INTRODUCTION

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IT IS WELL ESTABLISHED that stimulation of arterial high-pressure and cardiopulmonary low-pressure receptors suppresses the release of arginine vasopressin (AVP) (12, 17, 19, 21). The interaction, however, and relative influence of each type of receptor (high vs. low) still has to be determined in particular in humans (15). Share and Levy (20) observed more than 30 years ago that the amount of bioactive antidiuretic hormone in plasma increased during unloading of carotid baroreceptors in dogs. In accordance with this observation, Kamegai et al. (9) observed that the AVP release induced by head-up tilt in humans was attenuated by simultaneous application of static neck suction. Thus stimulation of carotid baroreceptors during an orthostatic stress attenuates the stimulation of AVP release.

Results of various studies from our laboratory (9, 12, 14) indicate that inhibition of arterial baroreceptors plays an important role for release of AVP in humans. In particular, we have observed that narrowing of arterial pulse pressure (PP) might stimulate AVP secretion in supine humans during lower body negative pressure (LBNP) (14). It is not known, however, to what degree an increase in the hydrostatic pressure in the carotid sinus is involved. Results of Kamegai et al. (9) indicate that a decrease in static carotid sinus pressure stimulates AVP release during a whole-body head-up tilt maneuver. Therefore, in the present study we set out to investigate whether the opposite, static carotid baroreceptor stimulation, participates in regulation of release of AVP.

To test this hypothesis, we used a model previously described by us (18). In this model, left atrial diameter (LAD), and thus cardiopulmonary low-pressure receptor stimulation, is maintained unchanged by LBNP during a moderate antiorthostatic posture change from upright seated (Seat) to horizontal supine (Sup). In this way, carotid baroreceptors can be hydrostatically stimulated by the posture change without simultaneously affecting the low-pressure receptors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ten male subjects (age 25 ± 1 yr, height 185 ± 1 cm, and weight 82 ± 2 kg) completed the experiment. Two additional subjects entered the study but did not complete the protocol because of occurrence of presyncopal symptoms. Another subject exhibited excessive sweating at the end of one study day during the final 10 min of the control session, but reported no nausea or other discomforts. He turned out, however, to have a 10-fold increase in AVP during this period, and therefore data from his control session of that particular day are not presented. All were nonsmokers, had a negative history of cardiovascular and kidney diseases, and were healthy as indicated by a physical examination, responses to a questionnaire (class 1 examination of professional pilots), and measurements of hematocrit (0.35-0.50), arterial blood pressure (<140/90 mmHg), electrocardiogram (ECG) (unipolar), and urine analysis for glucose, leukocytes, erythrocytes, and protein. None of the subjects took any medication. Informed consent was obtained after the subjects had read a description of the experimental protocol, which was in compliance with the declaration of Helsinki and approved by the Ethics Committee of Copenhagen (KF 01-347/93). No complications occurred apart from the ones described above.

For 4 days before each day of study the subject ingested standardized meals containing 65 mmol sodium/24 h. He was allowed to drink only tap water in this period and was instructed to drink in excess of thirst. For 24 h before the experiment, the subject collected his urine in a plastic container for the determination of urinary sodium excretion.

The subject fasted for 12 h before the experiment, spent the night at the laboratory, and was awakened at 7:30 AM on the day of study. Between 7:30 and 7:45 the subject went to the bathroom and walked one floor down to the experimental room. A short catheter (Venflon 2; 1.2 mm OD, length 45 mm) was then inserted to a cubital vein for blood sampling. Thereafter, the subject was seated (Seat) with the legs placed horizontally in an LBNP box. He was instrumented with electrodes for recording of transthoracic impedance and with a cuff around the right upper arm for determination of arterial pressures and rested in Seat for 1 h before the start of the experiment. Room temperature was kept between 24.7 and 27.6°C, and humidity was kept between 25 and 65%.

