Potassium\'s cardiovascular protective mechanisms

Potassium's cardiovascular protective mechanisms D. B. Young, H. Lin and R. D. McCabe

Am J Physiol Regul Integr Comp Physiol 268:R825-R837, 1995. ; You might find this additional info useful... This article has been cited by 7 other HighWire-hosted articles: http://ajpregu.physiology.org/content/268/4/R825#cited-by Updated information and services including high resolution figures, can be found at: http://ajpregu.physiology.org/content/268/4/R825.full

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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology publishes original investigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 1995 the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at http://www.the-aps.org/.

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Potassium’s

cardiovascular

protective

DAVID B. YOUNG, HUABAO LIN, AND RICHARD Department of Physiology and Biophysics, University Medical Center, Jackson, Mississippi 39216-4505

mechanisms D. MCCABE of Mississippi

atherosclerosis; free radical ; glomerular filtration kidney; thrombosis; vascular smooth muscle

ARTERY DISEASE, stroke, and other forms of cardiovascular disease are the leading causes of death in the populations of the industrialized nations, accounting for approximately one-half of all mortality. Arterial hypertension is a contributing factor to many form .s of cardiovascular d.iseaseand is present at some time in the lives of approximately one-third of the members of populations of industrialized societies. Through myocardial ischemia, heart failure, and cerebral infarction, cardiovascular diseases cripple, weaken, and otherwise rob the quality from the lives of tens of millions, sometimes for decades before the final morbid aspect of the disease We are all aware of these characteristics of the plague that kills and cripples so many, and we accept them CORONARY

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without the hue and cry that has arisen in response to diseasesthat affect only a small fraction of the numbers taken by those that afflict the cardiovascular system. Throughout modern history death and crippling from cardiovascular diseases have been recognized and accepted as nearly normal, common elements of the human condition. A belief that humans are genetically destined to suffer from the curse is implicit in the currently popular assumption that alteration of the genetic code through molecular biological approaches will be effective in preventing and treating cardiovascular disease. However, before modern history, before the creation of industrialized Western societies, the human genetic code provided for the development of a cardiovascular the American

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Young, David B., Huabao Lin, and Richard D. McCabe. Potassium’s cardiovascular protective mechanisms. Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R825-R837, 1995.-High rates of potassium intake are associated with protection from cardiovascular diseases in populations consuming primitive diets and in vegetarians living in industrialized cultures. In studies in humans and in animals, a strong inverse association between potassium intake and hypertension and stroke has been described. However, acceptance of the putative protective effect has been limited by inadequate understanding of 1) long-term potassium regulation, and 2) mechanisms by which small changes in plasma potassium concentration may affect development of cardiovascular diseases. In this review, we present results from analyses of long-term potassium regulation that indicated 1) changes in potassium intake may result in potassium concentrations from 3.1 to 4.6 mmol/l, and 2) when the initial rate is below normal, potassium concentration is very sensitive to changes in potassium intake rate. In addition, we present results that provide bases for possible mechanisms by which potassium may protect against cardiovascular diseases: 1) increases in potassium inhibit free radical formation from vascular endothelial cells and macrophages; 2) elevation of potassium inhibits proliferation of vascular smooth muscle cells; 3) platelet aggregation and arterial thrombosis are inhibited by elevation of potassium; and 4) renal vascular resistance is reduced and glomerular filtration rate is increased by elevation of plasma potassium. We propose that elevation of dietary potassium intake increases plasma potassium concentration, thereby inhibiting free radical formation, smooth muscle proliferation, and thrombus formation. As a result, the rate of atherosclerotic lesion formation and thrombosis will be diminished. In addition, we propose the increase in glomerular filtration rate will cause a shift in the relationship between arterial pressure and sodium excretion that will lead to a reduction in arterial blood pressure. By these actions, high levels of dietary intake of potassium could provide the observed protection against the cardiovascular diseases that have plagued humankind since we began eating a modern high-sodium, low-potassium diet.

