Alpha2A-adrenoceptor mediated tachypnea in awake goats.

To further elucidate the role of alpha2-adrenoceptors (alpha2-ARs) in the control of respiratory rhythm we examined the ventilatory effects of guanfacine (a preferentially selective alpha2A-AR agonist) and clonidine (a non-selective alpha2-AR agonist) in awake adult goats. Systemic administration of guanfacine in cumulative doses (20 microg/kg; 140-180 microg/kg total cumulative dose) increased breathing in all animals in a dose-dependent manner. The excitatory effect was entirely mediated by increases in respiratory frequency. The magnitude of the guanfacine-induced tachypnea was similar to that produced by systemic administration of cumulative doses of clonidine (1-2 microg/kg; 4-10 microg/kg total cumulative dose) in the same animals studied on a separate day. Both guanfacine- and clonidine-induced tachypnea was reversed by the preferentially selective alpha2A-AR antagonist RX821002 (2-6 microg/kg IV). Unlike clonidine however, guanfacine administration did not produce slow arrhythmic breathing episodes (irregular TE intervals and central apneas) that are characteristic of alpha2-AR stimulation with alpha2-AR agonists in the awake goat. The results suggest that alpha2-AR agonist-induced ventilatory excitation (tachypnea) requires the activation of alpha2A-ARs whereas clonidine-induced ventilatory depression (arrhythmic breathing) requires the activation of an alternate alpha2-AR subtype (presumably alpha2C-ARs). The results further demonstrate that alpha2-AR pathways exert an important influence on respiratory rhythm in the awake goat.

Ventilatory effects of alpha2-adrenoceptor blockade in awake goats.

We sought to determine the extent to which alpha2-adrenoceptor (alpha2-AR) pathways exert a tonic influence on respiratory rhythm under normal physiological conditions. The ventilatory effects of alpha2-AR blockade with SKF-86466 were examined in awake adult goats. The antagonist effect of SKF-86466 at alpha(2)-ARs was evident both as a reversal of the ventilatory disturbances produced by the alpha2-AR agonist clonidine (5 microg/kg IV) and as a rightward shift in the potency of the agonist-evoked response after SKF-86466 pretreatment. Systemic administration of SKF-86466 in cumulative doses (25-250 microg/kg) or as a single bolus injection (500 microg/kg) produced dose-dependent increases in breathing in all animals. The excitatory effect was primarily mediated by increases in respiratory frequency that persisted for at least 120 min after injection (500 microg/kg). The time course and magnitude of the ventilatory response to SKF-86466 was similar in carotid body denervated animals. The ventilatory response to isocapnic hypoxia but not normoxic hypercapnia was significantly elevated after SKF-86466 administration (500 microg/kg). SKF-86466 (25-1000 microg/kg) produced dose-dependent increases in heart rate in all animals but did not significantly change mean arterial blood pressure at any dose. The results demonstrate that alpha2-AR pathways exert a tonic inhibitory influence on respiratory rhythm in the awake goat.

Effect of 8-OH DPAT and ketanserin on the ventilatory acclimatization to hypoxia in awake goats.

We have previously reported that broad-spectrum serotonergic blockade increased the acute hypoxic ventilatory response in awake goats. The purpose of the present study was to examine the putative serotonin (5-HT) receptor subtype(s) that may have contributed to this response. Following the administration of the selective 5-HT(1A)-receptor agonist, 8-hydroxy-(2-di-n-propylamino) tetralin (8-OH DPAT, 0.1 mg x kg(-1)i.v.), there was an increase in normoxic expired minute ventilation (V(E)) that was due to an increased breathing frequency. V(E) increased during hypoxia but the change in V(E) (Delta V(E)) associated with hypoxic exposure was not different from the Delta V(E) of saline treated goats. The combination of 8-OH DPAT and a selective 5-HT(2A/2C) receptor antagonist, ketanserin (0.1 and 1.0 mg x kg(-1)i.v., respectively), also increased normoxic V(E) but did not alter the hypoxia induced Delta V(E). Both 8-OH DPAT alone and in combination with ketanserin attenuated the change in V(E) associated with sustained hypoxia but neither was able to attenuate the increased hypoxic ventilatory response that occurs following acclimatization. The augmented acute hypoxic ventilatory response that we previously reported does not appear to be mediated via the activation of the 5-HT(1A) receptor or through the combination of 5-HT(1A) activation and 5-HT(2A/2C) blockade. The results of this study further suggest that while 5-HT may modulate hypoxic ventilation it does not appear to be necessary for the development of ventilatory acclimatization to hypoxia.

Clonidine induces upper airway closure in awake goats.

