Adenosine A2A receptors in the rostral ventrolateral medulla participate in blood pressure decrease with electroacupuncture in hypertensive rats.

Acupuncture is increasingly used to manage high blood pressure (BP) as a complementary therapy. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36-37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). However, it is uncertain whether rVLM A2AR contributes to EA's BP-lowering effect in sustained hypertension. We hypothesized that a course of EA treatment lowers BP, in part, through the activation of adenosine A2AR in the rVLM in hypertensive rats. To mimic essential hypertension in the clinic, we performed EA in conscious Dahl salt-sensitive hypertensive rats (DSHRs). EA (0.1-0.4 mA, 2 Hz) was applied at ST36-37 for 30 min twice weekly for four weeks, while sham-EA was conducted in a similar manner but without electrical input. In hypertensive rats, BP was reduced by EA (n = 14) but neither by sham-EA (n = 14) nor in the absence of needling (n = 8). Following four weeks of eight treatments and then under anesthesia, EA's modulatory effect on elevated BP was reversed by unilateral rVLM microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n = 7; P < 0.05) but not the vehicle (n = 5) in EA-treated DSHRs. Activation of rVLM A2AR in DSHRs treated with sham-EA by an A2AR agonist, CGS-21680 (0.4 mM in 50 nl; n = 8), decreased BP. Unilateral administration of SCH 58261 or CGS-21680 into the rVLM did not alter basal BP in Dahl salt-sensitive rats fed a regular diet with normal BP. The A2AR level in the rVLM after EA was increased compared to the sham-EA and untreated DSHRs (n = 5 in each group; all P < 0.05). These data suggest that a 4-week twice weekly EA treatment reduced BP in salt-sensitive hypertensive rats likely through adenosine-mediated A2AR in the rVLM.

Sympathoinhibitory electroacupuncture (EA) interacts positively with anti-inflammatory EA alleviating blood pressure in hypertensive rats.

Elevated sympathetic activity and chronic inflammation are known contributory factors observed in hypertension. We have observed that sympathoinhibitory electroacupuncture (SI-EA) at acupoints ST36-37 alleviates sympathetic activity and hypertension. Additionally, EA at acupoints SP6-7 exerts anti-inflammatory (AI-EA) effects. However, it is not known whether simultaneous stimulation of this combination of acupoints attenuates or enhances individual effects. A 2 × 2 factorial design was used to test the hypothesis that combining SI-EA and AI-EA (cEA) leads to greater reduction of hypertension by decreasing sympathetic activity and inflammation in hypertensive rats than either set of acupoints alone. Dahl salt-sensitive hypertensive (DSSH) rats were treated with four EA regimens including cEA, SI-EA, AI-EA, and sham-EA twice weekly for five weeks. A group of normotensive (NTN) rats served as control. Systolic and diastolic BP (SBP and DBP) and heart rate (HR) were measured non-invasively by tail-cuff. Plasma norepinephrine (NE), high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) concentrations were determined with ELISA at the completion of treatments. DSSH rats on high salt diet progressively developed moderate hypertension within five weeks. DSSH rats treated with sham-EA showed continuous increase in SBP and DBP and elevations in plasma NE, hs-CRP, and IL-6 levels relative to NTN control. Both SI-EA and cEA decreased SBP and DBP, and had corresponding changes in biomarkers (NE, hs-CRP, and IL-6) compared with sham-EA. AI-EA prevented SBP and DBP elevation and decreased IL-6 and hs-CRP relative to sham-EA. Importantly in DSSH rats that received repetitive cEA treatment, SI-EA interacted positively with AI-EA leading to greater reduction of SBP, DBP, NE, hs-CRP, and IL-6 than SI-EA or AI-EA alone. These data suggest that by targeting both elevated sympathetic activity and chronic inflammation, cEA regimen results in a greater reduction of BP effects in treating hypertension compared to using individual SI-EA or AI-EA alone.

Neurogenic Hypotension and Bradycardia Modulated by Electroacupuncture in Hypothalamic Paraventricular Nucleus.

