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Cardiovascular Research 2005 66(2):384-392; doi:10.1016/j.cardiores.2005.01.015
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Copyright © 2005, European Society of Cardiology

Exaggerated coronary vasoreactivity to endothelin-1 in aged rats: Role of protein kinase C

Donna H. Korzicka,*, Judy M. Muller-Delpb, Patrick Doughertyb, Christine L. Heapsc, Douglas K. Bowlesc and Kevin K. Kricka

aDepartment of Kinesiology, The Pennsylvania State University, 106 Noll Laboratory, University Park, PA 16802, United States
bDepartment of Kinesiology, Texas A&M University, College Station TX, United States
cBiomedical Sciences, University of Missouri, Columbia MO, United States

* Corresponding author. Tel.: +1 814 865 5679; fax: +1 814 865 4602. Email address: dhk102{at}psu.edu

Received 1 September 2004; revised 29 December 2004; accepted 14 January 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The interaction between advanced age and increased susceptibility to ischemic insult is well documented. Age-related increases in coronary vascular resistance, in part due to impaired dilator responses, have been reported. Our aim was to determine the role of endothelin-1 (ET-1) on enhanced constrictor responses in aged coronary arteries (CAs) and whether protein kinase C (PKC) signaling mechanisms impact ET-1 responses.

Methods: Vasoreactivity was assessed in CAs isolated from aged (24 months; n=16) and adult (4 months; n=21) male F344 rats following ET-1 (10–10–10–8) with and without specific ETA/ETB receptor antagonists (BQ-123, 1 µM; BQ-788, 30 nM) or the PKC inhibitor bisindolylmaleimide (Bis; 10–6 M). Constrictor responses to KCl (80 mM) were also measured and voltage-gated Ca2+ channel (VGCC) determined in isolated coronary smooth muscle cells. Dilator responses to acetylcholine (ACH) and sodium nitroprusside (SNP) were assessed.

Results: Passive diameter was greater (357 ± 19 vs. 309 ± 9; p<0.02) while spontaneous tone was similar in 24 months vs. 4 months. ET-1 resulted in greater constriction in 24 months vs. 4 months (79% vs. 67%; p<0.01). Group differences persisted following selective ETB inhibition with BQ-788 (p<0.02), while BQ-123 abolished contractile responses to ET-1. Importantly, inhibition of ET-1 constriction by Bis occurred in 24 months but not 4 months (p<0.01). Constrictor responses to KCl and peak VGCC current density were similar in 24 months vs. 4 months (48% vs. 50%). No age-related differences were observed in ACH- or SNP-mediated dilation. Western blotting revealed increases in Ca2+-sensitive PKC{alpha}, -βI, and -βII levels with age, while eNOS and ETA receptor protein levels were unchanged.

Conclusion: Aberrant ETA constrictor responses and directional changes in PKC are likely to contribute to coronary vascular pathology with advanced age.

KEYWORDS Senescence; Vascular smooth muscle; Endothelium; ETA and ETB receptors; eNOS


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
A well-documented consequence of the aging process in both humans and animals is a reduction in coronary vascular reserve function [1–3]. Age-related declines in coronary blood flow [2] and increasing coronary vascular resistance [1,4] are frequent observations in the aged myocardium and may contribute to the genesis of age-related declines in cardiac contractility and increased incidence of congestive heart failure known to occur in the aged population [5]. While age-related alterations in coronary resistance may have a structural basis, i.e. vascular stiffening and vessel rarefaction [4], enhanced coronary vasoconstrictor responses may also provide a partial explanation for this phenomenon. Few studies have directly addressed this issue in isolated coronary arteries from aged rats [6–8] and involvement of specific post-receptor signaling mechanisms associated with enhanced vasoconstrictor responses in aged rat coronary arteries remain incompletely characterized.

