Effect of Jiawei Tongqiao Huoxue decoction in basilar artery dolichoectasia mice through yes-associated protein/transcriptional co-activator with PDZ-binding motif pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The Jiawei Tongqiao Huoxue decoction (JTHD), composed of Acorus calamus var. angustatus Besser, Paeonia lactiflora Pall., Conioselinum anthriscoides 'Chuanxiong', Prunus persica (L.) Batsch, Ziziphus jujuba Mill., Carthamus tinctorius L., Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep, Zingiber officinale Roscoe, Leiurus quinquestriatus, and Moschus berezovskii Flerov, was developed based on Tongqiao Huoxue decoction in Wang Qingren's "Yilin Gaicuo" in the Qing Dynasty. It has the effect of improving not only the blood flow velocity of vertebral and basilar arteries but also the blood flow parameters and wall shear stress. Especially in recent years, the potential efficacy of traditional Chinese medicine (TCM) for the treatment of basilar artery dolichoectasia (BAD) has attracted great attention as there are still no specific remedies for this disease. However, its molecular mechanism has not been elucidated. To identify the potential mechanisms of JTHD will help to intervene BAD and provide a reference for its clinical application. AIM OF THE STUDY: This study aims to establish a mouse model of BAD and explore the mechanism of JTHD regulating yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) pathway for attenuating BAD mice development. MATERIALS AND METHODS: Sixty post-modeling C57/BL6 female mice were randomly divided into sham-operated, model, atorvastatin calcium tablet, low-dose JTHD, and high-dose JTHD groups. After 14 days of modeling, the pharmacological intervention was given for 2 months. Then, JTHD was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). ELISA was utilized to detect the changes in vascular endothelial growth factor (VEGF) and lipoprotein a (Lp-a) in serum. EVG staining was conducted to observe the pathological changes of blood vessels. TUNEL method was employed to detect the apoptosis rate of vascular smooth muscle cells (VSMCs). Micro-CT and ImagePro Plus software were used to observe and calculate the tortuosity index, lengthening index, percentage increase in vessel diameter, and tortuosity of the basilar artery vessels in mice. Western blot analysis was performed to detect the expression levels of YAP and TAZ proteins in the vascular tissues of mice. RESULTS: Many effective compounds such as choline, tryptophan, and leucine with anti-inflammation and vascular remodeling were identified in the Chinese medicine formula by LC-MS analysis. The serum levels of VEGF in the model mice decreased significantly while the levels of Lp-a increased obviously compared with those in the sham-operated group. The intima-media of the basilar artery wall showed severe disruption of the internal elastic layer, atrophy of the muscular layer, and hyaline changes of the connective tissue. Apoptosis of VSMCs added. Dilatation, elongation, and tortuosity of the basilar artery became notable, and tortuosity index, lengthening index, percentage increase in vessel diameter, and bending angle remarkably improved. The expression levels of YAP and TAZ protein in blood vessels elevated conspicuously (P < 0.05, P < 0.01). JTHD group markedly reduced the lengthening, bending angle, percentage increase in vessel diameter, and tortuosity index of basilar artery compared with the model group after 2 months of pharmacological intervention. The group also decreased the secretion of Lp-a and increased the content of VEGF. It inhibited the destruction of the internal elastic layer, muscular atrophy, and hyaline degeneration of connective tissue in basilar artery wall. The apoptosis of VSMCs was decreased, and the expression levels of YAP and TAZ proteins were abated (P < 0.05, P < 0.01). CONCLUSIONS: The mechanism of inhibition of basilar artery elongation, dilation, and tortuosity by JTHD, which has various anti-BAD effective compound components, may be related to the reduction in VSMCs apoptosis and downregulation of YAP/TAZ pathway expression.

Role of ATP-sensitive potassium channels and inflammatory response of basilar artery smooth muscle cells in subarachnoid hemorrhage of rabbit and immune-modulation by shikonin.