Each subject underwent two experimental sessions: 1) upright seated for 30 min with the trunk vertical and the legs horizontal (Seat-1) followed by being supine (Sup) for 30 min and finally by being seated again for 30 min (Seat-2); and 2) 90 min with the same posture changes as in session 1 but with simultaneous application of LBNP during Sup (Sup + LBNP) to keep LAD unchanged compared with during Seat. The posture change from Seat to Sup was performed passively by tilting the back support from vertical to horizontal. The two sessions were performed on two separate study days with at least 3 wk in between. In addition, on each study day a seated control experiment for 90 min (Control) was conducted. Thus an experimental day consisted of two experiments of 90 min each: a control experiment with the subject seated and an experiment with the subject in the supine position with or without LBNP during the 30- to 60-min interval. The experiments were performed in a balanced, randomized order among the subjects and separated by 1 h of seated rest.

LBNP was carried out in an airtight Plexiglas box connected to a vacuum cleaner and a water manometer. The box surrounded the subject from below the iliac crest, where it was attached to the subject by a belt around a plastic seal. LBNP was initiated simultaneously with the passive tilting of the trunk and head of the subject from Seat to Sup and was varied between 22 ± 3 and 25 ± 1 mmHg to keep LAD unchanged from that of Seat. It should be noted that LBNP was effective from somewhere below the iliac crest and down, because the opening of the LBNP-box and the plastic seal had to have a certain size to allow the posture change (18). During Control and Sup, the vacuum cleaner was turned on but disconnected from the LBNP-box.

Arterial pressures and heart rate (HR) determinations were performed simultaneously at 5-min intervals and again 2.5 min after the posture changes. Blood sampling and echocardiography were performed simultaneously at 10-min intervals, and stroke volume (SV) and cardiac output (CO) were measured continuously by transthoracic impedance.

Systolic (SAP) and diastolic arterial pressures (DAP) were measured at heart level in the right brachial artery by conventional sphygmomanometry with the arm placed alongside the body. PP was calculated from SAP  DAP, and mean arterial pressure (MAP) was calculated from DAP + 1/3(PP). All arterial pressures were measured by the same observer.

To calculate MAP at the level of the carotid sinus (CSP) during Seat, the pressure exerted by a column of blood stretching from the level of the fourth intercostal space to the thyroid cartilage was subtracted from MAP measured at heart level (2, 18). Thus MAP and CSP were identical when the subjects were in the supine position. HR was determined by palpation of the radial artery over 1-min periods.

LAD was measured by echocardiography (Aloka SSD 500, Simonsen & Weel) in accordance with the criteria of Feigenbaum (4) at 10-min intervals during end-expiration as an average of measurements from three M-mode printouts obtained from the parasternal long axis view (LAD) and from three two-dimensional pictures obtained from the apical short axis view (LADa). The two-dimensional pictures were obtained immediately before opening of the mitral valves as determined from video recordings (Sony SVO-9500 MDP). All the measurements were performed in a blinded fashion by the same observer.

SV and CO were measured by transthoracic impedance by an NCCOM-3 cardiograph (BoMed Medical Manufacturing, Irvine, CA), and calculated automatically as a mean value over 12 cardiac cycles (1). Data were collected by computer for later calculation of mean values over 5- and 10-min periods. The reliability of this method has previously been confirmed by White et al. (22).

Seventeen milliliters of blood were sampled at 10-min intervals and immediately transferred into chilled tubes. The catheter was thereafter flushed with 15-20 ml of isotonic saline. Samples for determination of concentrations of plasma NE and plasma epinephrine (E) were transferred to polyethylene tubes containing 20 µl/ml blood of a mixture of reduced glutathione and EGTA (0.195 mol/l glutathione, 0.250 mol/l EGTA) adjusted to pH 6-7 with NaOH. The samples were immediately placed on ice and subsequently centrifuged at 4°C at 1,500 g for 10 min. Plasma was thereafter transferred to polyethylene tubes and frozen at 30°C for later analysis by a radioenzymatic assay as previously described by Knudsen et al. (10). Several standards and blanks were included in each assay. The intra-assay coefficient of variation for NE and E were 6 and 8%, respectively. Corresponding values of interassay coefficients of variation were 7 and 11%, respectively (10).

The tubes for determination of plasma concentration of AVP contained EDTA and aprotinine, and those for plasma osmolality determination contained 15 IU of lithium-heparin/ml blood. Plasma osmolality was measured in triplicate on fresh samples by freezing-point depression (Advanced Instruments; 3MO Plus).

