INTRODUCTION

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MICROGRAVITY IS KNOWN to alter the ability of the cardiovascular system to react to increased hydrostatic pressure induced by upright posture when astronauts return to Earth (12); it is called cardiovascular deconditioning (CVD). To investigate the pathophysiology of a phenomenon that, in many respects, is very important, a model has to be used to mimic the effects of microgravity; it is not possible to create long-lasting microgravity on Earth. The model commonly used by investigators implies an increase in central venous pressure (CVP) induced by head-down tilt (HDT) in humans or tail suspension (TS) in rats. In both species, CVP was shown to be restored within a few hours after starting HDT or TS, with no change in blood pressure (BP) or heart rate (HR). Such normalization of CVP appears to be realized at the expense of the extracellular fluid volume (11, 12, 14, 16). To ascertain that a deconditioning of the cardiovascular system was induced by several hours of HDT in humans or TS in rats, a stimulus has to be superimposed. In humans, after HDT, lower body negative pressure was shown to induce an increased tachycardia and deficient BP (14), as observed after spaceflight (12). In rats, simulated orthostatism (SO) was shown to be associated with transient episodes of hypotension and bradycardia (16). Thus differences exist between humans and rats, at least in response to stimuli expected to mimic the effects of upright posture. However, the CVD model in the rat is potentially very useful. Thus investigation of its pathophysiology was undertaken.

Increased CVP is known to activate baroreceptors in the low-pressure vascular bed, with appropriate messages sent to upper centers controlling the activity of the sympathetic system (21). Increased CVP in TS rats or inflation of a balloon located at the outlet of pulmonary veins in the dog is associated with increased excretion of sodium and water by the kidney (11, 16); this cardiorenal reflex is blocked by cooling of the neck afferent fibers (11). Furthermore, norepinephrine (NE) turnover rate was reduced in some brain cell groups involved in BP control and in the heart in TS rats (6). Thus a reduction in the activity of the sympathetic system during TS could have a positive effect in normalizing CVP and a negative effect in making the circulatory system less reactive to increased hydrostatic pressure induced by SO. Thus the first objective of the present work was to evaluate the activity of the sympathetic system in TS rats through spectral analysis of BP and HR variability, measurement of plasma catecholamines, and evaluation of reactivity of arterial -receptors.

Another mechanism was suspected in the rat, because TS was shown to induce episodes of hypotension and bradycardia (16). We wondered whether the serotonergic system could be involved. Phenylbiguanide, known to mimic selectively the effects of serotonin on mammalian neurons (4, 10), was shown to decrease BP, HR, and renal nerve activity after intrapericardial injection; such effects were no longer observed after vagotomy (23). On the contrary, vagal-mediated bradycardia and reduced renal nerve activity induced by hemorrhage in the rat were shown to be prevented by blockade of serotonin synthesis or receptors (18). If episodes of hypotension and bradycardia during SO are due to the hyperactivity of serotonergic receptors, their appearance should be prevented by pretreatment with 5-HT₃ receptor antagonists.

Thus the present study was carried out to investigate the mechanism(s) responsible for CVD induced by TS in the rat with two working hypotheses: 1) the activity of the sympathetic system should be reduced during TS and 2) the activity of the serotonergic mechanisms might be increased, accounting for BP and HR deficiency during SO.

	MATERIALS AND METHODS

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Experiments were performed on conscious, chronically instrumented rats. Male Wistar rats (Iffa-Credo) weighing 200-250 g were housed in separate cages and maintained on a 12:12-h light-dark cycle with temperature controlled at 20-25°C. Throughout the experimental period the animals were fed standard rat chow and tap water ad libitum.

General procedure. The procedure has been described elsewhere (16, 17). Briefly, animals were anesthetized with pentobarbital sodium (45 mg/kg ip). Polyethylene catheters were positioned in the femoral artery and vein for measurement of arterial pressure and intravenous infusion, respectively. They were routed subcutaneously, exteriorized behind the neck, and regularly flushed with heparinized saline.

Effect of TS on variability of BP and HR and plasma catecholamine concentration. Four groups of equipped rats (n = 10 in each group) were studied: animals in group 1 were TS for 24 h, while animals in group 2 remained in a horizontal position; animals in group 3 were were TS for 48 h, while animals in group 4 remained in a horizontal position. At the end of the experimental period, BP and HR were recorded and blood samples were obtained to measure plasma concentration of catecholamines.

