lack of involvement of the autonomic nervous system in early ventilatory and pulmonary vascular...

J Physiol 579.1 (2007) pp 215225 215

Lack of involvement of the autonomic nervous system inearly ventilatory and pulmonary vascular acclimatizationto hypoxia in humans

Chun Liu1, Thomas G. Smith1, George M. Balanos2, Jerome Brooks1, Alexi Crosby3, Mari Herigstad1,Keith L. Dorrington1 and Peter A. Robbins1

1Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK2School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK3Department of Respiratory Medicine, University of Cambridge, Level 5, Box 157, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK

The activity within the autonomic nervous system may be altered following sustained exposureto hypoxia, and it is possible that this increase in activity underlies the early acclimatizationof both ventilation and the pulmonary vasculature to hypoxia. To test this hypothesis, sevenindividuals were infused with the ganglionic blocker trimetaphan before and after an 8 h exposureto hypoxia. The short half-life of trimetaphan should ensure that the initial infusion doesnot affect acclimatization to the 8 h hypoxia exposure, and the use of a ganglion blockingagent should inhibit activity within all branches of the autonomic nervous system. Duringthe infusions of trimetaphan, measurements of ventilation and echocardiographic assessmentsof pulmonary vascular tone (Pmax) were made during euoxia and during a short period ofisocapnic hypoxia. Subjects were also studied on two control days, when a saline infusion wassubstituted for trimetaphan. Trimetaphan had no effect on either euoxic ventilation or thesensitivity of ventilation to acute hypoxia. Trimetaphan significantly reduced Pmax in euoxia(P < 0.05), but had no significant effect on the sensitivity of Pmax to acute hypoxia oncechanges in cardiac output had been controlled for. The 8 h period of hypoxia elevated euoxicventilation (P < 0.001) andPmax (P < 0.001) and increased their sensitivities to acute hypoxia(P < 0.001 for both), indicating that significant acclimatization had occurred. Trimetaphan hadno effect on the acclimatization response of any of these variables. We conclude that alteredautonomic activity following 8 h of hypoxia does not underlie the acclimatization observed inventilation or pulmonary vascular tone.

(Received 28 July 2006; accepted after revision 28 November 2006; first published online 30 November 2006)Corresponding author P. A. Robbins: Department of Physiology, Anatomy and Genetics, University of Oxford,Sherrington Building, Parks Road, Oxford OX1 3PT, UK. Email: [email protected]

Exposure of humans to a steady degree of hypoxia overa period of several hours leads to gradual increases inventilation (VE), pulmonary artery pressure (PAP), heartrate (HR) and cardiac output (CO) (Howard & Robbins,1995; Dorrington et al. 1997; Clar et al. 2001). In addition,the sensitivity of some or all of these variables to briefexposures to acute hypoxia is increased following sucha period of sustained hypoxia. Both of these kinds ofresponses are commonly subsumed under the heading

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early acclimatization to hypoxia, and the mechanismsunderlying them remain obscure.

The autonomic nervous system plays a major rolein regulating HR and CO, and a perhaps more subtlerole in modulating VE and PAP. Both sympathetic andparasympathetic activities have been shown to changeduring exposure to hypoxia in humans (Petersen et al.1974; Saito et al. 1988; Rowell et al. 1989). Furthermore, theelevation in sympathetic nervous system activity persistsfor a considerable period following relief from the hypoxicstimulus (Morgan et al. 1995). Chronic hypoxia causes astill more marked activation of both the parasympatheticand the sympathetic nervous system in healthy humans,despite a return towards normal of the arterial O2 content

C 2007 The Authors. Journal compilation C 2007 The Physiological Society DOI: 10.1113/jphysiol.2006.118190

216 C. Liu and others J Physiol 579.1

with acclimatization (Boushel et al. 2001; Calbet, 2003).In direct neurophysiological recording of sympatheticnerve activity, Hansen & Sander, (2003) found thatacclimatization to high altitude is accompanied by markedelevations in resting levels of sympathetic neural dischargeto the skeletal muscle vasculature. Furthermore, the highaltitude-induced sympathetic activation persisted for daysafter return to sea level, demonstrating a long-lastingsympathetic neural overactivity following relief fromhypoxia.

It has long been recognised that stimulation of thesympathetic nervous supply to the carotid body increasesthe frequency of sinus nerve chemosensory discharges(Floyd & Neil, 1952; Eyzaguirre & Lewin, 1961). Morerecently, two types of excitatory response have beendistinguished, and it has been suggested that these relateto two distinct components of sympathetic innervation,one arising from the innervation of the vasculature andthe other arising from direct sympathetic innervation ofthe type I cells of the carotid body (ORegan, 1981). Usingcirculating and urinary noradrenaline concentrations orspillover as indirect measures of sympathetic nerve activity,a progressive increase in sympatho-excitation in humansduring exposure to high altitude has been found, and thisincrease has been shown to correlate with the increase inVE (Asano et al. 1997).

