Atrial Injection Research Articles

A direct injection technique for investigation of lung sensory properties and reflex functions.

This article reviews a unique direct injection technique that complements the more conventional right atrial injection and aerosol delivery methods to study sensory and reflex effects of the lung sensors. Used in combination with other methods, this technique should contribute to the pulmonary sensory research. The lungs house sensory receptors (sensors) that mediate a variety of sensory and reflex responses to mechanical or chemical changes. These reflexes are mainly carried through pulmonary sympathetic and vagal afferent pathways. The chemosensors in the lung periphery are especially important in pulmonary diseases and their reflex responses have traditionally been studied either by aerosol delivery, which also activates receptors in the central airways, or by right atrial injection, which also activates receptors lying outside the lung. Thus, these techniques may confound the interpretation of sensory function. Our laboratory has developed a direct injection technique to deliver agents into the lung parenchyma, which complements the conventional techniques with some important advantages. This article reviews the technique.

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia.

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,β-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17β-estradiol (E2) at a low concentration (30 μg/ml) but not at high concentrations (50 and 150 μg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Mu-opioid receptors in the caudomedial NTS are critical for respiratory responses to stimulation of bronchopulmonary C-fibers and carotid body in conscious rats

We tested the hypothesis that mu-opioid receptors (MORs) in the caudomedial nucleus tractus solitarius (cmNTS) are important for the ventilatory responses to stimulation of bronchopulmonary C-fibers (PCFs), the carotid body-mediated hypoxia, and hypercapnia independent of the carotid body. First, we used immunohistochemistry to map MORs distribution in the caudal medulla. Then we compared the effects of intra-cmNTS microinjection of DAMGO (a MOR agonist) with or without a combination of CTAP (a MOR antagonist) on the ventilatory responses to: 1) right atrial injection of capsaicin (to stimulation of PCFs) and 2) acute hypoxia (HVR, to stimulate the carotid body) in awake intact rats; and 3) hypercapnia (HCVR) in the carotid body ablated rats. The cmNTS presented the highest MORs in the caudal medulla. Microinjection of DAMGO blocked the PCF-mediated apnea, attenuated HVR (70%) and HCVR (21%), while microinjection of CTAP+DAMGO failed to affect these chemoreflexes. Our data demonstrate a critical role of activation of cmNTS MORs in regulating these chemoreflexes and imply a presence of MORs in the synapse of the 2nd-order neurons receiving inputs from PCFs and the carotid body, and NTS chemosensitive neurons.

Hypersensitivity of vagal pulmonary C-fibers induced by increasing airway temperature in ovalbumin-sensitized rats.

Our recent study has shown that hyperventilation of humidified warm air (HWA) triggered cough and reflex bronchoconstriction in patients with mild asthma. We suggested that a sensitizing effect on bronchopulmonary C-fibers by increasing airway temperature was involved, but direct evidence was lacking. This study was carried out to test the hypothesis that HWA enhances the pulmonary C-fiber sensitivity in Brown-Norway rats sensitized with ovalbumin (Ova). In anesthetized rats, isocapnic hyperventilation of HWA for 3 min rapidly elevated airway temperature to a steady state of 41.7°C. Immediately after the HWA challenge, the baseline fiber activity (FA) of pulmonary C-fibers was markedly elevated in sensitized rats, but not in control rats. Furthermore, the response of pulmonary C-fibers to right atrial injection of capsaicin in sensitized rats was significantly higher than control rats before the HWA challenge, and the response to capsaicin was further amplified after HWA in sensitized rats (ΔFA = 4.51 ± 1.02 imp/s before, and 9.26 ± 1.74 imp/s after the HWA challenge). A similar pattern of the HWA-induced potentiation of the FA response to phenylbiguanide, another chemical stimulant of C-fibers, was also found in sensitized rats. These results clearly demonstrated that increasing airway temperature significantly elevated both the baseline activity and responses to chemical stimuli of pulmonary C-fibers in Ova-sensitized rats. In conclusion, this study supports the hypothesis that the increased excitability of these afferents may have contributed to the cough and reflex bronchoconstriction evoked by hyperventilation of HWA in patients with asthma.

NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism.

INHALATION OF HYDROGEN SULFIDE (H2S) AUGMENTS THE PULMONARY REFLEXES

We have previously reported that H2S exerted a sensitizing effect on pulmonary vagal C‐fiber afferents in anesthetized, artificially ventilated SD rats. However, whether the sensitizing effect of H2S in turn enhances pulmonary reflexes remains unknown. In anesthetized, spontaneously breathing rats, inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S) caused no significant changes in the baseline breathing pattern. However, the pulmonary chemoreflexes, characterized by apnea, bradycardia and hypotension, triggered by right atrial injection of capsaicin or adenosine, were potentiated after the NaHS inhalation. This potentiating effect was eliminated by pretreatment with perineural capsaicin (a selective blockade of the conduction of vagal C‐fibers) or HC‐030031 (an antagonist of TRPA1 receptors), but not affected by pretreatment with perineural sham surgery or HC‐030031 vehicle. Furthermore, in awake, restrained guinea pigs, the cough reflexes elicited by inhalation of aerosolized capsaicin were also potentiated after the NaHS inhalation. In conclusion, H2S enhanced pulmonary reflexes, and probably resulted from its action on pulmonary C‐fibers. (MOST 103‐2320‐B‐038‐014‐)

Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists.

Nucleus tractus solitarii A2a adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs

Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension.

Hydrogen sulfide induces hypersensitivity of rat capsaicin-sensitive lung vagal neurons: role of TRPA1 receptors

The sensitization of capsaicin-sensitive lung vagal (CSLV) afferents by inflammatory mediators is important in the development of airway hypersensitivity. Hydrogen sulfide (H2S) is an endogenous mediator inducing hyperalgesia through transient receptor potential ankyrin 1 (TRPA1) receptors located on nociceptors. We conducted this study to determine whether H2S elevates the sensitivity of rat CSLV afferents. In anesthetized, artificially ventilated rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a H2S donor) caused no significant changes in the baseline activity of CSLV afferents. However, the afferent responses to right atrial injection of capsaicin or phenylbiguanide and to lung inflation were all markedly potentiated after NaHS inhalation. By contrast, the inhalation of its vehicle or NaOH (with a similar pH to NaHS) failed to enhance the afferent responses. Additionally, the potentiating effect on the afferent responses was found in rats inhaling L-cysteine (a substrate of H2S synthase) that slowly releases H2S. The potentiating effect of NaHS on the sensitivity of CSLV afferents was completely blocked by pretreatment of HC-030031 (a TRPA1 receptor antagonist) but was unaffected by its vehicle. In isolated rat CSLV neurons, the perfusion of NaHS alone did not influence the intracellular Ca(2+) concentration but markedly potentiated the Ca(2+) transients evoked by capsaicin. The NaHS-caused effect was totally abolished by HC-030031 pretreatment. These results suggest that H2S induces a nonspecific sensitizing effect on CSLV fibers to both chemical and mechanical stimulation in rat lungs, which appears mediated through an action on the TRPA1 receptors expressed on the nerve endings of CSLV afferents.

Severe hemorrhage attenuates cardiopulmonary chemoreflex (CCR) control of renal and adrenal sympathetic nerves via adenosine operating in the nucleus of the solitary tract (NTS)

