INTRODUCTION

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DURING EXERCISE, there is a manifold increase in minute ventilation (E) in humans. This increase cannot be explained only by changes in parameters such as arterial pH (pH_a) and arterial blood gases [PCO₂ (Pa_CO2) and PO₂ (Pa_O2)] known to play a crucial part in the control of E under resting conditions. The accumulation of La and the accompanying decline in pH_a during metabolic acidosis in subjects while performing strenuous exercise have been known for many years and have been associated with the extra drive in E observed above the La threshold (25, 26). Little or no attention has been given to the possible role of La itself in the control of E. Results from studies of the effect of La on muscle afferents did not reveal any effect of the ion on the sensory neurons (20, 21). In other experiments, in which sustained venous infusions of La were applied, both pH_a and E were significantly altered (10, 12). The possibility that La could independently influence E was not considered. Therefore, we wanted to study whether raising the arterial La concentration ([La]_a) without changing the pH_a would affect E.

An increase in arterial plasma K⁺ concentration ([K⁺]_a) from ~4 to 7-8 mM has been found to occur during exercise (13, 19), caused by repeated muscle membrane depolarization and subsequent loss of intracellular K⁺ into the interstitium. The effect on E of infusion of KCl over a prolonged period, raising [K⁺]_a, has been studied by several investigators (2, 16, 18). The results suggest that K⁺ most likely plays a part in the increase of E during exercise. Supporting these findings are results from intravenous infusions of KCl, which have been shown to stimulate arterial chemoreceptors and induce E (11, 17), into anesthetized and decerebrate cats. Although studied in different species, the effects of raising [K⁺]_a in rats have not been investigated. An additional reason for infusing KCl into rats was that we wanted to test our model with a relatively known stimulus to enable us to better evaluate the effect La had on E. We used the anesthetized rat as a model, in which we increased both La and [K⁺] by venous infusions. The purposes of this study were therefore threefold: 1) to test the hypothesis that La had an independent effect on E; 2) to investigate the effect of sustained hyperkalemia on E; and 3) to compare the effect of these two variables on E.

	MATERIALS AND METHODS

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Basic surgical preparation. Twenty-four adult male rats (332-450 g, local Wistar strain) were used. The rats were initially anesthetized with methohexital sodium (70 mg/kg ip, Brietal, Lilly, Indianapolis, IN), tracheotomized, and prepared with catheters in the tail artery (PE-25) for arterial pressure recording, the left iliac vein (PE-50) for intravenous infusions, and the left iliac artery for blood sampling. After the tail artery was prepared, the rats received -chloralose, a bolus (50 mg/kg ia) at first and thereafter a continuous infusion (40 mg · kg¹ · h¹ in physiological saline, 1.3 ml/h) through a side branch of the tail artery catheter. Rectal temperature was continuously monitored and maintained at 38°C by external heating.

Experimental procedure. The rats were randomly divided into four experimental groups. The K⁺ group (n = 5) received 1,000 mM KCl solution at a constant infusion rate (0.127 mmol · min¹ · kg¹). The K⁺ control group (n = 5) received 1,000 mM NaCl solution at the same infusion rate as the K⁺ group. The La group (n = 6) received La solution made by mixing NaLa with (L⁺)-lactic acid (Sigma Chemical, St. Louis, MO), yielding a 2,000 mM solution at pH 4.0. The infusion rate was varied and controlled by a computer, starting at 0.0 and ending at 0.93 ± 0.04 (SE) mmol · min¹ · kg¹. Pilot studies had shown that this infusion protocol raised [La]_a to ~15 mM with minimal changes in pH_a and blood gases. The high concentration of the La solution was necessary to minimize the infused volume. The La control group (n = 6) received 1,620 mM NaCl solution at the same infusion rate as the La group.

Statistics. Results are shown as means ± SE. The change within groups and differences between groups were analyzed with repeated-measures analysis of variance, applying the Huynh-Feldt  adjustment of degrees of freedom. A P < 0.05 was regarded as significant. Two additional tests were applied: a t-test for comparing the increase in E at a single point between the experimental groups and a Pearson correlation coefficient for the relationship between E, on one hand, and [La]_a and [K⁺]_a on the other.

