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2002; 15:291–295
Original Contributions
A Link Between Polymorphonuclear Leukocyte Intracellular Calcium, Plasma Insulin, and Essential Hypertension Shifra Sela, Revital Shurtz-Swirski, Raymond Farah, Rivka Levy, Galina Shapiro, Judith Chezar, Shaul M. Shasha, and Batya Kristal Background: Intracellular ionized calcium ([Ca2⫹]i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca2⫹]i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca2⫹]i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP). Methods: The PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca2⫹]i was measured by flow cytometry in isolated cells stained with Fluo-3. Results: The EH PMNs showed significantly increased [Ca2⫹]i compared to NC PMN. Eighty percent of EH
H
patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca2⫹]i and mean arterial pressure (MAP) (r ⫽ 0.5, P ⬍ .006); 2) PMN [Ca2⫹]i and fasting plasma insulin (r ⫽ 0.7, P ⬍ .005); and 3) fasting plasma insulin and MAP (r ⫽ 0.4, P ⬍ .04). Conclusions: This study adds PMN to previously described cells exhibiting elevated [Ca2⫹]i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca2⫹]i and plasma insulin levels, together with the fact that elevated [Ca2⫹]i mediates PMN priming, suggest that elevated [Ca2⫹]i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH. Am J Hypertens 2002;15:291–295 © 2002 American Journal of Hypertension, Ltd. Key Words: Essential hypertension, hyperinsulinemia, intracellular ionized calcium, polymorphonuclear leukocytes.
ypertension is a well-established and independent risk factor for the development and progression of atherosclerosis.1,2 The mechanisms that predispose hypertensive subjects to organ injury and atherosclerosis are multifactorial. Abnormalities of endothelium function and morphology appear to play a central role in the pathogenesis of hypertension-related atherosclerosis.3–5 Among the mechanisms causing endothelial dysfunction that have been recently implicated in essential hypertension (EH) are insulin resistance and its accompanying compensatory hyperinsulinemia,6 oxidative stress (OS), and inflammation.7–9 We have previously reported that primed peripheral polymorphonuclear leukocytes (PMN) contribute to the OS and inflammation in EH.10 It is documented that degranulation of PMN with subsequent release of proteases such as elastases,
cytokines, and reactive oxygen species11,12 correlate with PMN intracellular ionized calcium [Ca2⫹]i concentration.13,14 Thus, [Ca2⫹]i has a role as a key mediator in the production of oxidants by PMN and in their degranulation, resulting in OS, inflammation, and damage to endothelial cells,11,12 vasoconstriction, and hypertension. Essential hypertensive patients have higher plasma insulin levels in response to glucose load, whether obese or of normal body weight, compared with age- and body weight-matched healthy subjects.15 The hyperinsulinemia associated with EH is a consequence of resistance to the effects of insulin on peripheral glucose utilization and to decreased hepatic uptake of insulin.16 Elevated [Ca2⫹]i has been described in various cells in insulin-resistant states such as uremia, diabetes, and EH.17–19 It has not
Received August 2, 2001. First Decision October 1, 2001. Accepted November 19, 2001. From the Nephrology Unit (SMS, BK), Hematology Unit (JC), Eliachar Research Laboratory (SS, RS-S, RL, GS, RF), Western Galilee Hospital,
Nahariya, Israel, Rappaport School of Medicine, Technion, Haifa, Israel. Address correspondence and reprint requests to Dr. Batya Kristal, Nephrology and Hypertension Unit, Western Galilee Hospital, Nahariya 22100, Israel; e-mail:
[email protected]
© 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
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been described in EH PMN. An elevation in [Ca2⫹]i in PMN from hemodialysis patients, diabetic patients, and diabetic rats has been previously described as playing an important role in the pathogenesis of their impaired phagocytosis.20 –22 In the light of these findings and the knowledge that [Ca2⫹]i is a key mediator in PMN priming, the present study investigates the levels of [Ca2⫹]i in peripheral EH PMN and relates these [Ca2⫹]i levels to plasma insulin and blood pressure (BP).