There is growing experimental evidence that alterations in endothelin-1 (ET-1) responses may contribute to enhanced constrictor responses and vascular complications associated with a variety of pathological states including atherosclerosis [9], diabetes [10], hypertension [8], as well as aging [11]. While the specific receptor subtype mediating endogenous ET-1 coronary responses in the rodent remains incompletely understood, studies from our laboratory [12,13] and others [14,15] suggest that modulation of voltage-gated calcium channels (VGCCs) by protein kinase C (PKC) in response to a variety of vasoactive stimuli, such as ET-1, is an important signal transduction mechanism underlying the maintenance of vascular tone. In diabetic coronary arteries (CA) and aorta, enhanced constrictor responses to ET-1 have been attributed to aberrant regulation of VGCCs by PKC-mediated processes [16]. However, whether enhanced vasoconstrictor sensitivity is mediated by alterations in VGCCs and/or PKC in conjunction with ET-1 responses in aged rat CAs is unknown.

In the aged vasculature, reductions in endothelium-dependent relaxation induced by cholinergic agonists (presumably mediated by endothelial release of nitric oxide) in large elastic arteries such as the thoracic aorta [17–19] and more recently the coronary resistance vasculature [20] have been demonstrated. However, we hypothesized that exaggerated ET-1 vasoconstrictor responses and associated signaling alterations may also be an important determinant of vascular contractile responses in the coronary circulation with senescence [7,8,11]. The experiments associated with the current study examined this possibility in greater detail and sought to determine whether alterations in PKC and VGCC-related signaling mechanisms impact CA ET-1 responses in rodents. Experiments were also conducted to determine the relative contribution of ETA vs. ETB receptor subtypes on ET-1-mediated vasoreactivity. Because age-related dysregulation of nitric oxide-dependent mechanisms may also impact ET-1 responses [11,21], we assessed acetylcholine-mediated dilator responses, endothelial nitric oxide synthase (eNOS) mRNA and protein levels, respectively.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Experimental animals
All experiments were approved by the Institutional Animal Care and Use committee of The Pennsylvania State University and were in agreement with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult (4 months; n=16) and aged (24–25 months; n=21) male Fisher 344 rats were obtained from Harlan Sprague–Dawley. All animals were housed in temperature- and humidity-controlled holding facilities with a 12 h dark–12 h light cycle and fed ad libitum standard rat chow.

2.2. Isolation of coronary arteries
The hearts of the animals were quickly excised and following removal of the right ventricular wall and atria, the heart was weighed and moved to a dissecting chamber containing cold (4 °C) physiological saline solution (PSS) [13]. Segments of the septal artery approximately 300 µm internal diameter (ID) and 0.5–1.0 mm in length were then dissected and cannulated according to standard procedures in our laboratory [12,13]. The vessels were pressurized at 100 cm H2O by two independent fluid-filled reservoirs which were attached to the micropipettes. Vessels that displayed leaks were discarded and excluded from the study; endothelium-intact vessels were used for all protocols associated with the current study. Vessels were allowed to equilibrate for 1 h (37 °C) and drugs were administered abluminally. A video tracking system (Colorado Video Calipers, Texas A&M University) was utilized to continuously monitor vessel ID throughout a given experiment (Power Lab software).

2.3. Experimental protocol for assessment of vascular reactivity
Vasoconstrictor responses to endothelin-1 (ET-1; 10–10 to 10–8 M; Peninsula Laboratories Inc.) were examined first based on known ET-1 receptor coupling to PKC [22]. ET-1 dose–response curves were repeated in a separate series of experiments following incubation with either the ETA antagonist BQ-123 (1 µM) or the ETB antagonist BQ-788 (30 nM). To determine the role of PKC on ET-1 vasoconstrictor responses, an additional set of experiments was performed in the presence of a specific PKC inhibitor, bisindolylmaleimide (Bis; 10–6 M). This inhibitor displays a greater relative specificity for the Ca2+-dependent conventional PKCs (cPKC) ({alpha}, βI, βII, {gamma}) compared to the novel PKCs (nPKC) ({delta}, {varepsilon}, {eta}, {theta}) which are Ca2+-independent [23]. Vessels were incubated in Bis for 30 min prior to the start of ET-1 concentration–response curves. Responses to KCl-induced depolarization were also assessed (80 mM), while endothelial-dependent dilation was evaluated by examining responses to acetylcholine (Ach; 10–9 to 10–5 M). Maximal passive diameter was obtained at the conclusion of each experiment by bathing the vessels in Ca2+ free PSS or sodium nitroprusside (SNP) at 100 cm H2O. Diameters were normalized to this measurement for the purpose of comparison as previously described by Tickerhoof et al. ([13] see below). Drug concentrations were increased after the response to the preceding dose was maximal (~5 min/dose), and only one experimental drug (± inhibitor) was used on a given vessel. Only vessels that exhibited at least 30% spontaneous tone were used for Ach concentration–response curves. All drugs were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified.

