The atrial volume reflex is attenuated in pregnancy. This may be mimicked by chronic administration of 5α-pregnan-3α-ol-20-one (pregnan). We investigated whether afferent output from sensory receptors may be suppressed at this time. Vagal afferent nerve activity was measured during discrete localized stimulation of the atrial volume receptors by inflation of a balloon at the superior vena caval-right atrial junction. The receptors were classified as high- (HF) or low- (LF) frequency subtypes on the basis of their response to graded atrial distension. Although both HF (regression coefficient = 0.50 ± 0.11 Hz/μl, r2 = 0.47, P < 0.001) and LF (regression coefficient = 0.03 ± 0.05 Hz/μl, r2 = 0.009, P = 0.613) subtypes could be identified in virgin rats, only LF (regression coefficient = 0.09 ± 0.05 Hz/μl, r2 = 0.044, P = 0.099) receptors were found in late-pregnant animals. Similarly, in virgin rats treated chronically with pregnan (500 μg/24 h for 2 days), only LF receptors were identified (regression coefficient = −0.004 ± 0.078 Hz/μl, r2 = 0.000, P = 0.962), whereas both subtypes were present in the vehicle-treated animals (HF regression coefficient = 0.626 ± 0.255 Hz/μl, r2 = 0.317, P = 0.029; LF regression coefficient = −0.012 ± 0.071 Hz/μl, r2 = 0.002, P = 0.866). By contrast, acute intracardiac pregnan (2.6 μg/kg) did not alter vagal afferent nerve activity. In conclusion, stretch-induced discharge of high-frequency atrial receptors is suppressed during pregnancy, whereas that of low-frequency receptors is preserved. This effect may be mimicked by chronic, but not acute, pregnan. We propose that, during pregnancy, pregnan alters the transducer properties of the atrial volume receptors, thus allowing blood volume to increase.
- cardiopulmonary receptors
- blood volume
plasma volume increases progressively throughout pregnancy, reaching a plateau of 40–50% above prepregnancy levels by the onset of the third trimester (1, 2, 4). This increase in circulating volume is one of the earliest and most striking cardiovascular changes observed during pregnancy (12).
Studies have been carried out to examine how the atrial volume receptor reflex, the primary mechanism for regulation of blood volume, is altered in pregnancy. This reflex arc is activated by stretch of mechanosensitive receptors located at the venoatrial junctions, it is processed in specific nuclei in the brain, and it induces changes in water and electrolyte output from the kidney (5). We have shown that reflex control of renal function by the atrial volume receptors is attenuated during pregnancy such that the increase in sodium excretion and urine volume normally seen in response to atrial distension does not occur (8). In addition, there is a loss of activation of central components of the reflex pathway in response to atrial receptor stimulation in pregnant animals (2). These observations have led us to suggest that the homeostatic mechanisms that operate to maintain fluid balance are attenuated in the pregnant animal, thus allowing blood volume to increase.
Pregnancy is a state of great hormonal flux, and many of the physiological adaptations to pregnancy occur because of changes in circulating levels of specific hormones. The progesterone metabolite pregnan (5-α-pregnan-3-α-ol-20-one), plasma levels of which increase progressively during pregnancy, has been shown to modulate the baroreflex (14). Pregnan also mimics the effect of pregnancy on volume regulation, i.e., it causes plasma volume to increase and the central response to atria distension to be attenuated (3, 13). It is therefore reasonable to believe that pregnan is one of the factors released during pregnancy that is responsible for suppressing the volume receptor reflex. Yet the question still remained whether inhibition of the reflex is confined to the brain, or whether it involves an alteration at the sensory receptor level. This study was carried out to determine whether atrial receptor discharge is altered during pregnancy and in response to acute or chronic administration of the progesterone metabolite pregnan.
MATERIALS AND METHODS
The experimental procedures in the following studies were approved by the local Animal Welfare Committee in accordance with the guidelines issued by the Canada Council on Animal Care. At the completion of each experiment, the animals were euthanized with an anesthetic overdose (0.3 ml iv Euthanyl, 240 mg/ml pentobarbital sodium, MTC Pharmaceuticals, Cambridge, Ontario, Canada).
Animals and housing.
Female Long Evans rats (250–350 g) were obtained from Charles River (St. Foy, Quebec, Canada) and housed in a temperature- and humidity-controlled animal facility with a 12:12-h light-dark cycle (light 0700–1900) for at least 1 wk before the experiments. They were maintained on a 0.3% sodium diet and water ad libitum. Animals were studied randomly with respect to the estrus cycle. We have previously confirmed that the rats continue to cycle normally during chronic pregnan treatment (13).
