The role of the sympathetic nervous system in hypertension has been investigated extensively, yet little research into the role of the parasympathetic nervous system has been reported (1,2). The parasympathetic nervous system has profound control over heart function, and its predominance over sympathetic control has long been recognized under normal physiological conditions. The parasympathetic nervous system may play a crucial role in hypertension (2,3). However, the contribution of the parasympathetic nerves has been thought mainly to be that the reduced activity of the parasympathetic nerve induces the sympathovagal imbalance.
The baroreceptor reflex may play an important role in the development and maintenance of hypertension (4). The parasympathetic limb of the baroreceptor reflex are consists of the baroreceptor itself, the central nervous system, the peripheral parasympathetic nerves, muscarinic receptors in cardiac tissue, and the post-receptor signaling system. Increased or decreased function of each component within the reflex are results in altered parasympathetic activity. This study involved investigation into the role of the peripheral parasympathetic nerves in the early phase of hypertension. Although some groups have reported attempts to determine the parasympathetic influence on hypertension in vitro (5-7), the significance in vivo remains speculative. The functional role of the parasympathetic system should be determined under physiological conditions in which endogenous acetylcholine is neurally released. Thus, a peripheral parasympathetic nerve was stimulated in decentralized animals, and the role of the peripheral parasympathetic nerves was determined in spontaneously hypertensive rats (SHR) at 5-7 weeks of age.
Sympathetic and parasympathetic nervous systems have opposing effects on cardiac function. Moreover, these nervous systems simultaneously affect each other. The interplay between the two nervous systems is not a linear summation of their respective effects. Complex mechanisms are evidently involved. For example, stimulation of the cardiac sympathetic nerve alone at 4 Hz increased the heart rate (HR) by 75 beats/min in dogs; vagal stimulation alone at 8 Hz decreased the HR by 75 beats/min (8). If the interplay between these two nervous systems was a linear summation, the net effect of combined stimulation would be zero; HR should neither increase nor decrease. However, combined stimulation decreased the HR by 75 beats/min. The effect of parasympathetic nerve activity on HR may be greater when sympathetic nerve activity is increased (accentuated antagonism) (9-11). Thus, the effect of interaction between the sympathetic and parasympathetic systems on HR in the early phase of hypertension was examined.
Experimental and control groups consisted of 5- to 7-week-old SHR (n = 11) and Wistar-Kyoto (WKY) rats (n = 14), respectively. All procedures were in accordance with institutional guidelines for animal care. Rats were anesthetized by intraperitoneal injection of 30 mg/kg pentobarbital. Experimental preparations have been described in detail elsewhere (12,13). Briefly, the trachea was exposed at the mid-cervical level. After artificial ventilation was begun, the chest was opened transversely at the second intercostal space. With the aid of a dissecting microscope (Natsui Co., Tokyo, Japan), both ansa subclaviae were carefully separated and crushed by tight ligature. Both cervical vagal trunks were separated and ligated tightly in order to interrupt almost all autonomic influence on the heart. Bipolar hook electrodes were placed in mineral oil on the cardiac end of the right ansa subclavia and on the right vagus nerve. The electrodes were connected to an electronic stimulator in parallel fashion with an isolation unit (Model SEN-3301; Nihon Koden Co., Tokyo, Japan). Thin needle electrodes were placed in the subcutaneous tissue of the limbs and connected to an electrocardiograph (ECG) amplifier (AC-600G; Nihon Koden Co.). A surface ECG (Lead II) was recorded on a thermal printer (WS-628G; Nihon Koden Co.) at a paper speed of 200 mm/s during measurements and 1 mm/s during the remainder of the experiment. Instantaneous HR was calculated from ECG tracings.
The right ansa subclavia and the right vagus nerve were stimulated at 4 Hz with a 5 V square-wave pulse (supramaximal voltage, 2 ms duration). Only the right ansa subclavia was stimulated for 90 s to determine the effect of sympathetic nerve stimulation alone. The vagus nerve was subsequently stimulated for 30 s to determine the effect of parasympathetic nerve stimulation alone. To determine the effect of combined sympathetic and parasympathetic nerve stimulation, the stimulation of the ansa subclavia was started and continued for 90 s. Thirty seconds after the initiation of the ansa subclavia stimulation, the vagus nerve was stimulated for 30 s concurrently. After the vagal stimulation was stopped, the ansa subclavia stimulation was continued for another 30 s.