An autonomic modulation of pulmonary vasculartone appears possible given that both sympathetic andparasympathetic branches of the autonomic nervoussystem innervate pulmonary vessels (Hebb, 1966;Downing & Lee, 1980). Levitzky et al. (1978) have shownin the dog that systemic arterial hypoxaemia induces achemoreflex-mediated pulmonary vascular dilatation. Alater study, employing experimental vagotomy in dogs,has suggested that the autonomic pulmonary vasodilatoreffect of systemic hypoxaemia has a parasympatheticorigin (Wilson & Levitzky, 1989). However, other literaturesuggests that these effects are mediated through thepulmonary sympathetic supply (Kazemi et al. 1972;Murray et al. 1986; Brimioulle et al. 1997).

Based on the observations described above, in particularthe persistence of an elevated sympathetic activityfollowing the relief of hypoxia and the ability of theautonomic nervous system to influence VE and pulmonaryvascular tone, we wished to test the hypothesis thatpersistently altered autonomic activity underlies partor all of early acclimatization to hypoxia. We studiedthe effects of a short period of exposure to isocapnichypoxia on ventilation and the pulmonary circulation,both before and after conditioning with 8 h of hypoxiato induce acclimatization to hypoxia (first control). Ona subsequent day, we repeated this sequence but weadministered the short acting ganglion-blocking agenttrimetaphan to abolish peripheral autonomic activitywhile we were testing the effects of acute exposure to

isocapnic hypoxia on ventilation and the pulmonarycirculation, before and after 8 h of hypoxia. On the thirdand final day, we undertook a second control sequence, thesame as the first.

On the test day, because of its short duration ofaction, trimetaphan should not have been active duringthe period of sustained hypoxia, and therefore shouldnot have prevented the acclimatization process fromoccurring. If it is the case that a persistent elevationin autonomic nervous activity is responsible for theacclimatization-induced increases in ventilatory andpulmonary vascular sensitivities to acute hypoxia, thenthe infusion of trimetaphan following sustained hypoxiawould be expected to remove the effects of acclimatization.In this case, under the conditions of trimetaphan infusion,the ventilatory and pulmonary vascular responses to acutehypoxia should be the same before and after exposure tosustained hypoxia.

Methods

Participants

Seven healthy volunteers (5 men, 2 women) with an ageof 28 4 years, height of 177.3 8.9 cm, and weight of71.1 12.7 kg (means s.d.) participated in the study.Each gave informed consent before participating. Thestudy was approved by the Oxfordshire Clinical ResearchEthics Committee and performed in accordance withthe Declaration of Helsinki. Each volunteer visited thelaboratory twice before undertaking any of the mainexperimental protocols to become familiar with thelaboratory and its procedures. On these visits we measuredthe normal end-tidal PCO2 (PET,CO2 ) of the participants andconfirmed that they were suitable for echocardiographicmeasurement of tricuspid regurgitation.

Protocols

Each volunteer attended the laboratory for threemain protocols. In the test protocol, trimetaphan wasadministered (Protocol T), which acts as a competitiveinhibitor at the cholinergic binding sites in autonomicganglia (Gilman et al. 1990). Trimetaphan also inducesa degree of histamine release through an independentmechanism (McCubbin & Page, 1952), although this doesnot appear to play an important role in the haemodynamicresponses to trimetaphan (Fahmy & Soter, 1985). The twoother protocols were control protocols in which placebowas administered (Protocol C1 and Protocol C2). Eachprotocol lasted 1 day and the protocols were separatedfrom each other by at least 1 week. The order of theseprotocols was fixed as C1, T and C2.

Participants reported to the laboratory at 08.00 h.After 30 min of supine rest, the participants performed

C 2007 The Authors. Journal compilation C 2007 The Physiological Society

J Physiol 579.1 Autonomic nervous system and acclimatization to hypoxia 217

a Valsalva manoeuvre with an expiratory strain of40 mmHg for 20 s. The strain pressure during the Valsalvamanoeuvre was monitored by a manometer. Typicalchanges in systemic arterial pressure (AP) and HRduring the Valsalva manoeuvre were observed in allsubjects before ganglion blockade (Fig. 1). In Protocol T,intravenous trimetaphan (trimetaphan camsylate,Cambridge Laboratories, Wallsend, UK) was administeredat an initial rate of infusion of 2 mg min1 afterperformance of the initial Valsalva manoeuvre. Threeminutes after starting the infusion, the Valsalva manoeuvrewas performed again to evaluate the HR and AP responsesto changes in intrathoracic pressure. The infusion ratewas increased incrementally until the tachycardia andAP recovery during phase II of the Valsalva manoeuvre,

