AJP - Heart AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H2986-H2994, 2007. First published August 31, 2007; doi:10.1152/ajpheart.00491.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/H2986    most recent
00491.2007v1
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 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 Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Du, Y.-M.
Right arrow Articles by Nathan, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Du, Y.-M.
Right arrow Articles by Nathan, R. D.

Simulated ischemia enhances L-type calcium current in pacemaker cells isolated from the rabbit sinoatrial node

Yi-Mei Du1,2 and Richard D. Nathan1

1Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas; and 2Department of Cardiology, Institute of Cardiovascular Diseases, Ion Channelopathy Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Submitted 24 April 2007 ; accepted in final form 29 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ischemic-like conditions (a glucose-free, pH 6.6 Tyrode solution bubbled with 100% N2) enhance L-type Ca current (ICa,L) in single pacemaker cells (PCs) isolated from the rabbit sinoatrial node (SAN). In contrast, studies of ventricular myocytes have shown that acidic extracellular pH, as employed in our "ischemic" Tyrode, reduces ICa,L. Therefore, our goal was to explain why ICa,L is increased by "ischemia" in SAN PCs. The major findings were the following: 1) blockade of Ca-induced Ca release with ryanodine, exposure of PCs to BAPTA-AM, or replacement of extracellular Ca2+ with Ba2+ failed to prevent the ischemia-induced enhancement of ICa,L; 2) inhibition of protein kinase A with H-89, or calcium/calmodulin-dependent protein kinase II with KN-93, reduced ICa,L but did not prevent its augmentation by ischemia; 3) ischemic Tyrode or pH 6.6 Tyrode shifted the steady-state inactivation curve in the positive direction, thereby reducing inactivation; 4) ischemic Tyrode increased the maximum conductance but did not affect the activation curve; 5) in rabbit atrial myocytes isolated and studied with exactly the same techniques used for SAN PCs, ischemic Tyrode reduced the maximum conductance and shifted the activation curve in the positive direction; pH 6.6 Tyrode also shifted the steady-state inactivation curve in the positive direction. We conclude that the acidic pH of ischemic Tyrode enhances ICa,L in SAN PCs, because it increases the maximum conductance and reduces inactivation. Furthermore, the opposite results obtained with rabbit atrial myocytes cannot be explained by differences in cell isolation or patch-clamp techniques.

acidosis; protein kinase A; calcium/calmodulin-dependent protein kinase II; calcium-induced inactivation; rabbit atrial myocytes

IT IS WELL KNOWN HOW ischemic-like conditions affect transmembrane currents in isolated atrial and ventricular myocytes (reviewed in Ref. 3), whereas little information exists for isolated cardiac pacemaker cells (PCs). As reported in an abstract, Han et al. (14) induced metabolic inhibition by exposing rabbit sinoatrial node (SAN) PCs to 3 mM cyanide. After 10 min, L-type Ca current (ICa,L) had declined; however, they did not mention if that effect was statistically significant. More recently, Ju and Allen (16) exposed nine toad sinus venosus PCs to 2 mM cyanide. ICa,L at 0 mV was reduced by 23%; however, the reduction was not statistically significant. One hallmark of ischemia is a marked decline in extracellular pH (17). When Satoh and Seyama (35) reduced extracellular pH to 6.5, they observed a voltage-independent reduction of ICa,L in small pieces of rabbit SAN tissue.

To explain the bradycardia (heart rate < 60 beats/min) experienced by patients following resuscitation from cardiac arrest (29), we recently isolated single PCs from the rabbit SAN and exposed them to ischemic-like conditions that consisted of a glucose-free, pH 6.6 Tyrode solution bubbled with 100% N2 (8). While recording the currents that contribute to SAN pacemaker activity, we made the surprising discovery that our "ischemic" Tyrode solution enhanced ICa,L significantly at potentials between –30 and +30 mV. At that time, we suggested that the greater ICa,L could account for a 6-mV increase in the action potential overshoot. We also proposed that the reductions of inward Na-Ca exchange current and T-type Ca current (ICa,T) that we observed were responsible for the slower pacemaker activity during "ischemia" (8). If ICa,L also increases during global ischemia, we speculate that such a response could reduce the action potential threshold and, thereby, counter the slower diastolic depolarization.

In contrast with our results in SAN PCs (8), previous studies of atrial and ventricular myocytes had shown that acidic extracellular pH, one of the hallmarks of global ischemia (reviewed in Ref. 3), reduces ICa,L significantly (reviewed in Ref. 28). Thus the goal of the current investigation was to explain why ICa,L in rabbit SAN PCs is increased by simulated ischemia. To attain that goal, we tested the hypothesis that ischemia enhances ICa,L by reducing Ca-induced inactivation of the current. However, we found that neither ryanodine, BAPTA-AM, nor replacement of extracellular Ca2+ (Cao) with Ba2+ could prevent the enhancement. Protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylate L-type Ca channels and enhance ICa,L in rabbit SAN PCs under basal conditions (31, 41). CaMKII plays a role in Ca-dependent facilitation of L-type Ca channels (42, 44), and PKA facilitates oscillations of local Ca2+ release that contribute to SAN pacemaker activity (40). Therefore, we also tested the hypothesis that ischemic Tyrode stimulates PKA and/or CaMKII, thereby enhancing ICa,L. Nevertheless, our results suggest that this hypothesis was also incorrect. After investigating the gating properties of ICa,L, we concluded that an increase in the maximum conductance and a positive shift of the steady-state inactivation curve, possibly due to H+ directly affecting L-type Ca channels, were responsible for the increased current.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of SAN PCs and right atrial myocytes. Standard procedures in our laboratory were used to isolate single SAN PCs from New Zealand White rabbits weighing 1.5–2.5 kg (8, 11). Precisely the same procedures were employed to isolate single myocytes from the right atrial appendage. The following is a brief description. Our animal protocol was approved by the Institutional Animal Care and Use Committee of the Texas Tech University Health Sciences Center, and the procedures complied fully with guidelines established in the Guide for the Care and Use of Laboratory Animals(NIH Publication 65–23, revised 1996). After a rabbit had been deeply anesthetized with 5% isoflurane, as confirmed by the absence of response to a normally painful stimulus, we removed the heart, excised the right atrium, and extracted a rectangular piece of tissue (1.5–2.0 x 3.0–3.5 mm), either from the SAN or from the right atrial appendage. Based on maps of the rabbit SAN reported in the literature (1), we assume that the excised nodal tissue included primary PCs located in the functional "center" of the SAN, as well as subsidiary PCs located in the "periphery." The tissue was exposed twice to a protease solution and then four or five times to a collagenase solution (37°C). Freed cells were transferred to a protease inhibitor solution after each exposure. After the last exposure, the cells were spun down, resuspended in a cold, modified Kraft-Brühe solution, and maintained at 4°C until use later the same day. Although we routinely isolated spider-shaped, spindle-shaped, and rod-shaped cells from the SAN (8), the current experiments were performed on mostly striated, beating spindle-shaped PCs or quiescent atrial myocytes. Because a TTX-sensitive Na current (INa) could be recorded in the spindle-shaped PCs when the holding potential was negative to –60 mV, those cells were most likely "transitional" (subsidiary) rather than "central" (primary) PCs (1). As reported recently (8), the beat rates of our freshly isolated PCs averaged 82 ± 13 beats/min (n = 6).

