The effect of hydrogen sulfide on the contractility of cerebral arterioles. A pilot study

Background and Aims: Endogenous gaseous substances, such as NO and CO have been found to be effective vasodilators earlier. H2S has been identified as an additional one, however, for that substance both vasodilatory and vasoconstrictor responses have been described in different vascular territories. Our aim was to examine the effect of hydrogen sulfide on the tone of cerebral arterioles and some aspects of its mechanism. Methods: The work was performed on excised rat anterior cerebral artery segments in vitro (diameter range 150–250 mm), using a pressure myograph system. We used NaHS as exogenous H2S donor, propargylglycine (PAG) to abolish the endogenous synthesis of hydrogen sulfide and 4,40-Diisothiocyanatostilbene-2,20-disulfonic acid (DIDS) to examine the potential role of Cl /HCO3 exchanger in the effects of H2S. The time course of the events after application of exogenous H2S was also evaluated. Results: Our findings revealed that in these pathologically important vessels (1) endogenously produced H2S is not a vasodilator, but a moderate vasoconstrictor; (2) H2S has a biphasic effect: low concentrations are moderate vasoconstrictors, while at higher concentrations the initial contraction is followed by dilatation; (3) that vasodilation is prevented by DIDS (4,40-Diisothiocyanatostilbene-2,20-disulfonic acid disodium, an inhibitor of the Cl /HCO3 exchanger). Conclusion: These studies confirm that H2S should be taken into consideration as a modulator of cerebral arteriolar tone in mammals.


INTRODUCTION
Research on the regulation of cerebrovascular tone has an outstanding significance from clinical point of view. Stroke and other cerebrovascular diseases are a serious burden for healthcare all over the world, their epidemiologic importance as a cause of chronic disability and death continues increasing despite improving preventive therapeutic means [1,2].
As it is known, cerebral vascular resistance is controlled by myogenic mechanisms, local metabolites and blood-borne substances. Its control is not homogenous throughout the brain, substantial territorial differences exist. In general, the acetylcholine-induced vasodilation is far more pronounced in the anterior circulation, while vasoconstrictors are more effective in the vertebrobasilar region [3]. There are other important findings, which show further territorial differences in the activity of cerebral the vessels. It was revealed that vasoconstrictor responses (to noradrenaline, dopamine, 5-HT, prostaglandin F2) were more effective in the anterior cerebral artery, while these vessels were less sensitive to bradykinineinduced vasodilation [3].
After centuries in the past, when hydrogen sulfide (H 2 S) was considered merely a hazardous gas, today it is widely accepted that this can be considered a third gasotransmitter in mammalians. It has widespread effects both in physiological and pathophysiological conditions [4]. H 2 S is a weak acid, therefore it is mostly dissociated (approx. in 80%) to hydrosulfide anions (HS À ) and hydrogen ions (H þ ) at physiological pH values [5]. The mechanism of action of hydrogen sulfide is diverse, (1) persulfidation/polysulfide generation, (2) interaction with reactive oxygen and nitrogen species, (3) reaction with metalloproteins [6]. Hydrogen sulfide is produced in the mammalian body by four enzymatic pathways, cystathionine-gamma-lyase (CSE) being the most important one [4,[7][8][9]. Although there are numerous attempts with different techniques for the correct and precise measurement of endogenous hydrogen sulfide production, the exact level of H 2 S is different tissue and, what is also very important, in different cell compartments, is still under debate. The serum and tissue concentrations are suspected to be in the nanomolar range [10]. It is also probable that although the tissue concentration of H 2 S is very low in general, in distinct intracellular microenvironments it can reach higher values and exert important biological effects [10]. Tissue levels can be different in the wall of larger and smaller arteries: H 2 S level was higher in the tail artery compared to the aorta of the rat [11,12].
It is known that this molecule has important effect not only on of vascular tone, but also on angiogenesis, vascular permeability and atherogenesis [13]. Considering vascular tone, mostly vasodilatation was observed, but two-phase effects and vasoconstriction also has been observed [6,14]. Vasorelaxation is more pronounced in the peripheral resistance arteries, compared with the large, conductive vessels [15]. In genetically modified mice lacking one of the H 2 S producing enzymes, CSE, cholinergic vasorelaxation and hyperpolarization was found to be reduced proving the physiological significance of that pathway [16]. H 2 S has even been suggested to be one of the endothelial hyperpolarization factor (EDHF) molecules by some authors [15,16].
The Cl À /HCO 3 channels were found to be part of the vascular response by one key publication [13]. The Cl-transport inhibitor DIDS ameliorated the H 2 S-induced vasorelaxation in murine models which further proves the role of this pathway [17,18]. Nevertheless, vasoconstrictor responses after H 2 S treatment were also observed on different specimens [19].
The aim of the present study was to test using pressure myography whether endogenous H 2 S produced in the wall of cerebral resistance vessels, and the same substance, given externally has a vasoactive effect and whether the anion exchanger plays an important role in its action.