OBJECTIVE: To investigate the role of inflammatory response, oxidative damage and changes of ATP-sensitive potassium channels (sKATP) in basilar artery (BA) smooth muscle cells (SMCS) of rabbits in subarachnoid hemorrhage (SAH) model. METHODS: Time course studies on inflammatory response by real-time PCR, oxidative process and function of isolated basilar artery after SAH in New Zealand White rabbits were performed. Basilar artery smooth muscle cells (BASMCs) in each group were obtained and whole-cell patch-clamp technique was applied to record cell membrane capacitance and KATP currents. The morphologies of basal arteries were analyzed. Protective effect of shikonin were also determine by same parameters. RESULTS: Inflammatory cytokines levels were highest at 24h compare to 72h after SAH whereas the oxidative damage and cell death marker were at highest peak at 72h. Oxidative damage peak coincided with significant alterations in cell membrane capacitance, KATP currents and morphological changes in basilar arteries. Shikokin pretreatment attenuated early inflammatory response at 24h and associated oxidative damage at 72h. Finally, shikonin attenuated morphological changes in basilar arteries and dysfunction. CONCLUSION: Currents of ATP-sensitive potassium channels in basilar smooth muscle cells decreased after SAH by putative oxidative modification from immediate inflammatory response and can be protected by shikonin pretreatment.

Sulforaphane activates the cerebral vascular Nrf2-ARE pathway and suppresses inflammation to attenuate cerebral vasospasm in rat with subarachnoid hemorrhage.

Nrf2-ARE pathway reportedly plays a protective role in several central nervous system diseases. No study has explored the role of the Nrf2-ARE pathway in cerebral vasospasm(CVS) after subarachnoid hemorrhage(SAH). The purpose of the present study was to investigate the activation of the cerebral vascular Nrf2-ARE pathway and to determine the potential role of this pathway in the development of CVS following SAH. We investigated whether the administration of sulforaphane (SFN, a specific Nrf2 activator) modulated vascular caliber, Nrf2-ARE pathway activity, proinflammatory cytokine expression, and clinical behavior in a rat model of SAH. A two-hemorrhage protocol was used to generate an animal model of SAH in male Sprague-Dawley rats. Administration of SFN to these rats following SAH enhanced the activity of the Nrf2-ARE pathway and suppressed the release of proinflammatory cytokines. Vasospasm was markedly attenuated in the basilar arteries after SFN therapy. Additionally, SFN administration significantly ameliorated two behavioral functions disrupted by SAH. These results suggest that SFN has a therapeutic benefit in post-SAH, and this may be due to elevated Nrf2-ARE pathway activity and inhibition of cerebral vascular proinflammatory cytokine expression.

Pharmacological characteristics of Artemisia vulgaris L. in isolated porcine basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE: In Vietnamese traditional herbalism, there are conflicting opinions about the effect of Artemisia vulgaris L. (AVL, English name: mugwort) on hypertension. Some ethnic doctors recommend the use of AVL for treatment of hypertension, whereas others advise against it. The purpose of this study was to clarify the pharmacological characteristics of AVL in isolated arteries to explain the conflicts surrounding the use of AVL for treatment of hypertension. MATERIALS AND METHODS: We initially performed a functional study using an organ bath system to investigate the effect of AVL extract on isolated porcine basilar artery. We then measured the change in intracellular free Ca(2+) concentration elicited by AVL using cultured smooth muscle cells loaded with the Ca(2+) indicator fluo-4. Finally, using HPLC, we determined the active components in AVL. RESULTS AND DISCUSSION: AVL induced vasoconstriction at resting tension, and endothelial removal enhanced this effect significantly. Pretreatment with PD123319 (an AT2 receptor antagonist), Nω-nitro-L-arginine (a nitric oxide synthase inhibitor), or both, also enhanced this effect. AVL-induced contraction was competitively inhibited by methiothepin (a 5-HT1 and 5-HT2 receptor antagonist) in the presence of ketanserin (a 5-HT2 receptor antagonist). Removal of extracellular calcium with nifedipine (an L-type Ca(2+) channel blocker) or ruthenium red (a ryanodine receptor blocker) significantly reduced AVL-induced contraction, whereas losartan (an AT1 receptor antagonist) and diphenhydramine (a H1 receptor antagonist) had no effect on this contraction. AVL increased the intracellular free Ca(2+) concentration in cultured cells, and this increment was inhibited by methiothepin. HPLC analysis revealed that the retention time of the first peak in the AVL profile was similar to that of the 5-HT standard, and that addition of 5-HT to the AVL sample enhanced this peak. On the other hand, AVL induced endothelium-independent relaxation under precontracted conditions with 60mM KCl. Captopril (an angiotensin converting enzyme inhibitor), atenolol (a ß1 receptor antagonist) and cimetidine (a H2 receptor antagonist) had no effect on this relaxation. In Ca(2+)-free 60mM KCl-containing solution, pretreatment with AVL significantly inhibited CaCl2-induced contraction. CONCLUSION: For the first time, the present study has demonstrated that AVL has two opposite effects, contraction and relaxation, on isolated artery, which may help to explain the conflicting indications for AVL in traditional herbalism. 5-HT is a significant factor affecting artery contraction in the presence of AVL.