During severe hemorrhage adrenal sympathoactivation is accompanied by paradoxical renal sympathoinhibition and bradycardia. Adenosine operating in the NTS via A1 and A2a receptors contributes to the paradoxical effects and it inhibits CCR mediated sympathoinhibition (Am J Physiol 291: H2453‐H2461, 2006; 303: R539‐R550, 2012; FASEB J 25: 844.3, 2011). Therefore, we hypothesized that adenosine released into the NTS during severe hemorrhage will attenuate the CCR and that blockade of NTS adenosine receptors will reverse this attenuation. We compared renal and adrenal sympathetic responses evoked by right atrial injections of 5HT3 receptor agonist, phenylbiguanide (PBG, 1–8 μg/kg), under control conditions, during hemorrhage and during hemorrhage after blockade of NTS adenosine receptors with bilateral microinjections of 8‐(p‐sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. Hemorrhage significantly attenuated the CCR responses and blockade of NTS adenosine receptors reversed this attenuation. We conclude that adenosine released into the NTS contributes to the paradoxical posthemorrhagic effects but it also serves as a negative feedback regulator of the CCR. HL‐67814

Hypersensitivity of bronchopulmonary C‐fibers induced by an increase in airway temperature in ovalbumin (Ova)‐sensitized Brown Norway rats.

Our recent study has reported that hyperventilation of humid warm air (WA) triggered cough and reflex bronchoconstriction in patients with mild asthma (AJRCCM 2012). We suggested that sensitization of the bronchopulmonary C‐fibers by increasing airway temperature is involved, but direct evidence is lacking. This study was carried out to determine whether hyperventilation of WA enhanced the pulmonary C‐fiber sensitivity in an animal model of asthma (Ova‐sensitized Brown‐Norway rats). Isocapnic hyperventilation of WA for 3 minutes rapidly raised airway temperature to a peak of ~42°C which significantly elevated the base‐line fiber activity (FA) of pulmonary C‐fibers in Ova rats immediately after the termination of WA challenge, but not in the control rats. Furthermore, the pulmonary C‐fiber response to right atrial injection of capsaicin was significantly higher in Ova rats than control rats before the WA challenge; this difference in the FA response to capsaicin was further amplified after WA in Ova rats (Δ=4.98±1.78 imp/s before, and 10.34±2.21 imp/s after WA; P<0.05, n=9). A similar pattern of the WA‐induced potentiation of the FA response to phenylbiguanide was also founded in Ova rats. These results suggest that Ova‐sensitization increases the excitability of pulmonary C‐fibers, and the hypersensitivity is further enhanced by an increase in the airway temperature. (Supported by NIH and DoD grants)

Abstract WP410: Comparison of Transcranial Doppler Ultrasound with Intracardiac Echocardiography in the Detection of Cardiac Right-to-Left shunt in Children and Young Adults

Background and Objectives: Right to left shunts (RLS) found with patent foramen ovale are implicated in the pathophysiology of cryptogenic stroke, migraine headaches as well as decompression illness. Our dysautonomia clinic has described a syndrome of neurocardiogenic syncope, stroke, migraine headaches and positive tilt table testing associated with atrial septal defects (ASD). All patients underwent transcranial Doppler ultrasound (TCD) and intracardiac echocardiogram (ICE) testing. Methods: We performed a restrospective analysis of a prospectively obtained registry of patients that underwent shunt testing ± endovascular ASD closure. All patients had previously undergone a transthoracic echocardiogram bubble study (TTE) to confirm a RLS. Patients underwent general anesthesia and bilateral TCD monitoring of the middle cerebral artery. Valsalva was produced by the anesthesiologist (40mm Hg). Simultaneous assessment of RLS was performed using ICE and TCD monitoring after injection of agitated saline into the femoral vein sheath. If negative, a direct right atrial injection was performed. The size of ASD was measured by ICE independent of TCD results. Results: Between 9/2010 and 7/2012, 112 patients underwent testing. Patient characteristics are displayed in the table. RLS was found in 82.1, 81.1, and 71.4% by TTE, ICE and TCD, respectively. After peripheral injection (n=85), when compared to ICE, TCD had a sensitivity, specificity, PPV, NPV and accuracy of 96, 100, 100, 64 and 95%, respectively. Combined peripheral and intracardiac injections (N=112) when compared to ICE, TCD had a sensitivity, specificity, PPV, NPV and accuracy of 89, 100, 100, 66 and 90%, respectively. Conclusion: Transcranial Doppler ultrasound detection of right to left shunts remains a highly accurate, non-invasive tool. If closure of cardiac shunts is proven to prevent stroke, migraines or treat neurocardiogenic syncope, TCD might be considered the preferred screening method.