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Figure 1. Typical changes in arterialpressure and heart rate during the Valsalvamanoeuvre in control and trimetaphanprotocolsThe efficacy of ganglion blockade bytrimetaphan was demonstrated not only by theabsence of heart rate response, but also by theabsence of BP recovery during phase II or BPovershoot during phase IV of the Valsalvamanoeuvre. Arrows show the time points whenValsalva manoeuvre was started or stopped.

and overshoot in AP during phase IV of the Valsalvamanoeuvre, were all abolished (Fig. 1). Once completeblockade was achieved, the infusion of trimetaphan wasmaintained at this peak dose throughout the time ofexposure to acute hypoxia. The maximum rate of infusionused was 4 mg min1. In Protocol C1 and C2, saline wasinfused instead. During the exposure to acute hypoxia,end-tidal PO2 (PET,O2 ) was held at 100 Torr in the first10 min, then at 50 Torr for 20 min, and at 100 Torr againfor the final 10 min. PET,CO2 was held constant at 2 Torrabove each volunteers normal baseline value throughoutthe exposures to acute hypoxia.

After the acute hypoxic exposure the infusion wasterminated and participants took a 30 min break. Theythen entered a purpose-built chamber to undergo an 8 h



period of sustained hypoxic exposure under isocapnicconditions. During this period, the composition of thechamber gas was adjusted so as to hold the subjects PET,O2at 50 Torr and to hold the subjects PET,CO2 at his or herinitial control air-breathing value.

At the end of the 8 h period of sustained hypoxia, thesubject left the chamber and was left to breathe room airfor 30 min. Following this, a second set of measurementswas made of the responses to acute hypoxia. The procedurethat was followed was identical to that used before the 8 hexposure to sustained hypoxia.

Control of end-tidal gases during the acute hypoxicexposure and respiratory measurements

During the acute hypoxic exposures, volunteers lay ontheir left side on a couch and breathed through amouthpiece with the nose occluded. Ventilatory volumeswere measured by a turbine volume-measuring deviceand flows by a pneumotachograph, both in series withthe mouthpiece. Respired gases were sampled via a finecatheter close to the mouth and analysed continuouslyfor PCO2 and PO2 by mass spectrometry. A pulse oximeterwas used to monitor arterial O2 saturation. Inspired andexpired volumes, end-inspiratory and end-expiratory PCO2and PO2 , and saturation were detected in real time bya computer and logged breath by breath. A dynamicend-tidal forcing system (Robbins et al. 1982) was usedto control the end-tidal gases in the manner specifiedfor the determination of the acute hypoxic ventilatoryresponse. Before the start of each experiment, a cardio-respiratory model was used to construct a forcing functionthat contained the breath-by-breath values for inspiratoryPCO2 and PO2 predicted to produce the desired end-tidalsequences. During the experiment, a computer-controlledgas-mixing system was used to generate this sequencein a modified manner. The modifications resulted fromfeedback control based on the deviations of the measuredvalues for PET,CO2 and PET,O2 from their desired values.

Measurements of pulmonary blood pressure, cardiacoutput and systemic arterial pressure

The majority of people have detectable regurgitationduring systole through their tricuspid valves. Dopplerechocardiography can be used to detect the presence ofthis jet of blood and measure the velocity with which itmoves back into the right atrium. On the assumptionthat the flow within the jet may be regarded as steady,Bernoullis equation can be used to calculate the maximumpressure difference between the right ventricle and rightatrium (Pmax) from the density of blood () and thepeak velocity of the jet (v). This gives the relationshipPmax = v2/2. Assuming right atrial pressure remainsconstant, changes in Pmax will be equal to changes inthe peak systolic PAP.

Echocardiographic measurements were performedusing a Hewlett-Packard Sonos 5500 ultrasound machinewith an S4 two-dimensional transducer (24 MHz). HRand respiratory waveform were also recorded.

A standard technique was used for the measurementof Pmax (Balanos et al. 2003). The tricuspid valve wasvisualized in an apical four-chamber view, and then colourDoppler format allowed the regurgitant jet to be detected.After proper alignment of the probe, continuous wavespectral analysis at a sweep speed of 50 mm s1 was usedto record the velocity profile of the jet. During analysis,the maximal velocity of the jet was measured using anelectronic caliper tool. Pmax values were then computedautomatically.

In order to measure CO, the flow through the aortic valveduring systole was identified using an apical five-chamberview. The velocity profile was then obtained in pulsedwave spectral mode at a display screen sweep speed of100 mm s1. Doppler sampling of the flow was madejust below the orifice of the aortic valve. An automatedprocedure was then used to calculate the velocity timeintegral (VTI) of the flow for each ventricular contraction.The diameter (D) of the aortic valve was measured froma parasternal long axis view. From these data, CO wascalculated as HR VTI (D/2)2.

