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(Circulation Research. 2001;88:210.)
© 2001 American Heart Association, Inc.

Cellular Biology

Decreased Expression of Voltage- and Ca²⁺-Activated K⁺ Channels in Coronary Smooth Muscle During Aging

Jure Marijic, QingXia Li, Min Song, Kazuhide Nishimaru, Enrico Stefani, Ligia Toro

From the Departments of Anesthesiology (J.M., Q.X.L., M.S., K.N., E.S., L.T.), Molecular & Medical Pharmacology (L.T.), and Physiology (E.S.), and the Brain Research Institute (E.S., L.T.), University of California Los Angeles, Los Angeles, Calif. The present affiliation for Q.X.L. is the House Ear Institute, Los Angeles, Calif.

Correspondence to Ligia Toro, Department of Anesthesiology, UCLA, BH-509A CHS, Box 957115, Los Angeles, CA 90095-7115. E-mail ltoro{at}ucla.edu

	Abstract

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Abstract—Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca²⁺-activated K⁺ channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52±9 channels/pF (3 months old) to 18±5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by 50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed 80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and 45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively (70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Key Words: vascular smooth muscle • coronary arteries • K⁺ channels • Ca²⁺-activated K⁺ channels • aging

	Introduction

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Aging is a main risk factor for coronary heart disease, which remains the leading cause of death in developed countries.¹ In human coronary arteries, spontaneous contractile activity seems to be more frequent in older subjects,^{2 3} increasing the risk of vasospasm. In addition, there is evidence that aging induces increased responses of rat coronary and mesenteric arteries to endothelial constricting factors and K⁺.^{4 5 6} The increase in K⁺-induced contractions in aging animals suggests a change in K⁺ channel function or expression as age progresses. Coronary arteries possess several types of K⁺ conductances; the large-conductance, voltage-dependent, and Ca²⁺-activated K⁺ channel (MaxiK, BK) is particularly abundant and plays a key role in regulating arterial tone.^{7 8 9 10}

MaxiK channels regulate membrane potential and intracellular Ca²⁺ ([Ca²⁺]_i) in various smooth muscles^{11 12} (compare with Archer et al¹³ and Cornfield et al¹⁴ ). Under basal conditions, MaxiK channels are tonically active and act as a hyperpolarizing force that reduces the activity of voltage-dependent Ca²⁺ channels and Ca²⁺ influx and, thus, oppose contraction.¹² In fact, MaxiK blockade causes smooth muscle constriction.^{10 11} Given the crucial role of MaxiK in setting the point of vascular contractility, we examined the possibility that these channels are diminished in number in aging coronary arteries. A decrease in MaxiK channel expression would provide a mechanism to explain coronary increased excitability as age progresses.

In the present study, we show that expression of MaxiK channel protein in aging coronary smooth muscle is substantially decreased not only in rats but also in humans. Functional (pharmacomechanical and electrophysiological) experiments were confirmed with immunochemistry using a polyclonal antibody raised against an intracellular epitope of the pore-forming subunit (slo) of MaxiK channels.⁷

	Materials and Methods

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F344 male rats (young, 3 months old; old, 25 to 30 months old) were killed according to a protocol approved by UCLA. Human coronary arteries from explanted hearts were used. The protocol received institutional review committee approval. Demographic data for human subjects are presented in the Table.

View this table: [in this window] [in a new window]   Table 1. Demographics of Human Subjects

Isometric Contraction
Arterial rings (inner diameter 0.15 to 0.3 mm, 3 mm long) were equilibrated 60 minutes in Krebs solution. Before experimentation, rings were progressively stretched to an optimal tension (maximal increase in tension in response to KCl). Optimal wall tension was 120 mg for young and 200 mg for old coronary arteries. KCl (80 mmol/L)-induced contraction was similar in 33 of 40 old rings (68±12%, n=33 rings) compared with young coronary rings (66±10%, n=45 rings). Consistent with previous studies,⁶ the mean value of all old samples gave significant differences (123±19% in old, n=40 versus 66±10% in young, n=45). Thus, data were normalized to the response of each ring to 80 mmol/L KCl. Equivalent results were obtained with or without normalization.

