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. 2006 May 4;574(Pt 2):565–572. doi: 10.1113/jphysiol.2006.110528

The carbonic anhydrase inhibitors methazolamide and acetazolamide have different effects on the hypoxic ventilatory response in the anaesthetized cat

Luc J Teppema 1, Hans Bijl 1, Babak Mousavi Gourabi 1, Albert Dahan 1
PMCID: PMC1817761  PMID: 16675491

Abstract

We compared the effects of the carbonic anhydrase inhibitors methazolamide and acetazolamide (3 mg kg−1, i.v.) on the steady-state hypoxic ventilatory response in 10 anaesthetized cats. In five additional animals, we studied the effect of 3 and 33 mg kg−1 methazolamide. The steady-state hypoxic ventilatory response was described by the exponential function:

graphic file with name tjp0574-0565-m1.jpg

where V̇I is the inspired ventilation, G is hypoxic sensitivity, D is the shape factor and A is hyperoxic ventilation. In the first group of 10 animals, methazolamide did not change parameters G and D, while A increased from 0.86 ± 0.33 to 1.30 ± 0.40 l min−1 (mean ± s.d., P = 0.003). However, the subsequent administration of acetazolamide reduced G by 44% (control, 1.93 ± 1.32; acetazolamide, 1.09 ± 0.92 l min−1, P = 0.003), while A did not show a further change. Acetazolamide tended to reduce D (control, 0.20 ± 0.07; acetazolamide, 0.14 ± 0.06 kPa−1, P = 0.023). In the second group of five animals, neither low- nor high-dose methazolamide changed parameters G, D and A. The observation that even high-dose methazolamide, causing full inhibition of carbonic anhydrase in all body tissues, did not reduce the hypoxic ventilatory response is reminiscent of previous findings by others showing no change in magnitude of the hypoxic response of the in vitro carotid body by this agent. This suggests that normal carbonic anhydrase activity is not necessary for a normal hypoxic ventilatory response to occur. The mechanism by which acetazolamide reduces the hypoxic ventilatory response needs further study.

Carbonic anhydrase (CA) catalyses the interconversion of CO2 and bicarbonate and plays a crucial role in respiration. To date, 14 isoenzymes have been characterized in higher vertebrates including humans (Chegwidden & Carter, 2000). A specific role of CA in respiration is indicated by the presence of various isoforms in tissues and cells that are directly or indirectly involved in ventilatory control, such as lung and brain capillary endothelium, kidney, muscle, carotid bodies and central chemosensitive areas in the rostroventrolateral medulla oblongata (see reviews by Maren, 1967; Chegwidden & Carter, 2000).

Inhibitors of CA have a profound influence on the control of respiration. Due to different physicochemical properties, various sulphonamide CA inhibitors have distinct dose-dependent effects. Acetazolamide (AZ), a moderately permeable sulphonamide, is the most frequently used inhibitor to study the role of CA in the hypercapnic response of the carotid body and it has been shown to diminish the steady-state CO2 sensitivity of the carotid body (Travis, 1971; Hayes et al. 1976). However, other studies reported only a reduced response speed and/or elimination of over- and undershoots in carotid sinus nerve activity upon (removal of) sudden hypercapnic stimuli (Gray, 1971; Black et al. 1971). This would be consistent with a role of CA in regulating the speed and magnitude of the initial CO2-induced fall in intracellular pH in type I carotid body cells (Buckler et al. 1991). In a superfused carotid body preparation from cat, AZ reduces both the release of dopamine and the increase in carotid sinus nerve discharge following acidic stimuli (Rigual et al. 1991). In humans and cats, AZ exerts inhibitory effects on carotid body-mediated reflexes. In both species, low intravenous doses reduce both steady-state hypoxic sensitivity and the O2–CO2 interaction (Teppema et al. 2001, 2006; Teppema & Dahan, 2004). In a dose that completely inhibits erythrocytic CA, it causes a much greater rise in ventilation in carotid body denervated cats than in intact animals (Teppema et al. 1988) while in the latter, the hypoxic ventilatory response (HVR) is abolished (Teppema et al. 1988, 1992; note that this has also been reported in man after an intravenous infusion of 500 mg (Swenson & Hughes, 1993)). To our knowledge, effects of AZ on the hypoxic response of the carotid sinus nerve or type I cells are unknown, but the above findings in intact organisms clearly suggest that it has inhibitory effects on both the O2 and CO2 response of the carotid bodies.

Methazolamide (MTZ) is a more liphophilic sulphonamide with an approximately equal KI as AZ for CA II and IV (Maren et al. 1993). MTZ does not seem to affect the magnitude of the cat carotid sinus nerve responses to hypercapnia and hypoxia (Gray, 1971; Ituriaga et al. 1991; Ituriaga et al. 1993). The failure of MTZ to reduce the magnitude of the steady-state hypoxic response of the carotid body in vitro (although it reduced basal chemosensory activity), suggests that full CA activity is not necessary for a normal hypoxic response to occur (Ituriaga et al. 1991, 1993). It also raises the question of whether the inhibiting effects of AZ (see above) may be exerted by a pharmacological action other than inhibition of CA.

Whether, as with AZ, MTZ affects the HVR in intact organisms is unknown. The aims of the present study in cats were to compare the effects of low-dose AZ and MTZ on the HVR and to examine the effect of MTZ in a dose that completely inhibits CA in all body tissues. This could allow us to determine whether the suggestion of an intact hypoxic response in vitro in the absence of (full) CA activity (see above) also applies to the HVR in an intact organism and whether AZ may exert inhibitory effects independent of CA inhibition. If the elimination of the HVR by high-dose AZ (see above) is due to inhibition of one or more CA isoenzymes, a high dose of the more permeable CA inhibitor MTZ should also abolish it. To study the effect of CA inhibition on the HVR in the first group of animals, we administered MTZ in a dose (33 mg kg−1) that would inhibit CA in all body tissues. To compare the effects of low-dose AZ and MTZ, we infused 3 mg kg−1 of both agents in a separate, second group of animals.