Electrophysiological techniques. A perforated-patch technique was used to record ICa,L because it minimized rundown of the current. We employed beta-escin (10–15 µM) (9) because it can be dissolved in water. In contrast, nystatin and amphotericin B must be dissolved in alcohol or dimethylsulfoxide (DMSO), solvents that are harmful to SAN PCs. Because beta-escin-induced channels are quite large (9), the pipette solution included important molecules that could leak from the cytoplasm into the patch pipette. It contained the following (in mM): 110 cesium-aspartate, 20 CsCl, 1 MgCl2, 5 Na2ATP, 5 Na2 creatine PO4, 0.1 Na2GTP, 0.05 cAMP, 5 EGTA, and 5 HEPES. The pH was adjusted to 7.2 with CsOH, and the osmolarity of the pipette solution was 283 ± 2 mosM (mean ± SE, n = 10). EGTA was included to buffer any Ca2+ that might leak from the pipette to the cytoplasm. To block time- and voltage-dependent currents other than ICa,L, we replaced potassium with cesium in the pipette solution and included 4 mM CsCl, 4 mM 4-aminopyridine, 5 µM chromanol 293 B, and 1 µM E-4031 in the Tyrode bathing solution. When recording current-voltage (ICa,L-V) relationships, we held the membrane potential at –40 mV to inactivate TTX-sensitive INa (26) and ICa,T (10). Measurements of steady-state inactivation employed a holding potential of –60 mV and prepulses (P1) that ranged from –90 to 0 mV, long enough (450 ms) to completely inactivate ICa,L (10). A 400-ms test pulse (P2) to +10 mV followed with no delay, and the paired pulses were presented once every 5 s, long enough for complete recovery from inactivation (32). With such negative prepulses, INa can be very large (26) and ICa,T is significant (10); therefore, INa was blocked by 10–20 µM TTX, and ICa,T was reduced by 0.5 µM mibefradil in such double-pulse experiments. Before each experiment, we waited 20–30 min for the series resistance to reach a minimum, suggesting that membrane perforation was complete. The series resistance averaged 8.8 ± 0.4 M{Omega} for 41 PCs and 8.6 ± 1.5 M{Omega} for 8 atrial myocytes after compensating > 50% of its initial value. Further compensation resulted in oscillation of the recorded current. Although we did not measure their dimensions, PC capacitance averaged 48 ± 2 pF (n = 41), and atrial myocyte capacitance averaged 68 ± 5 pF (n = 8). In most experiments, the bathing solutions were held within 1° of 33°C.

Solutions. The modified Kraft-Brühe solution contained the following (in mM): 70 L-glutamic acid, 25 KCl, 10 KH2PO4, 3 MgCl2, 20 taurine, 10 glucose, 2 Na2ATP, 0.3 EGTA, and 10 HEPES. The pH was titrated to 7.4 with KOH. The normal Tyrode solution contained the following (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.6 MgCl2, 0.6 NaH2PO4, 1.0 NaHCO3, 5.5 glucose, and 10 HEPES. The pH was titrated to 7.4 with NaOH. Before the addition of CaCl2 and glucose, the osmolarity of the normal Tyrode was 291 ± 1 mosM (mean ± SE, n = 12). Global ischemia can reduce extracellular pH to 6.6 in guinea pig hearts after only 8 min (17) and intracellular pH to 6.3 in rabbit hearts after only 10 min (24); therefore, we titrated our ischemic Tyrode solution to pH 6.6. Glucose was omitted, and the ischemic Tyrode was bubbled with 100% N2. Unfortunately, because the cell chamber was open to the air, the PO2 could not be reduced less than 147 Torr (8, 11). Previously, extracellular potassium was increased to 10 mM in some experiments to mimic ischemic conditions (8, 17). However, we did not do so in the present study because potassium concentration does not influence ICa,L during voltage clamp. Ryanodine (Invitrogen, Grand Island, NY, or Alomone Laboratories, Jerusalem, Israel) was dissolved in water to make a 5 mM stock solution; H-89 (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO to make a 20 mM stock solution; KN-93 (EMD Biosciences, La Jolla, CA) was dissolved in water to make a 10 mM stock solution; and BAPTA-AM (Invitrogen, Carlsbad, CA) was dissolved in 5% pluronic/DMSO to make a 25 mM stock solution.

Data analyses. Voltage-clamp recordings were performed with a model 3900A integrating patch-clamp amplifier (Dagan, Minneapolis, MN) and Clampfit 8.0 software (Molecular Devices, Sunnyvale, CA). We estimated the reversal potential of ICa,L and the maximum conductance by fitting the linear portion of the ICa,L-V relationship with a straight line. The activation curve was approximated by the normalized conductance-voltage relationship. Peak ICa,L was normalized to its maximum value during P2 to obtain a steady-state inactivation parameter (f{infty}) at each potential. Origin 7.0 (Microcal Software, Northampton, MA) and the appropriate Boltzmann equations (10) provided best fits of mean activation- and steady-state inactivation-voltage relationships. A single exponential was adequate to fit the inactivation phase of ICa,L. Data are expressed as means ± SE. ANOVA for repeated measures and a Tukey pairwise comparison posttest (SigmaStat 2.03; Systat Software, San Jose, CA) were used to determine whether there were significant differences among mean values for peak ICa,L or activation and inactivation parameters under control, ischemic, and washout conditions. Student's t-test for paired variants provided comparisons of mean inactivation time constants or maximum conductances under control and ischemic conditions. For all statistical tests, differences were considered significant only if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The experiments described below were designed to explain the previous observation that ischemic Tyrode enhances ICa,L (8). For example, when a PC was exposed to ischemic Tyrode for only 5 min, peak ICa,L at 0 mV was increased by >50% (Fig. 1A). On average, peak ICa,L was increased significantly at potentials between –30 and +30 mV (Fig. 1B), and the time course of its inactivation was shortened significantly (n= 6, P < 0.05). For example, at 0 mV, the mean time constants for ICa,L inactivation were 25.0 ± 2.6 and 21.1 ± 3.4 ms for control and ischemia, respectively. Washout of ischemic Tyrode resulted in complete recovery of ICa,L to its control value at each potential (Fig. 1B). In this example and the experiments described below, the close correspondence of ICa,L between control and washout (recordings performed some 15–20 min apart) suggests there was no significant rundown of the current during the experiments. Such rundown, which is often seen in ruptured-patch recordings but not perforated-patch recordings of ICa,L (9), results from leakage of important molecules from the cytoplasm to the pipette.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. "Ischemia" enhances L-type Ca current (ICa,L) in rabbit sinoatrial node pacemaker cells. A: ICa,L was recorded 30 min after obtaining the giga-ohm seal, 8 min after exposure to "ischemic" Tyrode (thick trace), and 5 min after washout of ischemic Tyrode. Cell capacitance was 52 pF. B: mean density of peak ICa,L for control, ischemia (thick trace), and washout. Holding potential was –40 mV. *P < 0.05, **P < 0.01, n = 6. [Reprinted with permission from Elsevier, from Du and Nathan (8).]

