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Hypertension. 1995;25:482-489

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(Hypertension. 1995;25:482-489.)
© 1995 American Heart Association, Inc.

Articles

pHi, [Ca2+]i, and Myosin Phosphorylation in Histamine- and NH4+-Induced Swine Carotid Artery Contraction

Xiao-Liang Chen; Christopher M. Rembold

From the Cardiovascular Division, Departments of Internal Medicine and Physiology, University of Virginia Health Sciences Center, Charlottesville.


*    Abstract
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 *Abstract
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Abstract We examined the interaction among changes in pHi, [Ca2+]i, myosin light-chain phosphorylation, and contraction in arterial smooth muscle stimulated by histamine, NH4+, Tris+, and/or changes in extracellular pH (pHo). We loaded swine carotid medial tissues with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to measure pHi or aequorin to measure [Ca2+]i. Incubation of tissues in NH4+ increased pHi, [Ca2+]i, myosin phosphorylation, and force. Washout of NH4+ decreased pHi and transiently further increased in [Ca2+]i and force. Incubation of tissues in a similar concentration of Tris+ or increasing pHo also increased pHi; however, there were only modest changes in [Ca2+]i and force. Increasing extracellular pH coincidentally with washout of NH4+ prevented the decrease in pHi but did not affect the NH4+ washout-induced contraction. These data suggest that NH4+ altered [Ca2+]i and contraction by mechanisms other than its effects on pHi. The type of pH buffer did not affect the [Ca2+]i, myosin phosphorylation, or stress response to histamine stimulation. The time course of changes in pHi was much slower than the time course of histamine-induced changes in [Ca2+]i, myosin phosphorylation, and stress. Addition of 10 mmol/L NH4+ concurrently with histamine aborted the histamine-induced decrease in pHi and significantly slowed the histamine-induced increase in [Ca2+]i, myosin phosphorylation, and stress. There was little effect on histamine-induced increases in [Ca2+]i, myosin phosphorylation, or contraction when three other protocols aborted the histamine-induced decrease in pHi. These data show that incubation in NH4+ can alter [Ca2+]i and contraction in both unstimulated and histamine-stimulated smooth muscle. However, these effects were not caused by NH4+-dependent changes in pHi. It appears that histamine-induced changes in pHi have at most minor effects on [Ca2+]i and contraction of swine carotid artery.


Key Words: aequorin • ammonium chloride • calcium • hydrogen-ion concentration • phosphorylation • muscle, smooth, vascular


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Stimulus-induced increases in [Ca2+]i are the primary determinants of arterial smooth muscle contraction. Ca2+, through activation of calmodulin, activates myosin light-chain kinase, which phosphorylates the 20-kd light chain of myosin.1 2 The phosphorylated myosin is active and interacts with the thin filament, inducing a contraction.3 Several processes modulate this signaling system. First, myosin light-chain kinase can be phosphorylated, and phosphorylated myosin light-chain kinase has a lower sensitivity to Ca2+.4 [Ca2+]i per se appears to regulate myosin light-chain kinase phosphorylation.5 This process is called Ca2+-induced desensitization. Second, contractile agonists can decrease the amount of [Ca2+]i required for increases in myosin phosphorylation, potentially by activation of a G protein.6 7 This is called agonist-induced sensitization and may represent inhibition of myosin light-chain phosphatase.8 9 Finally, some stimuli (nitrovasodilators, high extracellular [Mg2+], okadaic acid) can uncouple force generation from increases in myosin light-chain phosphorylation.10 11 12

Changes in pH affect many enzymatic processes, and the signal transduction pathway in smooth muscle is no exception. Depolarization and contractile agonist stimulation induced various degrees of intracellular acidosis in several types of intact smooth muscle tissue.13 14 15 16 In isolated smooth muscle cells, changes in pHi can alter [Ca2+]i or Ca2+ influx or efflux.17 18 19 20 21 Intracellular acidosis increased the [Ca2+]i sensitivity of force in several types of skinned smooth muscles.22 23 However, the interaction among changes in pHi, [Ca2+]i, and myosin phosphorylation has not been studied in an intact arterial smooth muscle.

