Intracellular hypertrophic NORBERTO Department Department
pH regulation of normal rat myocardium
and
C. GONZALEZ, HANS-GEORG WEMKEN, AND NORBERT HEISLER of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103; and of Physiology, Max-Planck-Institute for Experimental Medicine, Gottingen, Germany
GONZALEZ, NORBERTO C., HANS-GEORG WEMKEN, AND NORBERT HEISLER. IntracellularpI regulation of normal and hypertrophic rat myocardium. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 47(4): 651-656, 1979.-The myocardial cell pH (pHi) observed during breathing of 0, 7.5,or 10% CO2 in air for 3 h was studied in rats with myocardial hypertrophy due to aortic stenosis and in sham-operated rats. The change in pHi during hypercapnia was significantly smaller in the rats with myocardial hypertrophy, with the apparent nonbicarbonate buffer value (A[HCOy]i/ApHi) being almost three times that of the sham-operated rats. In vitro CO2 equilibration of myocardial tissue homogenates showed no difference in nonbicarbonate buffer value between homogenates obtained from normal rats and from rats with myocardial hypertrophy. Therefore, it appears that the increased ability of the myocardial cell to regulate its pH during hypertrophy is not due to an increase in the cellular level of nonbicarbonate buffers, but seems to be related to a larger bicarbonate uptake by the myocardial cell during hypercapnia.
present experiments were undertaken to determine if this factor played a role in the elevation of the apparent nonbicarbonate buffer value of myocardial hypertrophy. METHODS
Production of myocardial hypertrophy. Male SpragueDawley rats of 250-300 g body weight were placed in a large beaker containing cotton saturated with halothane. Once a light level of anesthesia was achieved, the trachea was intubated and anesthesia maintained with halothane delivered through a positive-pressure respirator. The abdominal aorta was exposed through a left flank incision and a ligature was passed around it. A l-mm-diameter wire was placed over the exposed section of the aorta and the ligature, which included the vessel and the wire, was fastened. The wire was then removed, leaving the aorta narrowed to a standardized diameter. An approximately equal number of rats were sham operated on the same intracellular buffering; cardiac nonbicarbonate buffer value; transmembrane HCOT flux; aortic stenosis-myocardial hyperday that aortic constriction was performed. The sham trophy operation included the same maneuvers, except for the constriction of the aorta. Experiments in intact animals. Six to 10 days after THE MYOCARDIAL CELL PH changes less than that of surgery, rats with aortic constriction and sham-operated skeletal muscle or blood when an intact animal is exposed rats were anesthetized with halothane, bilaterally neto hypercapnia of short duration (7, 16). It has become phrectomized, and a catheter was introduced in the left apparent that various factors contribute to the regulation carotid artery. The animals were aIlowed to recover from of cell pH; these include chemical buffering, exchange of the anesthesia, weighed, and placed in a plastic chamber H+ or HCO: ions with the extracellular fluid, and changes where they could breathe either air or 7.5 or 10% CO2 in in the production of acid metabolites. Part of the re- air. A maximum of six animals was used in each experimarkable ability of the myocardial cell to regulate its pH ment, and care was taken to include an approximately seems to be due to a H+ or HCO; exchange between equal number of sham-operated rats and rats with aortic cellular and extracellular fluid that takes part in the early constriction in every experiment. The animals could be stages of hypercapnia (30). introduced and removed from the chamber without apIt has been reported that in myocardial hypertrophy preciable change in the chamber’s gas composition. Each this ability to regulate cell pH is even more pronounced animal was exposed to a given gas mixture for 3 h. As (28). Consequently, the apparent nonbicarbonate buffer soon as they were placed into the chamber, the animals value of the cell fluid, i.e., the ratio of change in cell were injected with 5,5-[ 14C]dimethyloxazolidine-2,4bicarbonate concentration to change in cell pH, is higher dione (DMO) and “H[inulin]. Arterial blood samples were in myocardial hypertrophy than in normal hearts. This obtained 165 and 180 min after the animals were introobservation is of considerable interest in light of the duced into the chamber. Immediately after the last blood influence of acid-base changes on the mechanical per- sample was taken, the animals were killed by an overdose formance of the heart (5, 12, 22, 25). Hypertrophy is of pentobarbital sodium and removed from the chamber. accompanied by major biochemical changes that could The heart was quickly excised, blotted in filter paper, result in the apparent nonbicarbonate buffer value being and weighed. The blood samples were analyzed for pH, increased simply due to the elevation of the intracellular Pcoz, and POT with appropriate electrodes at 37°C. concentration of nonbicarbonate buffers (34, 35). The Plasma was separated by centrifugation immediately 0161-7567/79/0000-OOOOooo$Ol.25
Copyright
0 1979 the American
Physiological
Society
651
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652 after sampling. A weighed amount of plasma was placed on ftiter paper, oxidized in a model 306 Packard automatic sample oxidizer, and analyzed for 14C and 3H activity by liquid scintillation counting. Duplicate samples of the left ventricle were placed on filter paper, dried for 48 h at 100°C, and oxidized for later analysis of 14C and “H activity. Tissue water content was determined by weight difference before and after drying. Determination of the chemical nonbicarbonate buffer value of the myocardium. This was done by in vitro equilibration of myocardial tissue homogenates with various CO* tensions. Six to 10 days after surgery the animals were weighed and killed with an overdose of halothane. The heart was rapidly removed, rinsed in Ringer solution, blotted dry in filter paper, and frozen in liquid N2. The left ventricle was weighed while frozen and transferred to a mortar where it was ground to a fine powder under liquid N2. The tissue was diluted with Ringer solution (composition [meq/l]: Na+, 147.2; K’, 5.0; Ca’+, 2.6; Mg2+, 2.4; SO:-, 2.4; HCOT, 24.0; Cl-, 124.8). The ratio of homogenate water weight to tissue water weight averaged 3.73 t 0.076. The homogenate was placed in the cuvette of a tonometer where it was equilibrated with 2, 5, and 15% COn in 02 at 37OC. A single-unit double electrolyte bridge pH macroelectrode was introduced into the cuvette and pH of the homogenate was recorded continuously. Adequate mixing was ensured by intermittent rotation of the cuvette. The homogenate was initially exposed to 5% C02, and equilibration was maintained until a steady pH value was obtained, usually 20-30 min later. The gas mixture was switched to either 15 or 2% CO2 in 02. After equilibration with the new mixture was complete, the homogenate was again equilibrated with 5% CO2. In this way the acid drift that was occasionally observed as a function of time was taken into account by averaging the two values obtained during 5% CO2 equilibration before and after equilibration with 2 or 15% CO2. A typical run would consist of the following sequence of equilibrations: 5, 2, 5, 15, and 5% CO2 in 02. At least one of such runs was completed in each homogenate. In some experiments pH was altered by addition of small amounts of 0.5 M NaHC03, and another equilibration run was carried out. Calculations Experiments in intact animals. Intracellular pH (pHi) was derived from the distribution of DMO (33). Inulin was used as an extracellular marker, and the extracellular space (Qe) was expressed as the ratio of extracellular water to total tissue water. Intracellular HCOT concentration, [HCO-] 3 i was calculated from pHi and Pace, values, using pK’1 of 6.1 and cyco, of 0.03 mmol/(Torr x kg). Extracellular HCOi concentration, [ HCO;], was calculated from pH, and Pace, values using pW1 of 6.1 and (XCO,of 0.03 mmol/(Torr x kg). In vitro experiments. The HCO; concentration of the homogenate was calculated from pH and Pcoz values, using a pK’, of 6.1 and cyCO, of 0.03 mmol/(Torr x kg). The buffer value of the homogenate (Phomog) was calculated in the following manner.
