Stimuli-induced NOergic Molecules and Neuropeptides Mediated Axon Reflexes Contribute to Tracers along Meridian Pathways.

An abundance of studies from different international groups have demonstrated tracers along linear pathways resembling meridians over the body surface of humans. All experiments of the studies have been conducted by injection of a radiotracer solution or tracer dyes in a volume of solution into acupuncture points (acupoints). The solution injected into acupoints produces much stronger mechanical stimuli than acupuncture, which causes axon reflex. Anatomical studies have demonstrated that acupoints/meridians exist higher number of small nerve fibers and blood vessels with rich nitric oxide (NO) and neuropeptides in the cutaneous tissues as structures for the biomolecules mediated axon reflexes. Recent advances have determined that NO and calcitonin generelated peptides play crucial roles in the comprehension of the axon reflex. The stimuli-evoked axon reflex and NOergic biomolecules/neuropeptides increase local blood flow with higher levels in acupoints/meridians, which move radioactive substances or tracer dyes in the skin and subcutaneous tissue under a linear path resembling acupoints and meridians, the important phenomena of meridians induced by the stimuli. The evidence and understanding of the biomolecular processes of the tracers along linear pathways resembling meridians have been summarized with an emphasis on recent developments of NO and neuropeptides mediating stimuli-evoked axon reflexes to increase local blood flow with higher levels in acupoints/meridians, which move radioactive substances or tracer dyes in the skin and subcutaneous tissue contributing to tracers along linear pathways resembling meridians in this mini-review.

Low Electrical Resistance Properties of Acupoints: Roles of NOergic Signaling Molecules and Neuropeptides in Skin Electrical Conductance.

Early studies from several independent laboratories demonstrated that acupoints possess the characteristics of low electrical resistance. New devices are developing to increase the reliability of electrical skin impedance measurements for counteracting the factors including skin dryness, skin thickness, size of the sensing electrode, pressure applied on the electrode, interelectrode distance, room temperature, and humidity. Morphological studies have identified that blood vessels, hair follicles, and nervous components are enhanced in the meridians/acupoints, which represent areas of potentially high neuronal activity. Recent evidence shows that nitric oxide (NO) concentrations are enhanced in skin acupoints/meridians. L-arginine-derived NO synthesis modifies skin norepinephrine (NE) synthesis/release in acupoints/meridians, and NO-NE activations play an important role in mediating the skin conductance responses to electrical stimulation. NOergic signaling molecules interact with gap junction and transient receptor potential vanilloid type-1. Other studies reported that the high conductance at acupoints is a result of the release of the neuropeptides substance P and calcitonin gene-related peptide during neurogenic inflammation in the referred pain area. Pathological body conditions caused considerable changes in skin conductance or impedance at acupoints. Although systematic research with an improved equipment and research design to avoid the influencing factors are requested for a definite answer in this field, the results from anatomical and biochemical studies consistently show that acupoints exist higher levels of nervous components, and NOergic signaling molecules and neuropeptides involved in the skin low resistance at acupoints. The increased interest in the acupoints/meridians has led to an open-minded attitude towards understanding this system, which is fundamental important to establish the valid aspects of scientific basis of Chinese medicine mechanisms and therapies.

Nitric Oxide on Pathophysiology of SARS-CoV 19: Toward Possible Role of Acupuncture Treatment.

