AJP - Heart Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H732-H740, 2006; doi:10.1152/ajpheart.01357.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, M. T.
Right arrow Articles by Longo, L. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, M. T.
Right arrow Articles by Longo, L. D.

Modulation of BK channel calcium affinity by differential phosphorylation in developing ovine basilar artery myocytes

Mike T. Lin,1,2 David A. Hessinger,2 William J. Pearce,1,2 and Lawrence D. Longo1,2

1Center for Perinatal Biology and 2Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California

Submitted 22 December 2005 ; accepted in final form 4 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Large-conductance Ca2+-sensitive K+ (BK) channel activity is greater in basilar artery smooth muscle cells (SMCs) of the fetus than the adult, and this increased activity is associated with a lower BK channel Ca2+ set point (Ca0). Associated PKG activity is three times greater in BK channels from fetal than adult myocytes, whereas associated PKA activity is three times greater in channels from adult than fetal myocytes. We hypothesized that the change in Ca0 during development results from different levels of channel phosphorylation. In inside-out membrane patch preparations of basilar artery SMCs from adult and fetal sheep, we measured BK channel activity in four states of phosphorylation: native, dephosphorylated, PKA phosphorylated, and PKG phosphorylated. BK channels from adult and fetus exhibited similar voltage-activation curves, Ca0 values, and Ca2+ dissociation constants (Kd) for the dephosphorylated, PKA phosphorylated, and PKG phosphorylated states. However, voltage-activation curves of native fetal BK channels shifted significantly to the left of those of the adult, with Ca0 and Kd values half those of the adult. For the two age groups at each of the phosphorylation states, Ca0 and Kd produced linear relations when plotted against voltage at half-maximal channel activation. We conclude that the Ca0 and Kd values of the BK channel can be modulated by differential channel phosphorylation. Lower Ca0 and Kd values in BK channels of fetal myocytes can be explained by a greater extent of channel phosphorylation of fetal than adult myocytes.

patch clamp; inside-out patch; fetus; development

AN IMPORTANT QUESTION RELATES to the regulation of large-conductance, voltage-dependent, Ca2+-sensitive K+ (BK) channels and their change in the course of maturational development. BK channels are found in many cell types and help regulate tone in vascular smooth muscle cells (SMCs) (28). BK channel activity is modulated by a number of factors, including G proteins (16), intracellular Ca2+ concentration ([Ca2+]i) (28), oxygen tension (39, 40), membrane lipids (30), differential phosphorylation (4, 17, 19), and others (6, 20, 22, 42).

We and others have reported significant developmental differences related to the regulation of cerebral vascular smooth muscle tone. These include differences in K+ channel expression (35), K+ channel activity (18, 23), resting membrane potential (10), intracellular Ca2+ regulation (5, 24), sensitivity of contraction to [Ca2+]i (1, 12, 25), and others (26, 32). In the ovine fetal middle cerebral artery, contraction depends almost completely on the influx of extracellular Ca2+, whereas contraction in adult myocytes relies more on Ca2+ release from intracellular Ca2+ stores, including the inositol 1,4,5-trisphosphate-releasable and ryanodine-sensitive stores (1, 24, 27).

On the basis of these findings, the developmental differences in vascular SMC activity may be a function of differential regulation of plasma membrane potential. Membrane potential and [Ca2+]i are coupled to BK channel activity through an inhibitory feedback loop. BK channel activation hyperpolarizes the cell membrane and, in turn, reduces the activity of voltage-dependent L-type Ca2+ channels. This leads to a decrease in [Ca2+]i and vascular relaxation. On the other hand, at low [Ca2+]i, BK channels close, leading to depolarization (29), which activates voltage-gated Ca2+ channels and increases [Ca2+]i. In turn, this activates the BK channels, causing membrane hyperpolarization and decreased Ca2+ channel activity. Because of the high membrane resistance of SMCs, minute changes in K+ channel activity can dramatically affect membrane potential (29). Because of their high expression levels and high conductance, this effect applies particularly to the SMC BK channels. Thus conditions that alter BK channel Ca2+ sensitivity and/or the Ca2+ set point (Ca0) will, in general, affect the resting membrane potential and [Ca2+]i, and vice versa.

BK channels respond to [Ca2+]i in the micromolar range (15, 18, 33). However, in intact SMCs, global [Ca2+]i is in the submicromolar range and appears to resist increase to micromolar concentrations (38) largely because of Ca2+ sequestration and buffering (27). Moreover, BK channel activity is modulated by localized and transient Ca2+ elevations originating from ryanodine receptor-regulated intracellular stores (e.g., Ca2+ sparks) (34) and from activated L-type Ca2+ channels (9). Basilar artery SMCs exhibit much lower Ca2+ spark activity in the neonatal than the adult rat (13). This lowered spark activity in the neonate may be due to nonaggregation of ryanodine receptors and consequential unsynchronized Ca2+ release from the ryanodine-sensitive stores. Indeed, mature and immature arterial SMCs may rely on different intracellular Ca2+ stores (27).

Recently, we showed that the BK channels in fetal SMCs have a lower Ca0 and, as a consequence, are more active than those in adult SMCs at a given [Ca2+]i (18). Furthermore, SMC BK channels of the two age groups appear to be phosphorylated to different extents (19). These findings extend our previous observation that the BK channel Ca0 differs significantly with maturational age and suggest a biochemical mechanism for this difference. Additionally, the functional stoichiometry of the channel-associated phosphatases and kinases differs substantially between the two age groups (19). In view of these findings, we tested the hypothesis that the age-related increase in Ca0 of BK channels from adult SMCs results from the decreased phosphorylation of these channels.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Basilar arteries from near-term fetal (~140 days) and nonpregnant adult (≤2-yr-old) female sheep were obtained from Nebeker Ranch (Lancaster, CA). All surgical and experimental procedures were performed in accordance with the Animal Welfare Act, the National Institutes of Health "Guide for the Care and Use of Laboratory Animals," "Guiding Principles in the Care and use of Animals" approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of Loma Linda University.