Systemic arterial pressure was measured continuouslyusing finger plethysmography (Portapres, TNO-TPDBiomedical Instrumentation, the Netherlands) duringValsalva manoeuvres and the exposures to acute hypoxia.

Eight hour sustained exposure to hypoxia

The sustained hypoxic exposure was conducted using apurpose-built chamber, which was large enough to allowsubjects to sit or stand comfortably. Subjects were studiedindividually in the chamber so that the inspiratory gascomposition could be tailored to regulate their end-tidalgas composition accurately. Respired gas was sampledvia a fine nasal catheter at the opening of the subjectsnostril. The samples were continually analysed for PCO2and PO2 . Arterial O2 saturation was monitored using apulse oximeter. A computer sampled the PCO2 , PO2 andsaturation signals at a frequency of 100 Hz. It detectedend-inspiratory and end-expiratory values for PCO2 andPO2 and recorded these breath by breath along withvalues for saturation. The desired values for PET,CO2 andPET,O2 were entered into the computer. The computer thenautomatically adjusted the composition of the gas in thechamber every 5 min, or at manually overridden intervals,to minimize the error between desired and actual values forthe end-tidal gases. This system has been described in detailelsewhere (Howard et al. 1995). The subject was kept undercontinuous observation throughout the 8 h exposure. Inaddition, an environmental oxygen monitor was employedwithin the chamber as a further safety precaution.



Modelling of hypoxic ventilatory responses

To obtain numerical values for the sensitivity of VE to theacute hypoxic exposure, a respiratory model was fitted tothe data. The particular respiratory model employed wasModel I of those described by Liang et al. (1997). Thismodel separates the total ventilation, VE, into a central,hypoxia-independent component, Vc, and a peripheral,hypoxia-dependant component, Vp, and may be written:

VE = Vp + Vc

TpdVEdt

+ Vp = Gp[100 S(t Dp) + Kp]

ThdGpdt

+ Gp = G100 Gh[100 S(t Dp)],

where Tp is the time constant for development of the peri-pheral response, K p is the peripheral drive in the absence ofhypoxia, Gp is the peripheral chemoreflex sensitivity, and Sis the saturation function (Severinghaus, 1979) calculatedat time t delayed by the peripheral time delay, Dp. Th is thetime constant for the development of hypoxic ventilatorydecline, G100 is the steady-state chemoreflex sensitivity inthe absence of hypoxic ventilatory decline (i.e. followingconditioning at 100% arterial oxygen saturation), and Ghdefines the magnitude of hypoxic ventilatory decline as theratio of the decrease in peripheral chemoreflex sensitivityto the decrease in S.

In order to allow for the autocorrelation that existsbetween measurements of ventilation on successivebreaths, a model of the noise processes was fitted tothe data in parallel with the fitting of the model of theventilatory response to hypoxia (Liang et al. 1996). Themodel parameters were estimated by fitting the models tothe data using a standard subroutine to minimize the sumof squares of the residuals (subroutine E04FDF, NumericalAlgorithms Group, Oxford).

Modelling of pulmonary vascular responses

To quantify fully the pulmonary vascular responses toacute hypoxia observed in this study, a mathematicalmodel was fitted to the data. In this model, it was supposedthat the dynamics of the response could be represented bya simple, linear, first order process and that the changesin Pmax could be related in a linear manner to changesin saturation, at least of the range for PO2 values studied.Given these assumptions, a simple difference equation maybe written of the form:

Pmax(t) =P100 + GP(100 S(t)) [P100+GP(100S(t)) Pmax(t1)]e(t/TP)

where the difference between successive two time points(t) and (t 1) is t , S is the saturation (Severinghaus,1979), P100 is the steady-state value for Pmax in theabsence of hypoxia (i.e. when saturation is 100%), GPis the sensitivity of Pmax to a decrease in saturation andTP is the time constant for the response. The parametersof this model (P100, GP and TP) were estimated usingthe function LSQNONLIN in Matlab (version 7.0, TheMathWorks, Inc., Natick, MA, USA) to minimize the sumof squares of the residuals.

Quantification of systemic cardiovascular responses

The responses of the systemic circulation to acute hypoxiawere not sufficiently well defined to quantify through amodel fitting process. Accordingly simple averages of theseresponses were calculated under the conditions of euoxiaand hypoxia.