Animals were anesthetized with sodium pentobarbital (60 mg/kg body wt) plus atropine (0.1 ml, 0.4 mg/ml), followed by a supplementary dose of Inactin [ethyl-(1-methylpropyl)-malonyl-thio-urea; 60 mg sc] to maintain a level plane of anesthesia. During surgery, the rats were placed on a Deltaphase isothermic heating pad (Braintree Scientific, Braintree, MA) which maintained body temperature at ∼37°C. Animals were then implanted with intra-atrial balloons (7) and cannulas (polyethylene, 0.58 mm ID, 0.97 mm OD) in the femoral vein (for infusion of isotonic saline, 3 ml/h) and artery (for measurement of arterial blood pressure). Animals were ventilated at a rate of 60 strokes/min, and a midline sternotomy was performed for direct visualization of balloon placement at the superior vena caval-right atrial junction. The peculiar anatomy of the rat, whereby blood from the left jugular vein enters the inferior vena cava, enables one to stretch the venoatrial junction without interfering with venous return to the heart (7). Once the balloon was positioned and secured, the chest wall was closed and spontaneous breathing was resumed. The right cervical vagus was then dissected free of the carotid artery and placed on a platform for dissection under warm mineral oil.
Fiber bundles were dissected down to small filaments and placed on a bipolar silver electrode. The nerve signal was amplified and filtered between 100 and 1,000 Hz (Leaf Electronics QT-5B; World Precision Instruments LPF-30, Sarasota, FL). Output from the amplifier was then fed to an oscilloscope (Tektronix 7613, Wilsonville, OR) and loudspeaker and displayed on a computer (10-kHz sampling rate, Windaq, Dataq Instruments, Akron, OH) along with blood pressure and ECG waveforms.
Atrial receptor identification and localization.
Single fibers or small filaments containing three to four fibers were screened by an initial balloon inflation of 25 μl. Once a response had been established (increase in discharge frequency of >20%), fibers were stimulated with graded inflation of the intra-atrial balloon (15, 25, 35, and 50 μl, experiments A and C, or 25, 35, and 50 μl, experiment B) for periods varying from 10 to 60 s for each increment. In those cases in which activity was detected from more than one fiber in the bundle, output from the individual receptors was identified visually on the basis of amplitude and waveform and analyzed individually (Fig. 1). Baseline arterial pressure (AP), ECG, and atrial receptor (AR) afferent discharge were recorded for 20 s before and 20 s after balloon inflation (Fig. 2). After completion of the experimental protocol, the chest was reopened and the mechanosensitive receptive field of the nerve being recorded was confirmed by probing the venoatrial junction. The site that produced a high-frequency discharge when probed was accepted as the receptor location.
The receptors were classified as high frequency (HF) or low frequency (LF) on the basis of their response to atrial distention (15, 19, 20). The HF receptors exhibited spontaneous activity and a sustained, linear stimulus response to graded inflation of the intra-atrial balloon. By contrast, the LF receptors were often silent during baseline measurements and responded to balloon inflation with a burst of irregular discharge that, beyond the initial increase at the lowest level of balloon inflation, was unrelated to the degree of atrial distention. The analysis of the nerve discharge was based on average discharge rate (spikes/s or Hz). The balloon was inflated over a period of 1 s. The mean discharge frequency was then measured for 2 s after completion of atrial distension i.e., 3 s after initiation of balloon inflation. During this time, the increase in receptor activity and maximum change in tissue distention occurred (Fig. 2). This was then compared with the mean discharge frequency in the 2-s period immediately preceding balloon inflation. Receptors that attained a discharge frequency of >30 Hz at the highest level of balloon inflation (50 μl) were classified as HF. Those falling below this cutoff were classified as LF receptors.
Experiment A: Pregnancy.
Rats were randomly allocated to one of two groups: 1) pregnant (n = 8) or 2) age-matched virgin (n = 8). The rats in group 1 were subjected to vaginal smears and mated at proestrus. The success of pregnancy was estimated by the increase in body weight 7 days later. On day 20 of gestation the experiments were performed. To confirm viability of pregnancy, fetuses were counted at the end of each experiment.
Experiment B: Acute pregnan.
Virgin rats were prepared for vagal afferent nerve recording as described above. In addition, a cannula was implanted in the external jugular vein adjacent to the balloon cannula, for the administration of drug or vehicle. Once an atrial receptor had been identified, pregnan (0.2 ml, 2.6 μg/kg in 20% solution of 2-hydroxypropyl-β cyclodextrin in sterile distilled water, n = 8) or vehicle (n = 7) was infused over 10 s into the atrium. It was estimated that, at a cardiac output of 115 ml/min, this would raise plasma pregnan levels to high physiological levels (∼45 ng/ml). Afferent nerve activity was measured 3 min later.
