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Am J Physiol Heart Circ Physiol 283: H2169-H2176, 2002; doi:10.1152/ajpheart.00603.2002
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Vol. 283, Issue 6, H2169-H2176, December 2002

SPECIAL TOPICS
Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries

Thomas J. Heppner1,*, Adrian D. Bonev1,*, L. Fernando Santana2, and Mark T. Nelson1

1 Department of Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405-0068; and 2 Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of external pH (7.0-8.0) on intracellular Ca2+ signals (Ca2+ sparks and Ca2+ waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca2+ transients, or "Ca2+ sparks," and, at pH 7.6, significantly increased the frequency of Ca2+ waves. Alkaline pH-induced Ca2+ waves were inhibited by blocking Ca2+ release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca2+ channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca2+ waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca2+ channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca2+ waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.

ryanodine receptors; voltage-dependent calcium channels; arterial diameter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN SMOOTH MUSCLE, as in other cell types, Ca2+ signaling is central to nearly all cellular processes, including gene expression, neurotransmitter release, memory, and muscle contraction. In arterial smooth muscle, Ca2+ may have diverse effects depending on the spatiotemporal pattern of the Ca2+ signal. For example, increased Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) elevates intracellular Ca2+ concentrations ([Ca2+]i) globally (i.e., throughout the cell) and causes contraction by elevating myosin light chain kinase activity. In contrast, Ca2+ sparks, which represent transient, local release of Ca2+ through ryanodine-sensitive Ca2+ channels or ryanodine receptors (RyRs) located in the sarcoplasmic reticulum membrane, act to oppose arterial smooth muscle contraction. RyR-mediated Ca2+ sparks activate closely juxtaposed large conductance, Ca2+-sensitive K+ channels to promote membrane potential hyperpolarization and thus constitute an element in the negative feedback regulation of vascular tone (7, 18-20, 35, 40).

Another modality of Ca2+ signaling observed in arterial smooth muscle cells are asynchronous Ca2+ waves, which are usually defined as a change in internal Ca2+ concentration that travels the length of the cell. Ca2+ waves occur spontaneously in a small percentage of arterial smooth muscle (16, 18, 33), and their frequency and amplitude can be increased markedly by the addition of vasoconstrictor agonists such as norepinephrine, UTP, or phenylephrine (16, 18, 32, 33, 42). Whereas recent results suggest that Ca2+ waves induced by vasoconstrictors are caused by the release of Ca2+ from the sarcoplasmic reticulum through inosital 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors (17), the physiological role of Ca2+ waves is unclear. Indeed, recent reports on this topic are contradictory: several studies have suggested that Ca2+ waves contribute Ca2+ for vasoconstriction (2, 15, 22, 34, 42, 47), whereas other studies suggest that phenylephrine-induced Ca2+ waves do not contribute to arterial diameter regulation (33, 37).

In the present study, we demonstrate that extracellular alkalization constitutes a novel mechanism for the induction of Ca2+ waves in intact, pressurized cerebral arteries. We find that extracellular pH in the range of 7.0-7.4 has a minimal impact on Ca2+ spark or Ca2+ wave frequency. At pH 7.5, however, Ca2+ spark frequency is significantly increased, and, above pH 7.5, Ca2+ wave frequency is significantly increased. At pH 7.8 or higher, repetitive Ca2+ waves occurred in virtually every smooth muscle cell. The alkaline pH-induced Ca2+ waves occurred in the presence of inhibitors of VDCCs or Ins(1,4,5)P3 receptors but are blocked by inhibition of RyRs. These alkaline pH-induced Ca2+ waves appear to play a minor role in arterial vasoconstriction when VDCCs are inhibited.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Adult female rats (~250 g) were euthanized with pentobarbital sodium followed by thoracotomy as approved by the Office of Animal Care Management at the University of Vermont. Posterior cerebral arteries and cerebellar arteries were removed from the brain and placed in cold HEPES-buffered saline. After the connective tissue was removed, segments of the artery (2-4 mm in length; 50-120 µm) were placed in a chamber specially designed to measure Ca2+ responses in pressurized arteries. Arteries were tied to glass cannulas containing the same solution as the superfusing bath and pressurized to 60 mmHg. Arteries were superfused with either a HEPES-buffered solution or a physiological salt solution (PSS) at 2-3 ml/min (37°C). Drugs were dissolved in the superfusing saline and applied for at least 10 min before Ca2+ responses were recorded unless indicated otherwise.