Vascular SMC isolation. We enzymatically dissociated SMCs from dissected basilar arteries, as previously described (18). Briefly, basilar arteries from adult (200 to 400 µm diameter) and fetus (150 to 200 µm diameter) were cut into 1-mm2 pieces and placed in HEPES-buffered solution containing 0.3 mg/ml papain (Worthington Biochemical, Lakewood, NJ) and 1 mg/ml dithioerythritol for 20 min at 37°C. The tissues were incubated in HEPES-buffered solution containing 100 µM CaCl2, 0.6 mg/ml collagenase type F, and 0.3 mg/ml collagenase type H (Sigma Blend Collagenase, Sigma, St. Louis, MO) and again for 15 min at 37°C. Enzyme solutions also contained 1 mg/ml BSA to minimize effects on the cells. The digested tissues were triturated with a fire-polished siliconized (Sigmacote, Sigma) glass Pasteur pipette to yield intact, dissociated SMCs. The cells were kept on ice and used within 6 h.

Coverslips containing adherent vascular SMCs were mounted in a perfusion chamber containing HEPES-buffered solution on the stage of an inverted microscope (Axiovert 35M, Carl Zeiss, Thornwood, NY), where they were kept for 15 min while they were viewed and verified to have characteristic elongated shapes with axial ratios of ~10:1 for adult and 5:1 for fetal myocytes. The HEPES-buffered solution was then exchanged for the bath solution.

Whole cell current recordings. Using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and methods previously described (18), we obtained perforated whole cell preparations. Resting membrane potentials were recorded with Clampex 8 (Axon Instruments) in current-clamp mode. A programmable Flaming-Brown pipette puller and standard fire-polishing procedures were used to fabricate patch pipettes from borosilicate (Sutter Instrument, Novato, CA) glass capillary tubing (10 cm long, 1.5 mm OD, and 1.17 mm ID). The pipette resistance for the macroscopic recording was ~2–3 M{Omega}. Data were filtered at 1 kHz using an Axopatch 200B internal four-pole low-pass Bessel filter and digitized at 5 kHz. An agar-salt bridge was used to minimize the junction potential differences between the solutions.

Macroscopic recording. Macroscopic currents were recorded from excised, inside-out membrane patch preparations of vascular SMCs with Clampex 8, as previously described (19). The pipettes used for macropatch studies were the same as those used in whole cell recordings. Data from recorded currents were filtered at 1 kHz using an Axopatch 200B internal four-pole low-pass Bessel filter and digitized at 5 kHz.

For macropatch studies, the recording protocols differed depending on the Ca2+ concentration of the bath solution. For 3 x 10–7 and 10–6 M Ca2+, we held the voltage of the patch preparation at 0 mV and applied a series of +10-mV depolarization steps from –50 to +150 mV. For 3 x 10–6 M Ca2+, we held the patch preparation at 0 mV and applied a series of +10-mV depolarization steps from –100 to +100 mV. For 10–5 and 3 x 10–5 M Ca2+, we held the patch preparation at –50 mV and applied a series of +10-mV depolarization steps from –150 to +50 mV. All voltage pulses were applied for 500 ms at 5-s intervals.

Conductance (G) values, defined as G = I/Vm (where I is current and Vm is membrane potential), were normalized for each experiment by dividing G values by Gmax, where Gmax is defined as the largest G value obtained in each experiment. BK channel activity was expressed as relative conductance, i.e., G/Gmax. Voltage-conductance curves of G/Gmax vs. voltage were fitted with a form of the Boltzmann equation: G/Gmax = {1 + exp[(V0.5 – Vm)/K]}–1, where V0.5 is the membrane potential required for half-maximal activation of the channels and K is the logarithmic voltage sensitivity (i.e., {Delta}V required for an e-fold increase in activity), as previously described (19).

To study the effect of phosphorylation on the modulation of BK channel activity, we operationally defined four phosphorylation states of the BK channels: 1) the native state, i.e., the phosphorylation state of the channel best representing physiological phosphorylation, 2) the dephosphorylated state, i.e., the channel maximally dephosphorylated by exogenously applied alkaline phosphatase, 3) the PKA phosphorylation state, i.e., the channel maximally phosphorylated by the exogenous catalytic subunit of PKA (PKAc) after alkaline phosphatase dephosphorylation, and 4) the PKG phosphorylation state, i.e., the channel maximally phosphorylated by exogenous PKG after dephosphorylation as described above.

Chemical reagents and solutions. Papain was obtained from Worthington Biochemical (Lakewood, NJ); Ca2+ standard kits 2 and 3, fura 2, and fura 6 from Molecular Probes (Eugene, OR); and purified PKG, KT-5720, KT-5823, and okadaic acid from Calbiochem, EMD Biosciences (San Diego, CA). Dr. W. H. Fletcher (Dept. of Anatomy, Loma Linda University) generously provided purified PKAc. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Purified PKG was preactivated in bath solution containing 5 x 10–5 M cGMP. For isolation of SMCs, the HEPES-buffered solution contained (in mM) 55 NaCl, 80 sodium glutamate, 5.6 KCl, 10 HEPES, 2 MgCl2, and 10 glucose (with pH adjusted to 7.3 with NaOH). The recording bath solutions contained (in mM) 140 KCl, 10 HEPES, 1 Mg2+, and 5 EGTA (with pH adjusted to 7.2 with KOH), with several different free Ca2+ concentrations (0.3, 1.0, 3.0, 10, and 30 µM), which were first estimated with Max Chelator Sliders software (C. Patton, Stanford University; Ref. 31) and adjusted fluorometrically using fura Ca2+ indicators and Ca2+ standard kits. Composition of the patch pipette solution was the same as that of the bath solution that contained 3 x 10–6 M free Ca2+. Bath solution was changed by gravitational solution exchange.