Activation of NTS A1adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex

Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes integrated in the NTS.

Contribution of central μ-receptors to switching pulmonary C-fibers-mediated rapid shallow breathing into an apnea by fentanyl in anesthetized rats

Our previous study has shown that activating peripheral μ-receptors is necessary for switching the bronchopulmonary C-fibers (PCFs)-mediated rapid shallow breathing (RSB) into an apnea by systemic administration of fentanyl. The brainstem nuclei, such as the medial nucleus tractus solitarius (mNTS) and the pre-Botzinger complex (PBC), are required for completing the PCF-mediated respiratory reflexes. Moreover, these areas contain abundant μ-receptors and their activation prolongs expiratory duration (TE). Thus, we asked if central μ-receptors, especially those in the mNTS and PBC, are involved in fully expressing this RSB-apnea switch by fentanyl. In anesthetized rats, the cardiorespiratory responses to right atrial injection of phenylbiguanide (PBG, 3–6μg/kg) were repeated after: (1) fentanyl (iv), a μ-receptor agonist, alone (8μg/kg, iv); (2) fentanyl following microinjection of naloxone methiodide (NXM, an opioid receptor antagonist) into the cisterna magna (10μg/4μl); (3) the bilateral mNTS (10mM, 20nl); or (4) PBC (10mM, 20nl). Our results showed that PBG shortened TE by 37±6% (RSB, from 0.41±0.05 to 0.26±0.03s, P<0.01), but it markedly prolonged TE by 5.8-fold (an apnea, from 0.50±0.04s to 2.9±0.57s, P<0.01) after fentanyl (iv). Pretreatment with NXM injected into the cisterna magna or the PBC, but not the mNTS, prevented the fentanyl-induced switch. This study, along with our previous results mentioned above, suggests that although peripheral μ-receptors are essential for triggering the fentanyl-induced switch, central μ-receptors, especially those in the PBC, are required to fully exhibit such switch.Summary statement: Our results suggest that the activation of central μ-receptors, especially those in the pre-Botzinger complex, is required for switching the pulmonary C-fiber-mediated rapid shallow breathing into an apnea by systemic administration of fentanyl.

Canine coronary microvascular resistance at vasodilation depends on coronary perfusion pressure

ObjectiveWe tested the premise of a constant microvascular resistance at full vasodilation (Rm) that underlies the concept of fractional flow reserve.MethodsIn 7 anesthetized dogs, we measured aortic (Pa) and venous (Pv) pressure, flow in the left anterior descending (LAD) artery, distal pressure (Pd) in the left circumflex (LCX) artery. During LCX occlusion, wedge pressure (Pw) was measured and μspheres infused into the LAD at full vasodilation. In one dog, the RCA contribution was determined by left atrial injection of differently colored μsph. Hearts were analyzed using a high‐resolution 3D imaging cryomicrotome. The mass of LCX and LAD perfusion territories was obtained. Assuming equal resistance per gram tissue for LAD and LCX, the weight‐normalized Rm of LCX was determined at Pd and Pw. Collateral flow was calculated as the ratio of μsph in LCX and LAD territories multiplied by LAD flow.ResultsAccounting for the added 30%RCA contribution to the collateral flow obtained from LAD injection, Rm was 2.3 ± 1.9 times higher at low (Pw= 15.7+4.8 mmHg) compared to normal pressure (Pd=69.3+12.4 mmHg) (p&lt;0.05). Collateral flow from LAD to LCX was 8.6% + 5.3% of LAD hyperemic flow.ConclusionsOur data confirm the pressure‐dependence of coronary Rm at vasodilation, which suggests a reason for pressure‐based overestimation of collateral flow. Supported by the Netherlands Heart Foundation (2006B226)