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3) In experimental animals, potassium’s protective effect appears to be dramatic. Nearly 40 years ago, Meneely and co-workers (51) found that potassium supplementation to the diet of rats fed a very high sodium intake dramatically increased the longevity of the animals in which the cause of death was hypertensive renal failure. Dahl and co-workers (13) reported similar findings in Dahl salt-sensitive rats. More recently Tobian and co-workers and others have found that high dietary potassium intake protects against stroke (68, 69), hypertension (1, 13, 43, 50, 66, 68, 81), cardiac hypertrophy (69), renal glomerular lesions, and medial hypertrophy of the arterioles and kidneys (70) and that it reduces blood cholesterol and deposition of cholesterol in the aorta (68). 4) In humans from industrialized cultures, high rates of potassium intake have been reported to be inversely related to blood pressure (28, 29, 31, 32, 35, 39, 59, 60, 76, 82) and to the incidence of stroke (30) and other cardiovascular diseases. The protective effect of potassium does not appear to require exceptionally high levels of intake. For example, an increase in dietary intake from approximately 60 to 80 mmol/day was shown to be inversely and significantly related to the incidence of stroke mortality in adult women (30). An inverse effect on blood pressure has been reported with elevation of potassium intake from 23 to 54 (21), 38 to 71 (59), 61 to 81 (2), and 61 to 167 mmol/day (24). Potassium supplements to the diet ranging from 24 to 104 mmol/day have been reported to reduce blood pressure in hypertensive patients (4,34, 38,45,55,61,62, 75). Despite the evidence supporting an important cardiovascular protective effect of high rates of potassium intake, acceptance of the protective hypothesis remains tentative. Certainly, one significant impediment to acceptance is the consensus that plasma potassium concentration changes only slightly in response to large changes in potassium intake. Regulation of potassium concentration is complex in that it can be affected by several interacting variables, all of which may produce nonlinear responses. Therefore, we believed that potassium’s potential role in protection against cardiovascular disease could be evaluated more accurately if we first developed an understanding of the control of potassium excretion and concentration in the extracellular fluid. POTASSIUM

REGULATION

Under some conditions in which data have been collected in human studies, plasma potassium concentration changes only slightly in response to large changes in intake. For example, raising intake from 80 to 200 mmol/day for 8 wk raised fasting potassium from 4.2 to 4.4 mmol/l (55). However, under other conditions, plasma potassium concentration is more sensitive to changes in intake; lowering intake from 96 to 16 mmol/ day for 10 days has been reported to lower the plasma level from 4.2 to 3.4 mmol/l (34), and in another study, elevating intake from 0 to 300 mmol/day increased plasma potassium concentration by 0.8 mmol/l over a 5-day period (4). The change in potassium concentration associated with a change in the rate of intake is also a

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system that was successful for the conditions encountered by premodern humans. Today, several dozen isolated groups continue to live under conditions similar to those encountered by our prehistoric ancestors. All of these groups consume diets composed of unrefined foods, diets quite different from those of industrialized cultures. The fat content is much lower in primitive diets, the fiber content is much higher, the total caloric intake rate is lower, and the daily intake of potassium is much higher while that of sodium is much lower in the primitive than in the modern diet. The rates of intake for members of primitive cultures range from 20 to 40 mmol/day for sodium and from 150 to 290 mmol/day for potassium (14, 16). In contrast, the daily rates of intake of potassium and sodium for individuals of industrialized cultures eating processed foods are 80-250 mmol/ day for sodium and 30-70 mmol/day for potassium (19, 31, 36, 49). In the modern Western diet the potassiumto-sodium intake ratio on a molar basis is usually < 0.4 (3, 12, 21, 37, 78), whereas in primitive cultures the intake ratio is always > 3 and usually closer to 10. The diets and epidemiology of at least 17 of these primitive cultures from all corners of the globe have been studied in detail. In all of these the potassium-to-sodium intake ratio is high, while in none is the incidence of hypertension > l%, and coronary artery disease, heart failure, and strokes are rare even among the 5-10% of the populations that live into their sixth or seventh decades (8, 10, 15, 25-27, 44, 52-54, 56, 67, 80). These groups, which include considerable genetic diversity, have nearly complete protection from cardiovascular diseases. From these well-known but often ignored observations two conclusions can be drawn with confidence: 1) human beings are not genetically destined to suffer high rates of cardiovascular disease, and 2) factors or conditions associated with life in industrialized cultures greatly increase the incidence of cardiovascular diseases. Any one or all of the striking differences between modern and primitive diets may be important factors contributing to the increased incidence of the cardiovascular diseases. Evidence supporting a role for high potassium intake providing protection against hypertension and cardiovascular disease has been reported persistently during the last 90 years from a wide variety of disciplines including anthropology, epidemiology, clinical intervention trials, population-based research, and animal experimentation. The most persuasive and intriguing aspects of the evidence are the following. 1) As mentioned previously, < 1% of the primitive populations who consume a high-potassium, lowsodium intake develop hypertension, while nearly onethird of the population of industrialized cultures whose diet is low in potassium and high in sodium develop hypertension. 2) Analyses of subgroups of populations from diverse origins demonstrate a consistent increase in blood pressure when a subgroup relocates from its initial condition in which the diet is high in potassium and low in sodium to a new condition in which dietary sodium is high and potassium is low (11,58, 72, 77).

