# Hyperbaric-induced enhancement of noradrenaline-evoked contraction in rat thoracic aorta.

F. Guerrero, M. Lucciano, P. Joanny, Y. Jammes

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F. Guerrero, M. Lucciano, P. Joanny, Y. Jammes. Hyperbaric-induced enhancement of noradrenaline-evoked contraction in rat thoracic aorta.. Experimental Physiology, 1997, 82 (4), pp.687-95. hal-00743429

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# HYPERBARIC-INDUCED ENHANCEMENT OF NORADRENALINE-EVOKED CONTRACTION IN RAT THORACIC AORTA

FRANÇOIS GUERRERO, MICHEL LUCCIANO*, PIERRE JOANNY* AND YVES JAMMES*

UFR STAPS de Brest, Université de Bretagne Occidentale, BP 808, F 29285 Brest Cedex and *Laboratoire de Physiopathologie Respiratoire, URA 1630 CNRS, Institut Jean Roche, Faculté de Médecine-Nord, Boulevard P. Dramard, F 13916 Marseille Cedex 20, France

(MANUSCRIPT RECEIVED 30 JULY 1996, ACCEPTED 11 FEBRUARY 1997)

## SUMMARY

The aim of this study was to investigate the effects of a high ambient pressure of He on vascular contraction induced by noradrenaline and to distinguish the effects of ambient pressure *per se* from those of increased pressure of inert gas. Rings of thoracic aorta were isolated from male Wistar rats. Isometric tension was measured in preparations exposed to 7·1 MPa (absolute pressure) of He. Dose-response curves for noradrenaline and contractions elicited by 120 mM KCl were compared with time-matched experiments performed at atmospheric pressure. The same protocol was also carried out under 7·1 MPa of N₂. At the high pressure of He, the contraction elicited by noradrenaline was increased with no change in the response to K⁺-evoked depolarization. The tension developed in response to noradrenaline also increased under 7·1 MPa of N₂ but the effects were less marked than during the He experiments. Moreover, the response to KCl was reduced in this circumstance. Hyperbaric conditions enhance the noradrenaline-induced contraction of rat aorta *in vitro*. This effect probably results from an action of pressure *per se* on activation of adrenoceptors. However, the hyperbaric-induced increase in vascular smooth muscle contraction is partially counteracted by high pressures of inert gases (N₂, but also probably He), which impair the efficiency of the contractile machinery.

## INTRODUCTION

High ambient pressure is well known to alter a number of biological processes, including those involved in excitable tissues. The effects of elevated ambient pressure have already been investigated on various muscle types and revealed that pressure up to 13 MPa increases the strength of contraction. Although the mechanisms underlying these changes still remain unclear, data obtained from *in vitro* studies performed on both diaphragmatic (Kendig & Cohen, 1976) and cardiac muscle (Doubt & Hogan, 1978, 1979) showed pressure-induced decreases in neuromuscular transmission and muscle excitability, suggesting that the changes in strength of contraction could not be related to changes in these processes. Consequently, in rat atrium (Örnhagen & Sigurdsson, 1981; Gennser & Örnhagen, 1989a,b) and in diaphragm (Kendig & Cohen, 1976), other mechanisms have been proposed to account for the increased twitch tension at increased pressures; i.e. the lengthening of the muscular action potential, changes in the contractile process itself and/or in excitation-contraction coupling. Studies on skeletal muscle by Harris & Bennett (1983) showed that pressure increased the strength of a twitch contraction. Moreover, these results also suggested that the pressure-related increase in force of contraction was probably not due to the lengthening of the action potential, but due to changes in the contractile process itself and/or in excitation-contraction coupling.
To our knowledge, only two studies have focused on the effects of elevated ambient pressure on the strength of vascular smooth muscle (VSM) contraction (Sigurdsson & Örnhagen, 1980; Bratveit & Helle, 1989) and both were conducted in isolated rat portal vein. The work by Sigurdsson & Örnhagen (1980) showed a decrease in the spontaneous contractile activity as well as contractions elicited by electrical field stimulations under 10 MPa of hydrostatic pressure. It also showed that KCl-induced contractions were attenuated by elevated ambient pressure. However, using the same physiological model, Bratveit & Helle (1989) reported that a pressure of 3 MPa failed to modify the maximal contractile response to high $K^+$ as well as high $Ca^{2+}$ concentrations.

