Journal of Applied Physiology
Vol. 82, No. 1, pp. 292-297, January 1997
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

Effect of N-nitro-L-arginine on ventilatory response to hypercapnia in anesthetized cats

Luc Teppema, Aad Berkenbosch, and Cees Olievier

Department of Physiology, Leiden University, 2300 RC Leiden, The Netherlands

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

ABSTRACT

Teppema, Luc, Aad Berkenbosch, and Cees Olievier Effect of N-nitro-L-arginine on ventilatory response to hypercapnia in anesthetized cats. J. Appl. Physiol. 82(1): 292-297, 1997.The effect of intravenous administration of 40 mg/kg N-nitro-L-arginine (L-NNA), an inhibitor of the synthesis of nitric oxide (NO), on the ventilatory response to CO₂ was studied in anesthetized cats. The ventilatory response to CO₂ was assessed during normoxia by applying square-wave changes in end-tidal PCO₂ of ~1 kPa. Each CO₂ response was separated into a fast peripheral and slow central component characterized by a CO₂ sensitivity (S_p and S_c, respectively), time constant, time delay, and an offset (apneic threshold). L-NNA reduced S_p, S_c, and the apneic threshold significantly by ~30%. However, the ratio S_p/S_c was not changed. It is argued that the reduction in S_p and S_c, S_p/S_c remaining constant, may be due to a potent inhibitory action of L-NNA on the brain stem respiratory-integrating centers and on the neuromechanical link between these centers and respiratory movements. It is concluded that NO plays an important role in the control of breathing.

nitric oxide; nitric oxide synthase inhibitor; control of breathing; carbon dioxide response

INTRODUCTION

AN IMPORTANT FUNCTION of nitric oxide (NO) is its action as a biological messenger in the peripheral and central nervous system. In the brain, NO acts as a neurotransmitter to increase guanosine 3,5-cyclic monophosphate levels and to activate excitatory, mostly glutamatergic, pathways (2, 7, 16). In brain and endothelial cells, NO is synthesized from L-arginine (17). The enzyme responsible for this synthesis is nitric oxide synthase (NOS) (7, 17). Because NO is a short-lived, chemically unstable, and reactive molecule, its concentration is difficult to measure in vivo. Most studies on the physiological role of NO have, therefore, utilized analogues of L-arginine to block its synthesis by means of inhibition of NOS (for review, see Ref. 18).

It is well known that NO plays a role in the regulation of arterial blood pressure and cerebral blood flow (for reviews, see Refs. 12, 16). A normal cerebral vasodilatory response to hypercapnia is critically dependent on the presence of NO. However, a significant action of the molecule in cerebral (pressure) autoregulation has not been convincingly demonstrated (12).

The involvement of NO in the regulation of the cerebrovascular response to CO₂ must have implications for the control of breathing. Inhibition of the normal hypercapnic cerebral vasodilatory response by blockade of the synthesis of NO should cause larger changes in brain tissue PCO₂ at the site of the central chemoreceptors with a given change in arterial PCO₂ and, therefore, give rise to an increase in the slope of the ventilatory CO₂-response curve. Whether this occurs will also depend on a role of NO in the carotid bodies (cf. Refs. 3, 23) where the molecule has been shown to act as an inhibitory transmitter. Finally, there may be a role of NO in the central respiratory neural network because NOS is present in medullary and pontine respiratory regions. (5, 11, 29). From these data one might anticipate that NO plays a role in the chemical control of breathing.

To our knowledge, no studies have yet been performed in a whole animal preparation to investigate the effect of inhibition of NOS on the overall ventilatory response to changes in arterial PCO₂. Furthermore, it would be interesting to know if such an effect of NOS inhibition would be due to an action on the peripheral and/or central chemoreflex loops. The aim of the present study was to investigate this issue in anesthetized cats. To separate the contributions to the ventilatory CO₂ response of the peripheral and central chemoreflexes from each other, the dynamic end-tidal forcing (DEF) technique is an attractive tool to use because it enables one to extract the peripheral and central parts from the overall ventilatory response to a CO₂ challenge without interrupting neuronal pathways (4). To inhibit NOS, we used the analogue of L-arginine, N-nitro-L-arginine (L-NNA). Compared with other L-arginine analogues, this agent crosses the blood-brain barrier relatively easily and strongly inhibits NOS in the central nervous system (6, 13). The results indicate that brain NO plays an important role in the control of breathing.

METHODS

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RESULTS

The stability of our experimental model was tested in two cats receiving no L-NNA. The variability of the parameters between different runs within each animal is shown in Fig. 1. It appears that there are no systematic changes in S_c, S_p, and B over as long as 5-6 h. The average values of the estimated parameters together with their standard deviations are displayed in Table 1.