2.4. Protein expression in isolated coronary arteries
Segments of the left coronary artery (LCA) (~300 µm ID; ~1200 µm long) were dissected in cold PSS solution (4 °C). Vessels pieces (n=2 to 3 per tube) were snap frozen and stored at –70 °C until ready for use. Arteries were solubilized as previously described [12,13] and protein determination was assessed using NanoOrange (Molecular Probes). Equal amounts of sample per lane were electrophoresed on 7.5% (PKC, eNOS) or 10% (ETA, ETB receptors) SDS-polyacrylamide gels and transferred to PVDF membranes. Following blocking, membranes were incubated with primary antibodies for PKCβI (1:2100), PKCβII (1:1200), PKC{alpha} (1:1000), PKC{varepsilon} (1:500), PKC{delta} (1:1000; Santa Cruz), eNOS (1:1600; Transduction Labs), ETA or ETB receptors (1:1000; Sigma Aldrich) for 3 h at room temperature or overnight (4 °C). Antibody binding was assessed by enhanced chemiluminescence (ECL, Amersham) following incubation with secondary anti-rabbit antibodies (1 h). Densitometric analysis of immunoblot films was performed using NIH Scion Image Analysis Software (National Institutes of Health, Bethesda, MD). Randomly sampled membranes were Ponceau-stained to verify equivalent sample loading and protein transfer as described by Tickerhoof et al. [13]. Data were normalized by expressing aged values relative to adult controls.

2.4.1. RT-PCR for eNOS mRNA in isolated coronary arteries by RT-PCR
RNA was isolated from single, isolated CAs (n=6/group) and eNOS expression was assessed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) using previously published primers and cycling conditions [24]. All data were standardized by co-amplifying eNOS with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculating an eNOS-to-GAPDH ratio.

2.4.2. Whole-cell voltage clamp
Isolated CSM cells were obtained by enzymatic dispersion for voltage-clamp experiments as described previously [24–26]. Cell suspensions were stored in low Ca2+ (0.1 mM) buffer at 4 °C until use (0–6 h). Whole-cell VGCC currents were determined using a standard whole-cell voltage-clamp technique [24–27]. Following whole-cell configuration, the superfusate was switched to PSS with tetraethylammonium chloride (TEACl) substitution for NaCl and 10 mM Ba2+ as the charge carrier. Current densities (pA/pF) were obtained for each cell by normalization of whole-cell current to cell capacitance to account for differences in cell membrane surface area. Data acquisition and analysis were accomplished using pClamp 7.0 software (Axon Instruments, Foster City, CA). All experiments were conducted at room temperature (22–25 °C).

2.5. Data analysis
Vasomotor responses of CAs are presented as percent constriction, and expressed by the following equation, [(DSS–DB)/DB]*100, where DSS is the steady state diameter in response to the drug, and DB is the baseline diameter right before the start of the concentration–response curve. Diameter measurements were also expressed relative to passive diameter. Group comparisons for concentration–response curves to pharmacological agents or current–voltage (I–V) relationships were analyzed with a two-way ANOVA with repeated measures using the PROC Mixed General Linear Models program (Statistical Analysis Software; SAS). For immunoblotting or animal characteristics, an unpaired, two-tailed Student's t-test was used to determine group differences between mean values. The Least Significant Difference method was employed for all post hoc comparisons. All variables are reported as mean ± standard error (S.E.). Significance was defined as p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Physical and physiological characteristics of animals
The physical and physiological characteristics of the groups of rats are provided in Table 1. As expected, body weight was greater in aged vs. adult rats. Body weight, heart weight, and heart weight-to-body weight ratio were all significantly different in aged rats vs. adult rats (p<0.05). Mean maximum vessel diameter measured at 100 cm H2O in Ca2+-free PSS was also significantly greater in CCAs isolated from aged (357 ± 19 µm) vs. adult (309 ± 9 µm) rats.