Animals
All the applied procedures conformed to the Guide for the Care and Use of Laboratory Animals (8th edition, ELAR/NRC 2011), the legal and institutional guidelines for animal care and were approved by the Animal Care Committee of the Semmelweis University and Hungarian authorities (PE/EA/1430-7/2018).
The composition (in mmol l À1 ) of the Krebs-Ringer solution used was the following: Na þ 144; K þ 4.

Preparation of anterior cerebral artery segments
Male Wistar rats were anaesthetised with pentobarbital (Nembutal, Ceva Santé Animale, Libourne, France, 45 mg kg À1 body weight, administered i.p.). Then the right atrium was cut open, and all the systemic circulation was perfused from the left ventricle with heparinized cold Krebs-Ringer solution. The head was removed, the brain exposed and removed together and put in ice-cold Krebs solution. The course of the right anterior cerebral artery (ACA) was identified under the preparation microscope (Leica) and the A2 section carefully cleared from surrounding tissue and cut out. All preparation processes were performed under cold, oxygenated Krebs-Ringer solution. An approx. 2 mm long segment from the A2 section was used in the further experiments. Smaller side-branches, if any, were ligated, vessels were cannulated at both ends with plastic microcannulas and extended to their in vivo length. Vessels were pressurized with Krebs-Ringer solution as described below.

Pressure arteriography of arteriolar segments
The glass-bottomed organ chamber containing the already cannulated vessel segment was placed on the stage of an inverted microscope (Leica, Wetzlar, Germany). Servo-controlled pumps Physiology International (Living Systems, Burlington, VT, USA) were used to set the intraluminar pressure. Calibration of the experimental system was done with a mercury manometer. The vessels mounted in Krebs-Ringer solution were bubbled with a gas mixture containing 5% CO 2 , 20% O 2 and 75% N 2 , and were pressurized to 50 mmHg. Then the segments were left in the system for equilibration for 30 min. During the examinations, temperature of the bath solutions was kept at 37 8C. Pictures of the transparent, mounted segment were taken by a digital camera (Leica DFC 320) and were photographed using the Leica Qwin V3 software. The inner and outer diameters of the vessels were measured from these pictures. Calibration was made using a micrometer etalon (Wild, Heelbrugg, Switzerland). At the end of the drug tests, vessel segments were washed out, they were incubated in Ca 2þ -free Krebs solution for 30 min to determine passive, fully relaxed diameters. After 30 min equilibration at 50 mmHg, pressure-diameter in normal Krebs-Ringer solution, pressure-diameter curves were recorded (0-10-20-30-40-50-60-70-80-90-100 mmHg). Inner and outer diameters of the vessels were measured offline using the ImageJ software.

Pharmacological studies
Pressure diameter curves were recorded in myogenic contraction as above and the procedure was repeated with the PAG in the bath. In other specimens, at 50 mmHg intraluminal pressure the thromboxane-A2-receptor (T3A 2 r) agonist U46619 was added (3310 À6 M) and the contraction recorded. Than the H 2 S donor NaHS was added in increasing concentrations, 10-30-100-300-1,000 mM, and the diameter responses were recorded. In another group the specimens were pretreated with the anion exchanger inhibitor DIDS (300 mM), U46619 contraction was induced, and the vasodilatory action of NaHS (1,000 mM) was tested at this concentration.

Statistical analysis
Results are shown as mean ± SEM. One-way and two-way ANOVA was used with Tukey posthoc tests. Paired t test was applied when diameter changes from reference value were evaluated. The P value <0.05 was considered as statistically significant. Statistical analysis was performed with GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).