Experiment C: Chronic pregnan.
Virgin rats were randomly chosen to receive subcutaneous injections of either pregnan [500 μg in 20% 2-hydroxypropyl-β-cyclodextrin (Sigma) in sterile distilled water, n = 6], or vehicle (20% 2-hydroxy-β-cyclodextrin, n = 7) once daily (8 AM) for 2 consecutive days. Experiments were then carried out (in the afternoon of the second day of injection) as described in experiment A. It is estimated that this dose raises plasma pregnan levels to approximately the level seen in midpregnancy (20–30 ng/ml) (11, 13, 16).
Mean arterial pressure (MAP) was calculated from the 20-s baseline period preceding inflation and the 10- to 30-s period during balloon inflation. Hemodynamic measurements were compared between pregnant (or hormone-treated) and virgin (or vehicle-treated) groups by ANOVA or by unpaired Student's t-tests. Mean values of nerve discharge frequencies at the different degrees of balloon inflation were analyzed and compared by repeated-measures ANOVA followed by Student-Newman-Keuls post hoc test. Slopes and r2 values for the receptor stimulus-response curves were determined by linear regression analysis. Values were presented as means ± SE, and a P value < 0.05 was considered significant.
Baseline MAP in the virgin rats (control groups) was unaffected by 50-μl balloon inflation (preinflation: 94.6 ± 2.5 mmHg; postinflation: 90.7 ± 2.2 mmHg, n = 18, P > 0.05). In the pregnant animals, baseline MAP was significantly lower (76.9 ± 4.4 mmHg, n = 8) compared with the matched virgin rats (98.6 ± 3.6 mmHg, n = 8; P < 0.05); it also was not altered by balloon inflation (preinflation: 76.9 ± 4.4 mmHg; postinflation: 72.7 ± 4.7 mmHg, n = 8; P > 0.05). MAP in the virgin rats was unaffected by pregnan administration either acutely (pregnan: 95.2 ± 4.4 mmHg, n = 8; pretreatment baseline: 100 ± 7.5 mmHg, n = 8; vehicle: 90.1 ± 2.9, n = 6; P > 0.05) or chronically (pregnan: 94.3 ± 2.6 mmHg, n = 9; vehicle: 88.1 ± 4.8 mmHg, n = 7; P > 0.05).
Experiment A: Pregnancy.
Twenty-three functional single fibers were dissected in eight pregnant and eight virgin rats. Forty-five percent of atrial volume receptors identified in the virgin animals were deemed to be HF receptors on the basis of their discharge frequency exceeding 30 Hz at the highest level of atrial distention (50 μl). In these fibers, the frequency of afferent discharge increased with increasing atrial distention, in response to both the initial inflation of 15 μl and progressively increased volumes of inflation above 15 μl (regression coefficient = 0.50 ± 0.11 Hz/ml, r2 = 0.47, P < 0.001) (Fig. 3, A and C). Although the LF receptors also increased their rate of firing to 15-μl inflation, there was no further response with progressively larger volumes of inflation (regression coefficient = 0.03 ± 0.05 Hz/ml, r2 = 0.009, P = 0.613) (Fig. 3, A and C). The mean baseline rate of firing of the HF receptors (20.1 ± 3.3 Hz) was also higher than that of the LF receptors (6.8 ± 1.8 Hz, P < 0.001).
In contrast to the two populations of receptors localized in virgin rats, all receptors localized in pregnant rats exhibited a low resting discharge frequency that, although it increased in response to the initial 15-μl balloon inflation, failed to increase further with increased volumes of balloon inflation (regression coefficient = 0.09 ± 0.05 Hz/ml, r2 = 0.044, P = 0.099) (Fig. 3, B and C)
Experiment B: Acute pregnan.
Seventeen functional single fibers were dissected in a total of 17 virgin rats, with nine of the receptors stimulated in the presence of drug and eight in the presence of vehicle. All receptors responded to the lowest level of balloon inflation (25 μl) with increased afferent discharge (Fig. 4, A and B). Forty-one percent of the receptors were classified as HF receptors on the basis of their responses to 50-μl inflation (Fig. 4A). Pregnan did not alter either the spontaneous discharge frequency (Fig. 4A) or the stimulus-response characteristics of these HF receptors to increases in atrial distention above 25 μl (regression coefficient: baseline = 0.503 ± 0.23 Hz/μl, r2 = 0.230, P = 0.041; pregnan = 0.573 ± 0.267 Hz/μl, r2 = 0.397, P = 0.069; vehicle = 0.463 ± 0.131 Hz/μl, r2 = 0.641; P = 0.010) (Fig. 4A, Fig. 4C solid bars).