The ongoing outbreak of COVID-19 has quickly become a daunting challenge to global health. In the absence of satisfied therapy, effective treatment interventions are urgently needed. Previous studies have demonstrated that acupuncture is effective at relieving common symptoms of COVID-19 including breathlessness, nausea, insomnia, leukopenia, fatigue, vomiting, and abdominal pain. Experiments have shown that nitric oxide (NO) inhibits the replication cycle of severe acute respiratory syndrome (SARS) coronavirus with similar structures of COVID-19. Increase in level of NO by using NO gas inhalation has been shown to restore lung function by reducing airway resistance and improving virus-induced lung infections in SARS patients. Recent case report showed that a medical acupuncturist with symptoms consistent with severe COVID pneumonia achieved full recovery by self-administered medical acupuncture and cupping therapy at home. Clinical features and pathophysiology demonstrated that NO deficiency and endothelial dysfunction contribute to the development of COVID-19. Several studies from different groups consistently demonstrated that acupuncture increases NO synthase expression and induces an elevation of NO production and release in plasma and the local skin regions in both animals and humans. It is suggested that exogenous NO supplies or interventions that induce increasing levels of NO can play an important role in protective effects against inflammation and acute lung injury. This article reviews the rationale for mechanisms of NO induction induced by acupuncture in the possible treatment of COVID-19 and highlights its potential for contributing to better clinical outcomes and improving future clinical studies of acupuncture on treatment of COVID-19.

Establishing an adequate dose of acupuncture is essential for clinical trial studies.

Importance: A number of recent clinical trials have demonstrated that acupuncture is more effective for treating chronic pain conditions compared to sham and no acupuncture, but some research questions have remained unaddressed and are standing in the way of further progress. Observations and Advances: The effectiveness of acupuncture for pain conditions compared to usual care have been demonstrated, which has significantly enhanced the position of acupuncture in multiple pain guidelines following these studies. However, the studies also generated some conflicting results with difficulty in comparing each other. Research examining an adequate dose of acupuncture therapy with optimal intervention parameters and time table has also long been neglected and is now urgent. The dose of acupuncture depends on stimulation parameters: force/intensity and speed/frequency of manual acupuncture (MA) or electroacupuncture (EA) and time table (number of treatment sessions and duration). Various frequencies and intensities of MA and EA stimulation have been utilized in individual research. Different acupuncture treatment sessions (once, twice, three to five times per week) and periods (4, 6, 8, and 12 weeks) have been used in these clinical trials. One clinical trial using one session of needle acupuncture and laser at 12 weeks (8 to 12 treated sessions) did not improve pain in patients with chronic knee pain but similar trials of osteoarthritis knee have significant effects of pain improvement after biweekly sessions of needle acupuncture for 8 weeks of treatment. Conclusions and Relevance: Determining a right treatment regimen on correct acupuncture point(s) for acupuncture is a critical first step for acupuncture clinical trials. Appropriate acupuncture parameters such as acupuncture stimulation technique, treatment sessions, and treatment duration must be considered in acupuncture clinical trials. An adequate dose of acupuncture for clinical trials should be established following dose finding workshops for acupuncture before the studies, which not only improve the therapeutic effects of the therapies but also allow the comparisons between trials and between the acupuncture community/practice and trial studies.

Nitric oxide signaling molecules in acupoints: Toward mechanisms of acupuncture.

Recent clinical trial studies have demonstrated that the effects of acupuncture on pain improvement are small and no difference between acupoints and non-points. Whether acupuncture needles must be inserted in specific points depends on whether acupoint specificity exists that is still not resolved, and is now urgent. Previous anatomical studies have demonstrated that acupoints exist higher number of nerve fibers/trunks, blood vessels, hair follicles, and sweat glands as well as density of the gap junction. Recent evidence shows that nitric oxide (NO) level is elevated in the acupoints/ meridians and is associated with an enhanced expression of NO synthase endowed with transient receptor potential vanilloid type-1. There is growing evidence from international groups showing that acupuncture induces NO-mediated vasodilatation, which increases local blood flow and allows for a flush of algesic or sensitizing substances, leading to pain relief. Previous studies, using a novel biocapture system, have demonstrated that NOx- (total nitrite and nitrate) and cyclic guanosine monophosphate (cGMP) concentrations are consistently increased over skin acupoints compared to non-meridian control regions (NMCR) in humans. Dermal microdialysis in humans showed that NO-cGMP releases in the subcutaneous tissue of acupoint are higher than those in NMCR and increased by electroacupuncture (EA). Recent studies have demonstrated that low-frequency electrical stimulation and manual acupuncture with low stimulating force and rate produce an elevation of NO release predominantly over acupoints. In contrast, NO levels over the areas of the skin regions are moderately reduced by high-frequency EA stimulation. The results from anatomical and biochemical studies consistently show that acupoints exist higher levels of NO signaling molecules, and stimulus-evoked NO release is also with a higher level at acupoints. Results suggest that NO signaling molecules contribute to the specificity of acupoints, and selecting well-trained acupuncturetists for using correct acupoints and appropriate parameters should improve acupuncture clinical trial studies.