The most commonly used method to change pHi is the addition of NH4+ to the bathing solution. Addition of NH4+ to smooth muscle tissues increases pHi and induces a contraction.24 25 26 Washout of NH4+ transiently decreases pHi and further increases force. The proposed mechanism for the NH4+-induced change in pHi is as follows: NH3 migrates intracellularly, is protonated to form NH4+, and therefore increases pHi. Washout of NH4+ decreases pHi by deprotonation of NH4+, migration of NH3 across the plasma membrane, and trapping of protons intracellularly.

One recent study suggested that pHi regulation may have a role in modulating [Ca2+]i in smooth muscle. In cultured smooth muscle cells, Ganz et al27 found that contractile agonists induced sustained increases in [Ca2+]i only when cells were bathed in a physiological saline that contained a zwitterionic pH buffer without added bicarbonate. There was no sustained agonist-dependent increase in [Ca2+]i in those cells bathed in a physiological saline containing bicarbonate.

This study addressed the role of changes in pHi in determining changes in [Ca2+]i in both unstimulated and histamine-stimulated swine carotid artery. We first measured changes in pHi and [Ca2+]i with several treatments that alter pHi to correlate changes in pHi with contraction. We then measured pHi, [Ca2+]i, and myosin phosphorylation during histamine stimulation in physiological saline solutions both with and without bicarbonate. Finally, we altered histamine-induced changes in pHi and measured the effect on [Ca2+]i, myosin phosphorylation, and contraction. These experiments allowed us to test two hypotheses: washout of NH4+ induces a contraction by decreasing pHi, and histamine-dependent changes in pHi are important modulators of [Ca2+]i, myosin phosphorylation, and contraction in swine carotid artery. We measured pHi with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and [Ca2+]i with aequorin in intact swine carotid artery. Aequorin was chosen as a calcium indicator because it is relatively insensitive to changes in pH in the physiological range and has excellent stability during long experiments.


*    Methods
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 *Methods
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Tissues
Swine common carotid arteries were obtained from a slaughterhouse and transported at 0°C in physiological salt solution (PSS). The 3-[N-morpholino] propane sulfonic acid (MOPS) PSS contained (mmol/L) NaCl 140, KCl 4.7, MOPS 5, Na2HPO4 1.2, CaCl2 1.6, MgSO4 1.2, and D glucose 5.6, pH adjusted to 7.4 at 37°C. The Krebs' PSS (bicarbonate buffer) contained (mmol/L) NaCl 130, KCl 4.7, NaHCO3 15, Na2HPO4 1.2, CaCl2 1.6, MgCl2 1.2, EDTA 0.02, D glucose 5.6 (pH 7.4 at 37°C when gassed with 20% O2–5% CO2–75% N2). The pH buffering capacity of the Krebs' PSS (15 mmol/L HCO3-) was higher than that of the MOPS PSS (5 mmol/L MOPS). We did not use higher concentrations of MOPS because it decreased maximal force generation. Tissues were stored at 2°C in MOPS PSS (which was exchanged for fresh PSS daily) for up to 4 days before experiments. Most experiments were performed within 48 hours of tissue collection. This storage did not affect pHi, [Ca2+]i, myosin phosphorylation, or stress measurements. Fig 1 shows calculated resting pHi (see below for method) when tissues were incubated in the MOPS or Krebs' PSS, dependent on the number of days in cold storage. Dissection of medial strips and mounting and determination of the optimum length for stress development at 37°C were performed as described.28 The intimal surface was mechanically rubbed to remove the endothelium.



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  		Figure 1. Graph shows the dependence of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated resting pHi in tissues incubated in either 3- [N-morpholino] propane sulfonic acid physiological salt solution (MOPS PSS) ({bullet}, solid regression line) or Krebs' PSS ({circ}, dashed regression line) on the number of days that the tissues were stored at 4°C before warming to 37°C, loading with BCECF, and performance of experiments. For the MOPS PSS, n=9, 17, 18, 20, and 12 for days 0 through 4, respectively. For the Krebs' PSS, n=2, 4, 3, 6, and 5 for days 0 through 4, respectively. Neither regression line was significant, suggesting that storage had no effect on basal pHi.