As was found in experiment A, discharge of the LF receptors was only weakly related to the degree of atrial stretch, there being no further increase beyond the initial response to 25-μl balloon inflation (regression coefficient = −0.089 ± 0.12 Hz/μl, r2 = 0.019, P = 0.472) (Fig. 4, B and C). This characteristic discharge pattern of the LF receptors was unaltered by intracardiac infusion of pregnan (regression coefficient = −0.035 ± 0.10 Hz/μl, r2 = 0.007, P = 0.0.735) or vehicle (regression coefficient = 0.088 ± 0.188 Hz/μl, r2 = 0.021; P = 0.651) (Fig. 4C open bars). Neither was the mean spontaneous discharge of LF receptors altered by acute administration of pregnan (Fig. 4B).
Experiment C: Chronic pregnan.
Twenty-two functional single fibers were dissected in nine pregnan-treated and eight vehicle-treated virgin rats. In the vehicle-treated group, 36% of receptors were classified as HF receptors on the basis of their responses to 50-μl balloon inflation (Fig. 5A). The discharge frequency of these receptors increased progressively with increasing atrial stretch above the initial 15-μl balloon inflation (regression coefficient = 0.626 ± 0.255 Hz/ml, r2 = 0.317, P = 0.029) (Fig. 5, A and C). The remainder exhibited the characteristics of LF receptors, i.e., discharge frequency was unrelated to the degree of atrial stretch beyond 15 μl (regression coefficient = −0.012 ± 0.071 Hz/μl, r2 = 0.002, P = 0.866). In contrast to the two populations of receptors in the vehicle-treated rats, all receptors localized in pregnan-treated animals were classified as LF receptors: discharge frequency was unrelated to degree of atrial stretch beyond 15 μl (regression coefficient = −0.004 ± 0.078 Hz/μl, r2 = 0.000, P = 0.962) (Fig. 5, B and C), i.e., their characteristics did not differ from those of the LF receptors in the vehicle-treated rats. Although one fiber in the pregnan-treated group was found to have a discharge rate of 30 Hz at 50-μl balloon inflation (the cutoff point for HF receptors), further analysis revealed that it failed to respond to progressive atrial distension above the initial 15-μl inflation (regression coefficient = 0.271 ± 0.138, r2 = 0.657, P = 0.189). It was thus classified as type LF (Fig. 5B, dotted line).
The atrial volume receptor reflex is attenuated in pregnancy. Previous work has shown that activation of neurons in the hypothalamus in response to atrial receptor activation is reduced in pregnant rats compared with virgin rats (2). Thus the homeostatic mechanism that keeps fluid volume in check is suppressed during pregnancy. However, the question remained whether loss of central processing is due to an inhibition of areas of the brain and/or to a loss of afferent input from sensory receptors. We have now shown that the discharge characteristics of the right atrial volume receptors are indeed altered during pregnancy and that this may be mimicked by chronic administration of the progesterone metabolite pregnan.
Nonmedullated atrial receptors in rats have been divided into two subgroups: high and low frequency (6, 15, 19). Low-frequency receptors (also called rapidly adapting) respond to stretch of atrial tissue with a brief, irregular increase in discharge, whereas high-frequency receptors (slowly adapting) respond with a sustained discharge, often firing with the cardiac cycle (15, 19). Although some receptors showed cardiac rhythm in their spontaneous discharge activity, this was never observed during balloon inflation, probably because of the static nature of the stimulus.
We identified two clearly defined atrial volume receptor populations. We designated them “high-frequency” (HF) and “low-frequency” (LF) receptor subtypes as described by Thoren and colleagues (19, 20) and by Mifflin and Kunze (15). In our study, HF receptors showed a sustained and stretch-dependent frequency response and had a mean spontaneous resting discharge of ∼20 Hz. By contrast, LF receptors exhibited a burst of activity, the magnitude of which was independent of the size of the stimulus (distention), i.e., there was no further increase in afferent nerve activity in response to progressive atrial balloon inflation beyond the initial increase (15 μl in experiments A and C, and 25 μl in experiment B). Although the mean spontaneous resting discharge of the LF receptors (∼5 Hz) was less than that of the HF receptors, there was some overlap between the two subtypes. We chose therefore to make our classification based on the rate of discharge attained at the highest level of balloon inflation (50 μl). Those receptors that attained a discharge frequency of >30 Hz were classified as HF; this reflected their progressive rise in activity in response to increased atrial distension, as well as their higher average spontaneous rate of discharge. Those falling below this cutoff were classified as LF receptors; these receptors failed to increase firing beyond the initial response to the lowest degree of balloon inflation and generally had a lower spontaneous discharge rate. The data were quantified by linear regression analysis of the responses during progressive balloon inflation from the lowest (15 or 25 μl) to the highest (50 μl) volume. The HF receptors showed a significant regression slope, whereas the discharge of the LF receptors plateaued after the increase to the initial (lowest) balloon inflation, i.e., the regression coefficient was not significantly greater than zero.