Electroacupuncture (EA) stimulates somatic median afferents underlying P5-6 acupoints and modulates parasympathoexcitatory reflex responses through central processing in the brainstem. Although decreases in blood pressure and heart rate by the neural-mediated Bezold-Jarisch reflex responses are modulated by EA through opioid actions in the nucleus tractus solitarius and nucleus ambiguus, the role of the hypothalamus is unclear. The hypothalamic paraventricular nucleus (PVN) is activated by sympathetic afferents and regulates sympathetic outflow and sympathoexcitatory cardiovascular responses. In addition, the PVN is activated by vagal afferents, but little is known about its regulation of cardiopulmonary inhibitory hemodynamic responses. We hypothesized that the PVN participates in the Bezold-Jarisch reflex responses and EA inhibits these cardiopulmonary responses through the PVN opioid system. Rats were anesthetized and ventilated, and their heart rate and blood pressures were monitored. Application of phenylbiguanide every 10 min close to the right atrium induced consistent depressor and bradycardia reflex responses. Unilateral microinjection of the depolarization blockade agent kainic acid or glutamate receptor antagonist kynurenic acid in the PVN reduced these reflex responses. In at least 70% of the rats, 30 min of bilateral EA at P5-6 acupoints reduced the depressor and bradycardia responses for at least 60 min. Blockade of the CCK-1 receptors converted the non-responders into EA-responders. Unilateral PVN-microinjection with naloxone reversed the EA inhibition. Vagal-evoked activity of the PVN cardiovascular neurons was reduced by 30 min EA (P5-6) through opioid receptor activation. These data indicate that PVN processes inhibitory cardiopulmonary reflexes and participates in EA-modulation of the neural-mediated vasodepression and bradycardia.

Role of opioid receptors in modulation of P2X receptor-mediated cardiac sympathoexcitatory reflex response.

Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents' activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,ß-methylene ATP (α,ß-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,ß-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,ß-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.

Modulation of Neurally Mediated Vasodepression and Bradycardia by Electroacupuncture through Opioids in Nucleus Tractus Solitarius.

Stimulation of vagal afferent endings with intravenous phenylbiguanide (PBG) causes both bradycardia and vasodepression, simulating neurally mediated syncope. Activation of µ-opioid receptors in the nucleus tractus solitarius (NTS) increases blood pressure. Electroacupuncture (EA) stimulation of somatosensory nerves underneath acupoints P5-6, ST36-37, LI6-7 or G37-39 selectively but differentially modulates sympathoexcitatory responses. We therefore hypothesized that EA-stimulation at P5-6 or ST36-37, but not LI6-7 or G37-39 acupoints, inhibits the bradycardia and vasodepression through a µ-opioid receptor mechanism in the NTS. We observed that stimulation at acupoints P5-6 and ST36-37 overlying the deep somatosensory nerves and LI6-7 and G37-39 overlying cutaneous nerves differentially evoked NTS neural activity in anesthetized and ventilated animals. Thirty-min of EA-stimulation at P5-6 or ST36-37 reduced the depressor and bradycardia responses to PBG while EA at LI6-7 or G37-39 did not. Congruent with the hemodynamic responses, EA at P5-6 and ST36-37, but not at LI6-7 and G37-39, reduced vagally evoked activity of cardiovascular NTS cells. Finally, opioid receptor blockade in the NTS with naloxone or a specific µ-receptor antagonist reversed P5-6 EA-inhibition of the depressor, bradycardia and vagally evoked NTS activity. These data suggest that point specific EA stimulation inhibits PBG-induced vasodepression and bradycardia responses through a µ-opioid mechanism in the NTS.

Role of TRPV1 in acupuncture modulation of reflex excitatory cardiovascular responses.

