Liang-Wu Fu

Long-loop pathways in cardiovascular electroacupuncture responses

We have shown that electroacupuncture (EA) at P 5-6 (overlying median nerves) activates arcuate (ARC) neurons, which excite the ventrolateral periaqueductal gray (vlPAG) and inhibit cardiovascular sympathoexcitatory neurons in the rostral ventrolateral medulla (rVLM). To investigate whether the ARC inhibits rVLM activity directly or indirectly, we stimulated the splanchnic nerve to activate rVLM neurons. Micropipettes were inserted in the rVLM, vlPAG, and ARC for neural recording or injection. Microinjection of kainic acid (KA; 1 mM, 50 nl) in the ARC blocked EA inhibition of the splanchnic nerve stimulation-induced reflex increases in rVLM neuronal activity. Microinjection of d,l-homocysteic acid (4 nM, 50 nl) in the ARC, like EA, inhibited reflex increases in the rVLM neuronal discharge. The vlPAG neurons receive convergent input from the ARC, splanchnic nerve, P 5-6, and other acupoints. Microinjection of KA bilaterally into the rostral vlPAG partially reversed rVLM neuronal responses and cardiovascular inhibition during d,l-homocysteic acid stimulation of the ARC. On the other hand, injection of KA into the caudal vlPAG completely reversed these responses. We also observed that ARC neurons could be antidromically activated by stimulating the rVLM, and that ARC perikarya was labeled with retrograde tracer that had been microinjected into the rVLM. These neurons frequently contained beta-endorphin and c-Fos, activated by EA stimulation. Therefore, the vlPAG, particularly, the caudal vlPAG, is required for ARC inhibition of rVLM neuronal activation and subsequent EA-related cardiovascular activation. Direct projections from the ARC to the rVLM, which serve as an important source of beta-endorphin, appear also to exist.

Electroacupuncture modulates vlPAG release of GABA through presynaptic cannabinoid CB1 receptors.

Previous studies have demonstrated that electroacupuncture (EA) attenuates sympathoexcitatory reflex responses by activating a long-loop pathway involving the hypothalamic arcuate nucleus (ARC), midbrain ventrolateral periaqueductal gray (vlPAG), and rostral ventrolateral medulla (rVLM). Neurons in the ARC provide excitatory input to the vlPAG, whereas the vlPAG inhibits neuronal activity in the rVLM. gamma-Aminobutyric acid (GABA) and glutamate (Glu) have been identified in the vlPAG. Endocannabinoids (ECs), acting as atypical neurotransmitters, inhibit the release of both neurotransmitters in the hypothalamus and midbrain through a presynaptic cannabinoid type 1 (CB(1)) receptor mechanism. The EC system has been observed in the dorsal but not in the vlPAG. Since it is uncertain whether ECs influence GABA and Glu in the vlPAG, the present study tested the hypothesis that EA modulates the release of these neurotransmitters in the vlPAG through a presynaptic CB(1) receptor mechanism. We measured the release of GABA and Glu simultaneously by using HPLC to assess samples collected with microdialysis probes inserted unilaterally into the vlPAG of intact anesthetized rats. Twenty-eight min of EA (2 Hz, 2-4 mA, 0.5 ms) at the P5-6 acupoints reduced the release of GABA by 39% during EA and by 44% 15 min after EA. Thirty-five minutes after EA, GABA concentrations returned to pre-EA levels. In contrast, sham EA did not change the vlPAG GABA concentration. Blockade of CB(1) receptors with AM251, a selective CB(1) receptor antagonist, reversed the EA-modulated changes in GABA concentration, whereas microinjection of vehicle into the vlPAG did not alter EA-modulated GABA changes. In addition, we observed no changes in the vlPAG Glu concentrations during EA, although the baseline concentration of Glu was much higher than that of GABA (3,541 +/- 373 vs. 33.8 +/- 8.7 nM, Glu vs. GABA). These results suggest that EA modulates the sympathoexcitatory reflex responses by decreasing the release of GABA, but not Glu, in the vlPAG, most likely through a presynaptic CB(1) receptor mechanism.