Ca2+ imaging and analysis. Smooth muscle cells in the arterial wall were scanned with a laser scanning confocal microscope (OZ; Noran Instruments) controlled by an O2 workstation (Silicon Graphics) by using an Intervision software package. A Nikon Diaphot microscope with a ×60 water immersion lens (1.2 numerical aperature; Nikon) was used to visualize the artery. Cerebral arteries were incubated in an acetoxymethylester (10 µM) form of Ca2+-sensitive fluorescent dye fluo 4 and pluronic acid (2.5 µg/ml; Molecular Probes) dissolved in HEPES buffer. Arteries were maintained in the dark for 60 min at 21-23°C followed by a wash in HEPES buffer. A krypton-argon laser at 480 nm was used to excite the fluo 4, and the emitted light was detected at wavelengths >500 nm. Images were typically 103 × 106 µm (512 × 480 pixels) and acquired every 33.33 ms (30 images/s). In each field, ~10-15 smooth muscle cells could be simultaneously observed. Each field was scanned for 20 s, a time interval that was long enough to acquire a significant number of Ca2+ sparks and Ca2+ waves, yet short enough to prevent laser-induced damage to the tissue. Only cells with clearly identified edges were used for analysis. Image analysis was performed by using programs written in our laboratory with Interactive Data Language (Research Systems).

Cells used for Ca2+ spark analysis were also used to measure Ca2+ waves. Ca2+ sparks and waves were detected in individual cells offline by measuring an increase in the fractional fluorescence (F/Fo) >=  1.3, a value that could be distinguished above background noise. Baseline fluorescence (Fo) was determined by averaging 30 images with no Ca2+ events. Ca2+ sparks were detected in a rectangular area 1.5 × 1.5 µm (7 × 7 pixels) by custom software developed in our laboratory as described previously (18, 38, 50).

Ca2+ waves were detected by eye by examining three rectangular regions of interest (2.2 × 2.2 µm) located in the middle and at each end of the cell. Each region was separated from another region by 15-20 µm. Changes in fluorescence during the recorded file (20 s) were measured in each of the three regions. A nearly simultaneous or propagated change in the fluorescence above threshold for at least 1 s in at least two regions of interest (covering at least one-half the length of the cell) constituted a calcium wave.

Arterial diameter. Cerebral arteries were cannulated, mounted in an arteriograph (Living Systems Instruments), and superfused at ~2 ml/min with PSS at 37°C. The pressure was gradually increased in 10-mmHg increments until a final pressure of 60 mmHg was reached. Arteries were visualized with the use of a Panasonic CCD, model WV CD-20 camera connected to a videomonitor. Arterial diameter was measured by using calibrated edge-detection software and displayed by using Axotape software (sampling rate of 2 Hz) (Axon Instruments). Analysis of arterial diameter was made with customized software written in our laboratory.

Drugs and solutions. Ca2+ sparks and waves were measured in a HEPES solution of the following composition (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). NaOH was used to adjust the pH of HEPES solutions. Arterial diameter experiments used PSS of the following composition (in mM): 119 NaCl, 4.7 KCl, 23.8 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgCl2, 0.023 EDTA, and 11.0 glucose (pH 7.4). PSS was continuously bubbled with 95% O2-5% CO2. To adjust the pH of the PSS to a more alkaline pH, bubbling was stopped, and the container with PSS was opened to allow CO2 to escape. As CO2 slowly dissipated from the PSS, the pH gradually increased. When the desired alkaline pH value was reached, the container with PSS was tightly capped, preventing any further changes in pH. The alkalinized PSS was then superfused over the cannulated artery. Caffeine, nisoldipine, diltiazem, and U-73122 were obtained from Sigma. 2-Aminethoxydiphenylborate (2-APB) was obtained from Tocris, ryanodine was from LC Laboratories, and xestospongin C and verapamil were obtained from Calbiochem.

Statistics. Results are expressed as means ± SE where applicable. Unless otherwise noted, n is the number of cells. Significance between groups was evaluated with a one-way ANOVA followed by the appropriate multiple comparison test for significance (Dunn's pairwise multiple comparison procedure or Kruskal-Wallis analysis of variance on ranks).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of external pH from 7.0 to 7.5 on Ca2+ sparks. In pressurized cerebral arteries (60 mmHg; 37°C), all cells examined exhibited Ca2+ sparks at external pH values of 7.0, 7.4, and 7.5. Increasing the pH between 7.0 and 7.4 had no significant effect on Ca2+ spark frequency; however, increasing the pH to 7.5 induced a significant increase in Ca2+ spark frequency (Fig. 1, A and B). At higher pH values, longer-lasting Ca2+ events became so numerous that an accurate measurement of Ca2+ spark frequency was not possible.


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  		Fig. 1.   Elevating external pH from 7.4 to 7.5 increases Ca2+ spark frequency. A, top: images from pressurized (60 mmHg; 37°C) cerebral arteries in pH 7.4 (left) and 7.5 (right) that show vertically oriented individual smooth muscle cells. A, bottom: Ca2+ spark activity recorded from regions enclosed by the color boxes from the above images. B: number of Ca2+ sparks per smooth muscle cell at pH 7.0, 7.4, and 7.5. Number of cells analyzed is shown above each column. Ca2+ spark frequency was significantly increased at alkaline pH 7.5 (*P < 0.05). Images were acquired every 33.33 ms during a 20-s scan. F/Fo, fractional fluorescence.