Methods

Experiments were performed in 15 adult cats of either sex (body weight, 3.55 ± 0.98 kg) after approval by the Ethical Committee for Animal Experiments of the University of Leiden. The animals were sedated with 10 mg kg−1 ketamine hydrochloride (i.m.). Anaesthesia was induced with 2% sevoflurane in 30% O2 in N2. Both femoral arteries and the right femoral vein were cannulated, 20 mg kg−1 α-chloralose and 100 mg kg−1 urethane were slowly administered intravenously, and the volatile anaesthetic (sevoflurane) was gradually withdrawn. Then an infusion of a α-chloralose–urethane solution was started at a rate 1.0–1.5 mg kg−1 h−1α-chloralose and 5.0–7.5 mg kg−1 h−1 urethane to obtain stable but light anaesthesia (Teppema et al. 1997). The trachea was cannulated at midcervical level and connected to a respiratory circuit. Body temperature was maintained at 37.5°C with a heating pad. After termination of the experiments, the animals were killed humanely with an overdose of pentobarbital.

Tidal volume was measured electronically by integrating airway flow obtained from a pneumotachograph (number 0 flow transducer, Fleisch, Lausanne, Switzerland) connected to a differential pressure transducer (Statham PM 197, Los Angeles, CA, USA). The respiratory fractions of O2 and CO2 were continuously measured with a Datex gas monitor (Multicap, Helsinki, Finland), which was calibrated with gas mixtures of known composition. The inspiratory gas concentrations were made with computer-controlled mass flow controllers (type AFC 260, Bronkhorst High Tec Veenendaal, the Netherlands). End-tidal PCO2 (PET,CO2) and PO2 (PET,O2) were controlled independently using a PC by adjusting the inspiratory gas fractions.

Arterial blood samples were taken from the right femoral artery for blood gas analysis (ABL 700, Radiometer Copenhagen, Denmark). Arterial blood pressure was measured using a Statham pressure transducer (P23ac, Los Angeles, CA, USA). All signals were converted to digital values (sample frequency, 100 Hz), processed by a PC and stored breath-by-breath.

Study design

In the first group of 10 animals (group I), we measured the steady-state ventilatory O2 response at constant PET,CO2 in the control situation. Then, 3 mg kg−1 MTZ (Sigma; dissolved in NaOH, adjusting the pH to about 7.4 with HCl) was infused in a volume of about 5 ml (∼1 ml min−1). About 60 min later, a second isocapnic steady-state hypoxic response curve was determined. After making these measurements, 3 mg kg−1 acetazolamide (Diamox, AHP Pharma, Hoofddorp, the Netherlands; 2 mg ml−1 in saline) was infused and after another 60 min, a third steady-state isocapnic HVR was measured.

After control measurements in the second group of five animals (group II), an isocapnic HVR curve was determined after addition of 3 mg kg−1 MTZ as described above. Then, to obtain complete inhibition of CA in all tissues, these animals were given an additional dose of 30 mg kg−1 MTZ, after which another isocapnic hypoxic reponse curve was determined.

Isocapnic hypoxic ventilatory response

Near step-wise changes in PET,O2 were achieved by adjusting the O2 fraction of the inspired air; the PET,CO2 was kept constant by adjusting the inspired CO2 concentration (Berkenbosch et al. 1991). In this way, a new steady-state level of ventilation has established after 5–6 min (Berkenbosch et al. 1991). The last 20 breaths of this period were averaged to yield steady-state ventilation at a given PET,O2. Blood samples were taken at the end of the steady-state periods to analyse blood gases. Using a least-squares method, inspiratory ventilation V̇I was fitted to arterial PO2 (PaO2) according to the exponential function (Berkenbosch et al. 1991, 1997):

graphic file with name tjp0574-0565-m2.jpg

where G is the overall hypoxic sensitivity (in l min−1), D is a shape parameter (in kPa−1) and A is the ventilation during hyperoxia (in l min−1). None of these three parameters was fixed but all were estimated with the aid of an iteration method. The number of data points was not necessarily equal in all treatments (normally ∼4 points in hyperoxia, ∼2 in normoxia and ∼4–6 in hypoxia). However, the data points that were included were chosen such that within cats the PO2 range over which curve fitting was applied, was the same in all treatments. The maximum duration of uninterrupted hypoxic exposures did not exceed 20 min. The statistical analysis was performed using SPSS v11.0 for Windows (SPSS Inc., Chicago, IL, USA). To detect significant differences between the three treatments (control, AZ and MTZ), we performed a one-way repeated measures ANOVA with post hoc Bonferroni correction. P < 0.017 was considered significant. Unless otherwise indicated, data are presented as means ± s.d.

Results

In the animals of group I (n = 10), the HVRs were measured by forcing the PET,O2levels from hyperoxia to hypoxia resulting in Pa,O2levels that covered the range from 60.5 ± 4.1 to 5.6 ± 1.4 kPa in control, 57.1 ± 5.7 to 5.7 ± 1.0 kPa after application of MTZ and 57.6 ± 7.4 to 5.9 ± 1.2 kPa after application of AZ. The dose of 3 mg kg−1 MTZ did not cause a rise in the mean Pa,CO2 − PET,CO2 gradient, indicating the absence of effective erythrocytic CA inhibition (see Table 1). The addition of 3 mg kg−1 AZ caused a significant increase in the gradient by about 0.3 kPa, probably too small to cause appreciable tissue acidosis and a further rise in hyperoxic ventilation (see Table 1). Table 1 also shows that MTZ caused a mild acidosis that was more pronounced after the subsequent infusion of AZ.