 
We performed three experiments to test the hypothesis that ischemia enhances ICa,L by reducing Ca-induced inactivation of the current. First, we blocked Ca release from the sarcoplasmic reticulum (SR) with 40 µM ryanodine, thereby reducing the concentration of intracellular Ca2+ (Cai). Previously, we found 10 µM ryanodine to be sufficient for this purpose (20). Although 40 µM ryanodine did increase ICa,L in some PCs, the enhancement was not statistically significant at any potential (Fig. 2B), as we have shown previously (20). After the PCs had been exposed to 40 µM ryanodine, ischemic Tyrode increased ICa,L significantly at 0 mV (Fig. 2A) and all other potentials, except –30 and –20 mV (Fig. 2B). When we repeated the experiment with 2 µM ryanodine, which depletes the SR of Ca2+ in rabbit SAN PCs (19), we obtained the same result: ICa,L was enhanced by ischemic Tyrode in five PCs. Next we employed 25 µM BAPTA-AM to reduce Cai directly. This rapidly acting Ca2+ chelator did not increase ICa,L significantly, and it did not prevent significant enhancement of the current (at all potentials except –30 mV) when the six PCs were exposed to ischemia for 7 min (Fig. 3). Finally, we replaced Cao with Ba2+ to eliminate Ca-dependent inactivation of ICa,L (4). This slowed the current's inactivation dramatically. For example, the time constant for inactivation increased from 19 to 110 ms in one PC (compare L-type Ba2+ current and ICa,L in Fig. 4A). Nevertheless, even in the absence of Ca-dependent inactivation, Ba2+ current through L-type Ca channels was increased significantly by ischemic Tyrode in seven PCs and at all potentials, except –30 and –20 mV (Fig. 4B).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Ischemia enhances ICa,L in the presence of ryanodine. A: ICa,L was recorded 25 min after obtaining the giga-ohm seal, 10 min after exposure to 40 µM ryanodine, and 5 min after exposure to ischemic Tyrode (thick trace). Capacitance was 49 pF. B: mean density of peak ICa,L for control, ryanodine, and ischemia (thick trace). Holding potential was –40 mV. *P < 0.05, ***P < 0.001; n = 6.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Ischemia enhances ICa,L in the presence of BAPTA. A: ICa,L was recorded 20 min after obtaining the giga-ohm seal, 10 min after exposure to 25 µM BAPTA-AM, and 7 min after exposure to ischemic Tyrode (thick trace). Capacitance was 44 pF. B: mean density of peak ICa,L for control, BAPTA, and ischemia (thick trace). Holding potential was –40 mV. *P < 0.05, **P < 0.01; n = 6.

 

Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. Ischemia enhances ICa,L when extracellular Ca2+ is replaced by Ba2+. A: ICa,L was recorded in Tyrode containing 1.8 mM CaCl2 15 min after obtaining the giga-ohm seal, 10 min after replacing 1.8 mM CaCl2 with 1.8 mM BaCl2, and 7 min after exposure to ischemic Tyrode containing 1.8 mM BaCl2 (thick trace). Capacitance was 30 pF. B: mean density of peak L-type Ba2+ current (IBa,L) for control, IBa,L during ischemia (thick trace), and IBa,L during washout. Holding potential was –40 mV. *P < 0.05, **P < 0.01; n = 7.

 
Next, two inhibitors were employed to test the hypothesis that ischemia enhances ICa,L by activating protein kinases that phosphorylate L-type Ca channels. The rationale for using inhibitors was the following. If our hypothesis were correct, then ischemic Tyrode could not enhance ICa,L, if those kinases were inhibited. Activation of PKA is known to enhance ICa,L in SAN PCs and to be quite active, even under basal conditions (31, 40); therefore, we treated seven PCs with 10 µM H-89, a relatively selective inhibitor of PKA (30). After 10 min, H-89 had reduced peak ICa,L significantly at 0 mV (Fig. 5A) and at all other potentials (Fig. 5B). Nevertheless, even with H-89 present, ICa,L was enhanced significantly at all potentials, but at –30 and –20 mV when the PCs were exposed to ischemic Tyrode for 8 min (Fig. 5B). CaMKII plays a role in Ca-dependent facilitation of L-type Ca channels (44) and in regulating SAN pacemaker activity by modulating ICa,L inactivation and reactivation (41). Therefore, we used KN-93, a selective inhibitor of CaMKII (36), to determine whether that kinase plays a role in the ischemia-induced enhancement of ICa,L. After 10-min exposures to 1 µM KN-93, six PCs exhibited significant reductions of peak ICa,L at –10 and 0 mV (P < 0.05; data not shown). Nevertheless, even with KN-93 present, ICa,L was increased significantly at –20, –10, and 0 mV when the PCs were exposed to ischemic Tyrode for 5 min (data not shown).


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 5. Ischemia enhances ICa,L in the presence of H-89, a protein kinase A inhibitor. A: ICa,L was recorded 20 min after obtaining the giga-ohm seal, 10 min after exposure to 10 µM H-89, and 8 min after exposure to H-89 and ischemic Tyrode (thick trace). Capacitance was 59 pF. B: mean density of peak ICa,L for control, H-89, and ischemia (thick trace). Holding potential was –40 mV. #P < 0.05, ##P < 0.01, ###P < 0.001, n = 7 for comparisons of H-89 and control; *P < 0.05, **P < 0.01, n = 7 for comparisons of ischemia and H-89.

