We examined the releases of NO and cGMP in acupoint and non-meridian area along the PC meridian using dermal microdialysis in humans. The influences of EA stimulation on NO-cGMP releases were studied by chemical analysis of the dialysate samples collected from continuous dialysis of PC 4 and non-meridian area following EA PC 3. The major new findings of this study are that: 1) Baseline dialysate cGMP content was higher and NOx- concentration tended to be higher in PC 4 acupoint than in non-meridian area; 2) Dialysate NOx- concentrations were gradually reduced during a 120-min dialysis in all groups; 3) Reduced NOx- levels were attenuated predominantly in PC 4 acupoint at 20-40 min after EA PC 3 but not in the non-meridian area; and 4) EA-induced cGMP generation was parallel to an increased NOx- concentration. This is the first evidence showing that EA stimulation of PC 3 prevents reduction of dialysate NOx- release in the PC 4 acupoint but does not alter NO release in non-meridian area. The attenuation of NOx- reduction during dialysis reflects an increase in NO release in acupoint induced by EA stimulation. The time response to elevation of NO release in acupoints began at 0-20 min, reached a maximum at 20-40 min, and returned at 60-80 min after EA stimulation. The increased release of NO in PC 4 acupoint is parallel to the elevated amount of cGMP followed by EA stimulation. The elevation of both molecules induced by EA suggest that enhanced NO production, which stimulate guanylate cyclases to generate cGMP, which results in the subsequent biological effects through NO-cGMP signal system. These findings suggest that EA stimulation induced NO and cGMP release is specific to acupoints in humans. Enhanced chemicals following EA stimulation suggests a release/generation of NO-cGMP, which may mediate signaling pathway involved in the therapeutic effects of EA processes.
Dermal microdialysis has been successfully used to measure different substances in the extracellular space, such as NO, cGMP, histamine, glucose, and catecholamine in human skin [8
]. Present studies show that there is a fall in NOx-
concentrations during a 120-min dialysis especially in the initial dialysis period. The apparent fall in NO level during continued dermal dialysis has been reported previously [8
]. Possible explanations for this fall include: 1) Probe insertion and perfusion may cause changes in microvascular blood flow and/or interstitial environment resulting in an attenuation of NO production and release; and 2) Dialysis itself depletes the interstitial space of NO/nitrite [8
]. Our results are consistent with the previous studies using dermal microdialysis in humans [8
] and further suggest there are necessary to compare the treatment and control groups under the same dialysis period. In addition, our findings cannot exclude other factors against a falling baseline NOx-
concentrations involved in the interpretation of the EA effects. Although there is a large fall in NOx-
concentrations during 0-40 min dialysis resulting in large variability in dialysate recovery, the present results from the chemical analysis of the dialysate samples of acupoints suggest that dermal microdialysis is an effective approach to monitor chemical releases in acupoints along their meridian areas in vivo
and examine acupoint specificity in humans.
The mechanism responsible for an increase in NO and cGMP release in acupoints by EA stimulation is unclear. Anatomical studies have shown that most acupoints are located at a nerve trunk and situated at or adjacent to an artery and/or vein [7
]. Previous studies have also demonstrated that acupuncture induces the local releases of sensory neuropeptides [19
] and modified sensory function [36
] which cause peripheral vasodilation and increase in blood flow. Our results support these studies and further suggest that enhanced baseline and EA stimulation–evoked NO-cGMP concentrations in the acupoint may result from the rich distribution of blood vessels and neural fibers in the area. A number of international studies have demonstrated that, in both humans and animals, the majority of acupoints correspond to high electrical conductance and low skin resistance [6
]. Recent studies from our lab showed that NO content and nNOS expressions are consistently higher in the skin acupoints/meridians associated with low electric resistance in rats [30
]. L-arginine-derived NO synthesis modifies noradrenergic function, which contributes to low resistance characteristics of acupoints [4
]. Consistently, enhanced 3H-NE synthesis/release in acupoints/meridians is facilitated by the presence of an exogenous NO donor and inhibited by an inhibitor of NO synthesis [5
]. Recent studies have demonstrated that EA stimulation of ST 36 increases the production of NO in arterioles [22
]. It is well-demonstrated that three distinct forms of NO synthase (NOS)-nNOS, endothelial NOS (eNOS), and inducible NOS that catalyze the transformation of arginine to NO [3
], and the activities of L-arginine-derived NO synthesis, both neuronal and endothelial sources, are demonstrated in the skin tissues [4
]. The present results support the previous results reporting that activation of L-arginine-derived NO synthesis is high at acupoints and the local NO production is induced by EA stimulation. It is highly possible that elevation of NO-cGMP in the acupoint could be achieved through the activation of endothelial and/or neuronal NO synthesis/release system.
With regard to the potential role of EA-induced NO-cGMP release in acupoint, one view holds that acupoints are discrete sites on the body where needling or electrical stimulation can activate production via appropriate neural pathways to produce central effects [7
]. Dermal microdialysis studies show that NO release is essential in sustaining cutaneous dilation during heating [21
], and endogenous NO and noradrenaline contribute to the temperature threshold of the axon reflex response to heating on the skin [16
]. Endothelium-derived NO plays a role in reactive hyperemia [34
] and local anesthesia inhibits reactive hyperemia which suggests that postocclusion reactive hyperemia on forearm skin is mediated by a local reflex involved sensory nerve [25
]. In addition, acupuncture also works locally, and stimulation of the acupoint located on the ground of the pressure pain site (Ashi-point) has been often used for treatment of pain-related syndromes and soft-tissue damage in acupuncture clinical practices [1
]. Previous human studies have demonstrated that NO level is increased in the blood after warm needling [27
], and local circulation is enhanced by acupuncture stimulation correlated with NO increase in treated regions [41
]. It has been well-documented that NO-cGMP causes direct vasodilation and increase in blood flow [11
]. The present assay shows that NO and cGMP levels are consistently increased by EA stimulation in the PC acupoint but not in the non-meridian area. These results support the possibility that acupuncture induces local release of NO and cGMP [27
]. Elevated NO-cGMP results in peripheral vasodilation and improved local microcirculation, which contributes to the therapeutic effects of acupuncture.
In summary, these results show that EA stimulation induces a significant release of NO and cGMP in the acupoint, but not in non-meridian area. Time intervals of NO collections show that EA-induced increase in NO release in PC 4 are significantly higher at the 20-40 min interval than those collected from the non-meridian area. The results suggest that the elevation of both molecules induced by EA is specific to the acupoint, and acupoints with enriched blood vessels and neuronal components may contribute to NO generation, which stimulate guanylate cyclases to generate cGMP. Elevated NO-cGMP release evoked by EA stimulation mediates the therapeutic effects of acupuncture through local vasodilation and other possible mechanisms.