The atherosclerotic plaque.

Atherosclerosis is the most frequent cause of ischemic heart disease and cerebrovascular disorders. The condition is the leading cause of death in Western societies. At the core of this condition is the atherosclerotic plaque. It is within the structure of this lesion that multiple biochemical and cellular processes interact influencing its vulnerability to rupture and as a result acute ischemic events. This article will discuss the pathophysiology behind the atherosclerotic plaque, particularly those elements that lead to its instability and the medical tools currently available to counteract it.

Coronary artery abnormalities in Puerto Rico

A retrospective study was done to determine the frequency of coronary artery anomalies in terms of their origin, course, and structure. The clinical history, catheterization data and surgical reports of patients undergoing coronary angiography at the Cardiovascular Center of Puerto Rico and the Caribbean, from 1999 to 2004, were analyzed. Thirty-eight patients were identified with a coronary artery anomaly in this population. These anomalies were classified according to their clinical consequences and the need for surgical intervention.

Angiotensin II-dependent vascular alterations in young cardiomyopathic hamsters: role for oxidative stress.

Recent studies indicate the presence of vascular alterations in 2-month-old Syrian cardiomyopathic hamsters (SCH). These alterations include enhanced angiotensin-converting enzyme (ACE) activity in the aorta, increased contractile response to angiotensin II and impaired vasorelaxation to acetylcholine in norepinephrine-precontracted aortic rings. The mechanisms leading to these vascular alterations are not known nor has their relationship to the cardiac abnormalities been established. We assessed the status of the cardiovascular system of 2-month-old hamsters first to establish if the observed vascular alterations are secondary to cardiac dysfunction, and second to examine the role of oxidative stress in the etiology of vascular dysfunction. Cardiac function parameters evaluated by echocardiography included stroke volume (SV), left ventricular end-diastolic volume (LVEDV), left ventricular fractional shortening (LVFS), ejection fraction (EF), cardiac output index (COI), heart rate (HR) and left ventricular posterior wall thickness (LVPWT). In addition, heart/body weight (heart/BW) ratios and systolic blood pressure were determined in normal hamsters and SCH. Our results indicated that systolic blood pressure increased 56% in SCH when compared to control animals (P<0.05). The increased blood pressure coexisted with normal COI, SV, LVEDV, LVPWT, LVFS, EF, HR and heart/BW ratios. NAD(P)H oxidase activity increased 77% in SCH compared to control animals (P<0.02). The increased oxidase activity was abolished by pre-treatment of animals with the angiotensin II type 1 receptor blocker losartan (25 mg/kg BW/day) for 10 days. Losartan also abolished the increased blood pressure observed at 2 months of age. The antioxidant N-acetylcysteine (NAC) abrogated the increased blood pressure when administered for 30 days to 1-month-old animals. Altogether, these findings suggest that the angiotensin II-dependent vascular abnormalities present in young cardiomyopathic hamsters are associated with oxidative stress and precede the echocardiographic abnormalities characteristic of heart failure.

[Total plasma homocysteine concentrations in Puerto Rican patients with ischemic heart disease]. / Concentraciones totales de homocisteína plasmática en pacientes puertorriqueños con cardiopatía isquémica.