 
A double-pulse procedure was employed to determine whether steady-state inactivation played a role in the ischemia-induced enhancement of ICa,L (Fig. 6A, inset). Figure 6, A and B, illustrates the effect of ischemic Tyrode on ICa,L during the test pulse (P2). Although the maximum current was not enhanced by ischemia when the prepulse was –90 mV, there was proportionately less inactivation produced by the –40 and –20 mV prepulses following a 5-min exposure to ischemic Tyrode (e.g., note the smaller decrement of ICa,L between the two arrows when comparing current traces during ischemia and control). On average, f{infty} at potentials between –50 and –20 mV was increased significantly during ischemia (Fig. 6C; n= 5). On the other hand, f{infty} for control and washout conditions did not differ significantly at any potential. Ischemic Tyrode shifted the half-inactivation potential (V1/2) ~5 mV in the positive direction compared with the control (V1/2 = –26.9 ± 0.3 and –31.7 ± 0.6 mV, respectively; P < 0.01), and the slope factor (k) was reduced from 11.3 ± 0.5 to 9.8 ± 0.3 (P < 0.05). These results suggest that a positive shift of the steady-state inactivation curve (i.e., a reduction of inactivation) and the concomitant increase in ICa,L played a role in the ischemia-induced enhancement of ICa,L.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 6. Ischemia alters steady-state inactivation of ICa,L. A: ICa,L was recorded 25 min after obtaining the giga-ohm seal. Prepulse (P1) was –90 (arrow), –40 (arrow), –20, and 0 mV and 450 ms in duration. The 400-ms test pulse (P2) was +10 mV and 400 ms in duration. ICa,L during the end of P1 and most of P2 is illustrated. Capacitance was 57 pF. B: same as A, except ICa,L was recorded 5 min after exposure to ischemia. C: mean steady-state inactivation data for control, ischemia (thick line), and washout. *P < 0.05, **P < 0.01, ***P < 0.001; n = 5. Curves are Boltzmann fits of the mean values.

 
In contrast, there was no significant effect of ischemic Tyrode on the activation-voltage relationship. The mean values for V1/2 and k for control and ischemia did not differ significantly (V1/2 = –17.6 ± 0.3 and –17.3 ± 0.4 mV, respectively; k = 6.7 ± 0.3 and 6.5 ± 0.3, respectively). Nevertheless, the maximum conductance was increased significantly by ischemic Tyrode (from 3.9 ± 1.0 to 6.8 ± 1.5 nS; P < 0.01).

Our laboratory showed previously that the acidic pH of our ischemic Tyrode solution was responsible for ischemia's effects on SAN PC electrical activity (11); therefore, we repeated our measurements of steady-state inactivation using otherwise normal Tyrode solution titrated to pH 6.6. This pH 6.6 Tyrode solution increased the maximum amplitude of ICa,L and reduced its inactivation following prepulses to –30 and –20 mV (compare Fig. 7, A and B). On average, pH 6.6 Tyrode increased f{infty} significantly at potentials between –70 and –20 mV (Fig. 7C; n= 7) and shifted the steady-state inactivation curve ~7 mV in the positive direction compared with the control (V1/2 = –26.7 ± 0.6 and –34.1 ± 1.1 mV, respectively; P < 0.01). In this case, the k was reduced significantly (from 11.5 ± 1.0 to 8.4 ± 0.6; P < 0.001).


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. Acidosis alters steady-state inactivation of ICa,L, just like ischemia. A: ICa,L was recorded 25 min after obtaining the giga-ohm seal. P1 was –90 (arrow), –40 (arrow), –30, and 0 mV and 450 ms in duration. P2 was +10 mV and 400 ms in duration. ICa,L during the end of P1 and most of P2 is illustrated. Capacitance was 41 pF. B: same as A, except ICa,L was recorded 5 min after exposure to pH 6.6 Tyrode. C: mean steady-state inactivation data for control, pH 6.6 Tyrode (thick line), and washout. *P < 0.05, **P < 0.01, ***P < 0.001; n = 7. Curves are Boltzmann fits of the mean values.

 
To directly compare our results with the effects of ischemia or pH 6.6 Tyrode on atrial myocytes, we used exactly the same procedures to isolate single cells from the right atrial appendage and to record ICa,L. In a typical experiment, a quiescent atrial myocyte exhibited a marked reduction of ICa,L, rather than an increase, and slower, rather than faster, inactivation of ICa,L during a 5-min exposure to ischemic Tyrode (Fig. 8A). After washout of the ischemic Tyrode, ICa,L recovered and even exceeded its original amplitude, suggesting that its reduction during ischemia was not due to current rundown. On average, ischemic Tyrode reduced ICa,L significantly at potentials between –20 and +10 mV (Fig. 8B; n = 5) and increased the time constant for its inactivation at 0 mV (from 22.4 ± 1.7 to 28.9 ± 2.2 ms; P < 0.05). The values for control and washout were not significantly different at any potential.


Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Ischemia reduces ICa,L in right atrial myocytes. A: ICa,L was recorded 25 min after obtaining the giga-ohm seal, 5 min after exposure to ischemic Tyrode (thick trace), and 5 min after washout of ischemic Tyrode. Cell capacitance was 92 pF. B: mean density of peak ICa,L for control, ischemia (thick trace), and washout. Holding potential was –40 mV. *P < 0.05, ***P < 0.001; n = 5.

 
In agreement with our results for SAN PCs, pH 6.6 Tyrode reduced ICa,L inactivation at –20 and –10 mV (e.g., note the smaller decrement of ICa,L between the two arrows when comparing current traces during pH 6.6 and control in Fig. 9, B and A, respectively), thereby significantly increasing f{infty} mean values at those potentials (Fig. 9C) and shifting the steady-state inactivation curve ~4 mV in the positive direction compared with the control (V1/2 = –11.6 ± 1.3 and –15.8 ± 1.2, respectively; P < 0.01, n= 5). The k was not affected. In contrast with our results for SAN PCs, in atrial myocytes, ischemic Tyrode reduced the mean normalized conductance at –10, 0, and +10 mV (P < 0.001) and shifted the activation curve ~6 mV to more positive potentials compared with the control (Fig. 9D) (V1/2 = +1.9 ± 0.8 and –3.6 ± 0.4 mV, respectively; P < 0.001, n = 5). The k was unchanged. Also, in contrast, the maximum conductance was reduced significantly by ischemic Tyrode (from 7.7 ± 0.8 to 6.4 ± 0.8 nS; P < 0.05) in atrial myocytes.