Data analysis and statistics. Values are means ± SE. In all cases, n refers to the number of replicate samples. Statistical comparisons were performed at the 95% confidence level using two-sample, unpaired Student's t-tests or ANOVA. We verified that all sample populations were distributed normally. Curve fitting was performed with Prism 4 (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ca0 in the native state. To study the effect of phosphorylation on BK channel activity, we defined four phosphorylation states (see METHODS). For the native state, we determined the BK channel voltage-activation relations at different Ca2+ concentrations immediately after patch excision. Representative macropatch recordings of BK channels from adult and fetal myocytes are shown in Fig. 1, A and B. BK channel voltage sensitivities appeared similar at all tested Ca2+ concentrations for adult (Fig. 1C) and fetal (Fig. 1D) SMCs. Channel activity increased e-fold for a 24.8 ± 1.4 mV depolarization for adult (n = 5) and a 25.4 ± 1.0 mV depolarization for fetal (n = 5) myocytes, and the slopes of the fitted curves were 13.7 ± 0.8 for adult and 14.4 ± 0.5 for fetal myocytes (n = 5 each). V0.5 values for the different free Ca2+ concentrations for adult and fetal myocytes are shown in Table 1. In general, at the same free Ca2+ concentration, the V0.5 values for the adult BK channel were rightward shifted an average of 19.3 ± 3.1 mV relative to those for the fetus (range 26.0–8.2 mV).


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1. Native state large-conductance Ca2+-sensitive K+ (BK) channels from fetal myocytes (filled symbols) have lower Ca2+ set point (Ca0) and dissociation constant (Kd) values than those from adult myocytes (open symbols). A and B: representative native state recordings of macroscopic inside-out patches with 3.0 x 10–6 M free Ca2+ in bathing medium. Macroscopic currents were elicited by a series of 10-mV depolarization steps (–100 to +100 mV) of 500-ms duration from a holding potential of 0 mV. Vertical bars, 500 pA. C and D: voltage-activation curves in symmetrical 140 mM KCl for 0.3–30 µM Ca2+. Channel activities are expressed as channel conductance (G) relative to maximum G (Gmax). Solid lines, best-fit curves to Boltzmann equation (see METHODS). Vertical bars, SE (n = 5–10 for adult and n = 5–9 for fetus). E: effects of Ca2+ concentration ([Ca2+]) on BK channel. Membrane potential required for half-maximal channel activation (V0.5), Ca2+ sensitivity (i.e., slope), and Ca0 (i.e., x-intercept) were determined for adult and fetal myocyte BK channels. V0.5 values were obtained from data in C and D. Lines are linear regression fits to data (see Table 2 for linear fit parameters). F: effects of 0.1–100 µM free Ca2+ on mean BK channel activity at +20 mV from adult and fetal myocytes. Data were obtained from C and D. Solid lines, best-fit curves to Hill equation: G/Gmax = [Ca2+]Formula/(KdFormula+ [Ca2+]Formula), where nH is Hill coefficient (see Table 3 for nH and Kd values).

 

View this table:
[in this window]
[in a new window]
  		Table 1. V0.5 for adult and fetal myocytes

 
V0.5 values plotted as a function of the logarithm of Ca2+ concentration yielded linear and parallel relations for adult and fetal myocytes (Fig. 1E). The parameters for these linear fits are shown in Table 2. From the equations of the fitted lines, we estimated that {Delta}V0.5 or "Ca2+ sensitivity" of the BK channels ([Ca2+]i, µM i.e., change in V0.5 for a 10-fold change in Ca2+ concentration) did not change significantly (n = 4 for adult and fetus; Table 2). Ca0, defined as the Ca2+ concentration required for half-maximal activation at 0 mV, was 10.9 and 5.7 µM for adult and fetus, respectively.


View this table:
[in this window]
[in a new window]
  		Table 2. Linear fits for Ca0

 
For native state BK channels from adult SMCs, the mean apparent Kd for Ca2+ at +20 mV was 5.51 ± 0.41 µM, with a Hill coefficient (nH) of 1.71 ± 0.18 (Fig. 1F). In contrast, for channels from fetal SMCs, the mean apparent Kd for Ca2+ at +20 mV decreased by a factor of ~2 and was 2.27 ± 0.24 µM, and nH did not change significantly (1.99 ± 0.36; Fig. 1F). At 0 mV, Kd values averaged 11.61 ± 0.69 and 5.18 ± 0.49 µM for adult and fetal SMCs, respectively, and nH values were 2.64 + 0.47 and 1.80 + 0.44, respectively (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Best-fit values of Kd and nH from adult and fetal myocytes

 
Ca0 in the dephosphorylated state. To determine the effect of BK channel dephosphorylation on Ca0, alkaline phosphatase (350 U/ml) was added to the bath (i.e., cytosolic side) for 10 min to maximally shift the voltage-activation curves to the right, as previously described (19). Representative recordings of alkaline phosphatase-treated adult and fetal macropatch preparations are shown in Fig. 2, A and B. This treatment rightward shifted the BK channel voltage-activation curves from adult and fetal SMCs by 21.0 ± 5.4 mV (n = 5) and 36.5 ± 3.9 mV (n = 5), respectively (P < 0.05; Fig. 2, C and D). Although the extent of the rightward shift was significantly greater in the fetus, the final V0.5 values did not differ significantly between adult and fetal SMCs (Table 1). The Ca2+ sensitivities ({Delta}V0.5 values) of dephosphorylated BK channels did not differ significantly between the two age groups (Fig. 2E, Table 2).


