Journal of Applied Physiology Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 96: 1425-1432, 2004. First published December 12, 2003; doi:10.1152/japplphysiol.01077.2003
8750-7587/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
96/4/1425    most recent
01077.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Korzick, D. H.
Right arrow Articles by Bowles, D. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Korzick, D. H.
Right arrow Articles by Bowles, D. K.

Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries

D. H. Korzick,1,4 M. H. Laughlin,1,2,3 and D. K. Bowles1,3

Departments of 1Biomedical Sciences and 2Medical Pharmacology and Physiology and 3Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211; and 4Department of Kinesiology and Noll Physiological Research Center, The Pennsylvania State University, University Park, Pennsylvania 16802

Submitted 3 October 2003 ; accepted in final form 8 December 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The intracellular mechanisms underlying enhanced myogenic contraction (MC) in coronary resistance arteries (CRAs) from exercise-trained (EX) pigs have not been established. The purpose of this study was to test the hypothesis that exercise-induced alterations in protein kinase C (PKC) signaling underlie enhanced MC. Furthermore, we sought to determine whether modulation of intracellular Ca2+ signaling by PKC underlies enhanced MC in EX animals. Male Yucatan miniature swine were treadmill trained (n = 7) at ~75% of maximal O2 uptake for 16 wk (6 miles/h, 60 min) or remained sedentary (SED, n = 6). Diameter measurements in response to intraluminal pressure (60, 75, and 90 cmH2O) or 60 mM KCl were determined in single, cannulated CRAs (~100 µm ID) with and without the PKC inhibitor chelerythrine (CE, 1 µM). Confocal imaging of Ca2+ signaling [myogenic Ca2+ (Cam)] was also performed in CRAs of similar internal diameter after abluminal loading of the Ca2+ indicator dye fluo 4 (1 µM, 37°C, 30 min). We observed significantly greater MC in CRAs isolated from EX than from SED animals at 90 cmH2O, as well as greater reductions in MC after CE at all pressures studied. At intraluminal pressures of 75 and 90 cmH2O, CE produced greater decreases in Cam in CRAs from EX than from SED animals (64% vs. 25%, P < 0.05). Inhibition of KCl constriction and Cam by CE was also greater in EX animals (P < 0.05). Western blotting revealed significant increases in Ca2+-dependent PKC-{alpha} (~50%) but not Ca2+-independent PKC-{epsilon} levels in CRAs isolated from EX animals (P < 0.05). We also observed significant group differences in phosphorylated PKC-{alpha} levels. Finally, voltage-gated Ca2+ current (VGCC) was effectively blocked by CE, bisindolylmaleimide, and staurosporine in isolated smooth muscle cells from CRAs, providing evidence for a mechanistic link between VGCCs and PKC in our experimental paradigm. These results suggest that enhanced MC in CRAs from EX animals involves PKC-dependent modulation of intracellular Ca2+, including regulation of VGCCs.

protein kinase C; voltage-gated calcium channels

MYOGENIC RESPONSIVENESS is an inherent property of vascular smooth muscle and an important determinant of local blood flow autoregulatory responses in animals and humans (for review see Refs. 10, 17, and 31). The contribution of myogenic constriction to basal levels of vascular tone is also well established (36), and this mechanism has been implicated in the coordinated vascular response to a variety of vasoactive stimuli (8, 37). Interestingly, the myogenic response is amenable to adaptation by chronic exercise training in the coronary circulation (4). We previously demonstrated enhanced myogenic vasoconstriction in coronary resistance arteries (CRAs) isolated from female pigs subjected to chronic treadmill running (32); however, the intracellular signaling mechanisms underlying exercise-induced adaptations in coronary myogenic responses are poorly understood.

There is mounting experimental evidence to suggest that interactions between L-type voltage-gated Ca2+ channels (VGCCs) and the serine/threonine protein kinase C (PKC) play a central role in determining myogenic tone and myogenic reactivity under normal physiological conditions (2, 10, 15, 22, 29, 31, 43, 45). Depolarization of vascular smooth muscle in response to changes in intraluminal pressure and/or stretch and subsequent Ca2+ entry through VGCCs provides a potent mechanism for contractile protein activation (i.e., Ca2+-calmodulin/myosin light chain kinase) during myogenic constriction (for review, see Refs. 10, 17, and 18). Modulation of this basic scheme by the PKC signal transduction pathway is supported by several key experimental observations: 1) PKC inhibitors consistently block myogenic tone (15, 16, 20, 22, 31), 2) direct activation of PKC by phorbol esters potentiates myogenic constriction in an endothelium-independent manner (15, 16, 31), and 3) direct activation of PKC potentiates VGCC current, subsequent Ca2+ influx, and contraction (22, 29, 31). Importantly, PKC has been shown to phosphorylate the {alpha}1c-subunit of the VGCC (10, 35, 39, 42), resulting in increases in VGCC activity.