Response of plasma catecholamines to SO in TS rats. The present series of experiments was carried out with the following rationale. TS of the rat could reduce the activity of the sympathetic system with no statistically significant effect on plasma catecholamine concentration and/or because the magnitude of that effect was small or the sensitivity of our radioenzymatic assay was inappropriate. SO should increase plasma concentration of catecholamines. If TS was associated with reduction in sympathetic activity, the SO-induced response of catecholamines should be lower in TS rats than in controls.

Investigation of vascular receptors. Both ₁- and ₂-adrenergic receptors are known to be present in rat abdominal aorta (20). Briefly, TS (n = 24) or control (n = 24) rats were anesthetized with pentobarbital sodium (45 mg/kg ip); then the abdominal aorta was rapidly removed and gently cleaned of adherent connective tissue in an organ bath containing a Krebs-Ringer solution maintained at 4°C (in mM: 115 NaCl, 4.6 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 21.9 NaHCO₃, 11 glucose). Then vascular samples were dried and frozen in liquid nitrogen and stored at 80°C.

5

Effect of 5-HT₃ receptor blockade during stimulation by SO. Four groups of eight 48-h TS rats were tested. They were given ondansetron or MDL-72222, known to be selective 5-HT₃ receptor antagonists (1, 8), or their respective vehicles. After 48 h of TS the rats were gently restrained in a flexible hemicylinder fixed on a rocking support axis and kept in the horizontal position for 10 min. Then a 90° rotation was induced and sustained for 2 h, with permanent recording of BP and HR (16). Five minutes before rotation, ondansetron (300 µg/kg), MDL-72222 (1 mg/kg), or their respective vehicles were intravenously injected as a bolus. Cardiovascular effects of ondansetron were empirically shown to be short lasting; a second bolus was injected 1 h after SO.

Biochemical procedures. Plasma catecholamines (dopamine, NE, and epinephrine) were measured according to radioenzymatic techniques (3). The sensitivity of the assay was <1 pg for NE and epinephrine and <6 pg for dopamine. The interassay coefficients of variation (n = 36) were 11.8 and 10.2% for NE and epinephrine, respectively, and 15% for dopamine.

Statistical analysis. Values are means ± SE, except for plasma catecholamine concentrations, which are medians. Statistical differences were usually tested by one-way ANOVA followed by Newman-Keuls test, when needed. Differences in catecholamine results were assessed with the nonparametric Mann-Whitney test.

Drugs. 2-Methylserotonin maleate and MDL-72222 were obtained from Research Biochemicals and ondansetron from Glaxo. 2-Methylserotonin and ondansetron were dissolved in normal saline; MDL-72222 was dissolved in water with 2% ethanol. Bolus volume was 1 ml/kg.

	RESULTS

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Spectral analysis of BP and HR after TS. TS for 24 or 48 h had no effect on BP (Table 1) or HR (Table 2); this is in agreement with the basic definition of CVD. Spectral analysis into low-, mid-, or high-frequency segments disclosed no difference in the variability of systolic BP or HR between TS rats and controls.

Plasma concentrations of catecholamines after TS. There was no significant difference in plasma concentrations of catecholamines after TS between TS and control rats (Table 3).

Plasma catecholamine concentrations in response to SO. Plasma catecholamine concentrations in response to SO are shown in Fig. 1. As expected, plasma NE and epinephrine concentrations were elevated in control rats in response to SO compared with rats kept in the horizontal position; data obtained in horizontal rats have been reported previously (5). Also as expected, plasma NE and epinephrine concentrations during SO were significantly lower in TS than in control rats. Plasma concentration of dopamine was not significantly changed.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Plasma concentrations of -hydroxylated catecholamines in tail-suspended and control rats during simulated orthostatism. n, No. of rats.

Study of adrenergic receptors in abdominal aorta. The number of binding sites and affinity constants were determined for ₁- and ₂-adrenergic receptors in abdominal aorta. Results are shown in Table 4. Binding sites of ₁- and ₂-adrenergic receptors were significantly increased in TS rats compared with controls; ₁-adrenergic receptor binding sites were almost doubled, whereas ₂-adrenergic receptor binding sites were increased only 16%. The affinity constant of both groups of receptors was increased after 48 h of TS.

