Influence of calcium and magnesium on the respiratory response to changes in CSF pH

Respiration Physiology (1969) 7, 216-229 ; North-Holland Publishing Company, Amsterdam

INFLUENCE

OF CALClUM AND MAGNESIUM ON THE RESPIRATORY RESPONSE TO CHANGES IN CSF pH1

J. BERNDT, A. FENNER AND K. BERGER Institut fiir Physiologie der Ruhr-Universitiit, 463 Bochum, Germany

Abstract. The ventral surface of the medulla oblongata of anesthetized cats was superfused with fluids of different Gaff and Mg++ concentration and pH. The effects of altered composition of the superfusing fluids on respiration were studied. A three-fold increase of the reference Ca++ concentration (1.5 meq/l) in the fluids was followed by a decrease in the tidal volume and ventilation at pH 7.0, whereas at pH 7.6 an effect on respiration could not be observed. When CSF Ca++ was reduced to 30% of the reference concentration, an increase of respiratory parameters at pH 7.6 followed, while at pH 7.0 tidal volume and ventilation remained unchanged. Lower CSF Ca++ concentrations (0.15 meq/l) had no significant influence on respiration. Removal of Ca++ from the superfusing fluids caused respiratory irregularity which in some cases was accompanied by convulsions of skeletal muscles. Mean tidal volume and ventilation values were reduced. Effects of Ca++ removal were partially neutralized by adding equivalent amounts of Mg++ to the Ca++-free fluids, whereas Mg ++ when added to fluids containing physiological amounts of Ca++, had no significant effect on respiration. The possible mechanisms involved in the propagation of Ca++ and Mg+f effects are discussed. Medulla oblongata Respiratory centers

Cerebrospinal fluid Control of breathing

From

experiments

mainly

carried

out by LEUSEN and his group

(VERSTRAETEN, 1948,

cations when introduced into the CSF may exert an influence on autonomous systems such as respiration, circulation, and muscular tone. Nevertheless, the site of action of these ions was unknown. By the application of local cooling, electrical stimulation, and changes in CSF pH, several investigators have, during the past six years, localized in the cat certain areas on the ventral surface of the medulla oblongata which mediate an influence on respiration (MITCHELL et al., 1963 ; SCHLAEFKE and LOESCHCKE, 1967,1968 ; LOESCHCKE et al., 1968). 1949; LEUSEN, 1949; DEVOS, 1953) it has been demonstrated

Accepted for publication 8 April 1969. 1 To Professor Dr. L. LENDLE on the occasion of his 70th birthday. 216

that certain

EFFECTS OF CsF

H+

AND ca++

217

ON RESPIRATION

However, there exists little information concerning the nature of nervous elements which act as receptors to CSF hydrogen ions or to some parameter immediately dependent on CSF hydrogen ion concentration. The results of morphological investigations reported by PETROVICKY (1968) who described superficial nerve cells in an area medial and rostra1 to the roots of N. XII, will possibly prove to be important in this respect. In the present investigation, the chemical environment of these nervous structures approachabIe from the ventral surface of the medulla has been altered by superfusing this area with mock CSF containing varying Caf + and Mg+ + concentrations. The effects on the respiratory response to changes in CSF pH were studied and compared with known observations of the influence of these ions on nerve fibres and synaptic structures. Methods Experiments were carried out on 16 cats (1.8-3.6 kg) in chloraIose-urethane anesthesia (40 mg/kg chloralose, 200 mg/kg urethane). A femoral artery and vein and the trachea were cannulated. The ventral surface of the medulla oblongata was approached by means of a suboccipital craniotomy. Special attention was paid to complete hemostasis prior to the opening of the dura. The tracheal cannula was connected to a semiclosed circuit which contained a circulating pump, COz absorber, and a recording Krogh spirometer. The system was filled with 100% oxygen and its volume was kept constant by continuous addition

TABLE1 Composition

of superfusion fluids. The pH values were obtained during ~uilibration

with 6 % COa

in air at 37.0 ‘C. The fluids containing 1.5 meq/l Ca++ and no

Mg++are

referred to as “reference fluids” in the text.