Expression patterns of PKC isoforms in vascular smooth muscle are species specific and vary with vascular bed and intracellular location (i.e., membrane vs. cytoplasmic cell fractions) (38). We recently demonstrated expression of PKC-{alpha}, -{beta}I, -{beta}II, -{delta}, and -{epsilon} in porcine coronary resistance arterioles (unpublished observations). In the ferret coronary microcirculation, PKC-{alpha} has been recently identified as a key Ca2+-dependent PKC isoform involved in transducing myogenic responses (9); PKC-{alpha} translocation is observed in response to increases in transmural pressure and has been linked to Ca2+ sensitization effects and thin filament regulation. However, whether alterations in PKC underlie myogenic and, in particular, training-induced adaptations in myogenic contraction in the porcine coronary microcirculation is unknown. Therefore, the purpose of the present study was twofold: 1) to test the hypothesis that modulation of PKC signaling contributes to exercise-induced enhancements in myogenic contraction in CRAs isolated from male pigs and 2) to determine whether modulation of intracellular Ca2+-signaling mechanisms by PKC in CRAs is affected by exercise training.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals. Male Yucatan miniature swine (Charles River) weighing 25-40 kg were randomized to an exercise-training (EX) group (n = 7) or remained sedentary (SED, n = 6). The animal protocols associated with this study were approved by the University of Missouri Animal Care and Use Committee and carried out in accordance with the Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (US Government).

Exercise training program. Animals in the EX group were treadmill trained at ~75% of maximal O2 uptake for 16 wk, as described previously (3, 28, 32). Briefly, progressive exercise sessions (85 min) were conducted as follows: 1) 5-min warm-up run at 2.5 miles/h (mph), 2) 15-min sprint at 5-8 mph, 3) 60-min endurance run at 4-5 mph, and 4) 5-min cool-down run at 2 mph. To document the presence of an endurance-trained state, citrate synthase activity assays were performed on skeletal muscle homogenates, as previously described (44). Treadmill exercise performance tests were also performed in EX and SED animals before and at the conclusion of the training program and/or sedentary pen confinement (28, 32).

Evaluation of vasoreactivity in CRAs. Pigs were deeply anesthetized with ketamine (35 mg/kg im), xylazine (Rompun, 2.25 mg/kg im), and thiopental sodium (Pentothal Sodium, 10 mg/kg iv) and then treated with heparin (1,000 U/kg iv). Hearts were rapidly excised and placed in cold (4°C) Krebs solution containing (in mM) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 25.0 NaHCO3, and 11.2 glucose (pH 7.4) (32). CRAs (~100 µm ID) were isolated from the apex region of the anterior left ventricular free wall, cannulated with glass micropipettes (75-80 µm OD, resistance = 150-250 k{Omega}), and maintained in physiological saline solution containing 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.1 mM glucose, and 10 mg/ml BSA. Resistance arteries were viewed through an inverted microscope (Nikon Diaphot, x20 magnification, spatial resolution <1 µm), according to well-established methods in our laboratory (21, 32). Intraluminal diameter was monitored continuously utilizing a video tracking system (Microcirculation Research Institute, Texas A & M University) and recorded throughout the duration of the experiment utilizing PowerLab data-acquisition software (AD Instruments). During the initial equilibration period (1 h), vessels were monitored for leaks and warmed gradually to 37°C. Micropipettes were connected to two independent fluid-filled reservoirs of similar height, and arteries were pressurized at 60 cmH2O. Changes in intraluminal pressure were achieved by simultaneously raising or lowering the reservoirs, thus preventing changes in fluid flow within the vessel lumen (see below). Diameter measurements were expressed relative to passive diameter [after 100 µM sodium nitroprusside (SNP)] at 60 cmH2O. Vessels that failed to develop spontaneous tone during the initial equilibration period were eliminated from the study population.

Protocol of vasoreactivity assessment. CRAs (~100 µm ID) were isolated from EX (10 vessels from 7 pigs) and SED (6 vessels from 6 pigs) animals and cannulated with glass micropipettes, according to established procedures in our laboratory (21, 32), as described above. Vessels were pressurized at 60 cmH2O, and, after equilibration for 1 h at 37°C, vasoreactivity was assessed after administration of 60 mM KCl in the absence and presence of the specific PKC inhibitor chelerythrine (CE, 1 µM). We (25) and others (1, 6, 14, 23, 30) previously demonstrated the use of CE as a potent inhibitor of Ca2+-dependent and Ca2+-independent PKC isoforms with specific effects on the catalytic domain of PKC (IC50 = 0.7 µM) (14). In a separate series of experiments, myogenic responses were assessed in SED (9 vessels from 7 pigs) and EX (9 vessels from 6 pigs) animals after step increases in intraluminal pressure (60, 75, and 90 cmH2O), and CRAs were given 5 min after each experimental manipulation for diameter stabilization. These pressures were selected on the basis of our previous findings of training-induced differences in myogenic reactivity within this pressure range in female pigs (32). The experimental paradigm was repeated after abluminal administration of the PKC inhibitor CE (1 µM). At the conclusion of each experiment, passive vessel diameter was assessed after administration of 100 µM SNP (60 cmH2O). Diameter measurements were expressed relative to passive diameter.

Measurement of Ca2+ signaling by confocal microscopy. In a separate series of experiments, CRAs (~100 µm) were isolated from EX (6 vessels from 6 pigs) and SED (6 vessels from 6 pigs) animals, cannulated, and studied as described above. Before experimental manipulation and after equilibration at 37°C for 1 h, vessels were incubated with the Ca2+ indicator dye fluo 4 (1 µM) and 0.01% Pluronic for 30 min and washed three times with physiological saline solution. Coronary resistance vessels were visualized with a Nikon Diaphot inverted microscope and a x40 VLWD objective after confocal argon laser excitation at 488 nm (Noran Instruments). All acquisition parameters (e.g., exposure time and excitation intensity) were kept constant in all experiments.