Table 1. Mean parameters with standard deviation for repeated runs in 2 cats Cat 1  Cat 2  B, kPa 3.74 ± 0.28  3.63 ± 0.23  Stot, l · min1 · kPa1 0.675 ± 0.079  0.983 ± 0.118  Sc, l · min1 · kPa1 0.581 ± 0.087  0.804 ± 0.097  Sp, l · min1 · kPa1 0.076 ± 0.030  0.179 ± 0.042  Sp/Sc 0.136 ± 0.060  0.223 ± 0.054   on, s 78 ± 20  96 ± 34   off, s 124 ± 41  136 ± 26   p, s 3.2 ± 3.1  3.1 ± 3.1  Tc, s 5.3 ± 2.2  5.4 ± 2.9  Tp, s 4.0 ± 1.6  4.1 ± 1.5  C, ml/min2  1.4 ± 3.9   0.1 ± 8.9 Values are means ± SD; n, no. of runs (16 for cat 1, 17 for cat 2). B, apneic threshold; Stot, total chemoreflex CO2 sensitivities; Sc, Sp, CO2 sensitivities of central and peripheral chemoreflex loops, respectively; Sp/Sc, ratio of Sp to Sc; on, off, time constants of central chemoreflex loop, belonging to on-transit and off-transit, respectively; p, time constant of peripheral chemoreflex loop; Tc, Tp, delay times for transport of CO2 disturbance from lungs to central and peripheral chemoreceptors, respectively; C, drift term.

Infusion of L-NNA caused an increase in mean arterial blood pressure as illustrated in Fig. 2, which also shows that soon after the start of the infusion, ventilation rose. Blood pressure increased in all cats from 13.2 ± 1.0 to 16.5 ± 1.0 (SD) kPa. Infusions of L-NNA were performed at constant PET_CO2. Arterial pH did not change after the injections, indicating that the drug did not induce arterial acid-base disturbances. Generally, 2-2.5 h were allowed to pass after L-NNA infusion to let all parameters stabilize and to allow maximal inhibition of brain NOS to occur, which, after intravenous infusion of a NOS inhibitor, is the case after ~2 h (12).

Figure 3 shows examples of a CO₂ DEF run before and after administration of 40 mg/kg L-NNA. The points are the breath-by-breath ventilation data. The curve through the data is the least squares model fit, which uses the actual PET_CO2 as input. The total ventilation consisted of _c and _p component. Figure 3 illustrates that both S_c and S_p were depressed by L-NNA. The results of all experiments are illustrated in Fig. 4, which shows scatter diagrams of the averages per cat of S_c, S_p, the ratio S_p/S_c, and B before and after the administration of L-NNA. S_p, S_c, and B were significantly decreased, but S_p/S_c remained about the same. The results from the seven cats that received a dose of 40 mg/kg L-NNA are summarized in Table 2, which shows that of the other model parameters, the _p was shortened, and T_c and T_p were significantly increased. L-NNA did not change the breathing pattern; i.e., the relationship between ventilation and tidal volume remained unchanged.

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Table 2. Effect of L-NNA on ventilatory response to CO2 Control L-NNA P B, kPa 3.52 ± 0.18  2.42 ± 0.68  <0.0001 Sc, l · min1 · kPa1 0.780 ± 0.143  0.531 ± 0.118  <0.0001 Sp, l · min1 · kPa1 0.146 ± 0.034  0.100 ± 0.032  0.039 Sp/Sc 0.221 ± 0.068  0.217 ± 0.059  NS  on, s 89 ± 14  106 ± 45  NS  off, s 145 ± 14  139 ± 27  NS  p, s 9.5 ± 2.1  3.7 ± 1.4  0.0014 Tc, s 6.1 ± 1.3  12.8 ± 1.7  <0.0001 Tp, s 3.6 ± 0.4  6.5 ± 2.0  <0.0001 C, ml/min2  1.1 ± 1.6   1.2 ± 1.8  NS Values are means ± SD; n = 7 cats. Cats received a dose of 40 mg/kg N -nitro-L-arginine. (L-NNA). NS, not significant.

DISCUSSION

In this study we have used the DEF technique to evaluate the effects of L-NNA on the chemical control of breathing in the anesthetized cat. The time lapse between the DEF experiments before and after L-NNA infusion was often several hours. We, therefore, checked the stability of our anesthetic regime in two control animals by recording several ventilatory responses during a time span of ~5 h. Repeated measurements in the two animals show that the standard deviation of B is ~0.3 kPa and of S_c and S_p ~0.1 and 0.04 l ^· min^{1 ·} kPa¹, respectively. The variation coefficient of the total slope (S_c + S_p) was 12%. Other authors found similar values for repeated measurements in awake subjects (24, 25). The results indicate that the anesthetic regime we used allows measurements with a variability comparable to awake mammals and that this regime does not introduce systematic changes of the parameters in time (see Fig. 1). Previously, it has been shown that the DEF technique together with our two-compartment model correctly assesses S_c, S_p, and B (4).