Statistical analysis

A full factorial ANOVA was used to assess the statisticalsignificance of the results, with fixed factors of Protocol(control protocols (Protocols C1, C2) versus Protocol T)and Time (before the sustained hypoxic exposure (AMmeasurements) versus after the sustained hypoxic exposure(PM measurements)) together with a random factorof Subject. The null hypothesis of the experiment wasthat infusion of trimetaphan would not abrogate theacclimatization observed after sustained hypoxia. Theacceptance or rejection of this null hypothesis was assessedthrough the significance or otherwise of the interactiveterm between Protocol and Time.

Results

Control over the end-tidal gases during the acuteexposure to hypoxia

Figure 2A illustrates the average time course for PET,O2and PET,CO2 across all volunteers for both exposures toacute hypoxia in each protocol. The acute hypoxic stimuluswas generated accurately in all cases. The backgroundlevel of PET,CO2 was held very constant throughout eachexposure. Overall, no discernable differences among thethree protocols could be detected.

Ventilatory responses to hypoxia

Figure 2B illustrates the average responses for VE to acutehypoxia before (AM) and after (PM) exposure to the 8 hperiod of sustained hypoxia. Table 1 lists the parametersobtained from the model fitting. From these results, itis clear that trimetaphan had no discernable effect on



the ventilatory response to acute hypoxia. Significantacclimatization to sustained hypoxia was evident in allthree protocols, both through a significant increase in VEin euoxia (corresponding to parameter Vc of the model)and through a significant increase in the acute ventilatorysensitivity to hypoxia (corresponding to parameter G100of the model) in the PM measurements as compared withthe AM measurements. The presence of trimetaphan inProtocol T caused no discernable reduction in the degreeof acclimatization observed.

Pulmonary vascular responses to hypoxia

Figure 2C illustrates the average responses for Pmax toacute hypoxia before (AM) and after (PM) exposure to 8 hperiod of sustained hypoxia. Table 2 lists the parametersobtained from the model fitting process. Trimetaphansignificantly reduced Pmax under conditions of euoxia(corresponding to parameter P100 of the model) andsignificantly attenuated the sensitivity of Pmax to acutehypoxia (corresponding to parameter GP of the model).A significant acclimatization following the 8 h period of

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Figure 2. Mean values for end-tidal PO2 (PET,O2 ) (squares) and end-tidal PCO2 (PET,CO2 ) (triangles) (A),ventilation (B) and maximum systolic pressure difference across the tricuspid valve (Pmax) (C) duringacute hypoxic exposuresOpen symbols and dashed lines indicate responses before sustained hypoxic exposure and filled symbols orcontinuous lines indicate responses after sustained hypoxic exposure. Lines through data for ventilation indicatefit of respiratory model to data. Lines through data for Pmax indicate fit of pulmonary vascular response modelto data.

sustained hypoxia was observed through the increase inthe baseline value for Pmax in the absence of hypoxia(corresponding to parameter P100 of the model) andthrough the increase in sensitivity of Pmax to acutehypoxia (corresponding to parameter GP of the model)in the PM values as compared with the AM values. Nosignificant abrogation of these effects of sustained hypoxiawas observed through the administration of trimetaphanin Protocol T.

Cardiovascular responses to hypoxia

The systemic cardiovascular responses are illustrated inFig. 3 and the average values for the various variables underboth euoxic and hypoxic conditions are listed in Table 3.As expected, autonomic blockade with trimetaphan: (i)elevated HR in euoxia; (ii) abolished the normal increase inHR observed with acute hypoxia and (iii) reduced systemicvascular resistance (SVR), calculated as (mean AP)/CO.Trimetaphan did not block the reduction observed in SVRwith hypoxia. In euoxia, these effects led to the expectedreduction in mean AP and increase in CO. At the induction



Table 1. Ventilatory responses to hypoxia: parameter values (mean S.E.M) for the model of the ventilatory response to acute hypoxiafor the trimetaphan and control protocols before and after an 8 h period of sustained hypoxic exposure

Protocol/factor G100 Vc Tp Dp Gh Th Kp(l min1 %1) (l min1) (s) (s) (l min1%2) (s) (%)

AM measurements before sustained hypoxiaControl 1 0.85 0.20 7.58 1.51 11.77 4.40 5.83 147 0.028 0.011 537 135 3.48 1.41Trimetaphan 0.78 0.23 4.17 1.28 9.09 5.40 5.69 1.17 0.022 0.008 820 136 10.64 7.49Control 2 1.01 0.20 7.45 1.39 11.88 4.21 6.12 1.71 0.030 0.010 643 162 1.99 1.62

PM measurements after sustained hypoxiaControl 1 1.82 0.29 15.58 1.28 20.97 3.47 6.10 2.19 0.054 0.023 471 115 1.54 0.89Trimetaphan 1.40 0.23 12.03 2.95 12.17 3.60 4.63 0.91 0.045 0.021 880 114 2.77 1.57Control 2 1.75 0.15 16.61 0.99 12.83 3.96 2.74 0.63 0.030 0.020 594 134 0.83 0.77