Western blotting. CRAs (~100 µm ID) were isolated as described above, snap frozen on dry ice, and stored at -70°C until they were processed. Three to four vessels from each heart (n = 1 pig) were placed in 200-300 µl of extraction buffer (50 mM Tris·HCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% {beta}-mercaptoethanol, 1 mM PMSF, 2 µM leupeptin, 1 µM pepstatin A, and 20 mM 3-[(3-cholamidopropyl)dim-ethylammonio]-1-propanesulfonate) and sonicated. Samples were centrifuged at 100,000 g for 30 min at 4°C. Equal amounts of sample (~15 µg protein) were loaded per lane, electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and incubated with primary anti-rabbit antibodies for PKC-{alpha} or PKC-{epsilon} (1:400; Santa Cruz Biochemicals). Phosphospecific antibodies for PKC-{alpha} (Ser657) were also purchased from Santa Cruz Biochemicals (1:1,000). Antibody binding was detected by enhanced chemiluminescence (Amersham). Rat brain (~2 µg protein) was used as positive control. Densitometric analysis of immunoblot films (Hyperfilm, Amersham) was performed utilizing NIH Scion Image Analysis software. Care was taken to prevent saturation of the densitometric signal on film development.

Coronary myocyte isolation. CRAs were incubated in 200 µl of enzyme solution consisting of low-Ca2+ (0.1 mM) physiological saline solution plus 294 U/ml collagenase (CLS II, Worthington), 6.5 U/ml elastase (Worthington), 2 mg/ml BSA (fraction V, Sigma), 1 mg/ml soybean trypsin inhibitor (type I-S, Sigma), and 0.4 mg/ml DNase I (type IV, Sigma), as previously described (3). CRAs were incubated for 45-60 min in a water bath at 37°C and then incubated for 5 min at room temperature with enzyme-free low-Ca2+ solution. Isolated single cells were obtained by gentle trituration with a fire-polished Pasteur pipette.

Whole cell VGCC characteristics in isolated coronary myocytes. Macroscopic VGCC current (ICa) was determined by using dialyzing, whole cell voltage clamp, as previously described (3). Briefly, current-voltage relations were determined by 400-ms step depolarizations from a holding potential of -80 mV to test potentials from -60 to +60 mV. Voltage clamp and current amplification were performed with an Axopatch 200B patch-clamp amplifier. Current records were filtered at 1 kHz (-3 dB) and digitized at 5 kHz. Control of voltage protocols and analysis were performed by Clampex 7.0 software (Axon Instruments, Foster City, CA). All experiments were conducted at room temperature (22-25°C).

Statistics. All variables of interest are reported as means ± SE. Data were analyzed with the Statistical Analysis System (SAS). Specifically, group comparisons of vasoreactivity after changes in intraluminal pressure were analyzed with the SAS general linear models procedure for a two-way ANOVA with repeated measures on one factor (group x pressure). Higher order terms were employed to reveal significant interaction effects with respect to varying pressure on vasoreactivity between groups. For immunoblot data, a one-way ANOVA was employed. The Tukey-Kramer method was used for all post hoc comparisons. An alpha level of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Efficacy of the exercise training program. As we previously demonstrated (3, 28, 32), the exercise paradigm described here resulted in significant group differences in several markers associated with an endurance-trained state (Table 1). Specifically, chronic exercise was associated with increases of 17.5% and 13% in heart weight and heart weight-to-body weight ratio, respectively, whereas no group differences were observed for body weight. Furthermore, deltoid muscle citrate synthase activity was significantly greater in EX than in SED animals (Table 1), as was exercise run time to exhaustion (P < 0.05). Taken together, the physiological and biochemical data indicate exercise-induced enhancements in aerobic metabolic capacity as a result of chronic treadmill running.


View this table:
[in this window]
[in a new window]
  		Table 1. Efficacy of the exercise training program

 

Baseline vessel characteristics. Mean maximal intraluminal diameter of isolated coronary resistance vessels measured at 60 cmH2O after administration of 100 µM SNP was identical between groups: 100 ± 4 µm in EX (n = 20 vessels) and SED (n = 15 vessels). After vessel equilibration for 1 h at 37°C, mean spontaneous tone development across all series of experiments was also similar between experimental groups with normalized diameter: 0.78 ± 0.04 vs. 0.73 ± 0.04 in SED and EX groups, respectively (P > 0.05; Fig. 1, see Fig. 3).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. Dependence of myogenic contraction on PKC in sedentary (SED) and exercise-trained (EX) pigs. Myogenic responses to increasing intraluminal pressure in coronary microvessels isolated from SED (n = 9 vessels, ~100 µm ID) and EX (n = 9 vessels, ~98 µm ID) pigs are shown in the presence and absence of the PKC blocker chelerythrine (CE, 1 µM). Values are means ± SE. ANOVA revealed greater reductions in relative diameter in EX than in SED animals in response to PKC inhibition: *P < 0.05 vs. EX; {ddagger}P < 0.05 vs. EX + CE.

 


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 3. Dependence of KCl contraction on PKC. Vasoreactivity in response to 60 mM KCl in SED (n = 6 vessels, ~100 µm) and EX (n = 10 vessels, ~98 µm ID) pigs is shown in the presence and absence of 1 µM CE. Values are means ± SE. ANOVA revealed greater reductions in relative diameter in SED than in EX animals in response to KCl: *P < 0.05 vs. baseline; {dagger}P < 0.05 vs. SED.