GONZALEZ,
Phomog
W([HCG]1
WEMKEN,
+ [HCO&)
AND
HEISLER
- [HCO&
= W(pW
+
pH3)
-
pH2
where 1 and 3 refer to the values obtained in the initial and final equilibration with 5% COZ, and 2 refers to the values obtained equilibrating with either 2 or 15% CO2. Phomogwas expressed in mmol/(pH x kg homogenate). The buffer value of the tissue (Pti.ss) was
Ptiss
=
Phomog
x
weight homogenate weight tissue
Ptisy was expressed in mmol/(pH X kg tissue). The buffer value of the intracellular fluid (&) was calculated in the following way.
Pce11 =
Ptiss FH~O
X
(I-Qe)
where FH~O is the fractional water content of the tissue and Qe is the ratio of extracellular water to total tissue water. The values for FH~O and Qe employed were those obtained in the corresponding in vivo experiments. Pcell was expressed in mmol/(pH x kg intracellular water). RESULTS
Production of myocardial hypertrophy. The technique of abdominal aortic constriction was quite effective in producing myocardial hypertrophy, as evidenced by the heart-to-body weight ratios that were 2.45 t 0.03 x lo-” in the sham-operated rats and 2.93 t 0.08 x lo-” in the rats with aortic stenosis ( P < 0.01). These results agree with previous data using the same technique (28). Experiments with intact animals. Table 1 summarizes the data obtained with sham-operated and aortic stenosis rats breathing air and the hypercapni C gas mixtures. No differences can be observed between the intra- or extracellular acid-base parameters of the aortic stenosis and sham-operated groups breathing air. The only significant difference between these two groups was observed in Qe, which was significantly higher ( P < 0.01) in the aortic stenosis group. Breathing 7.5% CO2 in air resulted in slightly higher Pace, in the aortic stenosis group than in the shamoperated group. Although the difference was significant (P < O.Ol), it was not large and probably reflected a slightly different ventilatory response to inhaled COZ. In spite of the fact that PI o, was decreased with respect to the groups breathing air, Pao, increased. This was also evident in both groups of rats breathing 10% CO2 and is the result of the marked hyperventilation elicited by increased PICO,. Intracellular pH decreased less with hypercapnia in the rats with aortic stenosis than in the sham-operated rats; the difference is much more marked in the groups breathing 10% CO2, where pHi and [HCOT]i for the aortic stenosis group are significantly different from those of the sham-operated group ( P c 0.025). The ability of the heart to regulate its cell pH during acidosis is better demonstrated by Fig. 1, where the cell pH values are plotted as a function of the corresponding extracellular pH values for the vari .OUS ~ITOUPS. The slopes ApHi/ApHe,
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CELL
PH OF
NORMAL
1. In&a-
TABLE
AND
HYPERTROPHIC
and extracellular
%Insp CO?
acid-base
parameters
[HCO,I le, mmol/l
Pa,,
7.5 10
1.1 1.0 t, 0.7
21.7 24.4 26.6
t 0.46 t 0.57 t 0.41
100.2 114.5 111.8
t, 0.8 t, 0.7 79.8 t 0.9
21.9 24.1 26.2
t, 0.43 t 0.61 t 1.02
95.6 112.2 112.8
7.416 7.233 7.142
t, 0.008 t, 0.007 AI 0.006
35.2 60.7 80.0
7.413 7.247 7.140
t 0.007
35.6 57.3
t t
[ HCOZ
Ton-
Rats 0
653
MYOCARDIUM
with k 1.8 t 2.3 t, 1.7
aortic 6.765 6.757 6.739
Sham-operated 0 7.5
10 Values myocardial
t, 0.010
t 0.007
are means -+ SE. pH,, arterial cell bicarbonate concentration;
plasma pH; [HCO;],, Qe, ratio of extracellular
t, 1.9 k 1.8 t 1.6
6.790 6.740 6.703
]i, mmol/l HA&
FH~O
n
stenosis t, 0.016 I!I 0.015 t, 0.009
5.0 t 0.2 8.4 t, 0.3 10.5 t, 0.2
0.191 0.181 0.170
t, 0.005 Ifr 0.007 AI 0.004
0.808 0.803 0.799
5.2 t 0.3 7.6 t, 0.4 9.6 t 0.3
0.168 0.164 0.146
t 0.004 + 0.003 ?z 0.003
0.803 0.802
t 0.003 t 0.003 t, 0.004
27 16 27
rats t, 0.010 t 0.019 t, 0.015
t, 0.002 t 0.002 0.794 zk 0.004
arterial plasma bicarbonate concentration; pHi, myocardial cell pH; to total tissue water; FH~o, fractional concentration of tissue water.