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  		Table 1 Physical and physiological animal characteristics

 
3.2. Reactivity of coronary arteries
3.2.1. Vasoconstrictor responses
ET-1 produced dose-dependent constriction (Fig. 1) in isolated CAs from all groups. ANOVA revealed a significant group x drug interaction (p<0.01) in concentration–response curves. CAs from aged rats exhibited significantly greater constriction (p<0.01) than CAs isolated from adult controls in the dose–response range from 2.2 to 3.0 nM for ET-1. Inhibition of PKC with the specific antagonist, Bis, attenuated ET-1-mediated constriction in CAs from both adult and aged rats. However, the magnitude of attenuation was ~3-fold greater in aged vs. adult CA (group x drug interaction).


Figure 1
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  		Fig. 1 Cumulative addition of ET-1-induced constriction with and without the PKC inhibitor bisindolylmaleimide (Bis) in CAs isolated from adult (n=11) and aged rats (n=15). Values are means ± S.E. Arteries from aged rats exhibited greater constriction to ET-1 than did CAs from adult, aged+Bis, and adult+Bis. *p<0.01, aged vs. adult; {dagger}p<0.01, aged vs. aged+Bis; {ddagger}p<0.05, aged vs. adult+Bis.

 
To examine the relative contribution of ETA vs. ETB receptors to ET-1-mediated contraction in rodent CAs, ET-1 constrictor responses were also assessed in the presence of specific ET receptor subtype antagonists. Group differences in ET-1-mediated constrictor responses persisted in the presence of the specific ETB receptor antagonist BQ-788 (67% vs. 54%; Fig. 2A; p<0.01); whereas ET-1 constrictor responses were nearly abolished in the presence of the ETA antagonist BQ-123 (Fig. 2B). Taken together, these data support the conclusion that ETA receptors account for most of the ET-1 contractile responses in rodent CAs. These data also suggest that alterations in PKC contribute, at least in part, to enhanced vasoconstrictor responses to ET-1 in CAs isolated from aged rats. Group differences in depolarization-induced constriction following 80 mM KCl were not observed (Fig. 3).


Figure 2
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  		Fig. 2 Cumulative addition of ET-1-induced constriction in the presence of the specific ETA antagonist BQ-123 (M; Panel A) or the ETB antagonist BQ-788 (M; Panel B) in CAs isolated from adult (n=6/5) and aged rats (n=6/3). Values are mean ± S.E. Group differences in ET-1 constriction persisted in the presence of BQ-788 (p<0.02) while BQ-123 markedly inhibited ET-1 responses, suggesting a dominant role for ETA receptors in rodent CAs.

 

Figure 3
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  		Fig. 3 Depolarization-induced constriction by KCl. Relative diameter following abluminal administration of KCl (80 mM) was not statistically different in aged vs. adult CCA.

 
3.2.2. Endothelium-mediated vasodilator responses
Ach produced concentration-related increases in relative diameter (Fig. 4A) in CAs from both adult and aged rats. Maximal dilation was similar in CAs from aged vs. adult rats. Fig. 4B and C also presents PCR and immunoblot analysis of eNOS content in isolated CAs from adult and aged rats. Coronary arteries from aged rats had similar eNOS mRNA and protein levels compared to arteries from adult rats (p>0.01).


Figure 4
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  		Fig. 4 Effects of age on endothelium-mediated dilator responses and eNOS expression in CAs. Panel A: cumulative addition of Ach induced similar dilation in aged vs. adult CAs; Panel B: PCR products (2 µg/lane) were not different in aged vs. adult CAs; Panel C: eNOS immunoreactivity was not different in adult vs. aged CAs; equal amounts of protein were loaded per lane (10 µg/lane). Values are means ± S.E. (normalized by expressing all values relative to CTL).

