The effect of angiotensin-converting enzyme inhibition by captopril on respiratory mechanics in healthy rats

Abstract

Context: Angiotensin stimulates smooth-muscle contraction. Accordingly, angiotensin-converting enzyme (ACE) inhibition is expected to decrease airway resistance.

Objectives: To measure the effects of ACE inhibition on respiratory mechanics in healthy mammals.

Materials and methods: We measured respiratory mechanics before and after i.p. ACE inhibitor captopril (100 mg/kg) in normal anaesthetised rats. The end-inflation occlusion method allowed the measurements of respiratory system elastance and ohmic and viscoelastic pressure dissipations. Respiratory system hysteresis and the elastic and resistive work of breathing were calculated.

Results: Captopril induced a reduction of the ohmic and the total respiratory system resistances, while respiratory system hysteresis and elastance did not change. Accordingly, a reduction of the resistive and of the total work of breathing was observed.

Conclusions: The captopril-induced reduction of airway resistance indicates that angiotensin modulates bronchomotor tone in basal conditions. ACE inhibition may positively affect respiratory system mechanics and work of breathing.

Keywords::

• Captopril
• end-inflation occlusion method
• rats
• respiratory system mechanics
• work of breathing

Introduction

The angiotensin plasma concentration is increased and potentiates bronchoconstriction in asthmatic subjects^1,2. Angiotensin has also been shown to cause bronchoconstriction in guinea pigs³ and rats^4,5, and bronchial hyperresponsiveness to metacholine in human asthmatic subjects^2,6 and guinea pigs³.

Furthermore, the inhibition of angiotensin receptors of type 1, but not of type 2, has been shown to prevent in a dose-related manner the metacholine-induced bronchoconstriction in normal³ and inhalating antigen guinea pigs⁷. Angiotensin-elicited bronchoconstriction was also shown to be inhibited by type 1 angiotensin receptor antagonism⁴ and/or angiotensin-converting enzyme (ACE) inhibition in rats⁵.

Some data suggest that angiotensin may exert its biological effects in normal healthy subjects in basal conditions also. For example, it is well-known that low dosages of the ACE inhibitor captopril show their hypotensive effects in normal subjects⁸.This suggests that ACE inhibition might influence bronchial smooth muscle tone of normal subjects also. Moreover, angiotensin has been shown to cause contraction of bronchial rings taken from normal rats^4,5, and bronchoconstriction in healthy guinea pigs³.

However, Jamison and Glover⁹ observed no effect of ACE inhibition either on basal airway mechanics or on hypocapnic hyperventilation-induced bronchoconstriction in normal subjects. Moreover, ACE inhibition has been demonstrated to leave unaltered baseline lung function in normal subjects¹⁰, and basal airway resistance and bradychinine-induced bronchoconstriction in guinea pigs¹¹. Type 1 angiotensin receptor antagonism did not alter airway resistance in normal guinea pigs⁷. In addition, greater decreases of pulmonary conductance caused by substance P were observed in captopril-treated guinea pigs than in control animals, suggesting that angiotensin may even exert a bronchodilator effect¹².

Thus, the reported effects of angiotensin and/or of ACE or angiotensin receptors inhibition on the airway resistance of normal healthy mammals are conflicting.

No data are available describing the possible effects of ACE inhibition, hence indirectly of basal angiotensin plasma concentration, on other respiratory system mechanics parameters such as on viscoelastic resistance due to stress relaxation, and on elastance. These may be easily measured, together with the ohmic airway resistance, by the end-inflation occlusion method^13–16, which has never been applied to study the angiotensin’s effects on respiratory mechanics.

Stress relaxation is a complex phenomenon, exhibited by most tissues, including the lungs, whose molecular basis are essentially unclear. Due to their viscoelastic properties, the tissues do not maintain constant stress under constant deformation, but slowly relax. The viscoelastic pressure component of the inspiratory work of breathing contributes significantly to the overall breathing pressure dissipation^13–16. Hence, the study of the factors possibly affecting stress relaxation has practical connotations, because any effect reducing the viscoelastic component of the inspiratory work of breathing may suggest therapeutical implications. According to Potter et al.¹⁷ and Martin et al.¹⁸, the contractile activity of the peripheral cellular elements in the lungs may be changed by smooth muscle activity-influencing agents such as nitric oxide. Hence, a possible effect of angiotensin and/or ACE inhibition on respiratory system stress relaxation and elastance may not be excluded. These aspects have never been investigated before.