 

Myogenic responses and dependence on PKC. Myogenic responsiveness after step increases in intraluminal pressure in the absence and presence of the PKC inhibitor CE is presented in Fig. 1. Consistent with our previous findings in coronary resistance vessels isolated from exercise-trained female pigs (32), myogenic constriction was significantly greater in CRAs isolated from EX than from SED male pigs at 90 cmH2O (P < 0.05). After PKC inhibition with CE, myogenic tone was significantly attenuated at all intraluminal pressures studied in EX and SED animals; however, CE elicited greater reductions in myogenic constriction in EX than in SED animals (P < 0.05). Specifically, ANOVA revealed a significant interaction effect between group and drug, whereby EX increased the CE-sensitive component of myogenic tone by ~50% compared with vessels isolated from SED animals (28 ± 5% vs. 19 ± 6%) at 90 cmH2O (Fig. 1). In a separate series of experiments, confocal imaging of Ca2+ signaling [myogenic Ca2+ (Cam)] was performed on resistance arteries isolated from SED and EX animals using the Ca2+ indicator dye fluo 4 in response to changes in intraluminal pressure before and after PKC inhibition with 1 µM CE. It is clear from Fig. 2 that PKC inhibition resulted in the attenuation of Cam levels during myogenic constriction. At intraluminal pressures of 75 and 90 cmH2O, CE produced greater decreases in Cam in arterioles from EX than from SED animals (64 ± 8% vs. 25 ± 3%, P < 0.05).



View larger version (80K):
[in this window]
[in a new window]
  		Fig. 2. Dependence of myogenic contraction on Ca2+ and PKC. Confocal imaging of myoplasmic Ca2+ (Cam) using the Ca2+ indicator dye fluo 4 in response to changes in intraluminal pressure at 60 cmH2O (A) and during transition (B) to 90 cmH2O (C) is shown in a representative coronary arteriole (~100 µm ID) isolated from EX animals in the absence (top) and presence (bottom) of PKC inhibition with 1 µM CE.

 

Depolarization-induced constriction and dependence on PKC. To provide insight into the relation between depolarization, PKC, and VGCCs, vasoreactivity was assessed after abluminal administration of 60 mM KCl in the absence and presence of 1 µM CE (60 cmH2O). It is clear from Figs. 3 and 4 that constrictor responses induced by KCl were significantly attenuated in EX vs. SED animals (P < 0.05). However, inhibitory responses of CE on KCl-mediated constrictor responses were effectively greater in EX than in SED animals (Fig. 3). KCl constriction was completely blocked by CE in EX animals but only attenuated by CE in SED animals. Furthermore, the CE inhibition of vasoconstriction was accompanied by reductions in Cam (Fig. 4). To confirm a possible association between PKC and VGCCs, smooth muscle cells were isolated from CRAs, and whole cell VGCC current (ICa) was assessed before and after PKC inhibition with three different PKC inhibitors: CE (1 or 10 µM), bisindolylmaleimide (10 µM), and staurosporine (10 µM). Approximately 60% of ICa was effectively blocked by CE, bisindolylmaleimide, and staurosporine (Fig. 5), a finding similar to that in A7r5 cells, where PKC inhibition reduced ICa by 70-75% (34). Inclusion of a phosphatase inhibitor, okadaic acid, in the pipette prevented the CE effect, consistent with a PKC-dependent phosphorylation and activation of the VGCC. Together, these data support an important interaction between PKC and VGCCs in coronary vascular smooth muscle.



View larger version (62K):
[in this window]
[in a new window]
  		Fig. 4. Regulation of KCl contraction by Ca2+ and PKC in resistance arteries from SED and EX pigs. Confocal imaging of Cam using the Ca2+ indicator dye fluo 4 is shown in response to 60 mM KCl in the absence and presence of 1 µM CE in coronary resistance arteries (~100 µm ID) isolated from SED (A) and EX (B) male swine.

 


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5. Dependence of basal voltage-gated Ca2+ channel current (ICa) on PKC. Inhibition of ICa (10 mM external Ba2+) by the specific PKC inhibitors CE (10 µM), bisindolylmaleimide (Bis, 10 µM), and staurosporine (St, 10 µM) is shown. Superfusion with CE, Bis, or St produced inhibition of ICa compared with time control (C). Inset: inclusion of the phosphatase inhibitor okadaic acid [+OA ({circ}), 1 µM] in the pipette inhibited the effect of CE (; CE added at 4 min). *P < 0.05 vs. C; n = 3 cells (SED) per condition.

 