Rhinacanthin-C, A Fat-Soluble Extract from Rhinacanthus nasutus, Modulates High-Mobility Group Box 1-Related Neuro-Inflammation and Subarachnoid Hemorrhage-Induced Brain Apoptosis in a Rat Model.

OBJECTIVE: High-mobility group box 1 (HMGB1) was shown to be a major extracellular mediator involved in relayed neuro-inflammation in animals after subarachnoid hemorrhage (SAH). It is of interest to examine the effect of rhinacanthin-C (RCT-C, C25H30O5) on pro-inflammatory cytokines/HMGB1 in an SAH-related early brain injury model. METHODS: A rodent double SAH model was used. RCT-C was administered orally at 100, 200, and 400 µmol/kg/day. Cerebral spinal fluid samples were obtained to assess interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor α using a real-time polymerase chain reaction. Basilar arteries were harvested and cerebral cortex was examined for HMGB1 mRNA and protein expression (western blot) and caspases (real-time polymerase chain reaction). An intrathecal injection of 1 ng of HMGB-1 recombinant protein was given in the 400 µmol/kg/day RCT-C plus SAH groups. RESULTS: The levels of IL-1ß, IL-6, and tumor necrosis factor α mRNA were significantly increased in animals subject to SAH, compared with the healthy controls, but were absent in the RCT-C groups. Cleaved caspase-9a as well as HMGB-1 mRNA and protein were significantly reduced in the 400 µmol/kg/day RCT-C treatment groups. Similarly, administration of RCT-C reduced HMGB-1 mRNA and protein expression (P <0.01). CONCLUSIONS: RCT-C exerts a neuroprotective effect by reducing cleaved caspase-3- and caspase-9a-related apoptosis. Decreased HMGB-1 mRNA and protein expression in the RCT-C groups corresponds to its anti-inflammatory effect. HMGB-1 recombinant protein administration impaired the neuroprotective and immunosuppressive effect of RCT-C. This finding lends credence that RCT-C modulates the HMGB-1-related pathway and attenuates brain apoptosis in the pathogenesis of SAH.

Glycyrrhizin Attenuates Proinflammatory Cytokines through a Peroxisome Proliferator-Activated Receptor-γ-Dependent Mechanism and Experimental Vasospasm in a Rat Model.