INTRODUCTION AND OBJECTIVES: In Puerto Rico, it has been established that although coronary disease is the leading cause of death, the population has a lower incidence of coronary disease than in the continental United States. In addition, the severity of the disease is less aggressive in terms of a lower incidence of ventricular tachycardia and sudden death. One factor that could contribute to the lower incidence of coronary disease in Puerto Rico is that our population might have lower total plasma homocysteine concentrations (tHcys) than in the continental United States. Our main objective was to measure tHCys in the Puerto Rican population with atherosclerotic cardiovascular disease (ACD). METHODS: We randomly measured tHcys concentrations in seventy Puerto Rican patients who were hospitalized at the Cardiovascular Center of Puerto Rico and the Caribbean (UPR Division). RESULTS: The mean tHCys concentration in these patients is similar to those reported for the Framingham study when adjusted by age (11.2 vs. 11.8 micromol/l). In the Puerto Rican population, males had a higher tHcys concentration than females (11.7 vs 9.5 micromol/l, p = 0.07). In addition, we did not see an increase of tHcys concentrations in diabetic patients when compared with non-diabetics (10.1 vs. 11.2 micromol/l, p = 0.74). We did not see a direct correlation between tHcys concentrations and heart condition as measured by coronary angiography (normal = 11.1 micromol/l, light = 10.5 micromol/l, moderate = 10.9 micromol/l, severe = 10.5 micromol/l; Kruskal-Wallis = 0.45) either. CONCLUSION: These results suggest that tHcys concentration is not a good predictor of the seriousness of ACD in the Puerto Rican patient population.

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells.

The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B0,+ and y+) were evaluated in cells treated with IL-1beta or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y+ and B0,+ respectively, were examined. L-arginine transport was estimated with 3H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1beta depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]i < 10 microM) was 41.1 +/- 18 microM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y+ and B0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1beta or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools.

Total plasma homocysteine concentrations in Puerto Rican patients with presumptive atherosclerotic coronary disease.

BACKGROUND: In Puerto Rico, it has been established that although coronary heart disease is the leading cause of death, the population has a lower incidence of coronary disease than the continental United States. In addition, the severity of the disease is less aggressive in terms of a lower incidence of ventricular tachycardia and sudden death. A factor in the lower incidence of coronary disease in Puerto Rico could be a lower total plasma homocysteine concentration (tHcys) in our population. METHODS: We randomly measured tHcys concentrations in seventy-two Hispanic patients who were hospitalized for coronary angiography at the Cardiovascular Center of Puerto Rico and the Caribbean (UPR Division). RESULTS: The mean tHCys concentration in our patient population is similar than that reported for the Framingham study when adjusted by age (11.2 mumol/L vs. 11.8 mumol/L). In the Puerto Rican population, males had a higher tHcys concentration than females but this difference was not statistically significant (10.9 mumol/L vs. 9.4 mumol/L, p = 0.09). In addition, we did not see an increase of tHcys concentrations in diabetic patients when compared with nondiabetics (10.1 mumol/L vs. 10.3 mumol/L, p = 0.73). Neither we saw a direct correlation between tHcys concentrations and atherosclerosis as measured by coronary angiography (normal = 10.9 mumol/L, mild = 8.6 mumol/L, moderate = 10.9 mumol/L, severe = 10.5 mumol/L; ANOVA = 0.29). CONCLUSIONS: These results suggest that tHcys concentration is not a good predictor of atherosclerotic coronary disease in our patient population.

Altered vascular function in early stages of heart failure in hamsters.

BACKGROUND: Congestive heart failure is a clinical condition associated with alterations in the normal balance of neurohumoral agents and factors acting on the vascular wall. The etiology of this condition, however, remains largely undefined. To help elucidate the pathophysiology of this disease, vascular function and angiotensin-converting enzyme activity were evaluated in 2-month-old Syrian cardiomyopathic hamsters (SCHs) that had not yet developed heart failure. Age-matched normal hamsters were used as control hamsters. METHODS AND RESULTS: Vascular function studies included determinations of contractile responses of aortic rings to 0.1 microM angiotensin II and 0.1 microM norepinephrine. In addition, endothelial function was evaluated by the vasorelaxant action of acetylcholine on norepinephrine-precontracted aortic rings. The results indicate that the pressor effect of angiotensin II (0.1 microM) was 35% greater in aortic rings from SCRs than that observed in control animals. This effect is specific for angiotensin II because the contraction induced by NE (0.1 microM) was similar in both of these strains. Angiotensin-converting enzyme activity was three-fold higher in aorta homogenates from SCHs but normal in plasma and heart tissue when compared with control hamsters. Aortic ring preparations from SCHs also exhibited endothelial dysfunction because the maximal relaxation elicited by 10 microM acetylcholine was reduced 53%. Concentration-response curves with acetylcholine yielded EC50 values that were threefold lower in SCHs (97.2 +/- 0.1 nM) than in control animals (286 +/- 7 nM). Indomethacin (1 microM) increased the vasorelaxant effect of acetylcholine 28% in SCHs and shifted to the left the concentration-response curve of this agonist, suggesting an increased relaxation with the cyclooxygenase inhibitor. No effect of indomethacin on acetylcholine-induced relaxation was observed in control animals. Sodium nitroprusside induced similar relaxations in both control animals and SCHs, suggesting that the vascular smooth muscle response is normal in SCR. CONCLUSIONS: Altogether these results point to a state of enhanced vascular contractility in young SCHs that could predispose these animals to develop heart failure, the enhanced vascular contractility could result from increased activity of the local renin-angiotensin system, augmented vascular response to angiotensin II, reduced nitric oxide synthesis, and enhanced production of prostaglandins.