Table 1.

Effects of methazolamide (MTZ) and acetazolamide (AZ) on the steady-state hypoxic ventilatory response in 10 cats

Control MTZ AZ
G (l min−1) 1.93 ± 1.32 1.89 ± 0.90 1.09 ± 0.92*
D (kPa−1) 0.20 ± 0.07 0.22 ± 0.06 0.14 ± 0.06
A (l min−1) 0.86 ± 0.33 1.30 ± 0.401 1.32 ± 0.432
Pa,CO2 (kPa) 4.63 ± 0.75 4.55 ± 0.78 4.78 ± 0.81
Pa,CO2–PET,CO2 (kPa) −0.01 ± 0.30  −0.08 ± 0.38 0.29 ± 0.613
Arterial pH 7.338 ± 0.04 7.307 ± 0.054 7.253 ± 0.055
Base excess (mm) −6.75 ± 1.26  −8.56 ± 1.836 −10.53 ± 1.977
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G, hypoxic sensitivity; D, shape parameter; A, hyperoxic ventilation; Pa,CO2−PET,CO2, arterial–end-tidal PCO2 gradient.

*

P = 0.003 versus control and 0.010 versus MTZ;

1

P = 0.003 versus control;

2

P = 0.002 versus control;

3

P = 0.007 versus control;

4

P = 0.007 versus control;

5

P = 0.000 versus control;

6

P = 0.006 versus control;

7

P = 0.000 versus control.

Blood gas and acid–base data are means of the means per cat.

An example of the effect of both agents on the HVR in one animal is shown in Fig. 1. The overall results, summarized in Table 1, show that the mean control hypoxic sensitivity in the 10 animals studied was not different from that after addition of MTZ (P = 0.88 versus control) while AZ reduced it by 44%. Compared to the control situation, all animals except one had a lower hypoxic sensitivity after AZ administration. The shape parameter D was unaffected by MTZ, while AZ tended to reduce it (P = 0.023 versus control). The hyperoxic ventilation A increased after MTZ but did not rise further after addition of AZ.

Figure 1. Steady-state hypoxic ventilatory response curves in one animal before (▪) and after infusions of methazolamide (MTZ, ▵) and acetazolamide (AZ, ▾).

Figure 1

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Curves are optimal fits (least-squares method) to a mono-exponential equation with residual. Parameter values for G (l min−1), D (kPa−1) and A (l min−1) were 1.52, 0.19 and 1.17 in control, 1.1, 0.13 and 1.28 after addition of MTZ, and 0.68, 0.10 and 1.43 after addition of AZ, respectively. For further explanation see text.

The animals of group II (n = 5) were given 3 mg kg−1 MTZ, followed by 30 mg kg−1. HVRs in these animals were measured by forcing the PET,O2 levels from hyperoxia to hypoxia resulting in Pa,O2levels that covered the range from 58.1 ± 1.1 to 4.9 ± 0.8 kPa in control, 54.4 ± 4.3 to 4.9 ± 0.9 kPa after addition of 3 mg kg−1 MTZ and 56.6 ± 1.5 to 5.0 ± 0.9 kPa after 30 mg kg−1 MTZ. An example is shown in Fig. 2. In Table 2, note the appearance of a large Pa,CO2 − PET,CO2 gradient addition of after 33 mg kg−1, indicating effective inhibition of erythrocytic CA. The scatter diagram of Fig. 3 shows the individual effect of low-dose MTZ in all 15 animals (groups I and II) studied. Note the absence of a systematic increase or decrease of parameter G by MTZ. All five animals of group II showed a substantial response to hypoxia after 33 mg kg−1 MTZ (in three animals G was even larger than in control), which is in sharp contrast to our previous findings after high-dose AZ, when the HVR was totally abolished (Teppema et al. 1988, 1992). Overall, high-dose MTZ did not induce significant changes in hypoxic sensitivity, shape parameter and hyperoxic ventilation (Table 2).

Figure 2. Example of steady-state response curves in one animal in control (□), 3 mg kg−1 methazolamide (MTZ, ▵) and 33 mg kg−1 MTZ (▾).

Figure 2

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Curves are optimal fits (least-squares method) to a mono-exponential equation with residual. Parameter values for G (l min−1), D (kPa−1) and A (l min−1) were 3.99, 0.37 and 0.66 in control, 2.31, 0.31 and 0.45 after addition of low-dose MTZ and 3.70, 0.34 and 0.77 after addition of high-dose MTZ, respectively.

Table 2.

Effects of methazolamide (MTZ) (3 and 33 mg kg−1) on the steady-state hypoxic ventilatory response in five cats

Control MTZ 3 mg kg−1 MTZ 33 mg kg−1
G (l min−1) 3.26 ± 1.44 3.23 ± 1.16 3.00 ± 1.23
D (kPa−1) 0.27 ± 0.07 0.29 ± 0.10 0.27 ± 0.09
A (l min−1) 0.79 ± 0.16 0.59 ± 0.21 0.65 ± 0.21
Pa,CO2 (kPa) 5.75 ± 0.39 5.76 ± 0.31 6.14 ± 0.21
Pa,CO2−PET,CO2 (kPa) 0.40 ± 0.45 0.66 ± 0.72 2.40 ± 0.541
Arterial pH 7.262 ± 0.03 7.239 ± 0.04 7.217 ± 0.02*
Base excess (mm) −7.03 ± 0.88 −8.23 ± 1.27 −8.38 ± 1.09*
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G, hypoxic sensitivity; D, shape parameter; A, hyperoxic ventilation; Pa,CO2−PET,CO2, arterial–end-tidal PCO2 gradient. Note that after addition of 33 mg kg−1 MTZ, the Pa,CO2, pH and base excess represent equilibrium values in vitro; in vivo Pa,CO2 in the blood perfusing the carotid bodies must have been considerably lower and pH considerably higher than the in vitro values shown here.