The peroxisome proliferator-activated receptor (PPAR) is downregulated in the cortex of experimental subarachnoid hemorrhage (SAH) animals. This study is to examine the effect of glycyrrhizin on the alternation of PPARs and proinflammatory cytokines in a rodent SAH model. CSF cytokines were evaluated by RT-PCR. Basilar arteries (BAs) were harvested to examine PPARs (RT-PCR and Western blot), and a morphological examination was conducted. Deformed endothelium and tortuous elastic lamina were observed in the BAs of the SAH groups, but they were absent in the glycyrrhizin groups or the healthy controls. The PPAR-γ and -δ protein levels were reduced in the SAH groups (p < 0.01). Glycyrrhizin significantly increased the expressed PPAR-γ protein and mRNA (preconditioning) and PPAR-δ mRNA (both treatment and preconditioning), which corresponded to the reduced IL-1ß and TNF-α levels. The administration of a PPAR-γ inhibitor, BADGE, halted the reduction of IL-1ß and TNF-α in the glycyrrhizin groups. Conclusively, glycyrrhizin exerts anti-inflammatory effects on SAH-induced vasospasm and attenuates the expression of PPARs, especially PPAR-γ, which corresponds to the severity of SAH-related inflammation. These findings also offer credit to the antivasospastic effect of glycyrrhizin and its vasculoprotective effect in animals subjected to SAH.

Effects of tetramethylpyrazine on nitric oxide/cGMP signaling after cerebral vasospasm in rabbits.

Tetramethylpyrazine (TMP), an ingredient of Chinese herbal Szechwan lovage rhizome, shows vasorelaxant effect. Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) is associated with high mortality and morbidity. Here, we evaluated the effect of TMP in a model of CVS and sought to identify the underlying mechanisms of action. A rabbit SAH model was established by injection of the autoblood via cisterna magna. Cerebral blood flow and arterial diameter were measured by Transcranial Doppler (TCD) and Computed Tomography Angiography (CTA). Expression of eNOS and PDE-V in basilar artery (BA) was assessed by western blots. Levels of nitric oxide (NO) in plasma and cerebral spinal fluid, and of intra-endothelium Ca(2+) were measured. Significantly reduced diameter and accelerated blood flow velocity were detected in BAs of SAH animals (P<0.05 vs. sham group). Expression of eNOS and NO was increased, and PDE-V expression was reduced by TMP.TMP ameliorated cerebral vasospasm (P<0.05 vs. SAH group), and L-NAME (a NOS inhibitor) partly abrogated the effects of TMP. TMP induced a dose-dependent increase of intra-endothelium Ca(2+). The current results demonstrated that the vasorelaxant effect of TMP was at least in part via regulation of NO/cGMP signaling.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

Potential role for heat shock protein 72 in antagonizing cerebral vasospasm after rat subarachnoid hemorrhage.

BACKGROUND: Cerebral vasospasm can be defined as delayed-onset narrowing of the cerebral arteries that can occur after a spontaneous aneurysmal subarachnoid hemorrhage (SAH). Despite a large number of experimental and clinical investigations, the exact pathophysiology of vasospasm remains unknown. Using a fluorescence differential-display system, we have identified the gene encoding heat shock protein 72 (HSP72) as being highly upregulated by cerebral vasospasm. We therefore elucidated the role of the HSP72 gene in cerebral vasospasm in a rat experimental SAH model. METHODS AND RESULTS: By angiography, cerebral vasospasm was detected from day 1, with maximal narrowing detected on day 2. Intracisternal injection of antisense HSP72 oligodeoxynucleotide led to specific inhibition of HSP72 gene expression and significantly aggravated cerebral vasospasm on days 2 and 3 of the angiographic studies. Oral administration of geranylgeranylacetone (GGA), an antiulcer drug, enhanced HSP72 induction and reduced cerebral vasospasm. CONCLUSIONS: These results suggest HSP72 plays a novel role in antagonizing delayed cerebral vasospasm after SAH and that GGA provides protective effects against delayed cerebral vasospasm, at least partly via induction of HSP72.

Accumulation of calcium and phosphorus accompanied by increase of magnesium and decrease of sulfur in human arteries.