Cell Isolation
Single coronary myocytes were used within 6 to 8 hours after isolation. Vessels (1-mm pieces) were incubated for 15 minutes at 37°C with Ca²⁺-free Hanks’ solution containing 30 U/mL papain, 2 mg/mL BSA, and 1 mmol/L dithiothreitol followed by 10 minutes with 2.1 U/mL collagenase F and 0.2 U/mL collagenase H.

Electrophysiology
Whole-cell currents were measured using patch electrodes (3 to 5 M) filled with (in mmol/L) potassium methanesulfonate (Mes) 140, CaCl₂ 0.1, MgCl₂ 2, HEPES 10, glucose 10, and EGTA 0.146, pH 7.4, pCa 7, with/without 150 µg/mL nystatin. The bath solution was (mmol/L) KMes 5, NaMes 135, CaCl₂ 0.1, MgCl₂ 2, HEPES 10, and glucose 10, pH 7.4. Data acquisition and analysis were performed using pCLAMP (Axon Instruments).

Nonstationary variance analysis^{15 16} was performed using 1 mmol/L 4-aminopyridine and/or maintaining the cells at a depolarized holding potential (HP) to avoid a small Kv current component (<7%). To attain maximal open probability (Po), experiments were performed in the presence of the MaxiK channel opener NS1619 (50 to 100 µmol/L).¹⁷ NS1619 eliminated the need to use a high concentration of [Ca²⁺]_i or extremely high potentials (>200 mV) that would otherwise damage the cells. Data were fit using the following equation:

(1)

where ²_(t) is the variance as a function of time, |$$–_(t) is the mean current as a function of time, i is the unitary current, and N is the total number of functional channels. Limiting Po was calculated from Po=|$$–|EI_(t=)/iN; it was identical in young and old coronary arteries (0.64±0.06 versus 0.65±0.04).

Tissue Lysates, Membrane Preparations, and Immunochemistry
Rat coronary and pulmonary arteries were homogenized in 50 µL ice-cold hypotonic buffer containing 20 mmol/L HEPES-KOH and 1 mmol/L EDTA, pH 7.4, and supplemented with 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L pepstatin A, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 10 mmol/L CHAPS and centrifuged at 1000g. The solubilized protein was directly used for immunoblotting. Human membrane preparations, immunoblotting, and immunonohistochemistry were as described previously.¹⁸

	Results

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Contractile Response of Coronary Arteries Induced by a MaxiK Channel Blocker, Iberiotoxin
Contraction experiments on coronary arterial rings from F344 rats were performed using a specific blocker of MaxiK channels, iberiotoxin (IbTx).¹⁹ Consistent with their important role in the control of vascular tone, MaxiK channel blockade with IbTx induced a dramatic increase in tension of the coronary arterial rings (Figure 1). The increase in tension is dose-dependent and is maximal at 100 nmol/L IbTx for both young (3 months old) and old (26 months old) arteries. The EC₅₀ for young arteries (3 months old) was 9±1 nmol/L (n=13 rats) and for old (25 to 30 months old) coronary arteries was 15±6 nmol/L (n=11 rats); these values are in reasonable agreement with the EC₅₀ of IbTx blockade of MaxiK channels in reconstituted systems^{20 21} and with a role of MaxiK channels in IbTx-induced contractions. It was noteworthy that the increase in tension induced by IbTx was greatly reduced when we examined coronary arteries from aging rats (Figure 1B) versus young rats (Figure 1A). This dramatic difference is plotted in Figure 1C where the maximal change in tension induced by IbTx is nearly 100% of the KCl response in young coronary arteries (circles). In contrast, in old coronary arteries, IbTx induced only 25% increase in tension (squares). These results indicated that the function or numbers of MaxiK channels are decreased during aging in F344 rats.