*

P = 0.012 versus Control;

1

P = 0.000 versus control.

Blood gas and acid–base data are means of the means per cat.

Figure 3. Scatter diagram of the effect of 3 mg kg−1 methazolamide on hypoxic sensitivity G in 15 animals.

Figure 3

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Low-dose MTZ does not cause a systematic change in G.

Discussion

The main findings of this study are that: (1) at a dose (33 mg kg−1) that completely inhibits CA in all body tissues, MTZ did not alter the steady-state HVR in the cat; and (2) in a dose of 3 mg kg−1, the less lipohilic sulphonamide AZ reduced hypoxic sensitivity by 44%, while an equal low dose of MTZ lacked this effect. These results indicate that full inhibition of CA does not reduce the steady-state HVR in the cat and that the depressing effect of AZ may be caused by a pharmacological action other than on CA. Our results extend the observations in an in vitro carotid body preparation from cat, in which the necessity of CA activity for a normal hypoxic response was ruled out (Ituriaga et al. 1991, 1993).

Study design

Full physiological inhibition of CA is reached when 99.99% of the enzyme is inhibited (Maren, 1977). The concentration of CA II in cat erythrocytes is very high (Dodgson & Forster, 1983) providing a possible explanation of why 3 mg kg−1 MTZ did not widen the Pa,CO2 − PET,CO2 gradient. Because red cells contain much more CA than carotid bodies (Maren, 1967), the fractional inhibition of the enzyme after 3 mg kg−1 MTZ (which is evenly distributed) will be larger in the latter. In cat choroid plexus, also an organ with a relatively high CA concentration, this dose has been shown to result in 99.58% inhibition (Vogh, 1980). We thus speculate that in the carotid bodies, the inhibition will then be close to 99.99% if not more, so that a subsequent AZ dose would not exert a physiological effect via inhibition of local CA. Both MTZ and AZ have a high (and about equal) affinity for CA II and IV and there is no known interaction between the two agents (Maren, 1967; Maren et al. 1993). Although in the absence of concrete data we cannot exclude the possibility that the additional AZ dose (group I) could have made the difference between incomplete and full carotid body CA inhibition, our observation of an entirely intact HVR after a MTZ dose that completely inhibits CA in all tissues (Maren, 1967; Vogh, 1980) lends support to the view that the inhibiting effect of AZ in the animals of group 1 must be due to an effect unrelated to CA inhibition. Note that the AZ dose of 3 mg kg−1 was somewhat smaller than the dose previously shown to reduce the HVR (Teppema & Dahan, 2004).

The PET,CO2in group II was controlled at a higher level than in group I, explaining the higher hypoxic sensitivity G in this group (Tables 1 and 2). After complete inhibition of CA, a large Pa,CO2 − PET,CO2 gradient (2–3 kPa) develops, which, at constant PET,CO2, will result in a rise in the in vivo arterial PCO2 (Pa,CO2) unless this is prevented by reducing the PET,CO2 (via the inspired) to a sufficiently low level. Therefore, in order to be able to accomplish this, it was necessary to start the control experiments at a sufficiently high inspired PCO2 level. In an earlier study (Teppema et al. 1995), we estimated that with complete CA inhibition the arterial blood will enter the carotid arteries with a PCO2 that is approximately 0.6 kPa lower than the measured Pa,CO2in vitro at equilibrium. Viewed in this way, we estimate the mean in vivoPa,CO2after high-dose MTZ to be about 5.5 kPa (versus 5.75 and 5.76 kPa in control and after 3 mg kg−1, respectively, see Table 2).

Tables 1 and 2 show that 3 mg kg−1 MTZ caused a decrease in mean pH of 0.031 in group I and 0.023 in group II. This mild acidosis could have counteracted an inhibitory effect of the agent on the HVR. Low-dose AZ, however, causing a similar degree of mild acidosis, clearly reduces hypoxic sensitivity (Wagenaar et al. 1996; Teppema & Dahan, 2004), indicating that MTZ and AZ have different effects on the HVR indeed. In the group I animals, AZ enlarged the MTZ-induced acidosis, but this did not prevent a clear reduction in hypoxic sensitivity. Consequently, AZ must have a potent inhibitory effect on the HVR that is not shared by MTZ.

Effects of MTZ and AZ on the HVR: parameters A, D and G

In both man and animals, the HVR is biphasic, starting with an initial rise in ventilation mediated by the carotid bodies, followed by a secondary decrease called hypoxic ventilatory depression (HVD). HVD is a poorly understood phenomenon; it is related to the magnitude of the initial carotid-body stimulation, but possibly also to an increased washout of CO2 from the brain due to a rise in brain blood flow and/or a release of inhibitory neurotransmitters (Vizek et al. 1987; Dahan et al. 1996; Poulin & Robbins, 1998).

We examined the effects of MTZ and AZ on the steady-state HVR curve, described by the exponential equation given in the Methods, including hypoxic sensitivity G (comprising both the stimulation by the carotid bodies and HVD), shape factor D and hyperoxic ventilation A. Neither low- nor high-dose MTZ changed mean hypoxic sensitivity, while AZ, given after an equal initial dose of MTZ (3 mg kg−1), reduced it by 44%. The hyperoxic ventilation A increased after low- but not high-dose MTZ. AZ tended to reduce parameter D.