Mu‐Opioid Receptors (MORs) in Pre‐Botzinger Complex (PBC) Are Involved in Fentanyl Switching of Pulmonary C‐Fibers (PCFs)‐Mediated Rapid Shallow Breathing (RSB) into An Apnea in Anesthetized Rats

Our previous study showed that fentanyl (iv), a selective MOR agonist, could switch the PCFs‐mediated RSB into an apnea and that peripheral MORs are necessary for triggering this switch. Here we further tested if central MORs, especially those in the medial nucleus tractus solitarius (mNTS) receiving PCFs’ inputs and the PBC responsible for respiratory rhythmogenesis, were also involved in such switching. Right atrial injection of phenylbiguanide (3–5 μg/kg) was performed to stimulate PCFs in anesthetized rats before and after fentanyl (8 μg/kg, iv) without or with prior central administration of naloxone methiodide (M‐NLX, an opioid receptor antagonist). The central administration was achieved by injecting M‐NLX into the cisterna magna (8 μg/4μl), the mNTS or the PBC (10 mM, 15 nl in each side). Our results showed that phenylbiguanide alone shortened expiratory duration (TE) by 32 ± 6 % (RSB), while it markedly prolonged TE by 5.5‐fold (an apnea) after fentanyl. M‐NLX injected into the cisterna magna or the PBC, but not the mNTS, prevented the fentanyl switching with the TE of RSB slightly but significantly prolonged. This study, along with our previous results, suggests that although peripheral MORs are critical to trigger the fentanyl switching of PCF‐mediated RSB into an apnea, central MORs, especially those in the PBC, are necessary to establish this switching (Supported by ALA RG‐191095‐N and HL 107462).

IL-1β acutely increases pulmonary SP and permeability without associated changes in airway resistance and ventilation in anesthetized rats

Bronchopulmonary C-fibers (PCFs), when activated, promote substance P (SP) release, increase microvascular leak, and produce bronchoconstriction and apnea. IL-1β administered systemically or locally into the pulmonary parenchyma stimulates PCFs. Thus, we tested whether right atrial bolus injection or aerosol inhalation of IL-1β, to mainly stimulate PCFs, would acutely affect pulmonary SP level and vascular permeability, airway resistance (R(L)), and ventilation in anesthetized rats. Our results showed that 30min after IL-1β injection (2-6 μg kg⁻¹), SP levels and Evans blue extravasation in bronchoalveolar lavage fluid were markedly increased and these responses were eliminated or largely reduced in neonatal capsaicin-treated rats. In contrast, neither injection nor inhalation of IL-1β (5-15 μg ml⁻¹) significantly altered R(L) and ventilation. Additionally, the capsaicin-evoked (4 μg kg⁻¹, i.v.) apneic response was unaffected by IL-1β treatment. Our data suggest that IL-1β, as administered in this study, can acutely increase pulmonary SP and vascular permeability, likely via stimulating PCFs, with little impact on R(L) and ventilation.

Modulatory Effect of Fentanyl on Bronchopulmonary C‐fiber (PCF)‐mediated Ventilatory (VE) Responses Is Age‐dependent in Anesthetized Rats