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2. Dephosphorylated (Dephos) BK channels from fetal (filled symbols) and adult (open symbols) myocytes have similar Ca0 and Kd values but higher values than native state channels. Recording protocol and conditions are described in Fig. 1 legend. A and B: representative dephosphorylated state recordings of BK channels in macroscopic inside-out patches of adult and fetal myocytes. C and D: voltage-activation curves in symmetrical 140 mM KCl for 0.3–30 µM intracellular Ca2+. Vertical bars, SE (n = 5–7 for adult and fetus). E: effects of [Ca2+] on BK channel V0.5. Ca2+ sensitivity and Ca0 were determined for adult and fetal myocyte BK channels as described in Fig. 1E legend (see Table 2 for linear fit parameters). F: effects of 0.1–100 µM free Ca2+ on mean BK channel activity at +20 mV in adult and fetal myocytes as described in Fig. 1F legend (see Table 3 for nH and Kd values).

 
In the fully dephosphorylated state, the Ca0 values for adult and fetal BK channels were not significantly different (Fig. 2E, Table 2). For dephosphorylated BK channels from adult and fetal SMCs, the mean apparent Kd for Ca2+ at +20 mV also did not differ significantly (Fig. 1F; Table 3). We could not accurately calculate Kd and nH values at 0 mV for BK channels, because the current-voltage relations of the channels of the adult and fetus had shifted too far to the right (Fig. 2, C and D).

Ca0 in the PKA state. PKAc was used to determine the extent to which phosphorylation by PKA affects Ca0. Using alkaline phosphatase (350 U/ml for 10 min), we maximally dephosphorylated the channels in macropatch preparations. Then, after exchanging the bath solution to remove alkaline phosphatase activity, we added the selective PKG inhibitor KT-5823 (10–6 M), okadaic acid (10–8 M), Mg2+-ATP (5 x 10–4 M), and PKAc (30 U/ml) to adult and fetal preparations (Fig. 3, A and B) for 10 min. We previously showed that, under these conditions, KT-5823 and okadaic acid inhibit channel-associated PKG and protein phosphatase activities, respectively, and that the voltage-activation curves of the BK channels are maximally leftward shifted by PKAc (19). Addition of PKAc shifted the channel voltage-activation curves to the left by 63.7 ± 3.3 mV for adult and 54.5 ± 2.8 mV for fetal macropatches (n = 5 each; Fig. 3, C and D). Extent of the leftward shift, final V0.5 values, and {Delta}V0.5 values did not differ significantly between adult and fetal macropatches (Table 1).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. PKA-phosphorylated BK channels from fetal (filled symbols) and adult (open symbols) myocytes have similar Ca0 and Kd values but lower values than native state channels. Recording protocol and conditions are described in Fig. 1 legend. A and B: representative PKA-phosphorylated state recordings of BK channels in macroscopic inside-out patches of adult and fetal myocytes. C and D: voltage-activation curves in symmetrical 140 mM KCl for 0.3–30 µM intracellular Ca2+. Vertical bars, SE (n = 5–6 for adult and n = 5–7 for fetus). E: effects of [Ca2+] on BK channel V0.5. Ca2+ sensitivity and Ca0 were determined for adult and fetal myocyte BK channels as described in Fig. 1E legend (see Table 2 for linear fit parameters). F: effects of 0.1–100 µM free Ca2+ on mean BK channel activity at +20 mV from adult and fetal myocytes as described in Fig. 1F legend (see Table 3 for nH and Kd values).

 
Ca0 values for BK channels in the PKA state of 2.3 µM for adult and 2.9 µM for fetal myocytes were not significantly different (Fig. 3E, Table 2). Similarly, the mean apparent Kd values for Ca2+ at +20 mV for adult and fetal BK channels did not differ significantly (Fig. 1F, Table 3). At 0 mV, Kd values for adult and fetus did not differ significantly but were approximately twice the Kd values determined at +20 mV (Table 3).

Ca0 in the PKG state. To determine the extent to which PKG phosphorylation influences BK channel Ca0, we used commercially purified PKG. In a manner similar to our use of PKAc, we added PKG after BK channel dephosphorylation and subsequent removal of alkaline phosphatase. For adult and fetal macropatch preparations (Fig. 4, A and B), PKG (2,000 U/ml) was added to the bath solution in the presence of the selective PKA inhibitor KT-5720 (3 x 10–7 M), okadaic acid (10–8 M), Mg2+-ATP (5 x 10–4 M), and cGMP (5 x 10–5 M) for 10 min. We previously showed that, under these conditions, KT-5720 inhibits channel-associated PKG activity and that the voltage-activation curves of the BK channels are maximally leftward shifted by PKA (19). The voltage-activation curves of adult and fetal BK channels were leftward shifted by 51.8 ± 5.6 (n = 5) and 48.2 ± 1.4 mV (n = 5), respectively, by PKG (Fig. 4, C and D). After maximal PKG phosphorylation, neither the final V0.5 values for different Ca2+ concentrations nor the {Delta}V0.5 values differed significantly between the two age groups.