Alkaline pH increases Ca2+ waves. In pressurized cerebral arteries (60 mmHg, 37°C) at pH 7.4, a small percentage (7% of cells; n = 98) exhibited Ca2+ waves. At pH values of 7.8 or higher, virtually 100% of the cells (n = 207) exhibited Ca2+ waves (Fig. 2A). The frequency of waves in a given cell also increased markedly with alkaline pH (Fig. 2B). At pH 8.0, the mean Ca2+ wave frequency was 11.7 ± 0.6 waves · cell-1 · min-1, but ranged as high as 44 waves · cell-1 · min-1. These repetitive waves, which were asynchronous, originated near one end of the cell and then traveled to the opposite end of the cell (Fig. 2A) with an average velocity of 57.7 ± 0.9 µm/s (n = 7) at pH 8.0. 


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  		Fig. 2.   Alkaline pH induces Ca2+ waves in pressurized cerebral arteries. A: top left, pressurized cerebral artery (~100 µm in diameter; 60 mmHg; 37°C) with individual smooth muscle cells aligned vertically. Top right, steady Ca2+ levels measured in 4 boxes placed in four different cells. Middle left, artery exposed to a solution of pH 8.0. pH 8.0 induced Ca2+ waves in all cells (middle right). Bottom, progression of a Ca2+ wave from one end of the cell to the other (bracketed cell from middle). Images are color coded as indicated by the color bar. Images were acquired every 33.33 ms (for display in the bottom; every fifth image is shown). B: relationship between Ca2+ waves in analyzed cells and external pH 7.0 to 8.0. Ca2+ wave activity was virtually unchanged at pH values 7.0 and 7.4. At external pH 7.5, frequency of Ca2+ waves per cell increased dramatically reaching maximal values at external pH 7.8. Continuous line is a fit through the data. Number of cells analyzed is shown above each data point. *P < 0.05 compared with pH 7.4.

Interestingly, Ca2+ waves were not limited to the larger (60-100 µm) resistance arteries used throughout this study but were also found in arterioles. Figure 3 illustrates pH 8.0-induced Ca2+ waves propagating in smooth muscle cells in a pressurized cerebral arteriole (20-30 µm diameter). As with the larger arteries, 100% (n = 42) of the smooth muscle cells examined exhibited Ca2+ waves at pH 8.0 and had a similar frequency (Fig. 4).


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  		Fig. 3.   Alkaline pH-induced Ca2+ waves in cerebral arterioles. Repetitive Ca2+ waves were found in virtually all smooth muscle cells from arterioles when external pH was increased to 8.0. Every third image is displayed (~100 ms between images). First two rows are consecutive. Third row is from the same file recorded 12 s after the second row to show Ca2+ waves in additional cells. Color scale bar represents the percentage of the maximum fluorescence. Images were acquired every 33.33 ms.



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  		Fig. 4.   Ryanodine inhibited Ca2+ waves but not inhibitors of voltage-dependent Ca2+ channels (VDCCs), inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors, PLC, Ca2+-free solution, or the removal of the endothelium. Waves were induced by external pH 8.0 except for caffeine, which was in pH 7.4. * Significantly different from pH 8.0 control (P < 0.05). Numbers above bars refer to numbers of cells.

Ca2+ influx through VDCCs is not required for alkaline pH-induced Ca2+ waves. To determine the origin of the Ca2+ underlying Ca2+ waves, the effects of VDCC inhibitors on Ca2+ waves was examined. Application of the VDCC inhibitors verapamil (10 µM) (48), diltiazem (10 µM) (25), or nisoldipine (100 nM) (36) for up to 30 min did not reduce the frequency of ongoing Ca2+ waves (Figs. 4 and 5A). To examine the role of VDCCs in the initiation of Ca2+ waves, arteries were incubated with diltiazem (50 µM) for 10 min at pH 7.4 before the pH was increased to 7.8-8.0. However, preincubation with a VDCC inhibitor failed to prevent the induction of Ca2+ waves at pH 7.8-8.0 (Fig. 4). Alkaline pH-induced Ca2+ waves also persisted for at least 20 min (the longest time period cells were examined in Ca2+-free solution) in nominal absence of external Ca2+ (Fig. 4). These findings indicate that over the time of the experiments, external Ca2+ and Ca2+ entry through VDCCs or through other pathways do not play a significant role in the initiation or maintenance of alkaline pH-induced Ca2+ waves.


