Cytosolic pH in Cultured Cardiac Myocytes and Fibroblasts From Newborn Spontaneously Hypertensive Rats Elisabeth Devynck
Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHj) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pH in myocytes and fibro blasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 ± 0.10 ν 6.21 ± 0.10 and 5.98 ± 0.14 m g / g in 10, 5, and 7 groups of 20 to 40 animals; Ρ < .001 for both). Cytosolic pH, deter mined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in cul ture on confluent cells. The mean pHj was higher in myocytes from SHR than in those from W or WKY rats (7.19 ± 0.03, Ν = 30, ν 7.09 ± 0.03 and 7.11 ± 0.02, Ν = 25 and 30; Ρ = .008 and .024, respec tively). In contrast, pH was similar in fibroblasts from the three strains (7.21 ± 0.03, 7.18 ± 0.03, and 7.19 ± 0.02, Ν = 15,15, and 14, in SHR, W, and WKY £
£
I
ntracellular pH (pH ) plays a role in numerous bio logical and physiologic processes and, in particular constitutes a critical feature of cell activation. A small shift in pH modulates intracellular metabolic steps, such as glycolysis and DNA and protein synthe sis. A cytosolic alkalinization facilitates biosynthetic t
t
1
2,3
Received August 13, 1991. Accepted December 13, 1991. From the Department of Pharmacology, Centre National de la Re cherche Scientifique, Necker Medical School, Paris, France. This research was supported by INSERM grant 910408. Address correspondence and reprint requests to Dr C. Astarie, De partment of Pharmacology, CNRS URA1682, Necker Medical School, 156, Rue de Vaugirard, 75015 Paris, France.
Millanvoye-Van
Brussel,
rats, respectively). External acidification induced similar decreases in pHj from SHR and WKY myo cytes, maintaining higher pHj values in SHR myo cytes along the entire external pH ( p H J range studied. The inhibition of N a / H exchange by the amilor ide derivative, ethylisopropylamiloride, decreased the steady-state pHj of myocytes independently of the initial pH values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pres sure and in the absence of hemodynamic influences and specific plasma factors. Such cytosol alkalini zation present in myocytes, but not in fibroblasts, may participate in the mechanism of cardiac hyper plasia and hypertrophy previously reported in arte rial hypertension. Am J Hypertens 1992;5:281-287 +
+
£
KEY WORDS: Spontaneously hypertensive rats, cyto solic pH, cardiac myocytes, cardiac fibroblasts, fluo rescent pH indicator.
and mitotic events. In heart cells, changes in pH modu late cell excitability, cell-to-cell communication, and contractility. The established phase of arterial hypertension is espe cially associated with cardiac hypertrophy. In the spon taneously hypertensive rat from the Okamoto strain (SHR), cardiac hypertrophy is present before a signifi cant rise in blood pressure. The appearance of cardiac enlargement so early in the hypertensive process indi cated the involvement of non-pressure-dependent fac tors in the structural changes of heart. Recently, intracellular pH has become the focus of the pathophysiology of hypertension at the cellular level. 2
t
4
5
6
7
8
9
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Catherine Astarie, Monique David-Dufilho, Marianne Freyss-Beguin, and Marie-Aude
Berk et a l have proposed that the cytosolic alkalinization observed in vascular smooth muscle cells from SHR may reflect enhanced cell growth and contractile response. This study was designed to investigate cytosolic pH in spontaneously contractile myocytes and fibroblasts from SHR in the absence of hemodynamic influence. Experiments were performed on separate cultures of cardiac myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive (W) and Wistar-Kyoto controls (WKY). 10
10
METHODS
Cell Cultures Heart cell cultures were prepared as previously described. Cells were isolated from the minced hearts by repeated 5-min incubations at 25 °C with 0.05 to 0 . 