	RESULTS

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No significant difference was found between the experimental groups before infusions began, either in any of the measured blood variables, Pa_CO2, pH_a, Pa_O2, [K⁺]_a, and [La]_a, or the mean arterial pressure and heart rate, indicating that the animals were in a similar condition before the various infusion protocols (Table 1).

After a 10-min infusion, the mean [La]_a increased from 0.80 ± 0.2 to 13.2 ± 0.6 mM (Fig. 1). Equiosmolar infusions of NaCl had no effect on the [La]_a in the control group. During the first 10 min of the infusion period of the K⁺ solution, [K⁺]_a increased significantly (P < 0.01) from 4.2 ± 0.10 to 7.8 ± 0.11 mM (Fig. 1). No changes were observed in the control group.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Arterial blood concentration (mM) before and during infusion of KCl (top) and La solutions (bottom). Values are means ± SE.

The effects of the La infusions on E, pH_a, Pa_CO2, and Pa_O2 are summarized in Fig. 2. E increased significantly over the infusion period in the La group compared with in the control group (P < 0.01). After 10 min, E had increased from 112.3 ± 5.1 to 165.8 ± 11.0 ml/min, or by 47.0 ± 4.0%. During the infusion period, no significant changes were observed in pH_a in the La group compared with the preinfusion value. On the other hand, pH_a in the control group decreased from the preinfusion value after 7.5 min. The La and control groups did not differ with respect to changes in Pa_CO2. The partial pressure decreased initially, but after 5 min it had reached preinfusion values and did not vary much for the remainder of the infusion period. However, Pa_O2 in both groups increased during the infusion period and significantly more in the La group, reflecting the increased E. The K⁺ infusion resulted in an increase in E from 106.0 ± 7.41 to 126.2 ± 4.78 ml/min, or by 20.3 ± 5.28% (P < 0.05) (Fig. 3). No changes were observed in pH_a or Pa_O2 compared with the control group. Although Pa_CO2 tended to decline during the experiment, it did not reach a level of statistical significance. The relationship between [La]_a and [K⁺]_a and E is shown in Fig. 4. The correlation coefficients (r) for this relationship were 0.89 (P < 0.01) and 0.80 (P < 0.01), respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of La infusion on minute ventilation (E), arterial pH (pHa), and arterial PCO2 (PaCO2) and PO2 (PaO2). Values are means ± SE.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of KCl infusion on E, pHa, PaCO2, and PaO2. Values are means ± SE.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Relationship between arterial K+ concentration ([K+]a) and E (top) and arterial La concentration ([La]a) and E (bottom). Values are means ± SE.

The ventilatory increase was significantly greater (P < 0.01) in the La group compared with the K⁺ group (Fig. 5). The comparison was made after 10 min of infusion, when the arterial concentration had reached levels similar to those seen during severe exercise. In addition, the two experimental groups differed with respect to changes in their arterial blood-gas concentration (Figs. 2 and 3). The La group showed a significant increase in Pa_O2 but no change in Pa_CO2. However, the K⁺ group showed no significant changes in either Pa_CO2 or Pa_O2. No significant changes occurred in respiratory rate in either the La or the K⁺ group. The increase in E in both groups was thus primarily based on an increase in tidal volume. The increases in tidal volume in the K⁺ and La groups were 19.7 ± 7.33 and 34.7 ± 8.85%, respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Comparison of effects of raising [K+] and [La] on E within physiological limits. Values are means ± SE. [K+] = 7.8 ± 0.11 mM, and [La] = 13.2 ± 0.6 mM.

	DISCUSSION

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The main findings of the present study are that 1) La stimulates E despite a normal pH_a, 2) sustained hyperkalemia raises E in rats, and 3) La is a more potent stimulus to E than K⁺. The primary new finding of this study is that La infusion stimulates E without any changes in pH_a or Pa_CO2 (Fig. 2). Although the increase in E persists up to [La]_a = 20 mM, physiological limits were reached in ~10 min, resulting in a 47% increase in E.