Endothelial dysfunction in the San Juan hypertensive rat: possible role of the nitric oxide synthase.

Endothelial dysfunction is recognized as a critical event in the etiology of cardiovascular diseases, but its possible role during aging in arterial hypertension remains poorly defined. We evaluated the response of aortic rings precontracted with 0.1 microM norepinephrine (NE) to acetylcholine (ACh) in the San Juan hypertensive rats (SJH-Rs) (F19, F20) and Munich Wistar rats (MW). SJH-Rs is a model of inbred salt-sensitive hypertension, whereas similarly treated inbred MW rats are their normotensive counterpart. These experiments were performed with adult (6-7 months) and aged (11-13 months) rats to assess the effects of age and hypertension on endothelium-dependent relaxation. We generated dose-response curves by adding cumulative doses of ACh from 1.0 nM to 10.0 microM. In addition, we evaluated the Ca(2+)-dependent nitric oxide synthase (NOS) activity by increasing cell calcium with the ionophore A23187. The results indicate that hypertension induces a displacement to the right of the dose-response curve to ACh in both adults and aged SJH-Rs; IC50 for adult rats was 0.72 +/- 0.3 microM for SJH-Rs and 0.059 +/- 0.03 microM for MW (p < 0.05). Aged animals showed similar results: IC50 of 0.78 +/- 0.03 microM for SJH-Rs and of 0.043 +/- 0.01 microM in age-matched MW rats (p < 0.025). However, no difference was observed between hypertensive (SJH-Rs) adult and aged animals. Similarly, no difference was observed between adult and aged MW control animals. The displacement of the dose-response curve to ACh in SJH-Rs appears to be associated with a reduced activation of NOS since in precontracted aortas from aged animals 1 microM A23187 induced a relaxation of 51.2 +/- 12% in MW as compared with 34.4 +/- 7% in SJH-Rs (n = 5, p < 0.05). These results indicate that endothelial dysfunction exists in SJH-Rs. The data suggest that an alteration of the endothelial NOS may be the cause of this abnormality. Finally, the magnitude of the endothelial dysfunction is not age dependent within the range evaluated.

Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule.

We studied the Na+/K+ pump, Na+/K+ ATPase activity, and oxygen consumption (QO2) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na+/K+ pump activity was determined using 86Rb+ (a functional analog of K+) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na+/K+ pump and Na+/K+ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO2 was negligible in both of these cell preparations. However, OSQO2 was significantly lower in CH than in PH when the Na+/K+ pump was activated using the ionophore nystatin in a Na(+)-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.

Simvastatin releases Ca2+ from a thapsigargin-sensitive pool and inhibits InsP3-dependent Ca2+ mobilization in vascular smooth muscle cells.

