Jay K. Herman

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.

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.

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.

Dopaminergic excitation of the goat carotid body is mediated by the serotonin type 3 receptor subtype

The purpose of the present study was to use chemoafferent recordings from the goat carotid body (CB) to pharmacologically identify the putative low affinity excitatory receptor for dopamine (DA). Close arterial injections of DA (1-50 microg kg(-1)) induced a dose-dependent excitatory burst followed by inhibition of the CB chemoafferent activity. The inhibition is likely DA D(2) receptor-mediated as it was blocked by domperidone (0.5-1.0 mg kg(-1) iv). The initial high frequency burst of CB chemoafferent activity could not be attenuated by selective antagonists for the DA D(1-4) receptors but could be blocked by D-tubocurarine or the selective serotonin(3) (5-HT(3)) receptor antagonists, tropisetron and MDL72222. The selective nicotinic antagonists, hexamethonium and vecuronium, were without effect. Selective blockade of the 5-HT(3)-receptor subtype using tropisetron significantly reduced both normoxic and hypoxic unitary CB discharge. These results suggest that DA-mediated excitation of the goat CB chemosensitive afferents occurs via the 5-HT(3)-receptor subtype and that the 5-HT(3)-receptor may exert an excitatory modulation of CB output during normal physiological responses in the goat.

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.

Toxicity of Hexamoll® DINCH® following intravenous administration

Alternative plasticizers to di(2-ethylhexyl) phthalate (DEHP) for blood bags have been sought for many years. Cyclohexane-1,2-dicarboxylic acid, diisononylester (Hexamoll(®) DINCH(®)) is an alternative that has been evaluated in preliminary studies for compatibility and efficacy to preserve whole blood. While Hexamoll(®) DINCH(®) has an extensive database for mammalian toxicity via oral administration, data were needed to evaluate toxicity from intravenous (IV) administration to support the use of the plasticizer Hexamoll(®) DINCH(®) in blood bags. A series of studies was performed by slow IV injection or IV infusion of Hexamoll(®) DINCH(®), a highly viscous, hydrophobic substance, suspended in Intralipid(®) 20% (20% intravenous fat emulsion). Rats were injected once, followed by 14 days of recovery; injected daily for 5 days followed by 5 days of recovery, or infused for 29 days (4h/day) followed by 14 days of recovery. Dose levels were 0, 62, 125, and 250-300mg/kg body weight/day. These dose levels represent the limits of suspension and far exceed any anticipated exposures from migration out of plasticized blood bags. Animals were observed for signs of toxicity; body weight and feed consumption were measured; blood collected for clinical chemistry and hematology; and tissues collected and processed for histopathology. Special emphasis was placed on evaluating endpoints and tissues that are commonly associated with plasticizer exposure in rodents. Urine was collected during the 4-week study to quantify urinary metabolites of Hexamoll(®) DINCH(®). The results of the studies indicate that no substance-related toxicity occurred: no effects on behavior, no effects on organ weight, no effect on serum chemistry including thyroid hormones; and no effect on major organs, especially no testicular toxicity and no indication for peroxisome proliferation in the liver. The only effects seen were petechia and granulomas related to dissipation of suspended Hexamoll(®) DINCH(®) in the aqueous environment of the blood. However, the results of metabolite analyses demonstrate that Hexamoll(®) DINCH(®) was bioavailable. Therefore, based on the lack of Hexamoll(®) DINCH(®)-related systemic toxicity with the exception of the physical limitations, the no-observed-adverse-effect level for parenterally administered Hexamoll(®) DINCH(®) is considered to be 300mg/kg bw/day.

Ventilatory effects of α2-adrenoceptor blockade in awake goats

Abstract We sought to determine the extent to which α 2 -adrenoceptor (α 2 -AR) pathways exert a tonic influence on respiratory rhythm under normal physiological conditions. The ventilatory effects of α 2 -AR blockade with SKF-86466 were examined in awake adult goats. The antagonist effect of SKF-86466 at α 2 -ARs was evident both as a reversal of the ventilatory disturbances produced by the α 2 -AR agonist clonidine (5 μg/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 μg/kg) or as a single bolus injection (500 μg/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 μg/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 μg/kg). SKF-86466 (25–1000 μg/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 α 2 -AR pathways exert a tonic inhibitory influence on respiratory rhythm in the awake goat.

Nonvagal tachypnea following α2-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.

Dopaminergic excitation in goat carotid body may be mediated by serotonin receptors.

Dopamine (DA) is a prominent neurotransmitter that is found in relatively large quantities in type I cells of the carotid body (CB) of different mammals (Engwall et al. 1989; Gonzalez et al. 1994). The role of DA in CB function has been studied extensively yet it remains controversial (Gonzalez et al. 1994; Bisgard and Neubauer 1995). There is strong evidence indicating that DA has a modulatory role that is inhibitory to the chemosensory activity of the CB. Thus, exogenous administration of DA inhibits CB discharge in dogs (Bisgard et al 1979), cats (Black et al. 1972; Zapata 1975; Llados and Zapata 1978; Nishino and Lahiri 1981; Okajima and Nishi 1981), rabbits (Docherty and McQueen 1979), rats (Cardenas and Zapata 1981) and goats (Bisgard et al. 1997). Consistent with these observations exogenous DA depresses ventilation in animals (Black et al. 1972; Zapata and Zuazo 1980; Cardenas and Zapata 1981; Nishino and Lahiri 1981; Kressin et al. 1986; Janssen et al. 1998; O’Halloran et al. 1998) and human subjects (Welsh et al. 1978) whereas peripheral DA-receptor blockade stimulates ventilation and CB neural activity (Zapata and Torrealba 1984; Kressin et al. 1986; Janssen et al. 1998) and attenuates

Pharmacological and Immunochemical Evidence of the Dopamine D3 Receptor in the Goat Carotid Body

Dopamine (DA) represents the predominant amine in the carotid body (CB) of several species. Over 3 decades, numerous studies of the effects of DA on carotid chemoreceptor function have produced a variety of results, whether conducted in vivo or in vitro. The reported effects of DA on carotid chemoreflex function are mostly inhibitory, although several investigators have reported excitatory effects, or both inhibitory and excitatory effects1. DA D2 receptors have been found on the CB type I cells, petrosal ganglion and in afferent nerves2,3,4. It is possible that multiple DA receptor types may exist within the carotid chemoreflex pathway.