Response of Local Nitric Oxide Release to Manual Acupuncture and Electrical Heat in Humans: Effects of Reinforcement Methods.

This study was to examine the influences of manual acupuncture (MA) and electrical heat corresponding to reinforcing methods on nitric oxide (NO) release over the skin regions in humans. A device with collecting solution was taped to the skin surface along pericardium (PC) or lung (LU) meridian. Acupuncture needles were gently inserted into PC 4 with reinforcing stimulation (low force/rate) for 20 minutes in the MA group. LU11 on the finger was heated (43-44°C) by electrical heat for 20 minutes. Biocapture was consecutively conducted for two 20-minute intervals during and after each treatment. Total nitrite and nitrate (NO x-) in the collecting samples were quantified using chemiluminescence in blinded fashion. Baseline NO x- levels are higher and tended to be higher over PC and LU acupoints during the 1st biocapture. NO x- levels over PC regions were consistently increased by MA during both intervals. NO x- concentrations over LU acupoints were increased and tended to be increased by electrical heat in the 1st and 2nd biocapture. The results suggest that reinforcing MA and electrical heat induce NO released from the local skin regions with higher levels at acupoints, which improve local circulation and contribute to the beneficial effects of the therapies.

[Analysis of the Puzzle between Acupuncture Community and Acupuncture Clinical Trials].

Recently a number of acupuncture clinical trial projects mainly conducted by conventional scientists have generated many negative results. A large meta-analysis of patient-level acupuncture data for the treatment of chronic pain conditions have demonstrated that the effects of verum acupuncture on pain improvement have statistically significant, but small, differences compared with sham-acupuncture procedures and no difference between acupuncture points and non-points. These conclusions have puzzled the acupuncture community and made confusion for acupuncture research and practices. The purpose of this paper was to compare differences between acupuncture clinical practices and the trial studies, which include "acupuncture technical principles", "acupuncture clinical trial design", and "acupuncture practice based on the theory of traditional Chinese medicine". These factors contribute to the puzzle between the acupuncture community/practice and acupuncture clinical trials, which can be improved in future studies.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication.

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment. Animals were intoxicated by i.p. injection of 2,5-HD plus unilateral administration of 2,5-HD over the sciatic nerve. The mechanical thresholds and withdrawal latencies to heat and cold stimuli on the foot were measured at baseline and after intoxication. The medulla sections were examined by nNOS immunohistochemistry and NADPH-diaphorase histochemistry at the end of the treatments. The mechanical thresholds and withdrawal latencies were significantly decreased while nNOS immunostained neurons and NADPH-diaphorase positive cells were selectively reduced in the gracile nucleus at baseline in ZDF vs. LC rats. NADPH-diaphorase reactivity and nNOS positive neurons were increased in the ipsilateral gracile nucleus in LC rats following 2,5-HD intoxication, but its up-regulation was attenuated in ZDF rats. These results suggest that diabetic and chemical intoxication-induced nNOS expression is selectively reduced in the gracile nucleus in ZDF rats. Impaired axonal damage-induced nNOS expression in the gracile nucleus is involved in neuropathic pathophysiology in type II diabetic rats.

Influence of age, gender, and race on nitric oxide release over acupuncture points-meridians.