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Fig. 1. Simulated regulation of plasma potassium concentration over combined ranges of potassium and sodium intake under normal conditions is presented here in 3 dimensions. Intake units are normalized; for an adult eating a Western diet, potassium and sodium intakes are considered to be - 1 mmol kg-l. day-l. Gridlines on surface are in increments of 0.25 normalized units. Intake ranges simulated were from 0.25 to 4.0 times normal. [From Young (84).]

normal rates of potassium and sodium intake (1.0 on both the potassium and sodium intake axes) to the point of 4.0 (280 mm0 l/day) normal pota ssium intake and 1.0 normal sodium intake result 8 in a change in plasma potassium concentration from 4.20 to 4.60 mmol/l (a 10% increase in concentration over a 210 mmol/day increase in intake, or a 0.019 mmol/l increase in concentration for each 10 mmol/day increase in intake). Moving along the sodium intake axis from the point of normal sodium and potassium intake to the point of normal potassium intake and 4.0 normal sodium intake results in a decrease in plasma potassium concentration from 4.20 to 4.09 mmol/l. The model simulations suggest, therefore, that potassium concentration is closely regulated when potassium intake is normal or greater than normal. A corollary to this is that changes in intake (i.e., potassium supplements) will be ineffective in raising plasma potassium concentration if intake is initially normal or greater. In contrast to the precise control in the region of the surface above the normal potassium intake rate, control below the normal intake rate is predicted to be poor. Again starting from the point of normal (1.0) potassium and sodium intakes and moving to the point of 0.25 potassium intake and normal sodium intake results in a fall in plasma potassium concentration from 4.20 to 3.45 mmol/l (an 18% decrease in response to a 53 mmol/day decrease in potassium intake). This impaired control effectiveness results mainly from the decrease in sensitivity of the renal response to changes in plasma potassium concentration below the normal level. Therefore, the prediction from the model suggests that plasma potassium concentration will be sensitive to relatively small changes in intake if the initial intake rate is below normal. In the below normal range, potassium supplementation of the diet will increase plasma concentration sign ificantly ; the quantita tive estimate from the model is 0. 14 mmol/ ‘1 for each 10 mmol/day increase in i ntake,

