Influence of potassium on the respiratory response to changes in CSF pH

Resp~rut~o~~hys~oZogy(1970) 8, 177490;

I~L~N~E

forth-polkas

Publishing Company, Amsterdam

OF POTASS~~ ON THE ASPIRATORY CHANGES IN CSF pH

J. BERNDT, W.

RESPONSE

TO

BERGER AND IL BERGER

Institut fiir Physio~og~eder R~hr-~~~~ers~t~t,463 Bochu~, Germany

Abstract. The ventral surface of the medulla oblongata

of anesthetized cats was superfused with mock CSF of different K+ concen~ation and pH. The effects of the potassium concentration on the respiratory response to pH changes were studied. 50.0 meq/l K+ in the superfusing fluid cause respiratory arrest. This effect does not depend on the pH of the mock CSF. 15.0 meq/l Kf stimulate respiration. This excitatory action is strong at alkaline mock CSF pH (pH 7.6) and less pronounced at acid values (pH 7.0). Thus the differences between VT or 9~ at acid and at alkaline CSF pH values are diminished and as a consequence the respiratory sensitivity to changes in the CSF pH is reduced, when [K+]CSF is elevated to 15.0 meqjl. The stimulatory effect of 15.0 meq/l K+ on respiration and the depressant effect on the respiratory sensitivity to ~&SF changes are not abolished by a simultaneous increase of [ca++]CSF. No significant influence on respiration is observed during superfusion with fluids of decreased K+ content (1.5 and 0.5 meq/l). Removal of potassium from the mock CSF is followed by diminished respiration. The possible mechanisms involved in the interaction of hydrogen and potassium ions in the stimulation of respiration are discussed. Control of breathing

Central chemoreceptors Cerebrospinal fluid

In a previous communication, BERNDT, FENNER and BERGER (1969) reported that the CSF calcium concentration influences the respiratory reactions to changes in the CSF pH. Calcium effects on respiration were not counteracted by magnesium ions and the respiratory irregularity following total removal of calcium from the CSF was prevented by adding magnesium to the Ca’ ‘-free fluids. It was concluded that the nervous structure exposed to the different calcium concentrations was a nerve fibre or a receptor cell rather than a superficial cholinergic synapse. More impressive effects on respiration than those observed during application of Accepted for publication 6 August 1969. 177

178

J.BERNDT, W. BERGER AND K. BERGER

different calcium concentrations have been reported when increased amounts of potassium are introduced into the CSF spaces of the brain (SMOLIK, 1943; LEUSEN, 1949). Since these early investigations were performed by an injection or a ventriculocisternal perfusion technique, the action of the introduced potassium ions was not limited to well defined areas of the cerebral surface. Thus the interpretation of results is somewhat dificult. The present experiments have been undertaken to study the influence of the CSF potassium concentration on the respiratory reactions to altered CSF pH. The methods employed were designed to confine the induced changes of the CSF composition to the ventral and ventro-lateral surface of the medulla oblongata, a region which includes the chemosensitive areas described by MITCHELL etal.(1963) and SCHLAEFKE and LOESCHCKE(~~~~).

Methods The methods employed have been described in detail by BERNDT et al. (1969). The ventral surface of the medulla oblongata of anesthetized cats (2.19-3.86 kg; 40 mg/kg chloralose, 200 mg/kg urethane) was superfused with mock CSF of different ionic compositions (table 1). The superfusing fluids were kept in a water-bath at 37 “C and equilibrated with 6 % CO, in air. CaCl, was added to the solutions after at least one hour of equilibration. The pH of the solutions was measured immediately before starting the superfusion with a thermostated glass electrode calibrated against Soerensen phosphate buffers. There were two solutions for any one potassium conTABLE 1 Composition of superfusion fluids. pH values during equilibration with 6% CO2 in air. The fluids containing 5 meq/l potassium served as reference in the experiments and are referred to as “reference solutions” in the text. Cat+ meq/l __.--.._ 1.5 1.5 1.5 1.5 4.5 4.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

K+ meqjl 50.0 50.0 15.0 15.0 15.0 15.0 5.0 5.0 1.5 1.5 0.5 0.5 0.0 0.0

Na+ meq/l ---108.5 100.0 143.4 135.0 139.4 130.9 153.4 144.9 156.9 148.4 157.9 149.4 158.4 149.9