Figure 9
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 9. Acidosis alters steady-state inactivation of ICa,L, and ischemia alters its activation in right atrial myocytes. A: ICa,L was recorded 20 min after obtaining the giga-ohm seal. P1 was 450 ms in duration and equal to –50 (bottom arrow), –20 (top arrow), and –10 mV. P2 was 400 ms in duration and equal to +10 mV. Currents during the end of P1 and the first half of P2 are illustrated. Capacitance was 64 pF. B: same as A, except ICa,L was recorded after 5 min of exposure to pH 6.6 Tyrode. C: mean steady-state inactivation data for control and pH 6.6 Tyrode (thick line). *P < 0.05, **P < 0.01; n = 5. D: mean normalized conductance (activation) data for control and ischemia (thick line), which were derived from Fig. 8B as described in the METHODS. ***P < 0.001; n = 5. Curves are Boltzmann fits of the mean values.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Comparison of the current results with previous studies. We are aware of just a few reports describing how ICa,L is modified in isolated cardiac PCs exposed to ischemic-like conditions. However, it is difficult to compare the results of those investigations with the current one, because the pH of the bathing solution was not reduced in the previous ones. After treating rabbit SAN PCs with 3 mM cyanide for 10 min to produce metabolic inhibition, Han et al. (14) reported a depression of ICa,L, as well as delayed rectifier and hyperpolarization-activated currents. It is uncertain, however, if those changes were statistically significant. Similarly, Ju and Allen (16) exposed toad sinus venosus PCs to 2 mM cyanide. ICa,L at 0 mV was reduced by 23%; however, the reduction was not statistically significant. To simulate reperfusion of the ischemic SAN, Guo et al. (12) treated rabbit SAN PCs briefly (<10 min) with 0.1 mM H2O2 and observed a significant increase in peak ICa,L and a marked slowing of the latter phase of its inactivation. A comparison can be made with small multicellular preparations of rabbit SAN PCs that were exposed to acidic conditions (pH 6.5). Under those conditions, Satoh and Seyama (35) observed a 21% reduction of the maximum slope conductance, but no significant shift of the steady-state inactivation curve. In the present study, ischemic Tyrode (pH 6.6) produced a 74% increase in the maximum slope conductance and a 5-mV positive shift of the inactivation curve in isolated SAN PCs. Such differences might be due to the very different PC preparations employed. In the previous study (35), ICa,L was the averaged current flowing through a piece of nodal tissue (0.2–0.3 mm diameter). Depending on the location of the nodal tissue, that small piece could have contained up to four different cell types (39), with each cell having a different complement of {alpha}1C and {alpha}1D subunits (as discussed below) and a different density of L-type Ca channels, depending on its size (27).

Mechanisms for the ischemia-induced increase in ICa,L. The goal of this study was to explain why ischemic-like conditions enhance ICa,L in rabbit SAN PCs. The major findings are the following: 1) blockade of Ca-induced Ca release (CICR) with ryanodine, exposure of PCs to BAPTA-AM, or replacement of Cao with Ba2+ failed to prevent the ischemia-induced enhancement of ICa,L; 2) inhibition of PKA with H-89, or CaMKII with KN-93, reduced ICa,L but failed to prevent its augmentation by ischemia; 3) ischemic Tyrode or pH 6.6 Tyrode shifted the steady-state inactivation curve in the positive direction; 4) ischemic Tyrode increased the maximum conductance but did not affect the position of the activation curve; 5) in rabbit atrial myocytes isolated and studied with exactly the same techniques, ischemic Tyrode reduced the maximum conductance and shifted the activation curve in the positive direction; pH 6.6 Tyrode also shifted the steady-state inactivation curve in the positive direction.

The first set of results suggests that attenuation of Cai-dependent inactivation cannot account for the ischemia-induced enhancement of ICa,L. Our laboratory showed previously that 10 µM ryanodine was sufficient to block SR Ca release in rabbit SAN PCs (20); therefore, the 40 µM ryanodine used in the present study should have been more than sufficient. The fact that both 2 and 40 µM ryanodine failed to inhibit the ischemia-induced enhancement of ICa,L suggests that neither depletion of SR Ca2+ nor blockade of the ryanodine receptor was sufficient (9a). The rationale for such experiments was the following. If our ischemic-like conditions attenuate CICR in SAN PCs, as reported for the myocardium (25), and if the decreased CICR reduces Cai-dependent inactivation, then ischemia should not enhance ICa,L, if CICR is already blocked by ryanodine. Similar experiments were performed with 25 µM BAPTA-AM. Previously, our laboratory found that this concentration inhibited action potential-induced Cai transients and slowed spontaneous firing of SAN PCs by 28 ± 4% (20). Replacement of Cao with Ba2+ eliminates Cai-dependent inactivation of ICa,L (4), and the dramatic slowing of inactivation under those conditions (Fig. 4A) confirms this is also true for rabbit SAN PCs. Thus the fact that ischemic Tyrode greatly enhanced ICa,L under those conditions (Fig. 4) strongly suggests that reduced Cai-dependent inactivation did not contribute to the ischemia-induced enhancement of ICa,L.

PKA (31) and CaMKII (41) both phosphorylate L-type Ca channels and enhance ICa,L in rabbit SAN PCs under basal conditions. CaMKII also plays a role in Ca-dependent facilitation of L-type Ca channels (42, 44), and PKA facilitates oscillations of local Ca2+ release that contribute to SAN pacemaker activity (40). Based on those results, we assumed that further stimulation of PKA and/or CaMKII by our ischemic-like conditions might play a role in the enhancement of ICa,L. Our results with H-89 and KN-93 are in agreement with previous investigations (31, 41), because both inhibitors reduced ICa,L significantly under control conditions. It is unlikely that those reductions were due to inhibition of other kinases, because H-89 has an in vitro Ki for PKA that is 10-fold less than that for protein kinase G and 100-fold less than that for protein kinase C (PKC) (6), and KN-93 has a Ki for CaMKII that is 100-fold less than those for PKA and PKC (36). Although both PKA and CaMKII were active under our control conditions, their inhibition by H-89 and KN-93 did not prevent the enhancement of ICa,L by ischemic Tyrode. Those results suggest that neither PKA nor CaMKII played a role in the enhancement.