26 15 27
[HCOi]i,
A PHi / ApHe = -124 f 0007 l Stenosis obtained from linear regression analysis using individual experimental values are 0.337 t 0.010 for the shamoperated group and 0.124 t 0.007 for the group with o Sham Operated ApHi / ApHe -0337 f .010 aortic stenosis. These values correspond to apparent nonbicarbonate buffer values (A[HCOs]i/ApHi) of 56 and 228 mmol/(pH x liters cell water), respectively. 6.8 The total tissue water content did not show marked differences in the various groups. Qe, however, showed a tendency to decrease with hypercapnia, with Qe for the P” i aortic stenosis group being always higher than the shamoperated group. Chemical nonbicarbonate buffer value. Table 2 shows 6.7 the results of the in vitro CO2 equilibrations of myocardial homogenates. The values for Pcell were obtained using the average values of FH~O and Qe obtained in the experiments with intact rats. The average FH~O and Qe 7.4 7.1 7.2 7.3 used for the rats with aortic stenosis were 0.802 and 0.180, respectively; for the sham-operated rats they were 0.799 P”e and 0.158. The p values obtained after equilibration with FIG. 1. Changes in myocardial cell pH (pHi) as a function of the 2 and 5% CO2 are not different from those obtained extracellular pH (pH,) for rats with aortic stenosis (solid circles) and equilibrating with 5 and 15% COZ, i.e., p does not seem to sham-operated rats (open circles) breathing 0, 7.5, or 10% CO2 in air. change over a rather wide pH range. An acidification of Regression coefficients ApHi/ApH, were calculated from the individual the homogenate with time was observed in some of the PHI andpHi values. experiments, as it has been shown in other tissues using similar methods (13, 17). The acid shift, however,- was although the extracellular fluid volume was higher in the highly variable in magnitude and apparently not influenced by initial homogenate pH or Pcog, duration of rats with aortic stenosis than in the sham-operated rats. It is possible that aortic stenosis may have resulted in an equilibration, or type of heart (i.e., normal or hypertrophic) being equilibrated. When in approximately one- elevation of coronary capillary pressure with a resulting half of the experiments the HCO: concentration was increase in interstitial fluid volume. Both in the shamoperated and in the aortic stenosis rats, hypercapnia was increased by about 10 mM/kg homogenate, no appreciable change in the rate of acidification or of the homoge- accompanied by a decrease in Qe and a concomitant nate buffer value could be detected. It is evident that no increase in intracellular fluid volume. A similar finding appreciable difference exists in Ptis or Pcellbetween ho- was reported several years ago in dogs breathing 10% CO2 (29). These changes are probably due to a net mogenates of sham-operated rats and those of rats with movement of water from the extracellular to the intraaortic stenosis. Even though Qe was significantly higher cellular fluid in response to changes in osmolarity. The in stenosis (and consequently intracellular water content increase in PCOZ would result in generation of HCOi ions, was lower), this did not result in a significant difference in calculated Pcellbetween normal and hypertrophic my- both in the extracellular as well as in the intracellular fluid, according to their respective nonbicarbonate buffer ocardia. values. A given increase in PCO~ would result in more HCO; being formed in the intracellular fluid due to its DISCUSSION higher buffer value. Additional driving force for water The technique of subdiaphragmatic aortic stenosis re- shifts may have been provided by transmembrane HCO; sulted in a. prompt and reproducible inc rease in myocarfluxes (30) possibly accompanied by 0th .er ions. Although dial mass. The change in heart-to-body weight ratio was the exact mechanism of the changes in Qe cannot be not associated with an increase in total tissue water ascertained, the changes observed in Qe and in intracel-
I
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654 TABLE
---
GONZALEZ,
2. In vitro nonbicarbonate P I ,.A%,mmol/(pH
stenosis operated
27.5 26.8
t t
Values are means t SE. 2-5 and 5-15 normal and hypertrophic myocardium.
29.7 28.5
,&.II,mmol/(pH x kg
x kg t&we)
5-15 1.11 1.24
AND
HEISLER
buffer value
2-5
Aortic Sham
WEMKEN,
Avg.
t, 1.23 t_ 1.20
refer
28.6 27.7
to the percent
2-5
t 0.97 t, 1.13
41.8 39.8
CO2 concentrations
lular fluid volume both in sham-operated and aortic stenosis rats breathing 10% CO2 could have been produced by the generation of, or transfer to the intracellular fluid of approximately 10 mosmol/l of HaOi. The results obtained in the present experiments indicate that the ability of the heart to regulate its intracellular pH during hypercapnia is markedly increased in myocardial hypertrophy. These data in general agree with earlier observations of Saborowski et al. (28), although we did not observe, as they did, an increased intracellular pH in the hypertrophic hearts during normocapnia. The mechanism by which the hypertrophic myocardium maintains its pH within very narrow limits is not evident from these studies. However, the data of the in vitro CO2 equilibration of homogenates indicate that this increased capacity to regulate pH was not brought about by an elevation of the intracellular concentration of nonbicarbonate buffers because no difference was observed in &II or ptiss between homogenates obtained from normal and from hypertrophic hearts. CO? equilibration of tissue homogenates is thought to reflect the intracellular concentration of nonbicarbonate buffers and has been used before to estimate the role of passive chemical buffering in overall pHi regulation (1, 13, 17). Several assumptions were involved in the use of this method in the present experiments. First, for the calculation of ,&II, we assumed that all the nonbicarbonate buffers present in the homogenate are of intracellular origin. The only buffer in the suspending medium was HCOT, and the hearts were thoroughly rinsed free of blood. A small amount of phosphate from the interstitial space may have, however, introduced an error in this assumption. However, the ratio of total interstitial volume to homogenate volume makes this possible error smaller than 1%. Calculation of Ptiss, on the other hand, and comparison of p tisqof normal and hypertrophic hearts is not influenced by this assumption. Second, it was assumed that the buffer value of the homogenate was determined only by the concentration and pK of the buffers present and that other pH-regulating mechanisms that may contribute to control intracellular pH do not have any role in the homogenized tissue. Transmembrane ion fluxes can easily be ruled out because the cells were disrupted by the process of homogenization. Proton formation by tissue metabolism is taken into account by the serial equilibration regime followed in these experiments. Finally, the assumption was made that the nature and/ or concentration of the nonbicarbonate buffers present in the cell was not altered by the process of homogenization. Assessment of the validity of this assumption by comparison of our results with other methods is not easy
ICW)
5-15
Ifr 1.69 t, 1.84
45.1 42.4
t 1.87 & 1.78
Aw
43.5 41.0
used in the equilibration.
