INTRODUCTION

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THE ATRIAL RECEPTORS REGULATE intravascular volume through a cascade of neural and hormonal pathways (9, 24). Blood volume expansion stretches these receptors, which, through stimulation of cardiac afferent vagal fibers, activate key central neural areas for integration of cardiovascular homeostasis. The outcome is inhibition of sympathetic outflow, increased renal output, and restoration of blood volume to normal (11, 24). In addition to the reflex neural changes, atrial distension increases release of atrial natriuretic factor, which also acts to increase renal excretion of sodium and water (4, 7, 8).

Normal pregnancy is characterized by a marked increase in blood volume (10, 19) and cardiac output (17). Previous studies showed that localized distension of the superior venoatrial junction significantly increases the expression of the immediate early gene c-fos in the paraventricular region of the hypothalamus (3). The response is abolished in pregnant rats. These findings suggest two possibilities: that during pregnancy, central processing of signals from the atrial volume receptors is altered and/or neural afferent output from the atrial volume receptors is altered, i.e., the transducer properties of the mechanoreceptors are changed. Recent findings that discharge of the high-frequency type of cardiac receptors is suppressed in pregnant rats, while those of low-frequency type are maintained (5), support the latter possibility.

In light of the evidence that nitric oxide (NO) biosynthesis is significantly increased during pregnancy (1), NO has been shown to suppress baroreceptor activity (13), and central NO attenuates the baroreceptor reflex (14), we proposed that NO might also be responsible for blunting the atrial volume receptor reflex. We hypothesized that, by blocking NO biosynthesis during pregnancy, the central response to atrial distension would be restored to prepregnant levels. To this end, female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), or its inactive enantiomer D-NAME, were implanted subcutaneously. Seven days later, the atrial balloons were inflated for 1 h. The rats were then anesthetized, perfused with fixative, and brain sections were prepared for visualization of hypothalamic c-fos expression. To determine whether the observed changes were occurring at the level of the atrial mechanoreceptors, or during central neural processing of the afferent signals, the effect of NO on atrial receptor afferent activity (single-fiber recordings) was measured during distension of the atrial balloon.

	MATERIALS AND METHODS

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The experimental procedure was approved by the local Animal Welfare Committee in accordance with the guidelines issued by the Canada Council on Animal Care. All animals were killed with an anesthetic overdose of pentobarbital sodium at the completion of the studies.

Animal and housing. A total of 53 female Long-Evans rats (250-300 g) was used in this study (experiment A: 43; experiment B: 10). Long-Evans rats were obtained from Charles River Canada (St. Foy, Quebec) 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.

Experiment A

Surgery. Surgery was carried out under pentobarbital sodium anesthesia (62 mg/kg body wt ip), under sterile condition. Silastic cannulas (0.51-mm ID, 0.94-mm OD; Dow Corning) were implanted nonocclusively into the inferior vena cava. A small inflatable balloon cannula was passed down the right jugular vein and secured to the clavicle such that the tip of the balloon lay at the venoatrial junction (6). The anatomy of the rat is such that inflation of the balloon (50 µl) does not interfere with venous return to the heart. After the initial surgery, the rats were allowed to recover to their preoperative weights, which in this study took ~2 wk.

Experimental protocol. The rats were randomly allocated to the following groups: 1) virgin rats with atrial distension, 2) virgin rats without atrial distension, 3) 21-day pregnant rats with atrial distension, and 4) 21-day pregnant rats without atrial distension. The rats in groups 3 and 4 were subjected to vaginal smear and mated. The success of pregnancy was estimated by the increase in body weight 7 days later. On day 14 of pregnancy, osmotic minipumps (model 2ML1, DURECT) containing either D- or L-NAME (120 mg/2 ml at 10 µg/min; Calbiochem) were implanted subcutaneously in all animals (i.e., groups 1-4). Five days later (day 19), the rats were transferred to metabolic cages for habituation to the living conditions and for ease of accessing the cannula. On day 20, the rats were infused with saline (3 ml/h) via the inferior vena caval cannula for 2 h. The atrial balloons were then inflated for 1 h, during which time the saline infusion continued. (We found that, because rats consume water primarily at the start of their dark cycle in the early evening, they are slightly hypovolemic when the experiments start 15 h later. It has been our experience that, to elicit a robust renal response to atrial distention, we must slightly volume load them.) The rats were then deeply anesthetized with pentobarbital sodium and perfused intracardially with 4% paraformaldehyde. The fixed brains were processed for visualization and quantitation of c-fos expression.

Immunohistochemistry and quantitation of c-fos. The brains were prepared for visualization of c-fos-immunoreactive neurons in the medial preoptic area (MPO) and subdivisions of paraventricular region of the hypothalamus as previously described (3).