1 % (w/v) trypsin (Difco, Detroit, MI) in a C a - and Mg -free buffered solution (pH 7.4). Myocytes and fibroblasts were separated by differen tial attachment. Trypsinized cells were plated at a den sity of 3.2 Χ 1 0 cells/cm on a glass coverslip enclosed in a Leighton tube (Costar, Cambridge, MA) containing Eagle's minimum essential medium (MEM; Gibco-BRL, Cergy-Pontoise, France) supplemented with antibiotics and 10% newborn calf serum (Gibco-BRL). After incu bation for 20 to 30 min at 37°C, 9 5 % of the adherent cells were fibroblasts. We centrifuged the supernatant, which essentially contained myocytes, resuspended the pellet in MEM, and distributed it on glass coverslips. Myocytes and fibroblasts were grown for 8 days. The culture medium was renewed at the first and the sixth days in culture, and when necessary, the pH was adjusted to 7.4 with a sterile 8.4% N a H C 0 solution. The proliferation of the residual fibroblasts in the myocyte cultures was limited to less than 10% by addi tion, 48 and 72 h after plating, of /?-D-arabinofuranosyl-cytosine (AFC; Sigma, St. Louis, MO) at a final concentration of 1 //mol/L. As previously demon strated, this procedure did not affect the viability and metabolic activity of the cells. Myocytes retained several characteristic features of the intact heart and, in particu lar, a spontaneous beating activity. Contractile activity became synchronous at the fourth day in culture and reached 101 ± 9 beats per minute (bpm) (N = 21) in 6-day-old cells. The pH determinations were performed on confluent cells between the sixth and eighth days after plating. 11,12
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2+
5
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3
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14
2
2
4
4
3
15
5
Calibration of p H versus fluorescence intensities was performed using the K -ionophore, nigericin, as de scribed by Thomas et a l . BCECF-loaded cells were equilibrated into K -rich medium (buffer A containing 10 mmol/L NaCl and 130 mmol/L KC1, adjusted at 37°C with 1 Ν base-Tris to pH ranging from 6.65 to 7.65) and treated with 3 //g/mL nigericin, which al lowed the external pH to set ρΗ . As expected, the ratio I 5 0 3 / 1 4 4 0 changed linearly with pH values (Figure 2). The spectral properties of BCECF were similar for either type of cell (fibroblasts or myocytes) or rat strain (Wistar or SHR), thereby allowing the same calibration curve. :
+
16
+
4
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Pregnant spontaneously hypertensive rats and normo tensive W and WKY rats were supplied by Janvier (Le Genest-Saint-Isle, France). Three 4-day-old rats were weighed and anesthetized with ether. Their hearts were aseptically removed, washed with saline medium, and weighed.
Intracellular pH Determinations The pH-sensitive fluorescent probe, BCECF, was used to monitor cytoso lic p H in monolayered cardiac cells. We replaced the culture medium 30 min before each experiment with buffer A (in mmol/L: NaCl 108, KC1 5.4, CaCl 1.8, N a H P 0 0.8, M g S 0 0.8, N a H C 0 4, glucose 5.5, HEPES 25, pH 7.4 at 37°C) containing a mixture of amino acids and glutamine, and supplemented with 10% newborn calf serum. We then incubated cells at 37°C with 2 //mol/L BCECF-AM (Calbiochem, San Diego, CA) for 30 min. Just before fluorescence measure ments, cells were rinsed twice with buffer A containing 1 % serum and the coverslip mounted in a 3 mL quartz cuvette designed to allow frontal fluorescence measure ments (Figure 1). To minimize polarization and attenu ate the participation of reflected light in the emission signal light was measured at an angle under 56 ° perpen dicular to the coverslip plane. The fluorescence inten sities were recorded on a spectrofluorimeter Spex CM111 (Instrument-SA, Madison, NJ) equipped with a 450 W xenon lamp, two excitation monochromators, and a dual mirror chopping mechanism which allowed a rapidly alternating excitation at 440 and 503 nm, with an emission wavelength of 531 nm (1.8 and 4.5 nm excitation and emission bandwidth, respectively). Cyto solic pH was directly calculated from the ratio of excita tion fluorescence intensities ( Ι 5 0 3 / Ι 4 4 0 ) · All records were performed at 37° C on adherent cells bathed in buffer A supplemented with 1 % serum. The background fluores cence from cells represented 25.4 ± 2 . 4 and 4.1 ± 0.3% (N = 13) of the intracellular BCECF signal at 440 and 503 nm excitation wavelengths, respectively, and was systematically subtracted from each recording. The in tracellular BCECF concentration averaged 9.47 ± 0.89 10~ m o l / L and did not affect the spontaneous con tractile activity of myocytes evaluated by their beat ing frequency (115 ± 3 8 and 106 ± 33 beats/min, Ν = 4, before and after cell incubation with 2 //mol/L BCECF-AM, respectively). The rate of dye leakage from the cells, evaluated by the appearance of the BCECF characteristic spectra in the medium, was 10 to 20% after 10 min.