t t
1.47 1.59
Buffer
value
n
Homogenate Range
20 21
6.40-7.60 6.40-7.60
(/?=[AHCOJ/ApH)
pH
is for
due to the scarcity of data. Ellis and Thomas (9) attempted to measure the nonbicarbonate buffer value of rat ventricle using a pH microelectrode. Their value of 77 meq/(pH x liters celI water) is considerably higher than ours; however, as the authors acknowledge, it is possibly an overestimate due to the biphasic changes in pHi produced when Pco:! is altered. Heisler and Piiper obtained a nonbicarbonate buffer value of 67 meq/(pH x liters cell water) in homogenates of rat diaphragm (13). The same authors calculated an apparent nonbicarbonate buffer value of 20 meq/(pH x liters cell water), derived from DMO-based pHi calculations in intact rat diaphragms (14). However, when the amount of HCO; transferred from the intracellular to the extracellular fluid during hypercapnia (or that transferred in the opposite direction in hypocapnia) was taken into account, then the calculated buffer value was 68 meq/(pH x liters celI water) almost identical to that obtained in the homogenates. These data strongly argue in favor of the idea that equilibration of tissue homogenates gives a reasonable estimate of the passive chemical buffering of the cell. The preceding considerations indicate that a mechanism other than an increase in the role of passive chemical buffering must be sought to explain the enhanced ability of the hypertrophic myocardial cell to regulate its pH during hypercapnia. One important pH regulatory mechanism in the myocardial cell is a transfer of HCO; (or an opposite movement of H’) from the extracellular to the intracellular fluid when Pcoz is elevated (30). This HCO; exchange accounts for a sizable portion of the HCOS that accumulates in the myocardial cell in hypercapnia (32), is dependent to some extent on the extracellular HCOT concentration (31), and takes place in the early stages of hypercapnia (32). It is not apparent, however, how this mechanism is brought about. A HCO; for Cl- exchange has been postulated for other tissues (27), but evidence for this mechanism in the myocardial celI is lacking. It is interesting to speculate whether the uptake of K by the myocardium that is observed in hypercapnia is in some way related to the pHi regulation (2, 11, 20). The K uptake seems to be dependent on the sympathetic stimulation that occurs in hypercapnia (21). The observations that P-adrenergic stimulation promotes a net K uptake by the heart (4) and that catecholamines increase the nonbicarbonate buffer value of the myocardium (26) lend indirect support to the idea of a link between K and HCOT or H’ fluxes in the regulation of myocardial celI pH. From our data, however, we cannot ascertain if the apparent increased buffer value of hypertrophic myocardia was brought about by an enhancement of this mechanism.
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CELL
PH OF
NORMAL
AND
HYPERTROPHIC
655
MYOCARDIUM
It should be noted in this context that remarkable differences appear to exist between skeletal muscle and heart muscle with respect to the direction of bicarbonate transfers in hypercapnia. According to earlier reported high chemical and low apparent buffer values (13, 14), skeletal muscle releases at least two-thirds of the amount of bicarbonate produced by buffering of CO2 by the intracellular nonbicarbonate buffers and thus shares its buffering capacity with the extracelhrlar space. In contrast to this behavior, heart muscle takes up additional bicarbonate from the extracellular space and gains a higher apparent than chemical buffer value. A more complex, and in some way intermediate, pattern has been reported for rat diaphragm (15). Inasmuch as diaphragm behaves similarly to myocardium by taking up bicarbonate in hypercapnia with extracellular pH values between 7.4 and 7.15, bicarbonate is released to the extracellular space in the more acid extracellular pH range (15). The uptake of bicarbonate and the resultant reduction of changes in intracellular pH of both heart and diaphragm in the extracellular pH range of 7.4-7.15 can be seen as a protective mechanism for the maintenance of mechanical performance in tissues of vital importance. The postulated enhanced bicarbonate uptake into the intracellular space of hypertrophic hearts in hypercapnia may then be a compensation for the possibly increased sensitivity of the myocardium to changes in intracellular pH.
It has recently been shown that norepinephrine, glucagon, and other agents that lead to an increase in the concentration of CAMP produce an elevation of the nonbicarbonate buffer value of normal myocardial cells (8, 10, 26). It is interesting to consider this as a possible mechanism for the changes observed by us, in light of the recent data that suggest a central role for norepinephrine in the development of myocardial hypertrophy (l&23). Even though norepinephrine stores are depleted in the later stages of myocardial hypertrophy and failure (6,24), it has been suggested that norepinephrine release from the adrenergic nerve terminals in the myocardium is increased in the initial stage of hypertrophy (3, 19). This increased norepinephrine release would in some way trigger the biochemical events that would eventually lead to myocardial hypertrophy (l&19). Further research is necessary to ascertain the validity of this hypothesis and of the role of norepinephrine in the increased buffer value of hypertrophic myocardium. The skillful technical assistance of Mr. G. Forcht is gratefully acknowledged. This work was supported in part by a grant-in-aid from the American Heart Association and with funds contributed in part by the American Heart Association, Kansas Affiliate, Inc. N. C. Gonzalez is a fellow of the Alexander von Humboldt Stiftung, Bonn, West Germany. Received
2 January
1979; accepted
in final
form
11 May
1979.
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15. HEISLER, N. Intracellular pH of isolated rat diaphragm muscle with metabolic and respiratory changes of extracellular pH. Respir. Physiol. 23: 243-255, 1975. 16. LAI, Y. L., B. A. ATTEBERY, AND E. B. BROWN, JR. Intracellular adjustments of skeletal muscle, heart and brain to prolonged hypercapnia. Respir. Physiol. 19: 115-122, 1973. 17. LAI, Y. L., B. A. ATTEBERY, AND E. B. BROWN, JR. Mechanism of cardiac muscle adjustment to hypercapnia. Respir. Physiol. 19: 123-129, 1973. 18. LAKS, M., F. MORADY, AND H. J. C. SWAN. Myocardial hypertrophy produced by chronic infusion of subhypertensive doses of norepinephrine in the dog. Chest 64: 75-78, 1973. 19. LAKS, M. M., AND F. MORADY. Norepinephrine-the myocardial hypertrophy hormone? Am. Heart. J. 91: 674-675, 1976. 20. MITHOEFFER, J. C., H. KAZEMI, F. D. HOLFORD, AND I. FRIEDMAN. Myocardial potassium exchange during respiratory acidosis: the interaction of carbon dioxide and sympathoadrenal discharge. Respir. PhysioZ. 5: 91-107, 1968. 21. MORRIS, M. E., AND R. A. MILLER. Blood pH/plasma catecholamine relationships: respiratory acidosis. Br. J. Anaesth. 34: 672681, 1962. 22. NG, M. L., M. N. LEVY, AND H. A. ZIESKE. Effect of changes of pH and of carbon dioxide tension on left ventricular performance. Am. J. Physiol. 213: 115-120, 1967. 23. OSTMAN, I., N. SJOSTUND, AND G. SWEDIN. Cardiac norepinephrine turnover and urinary catecholamine excretion in trained and untrained rats during rest and exercise. Acta Physiol. Stand. 86: 29% 307, 1972. 24. POOLE, P., J. W. COVELL, M. LEVITT, J. GIBB, AND E. BRAUNWALD. Reduction of cardiac tyrosine hydroxylase activity in experimental congestive heart failure: its role in the depletion of cardiac norepinephrine stores. Circ. Res. 20: 349-361, 1967. 25. POOLE-WILSON, P. A., AND G. A. LANGER. Effect of pH on ionic exchange and function in rat and rabbit myocardium. Am. J. PhysioZ. 229: 570-581, 1975. 26. RIEGLE, K. M., AND R. L. CLANCY. The effect of norepinephrine on myocardial intracellular hydrogen ion concentration. Am. J. PhysioZ. 229: 344-349, 1975. 27. RUSSELL, J. M., AND W. F. BORON. Role of chloride transport in
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656 regulation 28.