A, the ventilation during hyperoxia, is a complex variable and is influenced by the CO2 sensitivity of the central chemoreflex loop, the x-intercept of the CO2 response curve (i.e. apnoeic threshold), the prevailing (arterial and brain stem tissue) PCO2/pH and a small, PCO2-dependent contribution of the carotid bodies to total ventilation. As with AZ (Wagenaar et al. 1996), low-dose MTZ causes a decrease in sensitivity of the central chemoreflex loop resulting in a less-steep CO2 response curve (Bijl et al. 2006) and this would tend to reduce the value of A. At the same time, however, also as with AZ (Wagenaar et al. 1996), it causes a large decrease in the apnoeic threshold (so CO2 response curves before and after MTZ intersect). The influence of the latter effect on A will thus depend on the prevailing PCO2: the lower the PCO2 the higher the tendency for low-dose MTZ to increase A. Therefore, because the experiments in the animals of group II were performed at a background PCO2 considerably higher than in group I, our finding of an unchanged value of A in this group by addition of low-dose MTZ is not necessarily conflicting with the increase in group I.

The physiological significance of the shape parameter D remains unknown and we cannot explain the tendency for AZ to decrease it. In our previous study, we did not find an influence of AZ on D (Teppema & Dahan, 2004).

The most important finding of this study is that neither low- nor high-dose MTZ changed hypoxic sensitivity G. Previously, we ascribed the elimination of the HVR by high-dose AZ solely to a total inhibition of CA isoenzymes in the carotid bodies (Teppema et al. 1988, 1992). Now, however, we may have to reconsider this view because the MTZ dose administered in group II (33 mg kg−1) will inhibit all extracellular membrane-bound CA IV as well as intracellular CA I and II (Maren, 1967, 1977; Vogh, 1980). An action of MTZ other than inhibition of CA is not known to us, so we cannot speculate about a scenario in which complete carotid body CA inhibition by MTZ would abolish the O2 response while at the same time an additional pharmacological action, not shared by AZ, would reverse this. However, the alternative, an action of AZ other than CA inhibition alone that is not shared by MTZ, may open a way to discuss our results against a background of recent studies showing unexpected actions of this agent.

Although there is evidence indicating that AZ has inhibiting effects on the carotid bodies (see Introduction), we will discuss possible different effects of MTZ and AZ on both components of the HVR, namely the initial carotid body-mediated increase in ventilation and the secondary (possibly centrally mediated) decrease (HVD).

Are there different effects of MTZ and AZ on HVD?

A hypoxia-induced release of inhibitory neurotransmitters may be one of the mechanisms that contributes to HVD (Vizek et al. 1987; Poulin & Robbins, 1998). AZ is known to reduce the excitability of neurons that are involved in seizures and as such is used as an anticonvulsant (Maren, 1967; Reiss & Oles, 1996). Changes in extra- and intracellular pH of neurons will influence their excitability, and it is possible that compared to AZ, MTZ, as a result of its higher permeability, may have different effects particularly on intracellular pH of CA-containing neurons. In hippocampal CA3 neurons, CA inhibitors cause intracellular acidosis, and this effect, which is at least partly responsible for their anticonvulsant action, is larger with membrane permeant inhibitors (Leniger et al. 2002). Compared to AZ, MTZ has a superior inhibiting effect on seizures (Maren, 1967; Barnish et al. 1980), and if this were to reflect a general decrease in activity in CA-containing neurons, the agent might be expected to promote rather than reduce HVD (compared to AZ), assuming that these neurons (or some of them) play a role in it.

Possible different effects of MTZ and AZ on the hypoxia-induced increase in cerebral blood flow (CBF) may also result in distinct effects on HVD. AZ has a reputation as a dilator of cerebral vessels, but this effect is clearly dose-dependent and does not seem to be operative with intravenous doses lower than 5 mg kg−1 (Grossmann & Koeberle, 2000). Also, it remains to been seen whether low-dose AZ would alter the hypoxia-induced rise in CBF. In humans, after a usual oral clinical dose this does not seem to be the case (Huang et al. 1988). We are not aware of studies showing a dilating effect of low-dose MTZ on cerebral vessels. At high-doses, CBF will rise by the increase in tissue PCO2 due to inhibition of erythrocytic CA.

Are there different effects of MTZ and AZ on the carotid bodies?

The most likely explanation for our results is a different effect of MTZ and AZ on the carotid bodies. One possibility is that AZ, with its proposed vasodilatory action, increases carotid body blood flow, while MTZ lacks this effect. Recently, Pickkers et al. (2001) showed a vasodilatory effect of AZ on the peripheral circulation (forearm) in humans. Because sulphonamides lacking CA inhibitory effects had a much smaller vasorelaxant effect, the authors argued that the inhibition of a CA isoenzyme results in intracellular alkalosis, followed by opening of large-conductance calcium-dependent potassium (BK) channels. In an in vitro carotid body preparation from cat, the specific BK channel inhibitor charybdotoxin has been reported to induce vasodilatation, indicating that this channel type may be involved in the regulation of carotid blood flow in this species (Osanai et al. 1997). Unfortunately, data on possible effects of a low intravenous dose of AZ and MTZ on the peripheral circulation are lacking. From the available data in the literature we would certainly expect high-dose AZ to cause a large increase in carotid body blood flow. Whether this could explain the elimination of the HVR that we showed previously, remains to be seen (Teppema et al. 1988, 1992). Would high-dose MTZ fail to induce changes in carotid body blood flow?