We examined the effects of the alpha(2)-adrenoceptor (alpha(2)-AR) agonist clonidine on pressure-flow relationships in the upper airway. Inspired and expired airflows, subglottic tracheal pressure (PTR), mask pressure and middle pharyngeal constrictor (MPC) and diaphragm electromyogram (EMG) activities were recorded in awake standing goats. Clonidine-induced central apneas were always associated with continuous tonic activation of the MPC. Subglottic PTR during expiration increased progressively in a dose-dependent manner after clonidine administration. In all cases, positive subglottic PTR was maintained throughout the duration of clonidine-induced apneas and was sufficient to retard or prevent expiratory flow during early and mid-expiration indicating complete airway closure. The effects of clonidine were reversed by selective alpha(2)-AR blockade with SKF-86466. Central apneas after spontaneous augmented breaths (sighs) were associated with continuous tonic activation of the MPC throughout the duration of the prolonged TE intervals. However, subglottic PTR during expiration was not significantly different from control breaths and there was no evidence of increased expiratory airway resistance or delayed expiratory flow. We conclude that continuous tonic activation of pharyngeal adductor muscles appears to be a constant feature of central apnea in the awake goat independent of the initiating cause of the apnea. However, our data suggest that MPC activation alone may not be sufficient to cause complete closure of the upper airway during central apnea.

Nonvagal tachypnea following alpha2-adrenoceptor stimulation in awake goats.

To assess the influence of vagal afferent feedback in the development of respiratory instabilities induced by alpha2-adrenoceptor (alpha2-AR) stimulation in the goat, we examined the ventilatory effects of clonidine, an alpha2-AR agonist, in awake tracheostomized goats before and after bilateral mid-cervical vagotomy. Prior to vagal section, systemic administration of clonidine (0.5-3.0 microg kg(-1)) induced a highly dysrhythmic pattern of breathing in all animals that was characterized by alternating episodes of tachypnea and slow irregular breathing patterns including prolonged and variable expiratory time (TE) intervals. Periods of apnea were commonly observed. Bilateral vagotomy resulted in a slower deeper breathing pattern and abolished the tachypnea evoked by intravenous administration of phenylbiguanide (PBG; 20-50 microg kg(-1)), a selective serotonin type 3 (5-HT3) receptor agonist. However, respiratory disturbances associated with alpha2-AR stimulation (including tachypnea) persisted after vagal section and were qualitatively and quantitatively similar to pre-vagotomy data demonstrating that vagal afferent feedback is not necessary for the development of respiratory disturbances induced by clonidine. The results suggest that respiratory dysrhythmias caused by alpha2-AR agonists in the goat are mediated by alpha2-ARs in the CNS.

Methysergide augments the acute, but not the sustained, hypoxic ventilatory response in goats.

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation that occurs during sustained hypoxia. As serotonin (5-HT) has been reported to be an important modulator of respiratory output, 5-HT may also play a role in VAH. Methysergide (a broad-spectrum 5-HT antagonist), was given to awake goats (1 mg kg(-1) i.v.) 30 min prior to being exposed to 4 h of isocapnic hypoxia. Although methysergide slightly decreased arterial pH, presumably due to a non-significant increase in arterial P(CO2), it did not alter normoxic ventilation. Following methysergide, the expired minute ventilation (VE) was significantly elevated above the control (saline) response after 30 min of hypoxia, but methysergide did not otherwise alter VAH. We repeated the study in the same goats using ketanserin, a specific 5-HT2A/2C receptor antagonist (1.2 mg kg(-1) i.v.). Ketanserin had no effect on the acute hypoxic ventilatory response, or on VAH. We conclude that while 5-HT modulates the acute hypoxic ventilatory response in goats, it does not appear to act through the 5-HT2A/2C receptor subtypes.

Differential effects of clonidine on upper airway abductor and adductor muscle activity in awake goats.

The purpose of this study was to determine the extent to which alpha(2)-adrenoceptor (alpha(2)-AR) pathways affect the central motor output to upper airway muscles that regulate airflow. Electromyogram (EMG) measurements were made from posterior cricoarytenoid (PCA), cricothyroid (CT), thyroarytenoid (TA), and middle (MPC) and inferior (IPC) pharyngeal constrictor muscles in awake standing goats. Systemic administration of the alpha(2)-AR agonist clonidine induced a highly dysrhythmic pattern of ventilation in all animals that was characterized by alternating episodes of tachypnea and slow irregular breathing patterns, including prolonged and variable expiratory time intervals. Periods of apnea were commonly observed. Dysrhythmic ventilatory patterns induced by clonidine were associated with differential recruitment of upper airway muscles. alpha(2)-AR stimulation preferentially decreased the activity of the PCA, CT, and IPC muscles while increasing TA and MPC EMG activities. Clonidine-induced apneas were associated with continuous tonic activation of laryngeal (TA) and pharyngeal (MPC) adductors, leading to airway closure and arterial oxygen desaturation. Tonic activation of the TA and MPC muscles was interrupted only during the first inspiratory efforts after central apnea. Laryngeal abductor, diaphragm, and transversus abdominis EMG activities were completely silenced during apneic events. Ventilatory and EMG effects were reversed by selective alpha(2)-AR blockade with SKF-86466. The results demonstrate that alpha(2)-AR pathways are important modulators of central respiratory motor outputs to the upper airway muscles.