McLoughlin et al. (12) used intravenous infusions of lactic acid. The pH_a was monitored, but [La]_a was not measured. In the study, the emphasis was placed on the acidic effect of lactic acid but not on La itself (12). In addition, Lee et al. (10) reported immediate apnea, bradycardia, and hypotension, followed by sustained hyperpnea, in response to intravenous bolus injections of lactic acid in rats and concluded that the cardiorespiratory depressor response was predominantly elicited by activation of vagal pulmonary C fibers because a selective block of the vagal afferents completely abolished the response. However, they concluded that the hyperpneic response that followed was mediated by peripheral chemoreceptors, as demonstrated in a study by Bainton (1). The above-mentioned authors (10, 12) did not discuss the possibility of whether La may have a role in stimulating E, as our results indicate, and they made no attempt to prevent changes in pH_a in their animal models, as was done in the present study.

The cause-and-effect relationship between metabolic acidosis and hyperventilation in heavy exercise in humans, as suggested by Wasserman et al. (26), has been questioned (7, 8, 14). In rats and several other species, hypocapnic alkalosis occurs during mild-to- moderate exercise and no acidosis occurs during heavy exercise (4, 15). As pointed out by Fregosi and Dempsey (4), possibly neither humans nor rats depend significantly on metabolic acidosis as the critical stimulus for hyperventilation during exercise.

Although the pH of the infusion solution is 4.0, no sensory mechanism is known for H⁺ in the venous circulation. By what mechanism La affects E is difficult to speculate. Muscle afferents (groups III and IV) have been proposed as candidates for stimulating E during exercise because of their proposed ability to monitor chemical changes in the muscle interstitium. A few experiments have tested the effect of lactic acid and La on the discharge rate of these afferents by raising their muscle concentration. An increased response of the chemoreceptors was found for lactic acid but not when either [NaLa] or [LiLa] was given (9, 20, 21). On the other hand, Rotto et al. (21) found that lactic acid had a stimulating effect on E in excess of that of an HCl infusion. Their findings suggested a potentiating role of La on the basis of the response evoked by the H⁺ activity in skeletal muscle.

To maintain a constant pH_a in the La group during the infusion period, the La solution had to be kept at pH 4.0. This acidity was necessary because pilot studies showed that, if the La infusate were at a higher pH, the rats would become alkaline. This effect was also found by Gladden and Yates (5), who varied the pH of an intravenously infused lactic acid solution in dogs. They concluded that the effect of lactic acid on blood pH was the result of two opposing effects: 1) an acidifying effect because of its dissociation properties as a weak acid and 2) an alkalinizing effect because of the oxidation of lactic acid to CO₂ and H₂O or the conversion of lactic acid to glycogen. Furthermore, in our study we found that during the infusion the rate had to be linearly increased to maintain a constant increase in [La]. This need for an increased La supply is presumably because of the accelerated oxidation effect or conversion to glycogen during the experiment.

By a comparison of the composition of the infusion solution and La plasma levels attained in rats, it was estimated that 36-43% of the infused La disappeared from the extracellular fluid. Presumably this La enters the cells, raises the resting level of lactic acid inside the cells, and is oxidized or converted to glycogen. From our data it cannot be estimated how much is oxidized or how much is converted to glycogen. If we assume that all of the La entering the cells in the present study were oxidized, it would have produced ~25 ml CO₂ · min¹ · kg¹ at the end of the infusion period. This could thus raise the metabolic rate comparably to that seen in mild exercise, during which a threefold increase in E was observed (4). However, in our study the E was only raised by 47%. It is thus apparent that more than enough La disappears from the plasma to produce enough CO₂ to account for the observed increase in E. Therefore, the effect of La on E could be mediated by an increase in CO₂ production (CO₂).