HCO, meq,‘l

Cl-

meq/l

PH range

42.0 9.5 42.0 9.5 42.0 9.5 42.0 9.5 42.0 9.5 42.0 9.5 42.0 9.5

118.0 142.0 117.9 142.0 116.9 140.9 117.9 141.9 117.9 141.9 117.9 141.9 117.9 141.9

7.532-7.599 6.930-7.026 7.520-7.622 6.939-7.010 7.501-7.553 6.935.-7.002 7.529-7.615 6.934-7.017 7.552-7.585 6.940-6.970 7.545-7.571 6.944-6.963 7.554-7.626 6.955-6.996

EFFECTSOF CSF H+ ANDK+ ON RESPIRATION

179

centration applied, one at about pH 7.0 and the other at about pH 7.6. The superfused area located between foramen occipitale and pons was approached by suboccipital oraniotomy. Superfusion (6-30 ml/min) was performed by gravity flow through a tube placed over the caudal part of the operated field; the fluids were removed by suction through a T tube inserted under the bone at the level of the pons. The animals breathed a mixture of oxygen (94-100 %) and carbon dioxide (O-6 %), the latter being adjusted manually in order to keep the end-tidal pC0, constant throughout the experiment. The experiments were carried out under iso-pC0, conditions at an end-tidal pCOZ which varied from experiment to experiment between 28 and 42 torr. The variation in a single experiment was not more than If: 1.0 torr. Femoral arterial blood pressure (strain gauge), end-tidal pC0, (infrared analyzer) and tidal volume (KROGH spirometer) were recorded by means of a direct recorder. The results described below were obtained during the steady state of each superfusing period (usually after three to four minutes of superfusion). When major changes in the mock CSF pH were induced (i.e. from 7.0 to 7.6 pH) 10 to 15 min might elapse before a new steady state could be reached. Since the responses of the respiratory frequency were small and not systematic, only VT and VE evaluations are presented. Results

EFFECTSOF CHANGES IN CSF pH ON RESPIRATION AT [K+],-sF 5.0 meq/l The effects of changes in the CSF hydrogen ion concentration on respiration are demonstrated in fig. 1. The arbitrary pH response curves of VE and VT are only an approximation of the actual relationship between VE or VT, and CSF pH. They have been calculated as linear regression lines from the data obtained from all experiments in this study during superfusion with reference fluids. The mean slopes of these curves which are regarded as a measure of the sensitivity of the respiratory system to changes in the CSF pH, approach the values reported by BERNDTet al. (1969) which were obtained under similar conditions (table 2). EFFECTS OF ALTEREDCSF POTASSIUM CONCENTRATION [K+lcsP 50 meq/l Superfusion of the ventral surface of the medulla with fluids containing 50 meq/l K+ was usually followed by a slowly developing respiratory arrest. The phase of respiratory depression was usually preceded by increased tidal volume and respiratory frequency. The effect resembles the respiratory arrest evoked by local cooling of a small area rostra1 to the roots of the hypoglossal nerves as described by SCHLAEFKE and LOESCHCKE (1967). Respiration was reestablished after replacing these fluids by reference solutions. Respiratory arrest occurred during superfusion with alkaline as well as with acid fluids containing 50 meq/l potassium, but it developed slightly faster at high pH values (pH 7.6) than at low values (7.0 pH). The effects on respiration were accompanied by bradycardia and an increase in blood pressure. Seven of the

J. BERNDT,

0+

6.8

IV. BERGER

AND K. BERGER

-I.0

1,

MOCK

CSF

2.6

pH

?A

LO

MOCK

CSF

7.6

pH

Fig. 1. Respiratory effect of a change in CSF pH at constant CSF potassium content. Each circle represents the mean from three to four superfusion periods during a single experiment. Points: mean values of the group of data & S.D. of pH (horizontal bars) 3 SD. of VT or ?E (vertical bars). The straight lines are arbitrarily linear pH response curves of VT and 9~ calculated from the data plotted in each graph. nine animals studied under these con~~ons reacted as described above (fig. 2) whereas two animals showed increased tidal volume and ventilation. In all cats a less