GONZALEZ, of intracellular
pH. Nature
London
264: 73-74,
1976.
SABOROWSKI, F., C. SCHOLAND, D. LANG, AND C. ALBERS. Intracellular muscle
pH and CO2 combining curve of hypertrophic cardiac in rats. Respir. Physiol. 18: 171-177, 1973. 29. SPURR, G. B., AND H. LAMBERT. Cardiac and skeletal muscle electrolytes in acute respiratory alkalemia and acidemia. J. Appl. PhysioZ. 15: 459-464, 1960. 30. STROME, D. R., R. L. CLANCY, AND N. C. GONZALEZ. Myocardial CO2 buffering: role of transmembrane transport of H’ or HC03ions. Am. J. PhysioZ. 230: 1037-1041, 1976. 31. STROME, D.R.,R.L. CLANCY,ANDN.C.GONZALEZ. Determinants of transmembrane bicarbonate flux during acid-base changes. J. AppZ. PhysioZ.: Respirat. Environ. Exercise Physiol. 43: 925-930, 1977.
WEMKEN,
AND
HEISLER
CLANCY,AND N.C.GONZALEZ. Contribution of a transmembrane HCOS- flux to intracellular acid-base regulation. J. AppZ. Physiol.: Respirat Environ. Exercise Physiol. 43: 931-935, 1977. 33. WADDELL, W. J., AND T. C. BUTLER. Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidine dione (DMO). Application to skeletal muscle of the dog. J. CZin. Invest. 38: 720-729, 1959. 34. ZAK, R. Protein metabolism in the overloaded myocardium. Adv. Cardiot. 18: 46-56, 1976. 35. ZIMMER, H. G., G. STEINKOPFF, AND E. GERLACH. Changes of protein synthesis in the hypertrophic rat heart. Pfluegers Arch. 336: 311-325, 1972.
32.
STROME, D.R.,R.L.
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pH regulation of normal rat myocardium
and
C. GONZALEZ, HANS-GEORG WEMKEN, AND NORBERT HEISLER of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103; and of Physiology, Max-Planck-Institute for Experimental Medicine, Gottingen, Germany
GONZALEZ, NORBERTO C., HANS-GEORG WEMKEN, AND NORBERT HEISLER. IntracellularpI regulation of normal and hypertrophic rat myocardium. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 47(4): 651-656, 1979.-The myocardial cell pH (pHi) observed during breathing of 0, 7.5,or 10% CO2 in air for 3 h was studied in rats with myocardial hypertrophy due to aortic stenosis and in sham-operated rats. The change in pHi during hypercapnia was significantly smaller in the rats with myocardial hypertrophy, with the apparent nonbicarbonate buffer value (A[HCOy]i/ApHi) being almost three times that of the sham-operated rats. In vitro CO2 equilibration of myocardial tissue homogenates showed no difference in nonbicarbonate buffer value between homogenates obtained from normal rats and from rats with myocardial hypertrophy. Therefore, it appears that the increased ability of the myocardial cell to regulate its pH during hypertrophy is not due to an increase in the cellular level of nonbicarbonate buffers, but seems to be related to a larger bicarbonate uptake by the myocardial cell during hypercapnia.
present experiments were undertaken to determine if this factor played a role in the elevation of the apparent nonbicarbonate buffer value of myocardial hypertrophy. METHODS
Production of myocardial hypertrophy. Male SpragueDawley rats of 250-300 g body weight were placed in a large beaker containing cotton saturated with halothane. Once a light level of anesthesia was achieved, the trachea was intubated and anesthesia maintained with halothane delivered through a positive-pressure respirator. The abdominal aorta was exposed through a left flank incision and a ligature was passed around it. A l-mm-diameter wire was placed over the exposed section of the aorta and the ligature, which included the vessel and the wire, was fastened. The wire was then removed, leaving the aorta narrowed to a standardized diameter. An approximately equal number of rats were sham operated on the same intracellular buffering; cardiac nonbicarbonate buffer value; transmembrane HCOT flux; aortic stenosis-myocardial hyperday that aortic constriction was performed. The sham trophy operation included the same maneuvers, except for the constriction of the aorta. Experiments in intact animals. Six to 10 days after THE MYOCARDIAL CELL PH changes less than that of surgery, rats with aortic constriction and sham-operated skeletal muscle or blood when an intact animal is exposed rats were anesthetized with halothane, bilaterally neto hypercapnia of short duration (7, 16). It has become phrectomized, and a catheter was introduced in the left apparent that various factors contribute to the regulation carotid artery. The animals were aIlowed to recover from of cell pH; these include chemical buffering, exchange of the anesthesia, weighed, and placed in a plastic chamber H+ or HCO: ions with the extracellular fluid, and changes where they could breathe either air or 7.5 or 10% CO2 in in the production of acid metabolites. Part of the re- air. A maximum of six animals was used in each experimarkable ability of the myocardial cell to regulate its pH ment, and care was taken to include an approximately seems to be due to a H+ or HCO; exchange between equal number of sham-operated rats and rats with aortic cellular and extracellular fluid that takes part in the early constriction in every experiment. The animals could be stages of hypercapnia (30). introduced and removed from the chamber without apIt has been reported that in myocardial hypertrophy preciable change in the chamber’s gas composition. Each this ability to regulate cell pH is even more pronounced animal was exposed to a given gas mixture for 3 h. As (28). Consequently, the apparent nonbicarbonate buffer soon as they were placed into the chamber, the animals value of the cell fluid, i.e., the ratio of change in cell were injected with 5,5-[ 14C]dimethyloxazolidine-2,4bicarbonate concentration to change in cell pH, is higher dione (DMO) and “H[inulin]. Arterial blood samples were in myocardial hypertrophy than in normal hearts. This obtained 165 and 180 min after the animals were introobservation is of considerable interest in light of the duced into the chamber. Immediately after the last blood influence of acid-base changes on the mechanical per- sample was taken, the animals were killed by an overdose formance of the heart (5, 12, 22, 25). Hypertrophy is of pentobarbital sodium and removed from the chamber. accompanied by major biochemical changes that could The heart was quickly excised, blotted in filter paper, result in the apparent nonbicarbonate buffer value being and weighed. The blood samples were analyzed for pH, increased simply due to the elevation of the intracellular Pcoz, and POT with appropriate electrodes at 37°C. concentration of nonbicarbonate buffers (34, 35). The Plasma was separated by centrifugation immediately 0161-7567/79/0000-OOOOooo$Ol.25