We also investigated the gating and conductance of L-type Ca channels to understand why ICa,L was potentiated. The positive shift of the steady-state inactivation curve and reduction of inactivation induced by ischemia (Fig. 6) or pH 6.6 Tyrode (Fig. 7) are consistent with an increase in ICa,L. Similar results were obtained when guinea pig (15) or rat (18, 43) ventricular myocytes were exposed to acidic conditions. However, the ICa,L activation curves were also shifted in the positive direction in those studies, whereas ischemic Tyrode had no significant effect on the activation curve in the present study. The 74% increase in maximum conductance when SAN PCs were exposed to ischemia is in marked contrast to the results of a previous study of guinea pig ventricular myocytes (37), in which the maximum conductance was reduced by 71% when the extracellular pH was decreased to 6.5. Thus, in SAN PCs, the increased maximum conductance and positive shift of the steady-state inactivation curve (reduced inactivation) probably account for the enhancement of ICa,L during ischemia. Because the positive shift occurred in both ischemic Tyrode and pH 6.6 (but otherwise normal) Tyrode, the low pH (6.6) of ischemic Tyrode is probably what influenced the channel's gating. Although pH 6.6 Tyrode also reduced the k (increased the slope) of the steady-state inactivation curve, ischemic Tyrode had no significant effect on the activation curve. Taken together, those results suggest a direct action of H+ on the L-type Ca channel rather than an indirect effect on extracellular negative surface charge, because such surface charge effects usually manifest as parallel shifts of the activation and inactivation curves, with little change in their slopes (18).

As described above, the results we obtained when SAN PCs were exposed to ischemic Tyrode or pH 6.6 Tyrode are very different from those reported by others who exposed ventricular myocytes to low pH. To eliminate our PC isolation procedures, the beta-escin perforated patch technique, and our pipette and Tyrode solutions as sources of such differences, we isolated myocytes from the rabbit right atrial appendage using exactly the same procedures, and we employed exactly the same perforated-patch technique and solutions in experiments with the atrial myocytes. We were surprised to find that our results with the atrial myocytes differed dramatically from those we obtained with SAN PCs. For example, ischemic Tyrode reduced ICa,L significantly at potentials between –20 and +10 mV, slowed its inactivation at 0 mV, reduced the maximum conductance, and shifted the activation curve to more positive potentials. Those results are in agreement with previous studies that exposed guinea pig (15), rat (18, 43), and rabbit (5) ventricular myocytes to an acidic pH. Also in our atrial myocytes, pH 6.6 Tyrode shifted the steady-state inactivation curve to more positive potentials, as observed previously in rat ventricular myocytes (7). Because both activation and inactivation curves were shifted to the right with no change in slope when our atrial myocytes were exposed to ischemic Tyrode and pH 6.6 Tyrode, respectively, we speculate that those shifts resulted from H+ binding to negative surface charge near the L-type Ca channel. Such parallel positive shifts are expected with increased extracellular H+, because only the electric field seen by the channel's gating sensor, and not the channel itself, is altered (18). Our previous recordings of ICa,T in SAN PCs (8) also suggest that the current results with ICa,L cannot be explained by differences in techniques. Using the same cell isolation procedures, perforated-patch technique, and solutions, we found just the opposite results for ICa,T: it was reduced by ischemic Tyrode (8). That result is consistent with those in which guinea pig ventricular myocytes (37) and HEK-293 cells transfected with human T-type Ca channels (7) were exposed to acidic pH.

Limitations of the study. We employed a holding potential of –40 mV to record ICa,L-V. At that potential, 20–30% of ICa,L is inactivated under steady-state conditions (10, 32). Therefore, the current-voltage relationships illustrated in Figs. 1–5 and Fig. 8 are underestimated and not representative of physiological conditions. Nevertheless, that holding potential inactivated 100% of TTX-sensitive INa (26) and 100% of ICa,T (10) in rabbit SAN PCs, allowing us to eliminate contributions from those currents to the measurement of ICa,L. For measurements of steady-state inactivation, we employed holding potentials of –60 or –80 mV and used TTX to block INa and mibefradil to block ICa,T. Protas and Robinson reported that mibefradil is not selective (33). For example, at 1.0 µM, it inhibits ICa,T and ICa,L almost equally in rabbit SAN PCs. Therefore, we employed 0.5 µM mibefradil to reduce ICa,T as much as possible while minimizing its reduction of ICa,L. Even though a small amount of ICa,T was present during some of the prepulses and ICa,L was partially inhibited during the test pulse, the results described below, and the fact that exactly the same protocol and inhibitors were used to measure ICa,L availability for both control and ischemic conditions, suggest that our conclusions are still valid. That is, ischemic-like conditions shift the steady-state inactivation of ICa,L to more positive potentials, allowing more L-type Ca channels to open. Because mibefradil's inhibition of ICa,L is enhanced with depolarization (9a), there could have been less entry of Ca2+ and, therefore, less Cai-induced inactivation of ICa,L as the prepulses became more positive. Such a mechanism might account for the more gradual slopes of the control inactivation curves illustrated in Figs. 6C and 7C.

The following results suggest that ICa,T was not blocked completely by 0.5 µM mibefradil. 1) The presence of a transient inward current (ICa) at –50 mV, when the holding potential was –80 mV (ICa = –0.4 ± 0.1 pA/pF, n = 12) or –60 mV (ICa = –0.1 ± 0.1 pA/pF, n = 12), is consistent with the presence of ICa,T in rabbit SAN PCs (8, 10, 32, 38). 2) Inactivation of ICa was best fit by a sum of two exponentials with fast ({tau}1) and slow ({tau}2) time constants. When P1 was 0 mV and P2 was +10 mV, {tau}1 = 17.0 ± 1.2 ms during P1 and 28.9 ± 3.4 ms during P2. Because ICa,T inactivates more rapidly than ICa,L in SAN PCs (13), the significantly shorter {tau}1 during P1 (n = 12, P = 0.003) is consistent with the presence of ICa,T during P1.

Our results also suggest that ICa,L was the predominant current during P1. 1) During P1, ICa reached a maximum at –13.3 ± 1.5 mV (n = 12), not between –20 and –40 mV, as reported for ICa,T in rabbit SAN PCs (8, 10, 32, 38; but see Ref. 13). 2) During P1, the maximum ICa (–12.6 ± 1.2 pA/pF; n = 12) was much larger than the maximum ICa,T in rabbit SAN PCs: –5.7 ± 0.9 pA/pF (8), –2.1 ± 0.7 pA/pF (13), –4.3 ± 0.7 pA/pF (32), and –1.9 ± 0.4 pA/pF (38). 3) At a holding potential of –60 mV (Figs. 6 and 7), much more ICa,T would have been inactivated than ICa,L, since 50–60% of ICa,T, but <5% of ICa,L, is inactivated at that potential under steady-state conditions in rabbit SAN PCs (10, 32).