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		Fig. 4. PKG-phosphorylated BK channels from fetal (filled symbols) and adult (open symbols) myocytes have similar Ca0 and Kd values but lower values than native state channels. Recording protocol and conditions are described in Fig. 1 legend. A and B: representative PKG-phosphorylated state recordings of BK channels in macroscopic inside-out patches of adult and fetal myocytes. C and D: voltage-activation curves in symmetrical 140 mM KCl for 0.3–30 µM intracellular Ca2+. Vertical bars, SE (n = 5–7 for adult and n = 5–8 for fetus). E: effects of [Ca2+] on BK channel V0.5. Ca2+ sensitivity and Ca0 were determined for adult and fetal myocyte BK channels as described in Fig. 1E legend (see Table 2 for linear fit parameters). F: effects of 0–100 µM free Ca2+ on mean BK channel activity at +20 mV from adult and fetal myocytes as described in Fig. 1F legend (see Table 3 for nH and Kd values).

 
Ca0 values for adult and fetal BK channels were 3.6 µM (Fig. 4E, Table 2). For BK channels in the PKG state from adult and fetal SMCs at +20 mV, the mean apparent Kd for Ca2+ did not differ significantly (Fig. 4F, Table 3). At 0 mV, Kd values for adult and fetal SMCs did not differ significantly but were approximately twice the Kd values at +20 mV (Table 3).

Ca0 and Kd values in relation to channel phosphorylation state. To determine the extent to which, if any, Ca0 is modulated by the state of channel phosphorylation, we plotted Ca0 values against V0.5 values (Fig. 5A) obtained at 3 µM Ca2+ for each of the four phosphorylation states (Tables 1 and 2) for both age groups. We selected 3 µM Ca2+, because it is in the middle of the tested Ca2+ concentration range. The r2 values of the best-fit linear regression lines were 0.985 and 0.998 for BK channels from adult and fetal myocytes, respectively. The slopes of the fitted lines were 1.39 ± 0.17 and 1.49 ± 0.05 log(M)/102 mV for adult and fetus, respectively.


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Ca0 values of BK channels are comodulated by channel phosphorylation with V0.5 and Kd values. Open symbols, adult myocytes; filled symbols, fetal myocytes. A: V0.5 values at 3.0 x 10–6 M Ca2+ for each phosphorylation state [native (circle), dephosphorylated (triangle), PKA (square), and PKG (diamond)] plotted against Ca0 values. Best linear fit is indicated for maximally dephosphorylated, PKA, and PKG states for adult (slope = 1.54 x 10–2 M/mV) and fetus (slope = 1.43 x 10–2 M/mV). B: Kd values for each phosphorylation state of adult and fetus plotted against corresponding Ca0 values determined at +20 and 0 mV. Solid and dashed lines, best linear fit.

 
If these data from adult and fetus are fitted to the same line, the calculated r2 value of the best-fitted line is 0.990, indicating that data from adult and fetus coincide. These results strongly suggest that the different Ca0 values of BK channels from native adult and fetal myocytes, as previously reported (18) and confirmed here (Fig. 1), result from different extents of channel phosphorylation, possibly due, in part, to differences in the functional ratios of the channel-associated protein kinases and phosphatases (19).

Kd values of BK channels for Ca2+ also differed between phosphorylation states. We plotted the calculated Kd values at +20 and 0 mV for each phosphorylation state of adult and fetal SMCs against the corresponding Ca0 values and found two nonparallel, linear alignments with a common intersection near the origin (Fig. 5B). The slope of the fitted line at 0 mV was 1.12, indicating that the calculated Kd values at 0 mV and the Ca0 values are close to parity at each of the tested phosphorylation states. The mean Hill coefficients for all phosphorylation states from adult and fetal myocytes were 2.03 ± 0.17 and 2.02 ± 0.11 mV, respectively.

Membrane potentials in the native state. In an effort to understand more completely the relation of BK channel activity to membrane potential, using the whole cell current clamp, we measured resting membrane potentials on freshly prepared SMCs. For adult and fetal SMCs, the membrane potentials were –33.4 ± 2.5 (n = 13) and –26.1 ± 1.4 mV (n = 17), respectively (P < 0.05). These values indicate somewhat more depolarization than values reported in other studies, probably because of the liquid junction potential of ~12 mV. Adjustment for this junction potential gives –45.4 ± 1 and –38.1 ± 3.4 mV for adult and fetal myocytes, respectively. Thus fetal SMCs are depolarized relative to adult myocytes.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In vascular smooth muscle, the BK channels play an important role in determining membrane potential, L-type Ca2+ channel activity, and tone (7, 29). To a great extent, activity of the BK channel is determined by its state of phosphorylation (19, 37). As shown in the present study, the phosphorylation state of the BK channel modulates the channel's affinity for Ca2+ (i.e., Ca0 and Kd) without affecting channel Ca2+ sensitivity (i.e., {Delta}V0.5). By regulating the channel's affinity for Ca2+, the channel phosphorylation state determines the concentration range of the channel's response to changes in [Ca2+]i. Our present findings indicate that the previously reported lower Ca0 for BK channels of the fetus than the adult (18) is consistent with the hypothesis that differences in the extent of channel phosphorylation are responsible for differences in BK affinity for Ca2+ between the two age groups.