Alternatively, one study investigated the effects of exposure to hyperbaric conditions on arterial smooth muscle contractions elicited by receptor agonists (Demchenko, Kostkin & Sechenov, 1994). Unfortunately, in this work, contractions were studied at atmospheric pressure after a 15 day exposure to 7·1 MPa of a $He-O_2$ mixture. However, the authors reported an increased sensitivity of femoral artery and aorta to noradrenaline (NA) associated with decreased contractility.

The aim of the present study was, therefore, to investigate the contractile response of arterial smooth muscle to stimulation of adrenoceptors by NA under increased ambient pressure of He. This work was conducted on aortic rings in order to eliminate the influence of spontaneous contractile activity and because the contractile processes of this physiological model, which have been widely used at atmospheric pressure, are well known. Furthermore, in an attempt to elucidate the mechanisms by which pressure *per se* and inert gases act on arterial smooth muscle contraction, contractions were also elicited under the same pressure of $N_2$, a more potent narcotic gas. High $K^+$-evoked depolarizations, which bypass the ligand-receptor coupling, were also performed in both hyperbaric conditions.

## METHODS

### Preparation of aortic rings

Male Wistar rats (350–400 g) were killed by a blow to the neck. The thoracic aorta was gently removed and immediately stored in cold Krebs–Henseleit bicarbonate (KHB) solution (4 °C) previously gassed with 95 % $O_2$–5 % $CO_2$. The composition of the KHB solution was (mM): 118·0 NaCl, 25·0 $NaHCO_3$, 1·18 $KH_2PO_4$, 4·74 KCl, 1·18 $MgSO_4$, 2·5 $CaCl_2$ and 10·0 glucose (pH 7·4). The use of 95 % $O_2$ protects the preparation from hypoxia, even in the core tissue, whereas 5 % $CO_2$ allows the so-called bicarbonate buffer to keep the pH of the KHB at a constant value. Indeed, in pioneering works, this milieu has been shown to give the best results (McIlwain, 1962). The aorta was carefully dissected to remove surrounding connective tissues and its midthoracic region was cut into rings of 2 mm length. Extreme care was taken during ring preparation to avoid the stretching of tissue or the touching of luminal surfaces in order to preserve intact endothelium, as assessed pharmacologically by the relaxation response to $10^{-6} M$ acetylcholine (Sigma) (Furchgott & Zawadski, 1980). The rings were then mounted between two fine nickel-chrome hooks and placed in a jacketed 2 ml organ bath continuously perfused with oxygenated KHB at a flow rate of 8 ml min⁻¹, allowing the complete change of solution four times per minute. In both normobaric and hyperbaric experiments we used a high-pressure pump (KONTRON LC 414) located outside the chamber. The upper hook was attached to an isometric force transducer (Gould Statham UC3) which was held by an electrically driven vernier control (type 850 EM; Hugo Sachs Elektronik KG) allowing fine vertical movements for tension adjustment of the aortic ring. We verified, in preliminary experiments, that the response of the force transducer was unaffected by pressure. Tension was recorded on a polygraph (BPN Servorac, SEFRAM, France). Bath temperature was measured with a PT100 thermocouple and maintained at 37 ± 0·5 °C. Although not recorded, this parameter was displayed continuously throughout all experiments, allowing us to ensure that no variation greater than ± 0·5 °C occurred during the stage at atmospheric pressure, the compression and the stage at maximal pressure.
Passive tension was maintained at 2·5 g (determined as the optimal resting tension during preliminary investigations) throughout all the experiments. Before any experiment was performed, rings were allowed to equilibrate for a 90–120 min period. Thereafter, aortic rings were depolarized twice with high-K⁺ (120 mM)-containing KHB. High-K⁺ solution refers to KHB in which NaCl was replaced with an equimolar concentration of KCl. Between trials, the rings were allowed to recover for a 30–45 min period. K⁺-induced stimulation provided us with a reference contraction and, thus, allowed us to control variability in contractile responsiveness between vessels. Therefore, all results were subsequently expressed as percentages of the KCl-induced contractions.