The following results suggest that ICa,T was negligible during P2, and its influx during P1 had a negligible effect on ICa,L during P2. 1) As illustrated in Figs. 6C and 7C, V1/2 was determined primarily by normalized ICa measured during P2 when P1 was –40 to –20 mV, potentials at which ICa,T is completely inactivated (10, 13, 32). 2) Under control conditions, V1/2 was –31.7 ± 0.6 mV (Fig. 6C) and –34.1 ± 1.1 mV (Fig. 7C), which is consistent with values for ICa,L but not ICa,T. For example, in rabbit SAN PCs,V1/2 for ICa,L = –35.4 ± 1.2 mV (10), –25 mV (13), and –28.3 ± 1.7 mV (32), whereas V1/2 for ICa,T = –58.7 ± 1.3 (10), –75 mV (13), and –65.1 ± 2.4 mV (32).

Under control conditions, our atrial myocytes exhibited a V1/2 = –15.8 ± 1.2 mV (Fig. 9C), which is far from the value for ICa,T in guinea pig ventricular myocytes, –54 mV (37). In fact, the following results suggest that ICa,T was absent in atrial myocytes during P1. 1) With a holding potential of –60 mV, ICa was zero, unless P1 was positive to –40 mV. 2) Between –30 and 0 mV, ICa was greatest at 0 mV (–6.6 ± 1.1 pA/pF, n = 6). 3) The shorter time constant for fitting ICa inactivation was not significantly different when comparing ICa during P1 and P2: {tau}1 = 12.7 ± 2.1 ms during P1 and 12.0 ± 1.6 ms during P2 (n = 8), suggesting that the same current flowed during both P1 and P2.

Conclusion. Our results suggest that the acidic pH of the ischemic Tyrode solution enhanced ICa,L in SAN PCs because of the increased maximum conductance and reduced inactivation. Although the intracellular pH of our isolated SAN PCs was not greatly affected by ischemic Tyrode, falling to no less than 7.2 during 3- to 5-min exposures (11), H+ could have displaced Mg2+ from the EF-hand of the channel's COOH-terminal, thereby reducing Ca2+-independent, voltage-dependent inactivation (2). As suggested below, further studies will be necessary to test this hypothesis.

Recently {alpha}1D [voltage-gated Ca (Cav) 1.3] L-type Ca channel transcripts were identified in the adult rabbit SAN, atrioventricular node, and atria, but not in the ventricles (34). In contrast to Cav1.2, Cav1.3 channels are activated at more negative potentials and contribute to diastolic depolarization in the mouse (22, 45). Thus, to explain the present results at a molecular level, future investigations could compare the characteristics of Cav1.2 or Cav1.3 channel mutants when they are expressed in a heterologous system and exposed to normal or acidic pH. To examine the influence of surface charge near the channels, one could remove those anionic residues with an enzyme such as neuraminidase (10) and then observe the effects of acidic pH.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by American Heart Association Texas Affiliate Grant-in-Aid 0355015Y and a seed grant from the Texas Tech Center for Cardiovascular Disease and Stroke.


   ACKNOWLEDGMENTS
 
We thank Dr. An-Ruo Zou (Amgen Inc.), and Drs. Ariel L. Escobar and Raul Martínez-Zaguilán (Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center) for helpful suggestions on the experimental design and interpretation of the results.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y-M Du, Dept. of Cell Physiology and Molecular Biophysics, Texas Tech Univ. Health Sciences Ctr., 3601 4th St., Lubbock, TX 79430 (e-mail: yimeidu{at}hotmail.com)