Copyright
0 1979 the American
Physiological
Society
651
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652 after sampling. A weighed amount of plasma was placed on ftiter paper, oxidized in a model 306 Packard automatic sample oxidizer, and analyzed for 14C and 3H activity by liquid scintillation counting. Duplicate samples of the left ventricle were placed on filter paper, dried for 48 h at 100°C, and oxidized for later analysis of 14C and “H activity. Tissue water content was determined by weight difference before and after drying. Determination of the chemical nonbicarbonate buffer value of the myocardium. This was done by in vitro equilibration of myocardial tissue homogenates with various CO* tensions. Six to 10 days after surgery the animals were weighed and killed with an overdose of halothane. The heart was rapidly removed, rinsed in Ringer solution, blotted dry in filter paper, and frozen in liquid N2. The left ventricle was weighed while frozen and transferred to a mortar where it was ground to a fine powder under liquid N2. The tissue was diluted with Ringer solution (composition [meq/l]: Na+, 147.2; K’, 5.0; Ca’+, 2.6; Mg2+, 2.4; SO:-, 2.4; HCOT, 24.0; Cl-, 124.8). The ratio of homogenate water weight to tissue water weight averaged 3.73 t 0.076. The homogenate was placed in the cuvette of a tonometer where it was equilibrated with 2, 5, and 15% COn in 02 at 37OC. A single-unit double electrolyte bridge pH macroelectrode was introduced into the cuvette and pH of the homogenate was recorded continuously. Adequate mixing was ensured by intermittent rotation of the cuvette. The homogenate was initially exposed to 5% C02, and equilibration was maintained until a steady pH value was obtained, usually 20-30 min later. The gas mixture was switched to either 15 or 2% CO2 in 02. After equilibration with the new mixture was complete, the homogenate was again equilibrated with 5% CO2. In this way the acid drift that was occasionally observed as a function of time was taken into account by averaging the two values obtained during 5% CO2 equilibration before and after equilibration with 2 or 15% CO2. A typical run would consist of the following sequence of equilibrations: 5, 2, 5, 15, and 5% CO2 in 02. At least one of such runs was completed in each homogenate. In some experiments pH was altered by addition of small amounts of 0.5 M NaHC03, and another equilibration run was carried out. Calculations Experiments in intact animals. Intracellular pH (pHi) was derived from the distribution of DMO (33). Inulin was used as an extracellular marker, and the extracellular space (Qe) was expressed as the ratio of extracellular water to total tissue water. Intracellular HCOT concentration, [HCO-] 3 i was calculated from pHi and Pace, values, using pK’1 of 6.1 and cyco, of 0.03 mmol/(Torr x kg). Extracellular HCOi concentration, [ HCO;], was calculated from pH, and Pace, values using pW1 of 6.1 and (XCO,of 0.03 mmol/(Torr x kg). In vitro experiments. The HCO; concentration of the homogenate was calculated from pH and Pcoz values, using a pK’, of 6.1 and cyCO, of 0.03 mmol/(Torr x kg). The buffer value of the homogenate (Phomog) was calculated in the following manner.
GONZALEZ,
Phomog
W([HCG]1
WEMKEN,
+ [HCO&)
AND
HEISLER
- [HCO&
= W(pW
+
pH3)
-
pH2
where 1 and 3 refer to the values obtained in the initial and final equilibration with 5% COZ, and 2 refers to the values obtained equilibrating with either 2 or 15% CO2. Phomogwas expressed in mmol/(pH x kg homogenate). The buffer value of the tissue (Pti.ss) was
Ptiss
=
Phomog
x
weight homogenate weight tissue
Ptisy was expressed in mmol/(pH X kg tissue). The buffer value of the intracellular fluid (&) was calculated in the following way.
Pce11 =
Ptiss FH~O
X
(I-Qe)
where FH~O is the fractional water content of the tissue and Qe is the ratio of extracellular water to total tissue water. The values for FH~O and Qe employed were those obtained in the corresponding in vivo experiments. Pcell was expressed in mmol/(pH x kg intracellular water). RESULTS
Production of myocardial hypertrophy. The technique of abdominal aortic constriction was quite effective in producing myocardial hypertrophy, as evidenced by the heart-to-body weight ratios that were 2.45 t 0.03 x lo-” in the sham-operated rats and 2.93 t 0.08 x lo-” in the rats with aortic stenosis ( P < 0.01). These results agree with previous data using the same technique (28). Experiments with intact animals. Table 1 summarizes the data obtained with sham-operated and aortic stenosis rats breathing air and the hypercapni C gas mixtures. No differences can be observed between the intra- or extracellular acid-base parameters of the aortic stenosis and sham-operated groups breathing air. The only significant difference between these two groups was observed in Qe, which was significantly higher ( P < 0.01) in the aortic stenosis group. Breathing 7.5% CO2 in air resulted in slightly higher Pace, in the aortic stenosis group than in the shamoperated group. Although the difference was significant (P < O.Ol), it was not large and probably reflected a slightly different ventilatory response to inhaled COZ. In spite of the fact that PI o, was decreased with respect to the groups breathing air, Pao, increased. This was also evident in both groups of rats breathing 10% CO2 and is the result of the marked hyperventilation elicited by increased PICO,. Intracellular pH decreased less with hypercapnia in the rats with aortic stenosis than in the sham-operated rats; the difference is much more marked in the groups breathing 10% CO2, where pHi and [HCOT]i for the aortic stenosis group are significantly different from those of the sham-operated group ( P c 0.025). The ability of the heart to regulate its cell pH during acidosis is better demonstrated by Fig. 1, where the cell pH values are plotted as a function of the corresponding extracellular pH values for the vari .OUS ~ITOUPS. The slopes ApHi/ApHe,
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CELL
PH OF
NORMAL
1. In&a-
TABLE
AND
HYPERTROPHIC
and extracellular
%Insp CO?