View larger version (19K): [in this window] [in a new window]   Figure 1. Aging coronary arteries are less responsive to IbTx, a MaxiK channel blocker. Cumulative dose-response contraction in coronary arteries from young (3 months old) (A) and old rats (27 to 30 months old) (B). Arrows mark the time of IbTx addition (numbers are in nmol/L). C, Percent change in contraction normalized to 80 mmol/L KCl response as a function of IbTx concentration. Percent changes in contraction force were calculated using [(Ff-Fi)/Fi]x100, where Ff is the final force and Fi is the initial force. Dose-response curves were well-fitted to a Hill function (continuous line): max/(1+(EC50/[IbTx])N, where N is the Hill coefficient. Mean values were as follows: young (•), EC50=9±1 nmol/L, N=1.3±0.15, maximum contraction=100±4% (n=13 rats, 13 to 25 rings for each point); old (), EC50=15±6 nmol/L, N=1.5, maximum contraction=19±3% (n=11 rats, 15 to 22 rings for each point). Maximum contraction was significantly reduced by 70% in old animals. Unless otherwise stated, in this and subsequent figures, values are mean±SE. Student’s t test was used. P<0.05 was considered statistically significant.

Functional MaxiK Channels Are Diminished in Coronary Myocytes Isolated From Old Rats
To test the hypothesis that during aging MaxiK channels are reduced in numbers, we directly quantified the number of functional MaxiK channels using electrophysiology (Figure 2) and measured protein expression of the pore-forming subunit with specific antibodies (see Figure 4). Figure 2A shows typical whole-cell MaxiK currents normalized to cell capacitance in single coronary myocytes from old and young rats. Under the same experimental conditions and with equivalent bulk Ca²⁺ levels, current density was significantly larger in young coronary myocytes. This result strongly supports the idea that changes in current density are likely due to differences in channel expression rather than changes in bulk [Ca²⁺]_i levels in young versus old coronary myocytes. The inset in Figure 2A shows the mean current density versus voltage plot, demonstrating that, at each potential, old coronary myocytes have a lower current density than young coronary myocytes.

View larger version (33K): [in this window] [in a new window]   Figure 2. Number of functional MaxiK channels decreases in aging coronary myocytes. A, Records illustrate whole-cell current density (100 nmol/L IbTx-sensitive component) from young and old rats. Pulses: -100 to 100 mV every 10 mV. HP=0 mV. Currents were normalized to the capacity C (in pF) of each cell. Inset, Mean MaxiK current density-voltage relationships: young (•), n=10; old (), n=7. B and C, Examples of variance versus mean current plots. Whole-cell perforated patches. Data were well-fitted (continuous line) to Equation 1 with young (B), N=1146 channels, i=10 pA (expected i=22 pA); old (C), N=411 channels, i=9.5 pA (expected i=28 pA). The reduced single-channel current can be explained by internal blockade by Mg2+.42 Insets, Mean current and variance during the pulse. Panel B inset, Limiting Po=0.77, C=16.5 pF, pulse=100 mV, and HP=-30 mV. Panel C inset, Limiting Po=0.8, C=16 pF, pulse=150 mV, and HP=-60 mV. Cell capacity was practically the same in young and old cells: young, 16±1 pF (n=9); old, 20±4 pF (n=10). D, Mean number of active channels/pF. Old coronary arteries have lower functional channel density than young coronary arteries. *Statistically different in this and subsequent figures.

View larger version (70K): [in this window] [in a new window]   Figure 4. Loss of MaxiK channel protein expression in aged rat coronary arteries. A, Confocal images of isolated myocytes from young and old coronary arteries labeled with anti-MaxiK -subunit antibody as in Figure 3. B, Arterial rings (10-µm sections) labeled with anti-MaxiK antibody and visualized with Alesa Fluor 488–conjugated secondary antibody. Controls are negative images of cell or tissue labeled with antibody preadsorbed to excess antigen. C, Pixel intensity bar plot of young versus old arterial rings in paired experiments. Images were digitized using a cooled CCD camera (Cambridge Research & Instrumentation) or using a confocal microscope and analyzed with ImagePro.