Changes in HR during the various treatments were evaluated statistically by analysis of variance and the Student-Newman-Keuls (SNK) test (a posteriori). p < 0.05 was considered significant. Values are expressed as mean ± standard error.
The body weight of WKY rats (175 ± 9 g) did not differ statistically from that of SHR (161 ± 10 g). The mean blood pressure (MBP) in WKY rats and SHR was 79 ± 4 and 113 ± 4 mmHg, respectively, and MBP in SHR was significantly greater (p < 0.001) than that in WKY rats. HR in SHR (422 ± 5 beats/min) was significantly greater than that in WKY rats (369 ± 14 beats/min; p < 0.01). However, HR in both groups was similar after rats were decentralized in the open-chest condition (330 ± 8 and 342 ± 11 beats/min for WKY and SHR, respectively).
Sympathetic nerve stimulation
When the cardiac sympathetic nerve was stimulated, HR in the WKY group increased 9 ± 1 beats/min at 30 s, and this increase remained fairly constant (11 ± 2 and 9 ± 1 beats/min at 60 and 90 s, respectively). In SHR, HR increased 14 ± 3 beats/min 30 s after sympathetic nerve stimulation and remained constant throughout the stimulation period (13 ± 2 and 14 ± 2 beats/min at 60 and 90 s, respectively). Although HR in SHR was slightly greater than HR in WKY rats, the difference was not statistically significant.
Vagal nerve stimulation
The mean decrease in HR evoked by the 30-s vagal stimulation was 23 ± 4 beats/min in WKY rats and 62 ± 8 beats/min in SHR (Fig. 1). The HR decrement in SHR was significantly greater than that in WKY rats (p < 0.01).
Thirty seconds after initiation of cardiac sympathetic nerve stimulation, HR increased 9 ± 2 beats/min in WKY rats and 9 ± 2 beats/min in SHR (Fig. 2, S − 30). These changes in HR did not differ statistically. After stimulation of the vagus nerve was initiated and maintained concurrent with sympathetic nerve stimulation for 30 s, HR decreased 31 ± 4 beats/min in WKY rats and 73 ± 9 beats/min in SHR (Fig. 2, S + V Int). The decrement evoked by vagal nerve stimulation concurrent with cardiac sympathetic nerve stimulation was much greater in SHR than it was in WKY rats (p < 0.01). When vagal nerve stimulation was terminated and cardiac sympathetic nerve stimulation was continued for an additional 30 s, HR increased again to above the pre-stimulation control level of 11 ± 2 beats/min in WKY rats and 13 ± 3 beats/min in SHR (Fig. 2, S − 90). These changes in HR were not statistically different and did not differ statistically from the HR increases observed after the first 30 s of sympathetic nerve stimulation.
The decrement in HR evoked by the combined stimulation of sympathetic and parasympathetic nerves was greater than that evoked by vagus nerve stimulation alone in both WKY rats and SHR (p < 0.05). The interaction of sympathetic and parasympathetic nerve stimulation may be different in SHR than in WKY rats. Thus, the difference between HR change in response to the combined stimulation and that in response to vagus nerve stimulation alone was compared in SHR and WKY rats. The difference in HR change for WKY rats was −8 ± 3 beats/min, and that for SHR was −11 ± 5 beats/min (Fig. 3). These values were not statistically different. For each rat, the difference between HR change in response to combined stimulation and HR change in response to vagal stimulation alone was also calculated as a percentage of the combined stimulation. The difference in WKY rats and in SHR was 24 ± 8 and 14 ± 6%, respectively, and it was not statistically different.