This study examined the influence of age, gender and race on nitric oxide (NO) release over acupuncture points, meridian without acupoint, and non-meridian regions of the Pericardium (PC) and Bladder (BL) meridian as well as aging on LU meridian in 61 healthy subjects. Biocapture tubes were attached to the skin surface, and total nitrite and nitrate was biocaptured and quantified using chemiluminescence. In elder ages compared to adults, NO levels over the ventral forearm were significantly decreased over LU on radial regions but not altered over PC on medial regions. Conversely, NO content was elevated over BL regions only in overweight/obesity of elder ages. NO levels over PC regions were marginally elevated in overweight/obese males compared to females but did not alter between races. These results suggest a selective reduction of NO release over LU meridian with aging, which is consistent with a progressive decline in lung function and increase in chronic respiratory disease in elder ages. Increased NO levels along the BL meridian in older obese subjects may reflect a modified NO level along somatic-bladder pathway for counteracting bladder dysfunctions with aging. Both of them support somatic-organ connections in the meridian system associated with potential pathophysiological changes with aging.

Transcutaneous Electrical Stimulation Increased Nitric Oxide-Cyclic GMP Release Biocaptured Over Skin Surface of Pericardium Meridian and Acupuncture Points in Humans.

OBJECTIVES: The purpose of this study was to consecutively capture and quantify nitric oxide (NO) and cGMP, the second messenger of NO, over the skin surface of acupuncture points (acupoints), meridian line without acupoint, and non-meridian control regions of the Pericardium meridian (PC) in humans, and investigate their response to transcutaneous electrical nerve stimulation (TENS) . DESIGN, SETTING, AND MAIN OUTCOME MEASURES: Adhesive biocapture tubes were attached to the skin surface along PC regions and injected with 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution, an NO-scavenging compound, contacting the skin surface for 20 minutes each during 4 consecutive biocapture intervals. TENS (1.0 mA, 6 Hz, 1.0 msec duration) was applied over acupoints PC 8 and PC 3 during the 2nd biocapture for 20 min. Total nitrite and nitrate (NO(x)-), the stable metabolic products of NO, and cGMP in biocaptured samples were quantified using chemiluminescence and ELISA. RESULTS: NO(x)- levels in the 1st biocapture over PC regions are almost two fold higher compared to subsequent biocaptures and are higher over PC acupoints versus non-meridian control region. Following TENS, NO(x)- concentrations over PC regions were significantly increased, and cGMP is predominantly released from the skin surface of PC acupoints. CONCLUSIONS: TENS induces elevations of NO-cGMP concentrations over local skin region with a high level at acupoints. The enhanced signal molecules improve local circulation, which contributes to beneficial effects of the therapy.

Effects of electroacupuncture Zusanli (ST36) on food intake and expression of POMC and TRPV1 through afferents-medulla pathway in obese prone rats.

OBJECTIVE: The purpose of this study was to determine the effects of electroacupuncture (EA) ST36 on food intake and body weight in obese prone (OP) rats compared to obese resistant (OR) strain on a high fat diet. The influences of EA on mRNA levels of pro-opiomelanocortin (POMC), transient receptor potential vanilloid type-1 (TRPV1), and neuronal nitric oxide synthase (nNOS) were also examined in the medulla regions and ST36 skin tissue. METHODS: Advanced EA ST36 was conducted in two sessions of 20 min separated by an 80 min interval for 7 days. Food intake and body weight were recorded in conscious rats every day. Real time PCR was conducted in the micropunches of the medulla regions and skin tissues at the end of the treatment. RESULTS: Food intake and body weight were significantly reduced by advanced EA ST36 in OP rats, but slightly decreased in OR strain and sham-EA rats. Advanced EA ST36 produced a marked increase in POMC mRNA level in the nucleus tractus solitarius (NTS) and hypoglossal nucleus (HN) regions. TRPV1 and nNOS mRNAs were simultaneously increased in the NTS/gracile nucleus regions and in the ST36 skin regions by the EA treatment in OP rats. CONCLUSIONS: We conclude that advanced EA ST36 produces an up-regulation of anorexigenic factor POMC production in the NTS/HN, which inhibits food intake and reduces body weight. EA-induced expression of TRPV1-nNOS in the ST36 and the NTS/gracile nucleus is involved in the signal transduction of EA stimuli via somatosensory afferents-medulla pathways.