The number (N) of functional MaxiK channels in single myocytes was directly assessed using the nonstationary variance analysis.^{15 16} In this method, a limiting Po value larger than 0.5 has to be reached to adequately fit the variance versus mean current curve and obtain N (see Materials and Methods). Hypothetical changes in [Ca²⁺]_i may change the pulse-voltage values necessary to reach limiting Po but will not modify the shape of the variance versus mean current plot and thus will not change the fitted N values. Therefore, N values obtained with this method are independent of [Ca²⁺]_i, and therefore possible changes in [Ca²⁺]_i levels in young versus old would not affect the results. To estimate the number of functional channels, cells were repetitively pulsed to 100 to 150 mV for 50 ms, and variance versus mean current curves were constructed. Figure 2B shows an example of the nonstationary noise analysis in a young cell. The inset shows superimposed traces of the corresponding mean current and variance. In this cell, we estimated a total of 1146 active channels. In comparison, a cell from an old coronary artery had 400 channels (Figure 2C). The number of functional channels obtained from the nonstationary variance analysis was normalized to the capacity of each cell (Figure 2D). It is clear that myocytes from old coronary arteries possess a lower number of functional channels (18±5 channels/pF, n=10 cells; 5 rats) than those from young arteries (52±9 channels/pF, n=9 cells; 4 rats), with a 65% reduction in the older population.

MaxiK Channel Antibodies Reveal Loss of Protein in Aging Coronary Arteries From F344 Rats and Humans
Western blot analysis was consistent with the functional measurements. Labeling was performed with an affinity-purified polyclonal antibody targeted to the carboxyl terminus of the MaxiK pore-forming subunit.⁷ Figure 3A demonstrates that the amount of MaxiK protein with a molecular mass of 120 kDa is less in old than in young coronary arteries. Normalized optical density (OD) was 0.74±0.08 (n=7) for young coronary arteries, whereas for old coronary arteries it was 0.4±0.05 (n=7). Interestingly, this age-dependent diminution was not observed in cell homogenates from pulmonary arteries (young, 0.8±0.1 versus old, 0.76±0.08; n=4). As an additional control, we used the same blots to determine the relative amount of protein kinase G (PKG)-I in old and young coronary arteries. A polyclonal antibody against the mammalian PKG-I and PKG-Iß isoforms recognized a doublet in coronary preparations (75 to 80 kDa); whereas, a single band was labeled in the pulmonary artery. These results suggest that rat coronary arteries express both PKG-I and ß isoforms.²² Quantification by densitometry showed that although there is a small tendency of PKG to diminish in old vessels (coronary and pulmonary), the differences were not significant. The blots in Figures 3A and 3B also show the specificity of the anti-MaxiK and anti-PKG antibodies when the antigenic peptides were preadsorbed to the antibodies.

View larger version (42K): [in this window] [in a new window]   Figure 3. Total amount of MaxiK -subunit protein diminishes in aging coronary arteries. A, Immunoblot of tissue homogenates from young (Y.) and old (O.) coronary (cor) (20 µg/lane) and pulmonary (pulm.) (10 µg/lane) arteries. The anti-MaxiK purified polyclonal antibody (raised against the sequence VNDTNVQFLDQDDD of the pore-forming subunit) recognized a band with the expected apparent molecular mass (120 kDa). The density of MaxiK -subunit protein diminished with aging in coronary arteries, but a change was not apparent in pulmonary arteries. +Antigen indicates anti- antibody preincubated with antigenic peptide. B, Same blot as in panel A but using anti-PKG purified polyclonal antibody (raised against the common sequence CDEPPPDDNSGWDIDF of the PKG-I/PKG-Iß isoforms). There were no differences in PKG expression in young versus old coronary arteries; similar results were obtained using anti-smooth muscle -actin. Signals are detected using enhanced chemiluminescence and exposed to Kodak BioMax film. Bands were quantified by densitometry and normalized to maximum OD.

In agreement with the Western blot and functional studies, immunocytochemistry of freshly isolated cells and of coronary rings demonstrated that aged coronary arteries expressed a reduced amount of MaxiK -subunit protein. Figure 4A shows examples of isolated cells used for patch-clamp recordings labeled with anti-MaxiK antibody, whereas Figure 4B illustrates images of the MaxiK channel signals obtained in coronary rings. Quantification of the pixel intensity in the smooth muscle layer was performed in paired experiments and demonstrated that the level of expression of MaxiK channels in aging coronary arteries is 50% of the channels expressed in young animals. Pixel intensity was 40±6 (n=4) in young coronary arteries versus 21±5 (n=4) in old coronary arteries.