Plasma concentrations of AVP were measured by RIA as previously described by Emmeluth et al. (3). The detection limit was 0.15 pg/ml, and recovery of unlabeled AVP added to plasma was within 72 ± 4%. The intra-assay coefficient of variation was 3.0% at 5.2 pg/ml and the interassay coefficient of variation was 7.9% at 2.4 pg/ml and 16.7% at 0.4 pg/ml. Results are not corrected for incomplete recovery. Two subjects had AVP values below detection limit throughout Seat-1 and Sup on one study day, so AVP data from these subjects are not included in the presentation. In the presented AVP data, 9 values out of 286 (6 values during Sup, 2 values during Seat, and 1 Control value) were undetectable, and in these cases the detection limit was used in the calculations. Therefore, the decrease in AVP during Sup could be underestimated. Using 0 pg/ml instead of 0.15 pg/ml for these values would, however, not change the conclusion.

An ANOVA (Statgraphics Plus for Windows, version 3.0) for repeated measures with the variable as main variate and time and subject as factors was used to evaluate the effects on a variable over time within each series of experiments compared with the mean value of Seat-1. Differences between mean values were evaluated by a post hoc multiple range test (Newman-Keuls). A paired Student's t-test was used to detect whether means differed at selected points in time of the two sessions (Sup and Sup + LBNP, respectively). If heterogeneity of variances was observed, logarithmic transformation of the data was performed before analysis. P < 0.05 was chosen as the level of significance.

In Figs. 1-3, the mean values at corresponding points in time of the two control sessions are presented as one Control for simplification. The statistics, however, were performed separately for each control session. When it is indicated that the values did not differ from Control, this implies that they did not differ from either of the two control sessions. Furthermore, values of arterial pressures in Table 1 are only depicted at 10-min intervals, except for immediately after the posture changes.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiovascular responses. MAP immediately decreased and remained so throughout Sup from a mean value of 98 ± 2 to between 91 ± 2 and 93 ± 2 mmHg (P < 0.05, Fig.1). During Sup + LBNP, MAP decreased during the initial 5 min from a mean value of 98 ± 2 to a nadir of 93 ± 2 mmHg and then increased toward the level of Seat (P < 0.05, Fig. 1). The decrease in MAP during Sup was more pronounced than that during Sup + LBNP (P < 0.05, Fig. 1). Calculated CSP increased during SUP, but the increase was more pronounced during Sup + LBNP (Fig. 1). DAP exhibited a pattern very similar to that of MAP. There were no significant changes in SAP (Table 1). PP increased during Sup from a mean value of 40 ± 2 to a maximum of 48 ± 2 mmHg (P < 0.05), whereas no significant changes occurred during any other session (Fig. 2).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Mean arterial pressure (MAP, solid lines) at level with aortic valve and at level with carotid sinus (CSP, dashed lines) and heart rate (HR) during a posture change from seated (Seat) to supine (Sup) with () and without () lower body negative pressure (LBNP) to maintain left atrial diameter (LAD) unchanged and during seated control (). CSP is calculated by subtracting from MAP the hydrostatic column from the carotid sinus to heart level (see METHODS). Therefore, MAP and CSP are identical in Sup position. Values are means ± SE; n = 9 (MAP) and 10 (HR) subjects. # Significant difference from Seat-1 (P < 0.05); * value of Sup significantly different from that of Sup + LBNP at same experimental point in time (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   LAD and arterial pulse pressure (PP) during Sup, Sup + LBNP, and Control as described in legend to Fig. 1. Values are means ± SE; n = 10 (LAD) and 9 (PP) subjects. # Significant difference compared with mean value of Seat-1 (P < 0.05).

HR exhibited a pattern very similar to that of MAP, with a decrease during Sup from a mean value of 63 ± 2 to between 55 ± 2 and 58 ± 2 beats/min (P < 0.05, Fig. 1). During Sup + LBNP there was a less pronounced decrease (P < 0.05) during the initial 2.5 min from a mean value of 62 ± 2 to a nadir of 58 ± 3 beats/min (P < 0.05), and HR thereafter increased toward the level of Seat (Fig. 1).

LAD increased from 30 ± 1 to 33 ± 1 mm during Sup and was unchanged during Sup + LBNP and Control (P < 0.05, Fig. 2). LADa exhibited the same pattern as LAD (Table 1).