Infrared heat treatment reduces food intake and modifies expressions of TRPV3-POMC in the dorsal medulla of obesity prone rats.

PURPOSE: Infrared heat, a transient receptor potential vanilloid type-3 (TRPV3) sensitive stimulus, may have potential physiological effects beneficial to treating metabolic syndrome. MATERIALS AND METHODS: Obesity prone (OP) and obesity resistant (OR) rats were fed for seven days on a high-fat diet. Heat treated OP rats were exposed twice daily to infrared light for 20 min each, separated by 80 min of rest. Food intake, blood pressure, blood glucose, and body weight measurements were taken daily and compared between treated OP rats, untreated OP rats, and OR controls. The animals were perfused with 4% paraformaldehyde, and immunohistochemistry was performed on the coronal brainstem sections with polyclonal antibodies against TRPV3 and pro-opiomelanocortin (POMC). The positive-staining cells in the medulla nuclei were quantified using a microscope with reticule grid. RESULTS: Food intake, body weight, and mean arterial blood pressure (MAP) were higher in OP rats, a diet-induced metabolic syndrome model, accompanied by a reduced expression of POMC, an anorectic agent, in the hypoglossal nucleus (HN) and medial nucleus tractus solitarius (mNTS). Food intake in heat-treated OP rats was significantly decreased. POMC positive neuron count was increased in the HN and mNTS of OP rats following treatment. TRPV3 positive staining neurons were increased in the HN and mNTS of OP control rats and decreased following the heat treatments. CONCLUSION: Lowered POMC and heightened TRPV3 expressions in the HN and mNTS are involved in development of hyperphagia and obesity in OP rats. Exposure to infrared heat modifies TRPV3 and POMC expression in the brainstem, reducing food intake.

TRPV1 expression in acupuncture points: response to electroacupuncture stimulation.

The present study was to examine the distribution of transient receptor potential vanilloid type-1 (TRPV1) receptor immunoreactivity in the acupuncture points (acupoint), and determine the influences of electroacupuncture (EA) stimulation on TRPV1 expression. EA stimulation of BL 40 was conducted in two sessions of 20 min separated by an 80 min interval in anesthetized rats. Sections of skin containing BL 40, and its non-meridian control were examined by immunolabeling with antibodies directed against TRPV1. Without EA, the number of subepidermal nerve fibers expressing TRPV1 was higher in the acupoint than in non-acupoint control skin (p<0.01). The subepidermal nerve fibers showed the co-localization of TRPV1 with peripherine, a marker for the C-fibers and A-δ fibers. The expression of TRPV1 in nerve fibers is significantly increased by EA stimulation in acupoints (p<0.01). However the upregulation in the non acupoint meridian and the non-meridian control skin was short of statistical significance. Double immunostaining of TRPV1 and neuronal nitric oxide synthase (nNOS) revealed their co-localization in both the subepidermal nerve fibers and in the dermal connective tissue cells. These results show that a high expression of TRPV1 endowed with nNOS in subepidermal nerve fibers exists in the acupoints and the expression is increased by EA. We conclude that the higher expression of TRPV1 in the subepidermal nerve fibers and its upregulation after EA stimulation may play a key role in mediating the transduction of EA signals to the CNS, and its expression in the subepidermal connective tissue cells may play a role in conducting the local effect of the EA.

Changes in nitric oxide, cGMP, and nitrotyrosine concentrations over skin along the meridians in obese subjects.

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NO(x)(-)) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semi-circular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NO(x)(-), cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NO(x)(-) and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4-7 compared with nonmeridian areas. NO(x)(-) concentration is enhanced over the bladder meridian (BL) 56-57, but cGMP level is similar between the regions. In overweight/obese subjects, NO(x)(-) contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.