Although some biochemical interactions between angiotensin and alveolar surfactant dipalmitoylphosphatidylcholine have been described¹⁹, the possible effects of angiotensin and/or ACE inhibition on respiratory system hysteresis have never been measured.

Thus, in the present report in vivo measurements of healthy normal rats respiratory system mechanics are described, taken before and after ACE inhibition by intraperitoneal captopril. The aim of the experiments is to measure by the end-inflation occlusion method the effects of basal angiotensin plasma concentration on the ohmic and viscous-elastic respiratory system resistances, and on respiratory system elastance, in anaesthetised healthy mammals in basal conditions. The possible effects on respiratory system hysteresis have been also for the first time investigated. In addition, the effects of ACE inhibition on the total inspiratory work of breathing and on its elastic and resistive components were studied, which has never been done before.

Methods

The experiments were carried out on 9 Wistar albino rats of both sexes (mean weight 307 ± 31 g, 4 males and 5 females).

Animals were housed and treated in accordance with the Italian law on animal experimentation (L. 116/92) and with the European Council (EC) provision 86/609/EEC. The experimental protocol received the approbation of the local ethical committee (CEASA, University of Padova, ref. no. 24bis/2011).

Rats were anesthetized with 50 mg/100 g i.p. chloralose and laid on a heated operating table. After a tracheostomy, a small polyethylene cannula (2 mm i.d, 5 cm long) was inserted through an incision in the second tracheal ring and firmly secured in place.

Positive pressure ventilation with a 10 mL/kg tidal volume and a 60/min breathing frequency (PEEP 3 cmH₂O) (Rodent Ventilator 7025, Basile, Italy) was begun, and constantly maintained throughout the experiment.

The level of anesthesia was tested by checking the suppression of the corneal reflex.

Limb ECG probes were placed and the rats were paralyzed (cis-atracurium 1 mg/100 g i.p.).

Additional doses of cis-atracurium, a drug that does not appear to substantially alter heart rate²⁰, were administered as needed whenever spontaneous breathing appeared.

Positive pressure ventilation was maintained for 10 min and respiratory mechanics were then measured using the end-inflation occlusion method^13–16.

The ventilator was disconnected, PEEP was discontinued, and the tracheal cannula was connected to a constant flow pump (SP 2000 Series Syringe Pump sp210iw, World Precision Instruments, USA) set to deliver a tidal volume (VT) of 3 mL with a square wave flow (F) of 4 mL/s. The time for the rise and the fall of the flow was approximately 30 ms. The pump setting was carefully checked by directly taking measurements before beginning the experiments.

The lateral tracheal pressure proximal to the tracheal cannula was monitored (142 pc 01d, Honeywell, USA) and continuously recorded (1326 Econo Recorder, Biorad, Italy). Because abrupt changes in diameters were not present in the circuit, errors in flow resistance measurements, such as those reported by Chang and Mortola²¹, were avoided. The frequency response of the transducer and the pressure measuring system was tested by sinusoidal forcing and found to be flat up to 20 Hz. In accordance with the literature^15,22, this frequency response was adequate to avoid mechanical artifacts in the pressure signal records.

The end-inflation occlusion method was utilised to determine the parameters of respiratory mechanics: the static elastic pressure of the respiratory system (P_el,rs), the total resistive pressure drop (P_max,rs) and the sudden Newtonian resistive pressure drop at flow interruption (P_min,rs) were measured on adequately magnified tracings (Figure 1). P_max,rs was calculated as the difference between the maximum value of pressure at end-inflation (P_dyn,max) and P_el,rs. P_min,rs was calculated as the difference between P_dyn,max and P1, the pressure value immediately after flow interruption (Figure 1).

Figure 1.  Representative tracings of lateral tracheal pressure at flow interruption. Panels A and B show tracings recorded before and 20 min after i.p. 100 mg/kg captopril administration, respectively, in the same rat. The relevant pressures used for the calculations of respiratory system mechanics are indicated: maximal pressure at end-inflation (P_dyn,max), pressure immediatly after flow interruption (P1), static elastic pressure of the respiratory system (P_el,rs), pressure drop due to the ohmic respiratory system resistance (P_min,rs) and total pressure drop including the effects of stress relaxation (P_max,rs).