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function of the rate of sodium intake. For example, changing the molar intake ratio of potassium to sodium from 0.343 to 0.245 was reported to be associated with a decrease in concentration of potassium from 4.26 to 3.85 mmol/l in a group of Japanese farmers (74). Clearly, the magnitude of change in plasma potassium concentration that may result from a given change in potassium intake is hard to predict. For a number of years we analyzed long-term potassium regulation and from those studies devel .oped a systems analysis-based math .ematical mode 1 that has been useful in understanding control of potassium over long time periods (83-85). Th .e model represents a complex set of hypotheses based on observations that included variability and scatter. Therefore, initially the validity of the hypotheses represented as the mathematical model was open to question, as are all hypotheses. Testing of the hypotheses has continued from the time of the model’s development by comparing the responses of the model to simulated perturbations with the responses to the same perturbations observed in experiments designed specifically for testing, and with the published responses of animals and humans to simil .ar perturbations. Agreement between model-si .mu .lated ou .tcomes with experimental data supports the validity of the hypotheses, while disagreement is evidence that the hypotheses are invalid or incomplete. Before its initial publication, the model was tested extensively for several-years, and in the published form no instance of significant disagreement has been found between model simulation and experimental observation. With each case of agreement confidence in the validity of the hypotheses has increased. Currently, we feel justified in presenting model predictions (hypothetical outcomes) concerning responses of the potassium control system to a variety of challenges that might be encountered during physiological and pathophysiological situations. For simulation purposes, basal potassium and sodium intakes in the model are both considered to be 1 mmol kg-l day-l for adults living in Western cultures. The maximum and minimum intake ranges for both sodium and potassium are considered to be 4 times normal and 0.25 times normal. The predicted plasma potassium concentrations over those ranges of intake are presented as a three-dimensional surface in Fig. 1. With basal intake rates of sodium and potassium, predicted plasma potassium concentration is 4.20 mmol/l. The plasma potassium concentrations expected at 4 times normal potassium intake combined with 0.25 normal sodium intake (280 mm01 I day potassium, 18 mmol/day sodiu m) would be ~4.6 mmol/l. The minimum value of potassium expected at 0.25 times normal potassium intake and 4 times normal sodium intake (18 mmol/day potassium, 280 mmol/day sodium) is 3.1 mmol/l. Several features of the three-dimensional surface are noteworthy. First, the surface is nearly flat above the normal level of potassium intake (1.0 on the potassium intake axis), which indicates that regulation of plasma potassium concentration is excellent above the normal level of potassium intake. Going from the point of

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PROSPECTIVE

PROTECTIVE

MECHANISMS

A second factor impeding acceptance of the proposed protective effect of potassium is the absence of potential mechanisms. On the basis of the analysis of potassium regulation and the previously reported observations, we predict that any protective mechanism dependent on changes in plasma potassium concentration related to changes in dietary potassium and sodium intake must be sensitive to changes in concentration within a 1.5 mmol/l range between approximately 3.1 and 4.6 mmol/l. Until recently, no mechanisms meeting this qualification that might protect against atherosclerosis and hypertension had been proposed. -We have initiated a- series of analyses designed to recognize potential cardiovascular protective mechanisms that could be mediated by small increases in plasma potassium concentration. To begin with we have made some assumptions. First, we propose that the cardiovascular diseases against which potassium appears to provide protection are those resulting from

atherosclerosis and its sequelae, and in the case of hypertension, resulting from changes in regulation of renal vascular resistance and other aspects of renal function. Second, we have assumed that changes in dietary potassium intake result in changes in plasma potassium concentration that affect the function of the cells involved in the complex processes of atherosclerotic lesion formation, and in regulation of vascular smooth muscle contractile function and growth. Mechanisms Affecting Development of Atherosclerotic Lesions Steinberg et al. (63) have proposed a model to account for the development of the atherosclerotic lesion that incorporates both the lipid infiltration hypothesis and the endothelial injury hypothesis. According to these authors, lesion development may be initiated by oxidation of low-density lipoproteins (LDLs) in the intima of the arteries. Macrophages and monocytes in the intima phagocytize the oxidized LDLs, and if a sufficient quantity of oxidized LDL accumulates in the cells, they are transformed to foam cells. In numbers these form the fatty streak lesion, the first microscopically visible element of the atherosclerotic lesion. Endothelial cell injury, which may be caused by the cytotoxic effect of oxidized LDLs, by products from the macrophage or monocytes in the intima, or by factors from the blood side, can result in changes in endothelial cell function that contribute to the progression of the lesion. Such changes may result in adherence and penetration by monocytes as well as adherence of platelets. Platelet adherence may then result in release of growthpromoting factors and in aggregation of other platelets. Vascular smooth muscle transformation, migration, and proliferation in the subintima may result from trophic factors from platelets, endothelial cells, and other cells in the developing lesion. Raised, advanced lesions may develop in time. The symptoms of atherosclerosis result either from ischemia due to reduction in the lumen area by advanced lesions or from thromboembolic events initiated by advanced lesions. We believed there was a good probability that potassium’s protective effect against development of diseases related to atherosclerosis included effects of small increases in potassium concentration on the function of the cells involved in lesion formation as described by Steinberg and co-workers (63). Changes in potassium concentration affect several aspects of cellular function, including the activity of Na+ -K+-adenosinetriphosphatase (Na+-K+-ATPase) and H+-K+-ATPase (47) located in the cellular membrane of vascular smooth muscle and other vascular tissue cells. Elevation of potassium concentration increases the activity of both enzymes, resulting in increased transport of Na+ and H+ out of the cells. These changes in transport reduce intracellular activities of Na+ and H+, ultimately affecting intracellular calcium activity by augmenting the transmembrane Na+ gradient that drives the Na+-Ca2+ exchanger. We believed that associated with elevation of extracellular potassium concentration, changes in intracellular activities of Na+, H+, and Ca2+ signal alterations in the