pHi Measurement
pHi was measured with BCECF loaded intracellularly by incubation of dissected tissues in the acetomethoxy ester of BCECF (BCECF-AM, Molecular Probes), a procedure similar to that previously described for Fura-2.29 The final loading solution was MOPS PSS containing 4 µmol/L BCECF-AM, 0.133 mg/mL Pluronic F127, and 0.66% dimethyl sulfoxide. Arterial strips were loaded at 37°C for 1 hour and washed in PSS for 1 to 1.5 hours before each experiment. The experimental apparatus consisted of a Nikon xenon light source, the output of which was directed through a rotating filter wheel that had 436±13-nm and 485±12-nm filters rotating at 3000 rpm. Excitation light passed through one arm of a bifurcated light guide, which was placed 0.5 to 1 mm from the luminal surface of the smooth muscle tissue. The tissue was isometrically mounted to a Harvard Bioscience capacitive force transducer and bathed in a 3-mL jacketed tissue bath. The second arm of the bifurcated light guide passed emission light through a 530±15-nm filter to a photomultiplier tube. Fluorescence signals were electronically demultiplexed, and the force, 436, and 485 fluorescence signals, were converted to digital signals and stored on a personal computer.

Calibration of BCECF Signals
After completion of pharmacological manipulations, tissues were incubated in a calibration solution containing (mmol/L) nigericin 0.003, KCl 145, MOPS 5, CaCl2 1.6, MgSO4 1.2, D glucose 5.6, and EDTA 0.02, pH adjusted to 6.8 or 7.4 at 37°C (this solution has no added Na+), and a two-point calibration was performed at pH 7.4 and 6.8 as described.13 We found that the relation between extracellular pH (pHo) and the 485/436 ratio was linear between pH 6.6 and 7.8 (n=5). Calculated pHi estimates are approximate because (1) some photobleaching and dye leakage occurred during the experiment, (2) nigericin may not totally equilibrate the pH gradient in all cells, and (3) protein binding can alter the fluorescent properties of BCECF.30 We were interested in the effects of changes in pHi; therefore, signals are reported as change in pHi rather than absolute pHi. This normalization decreased the variability caused exclusively by differences in estimates of basal pHi.

We entertained the possibility that the results obtained with changing pHo could result from the persistent presence of extracellular BCECF. If some BCECF were present in the extracellular space, a change in pHo would induce a change in BCECF fluorescence. We think that this possibility is unlikely because estimated pHi was substantially lower than pHo and because we washed out BCECF-AM for at least 70 minutes (much longer than the 10 minutes required for all the BCECF fluorescence to wash out of tissues loaded with 10 µmol/L BCECF free acid for 60 minutes).

[Ca2+]i Measurement
[Ca2+]i was measured in a second set of tissues loaded with aequorin as previously described.2 Simultaneous aequorin-emitted light and stress measurements were made in a light-tight enclosure. The light and force signals were collected on a personal computer. Stress was calculated as force per cross-sectional area estimated from measured length, weight, and a density of 1.050 g/cm.3 Aequorin light signals are presented as log L/Lmax, where L is the photon count (in counts per second) and Lmax is a measure of the total undischarged aequorin present in the tissue. Lmax was calculated at each time point, correcting for aequorin consumption. Aequorin light emission was calibrated in a series of Ca2+-EGTA buffers with [Mg2+]=0.5 mmol/L at 37°C.2 Basal [Ca2+]i was 69±6 nmol/L (n=25). Aequorin luminescence is insensitive to pH between 6.6 and 7.6.31

Myosin Light-Chain Phosphorylation
Phosphorylation was estimated in a third set of tissues. Prior studies revealed that aequorin loading did not alter phosphorylation time courses.2 Isometric force was measured with Grass FT.03 force transducers. Tissues were frozen by immersion in a dry ice–acetone slurry (20 g/20 mL) at -78°C. Phosphorylation of the smooth muscle–specific 20-kd myosin light chain was determined by two-dimensional electrophoretic separation.2 32 Phosphorylation is reported as moles of Pi per mole of total smooth muscle–specific light chain.