Ca++

Mg++

Kf

Na+

EIco;

Cl-

PH

meqjl

meqil

me&

meq/l

meq/l

mesil

range

4.5

-

5.0

149.6

42.0

121.6

7.479-7.650

4.5

-

5.0

141.1

145.6

6.871-6.959

1.5

-

5.0

153.5

9.5 42.0

119.5

7.518-7.589

1.5 0.45

-

5.0 5.0

145.1 154.6

9.5 42.0

143.6

6.910-7.027

118.5

7.566-7.631

0.45

-

5.0

146.2

142.6

6.90.5-7.094

0.15

-

5.0

153.1

9.5 42.0

118.4

7.577-7.631

0.15

-

5.0

146.6

6.940-7.110

-

5.0

155.2

9.5 42.0

142.4

0.0

118.2

7.579-7.674

0.0

-

5.0

146.8

142.3

6.928-7.047

1.5

1.5

5.0

152.0

9.5 42.0

121.0

7.544-7.599

1.5 0.0

1.5 1.5

5.0 5.0

143.6 153.5

9.5 42.0

145.1

6.888-7.045

119.5

7.535-7.696

0.0

1.5

5.0

145.1

9.5

143.6

6.937-7.055

218

J. BERNDT, A. FENNER AND K. BERGER

of oxygen and COZ, the latter being adjusted by hand to keep end-tidal Pco2 constant throughout the experiment. Continuous recording of tidal volume, end-tidal Pco2 (infrared analyzer), and arterial blood pressure (strain gauge) was performed on a direct recorder. Body temperature was kept at 37 + 1 “C by external heating or cooling. Mock fluids applied during the experiments are listed in table 1. They were kept in a water bath at 37 “C and equilibrated with 6% CO2 in air. CaCl, and MgCl, were added after one hour of equilibration, pH values of the fluids were measured immediately before starting the experiment in a thermostated MacInnes-Ingold glass electrode calibrated against Soerensen phosphate buffers. There were two solutions for any Ca” ’ and Mg + -i- concentration applied during this investigation, one at about pH 7.0 and another one near pH 7.6. Superfusion was performed by gravity flow from the mock CSF reservoirs through a jet placed in the caudal part of the operated area; the fluids were removed by suction at the level of the pons through a T tube, the lateral ends located about l-2 mm beneath the cut borders of the bone (fig. 1).

ROOT5 OF NN X]I Fotw ROOT5 OF NN VI

CAUDAL

Fig. I. View of the ventral surface of the medulla oblongata of the cat (schematic). The arrows indicate the direction of mock CSF flow (inflow caudal; outflow rostral) during superfusion.

Moderate differences in flow (6-30 ml/min) but cessation of flow was sometimes followed

did not influence the results obtained, by increased ventilation.

TIME COURSE OF EXPERIMENTS

In a first phase the fluids were superfused several times to find that one which caused the lowest ventilation and highest alveolar Pco2. All fluids were then applied during 3 or 4 periods, while end-tidal ‘Pco2 was adjusted to the previously obtained maximum value. Usually 2-3 min of superfusion was enough to reach a steady state. The duration of the experiments was kept below 2 hours following opening of the dura, in order to avoid alterations in the animals reactions to changed CSF com-

EFFECTS OF

CSF H+ AND Ca++ ON RESPIRATION

219

position. This meant that it was impossible to apply all test fluids in one experiment. Therefore the fluids containing 1.5 meq/l Ca+ + and no Mg+ + (see table 1) which were superfused in all experiments, served as reference. EVALUATIONOFDATA

1. DeJinition of symbols tidal volume and ventilation (ATPS),resp.

VT;eE

subscripts indicate VT and VE values at the respective CSF pH

V+., pH) or VE(6.8 pH) VTm,

subscripts indicate VT and 9~ values during superfusion with fluids containing the respective [Ca”] (in meq/l)

I. 5

irE[Cal0

relative difference

R.D. For example :

R.D. of VT values =

R.D. of -$$

Vrr,,x - Vrr,,,

values =

&a,

($i,c

s

I. 5 ] -(-i&,,

.5 .

dW

( APH 1rca11.s

2. Calculations The results presented below were derived from steady state measurements. For any single experiment and for each Ca+ + or Mgf + concentration applied, two VT and TE values (each being the mean of 3-4 superfusion periods) at two different pH levels (the one near pH 7.0, and the other near 7.6) were obtained. Using these data, the parameters of the two equations AVT VT=VTcO PH)+ --pH