To avoid a viscous pressure component in P_min,rs, P1 values were identified by extrapolating the pressure tracings to the time the flow stopped²³. Thus, P_min,rs represents the nearly instantaneous, Newtonian resistive pressure drop that theoretically occurs at infinite breathing frequency²³. P_min,rs does not include the viscoelastic pressure drop that results from stress relaxation which is, instead, included in the P_max,rs value.

The mean pressure data obtained from the three to five inflations for each rat were used to calculate the respiratory system static elastance (E_st,rs = P_el,rs/VT) and the total resistance of the respiratory system (R_max,rs = P_max,rs/F). The latter includes the ohmic inspiratory resistance to airflow offered by the airways and the resistance caused to the movement of respiratory system tissues (R_min,rs = P_min,rs/F), and the viscoelastic pressure drop resulting from the effect of stress relaxation. This last component of R_max,rs was isolated and quantified as “viscous” resistance (R_visc,rs = R_max,rs – R_min,rs).

The equipment resistance, including the tracheal cannula and the standard three-way stopcock, was measured separately at a flow rate of 4 mL/s and amounted to 0.0575 cmH₂O/(mL/s) (Req). All inflations were performed at a fixed flow rate of 4 mL/s, and Req was subtracted from the results, which thus represent intrinsic values.

The work of breathing (WOB_rs) values were calculated as previously described^16,24,25, and outlined in Figure 2. Static pressure-volume relationships were assumed to be linear up to an inflation volume of 3 mL. This measurement is in agreement with published data showing essentially linear pressure-volume relationships for the rat respiratory system within this volume range^16,24,26,31. The validity of this assumption has also been confirmed experimentally by the essentially linear pressure-volume relationships observed during static inflations performed for H_y,rs measurements up to a total inflation volume of 5 mL (see below). The total work of breathing (WOB_tot,rs) was calculated by measuring the surface areas delimited by the total pressure (P_dyn) tracings from which the resistive pressure due to the tracheal cannula and three-way stopcock was subtracted. The elastic work of breathing (WOB_el,rs) was obtained on the same diagram as the areas encompassing the static pressure/volume lines. The resistive work of breathing (WOB_res,rs) was obtained by subtraction: WOB_res,rs = WOB_tot,rs – WOB_el,rs. WOB_res,rs was also partitioned into the resistive work required to overcome ohmic airway resistance and respiratory system tissue movements (WOB_ohm,rs) and the resistive work required to overcome the effects of stress relaxation (WOB_visc,rs). WOB_ohm,rs was calculated as the area comprised between the P_dyn and P1 lines, where P1 is the pressure value immediately after flow interruption (see Figure 1). WOB_visc,rs was calculated by subtraction as WOB_visc,rs = WOB_res,rs – WOB_ohm,rs. Calculations were made for each rat and the mean values were statistically compared.

Figure 2.  Schematic drawing of the pressure/volume relationship during constant flow inflation and flow interruption. Area 1 represents the elastic work of breathing (WOB_el,rs), whereas areas 2 + 3 represent the total resistive work of breathing (WOB_res,rs).WOB_el,rs + WOB_res,rs = total inspiratory work of breathing (WOB_tot,rs). Area 3 represents the ohmic work of breathing (WOB_ohm,rs), dissipated to overcome ohmic airways resistance plus the viscous resistance to the movement opposed by the lungs and chest wall tissues. WOB_res,rs−WOB_ohm,rs = WOB_visc,rs, the mechanical work done to overcome the resistive effects of stress relaxation.

After respiratory mechanics were measured, which took approximately 1 min, mechanical ventilation was restored and maintained for 5 min. To obtain a constant volume history for the measurement of respiratory system hysteresis, the respiratory system was inflated with a 10-mL syringe to a static elastic pressure of 20–25 cmH₂O three times. After that, the respiratory system was inflated, and subsequently deflated, 5 times in 1-mL steps using a precision glass syringe starting from the functional residual capacity (FRC). Each volume was maintained at each step for approximately 5–6 s. The relevant static elastic pressures were measured, and the static inflation-deflation volume–pressure curves were determined. The hysteresis areas (H_y,rs) were quantified using electronic digital integration and expressed as cmH₂O·mL.

The ventilator was disconnected and PEEP was not present during the constant flow inflations to measure respiratory mechanics parameters nor during the 1-mL inflations and deflations to measure H_y,rs.