*    Results
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 up arrowMethods
 *Results
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Basal estimated pHi was 7.22±0.02 (n=76) in those tissues bathed in the MOPS PSS and 7.18±0.04 (n=20) in the Krebs' physiological saline. Incubation of swine carotid medial tissues in 20 mmol/L NH4+ (substituted stoichiometrically for Na+) induced a rapid and sustained increase in pHi (representative raw data in Fig 2; mean data in Fig 3, left). This increase in pHi was not caused by a change in pHo because pHo was held constant at 7.4 with 5 mmol/L MOPS in the MOPS PSS. The NH4+ treatment transiently decreased aequorin estimated myoplasmic [Ca2+] and did not significantly change myosin phosphorylation or stress (force normalized to cross-sectional area). An increase in NH4+ to 40 mmol/L induced a larger increase in pHi, [Ca2+]i, myosin phosphorylation, and stress. Washout of NH4+ transiently increased and then decreased pHi and further increased [Ca2+]i, myosin phosphorylation, and force. Two time points before and after washout of NH4+ (60 versus 76 minutes) had similar [Ca2+]i levels, yet pHi, myosin phosphorylation, and stress were lower after washout of NH4+ (the changes in pHi and stress were significant at P<.05). These data show that the [Ca2+]i sensitivity of force was higher during incubation in NH4+ than after washout of NH4+.



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  		Figure 2. Graph shows representative tracing of the effect of NH4+ on 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (top) and contractile stress (bottom) in tissues bathed in 3-[N-morpholino] propane sulfonic acid physiological salt solution (without bicarbonate). The tissue was stimulated with 20 mmol/L NH4+ at 10 minutes; the [NH4+] was increased to 40 mmol/L at 40 minutes; then the NH4+ was washed out at 60 minutes.



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  		Figure 3. Graphs show the effect of NH4+ or Tris+ on changes in 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (top), aequorin estimated myoplasmic [Ca2+] (second panel), myosin phosphorylation (third panel), and contractile stress (bottom) in tissues bathed in a physiological saline solution without bicarbonate. One set of tissues was stimulated with 20 mmol/L NH4+ at 10 minutes; the [NH4+] was increased to 40 mmol/L at 40 minutes; and then the NH4+ was washed out at 60 minutes (n=4 to 5). A second set of tissues was incubated in 20 mmol/L Tris+ at 90 minutes; Tris+ was increased to 40 mmol/L at 120 minutes; and then the Tris+ was washed out at 140 minutes (n=4 to 5). The mean is shown in solid lines with mean±SEM in dotted lines. Myosin phosphorylation values are shown as mean±SEM (n=4 to 6). Stress is from the BCECF-loaded tissues. Symbols without error bars reflect errors smaller than the size of the symbol. MOPS indicates 3-[N-morpholino] propane sulfonic acid.

If changes in pHi change [Ca2+]i and the [Ca2+]i sensitivity of force, other stimuli that change pHi should also alter [Ca2+]i and the [Ca2+]i sensitivity of force. We performed similar experiments with Tris+ rather than NH4+. Incubation of tissues in 20 mmol/L Tris+ (substituted stoichiometrically for Na+ at a constant pHo of 7.4) induced a rapid and sustained increase in pHi (Fig 3, right). Tris+ did not significantly change [Ca2+]i, myosin phosphorylation, or stress values. An increase in [Tris+] to 40 mmol/L further increased pHi, transiently increased [Ca2+]i, and did not change myosin phosphorylation or force. Washout of Tris+ decreased pHi some, transiently increased [Ca2+]i, and did not alter myosin phosphorylation or stress.

We evaluated the effect of changes in pHo on pHi, [Ca2+]i, and stress. Decreases in pHo did not significantly change [Ca2+]i and force (Fig 4). Increases in pHo increased pHi and induced a small contraction. Increasing pHo from 6.6 to 7.8 increased [Ca2+]i by a small but significant amount (0.11±0.03 log L/Lmax units comparing 30 to 50 minutes). The contraction induced by changes in increasing pHo was modest compared with the maximal response elicited by subsequent 145 mmol/L KCl stimulation (2.26±0.12x105 N/m2). These effects on [Ca2+]i, myosin phosphorylation, and force induced by Tris+ or changing pHo were smaller than those observed with NH4+. These results suggest that NH4+ may have effects on [Ca2+]i and contraction beyond those expected based on changes in pHi per se.



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  		Figure 4. Graphs show the effect of changes in extracellular pH (pHo) (top) on 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (second panel), aequorin estimated myoplasmic [Ca2+] (third panel), and contractile stress (bottom) in tissues bathed in a physiological saline solution without bicarbonate (n=4 to 5). The tissues were sequentially bathed in pHo 7.4, 7.0, 6.6, 7.4, and 7.8. The mean is shown in solid lines with mean±SEM in dotted lines.