APH

and %=\iTEcg PH) +

AVE --‘pH APH

(2)

were evaluated for each Ca+ + or Mg+ + concentration separately. Equations (1) and (2) describe a linear relationship between VT or VE, and CSF pH, which is an adequate approximation in the range between pH 7.0 and 7.6 (LOESCHCKE,1960; LOESCHCKE and MITCHELL, 1963). In order to eliminate the small pH differences between the fluids used during one experiment, VT and VE values at pH 7.0 and 7.6 were calculated by means of eqs. (1) and (2) and compared with the analogous data obtained for reference fluids in the same experiment. The results are presented as relative differences.

220

J.BERNDT,

A.FENNER

AND K.BERGER

In addition to the calculations described above, mean pH response curves (linear regression lines) of VT and VE from all experiments where a particular Ca+ + or Mg++ concentration was applied, were constructed. These were compared with the mean pH response curves obtained from measurements during superfusion with reference fluids in the same experiments (figs. 2 and 3). Results EFFECTS OF A CHANGEIN

CSF pH ON RESPIRATION (CSF[Ca++]

1.5 MEQ/L)

When the CSF hydrogen ion concentration on the ventral surface of the medulla is changed from about 7.6 pH to 7.0 pH, a marked increase of VT and VE is observed. The mean effects of a change in CSF pH during superfusion with reference fluids are demonstrated in table 2. The data presented there were derived from all experiments reported in this communication. End-tidal Pcol was kept constant (kO.5 mm Hg) during the course of an experiment but differed slightly between 39 and 42 mm Hg from experiment to experiment, mainly because of variations in the depth of anesthesia. TABLE 2 Steady state respiratory response to changes in CSF pH on the ventral surface of the medulla oblongata. CSF [Ca++] 1.5 meq/l. 26 experiments on 16 cats. S.D. = standard deviation. CSF pH

VT

S.D.

ml 1.6 7.0 Mean slopes of pH-response curves

EFFECTS OF INCREASED

29.6 70.4

i 10.2 & 15.5

~ 55.0

& 26.7

ml. (pH)-’

VE

S.D.

ml/min 578 1678 -1835

+ 324 ‘r 614 5 965

ml.min-l.(pH)-l

CSF [Ca++]

During superfusion with fluids of an increased [Ca”] (4.5 meq/l), a slight but significant reduction of VT~,., PHj and $7.0 Since VTC7.6 PH) and pH) is observed. VE(7.6 PHj remain unchanged, the slopes of the pH response curves (i.e. AVT/A~H and AVE/A~H, resp.) are distinctly depressed (table 3 ; fig. 2, upper traces). EFFECTSOF DECREASED CSF [Ca’ +] At pH 7.6, a reduction in the CSF [Ca’ ‘1 from 1.5 to 0.45 meq/l causes a considerable rise in VT and VE. Calculation of the statistical significance of this effect yields p < 0.05 in both cases, when the absolute differences are used, whereas the relative differences do not quite reach the same limit.

6b

5b

4a

3a

2

1

NO.

77.3

86.2

92.6

61.1

64.6

67.8

73.5

94.8

105.7

6.935

6.947

6.871

6.958

6.944

6.980

6.954

6.955

6.959

6.958

4.5

1.5

4.5

I.5

4.5

f-5

4.5

1.5

4.5

1.5

75.8

70.0

6.925

6.957

64.0

ml

VT

1.5

pH

1410

P

- 0.088

- 0.106

+pH

i 0.02

rtO.048

-0.182 &OB61

-0.064

-3393

-3177

- 0.041

-2088

-2004

- 0.336

- 1073

-692

_ 0.186

- 3239

- 2639

-0.186

-1577

--1285

- 0.258

-1411

-1074

APH mljmin

AirE

$0.025

PH)

-0.198

44.1

35.4 101.7

-0.100

23.0

20.7

91.0

< 0.01

- 0.035

-96.0

34.5 co.200

- 0,079

c 0.025

R.D.

1272

47.6

- 0.069 -92.7

70.0

&O.Ol?