The measurements and calculations described above were repeated for each rat 10 and 20 min following 100 mg/kg captopril dissolved in 1-mL saline i.p. administration.

Data in the literature and obtained in our laboratory indicate that mechanical ventilation parameters here adopted are not injurious to the rat respiratory system (ref. 24 and related references). This excludes the possibility that any observed alterations were due to a time-related effect.

The temporal sequence of the experimental procedure is summarised in Figure 3.

Figure 3.  Temporal sequence during experimental procedure: •: Start of mechanical ventilation, RM: respiratory mechanics measurements, *: electrocardiogram, Hy: H_y,rs measurement. ↑: i.p. injection.

Each rat being the control of itself, statistical analysis was performed by using the Student’s t test for paired data.

Results

We found that ACE inhibition by captopril caused a significant decrement of R_min,rs both at 10 and 20 min after i.p. administration (Figure 4), and similar results were observed for R_max,rs also (Figure 4). Although a trend indicating a time-related reduction of R_visc,rs was observed after captopril administration (Figure 4), the changes did not achieve statistical significance (although because of the result pertaining to one rat only of nine). No significant alterations in E_st,rs nor in H_y,rs mean values were observed after captopril (Figure 5).

Figure 4.  Mean values (n = 9) of R_max,rs (A), R_min,rs (B) and R_visc,rs (C) before and after 10 and 20 min captopril administration (time of injection indicated by the vertical arrow). *p < 0.05, **p < 0.01 and ^p = 0.07 with respect to values before captopril. Vertical bars represent one SE.

Figure 5.  Mean values (n = 9) of E_st,rs before and after 10 and 20 min (A) and of H_y,rs before and after 15 and 30 min (B) captopril administration (time of injection indicated by the vertical arrow). Vertical bars represent one SE. No significant difference was detected.

As expected from the above reported results, and depicted in Table 1, we observed significant reductions in WOB_ohm,rs mean values at both 10 and 20 min, and in WOB_res,rs value at 10 min from i.p. captopril administration (the reduction in WOB_res,rs at 20 min resulted almost only significant, p = 0.07). Although a trend indicating a time-related reduction in WOB_visc,rs mean values was observed after captopril (Table 1), these changes resulted not significant (see above), as well as the observed changes in WOB_el,rs (Table 1). The reduction of WOB_res,rs resulted, however, high enough to cause a significant decrement in WOB_tot,rs mean values both after 10 and 20 min after captopril (Table 1).

Table 1.  Mean values of respiratory work of breathing and its components.

Time (min)	0	10	20
WOBtot,rs	16.9 ± 0.9	14.9 ± 0.6*	14.6 ± 0.7*
WOBel,rs	9.9 ± 0.6	8.9 ± 0.3	8.9 ± 0.4
WOBres,rs	7 ± 0.5	6 ± 0.3*	5.7 ± 0.4^
WOBohm,rs	2.5 ± 0.2	1.9 ± 0.2**	1.9 ± 0.1**
WOBvisc,rs	4.5 ± 0.4	4.1 ± 0.4	3.8 ± 0.3

WOB_tot,rs, WOB_el,rs, WOB_res,rs, WOB_ohm,rs and WOB_visc,rs are the total, elastic, total resistive, newtonian and viscoelastic mean values (±SE, n = 9) of the work of breathing expressed in cm H₂O·mL.

^p = 0.07, *p < 0.05, and **p < 0.01 with respect to the time of captopril injection (time = 0).

A trend indicating a time-related significant reduction of heart rate was observed (Table 2).

Table 2.  Heart rate mean values (±SE, n = 9) over time from captopril injection (time = 0).

Time (min)	0	10	20
Heart rate (b/min)	328 ± 24	277 ± 28*	261 ± 25*

*p < 0.05 with respect to time = 0.

Discussion

Intraperitoneal administration of presently used captopril dosage is a well documented procedure in rat^27–29.

The values of respiratory mechanics parameters here measured and calculated are similar to those previously obtained in analogous experiments performed in different laboratories^{16,22,24,30,31}.

Our finding of reduced R_min,rs as an effect of ACE inhibition (Figure 4) confirms that angiotensin exerts a bronchoconstrictor effect even in basal conditions^3–5, suggesting that it may play a role in the modulation of airway calibre in healthy mammals.