Washout of NH4+ induces a decrease in pHi and a contraction. We evaluated whether the NH4+ washout contraction depends on the decrease in pHi. We washed out 10 mmol/L NH4+ from 3 sets of tissues at various levels of pHo. Washout of NH4+ at a constant pHo of 7.4 decreased pHi below basal levels and contracted the tissues (Fig 5, left). Washout of NH4+ into solutions with higher pHo (7.6 or 7.8) contracted the tissues despite attenuation of the decrease in pHi (Fig 5, center and right). In the tissues treated with pHo 7.8, 3 of the 10 tissues did not contract; however, the mean contraction induced by washout of NH4+ in all 10 tissues was not significantly different among the 3 groups. We observed similar results when 10 mmol/L Tris+ was added concurrently with washout of 10 mmol/L NH4+ (data not shown). These results suggest that factors beyond changes in pHi were responsible for NH4+ washout–dependent contraction.



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  		Figure 5. Graphs show the effect of extracellular pH (pHo) (top) on NH4+-induced changes in 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (middle) and contractile stress (bottom) in three sets of tissues bathed in a physiological saline solution without bicarbonate. The first set of tissues was incubated in 10 mmol/L NH4+ at 10 minutes, and the NH4+ was washed out at 20 minutes (left, n=5). The second set of tissues was incubated in 10 mmol/L NH4+ at 60 minutes, and the NH4+ was washed out concurrently with an increase in pHo to 7.6 at 70 minutes (middle, n=4). The third set of tissues was incubated in 10 mmol/L NH4+ at 110 minutes, and the NH4+ was washed out concurrently with an increase in pHo to 7.8 at 120 minutes (right, n=10). The mean is shown in solid lines with mean±SEM in dotted lines.

Fig 6 shows the effect of 10 µmol/L histamine stimulation on pHi, [Ca2+]i, myosin phosphorylation, and contraction in swine carotid artery. In the MOPS PSS, which contained the zwitterionic buffer, 10 µmol/L histamine transiently increased aequorin estimated [Ca2+]i, myosin phosphorylation, and stress (Fig 6, left). An intracellular acidosis developed gradually and persisted throughout the contraction. With sustained stimulation, aequorin estimated [Ca2+]i and myosin phosphorylation decreased to intermediate levels, while stress remained near maximal values. On removal of histamine, the intracellular acidosis resolved more slowly than the decrease in force ([Ca2+]i was not measured during relaxation).



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  		Figure 6. Graphs show the effect of buffer type on histamine (Hist.)-induced changes in 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (top), aequorin estimated myoplasmic [Ca2+] (second panel), myosin phosphorylation (third panel), and contractile stress (bottom) in two sets of four tissues. The first set of tissues was bathed in a physiological saline solution without bicarbonate (3-[N-morpholino] propane sulfonic acid [MOPS]) and stimulated with 10 µmol/L histamine at 10 minutes; the histamine was washed out at 20 minutes. The second set of tissues was bathed in a physiological saline solution with bicarbonate (Krebs') and stimulated with 10 µmol/L histamine at 40 minutes; the histamine was washed out at 50 minutes. The mean is shown in a solid line with mean±SEM in dotted lines. Myosin phosphorylation values are shown as mean±SEM (n=4 to 6). Symbols without error bars reflect errors smaller than the size of the symbol.

In the PSS containing bicarbonate (Krebs'), 10 µmol/L histamine also increased [Ca2+]i, myosin phosphorylation, and stress (Fig 6, right). Histamine stimulation induced a slight sustained intracellular acidosis. With sustained stimulation, [Ca2+]i and myosin phosphorylation decreased to intermediate values, while stress remained near maximal values. Changes in [Ca2+]i and myosin phosphorylation did not statistically differ from those observed with histamine stimulation in the PSS that did not contain bicarbonate (MOPS). In the PSS containing bicarbonate (Krebs'), 10 µmol/L histamine stimulation induced a more modest acidosis. Washout of histamine induced a transient enhancement of the acidosis. Myosin phosphorylation measurements revealed that loading with BCECF-AM did not change the time course of myosin phosphorylation (filled symbols represent data obtained in BCECF-AM–loaded tissues).