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  		Fig. 5.   Inhibitors of VDCCs or InsP3 receptors do not significantly alter pH 8.0-induced Ca2+ waves. A: pH 8.0-induced Ca2+ waves recorded from the same posterior cerebral artery before (left) and 10 min after the addition of nisoldipine (100 nM) (right). B: pH 8.0-induced Ca2+ waves recorded from the same posterior cerebral artery before (left) and 15 min after the addition of 2-aminethoxydiphenylborate (2-APB, 100 µM) (right).

It is conceivable that alkaline pH affects the release of factor(s) that would alter Ca2+ wave probability in the smooth muscle cells. To test the dependence of alkaline pH-induced Ca2+ waves on the endothelium, we removed the endothelium by pushing 3-5 ml of air through the vessel or by a combination of forcing air through the lumen and scraping the lumen with a hair. A lack of effect of the endothelial-dependent vasodilator acetylcholine was taken as evidence of endothelial denudation. Removal of the endothelium did not significantly affect Ca2+ wave activation induced by pH 8.0 (Fig. 4).

Ins(1,4,5)P3-mediated Ca2+ release is not involved in alkaline pH-induced Ca2+ waves. Previous studies have shown that Ca2+ waves induced in smooth muscle by vasoconstrictors are dependent on Ca2+ release through Ins(1,4,5)P3 receptors, reflecting agonist-induced activation of PLC and elevation of Ins(1,4,5)P3. Alkaline pH could also conceivably activate PLC and elevate Ins(1,4,5)P3 levels (1, 2). Inhibition of PLC (U-73122, 2 µM), after the induction of Ca2+ waves with external pH 7.8-8.0, however, was ineffective in blocking pH 8.0-induced Ca2+ waves (Fig. 4), suggesting that this pathway is not involved in the genesis and maintenance of Ca2+ waves. To directly test the dependence of Ca2+ waves on Ins(1,4,5)P3 receptors, two inhibitors of Ins(1,4,5)P3 receptors, xestospongin C (13) and 2-APB (31), were employed. Preincubation of arteries with xestospongin C (20 µM) for 20 min (37°C) in external pH 7.4 followed by wave induction by external pH 8.0 in the continued presence of xestospongin C was ineffective in abolishing Ca2+ waves (Fig. 4). 2-APB (100 µM) also failed to abolish Ca2+ waves at pH 8.0 (Figs. 4 and 5B). These results suggest that Ca2+ release through Ins(1,4,5)P3 receptors does not contribute to Ca2+ waves induced by alkaline pH.

Ca2+ waves originate from RyRs. Because Ca2+ influx or Ca2+ release through Ins(1,4,5)P3 receptors does not appear to be the immediate source of Ca2+ for Ca2+ waves, we examined the role of Ca2+ release through RyRs. In contrast to the results obtained with xestospongin C, 2-APB, and U-73122, treatment with the selective RyR inhibitor ryanodine (10 µM) abolished alkaline-induced Ca2+ waves within 10-15 min of application (Fig. 4 and Fig. 6A). Similarly, preincubation (20 min) of the artery with ryanodine (10 µM) completely prevented the induction of Ca2+ waves by the subsequent elevation of extracellular pH to 7.8 or higher (Fig. 4). These findings indicate that alkaline pH-induced Ca2+ waves originate from RyRs.


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  		Fig. 6.   Ca2+ waves originate from RyRs. A: inhibition of RyRs abolished alkaline-induced Ca2+ waves. pH 8.0-induced Ca2+ waves recorded from same posterior cerebral artery before (left) and 12 min after addition of ryanodine (10 µM) (right). Ryanodine abolished repetitive pH 8.0-induced Ca2+ waves. B: caffeine-induced Ca2+ waves. Ca2+ wave activity recorded at pH 7.4 from the same artery before (left) and 10 min after addition of 5 mM caffeine (right). There was no Ca2+ wave activity in control but appeared within 5 min following the addition of caffeine.

Because Ca2+ waves originate from RyRs, we hypothesized that caffeine, which elevates RyR activity by increasing the apparent Ca2+ sensitivity of the RyR (39), should be able to activate Ca2+ waves. We have previously shown that steady application of caffeine, in micromolar concentrations, increases Ca2+ spark and associated transient large conductance, Ca2+-sensitive K+ current frequency in cerebral artery smooth muscle (14, 18, 26, 40). Furthermore, bolus application of higher concentrations of caffeine (10 mM) induces a ryanodine-sensitive, global Ca2+ transient (34). Here, we demonstrate that the steady application of an intermediate caffeine concentration (5 mM) is capable of inducing repetitive Ca2+ waves at pH 7.4 (Figs. 4 and 6B). This finding indicates that activation of RyRs can induce and sustain repetitive Ca2+ waves.