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once a new steady state is reached. Attaining a steadystate condition may require 10 or more days. Therefore, when the initial potassium intake rate is below normal, plasma potassium concentration is - 7 times more sensitive to changes in intake than when the intake is initially normal or above. The problems with potassium control in response to less than normal potassium intake are intensified by greater than normal sodium intake. The final procesi determining the rate of potassium excretion is the diffusion of K+ across the apical membrane of the principal cells of the cortical connecting tubule into the lumen of the late distal tubule (20). Elevation of flow rate through this segment with elevation of sodium intake limits the increase in tubular K+ concentration associated with entry of K+ into the tubule. Therefore, the concentration gradient driving movement of K+ from cell to lumen tends to increase with high rates of flow. This interaction is reflected in the broadened area of the concentration surface, which is steeply sloped in the region below normal potassium intake and above two times normal sodium intake. Although the average rates of sodium and potassium intake for adult Americans are - 70 mmol/day for each element, a significant fraction of the population consumes much less potassium and much more sodium than the normal amounts. Sodium intake of greater than three times normal and potassium intake of less than one-third normal are reported for segments of the population in this country; for example, Watson and Langford (37, 78) found that excretion for young black the average daily potassium women in Jackson, Mississippi was 22 mmol/day, and similar values have been reported from population studies in other regions of the southern states (5). These low levels of potassium intake coupled with above normal sodium intake place these individuals in the region of the surface of Fig. 1 that is below the normal level of plasma potassium concentration and where plasma potassium concentration is very sensitive to changes in potassium intake.

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Fig. 2. Line graph shows rate of cytochrome c reduction, which was measured at 15min intervals after 3 groups of endothelial cells were exposed to 3.0 mmol/l potassium concentration. For the remaining measurements, potassium concentration of 2 of the groups was increased to 5.5 or 7.0 mmol/l, as indicated. The higher potassium concentration values had approximately the same effect, restoring the rate of cytochrome c reduction to the initial value. This restoration 1 h to reach the elevated was im .mediate, even though it had taken level after exposure to 3.6 mmol/l potassium concentration. [From McCabe et al. (46).]

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Fig. 3. Bar graph shows rate of cytochrome c reduction as a function of potassium concentration for endothelial cells (CPA 47) and monocyte/macrophages (TIB 186). This rate was measured in the 30- to 75min interval after exposure to different potassium concentrations. *P < 0 05 compared with 3.0 mmol/l potassium concentration. [From McCabe et al. (46).]