acid-base
parameters
[HCO,I le, mmol/l
Pa,,
7.5 10
1.1 1.0 t, 0.7
21.7 24.4 26.6
t 0.46 t 0.57 t 0.41
100.2 114.5 111.8
t, 0.8 t, 0.7 79.8 t 0.9
21.9 24.1 26.2
t, 0.43 t 0.61 t 1.02
95.6 112.2 112.8
7.416 7.233 7.142
t, 0.008 t, 0.007 AI 0.006
35.2 60.7 80.0
7.413 7.247 7.140
t 0.007
35.6 57.3
t t
[ HCOZ
Ton-
Rats 0
653
MYOCARDIUM
with k 1.8 t 2.3 t, 1.7
aortic 6.765 6.757 6.739
Sham-operated 0 7.5
10 Values myocardial
t, 0.010
t 0.007
are means -+ SE. pH,, arterial cell bicarbonate concentration;
plasma pH; [HCO;],, Qe, ratio of extracellular
t, 1.9 k 1.8 t 1.6
6.790 6.740 6.703
]i, mmol/l HA&
FH~O
n
stenosis t, 0.016 I!I 0.015 t, 0.009
5.0 t 0.2 8.4 t, 0.3 10.5 t, 0.2
0.191 0.181 0.170
t, 0.005 Ifr 0.007 AI 0.004
0.808 0.803 0.799
5.2 t 0.3 7.6 t, 0.4 9.6 t 0.3
0.168 0.164 0.146
t 0.004 + 0.003 ?z 0.003
0.803 0.802
t 0.003 t 0.003 t, 0.004
27 16 27
rats t, 0.010 t 0.019 t, 0.015
t, 0.002 t 0.002 0.794 zk 0.004
arterial plasma bicarbonate concentration; pHi, myocardial cell pH; to total tissue water; FH~o, fractional concentration of tissue water.
26 15 27
[HCOi]i,
A PHi / ApHe = -124 f 0007 l Stenosis obtained from linear regression analysis using individual experimental values are 0.337 t 0.010 for the shamoperated group and 0.124 t 0.007 for the group with o Sham Operated ApHi / ApHe -0337 f .010 aortic stenosis. These values correspond to apparent nonbicarbonate buffer values (A[HCOs]i/ApHi) of 56 and 228 mmol/(pH x liters cell water), respectively. 6.8 The total tissue water content did not show marked differences in the various groups. Qe, however, showed a tendency to decrease with hypercapnia, with Qe for the P” i aortic stenosis group being always higher than the shamoperated group. Chemical nonbicarbonate buffer value. Table 2 shows 6.7 the results of the in vitro CO2 equilibrations of myocardial homogenates. The values for Pcell were obtained using the average values of FH~O and Qe obtained in the experiments with intact rats. The average FH~O and Qe 7.4 7.1 7.2 7.3 used for the rats with aortic stenosis were 0.802 and 0.180, respectively; for the sham-operated rats they were 0.799 P”e and 0.158. The p values obtained after equilibration with FIG. 1. Changes in myocardial cell pH (pHi) as a function of the 2 and 5% CO2 are not different from those obtained extracellular pH (pH,) for rats with aortic stenosis (solid circles) and equilibrating with 5 and 15% COZ, i.e., p does not seem to sham-operated rats (open circles) breathing 0, 7.5, or 10% CO2 in air. change over a rather wide pH range. An acidification of Regression coefficients ApHi/ApH, were calculated from the individual the homogenate with time was observed in some of the PHI andpHi values. experiments, as it has been shown in other tissues using similar methods (13, 17). The acid shift, however,- was although the extracellular fluid volume was higher in the highly variable in magnitude and apparently not influenced by initial homogenate pH or Pcog, duration of rats with aortic stenosis than in the sham-operated rats. It is possible that aortic stenosis may have resulted in an equilibration, or type of heart (i.e., normal or hypertrophic) being equilibrated. When in approximately one- elevation of coronary capillary pressure with a resulting half of the experiments the HCO: concentration was increase in interstitial fluid volume. Both in the shamoperated and in the aortic stenosis rats, hypercapnia was increased by about 10 mM/kg homogenate, no appreciable change in the rate of acidification or of the homoge- accompanied by a decrease in Qe and a concomitant nate buffer value could be detected. It is evident that no increase in intracellular fluid volume. A similar finding appreciable difference exists in Ptis or Pcellbetween ho- was reported several years ago in dogs breathing 10% CO2 (29). These changes are probably due to a net mogenates of sham-operated rats and those of rats with movement of water from the extracellular to the intraaortic stenosis. Even though Qe was significantly higher cellular fluid in response to changes in osmolarity. The in stenosis (and consequently intracellular water content increase in PCOZ would result in generation of HCOi ions, was lower), this did not result in a significant difference in calculated Pcellbetween normal and hypertrophic my- both in the extracellular as well as in the intracellular fluid, according to their respective nonbicarbonate buffer ocardia. values. A given increase in PCO~ would result in more HCO; being formed in the intracellular fluid due to its DISCUSSION higher buffer value. Additional driving force for water The technique of subdiaphragmatic aortic stenosis re- shifts may have been provided by transmembrane HCO; sulted in a. prompt and reproducible inc rease in myocarfluxes (30) possibly accompanied by 0th .er ions. Although dial mass. The change in heart-to-body weight ratio was the exact mechanism of the changes in Qe cannot be not associated with an increase in total tissue water ascertained, the changes observed in Qe and in intracel-
I
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654 TABLE
---
GONZALEZ,
2. In vitro nonbicarbonate P I ,.A%,mmol/(pH
stenosis operated
27.5 26.8
t t
Values are means t SE. 2-5 and 5-15 normal and hypertrophic myocardium.
29.7 28.5
,&.II,mmol/(pH x kg
x kg t&we)
5-15 1.11 1.24
AND
HEISLER
buffer value
2-5
Aortic Sham
WEMKEN,
Avg.