Moreover, when we examined human coronary samples from explanted hearts, there was a correlation between age and expression levels of MaxiK channels as examined by Western blotting. Figure 5 shows that the density of MaxiK channel protein (normalized to PKG) is diminished with advancing age. Normalized OD was 0.55±0.1 (n=7) for the young (3 to 18 years old); it decreased to 0.3±0.04 (n=16) in the adult population (19 to 56 years old), and was further suppressed to 0.1±0.02 (n=10) for older subjects (61 to 70 years old). No correlation was found between males (OD=0.28±0.05, n =20) and females (OD=0.32±0.07, n=13). It is important to point out that coronary arteries from explanted hearts may not be the best model to study aging because other factors (eg, disease, medication) may influence protein expression. Nevertheless, our results are consistent with the view that aging per se diminishes MaxiK channel expression in humans as well. In addition, our results may explain the age-dependent decrease in the response to nitroglycerin in humans,²³ because MaxiK channels contribute to the therapeutic sodium nitroprusside²⁴ and nitroglycerin (authors’ unpublished results, November 1999) vasodilatory effects.

View larger version (39K): [in this window] [in a new window]   Figure 5. Human coronary arteries have depressed MaxiK channel expression with advanced age. Immunoblots of human coronary membrane preparations (5 µg protein/lane). MaxiK and PKG signals were measured in the same blots. Top, Immunoblots for 5-, 17-, 68-, and 70-year-old subjects. MaxiK OD values were normalized with respect to PKG signals and grouped according to age. Mean values were 0.55±0.1 (n=7), 3 to 18 years old; 0.3±0.04 (n=16), 19 to 56 years old; and 0.1±0.02 (n=10), 61 to 70 years old. Antibodies were as in Figure 3.

	Discussion

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Aging is known to affect cellular excitability, producing impaired cell function. Major age-related diseases are neurodegenerative, cardiovascular, and immunological. In the aging brain, the Ca²⁺ hypothesis postulates that aging induces sustained changes in cellular mechanisms of [Ca²⁺]_i regulation.²⁵ Cellular mechanisms that can alter both excitability and Ca²⁺ homeostasis are voltage-dependent ion channels. Consistent with this view, in hippocampal neurons, aging produces an increase in the number of L-type Ca²⁺ channels.²⁶ In addition to a massive increase in [Ca²⁺]_i that may lead to cell death, modification of Ca²⁺ entry through this class of channels may also promote altered gene transcription.^{27 28} In other systems, such as skeletal muscle where aging produces a decline in muscle performance, MaxiK channels are increased in aged animals.²⁹ However, their physiological role in skeletal muscle is unclear; therefore, the impact of MaxiK increased expression in the musculoskeletal system is difficult to predict.

In contrast to skeletal muscle, the role of MaxiK channels in the vasculature is much more defined, where they are thought to be key regulators of vascular tone.^{9 12} Despite their relevance in vascular physiology, and that aging is the main risk factor for coronary artery disease,¹ changes in MaxiK channel expression during aging had not been previously addressed. In this work, we demonstrate for the first time that aging induces a reduction in MaxiK channel expression in coronary myocytes. Because this class of channels regulates both [Ca²⁺]_i and membrane potential, their reduction in aged subjects is likely to produce a deleterious physiological impact, leading to vascular disease. In addition, MaxiK channels are important therapeutic targets that mediate, at least in part, the vascular effects of nitric oxide and nitroglycerin.^{24 30 31} Thus, the age-dependent decrease in density of MaxiK channels has implications in the treatment of cardiovascular disorders predominantly affecting the older population. The finding that MaxiK channels are reduced during aging prompts the question whether other coronary ion channels are affected during aging.