rise exponentially; however, the rate of reactive oxygen species formation by either of the other groups returned approximately to the initial value. By the end of 2 h the rate of reactive oxygen species formation by the group continually exposed to 3 mmol/l potassium was 8.4 times the initial rate, but the group that was returned to 5.5 mmol/l at 1 h had a rate of reactive oxygen species formation only 18% higher than the initial rate. Thus a 2.5 mmol/l increase in potassium concentration caused an 86% inhibition of reactive oxygen species formation, 3.4% for every 0.1 mmol/l increment in potassium. Potassium at 7 mmol/l had only a slightly greater effect than at 5.5 mmol/l. The monocyte/macrophage cells (cell line TIB 186) were only moderately adherent, and the cells began to detach after four or five solution changes; therefore, the protocol for these cells was modified so that data were taken between 30 and 75 min after the change in potassium concentration. Figure 3 presents data from both the endothelial cells and the monocyte/macrophage cells. Raising extracellular potassium concentration in 1 mmol/l increments from 3 to 7 mmol/l caused highly significant decreases in the rate of reactive oxygen species formation from both cell lines. Increasing potassium concentration from 3 to 7 mmol/l reduced reactive oxygen species formation by 55% in endothelial cells and by 49% in macrophage/monocytes over the period from 30 to 75 min. The greatest decrement in free radical formation was between 3 and 4 mmol/l. Freshly isolated human white blood cells were also used to test the effect of potassium on reactive oxygen species formation. In this case free radical formation was assessed by reaction of the superoxide anions with luminol. The photon emitted from the reaction of each free radical compound with luminol was counted using a scintillation counter set in the out-of-coincidence mode. Figure 4 presents the response of human polymorphonuclear neutrophils and monocytes to stimulation by

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function of the cells involved in lesion formation in ways that inhibit d.evelopment of atherosclerotic ch anges. Therefore, ou .r recent inves tigations have focused on the effects of changes in extracellular potassium on the cells involved in these processes. Inhibition of free radical formation by increases in potassium concentration. A very early and possibly rate-limiting step in the development of the atherosclerotic lesion is the oxidation of LDLs in the intima of the arteries. The LDLs are oxidized by reactive oxygen species produced by monocytes, macrophages, and possibly by endothelial cells and vascular smooth muscle cells (VSMCs). The oxidatively modified LDLs are recognized by receptors on macrophages and monocytes in the subintima, resulting in their rapid phagocytosis. We assessed the effects of potassium on reactive oxygen species formed by cultured endothelial and monocyte/ macrophage cells by measuring the rate of cytochrome c reduction in the medium (46). Time dependence of the effects of potassium on the rate of free radical formation from cultured endothelial cells (cell line CPA 47) is illustrated in Fig. 2. Three groups of cells were placed in medium with the potassium concentration of 3 mmol/l at time 0. One hour after exposure, the potassium concentration of the replacement solutions used for two of the groups was increased, whereas the potassium concen tration of the rep1 .acement solution of th .e third group was not than .ged. The rate of reactive oxygen species format !ion by all groups rose exponentially for about the first hour. During the second hour the rate of reactive oxygen species formation by the group with continued exposure to 3 mmol/l potassium continued to

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Fig. 4. Bar graph shows inhibitory effect of potassium concentration on reactive oxygen species formation (counts per minute) from freshly isolated human white blood cells (polymorphonuclear neutrophils and monocytes) stimulated by opsonized zymosan. Inhibition was consistent over the entire range of zymosan, although it was greatest at the highest levels of agonist. *P < 0.05 vs. lowest potassium concentration; #P < 0.05 vs. middle potassium concentration. [From McCabe et al. (46).]

cells were grown all night in medium without serum and in a potassium concentration of 4 mmol/l to induce quiescence. On day 0 the cells were exposed to medium with 5% fetal bovine serum and with final potassium concentrations ranging in 1 mmol/l increments from 3 to 7 mmol/l. The cells were harvested on days 1 to 6. Cell proliferation and growth were assessedby cell counts and thymidine incorporation. The effects of increasing potassium concentration are illustrated in Fig. 5, with cell counts in Fig. 5A and thymidine incorporation in Fig. 5B. In media with potassium concentration of 2 or 3 mmol/l, cell numbers increased from the original plating density of 10,000 to 60,000 cells/cm2 on day 7. Increasing potassium concentration from 3 to 4 mmol/l reduced the cell numbers on day 7 by 18%, and a further increase from 3 to 7 mmol/l reduced the count by 61%. The antiproliferative effects of elevation of potassium were confirmed by the thymidine incorporation data shown in Fig. 5B. Here again there is a strongly significant, consistent reduction in thymidine incorporaA