None of the receptors crossed over from a LF to a HF discharge pattern during the experiment. Whereas both types of receptors could be identified in the virgin rats, all of the receptors localized in the pregnant animals were typical of the LF subtype. It has been postulated that LF receptors in the rat signal the brain concerning a change in volume, whereas HF receptors signal absolute volume (6). The loss of HF response to the progressive stimulus of atrial distension therefore suggests that pregnant animals may retain the ability to signal central nuclei concerning acute changes in volume but lose the ability to signal absolute volume.
Our results from the pregnant animals are in agreement with those of Hines and Hodgson (6), who also found that the function of cardiac receptors with low-frequency discharge is preserved, whereas that of low-frequency receptors is suppressed. However, the nature of the stimulus (a bolus saline load) precluded their being able to distinguish whether the afferent nerve traffic derived from atrial or ventricular receptors. We have now confirmed that these are indeed atrial volume receptors. It is also important to emphasize that the balloon ensured that an equal degree of atrial wall distention was applied to all animals, pregnant or virgin. [We have previously confirmed that right atrial dimensions do not change during pregnancy in the rat (9)].
Plasma levels of pregnan, a metabolite of the sex steroid progesterone, increase progressively during pregnancy (16). Previous work has shown that pregnan increases plasma volume and that it suppresses central processing of the atrial volume receptor reflex, thus mimicking the effects of pregnancy (13). Although it had been shown that pregnan can directly inhibit central processing via GABA receptors (11, 16), it was unknown whether pregnan could also alter afferent signaling of sensory receptors in the periphery. The present study measured the effect of acute and chronic pregnan administration on stretch-induced changes in atrial volume receptor discharge.
Acutely administered pregnan, injected directly into the external jugular above the site of atrial distension, had no effect on discharge of either LF or HF receptors. There was no significant difference in spontaneous or stimulated discharge in response to graded balloon distension after intra-atrial injection of pregnan compared with injection of the vehicle. Thus it would appear that the ability of pregnan to rapidly suppress the atrial volume receptor reflex is limited to its effects on GABAA receptors in the brain (3). By contrast, pregnan administered over a 2-day period did alter the pattern of afferent discharge of the atrial volume receptors. Whereas both LF and HF receptors were identified in the control animals, only LF afferent discharge could be found in the animals administered pregnan. Thus chronic administration of pregnan suppresses the response of HF atrial receptors to distension of the venoatrial junction, i.e., it mimics the effect of pregnancy on atrial volume receptor discharge. The inability of acutely administered pregnan to alter the discharge characteristics of the atrial volume receptors is consistent with the observation that this treatment also fails to modulate afferent arterial baroreceptor discharge (10).
Ring-A-reduced metabolites such as pregnan have been shown to indirectly activate intracellular progesterone receptors via production of metabolites that are ligands of the receptor (17). Thus pregnan may effect long-term changes in expression of neurotransmitter receptor subunits and voltage-gated ion channels, as well as modulation of ligand-gated receptors such as G-protein-coupled receptors. We propose that pregnan modifies atrial receptor activation through long-term changes in neuronal excitability. In addition, we suggest that pregnan attenuates the processing of atrial receptor afferent signals in the brain, probably by rapidly potentiating GABAergic inhibition of central nuclei (3, 18).
This study has shown that, during pregnancy, discharge of HF volume receptors in response to atrial distension is suppressed, whereas that of LF receptors is preserved. This effect could be mimicked by chronic administration of the progesterone metabolite pregnan. It is not possible to say whether this occurs by rendering the HF receptors inactive or by converting them to LF receptors. It has been suggested that the HF receptors signal absolute intravascular volume (6). We conclude therefore that, during pregnancy, pregnan alters the transducer properties of the atrial volume mechanoreceptors, such that information regarding the gradual increase in blood volume is not received centrally and does not elicit reflex restoration to “normal.”
This research was supported by a grant from the Canadian Institutes of Health Research.
We gratefully acknowledge the assistance of Dr. David Bennett and of Leo Sanelli in setting up the system for nerve recording.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society