t, 1.23 t_ 1.20
refer
28.6 27.7
to the percent
2-5
t 0.97 t, 1.13
41.8 39.8
CO2 concentrations
lular fluid volume both in sham-operated and aortic stenosis rats breathing 10% CO2 could have been produced by the generation of, or transfer to the intracellular fluid of approximately 10 mosmol/l of HaOi. The results obtained in the present experiments indicate that the ability of the heart to regulate its intracellular pH during hypercapnia is markedly increased in myocardial hypertrophy. These data in general agree with earlier observations of Saborowski et al. (28), although we did not observe, as they did, an increased intracellular pH in the hypertrophic hearts during normocapnia. The mechanism by which the hypertrophic myocardium maintains its pH within very narrow limits is not evident from these studies. However, the data of the in vitro CO2 equilibration of homogenates indicate that this increased capacity to regulate pH was not brought about by an elevation of the intracellular concentration of nonbicarbonate buffers because no difference was observed in &II or ptiss between homogenates obtained from normal and from hypertrophic hearts. CO? equilibration of tissue homogenates is thought to reflect the intracellular concentration of nonbicarbonate buffers and has been used before to estimate the role of passive chemical buffering in overall pHi regulation (1, 13, 17). Several assumptions were involved in the use of this method in the present experiments. First, for the calculation of ,&II, we assumed that all the nonbicarbonate buffers present in the homogenate are of intracellular origin. The only buffer in the suspending medium was HCOT, and the hearts were thoroughly rinsed free of blood. A small amount of phosphate from the interstitial space may have, however, introduced an error in this assumption. However, the ratio of total interstitial volume to homogenate volume makes this possible error smaller than 1%. Calculation of Ptiss, on the other hand, and comparison of p tisqof normal and hypertrophic hearts is not influenced by this assumption. Second, it was assumed that the buffer value of the homogenate was determined only by the concentration and pK of the buffers present and that other pH-regulating mechanisms that may contribute to control intracellular pH do not have any role in the homogenized tissue. Transmembrane ion fluxes can easily be ruled out because the cells were disrupted by the process of homogenization. Proton formation by tissue metabolism is taken into account by the serial equilibration regime followed in these experiments. Finally, the assumption was made that the nature and/ or concentration of the nonbicarbonate buffers present in the cell was not altered by the process of homogenization. Assessment of the validity of this assumption by comparison of our results with other methods is not easy
ICW)
5-15
Ifr 1.69 t, 1.84
45.1 42.4
t 1.87 & 1.78
Aw
43.5 41.0
used in the equilibration.
t t
1.47 1.59
Buffer
value
n
Homogenate Range
20 21
6.40-7.60 6.40-7.60
(/?=[AHCOJ/ApH)
pH
is for
due to the scarcity of data. Ellis and Thomas (9) attempted to measure the nonbicarbonate buffer value of rat ventricle using a pH microelectrode. Their value of 77 meq/(pH x liters celI water) is considerably higher than ours; however, as the authors acknowledge, it is possibly an overestimate due to the biphasic changes in pHi produced when Pco:! is altered. Heisler and Piiper obtained a nonbicarbonate buffer value of 67 meq/(pH x liters cell water) in homogenates of rat diaphragm (13). The same authors calculated an apparent nonbicarbonate buffer value of 20 meq/(pH x liters cell water), derived from DMO-based pHi calculations in intact rat diaphragms (14). However, when the amount of HCO; transferred from the intracellular to the extracellular fluid during hypercapnia (or that transferred in the opposite direction in hypocapnia) was taken into account, then the calculated buffer value was 68 meq/(pH x liters celI water) almost identical to that obtained in the homogenates. These data strongly argue in favor of the idea that equilibration of tissue homogenates gives a reasonable estimate of the passive chemical buffering of the cell. The preceding considerations indicate that a mechanism other than an increase in the role of passive chemical buffering must be sought to explain the enhanced ability of the hypertrophic myocardial cell to regulate its pH during hypercapnia. One important pH regulatory mechanism in the myocardial cell is a transfer of HCO; (or an opposite movement of H’) from the extracellular to the intracellular fluid when Pcoz is elevated (30). This HCO; exchange accounts for a sizable portion of the HCOS that accumulates in the myocardial cell in hypercapnia (32), is dependent to some extent on the extracellular HCOT concentration (31), and takes place in the early stages of hypercapnia (32). It is not apparent, however, how this mechanism is brought about. A HCO; for Cl- exchange has been postulated for other tissues (27), but evidence for this mechanism in the myocardial celI is lacking. It is interesting to speculate whether the uptake of K by the myocardium that is observed in hypercapnia is in some way related to the pHi regulation (2, 11, 20). The K uptake seems to be dependent on the sympathetic stimulation that occurs in hypercapnia (21). The observations that P-adrenergic stimulation promotes a net K uptake by the heart (4) and that catecholamines increase the nonbicarbonate buffer value of the myocardium (26) lend indirect support to the idea of a link between K and HCOT or H’ fluxes in the regulation of myocardial celI pH. From our data, however, we cannot ascertain if the apparent increased buffer value of hypertrophic myocardia was brought about by an enhancement of this mechanism.
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CELL
PH OF
NORMAL
AND
HYPERTROPHIC
655
MYOCARDIUM
It should be noted in this context that remarkable differences appear to exist between skeletal muscle and heart muscle with respect to the direction of bicarbonate transfers in hypercapnia. According to earlier reported high chemical and low apparent buffer values (13, 14), skeletal muscle releases at least two-thirds of the amount of bicarbonate produced by buffering of CO2 by the intracellular nonbicarbonate buffers and thus shares its buffering capacity with the extracelhrlar space. In contrast to this behavior, heart muscle takes up additional bicarbonate from the extracellular space and gains a higher apparent than chemical buffer value. A more complex, and in some way intermediate, pattern has been reported for rat diaphragm (15). Inasmuch as diaphragm behaves similarly to myocardium by taking up bicarbonate in hypercapnia with extracellular pH values between 7.4 and 7.15, bicarbonate is released to the extracellular space in the more acid extracellular pH range (15). The uptake of bicarbonate and the resultant reduction of changes in intracellular pH of both heart and diaphragm in the extracellular pH range of 7.4-7.15 can be seen as a protective mechanism for the maintenance of mechanical performance in tissues of vital importance. The postulated enhanced bicarbonate uptake into the intracellular space of hypertrophic hearts in hypercapnia may then be a compensation for the possibly increased sensitivity of the myocardium to changes in intracellular pH.
It has recently been shown that norepinephrine, glucagon, and other agents that lead to an increase in the concentration of CAMP produce an elevation of the nonbicarbonate buffer value of normal myocardial cells (8, 10, 26). It is interesting to consider this as a possible mechanism for the changes observed by us, in light of the recent data that suggest a central role for norepinephrine in the development of myocardial hypertrophy (l&23). Even though norepinephrine stores are depleted in the later stages of myocardial hypertrophy and failure (6,24), it has been suggested that norepinephrine release from the adrenergic nerve terminals in the myocardium is increased in the initial stage of hypertrophy (3, 19). This increased norepinephrine release would in some way trigger the biochemical events that would eventually lead to myocardial hypertrophy (l&19). Further research is necessary to ascertain the validity of this hypothesis and of the role of norepinephrine in the increased buffer value of hypertrophic myocardium. The skillful technical assistance of Mr. G. Forcht is gratefully acknowledged. This work was supported in part by a grant-in-aid from the American Heart Association and with funds contributed in part by the American Heart Association, Kansas Affiliate, Inc. N. C. Gonzalez is a fellow of the Alexander von Humboldt Stiftung, Bonn, West Germany. Received
2 January
1979; accepted
in final
form
11 May
1979.
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