Statistical analysis ANOVATime (before/aftersustained hypoxia) < 0.001 < 0.001

Protocol (C1, C2 versus T) Interactive term

G100, steady-state chemoreflex sensitivity to hypoxia in absence of any hypoxic ventilatory depression (arterial oxygen saturation is100%); Vc, hypoxia independent (central chemoreflex) contribution to VE; Tp, time constant for the peripheral chemoreflex responsesto hypoxia; Dp, time delay for the peripheral chemoreflex; Gh, sensitivity to hypoxic ventilatory decline, expressed as the ratio of thedecrease in the sensitivity of the peripheral chemoreflex to the decrease in conditioning arterial oxygen saturation; Th, time constantassociated with the development of hypoxic ventilatory decline; Kp, bias term, representing peripheral drive in absence of hypoxia;AM, morning; PM, afternoon. ANOVA conducted on the parameter values with fixed factors Time (before/after sustained hypoxia)and Protocol (control protocols (Protocols C1, C2) versus Protocol T). Statistical significance was accepted at P < 0.05.

of hypoxia, the effects were associated with a loss of thenormal increase in CO that occurs with acute hypoxia andalso with a fall in mean AP distinct from the slight elevationin mean AP that was observed with acute hypoxia in theabsence of trimetaphan.

Following the 8 h period of hypoxia, a significantincrease in HR could be detected. This effect was greaterin hypoxia compared with euoxia, indicating that therewas also a significant increase in the sensitivity of HR toacute hypoxia. In contrast, no significant effects of the8 h period of hypoxia were detected on SVR. Infusion oftrimetaphan clearly abrogated much of the acclimatizationeffects of sustained hypoxia on HR in hypoxia, as evidencedby the significant interactive term between Protocol (C1,C2 versus T) and Time (AM versus PM) for HR. Thisinteractive effect was not, however, significant for HR inacute euoxia, and only of marginal significance (P = 0.05)for the increase in HR with acute hypoxia.

Discussion

The main finding of this study was that administrationof trimetaphan was unable to reverse theacclimatization-induced changes in both ventilatoryand pulmonary vascular control. From these observationswe infer that changes in autonomic function over an 8 hperiod of hypoxia are unlikely to underlie at least the earlystages of acclimatization to hypoxia in these systems. Incontrast, administration of trimetaphan abrogated most,

if not all, of the effect on HR of conditioning with an 8 hexposure to hypoxia.

Study design

Our study used control hypoxia protocols both before andafter the trimetaphan hypoxia protocol. As there was noeuoxia control protocol in the study design, in theory itis possible that there is a significant time of day effectincorporated within the acclimatization effect. In practicewe have conducted a considerable number of other studiesinvolving 8 h exposures to hypoxia, which have includedeuoxia control protocols, and in none of these controlshave we found a significant time of day effect (Howard &Robbins, 1995; Tansley et al. 1997; Fatemian & Robbins,1998; Clar et al. 1999, 2000; Ren et al. 2000).

The effect of trimetaphan on the ventilatoryresponse to hypoxia

The present study found no effect of ganglion blockade oneither the ventilatory sensitivity to acute hypoxia or theacclimatization-induced changes in respiratory control.We are unaware of any other studies in humans ofthe effects of either complete ganglionic blockade, orindeed-receptor blockade, on these ventilatory responsesto hypoxia. However, our results are consistent witha number of previous studies examining blockade of



Table 2. Pulmonary vascular responses to hypoxia: parametervalues (mean S.E.M.) for the model of the pulmonary vascularresponse to acute hypoxia for the trimetaphan and controlprotocols before and after an 8 h period of sustained hypoxicexposure

Protocol/factor GP P100 TP(mmHg %1) (mmHg) (min)

AM measurements before sustained hypoxiaControl 1 0.50 0.06 18.9 1.5 2.41 0.41Trimetaphan 0.41 0.04 17.4 1.2 3.06 0.32Control 2 0.58 0.06 17.9 1.2 3.24 0.20

PM measurements after sustained hypoxiaControl 1 0.95 0.10 22.2 1.8 3.90 0.64Trimetaphan 0.68 0.07 21.2 1.6 2.91 0.35Control 2 0.82 0.04 22.1 1.4 3.61 0.40

Statistical analysis ANOVATime (before/aftersustained hypoxia) < 0.001 < 0.001

Protocol (C1, C2 versus T) < 0.02 < 0.05 Interactive term < 0.05

GP is the sensitivity of maximum pressure difference acrosstricuspid valve during systole (Pmax) to a decrease in saturation;P100 is the steady-state value for Pmax in the absence of hypo-xia (i.e. when saturation is 100%); TP is the time constant for theresponse; AM, morning; PM, afternoon. ANOVA conducted on theparameter values with fixed factors Time (before/after sustainedhypoxia) and Protocol (control protocols (Protocols C1, C2) versusProtocol T). Statistical significance was accepted at P < 0.05.