7.563

3325

R.D. 1136

44.1

-78.3

64.5

~0.050

7.506

2874

473

26.5

-72.9

- 0.068

63.7

41.4

59.4

20.9 i-o.033

87.9 - 0.150

SE.

7.577

1726

499

27.5

-0.383

-48.6

- 30.0

- 0.207

-111.8

-0.187

7.507

1597

745

36.1 R.D.

903

44.4

R.D.

492

30.0

21.6

74.8

33.4 j-O.146

73.8 -0.024

c 0.070

32.7

35.0

ml

VT(7.6

38.3

,,H)

12.1

Mean relative difference

7,566

I.500

7.518

- 88.7

-61.3

692 527

-56.3

877

-0.100

67.5

-0.262

60.8

-58.1

ml

VT(7.0

-42.9

32.2

35.8

7.564

7.480

45.0

7.482

R.D.

683

651

-0.164

34.1

m]/pH

APH

R.D.

32.9

ml,/min

ml

7.650

VE

VT

7.575

1288 1314

2306

2134

1665

1580

1550

PH

isvT

relative difference. For further details see text. S.E. = standard error.

Alkaline superfusion fluid

R.D.:

VT and VE values: average of steady state evaluation of 3-4 superfusion periods.

ml/mm

VC7E

Acid superfusion &id

4.5

mecl/r

Ca++

MCCk

CsF:

Exp.

3

Effects of increased CSF [Ca++] on the respiratory response ta a change in CSF PH.

TABLE

018)

< 0.005

*0.018

--0.103

-0.138

3182

2743

-0.075

1632

1510

-0.076

1352

1250

-0.167

2150

1793

- 0.054

1581

14%

-0.109

1493

1331

ml/min

%.o

&O&75

-0.OO7

- 0.270

1146

837

-0.J90

380

308

+&I77

708

834

j-O.014

2%

209

+ OJ40

635

724

+ 0.086

641

703

ml/mm

hi”.~p~)

7

6a

Sa

4b

3a

No.

Exp.

89.6

92.6

0.45

101.2

98.7

70.7

69.8

6.967

6.946

6.939

0.45

1s

0.45

6.958

62.8

1.5

60.4

6.955

0.45

6.950

76.1

1.5

81.8

6.955

6.980

1.5

0.45

6.948

_1___1

4

I495 1295

62.2 53.3

7.602

7.566

1700

1570

506

-0.126

R.D. 294 455

19.6 25.0

7.631

7.572

1837

1789

.-

- 129.9

781 506

27.5 20.1

7.616

7.563

3131

2967

S.E.

Mean relative difference

R.D.

R.D.

10.099

-0.204

+0.053

-70.7

-74.5

-113.6

- 0.554

-54.2

519

29.1

-24.2

908

44.3

7.577

1419

7.615

- 0.222

-38.9

-30.3

-0.173

-111.8

-92.5

ml/pH

APH

AVT

1493

R.D.

R.D.

492

30-5 30.0

7.587

7.518

_____-

VE ml/min

VT ml

2208

PH

Alkaline superfusion fluid

_.____

f0.020

to.015

10.018

65.4

66.6

+0.045

93.3

97.5

- 0.020

60.4

59.2

+0.067

75.3

80.4

-0.036

87.9

84.4

VTt?.op~) ml

For further details see table 3 and text.

2306

VE

mI/min

ml

fluid

VT

6.958

pH

Acid suffusion

1.5

me@

Ca++

Mock CSF

TABLE

f0.166

+0.412

- 0.053

23.0

21.8

$0.915

15.3

29.3

+0.604

27.8

44.6

+0.198

51.9

62.2

-0.397

20.9

29.2

VT(?.~PH) ml

_.

10.098

-0.211

+ 0.067

-2109

-2252

-O.IIO

-4068

-3621

-0.510

-1566

- 768

- 0.325

-469

-317

-0.178

- 3239

-2664

APH ml/min. pH

Airy

Effects of reduced CSF [Ca++] on the respiratory response to changes in CSF pH.