(7.56 ± 0.10 ν 6.21 ± 0.10 and5.98 ± 0.14 mg/g, mean values from 10, 5, and 7 groups of 20 to 40 animals respectively; Ρ < .001 for both). In each of the six groups studied, in myocytes or fibro blasts from SHR, W, or WKY rats, pH remained stable between the sixth and eighth days in culture. We ana lyzed results by pooling the pH determinations ob RESULTS tained during these days. In cardiac myocytes which At the third day after birth, the relative heart weight, were spontaneously contractile at that time, the steadydetermined as the ratio of heart weight to body weight, state pHj values ranged between 6.90 and 7.57. Despite was greater in SHR than in W and WKY rats this scattering, the mean steady-state pH value in creased in myocytes from newborn SHR (7.19 ± 0.03, Ν = 30), compared with W and WKY (F = 4.74, df (2,82), Ρ = .017), in which pH averaged 7.09 ± 0.03 and 7.11 ± 0.02, respectively (N = 25 and 30, Ρ = .008 and Ρ = .024) (Figure 3A). In fibroblasts, pH did not differ between the three rat strains (SHR 7.21 ± 0.03, Ν = 15; WKY 7.19 ± 0.02, Ν = 15; and W 7.18 ± 0.03, Ν = 14) (Figure 3B). The cytosol of fibroblasts was more alkaline than that of myocytes in the two normotensive strains (P < .01 for both), whereas in SHR, no pH dif ference was observed between the two types of cells. The similarities of pHj values in W and WKY led us to restrict further comparison to SHR and WKY only. To verify that the pH increase in SHR myocytes was not caused by an alteration in intracellular buffering capacity, we measured buffering power using the NH C1 titration technique. Cell incubation for 2 min with 20 mmol/L NH C1 allowed us to calculate the buf fering capacities for SHR, WKY, and W myocytes, which averaged 58.0 ± 13.8, 74.0 ± 7.2, and 57.9 ± 8.3 7.70 6.50 6.90 7.30 m m o l / L / p H units at basal pH values, Ν = 9, 8, and 8, EXTERNAL pH respectively. To analyze further the cytosolic alkalinization asso FIGURE 2. Intracellular calibration of BCECF fluorescence in ciated with genetic hypertension, we investigated the tensities in myocytes (O, · ) and fibroblasts (Δ, A) from SHR ( · , A) effect of external pH (pH ) on pH . Myocytes and fibro and WKYfO, Δ). The experiments were performed directly into the blasts were bathed in buffer A ranging from a pH of 6.60 cuvette on adherent cells bathed into a rich K -medium contain to 7.70, and pH was followed until it reached a new ing 3 μg/mL of nigericin and adjusted to various pH values by steady state level. Under these conditions, pH was a successive additions of IN Tris-base. Individual pH values were determined for 10 monolayered myocytes and fibroblasts from two linear function of the imposed pH both in myocytes and fibroblasts (Figure 4). The linear regression analysis independent cultures. t
A
t
4
4
4
4
17
4
4
t
D
4
+
t
4
t
G
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Statistical Analysis All results are expressed as mean ± SEM. Experiments involving multiple groups were first submitted to analysis of variance. Statistical significance was assessed by the Mann-Whitney U test.
MYOCYTES 7.80
p=0.008 • =0.024-j
Γ
7.60
FIGURE 3. Individual values of intra cellular pH in cardiac myocytes (A) and fibroblasts (B) from SHR, WKY, and W. Resting pH values were determined in BCECF-loaded cells. Values were obtained from six independent cultures. The signifi cances of the differences between strains were calculated by the nonparametric Mann-Whitney test.