 
3.3. Expression of PKC isoforms and ETA receptors in conduit coronary arteries
Fig. 5 presents results of immunoblot analysis of PKC{alpha}, PKCβI, PKCβII, PKC{delta}, and PKC{varepsilon}. PKC{alpha} levels were elevated in CAs from aged rats by 51.5% (p<0.01) compared to adult rats. PKCβI and PKCβII expression was also significantly elevated (p<0.01) in CAs from aged rats by 54.9% and 123.7%, respectively. In contrast, PKC{varepsilon} and PKC{delta} levels were similar (p>0.05) in aged and adult rats. These results suggest a significant increase in cPKC levels compared to nPKC levels in isolated CAs from aged vs. adult control rats. Fig. 6 represents immunoblot analysis results for ETA receptor protein levels assessed in CAs isolated from aged and adult rats. Group differences were not observed in ETA receptor protein levels (p>0.01) and immunoreactivity for ETB receptors was not observed in CAs isolated from either aged or adult rats.


Figure 5
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  		Fig. 5 Effects of age on various PKC isoforms in CAs. PKC isoform content from representative immunoblots (top) prepared from CAs as described in the text. Equal amounts of protein were loaded per lane for CAs for PKC{alpha} (7 µg/lane), PKCβI (7 µg/lane), PKCβII (8 µg/lane), PKC{delta} (8 µg/lane), and PKC{varepsilon} (8 µg/lane). Bar graphs (bottom) are presented for average PKC isoform protein content of CAs from aged and adult rats for PKC{alpha}, PKCβI, PKCβII, PKC{delta}, and PKC{varepsilon}. Values are means ± S.E. (normalized by expressing all values relative to adult); n=10, adult; n=8, aged. *p<0.01.

 

Figure 6
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  		Fig. 6 Effects of age on ETA receptor protein levels in CAs. ETA receptor immunoreactivity (top, ~45 kDa) prepared from CA samples as described in the text. Equal amounts of protein were loaded per lane for CAs (12 µg/lane). The bar graph (bottom) presented is for average ETA receptor protein levels of CAs from n=4 aged and n=4 adult rats. Values are means ± S.E. (normalized by expressing all values relative to adult). There was no statistical difference between aged and adult ETA levels. Immunoreactivity for ETB receptor protein levels (see Methods) was not observed in adult or aged CAs.

 
3.3.1. VGCC in coronary smooth muscle
Fig. 7 shows current–voltage (I–V) relationships obtained in coronary smooth muscle from adult and aged animals. Successive depolarization steps from –60 to +70 mV produced inward currents showing a peak near +20 mV. The absolute magnitude of inward current was greater in cells from aged compared to adult animals (Fig. 7A). However, when VGCC current was normalized to cell membrane surface area (pA/pF; i.e. current density), ICa was not significantly different between experimental groups (Fig. 7B). Similarly, both the membrane potential producing half-maximal activation (V0.5) and voltage-sensitivity (k) were unaffected by aging (data not shown).