* 0.025

+0.025

+ 0.033

1660

1715

+0.077

2769

3012

- 0.031

1423

1380

+0.082

1560

1686

- 0.038

2150

2069

ml/min

%,i~ gm

--__

hO.292

+ 0.728

- 0.079

394

363

+ 1.359

356

840

+0.904

483

920

$0.170

1278

1496

+I.286

206

471

ml/min

+Ew pm

EFFECTS OF

CSF H+ AND Ca+ + ON RESPIRATION

223

reduction of the CSF VT(,., pH) and %.o pH) are not influenced by the moderate [Ca”]. These findings involve a marked decrease in the slopes of the pH response curves (table 4; fig. 2, second traces from above). These effects disappear when the CSF [Ca”] is further reduced to 0.15 meq/l. VT~,., PHJ is somewhat smaller than the reference value while VT(,.~ PH)seems to be CSF Ca” m&l

ri [bnh

ATPS]

VT [hl ATPS]

I_

0

0

Fig. 2. Mean pH response curves of tidal volume (right) and ventilation (left). The curves were calculated as linear regression lines from data of all experiments, in which solutions of the indicated Ca++ concentration (between 0 and 4.5 meq/l) were applied. Solid lines: altered Ca++ concentration Dashed lines : control, reference Ca ++ concentration (1.5 meq/l) in same experiments.

O(A)0

2103 2514 2413

6.928 72.9 6.937 77.9 6.910 76.4

O(A)0

0 (B) 1.5 1.5(C) 0

41.5 641 646

4.948 30.6 6.940 39.6 6.948 38.2

O(A)0 0 03) 1.5 1.5(C)O

la

2a

1638 2171

7.047 64.6 7.027 70.5

1456 1278

1502 2147

O(A)0

7.Q47 60.8 7.027 52.7

7.029 49.5 6.928 89.4

Acid superfusion fluid pH VT irE ml ml/mm

1.5(C)O

1.5 (C) 0

0690

1.5 (C) 0

TABLE 5

7.607 7.592 7.585

7.602 7.604 7.584

7.676 7.720

7.676 7.720

7.604 7.577

PH

418 487

438 977

190 208 321

1206 1215 1070

R.D. (B)-(C)

R. D. (A)-(C)

51.4 50.5 43.4

R.D. @j(C)

R.D. (A)-_(C)

13.3 14.4 21.9

R.D. (A)-(C)

19.0 37.3

R.D. (A)-(C)

18.9 22.1

R.D. (A)-(C)

436 591

ml/min

ml 20.0 32.0

i7E

VT

Alkaline superfusion fluid

- 0.353 -0.144

-31.6 -41.8 -48.8

t 0.03z + 0.480

-26.4 -37.9 -25.6

t 0.511

-72.4 -47.9

+ 0.510

-66.6 -44.1

- 0.438

-51.3 -91.2

APH ml/W

AVT

-0.020 fO.045

70.5 75.2 71.9

- 0.208 + 0.031

29.1 37.2 36.7

+0.211 f0.176

51.6 50.1 42.6

-0.384 -0.323

13.2 14.5 21.4

- 0.433

24.4 43.0

67.9 71.7 - 0.053

-0.125

23.9 27.3

-0.283

20.1 28.0

ml

VT(7.6pW

+o.m

63.8 53.8

- 0.386

50.8 82.7

ml

VT(7.0pH)

~ 0.336 - 0.003

.I”-1322 -1984 -- 1988

- 0.325 + 0.884

~ 345 - 963 -511

to.108

-1907 -1721

+ 0.445

-1651 -1142

- 0.250

- 1855 - 2473

APH mlimin~ pH

ALE

- a.101 + 0.069

2007 2388 2232

- 0.361 - 0.062

396 581 619

- 0.054

1727 1825

+0.172

1533 1308

-0.210

1555 1967

mljlmin

irEt7.0~15)

CSF (A) and of fiuids containing Mg++ instead of Ca++ (B) on the respiratory response to changes in CSF pH. For further details see table 3 and text.

0

9

8

JO.

:xp. Mock CSF Ca++ Mg++ meqjl meq/l

Effects of Cat+-free

vb7.611H)

+ 0.168 +0.152

1214 1197 1039

- 0.395 -0.869

189 41 312

- 0.266

582 792

-0.104

542 623

-0.087

442 484

ml/min

4a

0 (A) 0 0 m 1.5 l.SjG)O

365 731 636

14.5 16.9 29.8

273 239 510

RD. (A)-(C) RD. (BjK)

lS,j 31.6 29.5

Mean relative differexxce (B)-_(C) SE.