FIBROBLASTS
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χ CL
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Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHj) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pH in myocytes and fibro blasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 ± 0.10 ν 6.21 ± 0.10 and 5.98 ± 0.14 m g / g in 10, 5, and 7 groups of 20 to 40 animals; Ρ < .001 for both). Cytosolic pH, deter mined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in cul ture on confluent cells. The mean pHj was higher in myocytes from SHR than in those from W or WKY rats (7.19 ± 0.03, Ν = 30, ν 7.09 ± 0.03 and 7.11 ± 0.02, Ν = 25 and 30; Ρ = .008 and .024, respec tively). In contrast, pH was similar in fibroblasts from the three strains (7.21 ± 0.03, 7.18 ± 0.03, and 7.19 ± 0.02, Ν = 15,15, and 14, in SHR, W, and WKY £
£
I
ntracellular pH (pH ) plays a role in numerous bio logical and physiologic processes and, in particular constitutes a critical feature of cell activation. A small shift in pH modulates intracellular metabolic steps, such as glycolysis and DNA and protein synthe sis. A cytosolic alkalinization facilitates biosynthetic t
t
1
2,3
Received August 13, 1991. Accepted December 13, 1991. From the Department of Pharmacology, Centre National de la Re cherche Scientifique, Necker Medical School, Paris, France. This research was supported by INSERM grant 910408. Address correspondence and reprint requests to Dr C. Astarie, De partment of Pharmacology, CNRS URA1682, Necker Medical School, 156, Rue de Vaugirard, 75015 Paris, France.
Millanvoye-Van
Brussel,
rats, respectively). External acidification induced similar decreases in pHj from SHR and WKY myo cytes, maintaining higher pHj values in SHR myo cytes along the entire external pH ( p H J range studied. The inhibition of N a / H exchange by the amilor ide derivative, ethylisopropylamiloride, decreased the steady-state pHj of myocytes independently of the initial pH values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pres sure and in the absence of hemodynamic influences and specific plasma factors. Such cytosol alkalini zation present in myocytes, but not in fibroblasts, may participate in the mechanism of cardiac hyper plasia and hypertrophy previously reported in arte rial hypertension. Am J Hypertens 1992;5:281-287 +
+
£
KEY WORDS: Spontaneously hypertensive rats, cyto solic pH, cardiac myocytes, cardiac fibroblasts, fluo rescent pH indicator.
and mitotic events. In heart cells, changes in pH modu late cell excitability, cell-to-cell communication, and contractility. The established phase of arterial hypertension is espe cially associated with cardiac hypertrophy. In the spon taneously hypertensive rat from the Okamoto strain (SHR), cardiac hypertrophy is present before a signifi cant rise in blood pressure. The appearance of cardiac enlargement so early in the hypertensive process indi cated the involvement of non-pressure-dependent fac tors in the structural changes of heart. Recently, intracellular pH has become the focus of the pathophysiology of hypertension at the cellular level. 2
t
4
5
6
7
8
9
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Catherine Astarie, Monique David-Dufilho, Marianne Freyss-Beguin, and Marie-Aude
Berk et a l have proposed that the cytosolic alkalinization observed in vascular smooth muscle cells from SHR may reflect enhanced cell growth and contractile response. This study was designed to investigate cytosolic pH in spontaneously contractile myocytes and fibroblasts from SHR in the absence of hemodynamic influence. Experiments were performed on separate cultures of cardiac myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive (W) and Wistar-Kyoto controls (WKY). 10
10
METHODS