Activated platelets contribute to stimulation of cardiac afferents during ischaemia in cats: role of 5‐HT3 receptors

Myocardial ischaemia activates blood platelets and cardiac sympathetic afferents, which mediate chest pain and cardiovascular reflex responses. We have demonstrated that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents. Platelets absorb and release 5‐hydroxytryptamine (5‐HT) when they are activated. In the present study we hypothesized that, by releasing 5‐HT, activated platelets stimulate cardiac afferents during ischaemia through a 5‐HT3 receptor mechanism. Platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP) were obtained from cats. Activation of platelets in PRP was induced by thrombin (5 units ml−1) or collagen (2 mg kg−1). Using high‐performance liquid chromatography, we observed that the concentration of 5‐HT was increased significantly in suspensions of platelets activated with thrombin (PRP+thrombin, 28 ± 1.7 μm) or collagen (PRP+collagen, 27 ± 2.5 μm) compared with suspensions of unactivated platelets (PRP+saline, 2.3 ± 0.8 μm) and PPP. During myocardial ischaemia and reperfusion, tirofiban, a specific inhibitor of platelet glycoprotein (GP) IIb‐IIIa receptors (100 μg kg−1, I.V., followed by 5 μg kg−1 min−1), significantly reduced the increase in the concentration of 5‐HT in cardiac venous plasma from ischaemic region. Nerve activity of single‐unit cardiac afferents was recorded from the left sympathetic chain (T2‐T5) in anaesthetized cats. Eighty ischaemically sensitive and seven ischaemically insensitive cardiac afferents were identified. Tirofiban reduced the ischaemia‐related increase in activity of seven cardiac sympathetic afferents by 50 %. Injection of 1.5 ml of PRP+collagen or PRP+thrombin into the left atrium (LA) increased activity of 16 cardiac afferents. Tropisetron (300 μg kg−1, I.V.), a selective 5‐HT3 receptor antagonist, eliminated the afferents responses to platelets activated with collagen or thrombin. Moreover, LA injection of 5‐HT (20‐40 μg kg−1) and PBG (100 μg kg−1), a 5‐HT3 receptor agonist, stimulated nine ischaemically sensitive cardiac sympathetic afferents, significantly increasing the activity of these afferents. However, injection of α‐M‐5‐HT (100 μg kg−1, LA), a 5‐HT2 receptor agonist, stimulated only two of the nine ischaemically sensitive cardiac afferents, and thus did not significantly alter impulse activity of this group of afferents. Both the 5‐HT1 (5‐CT, 100 μg kg−1, LA) and 5‐HT4 receptor agonists (SC53116, 100 μg kg−1, LA) did not stimulate any of the nine afferents tested. Tropisetron (300 μg kg−1, I.V.) also eliminated the response of seven ischaemically sensitive cardiac afferents to exogenous 5‐HT and attenuated the ischaemia‐related increase in activity of nine cardiac sympathetic afferents by 41 %. Conversely, LA injection of 5‐HT (40 μg kg−1) did not stimulate any of seven ischaemically insensitive cardiac afferents, although this group of afferents consistently responded to bradykinin (3 μg, LA). These data indicate that during myocardial ischaemia the activated platelets stimulate cardiac sympathetic afferents, at least in part, through a 5‐HT3 receptor mechanism.

Role of unmyelinated fibers in electroacupuncture cardiovascular responses

The afferent fiber type responsible for the transmission of sensory neural traffic to the central nervous system during acupoint stimulation is uncertain. Several early studies evaluating compound action potentials have suggested that myelinated fibers contribute to the afferent input of the autonomic reflex adjustments during electroacupuncture (EA). Our more recent data, employing single unit recordings of somatic afferents, show that both myelinated and unmyelinated fibers are stimulated by EA, although more finely myelinated than unmyelinated fibers are activated by low frequency, low current stimulation. We hypothesized in this study that unmyelinated group VI fibers also contribute significantly to the inhibitory influence of EA on cardiovascular pressor responses. We found that neonatal capsaicin-treated rats depleted of substance P from primary afferents were insensitive to the inhibitory EA effect during gastric distention. Thus, EA at P5-P6 reduced gastric distention-induced pressor responses from 19+/-3 to 11+/-2 mmHg in eight untreated rats while capsaicin-treated rats (n=9) were unresponsive to EA. Substance P containing neurons in dorsal root ganglion cells at Ti-T5 were significantly decreased in the capsaicin-treated rats from 47+/-4 to 22+/-4 cells. Treated compared to untreated rats also demonstrated a significantly (P<0.03) reduced number of group IV fibers identified with single unit recording techniques. This study demonstrates that the inhibitory effect of EA at P5-P6 on cardiovascular autonomic excitatory reflexes involves unmyelinated group IV fibers of the median nerves.