Our findings also demonstrate that Ca0 and Kd are numerically equivalent when the Kd values for Ca2+ are calculated at 0 mV. Ca0 is defined as the Ca2+ concentration that produces half-maximal channel activation at 0 mV (8). Kd for Ca2+, when calculated from values of relative conductance (G/Gmax) or open probabilities (Po) recorded at 0 mV, is operationally defined as the Ca2+ concentration that produces half-maximal channel activation. Thus Ca0 and Kd measure the affinity of the channel for Ca2+. The present study shows that differences in the extent of BK channel phosphorylation among the four defined phosphorylation states produce up to a 10-fold, graded change in the affinity of the channel for Ca2+, as reflected in the values for Ca0 and Kd (Table 2). However, when the channels from the adult and fetus are manipulated into the same phosphorylation state by exogenous treatment with alkaline phosphatase or PKA or PKG, they exhibit similar Ca0 and Kd values. Thus the phosphorylation state of the channel, and not the developmental age per se, is the primary determinant of Ca2+ affinity.

Effects of phosphorylation state. Using the Boltzmann equation and linear regression for each of the four phosphorylation states, we found that changes in phosphorylation state affected neither BK channel voltage sensitivity (Table 1) nor Ca2+ sensitivity (Table 2). Nonetheless, phosphorylation significantly altered channel activity (V0.5) and Ca2+ affinity (Ca0; Fig. 5A). The three manipulated phosphorylation states (i.e., dephosphorylated by alkaline phosphatase and phosphorylated by PKA or PKG) exhibit a linear relation between V0.5 and Ca0. The native phosphorylation states from adult and fetal myocytes differ from each other but reside within the best-fit linear regression of this relation, suggesting that the extent of channel phosphorylation elicits a colinear relation between V0.5 and Ca0. This may serve as a convenient tool to study and predict the extent and state of BK channel phosphorylation.

Physiological changes during development. The regulation of cerebral artery BK channel activity changes during development. Iberiotoxin, a selective BK channel inhibitor, produced a significant increase in [Ca2+]i and in tension responses of adult and fetal midcerebral arteries (23, 41). Although the increase in tension in the two age groups was similar, the fetal vessels exhibited twice the increase in [Ca2+]i of the adult (23). These results reflect the age-related difference in physiological, resting BK channel properties and how this may contribute to Ca2+ handling in vascular smooth muscle.

Fetal myocytes exhibit higher outward current density when depolarized because of the greater BK channel activity in fetal than in adult SMCs (18). As shown by others, Ca2+ spark frequency is higher in adult than in neonatal rat myocytes (13). Additionally, regulation of intracellular stores for Ca2+ release varies between adult and fetal SMCs (24, 27). Because the arrangement of ryanodine receptor channel and Ca2+ spark frequency directly influence BK channel activity (13, 34), the lower Ca2+ spark frequency in fetal myocytes may be compensated by an increase in BK channel activity (18). This increase in fetal BK channel activity may be caused by channel phosphorylation and/or membrane depolarization, both of which we have demonstrated to change during development in the present study. Such increased channel activity may provide an important mechanism for modulation of vascular tone in fetal myocytes, despite a lower level of spark activity.

As we reported previously, the unitary conductance of BK channels in adult and fetal myocytes does not differ significantly (18). Moreover, on the basis of Western immunoblotting, the expression levels of the {alpha}-subunit of BK channels in fetal and adult myocytes appear to be similar (data not shown). In addition, the number of functional BK channels on adult and fetal inside-out membrane patches of similar area do not differ significantly (18, 19). Hence, neither the changes in unitary conductance nor the level of channel protein expression appear to contribute to the higher BK channel current density in fetal myocytes. Thus the basis of the different BK channel current density most likely reflects an intrinsic difference in channel activity.

Three intrinsic factors, a lower channel voltage sensitivity, a higher Ca2+ sensitivity, and a lower Ca0 set point, can contribute to the higher Po of fetal myocytes (8). Of these three factors, our present findings, for which ovine basilar SMCs were used as a developmental model, indicate that only Ca0 is modulated and that such modulation is controlled through differential phosphorylation of the channel. How BK channel phosphorylation is differentially controlled during development is probably quite complicated. In addition to activity of different signaling pathways in adult and fetal SMCs, the BK channels in adult and fetal SMCs associate with distinctly different functional ratios of channel-associated phosphatases and kinases (19).

Ca0 of BK channels from different SMCs has been reported to range from 5 x 10–7 M in guinea pig mesenteric artery (3) to 10–6 M in rabbit portal vein (14) and 1.5 x 10–6 M in rat pulmonary artery (2) to 9 x 10–6 M in hamster cremasteric arteriole (15). With use of micropatch preparations from ovine basilar artery SMCs, the BK channel Ca0 was found to be 8.8 and 4.7 x 10–6 M in adult and fetus, respectively (18). In the present study, with use of macropatch preparations from the same tissue, Ca0 was 11.0 and 5.7 x 10–6 M for adult and fetus, respectively. Thus our Ca0 estimates from macropatch recordings not only correspond to the micropatch values, but they also fit within the higher range reported by others. For fetal myocytes, the lower Ca0 values imply that, at a given [Ca2+]i and voltage, these channels are more activated than those of the adult. We propose that this lower Ca0 value in the fetus is an adaptation to a lower resting [Ca2+]i (1, 12, 18) and to a higher resting membrane potential (10, and present findings) to compensate for greater dependence on extracellular Ca2+ (5, 24).