Electroacupuncture Zusanli (ST36) on Release of Nitric Oxide in the Gracile Nucleus and Improvement of Sensory Neuropathies in Zucker Diabetic Fatty Rats.

The purpose of these studies was to examine the effects of electroacupuncture (EA) Zusanli (ST36) on release of nitric oxide (NO) in the gracile nucleus (GN) and determine if functional neuropathic changes were modified by EA ST36-induced NO in the nucleus in Zucker diabetic fatty (ZDF) rats. The foot withdrawal responses to mechanical, thermal and cold stimuli were measured before and after EA stimulation. A microdialysis probe was implanted in the GN and dialysate samples were collected 20 min before, during and after EA ST36. Total nitrate and nitrite (NO(x) (-)) concentrations in the samples were quantified by using chemiluminescence. The baseline dialysate NO(x) (-) concentrations in the GN were decreased in ZDF rats compared to lean control (LC) rats (P < .05). In ZDF rats, dialysate NO(x) (-) releases in the GN were markedly increased during EA ST36, whereas in LC rats, the releases were moderately enhanced at 20-40 min after EA ST36. The withdrawal latencies to mechanical, cold and thermal stimuli were significantly improved 20 min after EA ST36 both in LC and ZDF rats, but not altered by non-acupoint stimulation. The withdrawal latencies to EA ST36 were further potentiated by 3-morpholinyl-sydnoneimine and inhibited by N(G)-Propyl-l-arginine infused into the GN in ZDF rats (P < .05). These results show that EA ST36 increases NO release in the GN, and NO in the nucleus modifies withdrawal latencies to mechanical, cold, and thermal nociception stimuli. Data suggest that EA ST36 induces NO release in the GN, which contributes to improvement of sensory neuropathies in rats.

Responses of nitric oxide-cGMP release in acupuncture point to electroacupuncture in human skin in vivo using dermal microdialysis.

OBJECTIVES: The aim of this study was to examine the release of nitric oxide (NO) and cGMP in response to electroacupuncture (EA) stimulation in the acupuncture point (acupoint), compared to the non-meridian control area. METHODS: Thirty samples of dermal microdialysis data were collected from 24 volunteers at pericardium (PC) 4 and control area. EA was applied to PC 3 by using a 5-V pulse with a duration of 1.0 milliseconds at 10 Hz for 15 minutes. Dialysate samples were continuously collected 20 minutes each before, during, and after EA for two hours. Total nitrite and nitrate (NO(x)(-)) and cGMP in the dialysate were quantified in a blinded fashion. RESULTS: Dialysate NO(x)(-) concentrations were decreased during a 120-minute dialysis in all groups, but reduced NO(x)(-) levels were attenuated predominantly in PC 4 acupoint at 20-40 minutes after EA PC 3. cGMP concentrations were significantly enhanced in acupoint PC 4 by EA PC 3, but not in the non-meridian area. CONCLUSION: We suggest that the attenuation of NO(x)(-) reduction during dialysis reflects an increase in NO release induced by EA stimulation in acupoint and that cGMP mediates the signaling functions of NO to improve local microcirculation, which, at least in part, contributes to the effects of acupuncture.

[Effect of nitric oxide on noradrenergic function and skin electric resistance of acupoints and meridians].

Most acupuncture points correspond to low skin-resistance points (LSRP) on the body surface along the meridians. We did three experiments which conformed that the skin nitric oxide (NO) concentration and expression of neuronal nitric oxide synthesis (nNOS) were higher than those in non-acupoints, and non-meridian control points. Noradrenaline (NE) synthesis/release was modulated by exogenous NO donor and selective inhibitor of nNOS in the skin acupoints/meridians. Skin electrical currents in low skin resistance points were modified by L-arginine-derived NO synthesis and NE. As a conclusion, NO-NE contribute to low resistance characteristics of acupoints and meridians.

[Biochemical physiology of nitric oxide over acupuncture points and meridians: new approach and perspectives].