elevated [KilCsF brings about hyperventilation (see below). The fact that two cats continued to react with steady state hyperventilation even at a [K”JCsF of 50 meq/l implies that this concentration is not enough to cause respiratory arrest with absolute certainty. [K”fCsF 15.0 meq/l During superfusion with fluids containing 15.0 meq/l K+, both tidal volume and ventilation were increased above the reference values (fig. 3). In the alkaline pH range, the increase amounted to about 100 per cent of the tidal volume and ventilation obtained during superfusion with reference fluids of similar pH, while at acid pH values a slighter but still significant effect was observed. In two cases among nine the excitatory effect of these solutions near pH 7.6 was strong enough to produce higher tidal volume and ventiIation values than in the acid range at same elevated [K’], thus reversing the respiratory sensitivity to CSF pH changes. Seven animals only showed a markedly reduced difference between tidal volume or ventilation near

EFFECTS

OF

CSF H+

AND

K+

ON RESPIRATION

181

TABLE 2 Slopes of pH-response

curves of VT and VE at constant (*

1 torr) PACO,.

AVT apH (ml . PH-9

-69.2

- 68.0*

Airy apH

-1583

- 1835*

* Values reported by

(ml. min-l .pH-r)

BERNDT et al.

(1969).

Fig. 2. Respiratory arrest caused by superfusion of the medulla with fluid containing 50.0 meq/l K+. The arrows indicate the instant of fluid exchange. A: During superfusion with reference mock CSF (5.0 meq/l K+, pH 6.936) respiration is regular and deep (expiration upwards). B: Superfusion with mock CSF of high potassium content (50.0 meq/l K+, pH 6.938) causes a slight transitory increase of VT. Then both VT and f slowly decrease. Resipratory arrest in expiratory position is developed after 170 seconds. C: Replacing fluid B by reference solution restablishes respiration after a period of 75 set during which only minute respiratory movements occur.

pH 7.6 and pH 7.0. In most cases bradycardia and an increase of the arterial blood pressure accompanied the respiratory reactions. The depressing effect on the respiratory sensitivity to changes in the CSF pH was not abolished when the three-fold increase of the CSF potassium content was accompanied by a simultaneous increase of the CSF calcium concentration from 1.5 to 4.5 meq/l (fig. 4). In the alkaline range, the excitatory action on respiration was even stronger while in the acid range both tidal volume and ventilation remained unchanged. The mean absolute and relative effects on respiration of a three-fold augmented

182

J. BERNDT,

w. BERGER

AND

K. BERGER

EFFECTS OF

CSF H+

AND K+

ON RESPfRATlON

183

184

J.

BERNDT,W. BERGERAND

K. BERGER

CSF K’ content without and with simultaneous increase of the calcium concentration are demonstrated in table 3. [K”lCsF 1.5, 0.5, or 0.0 meq/l A reduction in the CSF potassium content to 1.5 or 0.5 me@ did not visibly affect respiration. However, total removal of potassium from the CSF was followed by a slight but significant depression of VT and VE (fig. 5 and table 4), while the arterial blood pressure remained unchanged. The action of K+-free solutions on respiration was similar in both the alkaline and the acid pH range. Thus the respiratory sensitivity to alterations of CSF pH was not distinctly affected. The effects of the CSF potassium concentration on the respiratory sensitivity to changes in CSF pH are summarized in fig. 6. The distance between each two curves of either VT/VT~ or VE/VE~is a measure of the pH effect on respiration.