Simvastatin (SV), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity inhibits migration and proliferation of vascular smooth muscle cells (SMC). To investigate whether these effects of SV are related to inhibition of cell calcium mobilization, cultured SMC obtained from rat aorta were loaded with Fura-2 to determine the basal cytosolic free calcium levels ([Ca2+]i) and the agonist-stimulated Ca2+ mobilization. SV (20 mu M) transiently increased cytosolic free calcium, an effect that depends mainly on intracellular calcium release (68%). This effect of SV was markedly reduced (75%) by thapsigargin, an inhibitor of the Ca2+ ATPase of inositol 1,4,5-triphosphate (InsP3)-sensitive calcium pools. Incubation of cells with SV (15 min) inhibited the mobilization of Ca2+ by angiotensin II, platelet-derived growth factor, and vasopressin (IC50 = 5 mu M). SV did not affect inositol trisphosphate (InsP3) levels or modify its generation by angiotensin II (Ang II) and vasopressin. Furthermore, in saponin-permeabilized cells, SV abolished the release of calcium by 2,3-dideoxy-InsP3. SV reduced the effect of thapsigargin on InsP3-sensitive stores by 67%, suggesting that SV depletes these calcium pools. The inhibitory effect of SV on calcium mobilization was prevented by coincubation of cultured cells (24 h) with 1 mM mevalonic acid, the product of HMG-CoA reductase activity. These results support the notion that SV inhibits [corrected] the migration and proliferation of SMC by directly affecting cell Ca2+.

Inhibition of smooth muscle cell calcium mobilization and aortic ring contraction by lactone vastatins.

OBJECTIVE: To determine the effects of vastatins on the contraction of rat aortic rings and to assess their effects on calcium mobilization using cultured smooth muscle cells from rat aorta. METHODS: Aortic rings from Sprague-Dawley rats were mounted on stainless steel wires to determine the generation of tension using force-displacement transducers. The tension (g) developed by angiotensin II (100 nmol/l) was measured under basal conditions and after 45 min incubation with 20 micromol/l simvastatin. The effect of 20 mol/l simvastatin, lovastatin, mevastatin and pravastatin on noradrenaline concentration-response curves and the angiotensin II-induced calcium mobilization was also evaluated. RESULTS: Addition of angiotensin II to aortic rings incubated in Krebs' Ringer bicarbonate medium produced tension generation (0.9 +/- 0.12 g = 100%). Treatment of aortic rings with simvastatin inhibited the angiotensin II-induced contraction 58 +/- 0.06%. To evaluate this effect further, dose-response curves with noradrenaline were measured in the presence and absence of 20 micromol/l simvastatin, lovastatin, mevastatin and pravastatin. The results indicate that simvastatin, lovastatin and mevastatin inhibited the contraction induced by noradrenaline (10 micromol/l) by about 50%. Pravastatin did not inhibit aortic ring contraction. Furthermore, the concentration required for 50% of the maximal contraction (EC50) by noradrenaline (6.2 +/- 0.1 nmol/l) was significantly increased by simvastatin, lovastatin, mevastatin and pravastatin. The inhibition of vascular contraction by vastatins appears to involve inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity because the inhibitory effect of simvastatin was reduced 50% by 10 mmol/l mevalonic acid. To determine whether the depression of vascular contraction by these agents was correlated with cell calcium changes, the angiotensin II-induced calcium mobilization was determined in Fura-2 loaded cells, before and after treatment with these inhibitors. Simvastatin, lovastatin and mevastatin significantly reduced the angiotensin II-induced calcium mobilization. The concentration that induced 50% inhibition was 3.3 micromol/l for simvastatin, 17.4 micromol/l for mevastatin and 21.7 micromol/l for lovastatin. No effect of pravastatin on calcium mobilization was observed. CONCLUSIONS: These findings suggest that lactone vastatins depress vascular contraction by reducing cytosolic calcium release in vascular smooth muscle cells. These agents also appear to exert competitive and non-competitive type antagonisms on noradrenaline action.

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C.

Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na(+)-independent (≈60%) and Na(+)-dependent pathways (≈40%). The Na(+)-independent arginine uptake appears to be mediated by system y(+) because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na(+)-dependent pathway is consistent with system B(0,+) since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li(+) as a Na(+) replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na(+)-dependent arginine transport without affecting systems y(+), A, and L. This effect occurred in a dose-dependent manner (IC50 of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B(0,+) by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1ß induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.