Possible different effects of MTZ and AZ on type I carotid body cells

Type I cells contain CA isoenzymes of which the precise subcellular locations remain to be elucidated except for the cytosolic isoforms CA II and III (possibly a membrane-bound isoform is also involved Rigual et al. 1985; Nurse, 1990; Botrè et al. 1994; Ridderstråle & Hanson, 1984; Yamamoto et al. 2003). There are ample data to indicate that in the carotid bodies, CA regulates the speed and magnitude of changes in intracellular pH of type I cells upon (removal of) sudden hypercapnic stimuli (Gray, 1971; Black et al. 1971). Hypoxia, however, does not cause a fall in intracellular pH (Ituriaga et al. 1992) and in this respect, and from the perspective of the absence of an effect of MTZ on the hypoxic response of the in vitro carotid body (Ituriaga et al. 1991, 1993), the failure of MTZ to reduce the HVR is not unexpected. Because even at low dose, AZ appears to reduce the HVR, while MTZ, even at high dose, fails to do so, we conclude that the inhibiting effects of AZ on the HVR are not related to inhibition of CA. But what other pharmacological effect of AZ could then explain our observations? Recently, Tricarico et al. (2004) showed a direct, stimulating action of AZ on voltage-sensitive BK channels in muscle cells from K+-depleted rats, an effect that is not shared by MTZ. Whereas the existence of specific oxygen-sensitive potassium channels in the carotid bodies of other species is well documented (e.g. Kv channels in rabbit and mouse, and TASK-1 and BK channels in rat), the information from cat is scarce (Peers & Green, 1991; Perez-Garcia & Lopez-Lopez, 2000; Buckler et al. 2000; Riesco-Fagundo et al. 2001; Perez-Garcia et al. 2004; Williams et al. 2004). In type I cells from adult cats, a role of BK channels in oxygen sensing could not be demonstrated (Chou & Shirahata, 1996). In one report, it was found that a voltage-sensitive potassium current (inhibited by hypoxia) recorded in type I cells from adult cats was insensitive to charybdotoxin (Chou & Shirahata, 1996). Another study in an in vitro perfused carotid body preparation from cat reported a decrease in carotid sinus nerve activity by charybdotoxin during hypoxia (Osanai et al. 1997). Consequently, at this stage it is not possible to say whether our findings may be related to a potential specific effect of AZ on BK, or on other oxygen-sensitive potassium channels. In addition, because AZ also appears to inhibit (directly or indirectly) various types of voltage-sensitive Ca2+ channels (McNaughton et al. 2004) it is also possible that this has contributed to the difference in effect of MTZ and AZ on the HVR.

Summary

In conclusion, we have shown different effects of MTZ and AZ on the steady-state HVR in the cat that in our opinion are best explained by an action of AZ on carotid body blood flow or type I cells that is not related to inhibition of CA. Our data indicate that even at the level of pulmonary ventilation, normal CA activity in the carotid bodies is not a prerequisite for a normal hypoxic response to occur.