*Exposure to pressure*

The organ bath was placed in a 30 l steel hyperbaric chamber equipped for *in vitro* studies. The chamber consisted of two half-cylinders held vertically together by a crown-shaped clamp. It was provided with electrical through-connectors, brass valves for gas flow and stainless-steel valves for liquid circulation. After the reference contractions elicited by high K⁺, ambient pressure was increased using pure gas (either He or N₂). Each compression was made to 7·1 MPa (absolute pressure) at a rate of 0·2 MPa min⁻¹ (time duration, 35 min), i.e. following the same hyperbaric schedule that we used previously for *in vivo* experiments (Guerrero & Burnet, 1996). After the maximal pressure was reached, the preparation was allowed to equilibrate for a 90 min period before the effects of high ambient pressure on VSM contraction were investigated.

*Effects of high ambient pressure on VSM contraction*

The major aim of the present study was to assess whether high ambient pressure could change the VSM response to NA. Concentration-response curves were obtained by perfusing the organ bath with increasing agonist concentrations, a plateau tension being reached at each dose. Drugs were purchased from Sigma and solutions were prepared in KHB no longer than 1 h before use and kept at 4 °C in the dark in order to prevent drug decomposition due to oxidizing reactions. To ensure that changes in the NA-evoked contraction could not result from modified contractile properties of aortic rings exposed to hyperbaric conditions, VSM contractions were also elicited under high ambient pressure by perfusing the organ bath with high-K⁺ KHB solution. The final aim of the present study was to distinguish, if possible, the effects of pressure *per se* from those of the narcotic potency of inert gas. Thus, both NA- and K⁺-induced contractions were performed on rings exposed to either He or N₂, a more potent narcotic gas, all other hyperbaric conditions (i.e. compression rate and maximal pressure) being the same. Control experiments were conducted in a time-matched manner on rings maintained at atmospheric pressure.

*Statistical analysis*

Data are expressed as means ± S.E.M.; *n* indicates the number of experiments performed. The differences between NA concentration-response curves were analysed using two-way analysis of variance, followed by Student-Newman-Keuls post-ANOVA tests to compare data for each NA concentration (Zar, 1984). Contractions elicited by high K⁺ in different environmental conditions were compared using one-way ANOVA and Student-Newman-Keuls post-ANOVA tests. The same procedure was used for comparisons among the EC₅₀ values, which represent the NA concentration eliciting 50% of maximal contraction. Values where *P* < 0·05 were considered to be significant.

## RESULTS

The strength of the contractions elicited before compressions (i.e. at atmospheric pressure) by 120 mM K⁺ was 1153 ± 71 g (mg ring dry weight)⁻¹; no difference was detected between groups.

Figure 1 shows representative examples of KCl- and NA-induced contractions recorded under control conditions (i.e. 0·1 MPa of air).

The dose-response curve for NA was significantly modified under 7·1 MPa of He (*n* = 4) compared with that for air at atmospheric pressure (*n* = 6) (Fig. 2). The contraction was enhanced over the entire range of the agonist concentrations tested, except for the lowest ones
<figure><img src="image_2.png" /><figcaption>Fig. 1. Representative recordings of tension changes in rat thoracic aorta induced by both high KCl and cumulative NA concentrations at atmospheric pressure.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2. NA concentration-response curves obtained in aortic rings exposed to either 0·1 MPa of air (●) or 7·1 MPa of He (■). Values are means ± S.E.M. Asterisks indicate significant differences.</figcaption></figure>

(10⁻⁸ and 10⁻⁷ M). However, no significant change in the EC₅₀ values was found (0·219 ± 0·071 vs. 0·168 ± 0·027 µM).

Exposure to 7·1 MPa of N₂ (*n* = 4) enhanced the NA-induced contraction of aortic rings compared with control conditions (*n* = 6) (Fig. 3). However, although the concentration-response curve was significantly modified under the high pressure of N₂, NA-induced active
<figure><img src="image_4.png" /><figcaption>Fig. 3. NA concentration response curves obtained in aortic rings exposed to either 0·1 MPa of air (●) or 7·1 MPa of N₂ (■). Values are means ± S.E.M. Asterisks indicate significant differences.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 4. KCl-evoked contraction of aortic rings during time-matched experiments conducted under 7·1 MPa of either He or N₂ (means ± S.E.M.). Asterisk indicates that the high pressure of N₂ significantly reduced the tension change compared with corresponding control trials at atmospheric pressure.</figcaption></figure>

tension was significantly increased with the highest agonist concentrations (≥100 µM) only. Furthermore, EC₅₀ values did not change significantly (0.219 ± 0.071 vs. 0.285 ± 0.043 µM).