Cell Cultures Heart cell cultures were prepared as previously described. Cells were isolated from the minced hearts by repeated 5-min incubations at 25 °C with 0.05 to 0 . 1 % (w/v) trypsin (Difco, Detroit, MI) in a C a - and Mg -free buffered solution (pH 7.4). Myocytes and fibroblasts were separated by differen tial attachment. Trypsinized cells were plated at a den sity of 3.2 Χ 1 0 cells/cm on a glass coverslip enclosed in a Leighton tube (Costar, Cambridge, MA) containing Eagle's minimum essential medium (MEM; Gibco-BRL, Cergy-Pontoise, France) supplemented with antibiotics and 10% newborn calf serum (Gibco-BRL). After incu bation for 20 to 30 min at 37°C, 9 5 % of the adherent cells were fibroblasts. We centrifuged the supernatant, which essentially contained myocytes, resuspended the pellet in MEM, and distributed it on glass coverslips. Myocytes and fibroblasts were grown for 8 days. The culture medium was renewed at the first and the sixth days in culture, and when necessary, the pH was adjusted to 7.4 with a sterile 8.4% N a H C 0 solution. The proliferation of the residual fibroblasts in the myocyte cultures was limited to less than 10% by addi tion, 48 and 72 h after plating, of /?-D-arabinofuranosyl-cytosine (AFC; Sigma, St. Louis, MO) at a final concentration of 1 //mol/L. As previously demon strated, this procedure did not affect the viability and metabolic activity of the cells. Myocytes retained several characteristic features of the intact heart and, in particu lar, a spontaneous beating activity. Contractile activity became synchronous at the fourth day in culture and reached 101 ± 9 beats per minute (bpm) (N = 21) in 6-day-old cells. The pH determinations were performed on confluent cells between the sixth and eighth days after plating. 11,12
2+
2+
5
2
3
13
14
2
2
4
4
3
15
5
Calibration of p H versus fluorescence intensities was performed using the K -ionophore, nigericin, as de scribed by Thomas et a l . BCECF-loaded cells were equilibrated into K -rich medium (buffer A containing 10 mmol/L NaCl and 130 mmol/L KC1, adjusted at 37°C with 1 Ν base-Tris to pH ranging from 6.65 to 7.65) and treated with 3 //g/mL nigericin, which al lowed the external pH to set ρΗ . As expected, the ratio I 5 0 3 / 1 4 4 0 changed linearly with pH values (Figure 2). The spectral properties of BCECF were similar for either type of cell (fibroblasts or myocytes) or rat strain (Wistar or SHR), thereby allowing the same calibration curve. :
+
16
+
4
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Pregnant spontaneously hypertensive rats and normo tensive W and WKY rats were supplied by Janvier (Le Genest-Saint-Isle, France). Three 4-day-old rats were weighed and anesthetized with ether. Their hearts were aseptically removed, washed with saline medium, and weighed.
Intracellular pH Determinations The pH-sensitive fluorescent probe, BCECF, was used to monitor cytoso lic p H in monolayered cardiac cells. We replaced the culture medium 30 min before each experiment with buffer A (in mmol/L: NaCl 108, KC1 5.4, CaCl 1.8, N a H P 0 0.8, M g S 0 0.8, N a H C 0 4, glucose 5.5, HEPES 25, pH 7.4 at 37°C) containing a mixture of amino acids and glutamine, and supplemented with 10% newborn calf serum. We then incubated cells at 37°C with 2 //mol/L BCECF-AM (Calbiochem, San Diego, CA) for 30 min. Just before fluorescence measure ments, cells were rinsed twice with buffer A containing 1 % serum and the coverslip mounted in a 3 mL quartz cuvette designed to allow frontal fluorescence measure ments (Figure 1). To minimize polarization and attenu ate the participation of reflected light in the emission signal light was measured at an angle under 56 ° perpen dicular to the coverslip plane. The fluorescence inten sities were recorded on a spectrofluorimeter Spex CM111 (Instrument-SA, Madison, NJ) equipped with a 450 W xenon lamp, two excitation monochromators, and a dual mirror chopping mechanism which allowed a rapidly alternating excitation at 440 and 503 nm, with an emission wavelength of 531 nm (1.8 and 4.5 nm excitation and emission bandwidth, respectively). Cyto solic pH was directly calculated from the ratio of excita tion fluorescence intensities ( Ι 5 0 3 / Ι 4 4 0 ) · All records were performed at 37° C on adherent cells bathed in buffer A supplemented with 1 % serum. The background fluores cence from cells represented 25.4 ± 2 . 4 and 4.1 ± 0.3% (N = 13) of the intracellular BCECF signal at 440 and 503 nm excitation wavelengths, respectively, and was systematically subtracted from each recording. The in tracellular BCECF concentration averaged 9.47 ± 0.89 10~ m o l / L and did not affect the spontaneous con tractile activity of myocytes evaluated by their beat ing frequency (115 ± 3 8 and 106 ± 33 beats/min, Ν = 4, before and after cell incubation with 2 //mol/L BCECF-AM, respectively). The rate of dye leakage from the cells, evaluated by the appearance of the BCECF characteristic spectra in the medium, was 10 to 20% after 10 min.