Increased erythrocyte calcium pump activity in a kindred with familial benign hypercalcaemia.

The hypothesis that a global defect in cellular calcium transport may be critical in the development of familial benign hypercalcaemia (FBH) was investigated. Nine hypercalcaemic patients from a kindred with FBH and nine normal subjects were evaluated. Our results indicate that calcium pump activity in the FBH kindred was significantly higher (28%, P < 0.005) when compared to normal subjects. These findings suggest that alterations in calcium pump activity in target tissues may play a role in the development of FBH.

[Enalapril reduces the degree of left ventricular remodeling after acute myocardial infarction and reduces the incidence of arrhythmia in ischemic disease]. / Enalapril reduce el grado de remodelamiento del ventrículo isquierdo después de un infarto agudo de miocardio y reduce la incidencia de arritmias en la enfermedad isquémica.

The present study shows that enalapril prevents the excessive remodeling of the left ventricle after acute myocardial infarction. This randomized and double blind clinical study analysed 50 patients with an inferior myocardial infarction. The effect of enalapril was evaluated through cardiac volumes, ejection fraction, neurohormonal levels and incidence of the left ventricle disfunction after acute myocardial infarction. The patients treated with enalapril showed a significant reduction on the values of nor-epinefrine, angiotensine II, natriuretic hormone and vasopressine, four weeks after initiation of treatment. The ejection fraction and the level of the wall movement was more favourable, four weeks after infarction, in the group treated with enalapril. The incidence of congestive heart failure and arrhythmias was lower in the group treated with enalapril. So, we conclude that enalapril is a drug that prevents the excessive remodelling of the left ventricle after an acute myocardial infarction.

Brevetoxin-3 (PbTx-3) inhibits oxygen consumption and increases Na+ content in mouse liver slices through a tetrodotoxin-sensitive pathway.

To have insights into possible non-neural effects of PbTx-3 (0.07 microgram/ml), its effects on various parameters of hepatic metabolism were evaluated. PbTx-3 inhibits oxygen consumption (QO2) in liver slices by 25 +/- 1%, and increases hepatocyte Na+/K+ ratio by 72 +/- 4%. Ouabain, a Na(+)-K(+)-pump inhibitor, also reduced QO2 by 19 +/- 2% and raised the Na+/K+ ratio by 77 +/- 4%. The effects of PbTx-3 and ouabain on QO2 and Na+/K+ ratio were not additive, suggesting a role for the Na(+)-K(+)-pump in these actions of PbTx-3. However, Na(+)-K(+)-pump activity determinations, using 86Rb as a K+ tracer, did not reveal inhibitory effects of this toxin on the transport system. This indicates that the pump is not a target of PbTx-3. The effect of PbTx-3 on liver slices Na+ content was abolished by the sodium channel blocker tetrodotoxin (0.1 microM). Tetrodotoxin also antagonized the inhibition of oxygen consumption by PbTx-3. These results suggest that in the liver PbTx-3 can induce effects that appear to be similar to those observed in excitable tissue.

Upregulation of Na(+)-dependent alanine transport in vascular endothelial cells by serum: role of intracellular Ca2+.