PKC-{alpha} and PKC-{epsilon} immunoreactivity in CRAs. Figures 6 and 7 present results of immunoblot analysis of PKC-{alpha} (Ca2+-dependent PKC) and PKC-{epsilon} (Ca2+-independent PKC) levels in isolated CRAs. We observed no significant group differences for PKC-{epsilon} (Fig. 6). However, PKC-{alpha} levels were elevated in CRAs from EX pigs and were significantly increased by 45% (P < 0.01) compared with CRAs isolated from SED pigs. Interestingly, increases in phosphorylated PKC-{alpha} levels were also observed in EX compared with SED animals (Fig. 6). These results are consistent with previous studies implicating a role for PKC-{alpha} in myogenic responses (11, 30).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6. PKC-{epsilon} immunoreactivity in coronary resistance arteries (CRAs). Equal amounts of protein were loaded per lane (15 µg). Lanes 1-4, CRAs isolated from SED (n = 4) pigs; lanes 5-8, CRAs isolated from EX (trained) pigs (n = 4). Each lane represents 3-4 CRAs per pig (~100 µM ID, see METHODS); isolated rat brain (RB) was utilized as a positive control. Values are means ± SE. PKC-{epsilon} (Ca2+-independent PKC) levels were similar in CRAs isolated from EX and SED animals.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. PKC-{alpha} and phosphorylated PKC-{alpha} immunoreactivity in CRAs. Equal amounts of protein were loaded per lane (15 µg). Lanes 1-4, microvessels isolated from SED pigs (n = 4); lanes 5-8, CRAs isolated from EX pigs (n = 4). Each lane represents 3-4 CRAs per pig (~100 µM ID, see METHODS); isolated RB was utilized as a positive control. Values are means ± SE. *P < 0.01. PKC-{alpha} (Ca2+-dependent PKC) levels were significantly greater in CRAs isolated from EX animals (A). Phosphorylated PKC-{alpha} levels (pSer657) were similarly increased in EX and SED animals (B).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
A major focus of the present study was that alterations in PKC signal transduction mechanisms underlie, in part, adaptations in CRAs isolated from EX pigs. Accordingly, we investigated the role of PKC as an important determinant of myogenic contraction in coronary arterioles isolated from porcine myocardium. Novel findings include the following: 1) myogenic constriction and inhibitory responses after PKC blockade were greater in CRAs isolated from EX than from SED pigs, 2) KCl constriction was significantly less in resistance arteries isolated from EX hearts and blocked to a greater extent by PKC inhibition, 3) PKC-{alpha} (Ca2+-dependent PKC) but not PKC-{epsilon} (Ca2+-independent PKC) levels were significantly greater in CRAs isolated from EX animals, and 4) ICa was effectively blocked by PKC inhibition, as was the Ca2+ signal induced by myogenic constriction and KCl. These results suggest, for the first time, a potential mechanism for enhanced myogenic constriction known to occur in CRAs isolated from trained myocardium. Our data also provide an important circumstantial link between VGCC and PKC-{alpha} as a basis, at least in part, for training-induced adaptations in coronary myogenic responses.

An important role for PKC signal transduction pathways in the modulation of vascular smooth muscle myogenic responses is well established (for review see Refs. 10, 17, 18, and 24). Recent experimental evidence has identified multiple roles for PKC activation in the genesis of myogenic responses, including depolarization-induced VGCC channel activation, Ca2+ sensitization, and/or myosin light chain phosphatase inhibition (5, 33, 40, 47). Activation of a G protein-coupled receptor-dependent mechanism may also lead to activation of PKC under myogenic conditions, whereby activation of G protein-coupled receptors results in Ca2+ release from inositol trisphosphate-sensitive stores and activation of PKC (18). In this regard, recent experimental evidence suggests a key role for the phospholipase C-diacylglycerol-PKC signal transduction axis as a basis for cation channel activation in the generation of myogenic tone (43). Therefore, mechanisms involving pressure-induced increases in intracellular Ca2+ and ion channel phosphorylation seem likely, and a final common pathway for the modulation of myogenic contraction via receptor-dependent and receptor-independent mechanisms occurs at the level of PKC.

In the present study, we observed significant increases in myogenic tone at 90 cmH2O in coronary resistance arterioles isolated from male EX pigs relative to SED control animals, extending our previous findings in female pigs (32). In both studies, a significant training effect became apparent only at intraluminal pressures >60 mmHg (~80 cmH2O). The myogenic mechanism that underlies the "threshold" for the training response is yet to be determined; however, this intraluminal pressure range coincides with the transition to a phase where depolarization, intracellular Ca2+ increases, and VGCC activation occur (36). Thus this range of pressures would be especially sensitive to training-induced changes in Ca2+-dependent PKC (e.g., PKC-{alpha}) and VGCC activity. The enhanced myogenic response may be a compensatory mechanism to the increased flow-mediated dilation that occurs with exercise training (26). An increased myogenic response would be necessary to allow training-induced increases in coronary flow reserve in the presence of increased flow-mediated dilation (8).

In response to PKC blockade, we observed significant attenuation of myogenic tone at all intraluminal pressures studied in SED and EX pigs, consistent with previous studies suggesting an important role for PKC in the genesis of myogenic responses in a variety of vascular beds, including cerebral (2, 22), mesenteric (45, 46), and skeletal (16, 30) circulations. Of notable interest was the finding that the degree of inhibition of myogenic contraction after CE was significantly greater in CRAs isolated from EX than from SED animals. Confocal microscopy revealed that inhibitory effects of PKC blockade on myogenic tone were associated with reductions in Cam (Fig. 2). Our data are consistent with previous studies (9, 22, 36); however, they are at odds with experimental evidence attributing PKC effects on myogenic contraction to Ca2+ sensitization (11, 33, 40, 47).