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. Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res 47: 658–687, 2000.[Abstract/Free Full Text]
  2. Brunet S, Scheuer T, Klevit R, Catterall WA. Modulation of Cav1.2 channels by Mg2+ acting at an EF-hand motif in the COOH-terminal domain. J Gen Physiol 126: 311–323, 2005.[Abstract/Free Full Text]
  3. Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmias. Physiol Rev 79: 917–1017, 1999.[Abstract/Free Full Text]
  4. Cens T, Rousset M, Leyris JP, Fesquet P, Charnet P. Voltage- and calcium-dependent inactivation in high voltage-gated Ca2+ channels. Prog Biophys Mol Biol 90: 104–117, 2006.[CrossRef][Web of Science][Medline]
  5. Chen F, Wetzel GT, Friedman WF, Klitzner TS. Developmental changes in the effects of pH on contraction and Ca2+ current in rabbit heart. J Mol Cell Cardiol 28: 635–642, 1996.[CrossRef][Web of Science][Medline]
  6. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265: 5267–5272, 1990.[Abstract/Free Full Text]
  7. Delisle BP, Satin J. pH modification of human T-type calcium channel gating. Biophys J 78: 1895–1905, 2000.[Web of Science][Medline]
  8. Du YM, Nathan RD. Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node. J Mol Cell Cardiol 42: 315–325, 2007.[CrossRef][Web of Science][Medline]
  9. Fan JS, Palade P. Perforated patch recording with beta-escin. Pflügers Arch 436: 1021–1023, 1998.[Web of Science][Medline]
  10. Fang LM, Osterrieder W. Potential-dependent inhibition of cardiac Ca2+ inward currents by Ro 40–5967 and verapamil: relation to negative inotropy. Eur J Pharmacol 196: 205–207, 1991.[CrossRef][Web of Science][Medline]
  11. Fermini B, Nathan RD. Removal of sialic acid alters both T- and L-type calcium currents in cardiac myocytes. Am J Physiol Heart Circ Physiol 260: H735–H743, 1991.[Abstract/Free Full Text]
  12. Gryshchenko O, Qu J, Nathan RD. Ischemia alters the electrical activity of pacemaker cells isolated from the rabbit sinoatrial node. Am J Physiol Heart Circ Physiol 282: H2284–H2295, 2002.[Abstract/Free Full Text]
  13. Guo J, Giles WR, Ward CA. Effect of hydrogen peroxide on the membrane currents of sinoatrial node cells from rabbit heart. Am J Physiol Heart Circ Physiol 279: H992–H999, 2000.[Abstract/Free Full Text]
  14. Hagaiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol 395: 233–253, 1988.[Abstract/Free Full Text]
  15. Han X, Habuchi Y, Giles WR. Effects of metabolic inhibition on action potentials and ionic currents in cardiac pacemaking cells (Abstract). Circ 90, Suppl I: 582, 1994.
  16. Irisawa H, Sato R. Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells. Circ Res 59: 348–355, 1986.[Abstract/Free Full Text]
  17. Ju YK, Allen DG. Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores. Am J Physiol Heart Circ Physiol 284: H1087–H1094, 2003.[Abstract/Free Full Text]
  18. Kléber AG, Riegger CB, Janse MJ. Extracellular K+ and H+ shifts in early ischemia: mechanisms and relation to changes in impulse propagation. J Mol Cell Cardiol 19, Suppl 5: 35–44, 1987.[CrossRef][Web of Science][Medline]
  19. Krafte DS, Kass RS. Hydrogen ion modulation of Ca channel current in cardiac ventricular cells: evidence for multiple mechanisms. J Gen Physiol 91: 641–657, 1988.[Abstract/Free Full Text]
  20. Lancaster MK, Jones SA, Harrison SM, Boyett MR. Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control. J Physiol 556: 481–494, 2004.[Abstract/Free Full Text]
  21. Li J, Qu J, Nathan RD. Ionic basis of ryanodine's negative chronotropic effect on pacemaker cells isolated from the sinoatrial node. Am J Physiol Heart Circ Physiol 273: H2481–H2489, 1997.[Abstract/Free Full Text]
  22. Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, Nargeot J. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci USA 100: 5543–5548, 2003.[Abstract/Free Full Text]
  23. Meissner G. Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J Biol Chem 261: 6300–6306, 1986.[Abstract/Free Full Text]
  24. Mohabir R, Lee HC, Kurz RW, Clusin WT. Effects of ischemia and hypercarbic acidosis on myocyte calcium transients, contraction, and pHi in perfused rabbit hearts. Circ Res 69: 1525–1537, 1991.[Abstract/Free Full Text]
  25. Mubagwa K. Sarcoplasmic reticulum function during myocardial ischaemia and reperfusion. Cardiovasc Res 30: 166–175, 1995.[CrossRef][Web of Science][Medline]
  26. Muramatsu H, Zou AR, Berkowitz GA, Nathan RD. Characterization of a TTX-sensitive Na+ current in pacemaker cells isolated from rabbit sinoatrial node. Am J Physiol Heart Circ Physiol 270: H2108–H2119, 1996.[Abstract/Free Full Text]
  27. Musa H, Lei M, Honjo H, Jones SA, Dobrzynski H, Lancaster MK, Takagishi Y, Henderson Z, Kodama I, Boyett MR. Heterogeneous expression of Ca2+ handling proteins in rabbit sinoatrial node. J Histochem Cytochem 50: 311–324, 2002.[Abstract/Free Full Text]
  28. Orchard CH, Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle. Am J Physiol Cell Physiol 258: C967–C981, 1990.[Abstract/Free Full Text]
  29. Ornato JP, Peberdy MA. The mystery of bradyasystole during cardiac arrest. Ann Emerg Med 27: 576–587, 1996.[CrossRef][Web of Science][Medline]
  30. Pearman C, Kent W, Bracken N, Hussain M. H-89 inhibits transient outward and inward rectifier potassium currents in isolated rat ventricular myocytes. Br J Pharmacol 148: 1091–1098, 2006.[CrossRef][Web of Science][Medline]
  31. Petit-Jacques J, Bois P, Bescond J, Lenfant J. Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes. Pflügers Arch 423: 21–27, 1993.[CrossRef][Web of Science][Medline]
  32. Protas L, DiFrancesco D, Robinson RB. L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node. Am J Physiol Heart Circ Physiol 281: H1252–H1259, 2001.[Abstract/Free Full Text]
  33. Protas L, Robinson RB. Mibefradil, an ICa,T blocker, effectively blocks ICa,L in rabbit sinus node cells. Eur J Pharmacol 401: 27–30, 2000.[CrossRef][Web of Science][Medline]
  34. Qu Y, Baroudi G, Yue Y, El-Sherif N, Boutjdir M. Localization and modulation of {alpha}1D (Cav1.3) L-type Ca channel by protein kinase A. Am J Physiol Heart Circ Physiol 288: H2123–H2130, 2005.[Abstract/Free Full Text]
  35. Satoh H, Seyama I. On the mechanism by which changes in extracellular pH affect the electrical activity of the rabbit sino-atrial node. J Physiol 381: 181–191, 1986.[Abstract/Free Full Text]
  36. Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181: 968–975, 1991.[CrossRef][Web of Science][Medline]
  37. Tytgat J, Nilius B, Carmeliet E. Modulation of the T-type cardiac Ca channel by changes in proton concentration. J Gen Physiol 96: 973–990, 1990.[Abstract/Free Full Text]
  38. Verheijck EE, Van Ginneken ACG, Wilders R, Bouman LN. Contribution of L-type Ca2+ current to electrical activity in sinoatrial nodal myocytes of rabbits. Am J Physiol Heart Circ Physiol 276: H1064–H1077, 1999.[Abstract/Free Full Text]
  39. Verheijck EE, Wessels A, van Ginneken ACG, Bourier J, Markman MWM, Vermeulen JLM, de Bakker JMT, Lamers WH, Opthof T, Bouman LN. Distribution of atrial and nodal cells within the rabbit sinoatrial node: models of sinoatrial transition. Circulation 97: 1623–1631, 1998.[Abstract/Free Full Text]
  40. Vinogradova TM, Lyashkov AE, Zhu W, Ruknudin AM, Sirenko S, Yang D, Deo S, Barlow M, Johnson S, Caffrey JL, Zhou YY, Xiao RP, Cheng H, Stern MD, Maltsev VA, Lakatta EG. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res 98: 505–514, 2006.[Abstract/Free Full Text]
  41. Vinogradova TM, Zhou YY, Bogdanov KY, Yang D, Kuschel M, Cheng H, Xiao RP. Sinoatrial node pacemaker activity requires Ca2+/calmodulin-dependent protein kinase II activation. Circ Res 87: 760–767, 2000.[Abstract/Free Full Text]
  42. Xiao RP, Cheng H, Lederer WJ, Suzuki T, Lakatta EG. Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci USA 91: 9659–9663, 1994.[Abstract/Free Full Text]
  43. Yatani A, Goto M. The effect of extracellular low pH on the plateau current in isolated, single rat ventricular cells: a voltage clamp study. Jpn J Physiol 33: 403–415, 1983.[Web of Science][Medline]
  44. Yuan W, Bers DM. Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase. Am J Physiol Heart Circ Physiol 267: H982–H993, 1994.[Abstract/Free Full Text]
  45. Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS, Chiamvimonvat N. Functional roles of Cav1.3 ({alpha}1D) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res 90: 981–987, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/H2986    most recent
00491.2007v1
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 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 Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Du, Y.-M.
Right arrow Articles by Nathan, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Du, Y.-M.
Right arrow Articles by Nathan, R. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.