We have found that nitric oxide (NO) contents are increased in skin acupoints associated with enhanced neuronal NO synthase (nNOS) protein level in rats. Our recent studies show that L-arginine-derived NO synthesis increases skin electric currents over acupoints in rats. Consistently, norepinephrine (NE) turnover rate is decreased in acupoints with high skin electric currents, and enhanced 3H-NE synthesis/release in acupoints/meridians is facilitated by presence of an exogenous NO donor and inhibited by an inhibitor of NO synthesis. We have developed an innovative method to collect and measure NO metabolites from acupoints and meridians on the skin surface in humans. NO contents are consistently increased over PC and BL acupoints in humans, and increased NO content is reduced by anti-bacteria on skin surface. We conclude that the nonenzymatic reduction of nitrate by bacteria is involved in chemical generation of NO on skin acupoints/meridians in addition to neuronal NOergic system. NO mediates noradrenergic function on skin sympathetic nerve activation, which contributes to low resistance characteristics of acupoints and meridians.

Effects of nitric oxide and noradrenergic activation in the posterior hypothalamus on arterial pressure tolerance to nitroglycerin in rats.

The effects of nitric oxide (NO) and noradrenergic activation in the posterior hypothalamus on arterial pressure tolerance induced by subcutaneous injection of nitroglycerin (NTG) was investigated in anesthetized Sprague-Dawley rats. Intravenous injections of NTG (3, 10, and 30 microg/kg) and sodium nitroprusside (1, 3, and 10 microg/kg) produced dose-dependant decreases in arterial blood pressure. Tolerance to NTG was produced by subcutaneous administration of 4.0 mg of NTG as 4 separate hourly injections of 1.0 mg each, affecting the dose-dependent response of NTG IV injection. The 4 high-dose NTG pulse injections produced a marked shift in the dose-response curves for arterial pressure depression induced by intravenous injection of the challenge doses of NTG, but did not alter hypotensive responses to sodium nitroprusside. The tolerance responses to arterial pressure depression were enhanced by a bilateral microinjection of NTG (1 nmol) and by diethylamine NONOate (1 nmol), an NO donor, into the posterior hypothalamus. Bilateral microinjection of guanethidine (1.5 nmol), a noradrenergic blocker, into the posterior hypothalamus inhibits NTG tolerance in a period of time within 2 hours. We conclude that exogenous NO and noradrenergic activation in the posterior hypothalamus play an important role in arterial pressure tolerance to systemically administered NTG.

Evidence of enhanced non-enzymatic generation of nitric oxide on the skin surface of acupuncture points: An innovative approach in humans.

The present study quantified total nitrate and nitrite (NOx-) collected from the skin surface along acupuncture points (acupoints) and determined whether non-enzymatic reduction of nitrate by bacteria is involved in chemical generation of nitric oxide (NO) on acupoints. A small plastic tube (0.5 x 7 cm) cut in half lengthwise was taped to the forearm or leg in 50 healthy volunteers. NO-collecting solutions with NO-scavenging compounds, hemoglobin or 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, was placed inside the tubing attached to the skin surface for 20 min. The concentrations of NOx- in the collected samples were quantified by using chemiluminescence. NOx- concentration was significantly enhanced in four acupoints on the pericardium meridian and in two acupoints on the bladder meridian compared with those collected on non-meridian control areas. The time intervals of NOx- levels were significantly higher at the first 20 min of acupoint collection, but the concentrations were similar among the study groups collected at 20-40, 40-60, and 60-80 min. NOx- concentrations and numbers of bacteria colonies detected on the skin surface were markedly reduced by pretreatment of skin with sodium hypochlorite compared to water treatment. This is the first evidence showing that NO has been successfully quantified on skin acupoints by a non-invasive device in humans. We conclude that NO is physiologically released from the skin surface with a higher level at acupoints, and that the non-enzymatic reduction of nitrate by bacteria is involved in chemical generation of NO on skin acupoints in addition to l-arginine-derived NO synthesis.