To elucidate the accumulation of elements in the arteries with aging, the authors investigated age-related changes of elements in human arteries, such as the thoracic aorta, femoral, basilar, coronary, radial, and common iliac arteries by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 17 men and 9 women, ranging in age from 55 to 92 yr in the cases of the five arteries, except for the common iliac arteries, in which the subjects consisted of 16 men and 8 women, ranging in age from 65 to 93 yr. It was found that there were significantly direct correlations between calcium and phosphorus contents and between calcium and magnesium contents in all of the six arteries: thoracic aorta, femoral, basilar, coronary, radial, and common iliac arteries. Significantly direct correlations were also found between phosphorus and magnesium contents in the five arteries, except for the basilar artery. In contrast, significantly inverse correlations were found between calcium and sulfur contents and between phosphorus and sulfur contents in the four arteries, except for the coronary and radial arteries. These revealed that the accumulation of calcium and phosphorus in the arteries was accompanied by an increase of magnesium in the arteries and by a decrease of sulfur in the arteries.

Selective accumulations of aluminum in five human arteries.

The aim of the present study was to determine variability of aluminum (Al) accumulation in human arteries and to observe the relationship between Al and five other elements (Ca, Fe, Mg, P, and Si) in the arteries. The Al contents in the thoracic aorta, basilar, coronary, femoral, and radial arteries of 26 human subjects were estimated by an inductively coupled plasma-atomic emission spectrometer and compared quantitatively to five elements. Al was detected in 88% of the cases in both the femoral and radial arteries, 73% in the coronary artery, 58% in the aorta, and 31% in the basilar artery. The average Al content was highest in the femoral artery (48.3 +/- 15.0 microg/g dry weight) and lowest in the basilar artery (8.1 +/- 3.6 microg/g). The Al had positive correlations with P, Ca, or Mg in both the aorta and femoral artery, and with Ca or P in the basilar artery. In the coronary artery, a correlation was found between Al and Si. No relationships were found between Al and each of the five elements in the radial artery. From these results, Al varied widely among the five arteries and accumulated more in the femoral and radial arteries but less in the basilar artery. These accumulations of Al were positively correlated with Ca or P in several arteries, but not sufficiently to explain the accumulation of Al. Further investigations are required to understand the mechanism of the variability of Al accumulation in the arteries.

Extracts of Ginkgo biloba and ginsenosides exert cerebral vasorelaxation via a nitric oxide pathway.

1. Extracts from the leaves of Ginkgo biloba (EGb) and ginsenosides (GS) have been reported to be effective at increasing vascular relaxation. In the present study, the actions of EGb and GS on the vascular functions of porcine basilar arteries were investigated in vitro using tissue bath techniques. 2. Both EGb and GS relaxed the basilar artery in a concentration-dependent and partly endothelium-dependent manner. However, EGb appeared to be more potent than GS. Relaxation induced by transmural nerve stimulation (TNS) was significantly enhanced by EGb (7.5, 15 and 30 micrograms/mL) and GS (20, 40 and 80 micrograms/mL) in both endothelium-intact and -denuded basilar arteries. Enhanced TNS-induced relaxations were abolished by 0.3 mmol/L N-L-arginine. 3. The present study demonstrates that nitric oxide plays a primary role in TNS-induced relaxation as well as in EGb- and GS-enhanced relaxation within the cerebral vasculature. In addition, our data support the potential of these compounds as therapeutic strategies in cerebral ischaemia and other related vascular dysfunctions.

Analysis of rabbit vascular responses to DBI, an ingol derivative isolated from Euphorbia canariensis.