SV increased during Sup from between 95 ± 8 and 100 ± 10 ml to a maximum of 118 ± 9 ml (P < 0.05, Table 1). During Control there were no significant changes in SV, whereas it decreased at one point in time at the end of Sup + LBNP (Table 1). CO decreased during Sup + LBNP from between 6.2 ± 0.4 and 6.3 ± 0.3 l/min to a nadir of 5.3 ± 0.3 l/min (P < 0.05), whereas it was unchanged during Sup and Control (Table 1).

Endocrine responses. Plasma concentration of AVP (n = 8 subjects) decreased during Sup from between 0.8 ± 0.1 and 1.0 ± 0.3 to 0.5 ± 0.1 pg/ml (P < 0.05), whereas it was unchanged during Sup + LBNP and Control (Fig. 3). Plasma NE likewise decreased during Sup from between 170 ± 22 and 182 ± 20 to a nadir of 118 ± 16 pg/ml, and was unchanged during Sup + LBNP and Control (P < 0.05, Fig.3). Plasma E exhibited a pattern similar to that of NE (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Plasma concentrations of arginine vasopressin (AVP) and norepinephrine (NE) during Sup, Sup + LBNP, and Control as described in legend to Fig. 1. Values are means ± SE; n = 8 (AVP) and 10 (NE) subjects. # Significant difference from Seat-1 (P < 0.05).

Plasma osmolality varied insignificantly between 284 ± 1 and 287 ± 1 mOsm/kgH₂O (Table 1).

Urinary sodium excretion for 24 h before the two experimental days was 66 ± 8 and 72 ± 7 mmol/24 h, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results indicate that static carotid baroreceptor stimulation per se is not sufficient to reduce vasopressin secretion during a moderate posture change from seated to supine. By LBNP we maintained LAD and PP unchanged, whereas carotid baroreceptors were stimulated. This maneuver prevented plasma vasopressin concentration from decreasing. Thus the changes in atrial dimension, pulsatile baroreceptor input, or both appear to be necessary for the inhibition of vasopressin secretion during a moderate antiorthostatic maneuver. Together with results of other studies (13, 14, 21) our data suggest that the combined stimulation of the said receptors are required.

Numerous investigations have focused on the relative importance of arterial high-pressure vs. cardiopulmonary low-pressure receptors in the release of AVP in humans and animals (for reviews, see Refs. 12, 21). For decades it has been anticipated that the regulation of AVP release during central hypo- and hypervolemia is primarily governed by cardiopulmonary low-pressure receptors (5, 6, 8). This concept was put in doubt already in 1975 by Goetz et al. (7), who questioned the experimental models used at the time for not being sufficiently selective to stimulate or inhibit the low-pressure receptors.

To selectively stimulate the low-pressure receptors without affecting those of the arterial system, Norsk et al. (13) used low-level lower body positive pressure and observed that plasma AVP did not change even though central venous pressure (CVP) and MAP increased, but PP remained unchanged. Using low-level LBNP where CVP decreased, but with no changes in PP and MAP, plasma AVP was likewise unchanged (14). Higher levels of LBNP, however, where PP narrowed but MAP was unaffected, induced more than a threefold increase in plasma AVP (14). These results suggest that isolated, moderate low-pressure receptor inhibition or stimulation in humans is not sufficient to modulate AVP release. Thus regulation of AVP release in humans could be determined by changes in stimulation of arterial baroreceptors or, more likely, by an interaction between cardiopulmonary low- and arterial high-pressure receptors during ortho- and antiorthostatic maneuvers.

In accordance with results of the above-mentioned studies (13, 14), we in the present experiment observed that suppression of AVP release was abolished when an increase in LAD and PP was prevented. Thus the effects of static carotid baroreceptor stimulation could be isolated from those of high-pressure dynamic and low-pressure receptor stimulation, because carotid sinus pressure increased during the antiorthostatic posture change combined with LBNP (Sup + LBNP), whereas PP and LAD were unchanged. It is noteworthy that the increase in calculated mean CSP actually was more pronounced during Sup + LBNP, where AVP release was unchanged, than during Sup (Fig. 1). Thus isolated, static carotid baroreceptor stimulation is not capable of suppressing AVP release during a moderate antiorthostatic maneuver.