Nitric oxide in rostral ventrolateral medulla regulates cardiac-sympathetic reflexes: role of synthase isoforms

Our previous studies have shown that nitric oxide (NO) synthase (NOS)-containing neurons in the rostral ventrolateral medulla (rVLM) are activated during cardiac sympathoexcitatory reflexes (Refs. 12 and 13). However, the precise function of NO in the rVLM in regulation of these reflexes has not been defined. Three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), are located in the rVLM. We explored the role of NO, derived from different NOS isoforms in the rVLM, in processing cardiac-sympathetic reflexes using whole animal reflex and electrophysiological approaches. We found that, in anesthetized cats, increased mean arterial blood pressure and renal sympathetic nerve activity elicited by epicardial application of bradykinin (BK; 1-10 microg/ml, 50 microl) were significantly attenuated following unilateral rVLM microinjection of the nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (50 nmol/50 nl), or a specific nNOS inhibitor, 7-nitroindazole (7-NI; 5-10 pmol/50 nl; both P < 0.05). In contrast, the responses of mean arterial blood pressure and renal sympathetic nerve activity to cardiac BK stimulation were unchanged by unilateral rVLM microinjection of N(omega)-nitro-D-arginine methyl ester (inactive isomer of N(omega)-nitro-L-arginine methyl ester, 50 nmol/50 nl), 3-6% methanol (7-NI vehicle), N(6)-(1-iminoethyl)-L-lysine (250 pmol/50 nl; iNOS inhibitor), or N(5)-(1-iminoethyl)-L-ornithine (250 nmol/50 nl; eNOS inhibitor). Furthermore, in separate cats, we noted that iontophoresis of 7-NI (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory rVLM neurons in response to cardiac stimulation with BK (P < 0.05). These neurons were characterized by their responses to inputs from baroreceptors, and their cardiac rhythmicity was determined through frequency and time domain analyses, correlating their discharge to arterial blood pressure and cardiac sympathetic efferent nerve activity. These data suggest that NO, specifically nNOS, mediates sympathetic cardiac-cardiovascular responses through its action in the rVLM.

Interactions between histamine and bradykinin in stimulation of ischaemically sensitive cardiac afferents in felines

Cardiac spinal afferents are activated during myocardial ischaemia. Our previous studies have shown that during ischaemia, histamine and bradykinin (BK) stimulate cardiac spinal afferents. Because the two mediators are released together during ischaemia, the present study examined the interactions between these two mediators with respect to their influence on ischaemically sensitive cardiac afferents. Single‐unit cardiac afferent activity was recorded from the left sympathetic chain or rami communicantes (T2–T5) in anaesthetized cats. Fifty‐five ischaemically sensitive cardiac afferents (conduction velocity (CV) = 0.2−5.6 m s−1, 8 Aδ‐ and 47 C‐fibres) were identified. Administration of histamine (10 μg kg−1) and BK (1 μg) in combination into the left atrium (LA) caused an additive response in 16 afferents compared with administration of either BK or histamine alone (2.62 ± 0.39 versus 1.67 ± 0.20 versus 1.24 ± 0.23 impulses s−1 (imp s−1), BK + histamine versus BK versus histamine). To further evaluate interactions between these mediators, we observed that injection of histamine (10 μg kg−1, LA) 4 min after the administration of BK (1 μg, LA) induced a significantly larger cardiac afferent response than the response to histamine before BK (1.24 ± 0.23 versus 1.96 ± 0.39 imp s−1, before versus after, n= 10). In contrast, six other afferents responded reproducibly to repeated injections of histamine (10 μg kg−1, LA) in the absence of BK. BK sensitization of the afferent response to histamine lasted for less than 10 min. Cyclooxygenase blockade with indomethacin (5 mg kg−1, i.v.) abolished BK sensitization of the response to histamine (1.09 ± 0.11 versus 1.11 ± 0.10 imp s−1, n= 10). Conversely, the response of most (7/9) cardiac afferents to repeat application of BK (1 μg, LA) 4 min after histamine (10 μg kg−1, LA) was attenuated compared with the BK response before histamine (1.84 ± 0.25 versus 1.31 ± 0.18 imp s−1, before versus after, P < 0.05). Repeat BK (1 μg, LA) induced a consistent response in five other afferents in the absence of histamine. Thus, BK interacts with histamine, and together they cause a larger response than either one alone. BK sensitizes cardiac afferents responding to histamine in a time‐dependent fashion, and the BK sensitization effect is dependent on an intact cyclooxygenase pathway. Conversely, histamine reduces the response of most afferents to BK.