We have analysed the effects of 7,12-O-diacetyl-8-O-benzoil-2,3-diepiingol (DBI), an ingol derivative isolated from E. canariensis, on isometric tension developed by isolated rabbit basilar and carotid arteries. Concentration-response curves to DBI (10(-8) - 3 x 10(-5) M) were obtained cumulatively in both arteries at resting tension and active tone (KCI, 50 mM). At resting tension, DBI induced a concentration-dependent contraction, which was not inhibited in Ca(2+)-free medium. H7 (1-(5-isoquinoline sulphonyl)-2-methylpiperazine dichloride) (10(-4) M) inhibited the DBI-induced contraction both in basilar and in carotid arteries. Calmidazolium (10(-4) M) inhibited the maximum contraction of the carotid artery to DBI, and completely abolished the response in the basilar artery. In pre-contracted basilar arteries DBI induced a concentration-dependent relaxation that was not modified by incubation with NG-nitro-L-arginine (L-NOARG; 10(-5) M) or indomethacin (10(-5) M). In the carotid artery with active tone DBI induced further contractions, which were not significantly modified by L-NOARG (10(-5) M) and were potentiated by indomethacin (10(-5) M). These results suggest that DBI contracts rabbit basilar and carotid arteries by a mechanism that is independent of extracellular Ca2+ and involves the participation both of protein kinase C and of calmodulin. DBI relaxes basilar but not carotid arteries by a mechanism independent of the liberation of nitric oxide and prostacyclin. In the carotid artery prostacyclin but not nitric oxide partially counteracts the contractile action of DBI.

High accumulation of elements in the human femoral artery.

The relative contents (RCs) of elements in the femoral arteries as well as the thoracic aorta, coronary, basilar, and radial arteries from 26 subjects within the age range between 55 and 92 yr old, were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of calcium and phosphorus in the femoral arteries started to increase before the age of 60 yr. The RCs of magnesium increased after the age of 70 yr. However, the RCs of sulfur did not change significantly within the age range between 55 and 92 yr. With regard to localization of the mineral accumulations in the femoral arterial wall, it was found that the accumulations of calcium and phosphorus occurred only in the tunica media, only in the tunica intima, or in both the tunica media and the tunica intima. The manner of accumulation of calcium and phosphorus in the femoral arterial wall was different from that in the aortic wall. The average RCs of calcium in the 26 specimens were the highest in the femoral artery, followed in descending order by the thoracic aorta, coronary, basilar, and radial arteries. The average RCs of phosphorus were highest in the thoracic aorta, followed by the coronary, femoral, basilar, and radial arteries. It is noted that the accumulation of mineral elements never occurred uniformly in all the arteries.

Biosynthesis of leukotrienes in canine cerebral vasospasm.

We produced cerebral vasospasm in 29 dogs by the "two-hemorrhage" method of intracisternal injections, 2 days apart, of autogenous arterial blood. Leukotriene (LT) C4, LTD4, and LTE4 were purified from incubated basilar artery, medulla oblongata, hypothalamus, median eminence, and blood clot from around the basilar artery using reverse-phase high-performance liquid chromatography, and the amount of each LT was quantified separately by bioassay with guinea pig ileum. The biosynthetic capacity for total LTs was approximately three times higher in the hypothalamus and median eminence than in the basilar artery and medulla oblongata in the eight normal dogs. In the dogs with subarachnoid hemorrhage, the biosynthetic capacity was increased significantly both before and 2 hours after the second injection of blood on Day 2 and was normal on Day 7 in the basilar artery and medulla oblongata, whereas the biosynthetic capacity was decreased significantly 2 hours after the first and second injections of blood and was increased significantly on Day 7 in the hypothalamus and median eminence. In blood clot the biosynthetic capacity was increased continuously after the first injection of blood. Thus, the biosynthetic capacity for total LTs showed a time- and tissue-specific change after subarachnoid hemorrhage.

Specificity of capsaicin treatment in the cerebral vasculature.

Capsaicin has been shown to specifically deplete substance P from primary sensory afferents, including sensory nerves innervating blood vessels of the cerebral circulation as well as other vascular beds. In order to further document the specificity of this treatment, we examined the effect of capsaicin treatment on 3 other types of nerves in the guinea pig. Four tissues were examined: cerebral arteries, the mesenteric artery, the heart and iris. Norepinephrine content was not altered after capsaicin treatment, confirming that adrenergic nerves are unaffected. As indices of cholinergic nerves, activities of choline acetyltransferase and acetylcholinesterase were also unchanged after capsaicin treatment. In addition, no significant differences in levels of vasoactive intestinal peptide in cerebral arteries and the heart were found in animals treated with capsaicin. These findings underscore the specificity of capsaicin treatment for substance P containing nerves.