The effects of changes in the CSF potassium concentration have been studied by several authors. HILAROWICZand SZAJNA(1928) observed in dogs an increase in the respiratory frequency and tidal volume after intracisternal injection of KC1 solutions (2-3 ml, 1%). An extensive study of the peripheral and central effects of potassium on respiration and circulation in cats is reported by VONEULER(1938). Intracisternal injection of KC1 (0.5-2.0 mg) was followed by only slight respiratory reactions, while the effects on circulation (increase of arterial blood pressure) were more pronounced. The effects on blood pressure could be abolished by simultaneous injection of CaCI, “in small doses”. SMOLIK(1943) investigated the effects of potassium phosphate applied intracisternally on the blood pressure during hemorrhage and shock. He described “and increase in the rate and amplitude of respiration” following an injection of 0.4 to 0.5 ml of a & molar solution of K2HP04 and KH&‘O,, which is especially remarkable since his solutions had a rather alkaline pH of about 7.6. LEUSEN(1949) and VERSTRAETEN (1950) employed a ventriculo-cisternal perfusion te~h~que in dogs. They observed stimulation of respiration when the amount of KC1 in the perfusing solutions was increased. VERSTRAETEN reports that fluids containing 1 g/l KC1 (which was a four-fold increase of his reference concentration) caused an increase of both the respiratory frequency and tidal volume, while the removal of potassium from the mock CSF had no effect on respiration. The effects of an excess of potassium were neutralized by an excess of calcium. All solutions employed by VERSTRAETEN have a pH between pH 7.3 and 7.4. The discrepancy between the results presented in this study and the observations of earlier authors concerns two main points. The first is that in most cases much higher concentrations than those applied in the described superfusion system were necessary to produce a stimulation of respiration. The same concentrations would have caused respiratory arrest in the present experiments. The second point is the neutralizing effect of excess calcium on the action of potassium, as reported by VON EULERand by VERSTR~TEN.

-

Relative difference

l.--

Relative difference: Reference values = 1.

N=8

10.006 10.007

Difference P

7.594 7.531

1.5 4.5

5.0 15.0

< 0.005

+ 1.564

140.2 < 0.005

25.7 65.9

10.904

+24.6

27.2 51.8

VT ml

P

i 0.008 10.011

SE.

Difference

7.559 7.551

pH

Relative difference

1.5 1.5

meq/t

CSF Ca++

l-j=9

15.0

5.0

Mock K’ meq/l

-

18.5

10.9 7tr8.9

k5.4

k3.1 f5.8

S.E.

+2.596

11119 < 0.01

431 1550

+469 < 0.001 +1.178

398 867

ml/mm

VE

6.979 6.918

6.983 6.969

pH

__~_.__.~_

5300

*55 & 342

+72

139 zt66

SE.

_

iO.008 & 0.007

10.014 & 0.008

SE.

10.0

69.8 69.8

-t&2 < 0.05 -t-O.118

69.3 77.5

ml

VT

il.3

“ro.010

1-14

1355 1369

l 5.0 14.3

1381 1554 $173 i 0.05 i-o.125

-.

ml!min

VE

h3.1

f7.0 h7.0

S.E.

&28

1160 &143

169

&206 j193

S.E.

Respiratory effects of a three-fold increase of the CSF [KC] at different CSF pH values. pH, VT and VE values are mean values + SE. from the indicated number of experiments.

TABLE 3

186

J. BERNDT, W. BERGER AND K. BERGER

Difference P Relative difference

N=8

10.006 10.009

Relative difference: reference values = 1.

1.594 7.592

5.0 0.0

-0.101

-2.6 < 0.01

25.7 23.1 ho.6

hO.9 51.3

- 0.093

-40 < 0.01

431 391 ill

&55 riz57

6.979 6.974

-+O.OOS &to.006

-0.138

-9.6 < 0.01

69.8 60.2

k2.5

k5.0 h3.5

-0.190

-257 < 0.01

1355 1048

-

zt69

5160 1102

Respiratory effects of the removal of potassium from the CSF at different pH values. pH, VT and ‘?E values are mean values from the indicated number of experiments. _.-.._ -__.-_ _.-_____I___ Mock CSF SE. VT SE. VE SE. pH SE. VT SE. VE S.E. K’ pH meq/t ml ml/min mlimin ml __

TABLE 4

188

J. BERNDT, W. BERGER AND K. BERGER

“0

+ VT/VT,,: SE {

VE/V&S.E,

0-1’5 MOCK

CSF [K+]

““’ \ 5.0

15.0

5010

meqll

Fig. 6. Relative respiratory effects of CSF K+ at two different CSF pH values. Ratio: VT or reference [K+] and alkaline pH = 1.