View this table: [in this window] [in a new window]   Table 4.   1- and 2-Adrenergic receptor binding profiles in rat abdominal aorta

Effects of 5-HT₃ antagonists on BP response to SO. Figure 2 shows recordings obtained in control TS rats (A) and in TS rats treated with ondansetron (B) during SO. Transient episodes of hypotension and bradycardia were no longer observed in ondansetron-treated TS rats. An identical profile of response was observed in data from TS rats treated with MDL-72222 (not reported).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Responses of blood pressure and heart rate (HR) to simulated orthostatism in tail-suspended rats treated with 5-HT3 antagonist vehicle (A) and with ondansetron (300 µg/kg; B). MAP, mean arterial pressure; bpm, beats/min.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Response of mean blood pressure (MBP) to simulated orthostatism in rats after 48 h of tail suspension. MBP, MBP before vs. after injection of 5-HT3 antagonists (hatched bars) or their respective vehicles (open bars). Ondansetron-induced effect on MBP was reproducible, as indicated by MBP response to second injection. * P < 0.05, ** P < 0.01, *** P < 0.001.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Mean blood pressure and heart rate responses in rats treated with 5-HT3 agonist 2-methylserotonin (3, 10, 30, and 100 mg/kg) in tail-suspended rats (hatched bars) and controls (open bars). * P < 0.05; ** P < 0.01.

	DISCUSSION

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In a previous study we reported that TS could be an interesting experimental model to decondition the cardiovascular system of the rat, although the response to hydrostatic stimulation was at variance with the response of humans after spaceflight or HDT (12, 14, 16). The present study was designed to investigate the pathophysiology of CVD in the rat with two working hypotheses: several lines of evidence indicate that any increase in CVP should lead to a decrease in sympathetic activity, and a decrease in sympathetic activity cannot fully account for the particular response of the rat circulatory system (transient episodes of hypotension and bradycardia) when challenged by an increased hydrostatic pressure. Whether serotonergic mechanisms could also be hyperactivated was a second working hypothesis.

The activity of the sympathetic system was assessed after 24 or 48 h of TS, when CVP was normalized (16) and when an apparent new equilibrium was established. Spectral analysis of BP and HR variability and measurement of plasma concentrations of catecholamines were carried out. Our results were negative, since no difference was observed between TS and control animals. This is in agreement with data obtained in humans after 24 h of HDT and in rats after 3 or 14 days of TS (6, 7, 14). Before any final conclusion is drawn, several technical aspects should be considered. Spectral analysis of BP and HR variabilities remains a difficult technique: whether it is sensitive enough to identify a reduced sympathetic drive cannot be determined. Regarding catecholamines, the radioenzymatic assay used to measure plasma concentrations was sensitive enough, since we have identified decreased NE concentration in rats given clonidine to reduce their sympathetic drive (5). However, a physiological mechanism could account for the lack of change of plasma catecholamines in TS rats: the expected reduced spillover rate of NE could be associated with a decreased clearance rate, in such a way that plasma NE concentration was not changed. Beyond technical limitations, normal NE concentration is frequently measured in recumbent patients who might present orthostatic hypotension (2, 15, 25).

The catecholamine response to SO was measured. As shown in Fig. 1, the level of plasma -hydroxylated catecholamines was significantly lower in TS than in control rats. Such a pattern of response was also observed in patients with orthostatic hypotension (2, 15, 25). This is probably due to a decreased neuronal reserve of neurotransmitter itself secondary to a TS-induced braking of the sympathetic activity with decreased release in the synaptic cleft during hydrostatic challenge. Reduced activity of the sympathetic system during TS was not identified with techniques (spectral analysis and plasma catecholamines) used in the present study but cannot be ruled out. Nonetheless, such a mechanism is supported by the lower level of catecholamines in TS rats during SO than in controls.