We have shown that acupuncture, including manual and electroacupuncture (MA and EA), at the P5-6 acupoints stimulates afferent fibers in the median nerve (MN) to modulate sympathoexcitatory cardiovascular reflexes through central regulation of autonomic function. However, the mechanisms underlying acupuncture activation of these sensory afferent nerves and their cell bodies in the dorsal root ganglia (DRG) are unclear. Transient receptor potential vanilloid type 1 (TRPV1) is present in sensory nerve fibers distributed in the general region of acupoints like ST36 and BL 40 located in the hindlimb. However, the contribution of TRPV1 to activation of sensory nerves by acupuncture, leading to modulation of pressor responses, has not been studied. We hypothesized that TRPV1 participates in acupuncture's activation of sensory afferents and their associated cell bodies in the DRG to modulate pressor reflexes. Local injection of iodoresiniferatoxin (Iodo-RTX; a selective TRPV1 antagonist), but not 5% DMSO (vehicle), into the P6 acupoint on the forelimb reversed the MA's inhibition of pressor reflexes induced by gastric distension (GD). Conversely, inhibition of GD-induced sympathoexcitatory responses by EA at P5-6 was unchanged after administration of Iodo-RTX into P5-6. Single-unit activity of Group III or IV bimodal afferents sensitive to both mechanical and capsaicin stimuli responded to MA stimulation at P6. MA-evoked activity was attenuated significantly ( P < 0.05) by local administration of Iodo-RTX ( n = 12) but not by 5% DMSO ( n = 12) into the region of the P6 acupoint in rats. Administration of Iodo-RTX into P5-6 did not reduce bimodal afferent activity evoked by EA stimulation ( n = 8). Finally, MA at P6 and EA at P5-6 induced phosphorylation of extracellular signal-regulated kinases (ERK; an intracellular signaling messenger involved in cellular excitation) in DRG neurons located at C7-8 spinal levels receiving MN inputs. After TRPV1 was knocked down in the DRG at these spinal levels with intrathecal injection of TRPV1-siRNA, expression of phosphorylated ERK in the DRG neuron was reduced in MA-treated, but not EA-treated animals. These data suggest that TRPV1 in Group III and IV bimodal sensory afferent nerves contributes to acupuncture inhibition of reflex increases in blood pressure and specifically plays an important role during MA but not EA.

Paraventricular Nucleus Modulates Excitatory Cardiovascular Reflexes during Electroacupuncture.

The paraventricular nucleus (PVN) regulates sympathetic outflow and blood pressure. Somatic afferent stimulation activates neurons in the hypothalamic PVN. Parvocellular PVN neurons project to sympathoexcitatory cardiovascular regions of the rostral ventrolateral medulla (rVLM). Electroacupuncture (EA) stimulates the median nerve (P5-P6) to modulate sympathoexcitatory responses. We hypothesized that the PVN and its projections to the rVLM participate in the EA-modulation of sympathoexcitatory cardiovascular responses. Cats were anesthetized and ventilated. Heart rate and mean blood pressure were monitored. Application of bradykinin every 10-min on the gallbladder induced consistent pressor reflex responses. Thirty-min of bilateral EA stimulation at acupoints P5-P6 reduced the pressor responses for at least 60-min. Inhibition of the PVN with naloxone reversed the EA-inhibition. Responses of cardiovascular barosensitive rVLM neurons evoked by splanchnic nerve stimulation were reduced by EA and then restored with opioid receptor blockade in the PVN. EA at P5-P6 decreased splanchnic evoked activity of cardiovascular barosensitive PVN neurons that also project directly to the rVLM. PVN neurons labeled with retrograde tracer from rVLM were co-labeled with µ-opioid receptors and juxtaposed to endorphinergic fibers. Thus, the PVN and its projection to rVLM are important in processing acupuncture modulation of elevated blood pressure responses through a PVN opioid mechanism.

elPBN neurons regulate rVLM activity through elPBN-rVLM projections during activation of cardiac sympathetic afferent nerves.

The external lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contributes to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases rVLM activity through elPBN-rVLM projections. Compared with controls, in rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P < 0.05). The majority of these elPBN neurons contain vesicular glutamate transporter 3. In cats, epicardial bradykinin and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was attenuated (n = 6, P < 0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn, 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced rVLM activity evoked by both bradykinin and electrical stimulation (n = 5, P < 0.05). Excitation of the elPBN with microinjection of dl-homocysteic acid (2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. However, the enhanced rVLM activity induced by dl-homocysteic acid injected into the elPBN was reversed following iontophoresis of Kyn into the rVLM (n = 7, P < 0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through a monosynaptic (glutamatergic) excitatory elPBN-rVLM pathway.