In human coronary vessels, MaxiK channels are mainly formed by the pore-forming subunit and regulatory ß subunits (likely ß₁).^{7 32 33} Recently, lacZ gene expression in a ß₁ gene–targeted mice demonstrated that ß₁ is also expressed in coronary vessels of this animal species.³⁴ ß₁ subunits do not form MaxiK channels themselves but increase their Ca²⁺/V sensitivity and slow down their activation kinetics.^{35 36} The increased Ca²⁺ sensitivity conferred by the ß₁ subunit is thought to allow the MaxiK subunit to open in response to local Ca²⁺ changes and maintain vascular tone.³⁷ In agreement, cerebral arteries of ß₁ knockout mice have higher tone and lose their ability to constrict in response to the pore blocker IbTx.³⁴ In this work, we have demonstrated the reduction in the number of functional MaxiK channels (Figures 1 and 2) and a reduction of the MaxiK -subunit protein in both rat and human coronary arteries from old subjects (Figures 3 through 5). At present, we do not know the nature of the MaxiK ß subunit(s) in rat coronary arteries or whether its (their) expression changes with aging. Nevertheless, it is likely that MaxiK channels from rat coronary myocytes are assembled by /ß₁ subunits because they do constrict in the presence of IbTx (Figure 1).³⁴ Moreover, it seems that the ratio of /ß subunits is unaltered in aged coronary arteries because MaxiK current activation kinetics were practically identical in both old and young populations (not shown).

It is interesting to note that aging seems to produce tissue-specific changes in channel expression. As mentioned above, in skeletal muscle, aging (5- to 7- versus 24- to 26-month-old rats) produced an increased MaxiK expression,²⁹ whereas aging produced a reduction of channel density in coronary arteries (Figures 2 through 5; 3 versus 25 to 30 months old). In corporal smooth muscle, MaxiK channels also seem to diminish with age because rat erectile dysfunction of retired breeders (>9 months old) can be restored by MaxiK gene transfection to normal levels.³⁸ Moreover, the age-dependent change is not uniform among different vascular beds, because in pulmonary artery MaxiK expression is not altered significantly when old versus young vessels were compared (Figure 3). The mechanism for this tissue-specific aging-related change in channel density is unknown. It would be interesting to determine whether sexual hormones that diminish during aging³⁹ trigger these changes.

The number of functional channels present in single coronary myocytes from young rats (3 months old) was estimated at 1000 channels/cell. Considering a rather even distribution of MaxiK channels, as observed in immunochemical experiments (Figure 4A), the channel density in rat coronary myocytes is 0.5 channels/µm². This channel density is smaller than that calculated for human coronary arteries (4 channels/µm²),⁷ toad stomach (1 channel/µm²),⁴⁰ and for rabbit jejunum (2 channels/µm²).⁴¹ The variation in channel density across species and smooth muscles may reflect differences in the relative contribution of MaxiK channels in the regulation of muscle tone. In this respect, it is noteworthy that humans possess 8 times more MaxiK channels than rats. However, other factors that alter MaxiK channel activity, such as resting membrane potential, intracellular pathways, and Ca²⁺ may also contribute to the regulatory role of these channels in different species and vascular beds.

The decrease in channel density of young versus old coronary arteries, as measured in single cells with electrophysiological methods (Figure 2), was corroborated with immunochemical experiments (Figures 3 through 5). The use of intact coronary vessels eliminated the possibility that variations in the enzyme treatments used to isolate old and young coronary cells were differentially affecting MaxiK density.

In summary, our results, using several independent measurements, demonstrate that MaxiK channels are diminished in aging coronary arteries in rats and in humans. We propose that a diminution in the numbers of MaxiK channels leads to a decrease in the normal tonic hyperpolarizing force provided by the activity of these channels in coronary arteries and, thus, may contribute to the increased risk of coronary spasm in older people. In general, modifications in expression of ion channels that regulate cell excitability most likely contribute to a disrupted (impaired) cell (organ) function (viability) in older people. Our results also reveal MaxiK channels as important therapeutic targets to alleviate vascular disease.

	Acknowledgments

 
This work was supported by National Institutes of Health Grants HL47382 (to L.T.) and GM52203 (E.S.). L.T. is an Established Investigator of the American Heart Association.

	Footnotes

 
Original received July 25, 2000; revision received December 22, 2000; accepted December 22, 2000.

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