In ovine cerebral arteries, maturation is associated with decreased Ca2+ release from ryanodine receptor channels (24, 27). In addition, in neonatal rat basilar SMCs, the ryanodine receptor channels do not act in a synchronized fashion to produce Ca2+ sparks, as in the adult (13). It is not unreasonable to postulate that the lower Ca0 of fetal ovine myocyte BK channels counters the much less frequent Ca2+ sparks. Thus fetal myocytes may respond to a smaller subplasmalemmal [Ca2+]i change than do adult myocytes. In light of this, the lower Ca0 of fetal BK channels may act as a protective mechanism to govern the membrane potential and vascular reactivity.

Perspectives

We have attempted to integrate the developmental aspects of this study with those of others (5, 13, 18, 19, 23, 24, 27, 33). Because of clustering and functional coupling of ryanodine receptors, Ca2+ spark frequency and Ca2+ release are greater in the adult SMCs. In fetal myocytes, as a result of the greater degree of BK channel phosphorylation, the BK channels conduct higher outward currents. Furthermore, in fetal SMCs, the greater expression of L-type Ca2+ channels probably results in greater inward Ca2+ current. Thus the tighter functional coupling of fetal SMC BK channels to Ca2+ may compensate for the lower density of ryanodine receptor channels. As an integrated system, these constituents of the fetal SMC modulate closely membrane potential and [Ca2+]i, important factors in many SMC signaling pathways.

The present study demonstrates that 1) BK channel Ca0 in freshly isolated SMCs is significantly lower in the fetus than in the adult, 2) BK channel Ca0 can be modulated by phosphorylation of the channel, 3) under appropriately controlled conditions, the BK channel Ca0 may be used as a measure of the extent of BK channel phosphorylation, and 4) phosphorylation plays a key role in modulating BK channel activity in vascular smooth muscle during development. The Ca2+ sensitivity and/or Ca0 of BK channels may vary among different vascular tissues as a result of any of several individual factors or combination of factors, including channel splice variation, differential phosphorylation, beta-subunit expression, and redox state. In the specific case of the developing ovine basilar artery, however, the state of BK channel phosphorylation plays a major role in determining the affinity of the channel for Ca2+ (i.e., Ca0 and Kd).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant HD/HL-03807-34 (to L. D. Longo).