Functional role of peripheral opioid receptors in the regulation of cardiac spinal afferent nerve activity during myocardial ischemia.

Thinly myelinated Aδ-fiber and unmyelinated C-fiber cardiac sympathetic (spinal) sensory nerve fibers are activated during myocardial ischemia to transmit the sensation of angina pectoris. Although recent observations showed that myocardial ischemia increases the concentrations of opioid peptides and that the stimulation of peripheral opioid receptors inhibits chemically induced visceral and somatic nociception, the role of opioids in cardiac spinal afferent signaling during myocardial ischemia has not been studied. The present study tested the hypothesis that peripheral opioid receptors modulate cardiac spinal afferent nerve activity during myocardial ischemia by suppressing the responses of cardiac afferent nerve to ischemic mediators like bradykinin and extracellular ATP. The nerve activity of single unit cardiac afferents was recorded from the left sympathetic chain (T2-T5) in anesthetized cats. Forty-three ischemically sensitive afferent nerves (conduction velocity: 0.32-3.90 m/s) with receptive fields in the left and right ventricles were identified. The responses of these afferent nerves to repeat ischemia or ischemic mediators were further studied in the following protocols. First, epicardial administration of naloxone (8 µmol), a nonselective opioid receptor antagonist, enhanced the responses of eight cardiac afferent nerves to recurrent myocardial ischemia by 62%, whereas epicardial application of vehicle (PBS) did not alter the responses of seven other cardiac afferent nerves to ischemia. Second, naloxone applied to the epicardial surface facilitated the responses of seven cardiac afferent nerves to epicardial ATP by 76%. Third, administration of naloxone enhanced the responses of seven other afferent nerves to bradykinin by 85%. In contrast, in the absence of naloxone, cardiac afferent nerves consistently responded to repeated application of ATP (n = 7) or bradykinin (n = 7). These data suggest that peripheral opioid peptides suppress the responses of cardiac sympathetic afferent nerves to myocardial ischemia and ischemic mediators like ATP and bradykinin.

Ionotropic glutamate receptors in the external lateral parabrachial nucleus participate in processing cardiac sympathoexcitatory reflexes.

Stimulation of cardiac sympathetic afferents during myocardial ischemia with metabolites such as bradykinin (BK) evokes sympathoexcitatory reflex responses and activates neurons in the external lateral parabrachial nucleus (elPBN). The present study tested the hypothesis that this region in the pons processes sympathoexcitatory cardiac reflexes through an ionotropic glutamate receptor mechanism. The ischemic metabolite BK (0.1-1 µg) was injected into the pericardial space of anesthetized and bilaterally vagotomized or intact cats. Hemodynamic and renal sympathetic nerve activity (RSNA) responses to repeated administration of BK before and after unilateral 50-nl microinjections of kynurenic acid (Kyn; 25 mM), 2-amino-5-phosphonopentanoic acid (AP5; 25 mM), and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol(F)quinoxaline (NBQX; 10 mM) into the elPBN were recorded. Intrapericardial BK evoked significant increases in mean arterial pressure (MAP) and RSNA in seven vagotomized cats. After blockade of glutamate receptors with the nonselective glutamate receptor antagonist Kyn, the BK-evoked reflex increases in MAP (50 ± 6 vs. 29 ± 2 mmHg) and RSNA (59 ± 8.6 vs. 29 ± 4.7%, before vs. after) were significantly attenuated. The BK-evoked responses returned to pre-Kyn levels 85 min after the application of Kyn. Similarly, BK-evoked reflex responses were reversibly attenuated by blockade of glutamate N-methyl-d-aspartate (NMDA) receptors with AP5 (n = 5) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with NBQX (n = 5). In contrast, we observed that the repetitive administration of BK evoked consistent reflex responses including MAP and RSNA before and after microinjection of 50 nl of the artificial cerebrospinal fluid vehicle into the elPBN in five animals. Microinjection of glutamate receptor antagonists into regions outside the elPBN did not alter BK-induced reflex responses. Microinjection of Kyn into the elPBN reversibly attenuated BK-induced reflex responses in four vagus intact animals. These data are the first to show that NMDA and AMPA ionotropic glutamate receptors in the elPBN play an important role in processing cardiac excitatory reflex responses.