Effect of Ca2+ waves on arterial diameter. Alkaline pH constricts cerebral arteries (3, 4, 10, 12, 28, 43). To test the contribution of Ca2+ waves to alkaline pH-induced vasoconstriction, arterial diameter was measured before and after the addition of ryanodine. External pH 8.0 solution constricted arteries to 73.6 ± 10.9% of a 60 mM K+-induced vasoconstriction. Inhibition of VDCCs (50 µM diltiazem) relaxed the pH 8.0-induced vasoconstriction by 82.1 ± 6.6% (n = 3 vessels). The addition of ryanodine (10 µM) further relaxed the pH 8.0-constricted arteries to control values (Fig. 7). This suggests that pH-induced Ca2+ waves make a small, direct contribution to alkaline pH-induced vasoconstriction.


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  		Fig. 7.   Inhibition of VDCCs and RyRs caused relaxation of posterior cerebral arteries. Diameter of a pressurized posterior cerebral artery (60 mmHg, 37°C) was recorded under different pH conditions and inhibition of VDCCs (diltiazem, 50 µM) and RyRs (ryanodine, 10 µM). pH 8.0 induced a large vasoconstriction. Block of VDCCs relaxed the artery 86% of the pH 8.0-induced constriction.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study identifies a mechanism whereby changes in extracellular pH are able to transform RyR-mediated Ca2+ signals from Ca2+ sparks to Ca2+ waves. Ca2+ sparks were present in all cells examined at pH 7.0, 7.4, and 7.5. Ca2+ spark activity was unchanged at external pH values of 7.0 and 7.4, but an increase in pH to 7.5 significantly increased Ca2+ spark frequency. At higher pH values, Ca2+ sparks could no longer be accurately identified but were replaced by longer-lasting, repetitive Ca2+ waves. Ca2+ wave frequency was pH dependent and reached a maximum at pH 7.8.

Source of Ca2+ release for alkaline pH-induced waves. Alkaline pH has been shown to increase Ca2+ influx into vascular smooth muscle cells through VDCCs (15, 23, 24, 44, 51). VDCC-mediated increases in intracellular Ca2+ should elevate the activity of Ca2+-sensitive RyRs (39) and might be predicted to contribute to the induction of Ca2+ waves. However, pharmacological inhibition of VDCC activity did not prevent the induction of pH-induced waves nor did it inhibit pH-induced waves after activation. Therefore, the additional Ca2+ influx through VDCCs that occurs under alkaline conditions does not appear to be the main factor in Ca2+ wave induction or maintenance. Alkalization can also increase the open probability of Ins(1,4,5)P3 receptors (21, 52), possibly through the activation of PLC (1, 2). However, our results do not support a role for Ins(1,4,5)P3 receptor activation underlying pH-induced Ca2+ waves, because selective inhibitors of Ins(1,4,5)P3 receptors (xestospongin C and 2-APB) and PLC (U-73122) failed to prevent or abolish alkaline pH-induced Ca2+ waves. Instead, our findings suggest that alkaline pH-induced Ca2+ waves originate from RyRs.

pH and apparent Ca2+ sensitivity of RyRs. RyR activity, measured at the single channel level in bilayers, increases steeply with alkaline pH (30, 41). With the use of Ca2+ as the charge carrier in skeletal or cardiac RyRs, the open probability of the RyR increases about twofold between pH 7.1 and pH 7.5 (41). Changes in external pH are directly translated into changes in internal pH values. In the mesenteric artery smooth muscle, internal pH has been shown to change with a ratio of 0.73 relative to the change in external pH (6). This change is quite rapid, reaching the half-peak intracellular response in strips of mesenteric artery in 38 s (6). From these observations and the results of the current study, we propose that an elevation in external pH acting through changes in intracellular pH, increases the apparent Ca2+ sensitivity of RyRs, resulting in an increase in the probability of regenerative Ca2+ waves. The observation that caffeine, which increases the apparent sensitivity of RyRs to Ca2+ also induces Ca2+ waves (Fig. 6B), is consistent with this model.

Function of Ca2+ waves. The physiological role of Ca2+ waves is unclear, with much attention in previous studies having been focused on their contribution to contraction. Several studies using alpha -adrenoceptor activation to induce Ca2+ waves suggest that waves contribute to contraction of vascular smooth muscle (15, 42), whereas other studies suggest that asynchronous Ca2+ waves have minimal or no impact on vascular tone (33, 37). Our findings suggest that vasoconstriction induced by extracellular alkaline pH depends primarily on influx of Ca2+ through VDCCs (see Fig. 7). From the effects of Ca2+ wave inhibition by ryanodine, Ca2+ waves account for a small percentage (~18%) of the pH 8.0-induced vasoconstriction. It should be noted that alkaline pH-induced Ca2+ waves represent activation of RyRs, whereas Ins(1,4,5)P3 receptors have a prominent role in agonist-induced Ca2+ waves. RyRs and Ins(1,4,5)P3 receptors communicate with different cellular targets and could explain differential effects of agonist and pH-induced Ca2+ waves.