?E

at

These differences in results are possibly explained by the differences in methods. Intracisternally injected electrolytes will mix with the genuine CSF and the concentration at their site of action will differ from the concentration in the injected solution. In addition they will reach different regions of the brain stem and cerebellar surface and thus influence different parts of the central nervous system. This is also a disadvantage of the ventriculo-cisternal perfusion technique. The ventricular surface reached by substances administered to the lateral ventricles and withdrawn from the cisterna magna will be even larger. Thus the results obtained from the superfusion of a well defined area of the brain with solutions of known electrolyte composition at their site of action may not be easily compared with the earlier observations described above. Some additional information is gained from the simultaneous evaluation of pH effects on respiration. Thus, our results show that the CSF potassium concentration influences not only the respiratory level but also the sensitivity to changes in the CSF pH. An analogous observation has been described by BERNDT et al. (1969) for the CSF calcium effects on respiration. Certainly all excitable cells with a membrane potential depending on the ratio of extracellular/intracellular potassium will alter their excitability when either the intracellular or the extracellular potassium content is changed. Each of the nervous

EFFECTSOF CSF H+ AND K+ ON RESPIRATION elements

exposed to the CSF may therefore

be responsible

for the respiratory

189 reactions

to an altered CSF composition. The question arises as to whether or not potassium ions may enter the adjacent brain tissue from the CSF, and approach the neurones of the respiratory centre. CSERR (1965), BRADBURY and DAVSON (1965), FENCL, MILLER and PAPPENHEIMER (1966), and KUFFLER (1968) suggest that free ionic exchange between the CSF and the brain extracellular fluid is possible. On the other hand, the concentration of potassium in the CSF and the brain extracellular fluid is regulated by a mechanism which seems to involve active transport since it can be blocked by ouabain (CAMERON, 1967). Thus excess potassium ions entering the extracellular space from the CSF will partially be removed by this mechanism and not easily reach deeper regions of the brain tissue. But at least superficial substrates may be able to participate in changes of the CSF composition. This is in good agreement with observations reported by SCHLAEFKE and LOESCHCKE (1967, 1968) and TROUTH (1969) who localized structures on the ventral and ventrolateral surface of the medulla of cats which mediate an influence on respiration exerted by chemical, electrical, and thermal stimuli. It is possibly these structures which react directly to the CSF ions. When the interactions of potassium and hydrogen ions are considered, a striking contradiction must be stated. On the one hand, the potassium effect on respiration is stronger at alkaline than at acid pH values. This is in good agreement with earlier observations. CORABOEUF and NIEDERGERKE (1953) demonstrated at the Ranvier node of frog nerve fibres a decrease of excitability (increase of the rheobase) with increasing extracellular hydrogen ion concentration. STRAUB (1956) described the influence of extracellular pH on the resting potential of myelinated nerve fibres. The latter increased with decreasing pH (0.5 mV/pH unit). Finally, DETTBARN and STAEMPFLI (1957) showed that the potassium-induced depolarization of frog nerve fibres is larger at alkaline than at acid extracellular pH. These observations caused SHANES (1958) to classify hydrogen ions among the stabilizing agents at excitable membranes. On the other hand, an increase in [H+] cSF which diminishes the respiratory effects of an elevated potassium concentration, causes hyperventilation at constant [K+]cSF. If in the latter case hyperventilation is the result of an enhanced stream of nerve impulses caused by the elevated hydrogen ion concentration, different sites of action of CSF ions on respiration should exist. Among them at least one should be endowed with membrane properties different from those described by the authors cited above. A suggestion how such a specialized membrane could work, is obtained from HAGIWARA et al. (1968), who studied the influence of external pH on the potassium and chloride conductances of Balanus nubilus muscle fibres. Between pH 10 and 5 the well known relationship was observed: the membrane resistance increased with decreasing pH. Below pH 5, however, a reverse relationship was obtained. The authors explained these findings by the amphoteric character of certain membrane constituents which are responsible for the Cl- conductance. The estimated pK of these amphoteric groups would then lie near 4.0.