other specific components of the autonomic system. Inparticular, Clar et al. (1999) found no effect of -blockadewith propranolol on ventilatory acclimatization to 8 h ofhypoxia and, similarly, Clar et al. (2001) found no effect ofparasympathetic blockade with glycopyrrolate. In neitherof these studies was any effect detected on the acuteventilatory response to hypoxia a finding consistent withthe earlier results of Koller et al. (1988).

In anaesthetized cats, carotid-body sympathectomydid not affect carotid-chemoreceptor afferent activityduring the first 10 min of hypoxia, but thereafterchemoreceptor discharge was augmented compared withcontrols (Prabhakar & Kou, 1994). However, in awakegoats, ventilation and the hypoxic-ventilatory responsewere found to be attenuated at 1 and 4 week aftercarotid-body sympathectomy (Ryan et al. 1995). In thatstudy, carotid-body sympathectomy was found to have noeffect on ventilatory acclimatization to hypoxia.

Overall, whilst it is recognized that alterations inautonomic activity can undoubtedly modify respiratorycontrol (Floyd & Neil, 1952; Eyzaguirre & Lewin, 1961),there is little evidence to suggest that either a normal levelof autonomic activity, or an altered level of autonomicactivity, such as can be induced by the level and durationof hypoxia in our experiments, affects respiratory controlin humans.

The effect of trimetaphan on pulmonary vascularresponses to hypoxia

The pulmonary circulation is innervated by bothadrenergic and cholinergic nerves (Hebb, 1966;Downing & Lee, 1980). The cholinergic system appearsto be vasodilatory (Nandiwada et al. 1983). Within theadrenergic system, stimulation of the receptors causesvasoconstriction and stimulation of the receptors causesvasodilatation (Silove & Grover, 1968; Howard et al. 1975;Rubin & Lazar, 1983). Numerically and functionally,the receptors tend to dominate such that sympatheticstimulation causes vasoconstriction and sympatheticblockade causes vasodilatation (Porcelli & Bergofsky,1973). Overall, ganglionic blockade would be expected toexert both vasodilatory and vasoconstrictive influenceswithin the lung, consistent with the variable natureof overall response observed in experimental animals(Murray et al. 1986; Lodato et al. 1988). In the presenthuman study, the net effect of blockade was vasodilatory.

In the present study, trimetaphan caused a modestreduction in the pulmonary vascular sensitivity to acutehypoxia. One possibility is that this finding is an artifactbecause trimetaphan also removed the normal rise inCO that occurs with the onset of acute hypoxia. Usinga value from the literature relating the increase in Pmaxto the increase in CO (Balanos et al. 2005), the valuesobtained for the increase in Pmax with acute hypoxia maybe corrected for the effect of any concomitant change inCO. Once this correction is made, the significance of theeffect of trimetaphan on the sensitivity of the pulmonaryvasculature to hypoxia is lost. This finding is consistentwith that of Lodato et al. (1988) in conscious dogs, whoconcluded that, once the confounding effects of changes inCO had been controlled for, neither sympathetic blockadenor total autonomic blockade (with hexamethonium)affected the pulmonary vascular sensitivity to acutehypoxia.

In the present studies, ganglionic blockade did notabrogate the increase in basal tone and sensitivity tohypoxia induced by acclimatization with 8 h of hypoxia.We are unaware of any previous studies with which thismay be reasonably compared.

The effect of trimetaphan on cardiovascularresponses to hypoxia

As anticipated, autonomic blockade with trimetaphanreduced SVR and increased HR, with a consequential risein CO and fall in mean AP. Trimetaphan blocked theincrease in HR that normally occurs with the inductionof acute hypoxia. This effect of trimetaphan is very similarto that observed with blockade of just the parasympatheticcomponent using muscarinic antagonists (Petersen et al.1974; Clar et al. 2001). Trimetaphan did not, however,



Table 3. Cardiovascular responses to hypoxia: values (mean S.E.M.) for heart rate (HR), cardiac output (CO), mean systemic arterialpressure (mean AP) and systemic vascular resistance (SVR) for the trimetaphan and control protocols before and after an 8 h periodof sustained hypoxic exposure

Protocol/Factor HR (beats min1) CO (l min1) Mean AP (mmHg) SVR (mmHg l1 min2)

Euoxia Hypoxia Increase Euoxia Hypoxia Increase Euoxia Hypoxia Increase Euoxia Hypoxia Increase