Figure 7
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  		Fig. 7 Effect of age on the VGCC current–voltage relationship. Current–voltage (I–V) relationships for whole-cell VGCC current in coronary myocytes from adult (solid symbols) and aged (open symbols) animals. Current data was obtained from the peak inward current measured during a 400 ms step depolarization to the membrane potential (Vm) indicated from a holding potential of –80 mV. Current plotted as absolute current (top graph; pA) and normalized to cell membrane capacitance (bottom graph; pA/pF). Data are means ± S.E. of 15 cells from 5 rats in each group. ANOVA indicated a significant main effect of age on absolute current, but not current density (pA/pF).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The purpose of this investigation was to determine whether age-related changes in PKC and VGCC-dependent mechanisms provide a basis for enhanced ET-1 mediated coronary vasoreactivity. We also examined the relative contribution of ETA and ETB receptor subtypes to ET-1 mediated coronary constriction utilizing specific receptor antagonists and antibodies. Our findings indicate that ET-1 mediated constriction in adult and aged rodent CAs is primarily mediated by ETA receptors. Our findings also indicate that ET-1-mediated vasoconstrictor responses were enhanced in the aged coronary vasculature and abolished by the specific PKC inhibitor, Bis. Age-related increases in Ca2+-dependent PKCs were observed in conjunction with enhanced ET-1 constrictor responses, however depolarization-induced vasoconstriction and VGCC current density were similar in aged vs. adult CA. Additional observations include unchanged NO-mediated vasodilation, eNOS protein or mRNA levels with advancing age. Collectively, our studies provide the first evidence for significant alterations in PKC-mediated signal transduction following ET-1 in the isolated coronary vasculature of aged rats, which may contribute to the increased coronary vascular resistance and diminished contractile reserve capacity known to occur in the senescent heart.

4.1. Effects of aging on constrictor responses
In the present study, we examined the mechanism of enhanced coronary vasoconstrictor responses to ET-1 in aged rat coronary arteries. Endothelin (ETA/ETB) receptors are Gq-coupled receptors and ET-1 is thought to induce potent vasoconstriction through activation of PKC [22]. Importantly, aberrant ET-1 responses have been implicated in age-associated increases in vasoreactivity in isolated aorta and the coronary circulation [8,11,28]. Our findings of enhanced coronary constrictor responses to ET-1 in aged CAs are in agreement with these previous studies [8,11,28], and provide novel evidence for an important regulatory role of PKC on ET-1-mediated responses. Specifically, administration of a PKC inhibitor caused significant reductions in constrictor responses induced by ET-1 in CAs isolated from aged but not adult rats. A similar phenomenon has been observed in the diabetic vasculature [29], and one plausible explanation for our results is that increased ET receptor number, combined with enhanced levels of PKC, converge to produce a contractile hypersensitivity of vascular smooth muscle. In the current study, however, group differences in coronary ETA/ETB receptor protein levels were not observed. Our findings of age-related increases in cPKC levels in isolated CAs (see below) do support a primary role for PKC in mediating enhanced ET-1 coronary vasoconstrictor responses in senescent rats.

While both ETA and ETB receptor subtypes have been implicated in ET-1 mediated constriction [21], recent studies suggest considerable heterogeneity in ET-1 responses across different circulations and a greater dependence of conduit coronary arteries on the ETA receptor subtype in a variety of species [9,30,31]. We now extend these findings to rodent CAs. Specifically, ET-1 constrictor responses persisted in the presence of the ETB receptor antagonist BQ-788, while the ETA receptor antagonist BQ-123 markedly inhibited ET-1 mediated responses. Consistent with previous studies demonstrating low levels of vascular ETB expression [31,32], we were unable to demonstrate immunoreactivity for the ETB receptor upon western blotting in isolated CAs. It is also important to note that we did not observe any vasodilatory properties of ET-1 as previously postulated to be mediated by the ETB receptor subtype [21]. While we cannot completely rule out the presence of ETB receptors in rodent CAs, our data do support a more dominant role for the ETA receptor in mediating constrictor responses to ET-1 in both adult and aged rodent CAs.

In contrast to ET-1-mediated responses, we observed no differences in KCl-induced coronary vasoconstriction in aged vs. adult rats. Furthermore, and contrary to our hypothesis, significant effects of aging on coronary VGCC current density were not observed. To the best of our knowledge, these results are the first to describe coronary smooth muscle ICa in senescence, and provide novel evidence that the underlying cellular mechanisms responsible for enhanced vasoreactivity in aged CAs apparently do not involve the VGCC. Other documented ion channel alterations with aging, such as decreased large-conductance, Ca2+-activated K+ (BKCa) channel expression [33,34] may contribute to our findings of enhanced ET-1 responses. Whether or not PKC plays a key regulatory role on BKCa channel activity under conditions of ET-1 stimulation in the aged coronary vasculature requires further study.