The next phase of this study was the evaluation of how changes in pHi alter [Ca2+]i, myosin phosphorylation, and contractility. Stimulation with 1 µmol/L histamine slowly decreased pHi and rapidly increased [Ca2+]i, myosin phosphorylation, and stress (Fig 7, left). After 10 minutes of stimulation, [Ca2+]i and myosin phosphorylation decreased to intermediate levels, and stress was maintained near peak values. Washout of histamine rapidly decreased both [Ca2+]i and stress.



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  		Figure 7. Graphs show the effect of NH4+, increased extracellular pH (pHo), and Tris+ on histamine-induced changes in 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (top), aequorin estimated myoplasmic [Ca2+] (second panel), myosin phosphorylation (third panel), and contractile stress (bottom) in tissues bathed in a physiological saline solution without bicarbonate (n=4 to 5). One set of tissues was stimulated with 1 µmol/L histamine at 10 minutes, and the histamine was washed out at 20 minutes. A second set of tissues was stimulated with 1 µmol/L histamine in the presence of 10 mmol/L NH4+ at 40 minutes, and both were washed out at 50 minutes. A third set of tissues was stimulated with 1 µmol/L histamine at pHo 7.6 at 70 minutes. The histamine was washed out and pHo returned to 7.4 at 80 minutes. A fourth set of tissues was stimulated with 1 µmol/L histamine in the presence of 10 mmol/L Tris+ at 100 minutes, and both were washed out at 110 minutes. The mean is shown in solid lines except that stress from the aequorin-loaded tissues is shown in a dashed line with mean±SEM in dotted lines. Myosin phosphorylation values are shown as mean±SEM (n=4 to 6). Symbols without error bars reflect errors smaller than the size of the symbol.

When 10 mmol/L NH4+ was added concurrently with the 1 µmol/L histamine, the histamine-induced decrease in pHi was aborted (pHo was held constant at 7.4, Fig 7, second panel from left). Aequorin estimated myoplasmic [Ca2+] acutely decreased and then gradually increased simultaneously with a very slow increase in force. The NH4+ treatment significantly decreased myosin phosphorylation values measured 1 minute after addition of histamine. After 10 minutes of histamine-NH4+ treatment, the [Ca2+]i estimate was higher than resting but less than histamine alone. Myosin phosphorylation and stress values were similar to histamine alone. Washout of NH4+ and histamine increased both [Ca2+]i and force transiently. These data suggest that treatment with NH4+ induced a biphasic effect on contraction. First, NH4+ initially decreased [Ca2+]i, which slowed the contraction by attenuating the increase in myosin phosphorylation. Second, NH4+ also increased the [Ca2+]i sensitivity of myosin phosphorylation, which enhanced contractility.

We performed a similar experiment with changes in pHo. An increase in pHo to 7.6 concurrently with the addition of 1 µmol/L histamine aborted the histamine-induced decrease in pHi (Fig 7, second panel from right). The initial [Ca2+]i and myosin phosphorylation transient were slightly attenuated compared with histamine alone. Sustained [Ca2+]i, myosin phosphorylation, and force values were similar to histamine alone. Because changes in pHo had only minor effects on [Ca2+]i, myosin phosphorylation, and contraction, we performed a similar experiment with Tris+. Addition of 10 mmol/L Tris+ concurrently with the 1 µmol/L histamine aborted the histamine-induced decrease in pHi (pHo was held constant at 7.4, Fig 7, right). The resulting increase in myosin phosphorylation and the contraction were similar to the control contraction. The preceding two experiments (Tris+ and changing pHo) suggest that abolishing the histamine-induced decrease in pHi had only minor effects on the resulting contraction. Only acute addition of NH4+ attenuated histamine-induced contractions (Fig 7, second panel from left).

We evaluated whether the time of NH4+ addition affected its inhibition of histamine-induced contraction. Potentially, the rate rather than the amount of pHi change affects the NH4+-dependent inhibition of contraction. Ten minutes before the second histamine stimulation, 10 mmol/L NH4+ was added. This treatment increased pHi (Fig 8). Addition of 1 µmol/L histamine at 40 minutes induced a contraction that was similar to the control (first) contraction despite attenuation of the histamine-induced decrease in pHi. This result suggests that either the rate of pHi change or some other effect of NH4+ affected the contraction rather than the total pHi change per se.