OBJECTIVE: Amino acid transport and its regulation in vascular endothelial cells remains a largely unexplored area. In this study, we evaluated alanine transport in bovine aortic endothelial cells to assess possible mechanisms of regulation. METHODS: Alanine transport into confluent monolayers of endothelial cells was measured using 100 microM [3H]alanine in the presence and absence of external Na+, in cells deprived of serum for 24 hr (SD), and in SD cells exposed to 10% serum (S) for 3 hr (SD + S cells). RESULTS: Our results indicate that although SD did not significantly affect the Na(+)-independent transport of alanine when compared to normal cells, serum addition to serum-deprived cells markedly stimulated the Na(+)-dependent uptake of this amino acid through system A. The stimulation of alanine transport pathway(s) by serum was totally abolished by pretreatment of endothelial cells with 10 microM cycloheximide, suggesting a role of protein synthesis. Serum also induced a marked increase in calcium recycling at the cell membrane, suggesting that calcium is a key element of the serum signaling pathway. Indeed, both BAPTA (20 microM), a cellular calcium chelator, and thapsigargin (1 microM), an agent that depletes intracellular calcium stores, prevented the stimulation of alanine uptake by serum. Finally, pertussis toxin (400 ng/ml), an agent known to inactivate certain G-protein-dependent pathways, significantly reduced the serum-dependent 45Ca uptake and [3H]alanine entry. However, the protein kinase C activator PMA (100 nM), significantly reduced the stimulation of alanine uptake by serum but did not affect the stimulation of calcium uptake. CONCLUSIONS: Altogether these findings suggest that cell calcium is involved in the regulation of system A by serum in vascular endothelial cells.

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: two distinct transport systems.

Rabbit erythrocytes are well known for possessing highly active Na+/Na- and Na+/H+ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na+ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethymaleimide (NEM) were used. Na+/Na+ exchange activity, determined as the Na+o-dependent 22Na efflux or Na+i-induced 22Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H+i-induced Na+ entry sensitive to amiloride (Na+/H+ exchange activity). Similarly, EIPA, a strong inhibitor of the Na+/H+ exchanger, did not inhibit Na+/Na- countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na+/Na+ exchanger could be involved in Na+/H+ countertransport was suggested by studies in which the net Na+ transport sensitive to NEM was determined. As expected, net Na+ transport through this transport system was zero at different [Na+]i/[Na+]o ratios when intracellular pH was 7.2. However, at pHi = 6.1, net Na+ influx occurred when [Na+]i was lower than 39 mM. Valinomycin, which at low [K+]o clamps the membrane potential close to the K+ equilibrium potential, did not affect the net NEM-sensitive Na+ entry but markedly stimulated the EIPA- and NEM-resistant Na+ uptake. This suggest that the net Na+ entry through the NEM-sensitive pathway at low pHi, is mediated by electroneutral process possibly involving Na+/H+ exchange. In contrast, the EIPA-sensitive Na+/H+ exchanger is not involved in Na+/Na+ countertransport, because Na+ transport through this mechanism is not affected by an increase in cell Na+ from 0.4 to 39 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic activation of Na(+)-H+ exchange in human endothelial cells.

Regulation of intracellular pH (pHi) via a Na(+)-H+ exchange-dependent mechanism was studied in cultured human umbilical vein endothelial cells (HEC) using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, as well as measuring 22Na influx. Basal pHi of HEC incubated in a bicarbonate-free Na+ medium was 6.99 +/- 0.03. In HEC that had been acid-loaded using nigericin or a NH4Cl prepulse, pHi recovery occurred via a Na(+)-dependent mechanism that was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The potency of amiloride derivatives to inhibit 22Na influx was EIPA greater than 5-(N,N-dimethyl)amiloride greater than amiloride [Ki (extracellular Na = 30 mM) = 17 nM, 150 nM, and 8.8 microM, respectively]. EIPA-sensitive 22Na influx in acid-loaded HEC was a saturable function of the external Na+ concentration (0-130 mM), exhibiting an approximate Km and Vmax of 19.70 +/- 0.14 mM and 34.01 +/- 2.2 nmol.10(6) cells-1.min-1, respectively. H+ efflux was also dependent on external Na+ and blocked by EIPA. At resting pHi, HEC Na(+)-H+ exchange was slightly stimulated by increases in medium osmolality. However, when HEC were acid-loaded in the presence of hypertonic (sucrose) medium, Na(+)-H+ exchange activity (22Na influx or pHi recovery) increased markedly. Overall, these data indicate that pHi in cultured HEC can be regulated by a Na(+)-H+ exchanger and that its activity can be markedly influenced by osmolality at acidic pHi.