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zymosan over a range from 1 to 10 mg/ml. The cells were suspended in buffer containing three different potassium concentrations ranging from 1.48 to 7.94 mmol/l. The inhibitory effect could be seen at all levels of zymosan stimulation (P < 0.001, analysis of variance). However, the inhibitory effect of potassium was not as great as in the experiments with the cultured cells. These results clearly demonstrate that small elevations in extracellular potassium concentration can strongly inhibit the rate of free radical formation from endothelial cells and white blood cells. If the effect we observed in vitro over several hours is also operative over years in vivo, then the inhibitory effect on free radical formation could account for a significant reduction in lesion formation in individuals consuming a high potassium intake. Given that oxidative modification of LDLs by free radicals appears to be the rate-limiting step in cholesterol deposition, Tobian’s finding of reduced cholesterol content in the aorta of rats given a high potassium intake (68) is consistent with a longterm effect of potassium to reduce free radical formation and therefore cholesterol deposition in the subintima. Inhibition of vascular smooth muscle proliferation in culture by elevation ofpotassium concentration. Progression from the fatty streak lesion to more advanced raised lesions involves transformation and migration of VSMCs from the media to the intima, and subsequently proliferation. We have begun a series of experiments designed to analyze the effects of changes in potassium concentration on these processes. The first experiment was designed to determine if elevation of extracellular potassium concentration reduced the rate of growth of VSMCs in culture (48). We used two types of cells: rat aortic VSMC line A7r5, and canine coronary artery VSMC (passages Z-3). The

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Fig. 5. A: effects of extracellular potassium concentration ([K],) on proliferation of canine coronary artery vascular smooth muscle cells (VSMCs) are highly significant (P < 0.001) by l-way analysis of variance (ANOVA). **P < 0.01 vs. 3 mmol/l and TP < 0.05 vs. 2 mmol/l. B: effects of [K], on [3H]thymidine incorporation by canine coronary artery are highly significant (P < 0.001) in l-way ANOVA. *P < 0.05, **P < 0.01 vs. 3 mmol/l potassium concentration. [Reprinted b y permission of Elsevier Science Inc. from McCabe and Young (48).]

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most common causes of death in industrialized societies are those resulting from coronary or cerebral artery occlusion by thromboembolic events initiated at advanced atherosclerotic lesions. We have begun a series of experiments to determine if elevation of plasma potassium concentration can inhibit arterial thrombosis (40). We used an in vivo model in the dog circumflex artery and in the rabbit carotid artery. The vessels were prepared in the anesthetized animals as described by Folts and co-workers (17, 18, 71). Briefly, the endothehum of the vessel was damaged by external compression of the artery after which a critical stenosis was produced under a Plexiglas occluder. The combination of the high shear rate and endothelial damage under the constrictor induced platelet aggregation and formation of a thrombus at the site of damage. The growth of the thrombus can be monitored by measuring the rate of flow through the vessel with an electromagnetic flow probe. When the vessel is occluded completely, the thrombus can be dislodged by gentle agitation or temporary removal of the constrictor. Immediately after the thrombus is dislodged, the platelets disaggregate and blood flow returns to the initial level. However, a new thrombus begins to form shortly after the previous one is dislodged. If the artery is prepared properly this pattern of cyclical flow reductions (CFRs) continues in a consistent manner for several hours. During this time the effect of interventions on the rate of CFRs can be used to assess the efficacy of their antithrombotic actions. Figure 7 presents polygraph records of circumflex artery blood flow during a 40-min control period and a 40-min period during which potassium chloride was infused intravenously to raise plasma potassium concentration. An increase in the potassium concentration in this manner drastically reduced the rate of CFRs. In a group of 10 dogs potassium infusion increased the concentration from 3.53 t 0.05 to 6.10 t 0.09 mmol/l, which resulted in a reduction in the rate of CFRs from 8 0 + 0 6 per 40-min observation period to 3.7 t 1.0 (b to.61) du ring the 40-min potassium chloride infusion. This dramatic reduction of more than 50% in the rate of thrombus formation was observed in the same model used by Folts and others to establish the antithrombotic efficacy of aspirin and other intervention subsequently shown to be clinically effective. We also analyzed the effects of acute increases of potassium concentration on the prothrombogenic effects of epinephrine. Intravenous infusion of epinephrine at 0.5 pg. kg-lemin-l increased the rate of CFRs more than 60%, from 7.1 t 0.5 to 11.5 t 0.7 in the 40-min period after epinephrine infusion. However, as a result of raising plasma potassium concentration to N 6 mmol/l while epinephrine was continuously administered, the frequency of CFRs was significantly reduced from 11.5 t 0.7 to 7.7 t 1.1 (P < 0.01) in the 40-min period. Elevation of plasma concentration was capable of opposing completely the thrombogenic effect of epinephrine infusion. A similar preparation was used to induce a pattern of cyclical flow reductions in the carotid artery of rabbits. In four of the five rabbits used. increasing potassium l