To investigate the possibility of key regulatory interactions between PKC and Ca2+ influx via VGCCs, depolarization-induced vasoreactivity was assessed after abluminal administration of KCl in the absence and presence of PKC inhibition. It is clear from Fig. 3 that vasoconstrictor responses induced by KCl depolarization were diminished by PKC inhibition, supporting a key role for PKC in modulating Ca2+ influx via depolarization-induced activation of VGCCs. Confirmation of this association was observed by confocal imaging of Cam, whereby KCl-induced increases in Cam were significantly reduced by PKC inhibition (Fig. 4). Although KCl constriction was significantly less in resistance arteries isolated from EX than from SED animals, effects of PKC inhibition were greater in EX than in SED animals, indicating an increased reliance on a PKC-mediated mechanism for constriction. Although it is impossible to rule out training-induced alterations in PKC on myofilament Ca2+ sensitization in our experimental paradigm, one interpretation of the present results is that, because the PKC-dependent portion of depolarization-induced contraction was greater in EX animals, regulation of VGCC activation by PKC assumes a greater role in mediating CRA contractile responses after chronic exercise training. Indeed, confocal data presented in Fig. 6 provide evidence for this supposition, inasmuch as Cam increases were blocked to a greater extent by PKC inhibition in EX than in SED animals after KCl depolarization. It is also important to note that three different, general PKC inhibitors inhibited whole cell ICa (Fig. 5), providing additional circumstantial evidence for important regulatory interactions between PKC and VGCCs in porcine CRAs. Taken together, our data support the hypothesis that PKC plays an important role in determining VGCC activity and vasoconstrictor tone in CRAs and that training-induced adaptations in PKC contribute to alterations in myogenic regulation.

The functional significance of specific PKC isoforms known to be expressed in vascular smooth muscle on myogenic reactivity remains poorly understood. In cultured smooth muscle cells isolated from human aorta, PKC-{alpha}, -{beta}I, -{beta}II, -{delta}, and -{epsilon}, but not PKC-{gamma} and -{zeta}, are detected (13). In the porcine coronary arterial microcirculation, we have documented the presence of PKC-{alpha}, -{beta}I, -{beta}II, -{delta}, and -{epsilon} (unpublished observations). In the present investigation, we observed significant increases in the Ca2+-dependent PKC-{alpha}, whereas group differences in the Ca2+-independent PKC-{epsilon} were not observed. These findings are consistent with experimental observations from the laboratory of Dessy and colleagues (11) as well as Massett et al. (30), whereby PKC-{alpha} activation was identified as a key transducer of myogenic responses in the ferret coronary and rodent skeletal muscle microcirculations, respectively. Additionally, we provide evidence, for the first time that PKC-{alpha} phosphorylation is enhanced as a result of chronic endurance exercise training in porcine CRAs, possibly identifying an important modulator of PKC-{alpha}-dependent signaling in coronary vascular smooth muscle after exercise training. PKC-{epsilon} involvement in myogenic reactivity has been shown to be linked to MAPKs (30, 38) and/or RhoA/Rho kinase (7, 40). That group differences in PKC-{epsilon} were not observed suggests that MAPKs and/or RhoA/Rho kinase are less likely to be a target of exercise-induced adaptation in the porcine coronary circulation under conditions of myogenic contraction. Future studies are indicated to resolve this issue. Collectively, our data suggest that the amount and phosphorylation of PKC-{alpha} protein levels are enhanced by chronic exercise. One important limitation of the present study is use of the general PKC inhibitor CE in our experimental paradigm. Additional studies are necessary to more precisely define the role of PKC-{alpha} in mediating exercise-induced adaptations in myogenic responses. In this regard, it will be necessary to utilize isoform-specific PKC inhibitors to directly address the involvement of PKC-{alpha} in exercise-induced alterations in myogenic reactivity.

Chronic endurance exercise has been implicated in a myriad of beneficial effects on the coronary circulation, and, of particular clinical interest, are adaptive responses known to occur in vascular smooth muscle regulatory mechanisms (3, 4, 27, 28). One logical issue to be addressed is the apparent dichotomy between pathological increases in myogenic contraction, as observed in the hypertensive heart (12, 19, 41), vs. physiological adaptations in myogenic responses, as described here. It is likely that these adaptations represent only one component of an integrated, balanced process that, when expressed in a coordinated fashion, contributes to a beneficial adaptation, e.g., increased coronary reserve. Disruption or inhibition of one or more of these components in disease may prevent the beneficial adaptation and predispose to pathology. Alternatively, increased myogenic responses in pathological and physiological conditions may represent identical compensatory responses of varying magnitude, analogous to compensated vs. uncompensated cardiac hypertrophy. Further study to determine the precise mechanism of the exercise-induced response will allow identification of critical components that separate the physiological and the pathological response.