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  		Figure 8. Graphs show the effect of pretreatment with NH4+ on histamine-induced changes in 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) estimated pHi (top) and contractile stress (bottom) in four tissues bathed in a physiological saline solution without bicarbonate. Tissues were first stimulated with 1 µmol/L histamine at 10 minutes, and the histamine was washed out at 20 minutes. At 30 minutes, the tissues were incubated in 10 mmol/L NH4+ and stimulated with 1 µmol/L histamine 10 minutes later at 40 minutes. Both histamine and NH4+ were washed out at 50 minutes. MOPS indicates 3-[N-morpholino] propane sulfonic acid. Symbols and lines are as given in Fig 7.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The literature suggests that changes in pHi could potentially modulate [Ca2+]i and contraction of arterial smooth muscle. Contractile stimuli decreased pHi in some smooth muscles.13 14 15 16 Increased PCO2 and the subsequent acidosis hyperpolarized and relaxed cerebral blood vessels.18 33 34 Hyperpolarization probably closed Ca2+ channels, which decreased [Ca2+]i. In patch-clamp studies, one investigator18 but not another35 found that intracellular acidosis opened K+ channels that would produce hyperpolarization. Acidosis directly inhibited Ca2+ currents in patch-clamped vascular smooth muscle.19 A recent study suggests that there are at least two forms of smooth muscle–specific phospholipase C. [Ca2+] and pH differentially regulated these forms of phospholipase C.36 Therefore, pHi could potentially modulate 1,4,5-inositol trisphosphate levels (and thus Ca2+ release) and diacylglycerol formation. Acidosis may also increase [Ca2+]i by releasing passively bound [Ca2+]i.21 In skinned smooth muscle, large decreases in pHi (from 6.9 to 6.5 or 6.2) increased the [Ca2+]i sensitivity of force.22 23 These investigators used BAPTA (a Ca2+ chelator that is less sensitive to changes in pH than EGTA) to clamp [Ca2+]i at specific levels. Therefore, their results are less susceptible to artifacts from changes in pH on the EGTA buffer system. Acidosis also slowed the rate of force development by slowing changes in myosin phosphorylation.23 While these studies suggest a potential role for changes in pHi on [Ca2+]i and contraction, they cannot conclude that the changes in pHi observed with physiological stimulation have a role in contraction.

Our estimates of resting pHi are slightly higher than previously described basal pHi values. In bicarbonate-based physiological saline, measurement with double-barreled microelectrodes revealed a pHi of 7.06 in guinea pig vas deferens.37 Measurement with 31P nuclear magnetic resonance revealed a pHi of 7.10 in rabbit bladder,38 an identical value in rat uterus,39 7.00 in rabbit portal vein,40 and 7.19 in rabbit blood vessels.41 Measurement with BCECF revealed a pHi of 7.11 in canine trachealis15 and 7.15 in rat mesenteric artery.13 In physiological salines without bicarbonate, measurement with BCECF revealed a pHi of 6.85 in canine trachealis15 and 7.19 in rat mesenteric artery.13

This study addressed the role of changes in pHi in determining changes in [Ca2+]i, myosin phosphorylation, and contraction in both unstimulated and histamine-stimulated swine carotid artery. We tested two hypotheses. The first hypothesis was that washout of NH4+ induces a contraction by decreasing pHi. We found that incubation of swine carotid artery in NH4+ alone increased pHi associated with biphasic effects on [Ca2+]i and contraction. Lower concentrations of NH4+ decreased [Ca2+]i, and higher concentrations of NH4+ increased both [Ca2+]i and stress (Fig 3). Similar changes in pHi induced by incubation in Tris+ or changes in pHo had smaller effects on [Ca2+]i and contraction (Figs 3 and 4). Washout of NH4+ in varying pHo induced contractions regardless of the change in BCECF estimated pHi (Fig 5). These data show a lack of correlation between changes in BCECF estimated pHi and changes in [Ca2+]i and/or stress. We cannot conclude that NH4+-dependent changes in pHi were responsible for NH4+-dependent changes in [Ca2+]i and stress. NH4+ appears to have other effects beyond changes in pHi on [Ca2+]i and contraction. Alternatively, changes in pHi induced by acute addition of NH4+ may have a different intracellular localization than changes in pHi induced by Tris+ or changes in pHo.

We also found that the [Ca2+]i sensitivity of force was higher during incubation in NH4+ than after washout of NH4+ (Fig 3). This result could be interpreted to suggest that increases in pHi increase the [Ca2+]i sensitivity of force. However, others found that increases in pHi decreased the [Ca2+]i sensitivity of force in skinned smooth muscle.22 23 A more likely explanation of our results is that NH4+ increases the [Ca2+]i sensitivity of force independent of its effects on pHi.