References

  1. Barnish IT, Cross PE, Dickinson RP, Gadsby B, Parry MJ, Randall MJ, Sinclair IW. Cerebrovasodilatation through selective inhibition of the enzyme carbonic anhydrase. 2. Imidazo[2,1-b]thiadiazole and imidazo[2,1-b]thiazolesulfonamides. Med Chem. 1980;23:117–121. doi: 10.1021/jm00176a003. [DOI] [PubMed] [Google Scholar]
  2. Berkenbosch A, DeGoede J, Ward DS, Olievier CN, VanHartevelt J. Dynamic response of the peripheral chemoreflex loop to changes in end-tidal O2. J Appl Physiol. 1991;71:1123–1128. doi: 10.1152/jappl.1991.71.3.1123. [DOI] [PubMed] [Google Scholar]
  3. Berkenbosch A, Teppema LJ, Olievier CN, Dahan A. Influences of morphine on the ventilatory response to isocapnic hypoxia. Anesthesiology. 1997;86:1342–1349. doi: 10.1097/00000542-199706000-00016. [DOI] [PubMed] [Google Scholar]
  4. Bijl JHL, Mousavi Gourabi B, Dahan A, Teppema LJ. Effect of low-dose methazolamide on the control of breathing in cats. Adv Exp Med Biol. 2006;580:257–262. doi: 10.1007/0-387-31311-7_40. [DOI] [PubMed] [Google Scholar]
  5. Black AMS, McCloskey DI, Torrance RW. The responses of carotid body chemoreceptors in the cat to sudden cahnges of hypercapnic and hypoxic stimuli. Respir Physiol. 1971;13:36–49. doi: 10.1016/0034-5687(71)90063-6. [DOI] [PubMed] [Google Scholar]
  6. Botrè F, Botrè C, Greco A, Data PG, Di Giulio C, Morelli L. Potentiometric determination of carbonic anhydrase activity in rabbit carotid bodies: comparison among normoxic, hyperoxic and hypoxic animals. Neurosci Lett. 1994;166:126–130. doi: 10.1016/0304-3940(94)90467-7. [DOI] [PubMed] [Google Scholar]
  7. Buckler KJ, Vaughan-Jones RD, Peers C, Nye PC. Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat. J Physiol. 1991;436:107–129. doi: 10.1113/jphysiol.1991.sp018542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buckler KJ, Williams BA, Honore E. An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells. J Physiol. 2000;525:135–142. doi: 10.1111/j.1469-7793.2000.00135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chegwidden WR, Carter ND. Introduction to the carbonic anhydrases. In: Chegwidden WR, Carter ND, Edward YH, editors. The Carbonic Anhydrases. New Horizons. Basel: Birkhauser Verlag; 2000. pp. 13–28. [Google Scholar]
  10. Chou CL, Shirahata M. Two types of voltage-gated K channels in carotid body cells of adult cats. Brain Res. 1996;742:34–42. doi: 10.1016/s0006-8993(96)00987-0. [DOI] [PubMed] [Google Scholar]
  11. Dahan A, Ward DS, Van den Elsen M, Temp J, Berkenbosch A. Influence of reduced carotid body drive during sustained hypoxia on hypoxic depression of ventilation in humans. J Appl Physiol. 1996;81:565–572. doi: 10.1152/jappl.1996.81.2.565. [DOI] [PubMed] [Google Scholar]
  12. Dodgson SJ, Forster RE. Carbonic anhydrase activity of intact erythrocytes from seven mammals. J Appl Physiol. 1983;55:1292–1298. doi: 10.1152/jappl.1983.55.4.1292. [DOI] [PubMed] [Google Scholar]
  13. Gray BA. On the speed of the carotid chemoreceptor response in relation to the kinetics of CO2 hydration. Respir Physiol. 1971;11:235–246. doi: 10.1016/0034-5687(71)90027-2. [DOI] [PubMed] [Google Scholar]
  14. Grossmann WM, Koeberle B. The dose–response relationship of acetazolamide on the cerebral blood flow in normal subjects. Cerebrovasc Dis. 2000;10:65–69. doi: 10.1159/000016027. [DOI] [PubMed] [Google Scholar]
  15. Hayes MW, Maini BK, Torrance RW. Reduction of the reponses of carotid chemoreceptors by acetazolamide. In: Paintal AS, editor. Morphology and Mechanisms of Arterial Chemoreceptors. Delhi: Vallabhbhai Petel Chest Institute University; 1976. pp. 36–47. [Google Scholar]
  16. Huang SY, McCullough RE, McCullough RG, Micco AJ, Maco-Johnson J, Weil JV, Reeves JT. Usual clinical dose of acetazaolamide does not alter cerebral blood flow velocity. Respir Physiol. 1988;72:315–326. doi: 10.1016/0034-5687(88)90090-4. [DOI] [PubMed] [Google Scholar]
  17. Ituriaga R, Lahiri S, Mokashi A. Carbonic anhydrase and chemoreception in the carotid body. Am J Physiol. 1991;265:C565–C573. doi: 10.1152/ajpcell.1991.261.4.C565. [DOI] [PubMed] [Google Scholar]
  18. Ituriaga R, Rumsey WL, Lahiri S, Spergel D, Wilson DF. Intracellular pH and oxygen chemoreception in the cat carotid body in vitro. J Appl Physiol. 1992;72:2259–2266. doi: 10.1152/jappl.1992.72.6.2259. [DOI] [PubMed] [Google Scholar]
  19. Iturriaga R, Mokashi A, Lahiri S. Dynamics of carotid body responses in vitro in the presence of CO2-HCO3−: role of carbonic anhydrase. J Appl Physiol. 1993;75:1587–1594. doi: 10.1152/jappl.1993.75.4.1587. [DOI] [PubMed] [Google Scholar]
  20. Leniger T, Wiemann N, Bingmann D, Widman G, Hufnagel A, Bonnet U. Carbonic anhydrase inhibitor Sulthiame reduces intracellular pH and epileptoform activity of hippocampal CA3 neurons. Epilepsia. 2002;43:469–474. doi: 10.1046/j.1528-1157.2002.32601.x. [DOI] [PubMed] [Google Scholar]
  21. McNaughton NCL, Davies CH, Randall A. Inhibition of alpha(1E) Ca2+ channels by carbonic anhydrase inhibitors. J Pharmacol Sci. 2004;95:240–247. doi: 10.1254/jphs.fp0040032. [DOI] [PubMed] [Google Scholar]
  22. Maren TH. Carbonic anhydrase: chemistry, physiology and inhibition. Physiol Rev. 1967;47:595–761. doi: 10.1152/physrev.1967.47.4.595. [DOI] [PubMed] [Google Scholar]
  23. Maren TH. Use of inhibitors in physiological studies of carbonic anhydrase. Am J Physiol. 1977;232:F291–F297. doi: 10.1152/ajprenal.1977.232.4.F291. [DOI] [PubMed] [Google Scholar]
  24. Maren TH, Wynns GC, Wistrand PJ. Chemical properties of carbonic anhydrase IV, the membrane-bound enzyme. Mol Pharmacol. 1993;44:901–905. [PubMed] [Google Scholar]
  25. Nurse CA. Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat. Cell Tissue Res. 1990;261:65–71. doi: 10.1007/BF00329439. [DOI] [PubMed] [Google Scholar]
  26. Osanai S, Buerk DG, Mokashi A, Chugh DK, Lahiri S. Cat carotid body discharge (in vitro) is insensitive to charybdotoxin. Brain Res. 1997;747:324–327. doi: 10.1016/s0006-8993(96)01313-3. [DOI] [PubMed] [Google Scholar]
