Polish Academy of Sciences, A. Pawinskiego Str. 5, 02-106 Warsaw, Poland

Katarzyna Klapczynska, Marta Aleksandrowicz & Ewa Kozniewska

Author contributions

Conception and design of the work: Kozniewska, E. Acquisition, analysis, and interpretation of experimental data: Klapczynska, K and Aleksandrowicz M. Drafting the paper: Kozniewska, E and Aleksandrowicz M.

Corresponding author

Correspondence to Ewa Kozniewska.

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No competing interests reported.

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Editorial decision: Major revision
25 Sep, 2022
Reviews received at journal
22 Sep, 2022
Reviewers agreed at journal
06 Sep, 2022
Reviewers invited by journal
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Editor assigned by journal
31 Aug, 2022
Submission checks completed at journal
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First submitted to journal
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Role of the endothelial reverse mode sodium-calcium exchanger in the dilation of the rat middle cerebral artery during hypoosmotic hyponatremia

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