Role of the parasympathetic nerves in the early phase of hypertension
Stimuli originating from baroreceptors activate cardiac effector cells. The resultant cardiac responses can conversely effect baroreceptor discharge to the central nervous system. The overall parasympathetic activity of the baroreceptor reflex may be reduced in the early phase as well as in the maintenance phase of hypertension when the reflex loop is intact (14-16). In the present experiments, the functional role of the peripheral parasympathetic nerves was investigated with respect to reduced activity of the parasympathetic limb of the baroreceptor reflex system in the pathogenesis of hypertension. For this purpose, rats were decentralized by ligating autonomic nerves tightly coupled to the heart (12,13). The effect of peripheral parasympathetic nerves per se on the cardiac response was augmented during the development of hypertension (Fig. 1). Muscarinic receptors activate the inward rectifying hyperpolarizing potassium channel via heterotrimeric inhibitory GTP-binding protein (Gi). Three different α subunits of Gi, Giα-1, Giα-2, and Giα-3 are encoded by three distinct genes (17). Expressions of Giα-2 and Giα-3 protein and mRNA have been shown to be significantly increased in SHR as early as 2 weeks after birth (7). This increase in Giα protein is observed before the development of high blood pressure. In DOCA-salt hypertensive rats, also, expression of Giα-2 and Giα-3 has been shown to precede the onset of hypertension (6,18). The physiological significance of this finding has not been clarified. Present results suggest that these early biochemical changes function to produce augmented cardiac responses evoked by parasympathetic nerve activity. Such early biochemical changes may explain the augmented response of peripheral parasympathetic nerves, which may be a contributing factor to the pathogenesis of hypertension or, at least, a prerequisite for the development of hypertension. The augmented cardiac response to parasympathetic nerve stimulation in SHR indicates that the normotensive condition tends to be preserved even though peripheral parasympathetic nerve traffic from the central nervous system is reduced.
Sympathetic-parasympathetic interaction in the early phase of hypertension
The experiments presented in this paper also examined whether the sympathetic and parasympathetic nerves play separate roles or whether they interact in the early phase of hypertension. Parasympathetic nerve stimulation was performed along with cardiac sympathetic nerve stimulation to examine potential sympathetic-parasympathetic interaction. The change in HR was significantly greater during combined stimulation of the sympathetic and parasympathetic nerves than it was during vagal stimulation alone (Fig. 2). After opening the chest under anesthesia, the heart was decentralized. HR tended to be the same level in both SHR and WKY rats. This may indicate that the influence of the sympathetic drive from the central nervous system was eliminated or, at least, reduced to the same level in both groups of rats. The greater reduction in HR during the combined stimulation could not have been produced by the change in cardiac responses evoked by cardiac sympathetic nerve stimulation, since the effect of cardiac sympathetic nerve stimulation remained constant. Therefore, combined stimulation produced a greater decrement in HR during the early phase of hypertension, possibly because parasympathetic nerves were potentiated when cardiac sympathetic nerves were stimulated concurrently; accentuated antagonism prevailed. This is the first study establishing a sympathetic-parasympathetic interaction during the early phase of hypertension.
The influence of the autonomic nervous system on the pathogenesis of hypertension is usually reported to be reduced parasympathetic nerve activity, and to produce the sympathovagal imbalance. Thus, relative predominance of sympathetic activity might occur. The accentuated antagonism observed in this study suggests that the two branches of the autonomic nervous system interact. Enhanced sympathetic nerve activity is present in the early phase of hypertension (1,3). In the very early phase of hypertension, baroreceptor reflex sensitivity may be reduced (14-16) and, as a consequence, peripheral parasympathetic nerve traffic may decrease. However, the cardiac response evoked by the parasympathetic nerve tends to be compensated for by the accentuated antagonism. Parasympathetic nerve activity can become more efficient. Further study is needed to determine the extent to which accentuated antagonism is present in the early phase of hypertension. The sympathetic-parasympathetic interaction does not differ between the normotensive state and the early hypertensive state.
In conclusion, the influence of the parasympathetic system in the early phase of hypertension is greater than that in the normotensive state, and this might be one of the causes of hypertension. Interaction between the two branches of the autonomic nervous system results in potentiated vagal inhibition (accentuated antagonism) of HR during the development of hypertension.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.