4.2. Effects of aging on PKC isoform expression
A number of studies have implicated specific PKC isoforms in the deleterious vascular effects of a variety of pathologies including diabetes and hypertension (for review see [35]). For example, PKCβ and PKC{alpha} have been identified as key isoforms in the development of diabetes-associated vascular complications including increased vascular permeability and proliferation [36,37]. In the aged coronary circulation, we observed significant increases in Ca2+ sensitive PKC{alpha}, PKCβI and PKCβII protein levels relative to adult controls. In contrast, PKC{delta} and PKC{varepsilon} protein levels were similar in CAs from aged vs. adult rats. These data are intriguing and provide novel support for the idea that similarities in vascular phenotype exist between the aged and diabetic heart. A previous report that endothelial release of ET-1 can increase PKCβ levels [38] also suggests potentially important regulatory interactions between ET-1 and cPKCs on coronary vascular responses and clearly merits further investigation.

4.3. Effects of aging on endothelial-dependent vasodilation
Because nitric oxide (NO) may exert important inhibitory effects on ET-1 mediated signaling in some vascular beds (for review see [21]), we speculated that aberrations in the functional cross-talk between ET receptors and NO might converge to exacerbate age-related enhancements in ET-1-mediated coronary constrictor responses. However, our results indicate preserved Ach-mediated vasodilation, steady-state levels of eNOS mRNA and eNOS protein levels in isolated CAs from aged vs. adult rats. While the findings of the current study are seemingly at odds with recent evidence supporting impaired NO-mediated signaling in CAs [20,39] with senescence, they are not without precedence. Previous studies in isolated rat CAs [6,40] from aged rats have also reported no difference in endothelium-dependent relaxation to Ach. One logical explanation for discrepant findings between studies may be attributed to differences in vessel internal diameter (ID). We utilized proximal CAs with IDs approximately 40% greater than those reported in the studies of Csiszar et al. [20]. As such, our findings are more likely related to macrovascular complications such as coronary artery vasospasm or the development of atherosclerotic lesions, processes known to occur to a greater extent in the aged vs. adult heart. Alternatively, it could be that Ach-mediated responses in rodent CAs are not solely dependent on NO generation and compensatory increases in endothelial factors such as EDHF masked endothelial dysfunction. However, this possibility is unlikely given recent findings demonstrating complete inhibition of Ach-mediated dilation by the NOS inhibitor L-NAME in both adult and aged CAs isolated from F344 rats (Muller-Delp). Age-related differences in the dependence of ET-1 mediated responses on NO is also unlikely given the absence of an ETB-mediated dilatory response in our experimental paradigm. However, future studies are indicated to address potential significant interactive effects between NO, PKC and ETA signal transduction pathways, respectively, on the vascular dysregulation associated with aging.

4.4. Conclusions
In summary, this study provides novel support for enhanced cPKC-mediated vasoconstriction in response to ET-1 in aged CAs, the effects of which could not be explained by alterations in VGCC-induced mechanisms. Findings of preserved NO-mediated vasodilation and eNOS levels in isolated CAs from aged vs. adult rats were also observed. Taken together, the data suggest that enhanced ETA receptor-mediated vasoconstriction in the coronary vasculature is, in part, mediated by PKC-dependent mechanisms localized to vascular smooth muscle. These results were further supported by immunoblot analyses revealing, for the first time, increased PKCβI, PKCβII, and PKC{alpha} in isolated CAs of aged vs. adult rats. The increased incidence of atherosclerotic heart disease and vulnerability for progression to congestive heart failure in the aged population is well-documented [5]. Future studies examining PKC translocation and activation and the use of specific PKC isoforms inhibitors in isolated CAs of aged rats are needed to determine the exact role and function of specific PKC isoforms in the vascular dysfunction associated with cardiovascular aging.


   Acknowledgements
 
We thank J.C. Kostyak for his assistance with this project. This work was supported in part by: NIH K01-AG00875 (DHK), AHA (JMD), NIH HL52840 (DKB), AHA SDG 0330252N and NIH F32 HL70500 (CLH).


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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