The measured changes in B and S_c after L-NNA are highly significant, but the change in S_p just reaches significance. We have no explanation for the finding that during control measurements _p was significantly larger than after L-NNA administration. Because the _p of the control runs is somewhat larger than that found in the two cats presented in Table 1, we are not sure whether the shortening of _p has a physiological meaning. From the observed changes in slope and intercept, it follows that the ventilatory CO₂-response curves before and after L-NNA infusion intersect at a PET_CO2 higher than the resting value of ~4 kPa, implying that the resting ventilation will be increased after infusion of the NOS inhibitor.

The transport delay times from the lungs to the central and peripheral chemoreceptors were significantly increased by L-NNA (see Table 2). Although the uncertainty of these parameters is at least the duration of a breath, this could indicate that the agent caused an appreciable vasoconstriction.

The mean increase in arterial blood pressure of 2.7 kPa we observed after administration of 40 mg/kg L-NNA is similar to that reported by Sandor et al. (27) after 30 mg/kg N^G-nitro-L-arginine methyl ester but much smaller than the rise in pressure of ~6 kPa observed by Kovach et al. (14). The latter authors, however, infused a much larger dose of L-NNA than we did. A rise in arterial blood pressure tends to decrease ventilation, which is probably a baroreceptor-mediated effect (e.g., Refs. 26, 30). The slope of the CO₂-response curve, however, is unaffected by blood pressure changes (10). We cannot exclude that the rise in blood pressure after L-NNA infusion was counteracting the mechanisms operating to increase the level of ventilation (by decreasing the level of B). The effectiveness of this possibly opposing mechanism, however, might be (partly) neutralized at the level of the brain stem because the glutamate-dependent baroafferent impulse transmission in the nucleus tractus solitarii (NTS) may also expected to be affected by L-NNA (Ref. 5; see also below).

In many studies it was shown that NOS inhibition decreases normocapnic cerebral blood flow in different brain regions and attenuates the specific cerebrovascular response to CO₂ (for references, see Ref. 12). At inhibitor concentrations comparable to the one used in the present study, a pronounced decrease in blood flow through the pontomedullary tissue was found (27). This should lead to an increase in brain tissue PCO₂, to a rise in ventilation, and thus to a shift of the ventilatory response to lower PET_CO2 values. Furthermore, baseline activity of the carotid bodies is reported to increase by NOS inhibition (3, 23). The observed decrease in B of ~1 kPa could be due to these effects. In addition to a decrease in cerebral blood flow, an almost complete inhibition of medullary vascular CO₂ sensitivity was found (27). This should result in an increase in the central ventilatory sensitivity to changes in PET_CO2. However, we observed a decrease in S_c. In the central nervous system, the action of NO is predominantly excitatory (7, 18, 28) and, therefore, an inhibitory role of L-NNA on the central respiratory neural network should be taken into consideration. In agreement herewith, we also observed a decrease in S_p by L-NNA, although the drug may have an excitatory effect on the carotid bodies. For example, inhibition of NOS within the in vitro carotid body results in an increase in chemoreceptor sensitivity to PO₂ changes in the perfusate and in an increase in baseline activity (3, 23). Further in vivo studies involving recording of chemosensory fiber activity are needed to determine the role of NO within the carotid bodies of intact animals.

Several data indicate that NO plays a modulatory role in respiratory brain stem nuclei. For example, clusters of neurons with NOS immunoreactivity have been identified in cardiorespiratory regions of the NTS, in (the vicinity of) the nucleus ambiguus and in the rostroventrolateral medulla, particularly within the lateral paragigantocellular nucleus (5, 11, 29). Generally, in the central nervous system, production of NO is linked to synaptic activation involving glutamate (7, 8). In cats and rats, both N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate-receptor activation in the NTS and paragigantocellular nucleus are necessary to sustain normal levels of blood pressure and ventilation and also for a normal ventilatory CO₂ response to occur (9, 20-22). In the (rat) pons, the medial and lateral parabrachial nuclei contain clusters of neurons with NOS immunoreactivity (5, 29). Microinjection of L-NNA in this region in the cat produces a pronounced disruption of the pneumotaxic mechanism (15). These data indicate that NO plays an excitatory role in several respiratory brain stem nuclei. Besides these effects in the brain stem, it has also been reported recently (19) that NO is essential for an optimal function of myofilament function in the rat diaphragm.

Our finding that the ventilatory CO₂ sensitivities of the peripheral and central chemoreflex loops are depressed to the same extent suggests that the main effect of L-NNA is on that part of the pathways to ventilation common to both the peripheral and central chemoreceptors. In other words, the main inhibitory action resides in the integrating respiratory centers in the brain stem and in the respiratory muscles, despite a possible excitatory effect on the carotid bodies.

FOOTNOTES

Address for reprint requests: L. J. Teppema, Dept. of Physiology, Leiden Univ., P.O. Box 9604, 2300 RC Leiden, The Netherlands (E-mail: Teppema{at}rullf2.LeidenUniv.nl).

Received 6 May 1996; accepted in final form 3 September 1996.

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