The observed isocapnea shows that there is a balance between CO₂ and CO₂ elimination by breathing. This agrees with the strong correlation known to exist between CO₂ and E during exercise. However, the increase in E is more than that required to fulfill the need for O₂, as indicated by the increase in Pa_O2, which causes less stimulation of the arterial O₂ receptors. The mechanism responsible for monitoring and mediating information about increased CO₂, despite arterial iso- or hypocapnia, is not known. It has been suggested that the oscillations in Pa_CO2 in synchrony with breathing rhythm can be sensed by the arterial CO₂/H⁺ receptors and used by the brain to monitor CO₂ (3, 27). Recent experiments have not been able to support this hypothesis (23). In rats, CO₂ is proportional to the increase in [La] (4). Therefore, if a mechanism exists to monitor plasma [La], it could possibly be used to indicate CO₂ in rats.

An unexpected effect was the decrease in pH_a in the La control group after 10 min of infusions. This decrease in pH_a indicates that the changes in E in the control group do not underestimate the H⁺ stimulation in the La group and therefore cannot explain the E increase in this group. Why this decrease in pH_a occurs is not certain, but an increase in the Na⁺/H⁺ exchange between the extra- and intracellular fluid compartments could explain this effect (6). In addition, the increased concentration of Na⁺ and subsequent suppression of Na⁺/ reuptake cotransport in the kidneys could contribute to a lower pH_a.

The infusion of KCl resulted in a gradual increase in E during the infusion period (Fig. 1). The ~20% increase observed in E occurs within 10 min when [K⁺]_a reaches values seen in strenuous exercise in men (13), i.e., 8 mM. Compared with previous experiments in other species in which venous infusions of KCl have been used (2, 12, 16, 18, 22), the increase in E in the present study was moderate. Increases from 20 to 40% were commonly observed. A doubling in E has been reported by using brief (4- to 5-s) KCl stimuli (11). Although the change in Pa_CO2 did not differ significantly between the K⁺ group and its control group (P = 0.15 for the interaction term), there was a clear tendency for a decrease in Pa_CO2 from the preinfusion level in the K⁺ group (Fig. 3), which is in accordance with previous studies in cats (12, 22). Mediated by peripheral and central (medullar) chemoreceptors, the fall in Pa_CO2 will have inhibitory effects on E. Thus this hypocapnic breaking most likely diminishes the stimulatory effects of K⁺. Abolition of hypocapnic breaking by rebreathing of CO₂ would thus be expected to increase the observed response in E. Therefore, it is most likely that K⁺ is responsible for stimulating hyperventilation and producing the observed hypocapnia in the animal models studied thus far.

The stimulating effect of La on E in rats was more than twice that observed by using K⁺ (Fig. 5) when both ions were increased to levels similar to those observed during maximal exercise in men (13, 24). Although our results suggest that in rats La is a more potent ventilatory stimulant than K⁺, it is important to recognize that [K⁺]_a increases gradually during incremental exercise (19). The accumulation of [La]_a in humans, on the other hand, is only observed when the La threshold has been reached and therefore could only contribute to an increase in E during exercise above 50-70% of maximal O₂ uptake and/or to a sustained high E during the recovery period. In a similar experiment, both lactic acid and KCl were infused into cats, which resulted, after 40-60 s, in an 85 and 20% increase in E, respectively (12). The increase in the lactic acid group increased E substantially more than was observed in our experiment, not surprisingly because the pH_a decreased significantly.

Compared with the manifold increase in E during exercise, the increases observed in E during infusions of either La or K⁺ in our experiments are modest. This does not imply that these ions are of minor consequence in the control of exercise hyperpnea. During exercise many factors affect E, most probably synergistically. The present study only focuses on the isolated effect of these ions and confirms that they may contribute to the increase in E observed during exercise.

The present study shows that La can stimulate E without any accompanying changes, in either pH_a or Pa_CO2. This finding further supports the notion that [H⁺]_a may not be as important in stimulating hyperventilation observed in heavy exercise as previously thought. The possibility that La may be even more important is suggested by the present study. Furthermore, the idea that plasma [K⁺] is responsible for the induced hypocapnia (and accompanying alkalosis or reduced acidosis) observed in various stages of exercise is supported.