Because hypersensitivity to a low dose of NE was reported in patients with orthostatic hypotension (15), we wonder whether reduced activity of the sympathetic system in TS rats could be associated with alteration of -adrenergic receptors. In vitro studies were carried out on rat aorta. Binding sites and affinity constants of prazosin and RX-821002 radioligands, used to investigate ₁- and ₂-adrenergic receptors, respectively, were studied. TS was shown to induce an increase in the number of binding sites and the affinity constant of ₁- and ₂-adrenergic receptors (Table 4). Although it is difficult to extrapolate in vitro to in vivo data, it is tempting to consider that the increased number of binding sites and increased affinity represent adaptation of postsynaptic structures to the reduced amount of neurotransmitter in their vicinity, the reduced amount being the final step in a cascade of events starting with reduced activity of the sympathetic system. Once again, human and rat models appear different, although both are initiated with an increased CVP. In humans we have reported data suggesting an increased sympathetic drive with deficient response of peripheral resistances during lower body negative pressure in volunteers in the HDT position for 24 h, and we have proposed a downregulation of adrenergic receptors to account for the final pathophysiological picture (14). In rats, if decreased activity of the sympathetic system after TS is confirmed, one may wonder whether an increase in -adrenergic receptor binding sites and affinity could not be an attempt by postsynaptic mechanisms to counterbalance a reduced level of the neurotransmitter.

Although some of the data are compatible with decreased activity of the sympathetic system during CVD induced by TS in the rat, it was necessary to seek other mechanism(s) to take into account the transient episodes of hypotension and bradycardia during SO after 48 h of TS. A pure vasovagal response in rats was not thought to be an appropriate hypothesis compared with humans: hypotension and bradycardia in humans may be progressive and result in fainting (22), whereas in rats they break down (Fig. 2). A second working hypothesis was developed on the basis of the following reports. Higuchi et al. (13) proposed that cardiac vagal afferents could be activated by mechano- or chemostimuli with identical BP, HR, and renal sympathetic outflow responses (decreased) but a different adrenal sympathetic outflow response (increased). Thus a TS-induced increase in CVP could trigger two series of cardiovascular responses. The same group reported an inhibition of hemorrhage-induced bradycardia in the rat by blocking serotonin synthesis or serotonin receptors (18). Thus we have speculated on TS-induced hypersensitivity of serotonergic mechanisms. It could be interesting to recall that hypotension and bradycardia episodes were identified after 48 but not 24 h of TS (16), suggesting that serotonergic alterations, if any, take place after a certain period of time only. According to our working hypothesis, pretreatment with 5-HT₃ receptor antagonists should block hypotension and bradycardia episodes induced by SO in rats after 48 h of TS, whereas hypotension and bradycardia induced by 5-HT₃ receptor agonists should be greater in TS rats than in controls. The data tended to support our hypothesis. As shown in Fig. 3, two different 5-HT₃ antagonists blocked hypotension episodes induced by SO in TS rats. Data reported in Table 5 indicate that expected tachycardia induced by SO was restored in TS rats treated with 5-HT₃ receptor antagonists. Figure 4 shows that hypotension and bradycardia were slightly but significantly more marked in TS rats given low doses of 2-methylserotonin, suggesting a hypersensitivity of 2-methylserotonin receptors. Such a conclusion deserves to be confirmed. Thus, taken together, our data support a serotonergic involvement in circulatory dysregulation during SO in TS rats; if there is such an involvement, it remains to be understood why the decrease in BP and HR was transient.

In conclusion, the present study was carried out to test whether the activity of the sympathetic system was reduced when the cardiovascular system was deconditioned by TS. Direct assessment by spectral analysis of BP and HR variability or measurement of plasma catecholamines provided apparently negative results. However, when the rat cardiovascular and sympathetic systems were challenged by SO, the response of plasma catecholamine concentrations was significantly lower in TS rats than in controls: a reduced increase could be secondary to reduced reserve of neuronal catecholamine, itself secondary to reduced activity of the sympathetic system. To some extent, data obtained during study of aortic adrenergic receptors support that notion: an increased number of -adrenergic receptor binding sites and increased affinity to radioligands observed in vitro in TS rats could be secondary to a reduced level of neurotransmitter in the synaptic cleft. A second series of experiments with 5-HT₃ receptor antagonists (ondansetron or MDL-72222) and agonist (2-methylserotonin) leads us to suggest a hypersensitivity of serotonergic mechanisms in TS rats: hypotension and bradycardia episodes observed during SO in rats after 48 h of TS were blocked by pretreatment with 5-HT₃ receptor antagonists, whereas hypotension and bradycardia induced by the 5-HT₃ receptor agonist are likely to be more marked in TS rats than in controls. Taken together, our data suggest that CVD induced by TS in the rat could be a combined effect of a hypoactivity of the sympathetic system and a hypersensitivity of serotonergic mechanisms.