In adult rats, opioids administered into the pulmonary circulation produces an apnea and given systemically turns the phenylbiguanide (PBG)‐evoked rapid shallow breaths (RSBs) to an apnea by stimulating and facilitating PCFs, respectively. Fentanyl, an opioid μ‐receptor agonist, clinically has a lesser inhibitory effect on ventilation in children (&lt; 3 years old) than in adults (Barker, Pediatric Anesthesia 2007). Thus, we tested if the modulatory effect of opioids on the PCF‐mediated VE responses in rats were age‐dependent. VE responses to a right atrial bolus injection of PBG (before and after systemic administration of fentanyl) or fentanyl were recorded in anesthetized rat pups (3 week‐old) and adults. We found that: 1) PBG (5 μg/kg) evoked 5–10 RSBs in adults, but caused no change or 2–3 rapid breaths in pups; 2) systemic fentanyl (6 μg/kg, iv) reduced VE more greatly in adults (60%) than pups (27%, P &lt; 0.01) and switched the PBG‐induced RSB to an apnea in adults, but failed to alter the PBG effects on VE in pups; 3) right atrial injection of fentanyl (2 μg/kg) alone produced an apnea in adults but not in pups; and 4) the VE responses to right atrial injection of PBG or fentanyl were eliminated by bilateral vagotomy. Our data suggest that the PCF‐mediated RSBs and inspiratory inhibition in response to PBG and fentanyl are age‐dependent in rats, indicating a plasticity of PCFs as development. (ALA RT‐83131‐N).

Activation of NTS A2a Adenosine Receptors Impairs Cardiopulmonary Chemoreflex Control of Renal (RSNA), Adrenal (ASNA) and Lumbar (LSNA) Sympathetic Nerve Activity

Activation of NTS A2a adenosine receptors increases ASNA, decreases RSNA and does not alter LSNA (Clin Exp Physiol Pharmacol 28: 120–124, 2001). A similar pattern of regional sympathetic responses was occasionally observed following activation of cardiopulmonary chemoreceptors. As A2a adenosine receptors may facilitate neurotransmitter release it was likely that these receptors may facilitate the cardiopulmonary chemoreflex. In the present study we compared the regional sympathetic responses evoked by right atrial injections of 5HT3 receptor agonist, phenylbiguanide (PBG, 1–8 μg/kg), before and after stimulation of NTS A2a adenosine receptors (microinjections of CGS21680, 20 pmol/50 nl) in urethane/chloralose anesthetized rats (n=7). Activation of cardiopulmonary chemoreceptors evoked differential, dose dependent sympathoinhibition RSNA&gt;ASNA&gt;LSNA). Surprisingly, the responses in all sympathetic outputs (Δ%) were similarly attenuated following stimulation of NTS A2a adenosine receptors (−95.5±1.8 vs. −60.3±5.3, −74.9±7.1 vs. −44.6±8.6, −56.4±3.6 vs. −34.61±5.6, for RSNA, ASNA and LSNA, respectively, at the maximal dose of PBG). We conclude that A2a adenosine receptors may facilitate NTS neurons/terminals which inhibit cardiopulmonary chemoreflex pathway. NIH HL‐67814

Pulmonary Chemoreflex Responses Are Potentiated by Chronic Treatment of Tumor Necrosis Factor Alpha (TNFα) in Mice

TNFα, a pro‐inflammatory cytokine, is involved in the pathogenesis of asthma and also known for its sensitizing effect on somatic nociceptive afferents. This study was carried out to test whether chronic treatment of TNFα enhances pulmonary chemoreflex responses. Mice were divided into two matching groups (n=9 each), TNFα (10.08 μg/kg; 1μg = ~105 units) and vehicle were administered by intratracheal (i.t.) instillation in treated and control group, respectively, during a brief anesthesia. 24 h later, the dose‐related responses of apnea to right atrial injection of capsaicin (a TRPV1 activator; 0.25–1.0 μg/kg), allyl isothiocyanate (a TRPA1 activator; 0.3–1.2 mg/kg) and phenylbiguanide (a 5‐HT3 activator; 30–120 μg/kg) were all markedly elevated in TNFα‐treated group as compared to that in the control group. Furthermore, the elevated sensitivity was absent when the same TNFα treatment was applied to p55/p75‐null mice, in which both types of TNFα receptors were mutated. These results suggest a positive interaction between TNFα and pulmonary C‐fiber receptors, which is responsible for elevating the pulmonary chemoreflex sensitivity. (Supported by NIH grant HL96914)