P

68.1 78.8 73.7

- lOQ.6 - 83.2 - 7x?

+ 0,188 $#.008

- 85.3 -72.4 -71.8

+5,180 10,095

f-O.542 AO.025

-0.552 &0.556

10.126 -0.070

80.8 66.7 71.7

-0.040 +-0.047

68S-J 70.8 75.6

--o.J7S -0.076 -+a. A90 fO.069

-555.8 -%5*5 - 67.6

i-0.113 f5.124

7.615 7.655 7.581

529 55% 553

RD. ~A)--@) RJ. (B)-_(C)

33.2 3b.l 340

wJ.00~ l FO.fUB

-58.6 -42.0 -t_0.230 -i-o,395

n2.5

47.1 42-S

-Sk7

Mean relative difference (AHC) SE.

1838 1327 lSl0

6.959 85.4 6.980 68.5 6.947 75.7

7,422 7.659 7.577

167 201 241

R.D, (A)---&) R.D. @q-~C)

12.7 1S,6 18.3

+0,515 +O.I$o

175% 2509 i 940

6.944 73.5 6.970 77.8 6.Q45 74.9

7.627 7.594 7.588

7.540 7.577

1,519

I&D. (A j(C) RD. (B)-(C)

1413 2188 2027

676 627

6.964 49.4 6.921 45.9

6.955 70.7 6.973 81.1 6.955 76.9

422

6.961 44.7

f 0.254 f0S41

---Q&O2 15.119

-0,241 f5_57% < 0.02 -0.131 &5.154

+i?*s22 -k&111

- 2403 -1754 - 157%

- 0,005 - O.fIJ1

- 2554 -2551 - 2063

-0,435 -to,163

-1315 -2755 -2324

+ O..w _i-0,402

-737 -%22 -586

-0.469 - 0.409

15.1. 16.8 28.4

16.9 27.7 32.1

+0.042 -0.082

34.6 30.5 33.2

- 0.336 --a.3#7

17.3 12.0

11.5

+ 0.032 iO.036

- 0.079 10*56S

to.21s - O.OP5

1737 1291 1425

- 0.101 $0.0~7

1641 1948 1825

-O,Z96 +O,IWO

1353 2114 1921

$0,024 +0.114

592 644 578

- 0.225 &OS15

-0.183 i0.m c 0.05

-0,381 - 0,503

296 238 478

-0,307 co.270

408 747 588

+0.072 --oil67

s64 491 526

- 0,344 - 0.335

149 151 227

J. BERNDT, A. FENNER AND K. BERGER

226

slightly increased, but the differences are not significant. In fig. 2 (second traces from below), the mean pH response curves calculated for the CSF [Ca”] of 0.15 meq/l are shown to be not distinctly different from the reference curves. If no Ca+” is added to the mock CSF, i.e. when the fluids only contain traces of this ion, VT and 9~ are reduced in the entire range between pH 7.0 and 7.6. The slopes of the pH response curves are not significantly different from those of the reference curves (table 5; fig. 2, lower traces). During superfusion with Ca++-free fluids, r esp iration is irregular and a steady state is not reached. The muscular tone seems to be increased, and convulsive contractions of skeletal muscles occasionally occur. In such experiments VT and VE can be increased above the reference values. Thus the mean effect of these solutions (depressed respiration) cannot be proved to be statistically signi~cant over the whole pII range. EFFECTS OF

Mg+ +

ADDED TO MOCK

CSF

When Mg’ ’ is added to reference solutions in a concentration equivalent to that of Ca+ + (i.e. [Ca”] 1.5 meq/l; [Mg+ +] 1.5 meq/l), no significant influence on VT

m<-

\ A

A

Fig. 3. Influence of Mg++ (1.5 meq/l) on the mean response curves of tidal volume and ventilation. Mg++ added to mock CSF containing 1.J meqjl Ca++ (upper traces, solid tines) does not signi%antly influence the pH response of tidal volume (right) and ventilation (left). Addition of Mg++ to Ca++ -free solution neutralizes the effect of Cat+ removal (lower traces). Curves were calculated as in fig. 2. A --- Ca++ 1.5 meq/l‘ ‘Mg++ 0 meqji - Ca++ 1.5 meq/I;p Mg++ 1.5 meq/l B --- Cat+ 1.5 meq/l Mg++ 0 meq/lJ - Ca++ 0 meq/l Mg++ 1.5 meq/l v-w-. Ca++ 0 meq/l Mg++ Ozmeq/l