(7.56 ± 0.10 ν 6.21 ± 0.10 and5.98 ± 0.14 mg/g, mean values from 10, 5, and 7 groups of 20 to 40 animals respectively; Ρ < .001 for both). In each of the six groups studied, in myocytes or fibro blasts from SHR, W, or WKY rats, pH remained stable between the sixth and eighth days in culture. We ana lyzed results by pooling the pH determinations ob RESULTS tained during these days. In cardiac myocytes which At the third day after birth, the relative heart weight, were spontaneously contractile at that time, the steadydetermined as the ratio of heart weight to body weight, state pHj values ranged between 6.90 and 7.57. Despite was greater in SHR than in W and WKY rats this scattering, the mean steady-state pH value in creased in myocytes from newborn SHR (7.19 ± 0.03, Ν = 30), compared with W and WKY (F = 4.74, df (2,82), Ρ = .017), in which pH averaged 7.09 ± 0.03 and 7.11 ± 0.02, respectively (N = 25 and 30, Ρ = .008 and Ρ = .024) (Figure 3A). In fibroblasts, pH did not differ between the three rat strains (SHR 7.21 ± 0.03, Ν = 15; WKY 7.19 ± 0.02, Ν = 15; and W 7.18 ± 0.03, Ν = 14) (Figure 3B). The cytosol of fibroblasts was more alkaline than that of myocytes in the two normotensive strains (P < .01 for both), whereas in SHR, no pH dif ference was observed between the two types of cells. The similarities of pHj values in W and WKY led us to restrict further comparison to SHR and WKY only. To verify that the pH increase in SHR myocytes was not caused by an alteration in intracellular buffering capacity, we measured buffering power using the NH C1 titration technique. Cell incubation for 2 min with 20 mmol/L NH C1 allowed us to calculate the buf fering capacities for SHR, WKY, and W myocytes, which averaged 58.0 ± 13.8, 74.0 ± 7.2, and 57.9 ± 8.3 7.70 6.50 6.90 7.30 m m o l / L / p H units at basal pH values, Ν = 9, 8, and 8, EXTERNAL pH respectively. To analyze further the cytosolic alkalinization asso FIGURE 2. Intracellular calibration of BCECF fluorescence in ciated with genetic hypertension, we investigated the tensities in myocytes (O, · ) and fibroblasts (Δ, A) from SHR ( · , A) effect of external pH (pH ) on pH . Myocytes and fibro and WKYfO, Δ). The experiments were performed directly into the blasts were bathed in buffer A ranging from a pH of 6.60 cuvette on adherent cells bathed into a rich K -medium contain to 7.70, and pH was followed until it reached a new ing 3 μg/mL of nigericin and adjusted to various pH values by steady state level. Under these conditions, pH was a successive additions of IN Tris-base. Individual pH values were determined for 10 monolayered myocytes and fibroblasts from two linear function of the imposed pH both in myocytes and fibroblasts (Figure 4). The linear regression analysis independent cultures. t
A
t
4
4
4
4
17
4
4
t
D
4
+
t
4
t
G
Downloaded from http://ajh.oxfordjournals.org/ at University of Otago on December 7, 2016
Statistical Analysis All results are expressed as mean ± SEM. Experiments involving multiple groups were first submitted to analysis of variance. Statistical significance was assessed by the Mann-Whitney U test.
MYOCYTES 7.80
p=0.008 • =0.024-j
Γ
7.60
FIGURE 3. Individual values of intra cellular pH in cardiac myocytes (A) and fibroblasts (B) from SHR, WKY, and W. Resting pH values were determined in BCECF-loaded cells. Values were obtained from six independent cultures. The signifi cances of the differences between strains were calculated by the nonparametric Mann-Whitney test.
FIBROBLASTS
Ο
χ CL
7.40
8
{
ο LU ZD Ο