Statistics. Unpaired Student's t-test was used to examine statistical significance of changes in c-fos expression in response to atrial distension. Significance was accepted with P < 0.05.

Experiment B

Surgical procedures. Under pentobarbital sodium anesthesia (62 mg/kg body wt) plus atropine (0.1 ml, 0.4 mg/ml), the rats were implanted with intra-atrial balloons and cannulas in the femoral vein (for infusion of saline at 3.0 ml/h) and artery (for measurement of blood pressure). The rat was ventilated, and a midline sternotomy was performed for direct visualization of the intracardiac balloon. Once the balloon was positioned and secured at the clavicle, the chest wall was closed and spontaneous breathing was resumed. A supplementary dose of Inactin [ethyl-(1-methylpropyl)-malonyl-thio-urea; 60 mg sc] was administered to maintain a level plane of anesthesia. The right cervical vagus nerve was then dissected free of the carotid artery and placed on a platform for dissection.

Recordings. The vagus nerve was dissected under warm mineral oil, and thin filaments were 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 was displayed on a PC (10-kHz sampling rate, Windaq, Dataq Instruments, Akron, OH) along with blood pressure and ECG waveforms.

Experimental protocol. 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, infusion of the NO donor [S-nitroso-N-acetyl-penicillamine (SNAP), Sigma Canada: 10 µg · 50 µl¹ · min¹] or saline (50 µl/min) was initiated. Three recording sessions were carried out: 1, 3, and 5 min following the commencement of drug/saline infusion. In the course of one recording session, fibers were stimulated with inflation of the balloon (25 and 50 µl) with a period of 10 s for each increment. Baseline mean arterial pressure (MAP), ECG, and atrial receptor afferent discharge were recorded for 10 s before and 20 s after balloon inflation. After completion of the experimental protocol for each fiber, the chest was reopened and the mechanosensitive receptive field was confirmed by probing the venoatrial junction. The site that, when probed, produced a high-frequency discharge was accepted as the receptor location.

	RESULTS

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Experiment A

Spatial analysis of the paraventricular nucleus revealed that atrial distension increased c-fos expression in all subdivisions (periventricular, parvocellular, and magnocellular nuclei) in the virgin rats (Fig. 1) and that the increase was abolished in the D-NAME- (Fig. 1C) but not the L-NAME-treated pregnant rats (Fig. 1D). The increase in c-fos expression was most pronounced in the parvocellular subdivision (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of atrial distension and nitric oxide (NO) synthase inhibition on c-fos expression in subdivisions of the paraventricular nucleus in virgin [A: D-NAME, B: NG-nitro-L-arginine methyl ester (L-NAME); n = 6] and pregnant (C: D-NAME, D: L-NAME; n = 5) rats. Open bars represent without balloon inflation, and hatched bars represent after balloon inflation. Vertical bars delineate SE of mean. N equals number of animals studied. *P < 0.05.

Atrial distension also increased the number of c-fos-expressing neurons in the MPO in virgin rats (Fig. 2A). In the pregnant animals, the response to atrial distension was greatly reduced in the D-NAME-treated animals and restored by L-NAME (Fig. 2B).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of atrial distension and NO synthase inhibition on c-fos expression in the medial preoptic region in virgin (A, n = 6) and pregnant (B, n = 5) rats. Open bars represent without balloon inflation, and hatched bars represent after balloon inflation. Vertical bars delineate SE of mean. N equals number of animals studied. *P < 0.05.

Experiment B

Eight of the receptors had low spontaneous frequency of 3.07 ± 0.45 Hz with a mean discharge frequency of 11.90 ± 0.60 and 10.10 ± 1.30 Hz in response to balloon inflation at 25 and 50 µl, respectively. The response of these receptors was unrelated to the degree of atrial distension. Two receptors had a higher spontaneous frequency of 12.00 ± 5.00 Hz and responded to 25-µl inflation with a discharge frequency of 18.50 ± 2.50 Hz and to the 50-µl inflation with a discharge frequency of 24.50 ± 1.50 Hz (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Afferent discharge of lower (solid lines) and higher (interrupted lines) frequency atrial receptors in animals infused with S-nitroso-N-acetyl-penicillamine (, n = 6) or saline (, n = 5) in response to graded inflation of indwelling atrial balloon. N refers to number of atrial receptor afferent fibers studied.

Five receptors were studied during SNAP infusion, four during saline infusion, and one with both vehicle and SNAP. Mean spontaneous and stimulated discharge in both the lower and higher frequency atrial receptors from rats given SNAP did not differ from that observed in the saline-injected controls (Fig. 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously observed that reflex central neural activation in response to atrial distension is attenuated during pregnancy (3). We postulated that NO was, at least in part, responsible for this change. The results of the current study, that L-NAME restores the central response to atrial distension in pregnant rats, support our hypothesis. This cannot be attributed to an alteration in the transducer properties of atrial volume receptors, because administration of the NO donor SNAP did not suppress either spontaneous or stimulated discharge of the atrial receptors. We conclude therefore that NO alters central neural processing of afferent signals from the atrial volume receptors.

