The present study, for the first time, provides quantitative data on the expression of preproenkephalin mRNA in the rVLM over a 4 hour period of following acupuncture stimulation using real-time PCR. The main findings of this study were: Preproenkephalin mRNA in the rVLM was increased 1.5 h after EA relative to baseline, while preproenkephalin in the sham and baseline group was unaltered. Conversely, preproenkephalin mRNA was unchanged both 20 min and 4 h after EA, compared with sham or surgery group at the same time points.
Acupuncture is distinguished from other somatosensory autonomic responses by the relative specificity of acupoints in treating a number of clinical conditions and by the prolonged nature of its influence. In this regard, our laboratory has demonstrated point specific EA regulation of cardiovascular sympathoexcitatory responses related to the extent of afferent stimulation and convergent input to cardiovascular neurons in the rVLM [25
]. One well-recognized set of acupoints, Jianshi and Neiguan, P5-P6 (located along the pericardial meridian), positioned directly over the median nerve on the wrist, when stimulated has been shown experimentally to be able to effectively treat hypertension and symptomatic coronary heart disease [3
]. Our studies also have provided a frame-work to understand the mechanism of EA’s action on cardiovascular function. Long-loop pathways contributing to prolonged inhibition of blood pressure by EA involve the arcuate nucleus (ARC) in the ventral hypothalamus, the ventrolateral periaqueductal gray (vlPAG) in the midbrain, and the nucleus raphe pallidus (NRP) and rVLM in the brain stem [18
]. These nuclei and pathways underlie acupuncture’s influence on sympathetic outflow. Thus EA at P5-P6 involves stimulation of the median nerves, which activates the ARC and vlPAG both of which ultimately inhibit premotor sympathoexcitatory neurons in the rVLM and blood pressure increases induced by visceral afferent stimulation. Our previous physiological studies have shown that inhibitory neuromodulators, including the opioid system, play a major role in the prolonged inhibition of neuronal activity in the rVLM [24
]. In the present study, which focused on the rVLM, we concentrated on the precursor for enkephalin rather than β-endorphin because our earlier data indicated that while enkephalins are produced in the rVLM, endorphins are synthesized in the arcuate nucleus and then transported by a long pathway to the rVLM [18
Because the EA inhibitory effect in anesthetized reflex-induced hypertensive animals can last for between 60 and 290 min [16
] and because our previous study has shown that 1.5 h following 30 min of EA activated nuclei are present in perikarya of rVLM neurons containing enkephalin peptides [8
], we chose time points of 20 min, 1.5 and 4 h following EA to study the influence of EA on preproenkephalin gene expression. Consistent with our laboratory’s previous anatomical study [8
], we noted that preproenkephalin mRNA levels in the rVLM were increased 1.5 h after EA. However, preproenkephalin mRNA was not altered 20 min or 4 h following EA compared to control values, thus demonstrating that with a single application of acupuncture for 30 min there is transient activation of preproenkephalin gene expression in the rVLM.
A previous study [6
] using a semi-quantitative technique, Northern blotting, to assess preproenkephalin mRNA level in the whole brain less cerebral cortex and cerebellum, has shown that 30 min of EA (2-15 Hz) in the awake Wistar rats induces an increase of preproenkephalin mRNA, which begins at 4 h, and peaked at 48 h after the termination of EA. The same research group using in situ
] also showed that 2 Hz EA for 30 min in awake Wistar rats increases mRNA expression of preproenkephalin in paragigantocellular nucleus, which is adjacent to rVLM, compared with a naïve group which did not receive treatment. Given the large differences in techniques and areas of the brain that were studied, it is difficult to compare these earlier studies with the present investigation. However, they do suggest that enkephalins may be increased in the brain following acupuncture and form an important backdrop for our investigation.
As noted earlier, the rVLM serves a crucial role in regulating premotor sympathetic outflow to the spinal cord and ultimately the cardiovascular system [5
]. Preproenkephalin mRNA is present in bulbospinal rVLM neurons with putative sympathoexcitatory and vasomotor functions, including approximately 20% of C1 and most of non-C1 neurons [23
]. The presence of increased preproenkephalin mRNA does not necessarily mean that the cells make more enkephalin peptide. However, the regulation of enkephalin peptide synthesis is mainly at the mRNA level [2
] and certainly increased preproenkephalin mRNA suggests that there is the potential for more peptide to be produced and thus ultimately to be available for release following electroacupuncture [17
Even though our previous studies have shown that the opioid system is activated by EA [17
], the situations under which enkephalin peptide are likely to be released for the modulation of inputs to sympathetic preganglionic neurons are not fully known [11
]. Acupuncture does not significantly influence blood pressure in normotensive humans [13
] or animals [8
], consistent with our present observations. An interesting finding was that preproenkephalin mRNA was increased without concomitant changes in blood pressure in normal rats, indicating that EA activation of somatic nerves is capable of inducing preproenkephalin mRNA synthesis in the absence of any blood pressure changes and secondary reflex effects from other regions like the arterial baroreceptors. Furthermore, these data suggest that the extent and duration of increase in preproenkephalin during and after a single 30 min period of acupuncture is not sufficient to alter resting sympathetic outflow and ultimately blood pressure.
We were surprised to note that preproenkephalin mRNA was not altered 20 min post EA when the inhibitory effect of EA in reflex-induced hypertension in anesthetized animals is most profound [17
]. Rather, the increase in preproenkephalin occurred later, 90 min following EA. These data suggest that neurons in the rVLM release existing enkephalin peptide during acute hypertension and that the new preproenkephalin simply helps to recharge the cellular content of this opioid peptide.
In summary, the present study provides the first quantitative evidence that EA applied at the P5-P6 EA can induce preproenkephalin mRNA expression 1.5 h after termination of the procedure. This study complements our previous anatomical, electrophysiological and pharmacological findings that EA evokes activation of enkephalinergic neurons in the rVLM, which contribute to reductions in sympathetic outflow and blood pressure when it is elevated.