The observed time-related reduction of heart rate (Table 2), probably due to a deepening of anaesthesia level in time, suggests a time-related reduction of sympathetic tone also. This would led to an increase of R_min,rs in time, while an actual decrement was observed, suggesting that the bronchoconstrictor effect of angiotensin has to be considered rather strong.

The decrement of R_min,rs we observed is agreement with most previously published results (see above), but in contrast with other experiments. However, the authors of the latter used clinically hypotensive low dosages of ACE inhibitors different from captopril^9,10, or tested much lower captopril dosages¹¹.

By applying the end-inflation occlusion method, we have been able also to measure the overall inflation inspiratory pressure dissipation including the viscoelastic effects due to stress relaxation. It has been shown that ACE inhibition significantly reduces the overall respiratory system resistance to inflation (Figure 4), hence the overall resistive inspiratory work of breathing (see below).

The analysis of the data describing the ACE inhibition effects on the isolated R_visc,rs demonstrates a trend to a time-related reduction, which however does not achieve statistical significance (Figure 4). This is probably due to a lack of statistical power secondary to the dispersion in the individual data. Nevertheless, R_visc,rs is included in R_max,rs, which has been here shown to decrease significantly after ACE inhibition, strongly suggesting that angiotensin effects in basal conditions are to increase not only the ohmic airway resistance but also the viscoelastic one, which is measurable as stress relaxation^13–16.

In basal conditions, the viscous component of the inspiratory pressure dissipation represented by stress relaxation substantially contributes to the total resistive work of breathing^13–16,32, but its molecular mechanism(s) remain largely unclear and poorly understood.

It has been shown that it is increased as an effect of increased lung blood volume and flow³¹, in inflamed lung tissue^24,33,34, and decreased as an effect of body warming¹⁶, but it is not influenced by oestrous cycle in the rat³⁵. Here, a possible effect of basal angiotensin plasma concentration on stress relaxation is hypothesised. In fact, it has previously been shown that smooth muscle cells contraction influencing agents such as nitric oxide may change the activity of the contractile peripheral lung cellular elements^17,18. Influences of these cellular elements activity on the viscoelastic characteristics of the respiratory system can not be excluded³². Angiotensin is known to exert its stimulating activity in a wide population of contractile cells, and captopril has been shown to induce nitric oxide release³⁶, so that an effect on stress relaxation may be hypothesized, although more work is needed to verify this working hypothesis.

Although biochemical interactions between angiotensin and alveolar surfactant have been described¹⁹, no data are available describing the effect of angiotensin or ACE inhibition on respiratory system hysteresis and elastance. In the present experiments, no significant effect was found. Hence, ACE inhibition and basal angiotensin plasma concentration do not influence the elastic mechanical properties of the respiratory system in healthy mammals (Figure 5).

The functional effect of the ACE inhibition-linked reduction of R_min,rs and R_max,rs is a significant decrement of the related resistive inspiratory work of breathing (see Table 1). Our data confirm that WOB_visc,rs represents the major part of WOB_res,rs^13,14,16,24. ACE inhibition significantly reduces both WOB_res,rs and WOB_ohm,rs while the reduction of the isolated WOB_visc,rs does not achieve statistical significance (see above). The changes of WOB_res,rs are high enough to cause a significant reduction of the overall inspiratory work of breathing (WOB_tot,rs), even in the presence of a not significant reduction of the elastic component (WOB_el,rs).

The procedure adopted to calculate the work of breathing and its components introduced an approximation due to the well-known volume-dependence of resistive pressures in the respiratory system, the effects of which were not considered here. However, on the basis of data published previously¹⁵, this approximation would have only minor effects, also due to the small volume change in the current study. Moreover, these effects would be expected to affect the results obtained before and after ACE inhibition equally and thus would not be predicted to introduce systematic error.

Conclusions

Our results indicate that the renin-angiotensin system affects the basal bronchomotor tone in anaesthetised healthy mammals in basal conditions.

The observed reductions of the resistive pressures dissipations and of the related WOB_res,rs and WOB_tot,rs values suggest that substantial ACE inhibition may add beneficial effects on respiratory mechanics to the well-known hypotensive effect in patients assuming captopril. This effect, if confirmed, could determine a substantial reduction of the inspiratory effort and of the work of breathing in hypertensive patients assuming captopril. Thus, a reduction of the frequently observed respiratory failure signs in hypertensive patients might ensue.

Declaration of interest

The authors report no declarations of interest.