In conclusion, this study provides novel evidence that endurance exercise training increases the myogenic response in the coronary microcirculation through a PKC-dependent mechanism. Increased expression and phosphorylation of PKC-{alpha} by exercise training suggests that this PKC isoform may mediate this training adaptation. The increased reliance on PKC in mediating Cam responses further suggests a training-induced increase in VGCC regulation by PKC. Together, these results suggest that enhanced myogenic responses in coronary arterioles by exercise training involve PKC-dependent modulation of intracellular Ca2+, including regulation of VGCCs.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was funded by National Institutes of Health Grants PO1 HL-52490 (to M. H. Laughlin, D. K. Bowles, and D. H. Korzick) and KO1 AG-00875 (to D. H. Korzick).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Pam Thorne for expert technical assistance with the isolated cannulated vessel studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Bowles, E102 Veterinary Medicine Bldg., Univ. of Missouri, Columbia, MO 65211 (E-mail: bowlesd{at}missouri.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
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Bakker ENTP, Kerkhof CJM, and Sipkema P. Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle. Cardiovasc Res 41: 229-236, 1999.
  2. Bonev AD, Jaggar JH, Rubart M, and Nelson MT. Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries. Am J Physiol Cell Physiol 273: C2090-C2095, 1997.
  3. Bowles DK. Gender influences coronary L-type Ca2+ current and adaptation to exercise training in miniature swine. J Appl Physiol 91: 2503-2510, 2001.
  4. Bowles DK, Woodman CR, and Laughlin MH. Coronary smooth muscle and endothelial adaptations to exercise training. Exerc Sport Sci Rev 28: 57-62, 2000.
  5. Buus CL, Aalkjaer C, Nilsson H, Juul B, Moller JV, and Mulvany MJ. Mechanisms of Ca2+ sensitization of force production by noradrenaline in rat mesenteric small arteries. J Physiol 510: 577-590, 1998.
  6. Chen CH, Gray MO, and Mochly-Rosen D. Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: role of {epsilon} protein kinase C. Proc Natl Acad Sci USA 96: 12784-12789, 1999.
  7. Civelek M, Ainslie K, Garanich JS, and Tarbell JM. Smooth muscle cells contract in response to fluid flow via a Ca2+-independent signaling mechanism. J Appl Physiol 93: 1907-1917, 2002.
  8. Cornelissen AJ, Dankelman J, VanBavel E, and Spaan JA. Balance between myogenic, flow-dependent, and metabolic flow control in coronary arterial tree: a model study. Am J Physiol Heart Circ Physiol 282: H2224-H2237, 2002.
  9. D'Angelo G, Davis MJ, and Meininger GA. Calcium and mechanotransduction of the myogenic response. Am J Physiol Heart Circ Physiol 273: H175-H182, 1997.
  10. Davis MJ and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387-423, 1999.
  11. Dessy C, Matsuda N, Hulvershorn J, Sougnez CL, Selke FW, and Morgan KG. Evidence for involvement of the PKC-{alpha} isoform in myogenic contractions of the coronary circulation. Am J Physiol Heart Circ Physiol 279: H916-H923, 2000.
  12. Garcia SR, Izzard AS, Heagerty AM, and Bund SJ. Myogenic tone in coronary arteries from spontaneously hypertensive rats. J Vasc Res 34: 109-116, 1997.
  13. Grange JJ, Baca-Regen LM, Nollendorfs AJ, Persidsky Y, Sudan DL, and Baxter BT. Protein kinase C isoforms in human aortic smooth muscle cells. J Vasc Surg 27: 919-927, 1998.
  14. Herbert JM, Augereau JM, Gleye J, and Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172: 993-999, 1990.
  15. Hill MA, Davis MJ, Song J, and Zou H. Calcium dependence of indolactam-mediated contractions in resistance vessels. J Pharmacol Exp Ther 276: 867-874, 1996.
  16. Hill MA, Falcone JC, and Meininger GA. Evidence for protein kinase C involvement in arteriolar myogenic reactivity. Am J Physiol Heart Circ Physiol 259: H1586-H1594, 1990.
  17. Hill MA, Zou H, Potocnik SJ, Meininger GA, and Davis MJ. Arteriolar smooth muscle mechanotransduction: Ca2+ signaling pathways underlying myogenic reactivity. J Appl Physiol 91: 973-983, 2001.
  18. Horowitz A, Menice CB, LaPorte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967-1004, 1996.
  19. Hughes JM and Bund SJ. Arterial myogenic properties of the spontaneously hypertensive rat. Exp Physiol 87: 527-534, 2002.
  20. Jain M, Dai HB, Carrozza JP, Sellke FW, and Morgan KG. Intrinsic tone as potential vascular reserve in conductance and resistance vessels. Circulation 94: 1083-1088, 1996.
  21. Jasperse JL and Laughlin MH. Flow-induced dilation of rat soleus feed arteries. Am J Physiol Heart Circ Physiol 273: H2423-H2427, 1997.
  22. Karibe A, Watanabe J, Horiguchi S, Takeuchi M, Suzuki S, Funakoshi M, Katoh H, Keitoku M, Satoh S, and Shirato K. Role of cytosolic Ca2+ and protein kinase C in developing myogenic contraction in isolated rat small arteries. Am J Physiol Heart Circ Physiol 272: H1165-H1172, 1997.
  23. Kirton CA and Loutzenhiser R. Alterations in basal protein kinase C activity modulate renal afferent arteriolar myogenic reactivity. Am J Physiol Heart Circ Physiol 275: H467-H475, 1998.
  24. Koller A. Signaling pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension. Microcirculation 9: 277-294, 2002.
  25. Korzick DH, Holiman DA, Boluyt MO, Laughlin MH, and Lakatta EG. Diminished {alpha}1-adrenergic-mediated contraction and translocation of PKC in senescent rat heart. Am J Physiol Heart Circ Physiol 281: H581-H589, 2001.
  26. Laughlin MH. Endothelium-mediated control of coronary vascular tone after chronic exercise training. Med Sci Sports Exerc 8: 1135-1144, 1995.