Undiscovered role of endogenous thromboxane A2 in activation of cardiac sympathetic afferents during ischaemia

Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A2 (TxA2) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA2 stimulates cardiac afferents during ischaemia through direct activation of TxA2 (TP) receptors coupled with the phospholipase C–protein kinase C (PLC–PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T2–T5) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27–3.65 m s−1) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 Aδ‐, 31 C‐fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 μg of the TxA2 mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose‐dependent manner. Second, BM13,177, a selective TxA2 receptor antagonist, abolished the responses of six afferents to 5 μg of U46619 injected into the left atrium and attenuated the ischaemia‐related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n= 6) and to recurrent myocardial ischaemia (n= 7). In the fourth protocol, administration of PKC‐(19–36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38%. Finally, using an immunohistochemical staining approach, we observed that TP receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous TxA2 contributes to the activation of cardiac afferents during myocardial ischaemia through direct stimulation of TP receptors probably located in the cardiac sensory nervous system and that the stimulating effect of TxA2 on cardiac afferents is dependent, at least in part, upon the PLC–PKC cellular pathway.

Myocardial ischemia-mediated excitatory reflexes: a new function for thromboxane A2?

Clinical and experimental evidence has shown that myocardial ischemia activates cardiac spinal afferents that mediate sympathoexcitatory reflex responses. During myocardial ischemia, thromboxane A2 (TxA2) is released in large quantities by activated platelets in the coronary circulation of patients with coronary artery disease. We hypothesized that endogenous TxA2 contributes to sympathoexcitatory reflexes during myocardial ischemia through stimulation of TxA2/prostaglandin endoperoxide (TP) receptors. Regional myocardial ischemia was induced by occlusion of a diagonal branch of left anterior descending coronary artery of anesthetized cats. Hemodynamic parameters and renal sympathetic nerve activity were recorded after sinoaortic denervation and bilateral vagotomy. Regional myocardial ischemia evoked significant increases in mean blood pressure (122+/-10 vs. 139+/-12 mmHg, before vs. ischemia), aortic flow (153+/-18 vs. 167+/-20 ml/min), first derivative of left ventricular pressure at 40-mmHg developed pressure (2,736+/-252 vs. 2,926+/-281 mmHg/s), systemic vascular resistance (0.6+/-0.1 vs. 0.9+/-0.12 peripheral resistance units), and renal sympathetic nerve activity (by 22%). The reflex nature of the excitatory responses was confirmed by observing its disappearance after blockade of cardiac nerve transmission with intrapericardial 2% procaine treatment. Moreover, application of U-46619 (2.5-10 microg), a TxA2 mimetic, on the heart caused graded increases in mean arterial pressure and renal nerve activity, responses that were abolished 3 min after local blockade of cardiac neural transmission with intrapericardial procaine. BM 13,177 (30 mg/kg iv), a selective TP receptor antagonist, eliminated the reflex responses to U-46619 and significantly attenuated the excitatory responses during brief (5 min) regional myocardial ischemia. The sympathoexcitatory reflex responses to U-46619 were unchanged by blockade of histamine H1 receptors with pyrilamine and serotonin 5-HT3 receptors with tropisetron, indicating specificity of this TP receptor agonist. These data indicate that endogenous TxA2 participates in myocardial ischemia-mediated sympathoexcitatory reflex responses through a TP receptor mechanism.