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Fig. 6. Effects of [K], on intracellular sodium concentration in canine coronary artery VSMC. Effects of time and [K], are highly significant (P < 0.001) and interact significantly (P < 0.01) by 2-way ANOVA. *P < 0.05, **P < 0.01 vs. 3 mmol/l on respective day. [Reprinted by Permission of Elsevier Science Inc. from McCabe and Young: (48M

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tion across the whole range of potassium concentration from 2 to 7 mmolll. One of the earliest events in cell proliferation is a dramatic increase in cellular sodium content. We analyzed the effect of increasing potassium concentration on cell sodium content after stimulation by serum. These results are depicted in Fig. 6. One day’s exposure to serum more than doubled intracellular sodium content, whereas increased potassium concentration reduced sodium content by 64-74% either before or after exposure to serum. Both the newly passaged cells from the coronary arteries of dogs and the rat aortic cell line were proliferative/synthetic cells, the phenotype that participates in the formation of advanced atherosclerotic lesions. In our experiments the cells received strong stimulation from 5% serum, which contains growth factors and cytokines derived from platelets. The strongly antiproliferative effect of small increases in potassium concentration in this system indicates that potassium can exert a direct effect on smooth muscle growth over at least a 7-day period in vitro and that the effect of as small as a 1 mmol/l increase in potassium concentration from 3 to 4 mmol/l has an effect that may be functionally significant. Tobian’s finding that dietary potassium supplementation prevents the renal arteriolar medial hypertrophy in hypertensive Dahl salt-sensitive rats (70) provides some indication that the antiproliferative effect of potassium may be active in vivo over periods of weeks as well as in vitro. Additional studies are required to analyze the potential long-term effectiveness of elevation of potassium concentration in reducing the rate of cellular proliferation in vascular lesions. Elevation of potassium concentration inhibits arterial thrombosis in vivo andplatelet aggregation in vitro. The

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Phasic Blood Flow in Circumflex Artery (mUmin)

Mean Blood Flow in Circumflex Artery (mUmin) 5 min

IS

Phasic Blood Flow in Circumflex Artery (mUmin)

)o *I 0 .. I I--

Mean Blood Flow in Circumflex Artery (mUmin)

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Fig. 7. Blood flow recording from circumflex artery of a dog prepared produce cyclical blood flow reductions (CFRs). A is from control period; infusion. The reduction in rate of CFRs during potassium infusion elevation of potassium concentration. [From Lin and Young (4Oj.l

with endothelial damage and B is from period of intravenous demonstrates the antithrombotic

stenosis to potassium effect of

concentration from approximately 3.5 to 5.5 mmol/l completely inhibited thrombus formation and reduced the rate of cyclical flow reductions to zero. Results from one of these rabbit experiments are illustrated in Fig. 8.

In this case the potassium concentration before potassium chloride infusion was 3.54 mmol/l. In