Different spatiotemporal patterning of Ca2+ events may selectively activate specific cellular processes. For example, increases in "global Ca2+" bring about vasoconstriction, whereas Ca2+ sparks play a key role to reduce cell excitability through the activation of Ca2+-sensitive K+ channels and an attendant decrease in Ca2+ entry through VDCCs (19, 34, 35, 38, 53). Ca2+ waves evoked by caffeine have been shown to activate Ca2+ dependent Cl- channels in rat portal vein (34). Ca2+ mobilization may also contribute to the modulation of transcription factors such as nuclear factor of activated T cells and cAMP-responsive binding protein in smooth muscle (8, 45, 46, 49). In nonexcitable cells, Ca2+ waves have been shown to increase the efficiency, as well as the selectivity, of Ca2+-dependent transcription factor(s) (11, 29). Although the function of alkaline pH-induced Ca2+ waves is unclear at this time, it is conceivable that they may regulate both Ca2+-sensitive ion channels and transcription factors.

pH plays an important role in the regulation of the cerebral vasculature (3, 4, 10, 12, 28, 43). Arterial pH values >7.44 are observed under clinical conditions of alkalemia (9), and arterial pH can increase to 7.8 before death results (27). This study describes a unique pH-induced shift in Ca2+ signaling in cerebral arterial smooth muscle cells; a shift that may contribute to alkaline pH-induced vasoconstriction. This direct contribution appears to be relatively minor, suggesting that alkaline pH-induced Ca2+ waves may serve additional functions. Although the function of these waves is not entirely clear, the fact that Ca2+ signaling shifts dramatically between sparks and waves within a narrow pH range that corresponds to clinical alkalemia is provocative.


    ACKNOWLEDGEMENTS

The authors thank Drs. B. Etherton, M. Gomez, L. Gonzalez Bosc, D. Hill-Eubanks, G. Petkov, K. Thorneloe, M. Talyor, and G. Wellman for critical comments on this manuscript.


    FOOTNOTES

* T. J. Heppner and A. D. Bonev contributed equally to this study.

This work was supported by National Institutes of Health Grants HL-44455, HL-63722 (to M. T. Nelson), and NS-39405 (to L. F. Santana, University of Puerto Rico, and M. T. Nelson), and the Totman Trust for Human Cerebrovascular Research.

Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, Univ. of Vermont College of Medicine, Burlington, VT 05405-0068 (E-mail: mark.nelson{at}uvm.edu).

This article belongs to a collection of papers accepted in response to the Editor's special call for papers entitled "Mechanisms of vascular myogenic tone."

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00603.2002

Received 16 July 2002; accepted in final form 15 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albuquerque, MLC, and Leffler CW. pHo, pHi, and Pco2 in stimulation of IP3 and [Ca2+]c in piglet cerebrovascular smooth muscle. Proc Soc Exp Biol Med 219: 226-234, 1998[Medline].

2.   Albuquerque, ML, Lowery-Smith L, Hsu P, Parfenova H, and Leffler CW. Low CO2 stimulates inositol phosphate turnover and increased inositol 1,4,5-trisphosphate levels in piglet cerebral microvascular smooth muscle cells. Proc Soc Exp Biol Med 209: 14-19, 1995[Medline].

3.   Aoyama, Y, Ueda K, Setogawa A, and Kawai Y. Effects of pH on contraction and Ca2+ mobilization in vascular smooth muscles of the rabbit basilar artery. Jpn J Physiol 349: 55-62, 1999.

4.   Apkon, M, and Boron WF. Extracellular and intracellular alkalinization and the constriction of rat cerebral arterioles. J Physiol 484: 743-753, 1995[Abstract/Free Full Text].

5.   Asada, Y, Yamazawa T, Hirose K, Takasaka T, and Iino M. Dynamic Ca2+ signaling in rat arterial smooth muscle cells under the control of local rennin-angiotensin system. J Physiol 521.2: 497-505, 1999.

6.   Austin, C, and Wray S. Extracellular pH signals affect rat vascular tone by rapid transduction into intracellular pH changes. J Physiol 466: 1-8, 1993[Abstract/Free Full Text].

7.   Bonev, AD, Jaggar JH, Rubart M, and Nelson MT. Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries. Am J Physiol Cell Physiol 273: C2090-C2095, 1997[Abstract/Free Full Text].

8.   Cartin, L, Lounsbury KM, and Nelson MT. Coupling of Ca2+ to CREB activation and gene expression in intact cerebral arteries from mouse: roles of ryanodine receptors and voltage-dependent Ca2+ channels. Circ Res 86: 760-767, 2000[Abstract/Free Full Text].