A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia.

Myocardial ischemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Brief myocardial ischemia leads to ATP release in the interstitial space. Furthermore, exogenous ATP and α,ß-methylene ATP (α,ß-meATP), a P2X receptor agonist, stimulate cutaneous group III and IV sensory nerve fibers. The present study tested the hypothesis that endogenous ATP excites cardiac afferents during ischemia through activation of P2 receptors. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anesthetized cats. Single fields of 45 afferents (conduction velocities = 0.25-4.92 m/s) were identified in the left ventricle with a stimulating electrode. Five minutes of myocardial ischemia stimulated 39 of 45 cardiac afferents (8 Aδ, 37 C fibers). Epicardial application of ATP (1-4 µmol) stimulated six ischemically sensitive cardiac afferents in a dose-dependent manner. Additionally, epicardial ATP (2 µmol), ADP (2 µmol), a P2Y agonist, and α,ß-meATP (0.5 µmol) significantly activated eight other ischemically sensitive afferents. Third, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist, abolished the responses of six afferents to epicardial ATP (2 µmol) and attenuated the ischemia-related increase in activity of seven other afferents by 37%. In the absence of P2 receptor blockade, cardiac afferents responded consistently to repeated application of ATP (n = 6) and to recurrent myocardial ischemia (n = 6). Finally, six ischemia-insensitive cardiac spinal afferents did not respond to epicardial ATP (2-4 µmol), although these afferents did respond to epicardial bradykinin. Taken together, these data indicate that, during ischemia, endogenously released ATP activates ischemia-sensitive, but not ischemia-insensitive, cardiac spinal afferents through stimulation of P2 receptors likely located on the cardiac sensory neurites.

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 (ET(A)Rs). Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) 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 Adelta, 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 microg) into the left atrium (LA) stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BQ-123, a selective ET(A)R antagonist, abolished the responses of nine afferents to 2 microg 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 ET(B) receptors caused inconsistent responses to exogenous ET-1 as well as to ischaemia. Furthermore, in the absence of ET(A)R 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 ET(A) 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 ET(A) receptors likely to be located in the cardiac sensory nervous system.

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.

Regulation of cardiac afferent excitability in ischemia.

The heart at the time of Sir William Harvey originally was thought to be an insensate organ. Today, however, we know that this organ is innervated by sensory nerves that course centrally though mixed nerve pathways that also contain parasympathetic or sympathetic motor nerves. Angina or cardiac pain is now well recognized as a pressure-like pain that occurs during myocardial ischemia when coronary artery blood flow is interrupted. Sympathetic (or spinal) afferent fibers that are either finely myelinated or unmyelinated are responsible for the transmission of information to the brain that ultimately allows the perception of angina as well as activation of the sympathetic nervous system, resulting in tachycardia, hypertension, and sometimes arrhythmias. Although early studies defined the importance of the vagal and sympathetic cardiac afferent systems in reflex autonomic control, until recently there has been little appreciation of the mechanisms of activation of the sensory endings. This review examines the role of a number of chemical mediators and their sources that are activated by the ischemic process. In this regard, patients with ischemic syndromes, particularly myocardial infarction and unstable angina, are known to have platelet activation, which leads to release of a number of chemical mediators, including serotonin, histamine, and thromboxane A(2), all of which stimulate ischemically sensitive cardiac spinal afferent endings in the ventricles through specific receptor-mediated processes. Furthermore, protons from lactic acid, bradykinin, and reactive oxygen species, especially hydroxyl radicals, individually and frequently in combination, stimulate these endings during ischemia. Cyclooxygenase products appear to sensitize the endings to the action of bradykinin and histamine. These studies of the chemical mechanisms of activation of cardiac sympathetic afferent endings during ischemia have the potential to provide targeted therapies that can modify the angina and the deleterious reflex responses that have the potential to exacerbate ischemia and myocardial cell death.

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.

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.

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.