The contractile response to 120 mM KCl (Fig. 4) was not significantly modified at 7·1 MPa of He (108.9 ± 9.78%; n = 4) compared with air at atmospheric pressure (103.5 ± 2.39%, n = 6). Conversely, the same pressure of N₂ significantly decreased the contractile response of aortic rings to high K⁺ (75.8 ± 10.84%; n = 4).
DISCUSSION

The present study provides evidence that high ambient pressure of inert gas leads to a striking increase in the strength of contraction elicited by NA in rat aortic smooth muscle *in vitro*.

It is of note that in our control experiments, a 2 h incubation in KHB failed to modify the strength of the contraction elicited by high K⁺, suggesting that aortic rings are not damaged over time by our incubation conditions. Another point is whether changes in tissue temperature could occur as a result of differences in our various experimental conditions. In this regard, it should be emphasized that the heat exchanges between the preparation and the KHB are related to VρCₚ, where V is the volume flow of the KHB, ρ and Cₚ are the density and the constant pressure specific heat of the fluid, respectively. In our protocol, V remained constant, while the ρCₚ product was not significantly modified either by 7·1 MPa ambient pressure or by the changes in the amount of dissolved gas; the ρCₚ product of both He and N₂ was negligible compared with the KHB product. Furthermore, because of the very high ρCₚ product of the liquid environment, and because isolated VSM cannot regulate its temperature, the temperature of the preparation must be the same as the KHB; every modification of the latter leading to a rapid and equal change of the former. Consequently, although marked variations occurred in the various experimental conditions for heat exchange between the bath and the surrounding gas mixture, thermal flux between the KHB and the preparation must have remained unaltered under 7·1 MPa of both He and N₂. Thus, the changes in VSM contraction we measured during both He and N₂ hyperbaric experiments could not result from changes in the organ temperature since no modification of this environmental parameter was detected in the bath during either the compression or the stage at maximal pressure. Finally, the increase in NA-induced contractions could result from modified O₂ and/or CO₂ concentrations in the KHB under elevated ambient pressure. In the present work we did not measure either the O₂ or CO₂ content of the KHB. Nevertheless, it should be emphasized that the O₂ partial pressure (P_O₂) in the hyperbaric chamber was the same during control and hyperbaric experiments, i.e. 210 hPa, which represents the P_O₂ in the air present when the pressure chamber was closed. Thus, according to the laws of Dalton and Henry (for details see Dejours, 1981), no modifications occurred in O₂ and CO₂ diffusion between KHB and the surrounding gas mixture in hyperbaric conditions compared with atmospheric pressure. Therefore, it appears that O₂ and CO₂ concentrations in the perfusing liquid must have remained the same during both hyperbaric and control experiments. Consequently, the only environmental changes that occurred under our experimental conditions concerned the rise in ambient pressure itself and in the partial pressures of either He or N₂; each gas concentration also depending on its solubility in KHB. Thus, the changes in VSM contraction we measured in the present work could be a consequence of the increased ambient pressure *per se* and/or of the partial pressures of both He and N₂.

We found that high ambient pressure of He failed to modify the contraction elicited by high-K⁺-induced depolarization of aortic smooth muscle, which bypasses activation of adrenoceptors. This result agrees with the effect of 3 MPa pressure on the rat portal vein (Bratveit & Helle, 1989). Conversely, it has been reported that 10 MPa of hydrostatic pressure decreases the contractile response to 120 mmHg KCl (Sigurdsson & Örnhagen, 1980). However, it should be emphasized that in the study by Sigurdsson & Örnhagen (1980) VSM contraction was induced less than 20 min after rapid (1 MPa min⁻¹) compression to 10 MPa, while in the present work aortic rings were allowed to recover for 90 min after a 0·2 MPa min⁻¹ compression up to 7·1 MPa.
Considering the aortic smooth muscle sensitivity to NA, the present data show that 7·1 MPa of He led to an increased contractile response to this neurotransmitter when compared with time-matched experiments conducted with air at atmospheric pressure. Our results agree with the previous work by Demchencko *et al*. (1994) which showed enhanced VSM responsiveness to NA. Moreover, in hyperbaric conditions, we found that the contractile response to NA became larger than the response to maximal depolarization by K⁺. In this regard, it should be noted that at atmospheric pressure, the cytosolic Ca²⁺ concentration measured at a matched contraction level was found to be higher during K⁺- than NA-induced contraction of the rat aorta *in vitro* (Karaki, Sato & Osaki, 1981). According to the authors, the consequence is a greater contractile response to NA than to K⁺ when both stimulants increase the cytosolic Ca²⁺ concentration to the same level, and could reflect different sensitivities of the contractile elements to Ca²⁺ during high-K⁺- and NA-induced contractions. Therefore, modulation of the contractile elements by a similar mechanism may explain the occurrence of the larger contractions induced by NA than by K⁺ under high ambient pressure. Elsewhere, the existence of two separate and independent Ca²⁺ pathways for NA- and K⁺-induced stimulation (Meisheri, Hwang & Van Breenen, 1981) may provide further support for this hypothesis. Thus, there exists the possibility that hyperbaric conditions might selectively potentiate the NA-induced Ca²⁺ fluxes and hence increase the strength of NA-induced contraction without affecting the K⁺-induced one. In this regard, augmented availability of Ca²⁺ at the contractile site has been previously suggested for the pressure-related increase in force of contraction seen in rat atrium (Örnhagen & Sigurdsson, 1981; Gennser & Örnhagen, 1989*a*) and diaphragm (Kendig & Cohen, 1976).