The second hypothesis tested was that the histamine-dependent change in pHi is a modulator of [Ca2+]i, myosin phosphorylation, and contraction in swine carotid artery. We found that the presence of bicarbonate in the physiological saline altered histamine-induced changes in pHi. Similar effects on pHi were observed in rat mesentery stimulated with norepinephrine13 14 and in canine trachealis stimulated with carbachol.15 16 While the presence of bicarbonate in the physiological saline affected the pHi response to histamine, it did not alter the [Ca2+]i, myosin phosphorylation, or contractile response (Fig 6). Furthermore, histamine-induced changes in pHi occurred more slowly than histamine-induced contraction or the relaxation induced by washout of histamine. Therefore, the contractile behavior of smooth muscle did not correlate with changes in pHi. These data suggest that histamine-induced changes in pHi have at most minor effects on [Ca2+]i, myosin phosphorylation, and contraction in histamine-stimulated swine carotid artery. This result differs from that observed by Ganz et al,27 who found no sustained increase in [Ca2+]i in isolated smooth muscle cells stimulated by a variety of contractile agonists in a bicarbonate containing physiological saline. This difference is probably a manifestation of the dedifferentiation of smooth muscle observed in dissociated and cultured smooth muscle cells.42 Our data are consistent with the hypothesis that [Ca2+]i-dependent myosin phosphorylation is the major regulator of contractile stress in swine carotid artery.

We found several protocols that abolished the histamine-induced decrease in pHi (pretreatment with 10 mmol/L NH4+, changing pHo to 7.6, or addition of 10 mmol/L Tris+). These protocols had little effect on histamine-induced changes in [Ca2+]i, myosin phosphorylation, or stress (Fig 8 and right panels of Fig 7). From these data, it appears that the histamine-induced decrease in pHi had little effect on histamine-induced changes in [Ca2+]i, myosin phosphorylation, and contraction.

However, we found that histamine-induced increases in [Ca2+]i and myosin phosphorylation were attenuated and that the contraction was slowed when simultaneous addition of NH4+ abolished the histamine-induced acidosis (second panel from left in Fig 7). During the sustained phase of contraction, we observed an increase in the [Ca2+]i sensitivity of phosphorylation. Taken alone, these data suggest that pHi could have a role in signal transduction. However, our results with changing pHo and Tris+ (Fig 7) suggest an alternative explanation. NH4+ may alter [Ca2+]i by mechanisms other than its effects on pHi.

In conclusion, we dissociated the effects of NH4+ on pHi from its effects on contraction. It appears that NH4+ alters contractility by mechanisms other than its effects on pHi. Alternatively, NH4+-induced changes in pHi may have a different intracellular localization than changes in pHi induced by Tris+ or changes in pHo. The presence of bicarbonate in the physiological saline altered histamine-induced changes in pHi but did not alter histamine-induced changes in [Ca2+]i, myosin phosphorylation, and contraction. This finding suggests that the results of Ganz et al27 in isolated cells cannot be generalized to intact arterial smooth muscle tissues. Attenuation of a histamine-induced decrease in pHi did not alter histamine-induced changes in [Ca2+]i, myosin light-chain phosphorylation, and contraction. This suggests that histamine-induced changes in pHi have at most minor effects on [Ca2+]i, myosin phosphorylation, and contraction in histamine-stimulated swine carotid artery. However, this result does not rule out effects on [Ca2+]i and contraction from larger changes in pHi induced by changes in PCO2, pHo, or other stimuli. Additionally, other smooth muscle tissues may be more sensitive to changes in pHi.


*    Acknowledgments
 
Dr Rembold is a Lucille P. Markey Scholar, and grants from the Lucille P. Markey Charitable Trust, the PHS (1RO1 HL38918), and the Virginia Affiliate of the American Heart Association supported this research. Smithfield Co donated the swine carotid arteries. We would like to thank Barbara Weaver for technical support.


*    Footnotes
 
Reprint requests to Christopher M. Rembold, MD, Box 146, Cardiovascular Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908.

Received September 22, 1994; first decision December 1, 1994; accepted December 1, 1994.


*    References
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