Bradykinin and thromboxane A2 reciprocally interact to synergistically stimulate cardiac spinal afferents during myocardial ischemia

Myocardial ischemia is a complex process leading to the simultaneous release of a number of mediators, including thromboxane A(2) (TxA(2)) and bradykinin (BK), that activate cardiac spinal afferents. The present study tested the hypothesis that TxA(2) and BK reciprocally interact to excite ischemically sensitive cardiac afferents. Nerve activity of single cardiac afferent units was recorded from the left sympathetic chain or rami communicantes (T(2)-T(5)) of anesthetized cats. Fifty-two ischemically sensitive afferents (conduction velocity = 0.27-3.35 m/s, 7 Adelta-fibers and 45 C-fibers) were identified. Repeated injections (1 microg) of BK into the left atrium (LA) 4 min after the administration of U-46619 (5 microg into the LA), a TxA(2) mimetic, induced a significantly larger cardiac afferent response than the first response to BK (0.61 +/- 0.14 to 1.95 +/- 0.29 vs. 0.66 +/- 0.09 to 2.75 +/- 0.34 impulses/s, first injection vs. second injection, n = 8). Conversely, blockade of TxA(2) receptors with BM-13,177 (30 mg/kg iv) attenuated the responses of eight other afferents to BK (1 microg into the LA) by 45%. In contrast, repeated BK (1 microg into the LA) induced consistent discharge activity in six separate afferents. We then observed that the coadministration of U-46619 (5 microg) and BK (1 microg into the LA) together caused a total response that was significantly higher than the predicted response by the simple addition of the individual responses. BK (1 microg) facilitated eight cardiac afferent responses to U-46619 (5 microg into the LA) by 64%. In contrast, repeated U-46619 (5 microg into the LA) without intervening BK stimulation evoked consistent responses in seven other ischemically sensitive afferents. Finally, inhibition of cyclooxygenase with indomethacin (5 mg/kg iv) eliminated the potentiating effects of BK on the cardiac afferent response to U-46619 (5 microg into the LA) but did not alter the afferent response to U-46619. These data suggest that BK and TxA(2) reciprocally interact to stimulate ischemically sensitive cardiac afferent endings leading to synergistic afferent responses and that the BK sensitization effect is mediated by cyclooxygenase products.

Endogenous endothelin stimulates cardiac sympathetic afferents during ischaemia

Myocardial ischaemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Previous studies have shown that a brief period of myocardial ischaemia increases endothelin in cardiac venous plasma draining ischaemic myocardium and that exogenous endothelin excites cutaneous group III and IV sensory nerve fibres. The present study tested the hypothesis that endogenous endothelin stimulates cardiac afferents during ischaemia through direct activation of endothelin A receptors (ETARs). Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T2–T5) in anaesthetized cats. Single fields of 38 afferents (CV = 0.25–3.86 m s−1) were identified in the left or right ventricle with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 38 cardiac afferents (8 Aδ, 30 C‐fibres) and the responses of these 38 afferents to chemical stimuli were further studied in the following protocols. In the first protocol, injection of endothelin 1 (ET‐1, 1, 2 and 4 μg) into the left atrium (LA) stimulated seven ischaemically sensitive cardiac afferents in a dose‐dependent manner. Second, BQ‐123, a selective ETAR antagonist, abolished the responses of nine afferents to 2 μg of ET‐1 injected into the left atrium and attenuated the ischaemia‐related increase in activity of eight other afferents by 51%. In contrast, blockade of ETB receptors caused inconsistent responses to exogenous ET‐1 as well as to ischaemia. Furthermore, in the absence of ETAR blockade, cardiac afferents responded consistently to repeated administration of ET‐1 (n= 7) and to recurrent myocardial ischaemia (n= 7). Finally, using an immunocytochemical staining approach, we observed that ETA receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous endothelin contributes to activation of cardiac afferents during myocardial ischaemia through direct stimulation of ETA receptors likely to be located in the cardiac sensory nervous system.