   ACKNOWLEDGMENTS
 
We thank Michael E. Barish (Beckman Research Institute, City of Hope, Duarte, CA) for helpful discussions and suggestions, William H. Fletcher (Department of Anatomy, Loma Linda University) for providing purified protein kinases, and Brenda Kreutzer for assistance in preparing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. D. Longo, Center for Perinatal Biology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (e-mail: llongo{at}llu.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akopov SE, Zhang L, and Pearce WJ. Physiological variations in ovine cerebrovascular calcium sensitivity. Am J Physiol Heart Circ Physiol 272: H2271–H2281, 1997.[Abstract/Free Full Text]
  2. Albarwani S, Robertson BE, Nye PC, and Kozlowski RZ. Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat. Pflügers Arch 428: 446–454, 1994.[CrossRef][ISI][Medline]
  3. Benham CD, Bolton TB, Lang RJ, and Takewaki T. Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J Physiol 371: 45–67, 1986.[Abstract/Free Full Text]
  4. Bielefeldt K and Jackson MB. Phosphorylation and dephosphorylation modulate a Ca2+-activated K+ channel in rat peptidergic nerve terminals. J Physiol 475: 241–254, 1994.[Abstract/Free Full Text]
  5. Blood AB, Zhao Y, Long W, Zhang L, and Longo LD. L-type Ca2+ channels in fetal and adult ovine cerebral arteries. Am J Physiol Regul Integr Comp Physiol 282: R131–R138, 2002.[Abstract/Free Full Text]
  6. Braun AP, Heist EK, and Schulman H. Inhibition of a mammalian large-conductance, calcium-sensitive K+ channel by calmodulin-binding peptides. J Physiol 527: 479–492, 2000.[Abstract/Free Full Text]
  7. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, and Aldrich RW. Vasoregulation by the beta1-subunit of the calcium-activated potassium channel. Nature 407: 870–876, 2000.[CrossRef][Medline]
  8. Carl A, Lee HK, and Sanders KM. Regulation of ion channels in smooth muscles by calcium. Am J Physiol Cell Physiol 271: C9–C34, 1996.[Abstract/Free Full Text]
  9. Collier ML, Thomas AP, and Berlin JR. Relationship between L-type Ca2+ current and unitary sarcoplasmic reticulum Ca2+ release events in rat ventricular myocytes. J Physiol 516: 117–128, 1999.[Abstract/Free Full Text]
  10. Evans AM, Osipenko ON, Haworth SG, and Gurney AM. Resting potentials and potassium currents during development of pulmonary artery smooth muscle cells. Am J Physiol Heart Circ Physiol 275: H887–H899, 1998.[Abstract/Free Full Text]
  11. Fox AJ, Barnes PJ, Venkatesan P, and Belvisi MG. Activation of large-conductance potassium channels inhibits the afferent and efferent function of airway sensory nerves in the guinea pig. J Clin Invest 99: 513–519, 1997.[ISI][Medline]
  12. Geary GG, Osol GJ, and Longo LD. Development affects in vitro vascular tone and calcium sensitivity in ovine cerebral arteries. J Physiol 558: 883–896, 2004.[Abstract/Free Full Text]
  13. Gollasch M, Wellman GC, 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]
  14. Inoue R, Kitamura K, and Kuriyama H. Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein. Pflügers Arch 405: 173–179, 1985.[CrossRef][ISI][Medline]
  15. Jackson WF and Blair KL. Characterization and function of Ca2+-activated K+ channels in arteriolar muscle cells. Am J Physiol Heart Circ Physiol 274: H27–H34, 1998.[Abstract/Free Full Text]
  16. Kume H, Mikawa K, Takagi K, and Kotlikoff MI. Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L221–L229, 1995.[Abstract/Free Full Text]
  17. Levitan IB. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56: 193–212, 1994.[CrossRef][ISI][Medline]
  18. Lin MT, Hessinger DA, Pearce WJ, and Longo LD. Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery. Am J Physiol Heart Circ Physiol 285: H701–H709, 2003.[Abstract/Free Full Text]
  19. Lin MT, Longo LD, Pearce WJ, and Hessinger DA. Ca2+-activated K+ (BK) channel-associated phosphatase and kinase activities during development. Am J Physiol Heart Circ Physiol 289: H414–H425, 2005.[Abstract/Free Full Text]
  20. Ling S, Woronuk G, Sy L, Lev S, and Braun AP. Enhanced activity of a large-conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase. J Biol Chem 275: 30683–30689, 2000.[Abstract/Free Full Text]
  21. Lingle CJ, Solaro CR, Prakriya M, and Ding JP. Calcium-activated potassium channels in adrenal chromaffin cells. Ion Channels 4: 261–301, 1996.[Medline]
  22. Liu Y, Hudetz AG, Knaus HG, and Rusch NJ. Increased expression of Ca2+-sensitive K+ channels in the cerebral microcirculation of genetically hypertensive rats: evidence for their protection against cerebral vasospasm. Circ Res 82: 729–737, 1998.[Abstract/Free Full Text]
  23. Long W, Zhang L, and Longo LD. Cerebral artery KATP and KCa channel activity and contractility: changes with development. Am J Physiol Regul Integr Comp Physiol 279: R2004–R2014, 2000.[Abstract/Free Full Text]
  24. Long W, Zhang L, and Longo LD. Cerebral artery sarcoplasmic reticulum Ca2+ stores and contractility: changes with development. Am J Physiol Regul Integr Comp Physiol 279: R860–R873, 2000.[Abstract/Free Full Text]
  25. Long W, Zhang L, and Longo LD. Fetal and adult cerebral artery KATP and KCa channel responses to long-term hypoxia. J Appl Physiol 92: 1692–1701, 2002.[Abstract/Free Full Text]
  26. Longo LD, Zhao Y, Long W, Miguel C, Windemuth RS, Cantwell AM, Nanyonga AT, Saito T, and Zhang L. Dual role of PKC in modulating pharmacomechanical coupling in fetal and adult cerebral arteries. Am J Physiol Regul Integr Comp Physiol 279: R1419–R1429, 2000.[Abstract/Free Full Text]
  27. Nauli SM, Williams JM, Akopov SE, Zhang L, and Pearce WJ. Developmental changes in ryanodine- and IP3-sensitive Ca2+ pools in ovine basilar artery. Am J Physiol Cell Physiol 281: C1785–C1796, 2001.[Abstract/Free Full Text]
  28. 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]
  29. Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]
  30. Park JB, Kim HJ, Ryu PD, and Moczydlowski E. Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis. J Gen Physiol 121: 375–397, 2003.[Abstract/Free Full Text]
  31. Patton C, Thompson S, and Epel D. Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium 35: 427–431, 2004.[CrossRef][ISI][Medline]
  32. Pearce WJ, Hull AD, Long DM, and Longo LD. Developmental changes in ovine cerebral artery composition and reactivity. Am J Physiol Regul Integr Comp Physiol 261: R458–R465, 1991.[Abstract/Free Full Text]
  33. Perez GJ, Bonev AD, and Nelson MT. Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle. Am J Physiol Cell Physiol 281: C1769–C1775, 2001.[Abstract/Free Full Text]
  34. 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]
  35. Reeve HL, Weir EK, Archer SL, and Cornfield DN. A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent. Am J Physiol Lung Cell Mol Physiol 275: L1019–L1025, 1998.[Abstract/Free Full Text]
  36. Robitaille R, Garcia ML, Kaczorowski GJ, and Charlton MP. Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11: 645–655, 1993.[CrossRef][ISI][Medline]
  37. Schubert R and Nelson MT. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22: 505–512, 2001.[CrossRef][Medline]
  38. Szado T, McLarnon M, Wang X, and van Breemen C. Role of sarcoplasmic reticulum in regulation of tonic contraction of rabbit basilar artery. Am J Physiol Heart Circ Physiol 281: H1481–H1489, 2001.[Abstract/Free Full Text]
  39. Tang XD, Garcia ML, Heinemann SH, and Hoshi T. Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing. Nat Struct Mol Biol 11: 171–178, 2004.[CrossRef][ISI][Medline]
  40. Tang XD, Xu R, Reynolds MF, Garcia ML, Heinemann SH, and Hoshi T. Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels. Nature 425: 531–535, 2003.[CrossRef][Medline]
  41. Teng GQ, Nauli SM, Brayden JE, and Pearce WJ. Maturation alters the contribution of potassium channels to resting and 5HT-induced tone in small cerebral arteries of the sheep. Brain Res Dev Brain Res 133: 81–91, 2002.[Medline]
  42. Zhang F, Kaide JI, Yang L, Jiang H, Quan S, Kemp R, Gong W, Balazy M, Abraham NG, and Nasjletti A. CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin. Am J Physiol Heart Circ Physiol 286: H137–H144, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Y. Gao, A. D. Portugal, S. Negash, W. Zhou, L. D. Longo, and J. Usha Raj
Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia
Am J Physiol Lung Cell Mol Physiol, March 1, 2007; 292(3): L678 - L684.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, M. T.
Right arrow Articles by Longo, L. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, M. T.
Right arrow Articles by Longo, L. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.