9.  Deshpande GG. Alkalosis Respiratory [Online] (July 2 GG, 2002). Mecosta County General Hospital. http://www.emedicine.com/ped/topic70.htm (July 10, 2002).

10.   Dietrich, HH, and Dacey RGJ Effects of extravascular acidification and extravascular alkalinization on constriction and depolarization in rat cerebral arterioles in vitro. J Neurosurg 81: 437-442, 1994[Web of Science][Medline].

11.   Dolmetsch, RE, Xu K, and Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392: 933-936, 1998[Medline].

12.   Edvinsson, L, and Sercombe R. Influence of pH and pCO2 on alpha-receptor mediated contraction in brain vessels. Acta Physiol Scand 97: 325-331, 1976[Web of Science][Medline].

13.   Gafni, J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, and Pessah IN. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19: 723-733, 1997[Web of Science][Medline].

14.   Gollasch, M, Wellman GD, Knot HJ, Jaggar JH, Damon DH, Bonev AD, and Nelson MT. Ontogeny of local sarcoplasmic reticulum Ca2+ signals in cerebral arteries: Ca2+ sparks as elementary physiological events. Circ Res 83: 1104-1114, 1998[Abstract/Free Full Text].

15.   Iino, S, Hayashi H, Saito H, Tokuno H, and Tomita T. Effects of intracellular pH on calcium currents and intracellular calcium ions in the smooth muscle of rabbit portal vein. Exp Physiol 79: 669-680, 1994[Abstract].

16.   Iino, M, Kasai H, and Yamazawa T. Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ. EMBO J 13: 5026-5031, 1994[Web of Science][Medline].

17.   Iino, M, Yamazawa T, Miyashita Y, Endo M, and Kasai H. Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells. EMBO J 13: 5287-5291, 1993.

18.   Jaggar, JH, and Nelson MT. Differential regulation of Ca2+ sparks and Ca2+ waves by UTP in rat cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 279: C1528-C1539, 2000[Abstract/Free Full Text].

19.   Jaggar, JH, Porter VA, Lederer WJ, and Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235-C256, 2000[Abstract/Free Full Text].

20.   Jaggar, JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, Kleppisch T, Rubart M, Stevenson AS, Lederer WJ, Knot HJ, Bonev AD, and Nelson MT. Ca2+ channels, ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 164: 577-587, 1998[Web of Science][Medline].

21.   Joseph, SK, Rice HL, and Williamson JR. The effect of external calcium and pH on inositol trisphosphate-mediated calcium release from cerebellum microsomal fractions. Biochem J 258: 261-265, 1989[Web of Science][Medline].

22.   Kasai, Y, Yamazawa T, Sakurai T, Taketani Y, and Iino M. Endothelium-dependent frequency modulation of Ca2+ signaling in individual vascular smooth muscle cells of the rat. J Physiol 504: 349-357, 1997[Abstract/Free Full Text].

23.   Klöckner, U, and Isenberg G. Intracellular pH modulates the availability of vascular L-type Ca2+ channels. J Gen Physiol 103: 647-663, 1994[Abstract/Free Full Text].

24.   Klöckner, U, and Isenberg G. Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance. J Gen Physiol 103: 665-678, 1994[Abstract/Free Full Text].

25.   Knot, HJ, and Nelson MT. Regulation of membrane potential and diameter by voltage-dependent K+ channels in rabbit myogenic cerebral arteries. Am J Physiol Heart Circ Physiol 269: H348-H355, 1995[Abstract/Free Full Text].

26.   Knot, HJ, Standen NB, and Nelson MT. Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels. J Physiol 508: 211-221, 1998[Abstract/Free Full Text].

27.  Kohan DE. Acid-Base Physiology [Online] (Dec. 2000). University of Utah. http://www.umed.med.utah.edu/ms2/renal/Word%20files/h)%20Acid_Base%20Physiology.htm. (March 5, 2002).

28.   Kontos, HA, Raper AJ, and Patterson JL. Analysis of vasoactivity of local pH, PCO2 and bicarbonate on pial vessels. Stroke 8: 358-360, 1977[Abstract/Free Full Text].

29.   Li, W, Llopis J, Whitney M, Zlokarnik G, and Tsien RY. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392: 936-941, 1998[Medline].

30.   Ma, J, and Zhao J. Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to change in proton concentrations. Biophys J 67: 626-633, 1994[Medline].

31.   Maruyama, T, Kanaji T, Nakade S, Kanno T, and Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem (Tokyo) 122: 498-505, 1997[Abstract/Free Full Text].

32.   Mauban, JRH, Lamont C, Balke CW, and Wier WG. Adrenergic stimulation of rat resistance arteries affects Ca2+ sparks, Ca2+ waves, and Ca2+ oscillations. Am J Physiol Heart Circ Physiol 280: H2399-H2405, 2001[Abstract/Free Full Text].