RESULTS
Segments had a typical for these small arterioles myogenic tone (approx. 20% at higher pressures, Fig. 2A). Addition of PAG induced a slight relaxation at higher intraluminal pressures, which did not reach the level of statistical significance ( Fig. 2A). All segments produced significant in vitro contraction with U46619 (3 mM, Fig. 2B pp , P < 0.01 with the paired t test). The H 2 S donor NaHS had a biphasic effect: a vasoconstriction at lower concentrations ( Fig. 1A and B; Fig. 2C), while if applied in higher concentrations (300-1,000 mM, an initial vasoconstriction was followed by vasorelaxation ( Fig. 2C and D) significant with the paired t test P < 0.05). Pretreatment of the vessel segments with DIDS inhibited the vasorelaxation exerted by the administration of 1,000 mM NaHS; this effect was statistically significant (Fig. 2E).

DISCUSSION
In our experiments, the inhibition of endogenous H 2 S production by the CSE-inhibitor molecule PAG resulted only moderate, non-significant relaxation of rat cerebral arterioles in the intraluminal pressure range of 50-100 mmHg in vitro. Our result strengthens the observation that moderate vasoconstrictor response might be the consequence of this substance which might be important when considering sulfide releasing molecules as drugs [20]. Vascular consequences of the presence of H 2 S in the brain tissue have been reviewed by us lately [21]. Compared to the previous publications in this field, our recent work has particular characteristics: while majority of the former studies were performed on rat middle cerebral artery segments, using mostly wire myography in the ex vivo experiments, we studied the effect of H2S on rat anterior cerebral artery segments, using pressure myography, which allows the direct examination of vasoconstriction or dilatation in resistance vessels. These differences in the type of the used specimens and methods underline the importance of our findings in this pilot study. When NaHS was administered in increasing concentrations to cerebral arteriolar segments precontracted with 3 mM U46619 (a T3A2 agonist), a biphasic effect was detected, which consisted of a vasoconstriction at lower doses of H 2 S, followed by vasorelaxation after the H 2 S concentration increased above the level of 300 mM. Vasoconstrictor effect of H 2 S is a well-known phenomenon from the related literature: it was formerly observed in other cerebral vessel studies as well, while the biphasic effect was proven by measurements on vascular segments from the Note moderate vasodilator effect at higher pressures (n.s.). B. Precontraction of rat cerebral arterioles with the TxA 2 agonist U46619 (3 mM, n 5 9, ** P < 0.01 with ANOVA), C. Effect of increasing concentrations of the H 2 S donor NaHS on the diameter of precontracted segments (2 min after addition of the drug to the bath). Note moderate contraction at 30-300 mM, but relaxation at 1,000 mM (n 5 5, P < 0.05 with the paired t test). D. Time course of the effect of 300 mM NaHS treatment on the diameter of precontracted segments, 1-5 min after addition of the drug. Note initial wave of contraction followed by relaxation (statistically significant with the paired t test). U46619-contracted diameters are considered as units. E. Effect of DIDS pretreatment (4,4 0 -Diisothiocyanatostilbene-2,2 0 -disulfonic acid disodium, an anion exchanger blocker) on the vasodilation exerted by 1,000 mM NaHS (n 5 5, paired t test *P < 0.05). U46619 contracted diameters are considered as unit. Note inhibition of H 2 S relaxation.
systemic circulation [6,21] A possible underlying mechanism behind this phenomenon is the regulatory effect of H 2 S on NO production and bioavailability, resulting in decreased NOmediated vasorelaxation [14,22]. After pretreatment with DIDS, NaHS-induced relaxation diminished. While the effect of DIDS on the vasorelaxant effect of NaHS has been described in other vessels earlier by other studies using wire myography on mouse mesenteric arteries, rat middle cerebral arteries or on rat thoracic aortic rings, the underlying mechanisms are still not clearly understood [18,23] and there are only few publications on this so far, especially related to the cerebrovascular circulation [21]. According to these previous studies it is possible that H 2 S acts directly on the Cl À /HCO 3 channels -supposedly through PKA or PKC activation [24,25]. However, based on results of other studies it is also possible that H 2 S treatment leads to the activation of the Cl À /HCO 3 channels not directly, but through intracellular acidosis, which is a result of the blocked ATP generation after inhibition of cytochrome c [23].

CONCLUSION
Taken together, the two most important aspects of our current work are (1) the description of the vasoconstrictive nature of endogenous H 2 S in the modulation of the tone of the rat cerebral circulation and (2) the biphasic vascular effect after exogenous H 2 S treatment, which was only reported in the systemic circulation previously. Furthermore, the H 2 S induced relaxation is significantly inhibited by an anionic exchanger blocker in these vessels as well which reveals one, albeit not deciphered, detail regarding the vasodilatory mechanism of H 2 S in the cerebral circulation.