  27. Laughlin MH and Korzick DH. Skeletal muscle blood flow during exercise, vascular smooth muscle: integrator of vasoactive signals. Med Sci Sports Exerc 33: 81-91, 2001.
  28. Laughlin MH and Muller JM. Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs. J Appl Physiol 75: 2677-2682, 1998.
  29. Litten RZ, Suba EA, and Roth BL. Effects of a phorbol ester on rat aortic contraction and calcium influx in the presence and absence of BAY K 8644. Eur J Pharmacol 144: 185-191, 1987.
  30. Massett MP, Ungvari Z, Csiszar A, Kaley G, and Koller A. Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists. Am J Physiol Heart Circ Physiol 283: H2282-H2287, 2002.
  31. Miller FJ, Dellsperger KC, and Gutterman DD. Myogenic constriction of human coronary arterioles. Am J Physiol Heart Circ Physiol 273: H257-H264, 1997.
  32. Muller JM. Exercise training alters myogenic responses in porcine coronary resistance arteries. J Appl Physiol 75: 2677-2682, 1993.
  33. Nakayama K, Obara K, Tanabe Y, Saito M, Ishikawa T, and Nishizawa S. Interactive role of tyrosine kinase, protein kinase C, and Rho/Rho kinase systems in the mechanotransduction of vascular smooth muscles. Biorheology 40: 307-314, 2003.
  34. Obejero-Paz CA, Auslender M, and Scarpa A. PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells. Am J Physiol Cell Physiol 275: C535-C543, 1998.
  35. O'Callahan C, Ptasienski J, and Hosey M. Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C. J Biol Chem 263: 17342-17349, 1988.
  36. Osol G, Brekke JF, McElroy-Yaggy K, and Gokina NI. Myogenic tone, reactivity, and forced dilatation: a three-phase model of in vitro arterial myogenic behavior. Am J Physiol Heart Circ Physiol 283: H2260-H2267, 2002.
  37. Petersen HH, Choy J, Stauffer B, Moien-Afshari F, Aalkjaer C, Leinwand L, McManus BM, and Laher I. Coronary artery myogenic response in a genetic model of hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol 283: H2244-H2249, 2002.
  38. Prewitt RL, Rice DC, and Dobrian AD. Adaptation of resistance arteries to increases in pressure. Microcirculation 9: 295-304, 2002.
  39. Puri TS, Gerhardstein BL, Zhao XL, Ladner MB, and Hosey MM. Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels. Biochemistry 36: 9605-9615, 1997.
  40. Schubert R, Kalentchuk VU, and Krien U. Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity. Am J Physiol Heart Circ Physiol 283: H2288-H2295, 2002.
  41. Shibuya J, Ohyanagi M, and Iwasaki T. Enhanced myogenic response in resistance small arteries from spontaneously hypertensive rats: relationship to the voltage-dependent calcium channel. Am J Hypertens 11: 767-773, 1998.
  42. Shistik E, Keren-Raifman T, Idelson GH, Blumenstein Y, Dascal N, and Ivanina T. The N terminus of the cardiac L-type Ca2+ channel {alpha}1C-subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated. J Biol Chem 274: 31145-31149, 1999.
  43. Slish DF, Welsh DG, and Brayden JE. Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone. Am J Physiol Heart Circ Physiol 283: H2196-H2201, 2002.
  44. Srere P. Citrate synthase. Methods Enzymol 13: 3-5, 1969.
  45. Su BY, Reber KM, Nankervis CA, and Nowicki PT. Development of the myogenic response in postnatal intestine: role of PKC. Am J Physiol Gastrointest Liver Physiol 284: G445-G452, 2003.
  46. Wesselman JP, Spaan JA, van der Meulen ET, and VanBavel E. Role of protein kinase C in myogenic calcium-contraction coupling of rat cannulated mesenteric small arteries. Clin Exp Pharmacol Physiol 28: 848-855, 2001.
  47. Yeon DS, Kim JS, Ahn DS, Kwon SC, Kang BS, Morgan KG, and Lee YH. Role of protein kinase C- or RhoA-induced Ca2+ sensitization in stretch-induced myogenic tone. Cardiovasc Res 53: 431-438, 2002.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Ghosh, M. Khazaei, F. Moien-Afshari, L. S. Ang, D. J. Granville, C. B. Verchere, S. R. Dunn, P. McCue, A. Mizisin, K. Sharma, et al.
Moderate exercise attenuates caspase-3 activity, oxidative stress, and inhibits progression of diabetic renal disease in db/db mice
Am J Physiol Renal Physiol, April 1, 2009; 296(4): F700 - F708.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
D. J. Duncker and R. J. Bache
Regulation of Coronary Blood Flow During Exercise
Physiol Rev, July 1, 2008; 88(3): 1009 - 1086.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
R. Schubert, D. Lidington, and S.-S. Bolz
The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction
Cardiovasc Res, January 1, 2008; 77(1): 8 - 18.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. A. Cobine, B. P. Callaghan, and K. D. Keef
Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery
Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3079 - H3088.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
G. C. Amberg, M. F. Navedo, M. Nieves-Cintron, J. D. Molkentin, and L. F. Santana
Calcium sparklets regulate local and global calcium in murine arterial smooth muscle
J. Physiol., February 15, 2007; 579(1): 187 - 201.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
D. H. Korzick, J. M. Muller-Delp, P. Dougherty, C. L. Heaps, D. K. Bowles, and K. K. Krick
Exaggerated coronary vasoreactivity to endothelin-1 in aged rats: Role of protein kinase C
Cardiovasc Res, May 1, 2005; 66(2): 384 - 392.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
96/4/1425    most recent
01077.2003v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Korzick, D. H.
Right arrow Articles by Bowles, D. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Korzick, D. H.
Right arrow Articles by Bowles, D. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.