33.   Miriel, VA, Mauban JRH, Blaustein MP, and Wier WG. Local and cellular Ca2+ transients in smooth muscle of pressurized rat resistance arteries during myogenic and agonist stimulation. J Physiol 518.3: 815-824, 1999.

34.   Mironneau, J, Arnaudeau S, Macrez-Lepretre N, and Boittin FX. Ca2+ sparks and Ca2+ waves activate different Ca2+-dependent ion channels in single myocytes from rat portal vein. Cell Calcium 20: 153-160, 1996[Web of Science][Medline].

35.   Nelson, MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract/Free Full Text].

36.   Nelson, MT, and Worley JF. Dihydropyridine inhibition of single calcium channels and contraction in rabbit mesenteric artery depends on voltage. J Physiol 412: 65-91, 1989[Abstract/Free Full Text].

37.   Peng, H, Matchkov V, Ivarsen A, Aalkjaer C, and Nilsson J. Hypothesis for the initiation of vasomotion. Circ Res 88: 810-815, 2001[Abstract/Free Full Text].

38.   Perez, GJ, Bonev AD, Patlak JB, and Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol 113: 229-238, 1999[Abstract/Free Full Text].

39.   Pessah, IN, Stambuk RA, and Casida JE. Ca2+-activated ryanodine binding: mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nucleotides. Mol Pharmacol 31: 232-238, 1987[Abstract].

40.   Porter, VA, Bonev AD, Knot HJ, Heppner TJ, Stevenson AS, Kleppisch T, Lederer WJ, and Nelson MT. Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am J Physiol Cell Physiol 274: C1346-C1355, 1998[Abstract/Free Full Text].

41.   Rousseau, E, and Pinkos J. pH modulates conducting and gating behaviour of single calcium release channels. Pflügers Arch 415: 645-647, 1990[Web of Science][Medline].

42.   Ruehlmann, DO, Lee CH, Poburko D, and van Breemen C. Asynchronous Ca2+ waves in intact venous smooth muscle. Circ Res 86: E72-E79, 2000[Web of Science][Medline].

43.   Smeda, JS, Lombard JH, Madden JA, and Harder DR. The effect of alkaline pH and transmural pressure on arterial constriction and membrane potential of hypertensive cerebral arteries. Pflügers Arch 408: 239-242, 1987[Web of Science][Medline].

44.   Smirnov, S, Knock GA, Belevych AE, and Aaronson PI. Mechanism of effect of extracellular pH on L-type Ca2+ channel currents in human mesenteric arterial cells. Am J Physiol Heart Circ Physiol 279: H76-H85, 2000[Abstract/Free Full Text].

45.   Stevenson, AS, Cartin L, Wellman TL, Dick MH, Nelson MT, and Lounsbury KM. Membrane depolarization mediates phosphorylation and nuclear translocation of CREB in vascular smooth muscle cells. Exp Cell Res 263: 118-130, 2001[Web of Science][Medline].

46.   Stevenson, AS, Gomez MF, Hill-Eubanks DC, and Nelson MT. NFAT4 movement in native smooth muscle. A role for differential Ca2+ signaling. J Biol Chem 276: 15018-15024, 2001[Abstract/Free Full Text].

47.   Swärd, K, Dreja K, Lindqvist A, Persson E, and Hellstrand P. Influence of mitochondrial inhibition on global and local [Ca2+]i in rat tail artery. Circ Res 90: 792-799, 2002[Abstract/Free Full Text].

48.   Terada, K, Kitamura K, and Kuriyama H. Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine. Pflügers Arch 408: 552-557, 1987[Web of Science][Medline].

49.   Wellman, GC, Cartin L, Eckman DM, Stevenson AS, Saundry CM, Lederer WJ, and Nelson MT. Membrane depolarization, elevated Ca2+ entry, and gene expression in cerebral arteries of hypertensive rats. Am J Physiol Heart Circ Physiol 281: H2559-H2567, 2001[Abstract/Free Full Text].

50.   Wellman, GC, Santana LF, Bonev AD, and Nelson MT. Role of phospholamban in the modulation of arterial Ca2+ sparks and Ca2+-activated K+ channels by cAMP. Am J Physiol Cell Physiol 281: C1029-C1037, 2001[Abstract/Free Full Text].

51.   West, GA, Leppla DC, and Simard JM. Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig. Circ Res 71: 201-209, 1992[Abstract/Free Full Text].

52.   Worley, PF, Baraban JM, Supattapone S, Wilson VS, and Snyder SH. Characterization of inositol trisphosphate receptor binding in brain. J Biol Chem 262: 12132-12136, 1987[Abstract/Free Full Text].

53.   ZhuGe, R, Sims SM, Tuft RA, Fogarty KE, and Walsh JV. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J Physiol 513: 711-718, 1998[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 283(6):H2169-H2176
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