J. BERNDT, W. BERGER AND K. BERGER

190

A membrane which shows a similar reverse reaction at more physiological pH values, i.e. whose amphoteric groups governing the anion movements through the membrane have a pK above 7.0 would probably supply an explanation for the “paradoxical” effects of CSF hydrogen ions on respiration. References BERNDT,J., A. FENNERand K. BERGER(1969). Influence of calcium and magnesium on therespiratory response to changes in CSF pH. Respir. Physiol. 7: 216-229. BRADBURY,M. W. B. and H. DAVSON(1965). The transport of potassium between blood, cerebrospinal fluid and brain. J. Physiol. (London) 181: 151-174. CAMERON,I. R. (1967). The respiratory response to injection of ouabain into the cerebral ventricles. Respir. Physiol.

3 : 55-63.

CORABOEUF,E. and R. NIEDERGERKE(1953). Kohlensgure- und pH-Wirkung an der markhaltigen Einzelfaser des Frosches. Ppiigers Arch. ges. Physiol. 258: 103-107. CSERR, H. (1965). Potassium exchange between cerebrospinal fluid, plasma, and brain. Am. J. Physiol.

209: 1219-1226.

DETTBARN,W.-D. and R. STAEMPFLI (1957). Untersuchungen iiber die pH-Wirkung auf das Membranpotential markhaltiger Nervenfasern. He/u. physiol. Acta 15: C 16-C 17. EULER, U. S. VON (1938). Reflektorische und zentrale Wirkungen von Kaliumionen auf Blutdruck und Atmung. Acta Physiol. Scand. 80: 94-122. FENCL, V., T. B. MILLER and J. R. PAPPENHEIMER (1966). Studies on the respiratory response to disturbances of acid-base balance, with deductions concerning the ionic composition of cerebral interstitial fluid. Am. J. Physiol. 210: 459-472. ~GIWARA, S., R. GRUENER,H. HAYASHI,H. SAKATAand A. D. GRINNELL(1968). Effect of external and internal pH changes on K and Cl conductances in the muscle fibre membrane of a giant barnacle. J. gen. Physiol. 52: 113-192. HILAROWICZ,H. and M. SZAJNA(1928). uber den Einlluss der subduralen Darreichung von Kaliumchlorid auf die Vasomotoren-, Herzhemmungs- und Atemzentren. Z. ges. exp. Med. 64: 772-786. KUFFLER, S. W. (1968). Studies on the physiology of neuroglia cells. Proc. Int. Un. Physiol. Sci. VII: 79. LEUSEN,I. (1949). The influence of calcium, potassium and magnesium ions in cerebrospinal fluid on vasomotor system. J. Physiol. (London) 110: 319-329. MITCHELL,R. A., H. H. LQESCHCKE,W. H. MASSIONand J. W. SEVERINGHALJS (1963). Respiratory responses mediated through superficial chemosensitive areas on the medulla. J. Appl. Physiol. 18: 523-533.

SCHLAEFKE,M. and H. H. LOESCHCKE(1967). Lokalisation eines an der Regulation von Atmung und Kreislauf beteiligten Gebietes an der ventralen Oberfllche der Medulla oblongata durch KPlteblockade. Pfliigers Arch. ges. Physiol. 297: 201-220. SCHLAEFKE,M. and H. H. LOESCHCKE (1968). Ventilatory response to alterations of H+ concentration in small areas of the ventral medullary surface. Proc. Int. Un. Physiol. Sci. VII: 390. SHANES,A. M. (1958). Electrochemical aspects of physiological and pharmacological action in excitable cells. Part I. The resting cell and its alteration by extrinsic factors. Pharmacol. Rev. 10: 59-164. SMOLIK, E. A. (1943). Effect of intracistemal injection of potassium phosphate in hemorrhagic hypotension and shock in the dog. Proc. Sot. exp. Biol. Med. 53 : 70. STRAUB,R. (1956). The action of CO2 and pH on the resting potential of myelinated nerve fibres. Abstr. Comm. XXth Internat. Physiol. Congr. Brussels 1956: 858-860. TROUTH,C. 0. (1969). Lokalisation oberfllchlicher und tiefer auf die Atmung wirkender Strukturen in der Medulla oblongata durch elektrischen Reiz. PjZgers Arch. ges. Physiol. 307: R 16. VERSTRAETEN, J.-M. (1950). Influences de la concentration en ions potassium, calcium et magnesium du liquide &phalo-rachidien sur la respiration. Rev. Be/g. Path. 20: 1-21.