Hines and Hodgson (5) found afferent discharge from the heart to be attenuated during pregnancy. On the basis of these results, and given our postulate that NO is responsible for the pregnancy-induced changes in reflex function, we would have expected to find that SNAP attenuated atrial receptor discharge in our experiments. It had no such effect. On the other hand, the nature of the stimulus was quite different in our study. Hines and Hodgson (5) injected an intra-atrial bolus of saline, which would have activated many receptor types in the ventricles as well as in the atria. We discretely distended the right vein/atrial junction. At the end of the experiment, we were able to localize the atrial receptor from which we had been recording. It is possible therefore that, whereas the activation properties of the atrial volume receptors are not altered during pregnancy, the characteristics of other cardiac receptors are changed. This suggestion would be consistent with the finding that arterial baroreceptor activity is modulated by NO (13).

There is precedent for suggesting that NO may influence central control of the cardiovascular system. It has been shown that baroreflex control of renal sympathetic nerve activity and heart rate is attenuated by intracerebroventricular infusion of an NO donor and that the hypertension associated with chronic administration of the NO synthase inhibitor L-NAME may be attributed, in part, to increased sympathetic outflow (14, 20). Whereas L-NAME suppresses excitability of neurons in the nucleus of the solitary tract (12), NO donors, microinjected into the rostral ventrolateral medulla, attenuate renal sympathetic nerve activity and lower blood pressure (18). Apart from implicating guanosine 3',5'-cyclic monophosphate (22), little progress has been made in determining the mechanism by which NO might influence central control of sympathetic outflow. Ours is the first study to investigate the role of NO in modulating reflex central neural control of blood volume.

Nonmedullated atrial receptor afferents in rats are generally classified into low- and high-frequency subsets (15, 23). In this study, nearly all of the receptors localized had low spontaneous discharge of <5 Hz and responded to atrial stretch with an increased, irregular discharge. This response was transient, beginning to fade on an average of 4 s following administration of the stimulus. In addition, discharge frequency of these receptors appeared to be unrelated to the degree of atrial stretch, i.e., differential degrees of atrial distension were marked by an increase in recruitment rather than an increase in firing frequency of any given receptor. Nevertheless, their stimulated discharge frequency was significantly greater than that reported by Hines and Hodgson (5). Again, this is most likely due to differences in the populations of cardiac receptors that were stimulated by balloon inflation vs. saline injection. Only two receptors were found that discharged with a higher frequency that appeared to be related to the degree of atrial stretch (14-µm circumference at 50 µl vs. 11 µm at 25 µl). Evidence indicates that receptors discharging with a higher frequency make up a significantly smaller component of the total atrial receptor population (5, 23).

It could be argued that the effect of L-NAME on central activation during pregnancy was influenced by the change in baseline blood pressure. However, we previously showed that this same dose of L-NAME causes an equipressor effect in virgin and pregnant rats (25), whereas the effects on blood volume and central neural activation are limited to the pregnant animals. Our results also confirm that neither D- nor L-NAME imposes direct intrinsic effects on the atrial volume receptors, because neither had any effect on baseline or stimulated c-fos expression in the virgin rats.

The paraventricular region of the hypothalamus in rat comprises a heterogenous population of nuclei that are generally categorized into subdivisions based on cytoarchitecture, afferent and efferent projections, and neurotransmitters (21). For the present study, we focussed on the periventricular, parvocellular, and magnocellular subdivisions of the paraventricular nucleus and the MPO. We showed that atrial distension increased activation in all these areas but in particular the parvocellular division. This area of the paraventricular nucleus is divided into several different regions, each of which receives targeted input from cell groups in the medulla and other nuclei in the hypothalamus. The region also has targeted output to the median eminence, posterior pituitary gland, and/or preganglionic neurons in the medulla or spinal cord. Activation of the parvocellular division of the paraventricular nucleus thus influences neurohormone secretion, the hypothalamo-pituitary-adrenal axis, and sympathetic outflow to organs such as the kidney, each of which contributes to volume regulation (2, 16, 21).

In conclusion, we confirmed our previous findings that, during pregnancy, the central reflex response to stimulation of the atrial volume receptors is attenuated. We also showed that this is mediated, at least in part, through NO. Furthermore, we showed that the reduction in atrial distention-induced activation of central neural pathways cannot be attributed to an alteration, by NO, of the transducer properties of the atrial volume receptors.