Our results obtained under 7·1 MPa of He suggest that high ambient pressure may act on VSM contraction by interfering with the binding of NA to its membrane receptors and/or the subsequent signal transduction pathways. Such a hypothesis could also be supported by a pioneering study conducted *in vitro* with skeletal muscle exposed to high ambient pressure (Brown, 1936). In this work, the total twitch tension developed by the penis retractor muscle exposed to high hydrostatic pressure was more than twice as great as that at atmospheric pressure. Furthermore, performing compressions at various times in the contraction cycle, Brown (1936) reported that pressure increased the twitch tension only when the compression was achieved during the first 0·04 s of a contraction phase of 0·28 s duration.

When ambient pressure was raised with N₂, instead of He, we observed that the hyperbaric-induced increase in the contractile response to NA was attenuated. Gennser & Örnhagen (1989*a,b*) have already investigated the effects of inert gases on the changes in strength of contraction of rat atrial preparations at 10 MPa of hydrostatic pressure. They noted that introduction of He, N₂ or H₂ in the superfusate counteracted the effects of hydrostatic pressure, the efficacy of inert gases to reduce the strength of contraction being related to their lipid solubility and, thus, their narcotic potency, the greatest being found with N₂. Interestingly, the results by Gennser & Örnhagen (1989*a,b*) also suggest that He may already attenuate the effect of raised pressure on vascular smooth muscle contraction.

In the present study, the use of N₂ reduced the effect of elevated ambient pressure on both the NA-induced smooth muscle contraction and the K⁺-evoked contraction. Moreover, the amplitude of this attenuation was nearly the same for both NA- and K⁺-induced contractions. This suggests that high ambient pressure of N₂ could impair the efficiency of the contractile machinery. If so, inert gases and raised pressure could modify vascular smooth muscle function through at least two opposite mechanisms. This hypothesis agrees well with the results of
Gennser & Örnhagen (1989a, b) on electrically stimulated atrial muscle and with a recent model of interactions between inert gases and raised pressure (Abraini, 1995) which suggests that narcotics and pressure act at different hydrophobic cell levels on narcotic- and pressure-sensitive sites, respectively. As a consequence, this model also proposed that in divers, inert gas narcosis and high pressure neurological syndrome can occur simultaneously or can be antagonists, depending on their respective effects in different, and as yet unidentified, sites of the central nervous system; moreover, the overall effect observed is the result of a balance between the action of inert gases and that of pressure on their respective sites.

In conclusion, high ambient pressure of He enhanced the VSM contraction mediated by adrenoceptor stimulation. This effect was not due to changes in the contractile properties of the smooth muscle cells since the contraction produced by high $K^+$ was not altered under these hyperbaric conditions. Conversely, the same level of ambient pressure with $N_2$, a more potent narcotic gas, counteracted the effects of high ambient pressure, via an action on the contractile machinery. This work also suggests that hyperbaric conditions could be a useful tool for investigations of VSM contraction processes. However, further investigations need to be performed to determine thoroughly the effect of pressure upon each type of adrenoceptor and the mechanisms by which hyperbaric conditions modify VSM contraction.

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