AM measurements before sustained hypoxiaControl 1 57 3 67 4 10 2 5.1 0.2 6.0 0.3 0.9 0.1 70 5 74 6 4 2 13.3 1.1 12.5 0.9 0.8 0.6Trimetaphan 83 5 85 3 2 2 6.8 0.3 7.2 0.3 0.4 0.3 59 3 54 2 5 2 8.7 0.5 7.6 0.4 1.1 0.2Control 2 57 2 69 3 12 2 4.8 0.2 5.9 0.3 1.1 0.2 69 3 74 3 5 2 14.5 1.1 12.7 0.8 1.8 0.6

PM measurements after sustained hypoxiaControl 1 67 4 85 5 18 1 5.8 0.4 7.5 0.5 1.7 0.2 79 7 81 7 2 2 12.8 0.7 11.0 0.9 1.8 0.4Trimetaphan 88 5 90 4 2 1 6.9 0.5 7.2 0.5 0.2 0.2 59 5 50 4 9 3 8.9 1.1 7.2 0.9 1.7 0.3Control 2 66 4 83 5 17 2 5.8 0.4 7.3 0.5 1.5 0.4 88 7 92 7 5 2 15.4 1.7 13.1 1.4 2.3 0.7

Statistical analysis ANOVATime (before/after sustainedhypoxia) < 0.002 < 0.01 < 0.05 < 0.05 < 0.05 Protocol (C1, C2versus T) < 0.001 < 0.01 < 0.01 < 0.001 < 0.05 < 0.01 < 0.01 < 0.001 < 0.01 < 0.001 < 0.001 Interactiveterm < 0.01 0.050 < 0.05 < 0.01 < 0.05

AM, morning; PM, afternoon. A one-sample t test was conducted on the increase from euoxia to hypoxia, with results indicated asfollows; P < 0.05; P < 0.01. ANOVA conducted on the parameter values with fixed factors Time (before/after sustained hypoxia)and Protocol (control protocols (Protocols C1, C2) versus Protocol T). Statistical significance was accepted at P < 0.05.

Control-1 Protocol Trimetaphan Protocol Control-2 Protocol

0

40

80

120

0369

-10 100 20 30 -10 100 20 30 -10 100 20 300

10

20

0

40

80

120

A

HR

(be

ats/m

in)

CO

(l/min)

C

B

mean A

P (m

mHg)

SVR

(mmH

g/l/m

in) D

Time (min)

-10 100 20 30 -10 100 20 30 -10 100 20 30Time (min)

-10 100 20 30 -10 100 20 30 -10 100 20 30Time (min)

-10 100 20 30 -10 100 20 30 -10 100 20 30Time (min)

Figure 3Mean values ( S.E.M.) for heart rate (HR) (A), cardiac output (CO) (B), mean systemic arterial pressure (AP) (C) andsystemic vascular resistance (SVR) (D) during acute hypoxia before ( ) and after () sustained hypoxic exposure.



block the reduction in SVR with acute hypoxia consistentwith hypoxia acting as a local vasodilator (Weisbrod et al.2001). As a result, a further rise in CO and fall in APoccurred.

Sustained hypoxia of 8 h duration induced a rise inHR under subsequent euoxic conditions and a rise in thesensitivity of HR to acute hypoxia that was similar to thatreported in previous studies (Clar et al. 2000; Clar et al.2001). Much of this was abrogated with the acute infusionof trimetaphan in Protocol T, consistent with the previousreport that much of the change in HR following sustainedhypoxia arises through alterations in parasympatheticfunction (Clar et al. 2001). Possible causes of any smallelevation in HR following 8 h of hypoxia that persistsduring trimetaphan infusion include both alterations inhumoral factors and alterations in intrinsic factors inducedby hypoxia, as well as the possibility that it somehowreflects an increase in the work of breathing caused by themore vigorous ventilatory response. Longer-term studiesof hypoxia report increases in both parasympathetic andsympathetic activities in regulating HR, with the resultthat HR in chronic hypoxia is only mildly elevated(Boushel et al. 2001; Calbet, 2003; Hansen & Sander,2003).

Of note in this study was the absence of any conditioningeffect of sustained hypoxia on SVR. This appears to besomewhat at variance with the results of Dorrington et al.(1997) where SVR appeared to remain below the controleuoxic value for at least 2 h following the relief of thesustained hypoxia. In the absence of any conditioningeffect of the sustained hypoxia on SVR, the hypothesis thattrimetaphan might modify such conditioning immediatelyfails.

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Acknowledgements

We thank David OConnor for skilled technical assistance andthe volunteers for enthusiastic participation. We thank Dr ShouYan Wang for assistance with modelling the pulmonary vascularresponses to hypoxia. This study was supported by the WellcomeTrust.


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