Respiratory-related pharyngeal constrictor muscle activity in awake goats.

Respiratory-related electromyogram (EMG) activities of the middle (MPC) and inferior (IPC) pharyngeal constrictor (PC) muscles were determined simultaneously with up to six additional upper airway abductor and adductor muscles in awake adult goats. Phasic PC activation began in late inspiration and persisted throughout expiration with a steady, an augmenting or a biphasic pattern of activity. Considerable differences were noted in the EMG responses of the MPC and IPC muscles to respiratory-related stimuli. During hypoxia and hypercapnia, phasic MPC activity decreased or was not recruited whereas phasic IPC activity was augmented with increased chemical drive. During spontaneous augmented breaths and peripheral chemoreceptor stimulation with sodium cyanide, the pattern of activation of the MPC was similar to that of the thyroarytenoid muscle (TA), a laryngeal adductor whereas IPC activity was strikingly similar to activity of the laryngeal and pharyngeal dilators. The expiratory portion of an augmented breath was associated with increased phasic MPC and TA but not IPC activities. Dopamine-induced apneas resulted in tonic activation of the MPC and TA at a level equal to or greater than control activity but no recruitment of IPC activity. The marked differences in MPC and IPC responses to respiratory-related stimuli suggests that these muscles may have different mechanical effects on pharyngeal airway caliber in the goat. The results suggest that the MPC may help brake expiratory flow thus helping to control expiratory timing and lung volume. In contrast, the IPC may promote pharyngeal airway patency by stiffening or dilating the pharyngeal airway. The results demonstrate that a variety of stimuli can influence respiratory-related PC activity and suggest that the PC muscles are important in the regulation of breathing and upper airway patency.

Surfactant regulates pulmonary fluid balance in reptiles.

Reptilian lungs are potentially susceptible to fluid disturbances because they have very high pulmonary fluid filtration rates. In mammals, pulmonary surfactant protects the lung from developing alveolar edema. Reptiles also have an order of magnitude more surfactant per square centimeter of respiratory surface area compared with mammals. We investigated the role of reptilian surfactant 1) in the entry of vascularly derived fluid into the alveolar space of the isolated perfused lizard (Pogona vitticeps) lung and 2) in the removal of accumulated fluid from the alveolar space of the isolated perfused turtle (Trachemys scripta) lung by both the pulmonary venous and lymphatic circulations. The flux of fluorescent (fluorescein isothiocyanate) inulin from the vasculature into the alveolar compartment increased 60% after the removal of surfactant, but capillary fluid filtration over a 10-min period was not affected. Surfactant removal decreased alveolar inulin clearance by both the pulmonary venous circulation and the pulmonary lymphatic system approximately 1.5- and 3-fold, respectively. In reptiles, fluid flux from capillary to air space must occur indirectly via the interstitium. In the absence of surfactant, this may result in interstitial drying, which affects both pulmonary venous and pulmonary lymphatic clearance of alveolar fluid.

Nucleoside triphosphate levels versus oxygen affinity of rattlesnake red cells.

Squamate hemoglobins are responsive to modulation by nucleoside triphosphates (NTP, generally ATP), and bind NTP with a 1-to-1 molar stoichiometry. However, red cells of nonpregnant rattlesnakes contain NTP-to-hemoglobin molar ratios of approximately 2.5 suggesting most NTP is supersaturating and should not influence the oxygen affinity directly. To test this hypothesis, we metabolically depleted red cells of NTP and determined the oxygen affinity. There was a significant linear relationship between red cell NTP concentrations and oxygen affinity over the NTP/Hb range examined. In contrast, intracellular pH, Mg2+ and C1- changed slightly, or not all, during depletion. These data indicate NTP concentrations represent the primary control of hemoglobin function within these cells. Purified hemoglobin was functionally sensitive to 5 mM GTP or inositol hexaphosphate but not sensitive to 5 mM ATP or pH. Together, these findings indicate rattlesnake hemoglobin, within red cells, is functionally controlled by NTP, but the binding affinity is low such that NTP is not saturating at NTP/Hb ratios below 3.5.