In a previous experiment from our laboratory, Kamegai et al. (9) observed that the augmented AVP release during head-up tilt in humans was attenuated by simultaneous application of neck suction. It was concluded that carotid baroreceptor unloading participates as a mechanism in AVP release during orthostasis. This conclusion and those of other investigators (20) made us hypothesize that carotid baroreceptor stimulation is an important mechanism for suppression of AVP during a moderate antiorthostatic maneuver. The hypothesis is, however, rejected by the present results, because plasma AVP was unchanged even though the carotid baroreceptors were stimulated. The discrepancy between these results and those of Kamegai could be a result of the fact that the carotid baroreceptors might play an important role in regulation of AVP release, when the aortic and low-pressure baroreceptors are simultaneously affected. In other words, static carotid stimulation might augment the effects of the low-pressure receptors on AVP-release. Another explanation could be that we only tilted the upper part of the body, whereas Kamegai et al. used a more powerful orthostatic stimulus (head-up tilt). The carotid baroreceptors probably play a role in regulation of AVP release during a more pronounced antiorthostatic stimulus such as passive whole-body tilting.

We have previously been unable to detect a significant change in plasma AVP during a similar posture change (18). The main purpose of the previous study, however, was not to evaluate neuroendocrine responses but to investigate the effects of low- and high-pressure receptor stimulation on arterial pressures and HR (18). In the present study, the main purpose was to gain further insight to baroreflex-mediated control systems by focusing on AVP release. Therefore, we prolonged the period of intervention, had access to a more precise RIA, increased the number of subjects (regarding AVP measurements), controlled the sodium intake, and introduced a seated control session. It turned out that in this case plasma AVP was unchanged during Sup + LBNP but significantly decreased during Sup.

The present results confirm our previous findings that the hypotensive response to an antiorthostatic maneuver is attenuated when LBNP is applied to prevent LAD and PP from increasing (18). The lack of stimuli from the latter two variables apparently caused an attenuated decrease in HR, and this in turn could have weakened the decrease in MAP. Thus we conclude that during carotid baroreceptor stimulation caused by a moderate antiorthostatic maneuver, the decrease in MAP is attenuated when LAD and PP are prevented from changing.

In compliance with our previous findings (18), forearm venous plasma concentration of NE decreased during Sup, whereas it was unchanged during Sup + LBNP. This is consistent with results of Mancia et al. (11) and results from our laboratory (B. Pump, T. Kamu, A. Gabrielsen, P. Bie, N. J. Christensen, and P. Norsk, unpublished observations), demonstrating that plasma concentration of NE did not change during stimulation of the carotid baroreceptors by a neck chamber technique. Thus results of this study confirm that NE release into forearm venous blood is unaffected by carotid baroreceptor stimulation.

SV increased during Sup, whereas the increase was abolished during Sup + LBNP. The lack of increase in SV during Sup + LBNP is consistent with the observation that the increase in PP was abolished. Thus when LAD is kept unchanged by application of LBNP, SV and PP are prevented from increasing.

In conclusion, static carotid baroreceptor stimulation per se does not suppress vasopressin release in humans during a moderate antiorthostatic posture change from seated to supine, when LAD and PP are prevented from increasing. On the basis of results of this study and those of others (13, 14, 21), we suggest that the combined increase in LAD and PP is pivotal for the decrease in vasopressin release to occur during a moderate antiorthostatic maneuver.

Perspectives

An elucidation of the relative role of high- and low-pressure receptors, respectively, on regulation of release of vasoactive hormones may provide a better understanding of the pathophysiology of diseases with disturbances in autonomic regulation of the cardiovascular system. Heart failure patients, e.g., exhibit elevated plasma levels of vasoconstrictor hormones and increase in systemic vascular resistance, leading to a vicious circle with increase in cardiac afterload and accumulation of fluid and electrolytes. Defining the relative contribution of each type of baroreceptor (cardiopulmonary, carotid, and aortic, respectively) and how they interact in regard to regulation of release of vasoactive hormones might have implications for development of fluid and sodium regimes in cardiovascular diseases and for positioning of the patients. Thus understanding the normal and pathophysiological mechanisms of regulation of vasoactive hormones may lead to new approaches in the treatment of diseases.