  27. Peers C, Green FK. Inhibition of Ca2+-activated K+ currents by intracellular acidosis in isolated type I cells of the neonatal rat carotid body. J Physiol. 1991;437:589–602. doi: 10.1113/jphysiol.1991.sp018613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Perez-Garcia MT, Colinas O, Miguel-Velado E, Moreno Dominguez A, Lopez-Lopez JR. Characterisation of the Kv channels of mouse carotid body chemoreceptors cells and their role in oxygen sensing. J Physiol. 2004;557:457–471. doi: 10.1113/jphysiol.2004.062281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perez-Garcia MT, Lopez-Lopez JR. Are Kv channels the essence of O2 sensing? Circ Res. 2000;86:490–491. doi: 10.1161/01.res.86.5.490. [DOI] [PubMed] [Google Scholar]
  30. Pickkers P, Hughes AD, Russel F, Thien T, Smits P. In vivo evidence for KCa channel opening properties of acetazolamide in the human vasculature. Br J Pharmacol. 2001;132:443–450. doi: 10.1038/sj.bjp.0703825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Poulin MJ, Robbins PA. Influence of cerebral blood flow on the ventilatory response to hypoxia in humans. Exp Physiol. 1998;83:95–106. doi: 10.1113/expphysiol.1998.sp004095. [DOI] [PubMed] [Google Scholar]
  32. Reiss WG, Oles KS. Acetazolamide in the treatment of seizures. Ann Pharmacother. 1996;30:514–519. doi: 10.1177/106002809603000515. [DOI] [PubMed] [Google Scholar]
  33. Ridderstråle Y, Hanson MA. Histochemical localization of carbonic anhydrase in the cat carotid body. Ann NY Acad Sci. 1984;429:398–400. doi: 10.1111/j.1749-6632.1984.tb12363.x. [DOI] [PubMed] [Google Scholar]
  34. Riesco-Fagundo AM, Perez-Garcia MT, Gonzalez C, Lopez-Lopez JR. O2 modulates large-conductance Ca2+-dependent K+ channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism. Circ Res. 2001;89:430–436. doi: 10.1161/hh1701.095632. [DOI] [PubMed] [Google Scholar]
  35. Rigual R, Iñiguez C, Carreres J, Gonzalez C. Carbonic anhydrase in the carotid body and the carotid sinus nerve. Histochemistry. 1985;82:577–580. doi: 10.1007/BF00489979. [DOI] [PubMed] [Google Scholar]
  36. Rigual R, Lopez-Lopez JR, Gonzalez C. Release of dopamine and chemoreceptor discharge induced by low pH and high PCO2 stimulation of the cat carotid body. J Physiol. 1991;433:519–531. doi: 10.1113/jphysiol.1991.sp018441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Swenson ER, Hughes JM. Effects of acute and chronic acetazolamide on resting ventilation and ventilatory responses in men. J Appl Physiol. 1993;74:230–237. doi: 10.1152/jappl.1993.74.1.230. [DOI] [PubMed] [Google Scholar]
  38. Teppema L, Berkenbosch A, Olievier CN. Effect of Nω-nitro-L-arginine on ventilatory responses to hypercapnia in anesthetized cats. J Appl Physiol. 1997;82:292–297. doi: 10.1152/jappl.1997.82.1.292. [DOI] [PubMed] [Google Scholar]
  39. Teppema LJ, Berkenbosch A, De Goede J, Olievier C. Carbonic anhydrase and control of breathing: different effect of benzolamide and methazolamide in the anaesthetized cat. J Physiol. 1995;488:767–777. doi: 10.1113/jphysiol.1995.sp021008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Teppema LJ, Bijl H, Romberg RR, Dahan A. Antioxidants reverse depression of the hypoxic ventilatory response by acetazolamide in man. J Physiol. 2006;572:849–856. doi: 10.1113/jphysiol.2005.104174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Teppema LJ, Dahan A. Low-dose acetazolamide reduces the hypoxic ventilatory response in the anesthetized cat. Respir Physiol Neurobiol. 2004;140:43–51. doi: 10.1016/j.resp.2004.01.001. [DOI] [PubMed] [Google Scholar]
  42. Teppema LJ, Dahan A, Olievier CN. Low-dose acetazolamide reduces CO2–O2 stimulus interaction within the peripheral chemoreceptors in the anaesthetised cat. J Physiol. 2001;537:221–229. doi: 10.1111/j.1469-7793.2001.0221k.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Teppema LJ, Rochette F, Demedts M. Ventilatory effects of carbonic anhydrase inhibition in cats: effects of acetazolamide in intact vs. peripherally chemodenervated animals. Respir Physiol. 1988;74:373–382. doi: 10.1016/0034-5687(88)90044-8. [DOI] [PubMed] [Google Scholar]
  44. Teppema LJ, Rochette F, Demedts M. Ventilatory effects of acetazolamide in cats during hypoxemia. J Appl Physiol. 1992;72:1717–1723. doi: 10.1152/jappl.1992.72.5.1717. [DOI] [PubMed] [Google Scholar]
  45. Travis DM. Molecular CO2 is inert on carotid chemoreceptor: demonstration by inhibition of carbonic anhydrase. J Pharmacol Exp Ther. 1971;178:529–540. [PubMed] [Google Scholar]
  46. Tricarico D, Barbieri M, Mele A, Carbonara G, Camerino DC. Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats. FASEB J. 2004;18:760–761. doi: 10.1096/fj.03-0722fje. [DOI] [PubMed] [Google Scholar]
  47. Vizek M, Picket CV, Weil JV. Biphasic ventilatory response to hypoxia has central origin. J Appl Physiol. 1987;93:1658–1664. doi: 10.1152/jappl.1987.63.4.1658. [DOI] [PubMed] [Google Scholar]
  48. Vogh B. The relation of choroid plexus carbonic anhydrase activity to cerebral spinal fluid formation: study of three inhibitors in cat with extrapolation to man. J Pharmacol Exp Ther. 1980;213:321–331. [PubMed] [Google Scholar]
  49. Wagenaar M, Teppema L, Berkenbosch A, Olievier C, Folgering H. The effect of low-dose acetazolamide on the ventilatory CO2 response curve in the anaesthetized cat. J Physiol. 1996;495:227–237. doi: 10.1113/jphysiol.1996.sp021587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Williams SE, Wootton P, Mason MS, Bould J, Iles DE, Riccardi D, Peers C, Kemp PJ. Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science. 2004;306:2093–2097. doi: 10.1126/science.1105010. [DOI] [PubMed] [Google Scholar]
  51. Yamamoto Y, Fujimura M, Nishita T, Nishijima K, Atoji Y, Suzuki Y. Immunohistochemical localization of carbonic anhydrase isozymes in the rat carotid body. J Anat. 2003;202:573–577. doi: 10.1046/j.1469-7580.2003.00191.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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