EFFECTSOF CSF H+ ANDCa++ ON RESPIRATION

227

and VE is observed (fig. 3). There is a tendency to increased VT and \j~ values at pH 7.0 while VT(,., PHj and VEX,., PHj remain unchanged, but this is expressed in the arithmetic mean of all data only, while single measurements show an increased scatter. If Ca’ + is replaced in the superfusion fluids by equivalent amounts of Mg+ + (i.e. [Ca”] 0.0 meq/l; [Mg+ +] 1.5 meq/l), the effects of the removal of Ca+ + are neutralized to some extent, but reference conditions are not completely restored. Respiration is regular, and convulsions do not occur. The effect of adding Mg’+ to Ca++ -free solutions is more pronounced in the acid range, while at pH 7.6 only VT is shifted in the normal direction (table 5; fig. 3). Discussion

The influence of changes in the CSF Ca+ + on respiration has been reported by several authors. It was found that increased CSF [Ca++] depressed ventilation whereas a low concentration or the withdrawal of Caf + from the CSF caused a rise of respiratory parameters. However, the site of action of the electrolytes introduced into the CSF spaces and the effective [Ca”] there was unknown. In addition, the CSF [H+] was not considered (VERSTRAETEN, 1948, 1949; FELDBERGand SHERWOOD, 1957). There is general agreement between the observations in this study and that of the authors cited. On the other hand, it is demonstrated here that the effects of Ca’ + on respiration are seen when the induced changes of the CSF composition are confined to the ventral surface of the medulla. Furthermore, it could be shown that the sensitivity of the respiratory system to changes in the CSF pH is distinctly affected by variations of the CSF [Ca”]. It is known that the variations of the CSF [Ca”] and [Mg+ +] as applied in these experiments do not occur under physiological conditions (KEMENY,BOLDIZSARand PETHES, 1960; K_EWN, BOLGYORand PETAES, 1961). However, this study was not intended to demonstrate a physiological mechanism. Its purpose was to alter experimentally the chemical environment of an unknown nervous substrate approachable by CSF ions from the superfused region, and to find out whether the observed responses of respiration might provide some information on the nature of this substrate. One may first compare the reactions to changes in the CSF Ca+ + and Mg+ + concentrations. Since Ca+ + can be partially replaced by Mgf ‘, both ions have a similar influence on respiration, so that a counteraction between the two may be excluded. This result is not compatible with observations of the effects of these ions on cholinergic synapses; Ca ++ is essential for the release of acetylcholine whereas Mg++ blocks the transmission of impulses (KATZ and MILEDI, 1963; MILEDI and SLATER,1966; GINSBORGand RAY, 1963 a,b; ELMQVISTand FELDMAN,1965). On the other hand, the excitability of the myelinated nerve fibre depends on the external [Ca”]. It decreases when the external [Ca”] is raised and increases when the Caf + content of the extracellular fluid is reduced. Ca+ + can be replaced by

J. BERNDT, A. FENNER AND K. BERGER

228

Mg’+ which acts similarly but less effective (FRANKENHAEUSER, 1957; FRANKENHAEUSERand HODGKIN, 1957 ; FRANKENHAEUSERand MEVES, 1958). Considering these findings it does not seem very probable that cholinergic mechanisms are involved in the propagation of the effects of Ca+ + on respiration. A better correspondence with the results of this study is obtained when an action - possibly nonspecific - of Ca+ + and Mg+ + on superficial nerve fibres or receptors is suggested. The question as to whether or not Ca++, Mg++, and hydrogen ions act on the same mechanism involved in the respiratory drive must be a subject for further investigation. References BLACKMAN,J. G., B. L. GINSBORG

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DEVOS, J. (1953). Localisation

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