Methods Study Population Twenty untreated EH patients (11 men/9 women) with mild to moderate hypertension (age range, 20 to 60 years) and 20 age- and sex-matched healthy controls (NC) were enrolled in the study. Inclusion criteria in the EH group were: sitting diastolic BP ⬎90 mm Hg (average of three outpatient visits), sitting systolic BP ⬎140 mm Hg (average as above), body mass index ⬍30 kg/m2, no evidence of target organ damage and systemic diseases. Subjects with evidence of infection, inflammation, receiving medication, smoking, and secondary causes of hypertension were excluded. The selection of all participants was based on a clinical examination and laboratory confirmation. All the subjects had normal fasting (⬎14 h) serum cholesterol and glucose levels with normal kidney and liver function. All subjects signed an informed consent form, which was approved by the institutional review committee before their blood donation. Blood Withdrawal and PMN Separation Blood was withdrawn from all the subjects and patients after an overnight fast for biochemical and hematological parameters and for PMN isolation. The PMN isolation was carried out according to the method of Klebanoff and Clark,23 with modification by Kristal et al.10 The separated PMN (⬎98% pure) were resuspended in a minimal volume (0.1 to 0.3 mL) of cold phosphate-buffered saline solution (PBS), immediately counted and diluted with PBS containing 0.1% glucose, according to the different experimental needs. Due to some technical reasons not all tests were accomplished for all blood samples, nevertheless all data were incorporated into the results and the number of samples was included. Measurement of [Ca2ⴙ]i Determination of [Ca2⫹]i was carried out in 11 EH and matched 11 NC subjects. The assay was performed with acetoxymethyl (AM) ester of Fluo-3 (Fluo-3 AM)14,24, a calcium-sensitive dye, dissolved in a 0.1% solution of the nonionic detergent Pluronic F-127 by flow cytometry (Coulter EPICS XL—MCL; Coulter, Miami, FL), according to June and Rabinovitch25 and to Vandenberghe and Ceuppens.26 Separated PMN were loaded with the mem-
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brane-permeable Fluo-3 AM, which is converted intracellularly to the membrane impermeable Fluo-3 free acid.27 Fluo-3 AM and Pluronic F-127 were purchased from Molecular Probes, Europe BV, The Netherlands. Fluo-3 (excitation at 488 nm and emission at 530 nm) has a negligible fluorescence as a free acid, which is enhanced upon binding to Ca2⫹. Intracellular ionized calcium was calculated using the formula [Ca2⫹]i ⫽ Kd [F ⫺ Fmin]/[Fmax ⫺ F] as described by Vandenberghe and Ceuppens,26 where F is baseline fluorescence measured following a 10-min incubation of separated PMN in 37°C with Fluo-3 AM; Fmax is the fluorescence measured in the presence of 1 mmol/L CaCl2 and 5 mol/L of the calcium ionophore A23187 (Sigma Chemical Co., St. Louis, MO) after a 1-min incubation in 37°C; Fmin is determined immediately after the addition of 2 mmol/L MnCl2 to the ionophore-treated cells, as Mn2⫹ displaces Ca2⫹ from Fluo-3, as described.26,28 Kd is the dissociation constant of Fluo-3 bound to Ca2⫹ and equals 400 nmol/L in 37°C.26,28 Insulin Measurement Fasting plasma insulin levels at time 0 and 120 min after a 75-g glucose oral load were determined using RIA kit (Biodata S.P.A., Milano, Italy) in 10 EH patients and matched in 10 NC subjects (as not all subjects agreed to receive oral glucose load). Statistical Analysis Data are expressed as means ⫾ SEM. Differences between the study parameters of the two groups were analyzed by Student’s t test. Mean arterial pressure (MAP), plasma insulin, and [Ca2⫹]i values were correlated by linear regression analysis. P ⬍ .05 was considered statistically significant.
Results The clinical and biochemical parameters of the EH patients and the NC subjects are depicted in Table 1. The only parameters that significantly differ between the EH and NC groups are the BP values: systolic BP (SBP), diastolic BP (DBP), and MAP. [Ca2⫹]i in EH PMN were significantly higher than [Ca2⫹]i in NC PMN (P ⫽ .002; Fig. 1). PMN [Ca2⫹]i from both EH and NC exhibits a linear correlation with MAP (P ⫽ .006; r ⫽ 0.5; Fig. 2). A linear significant correlation (P ⫽ .04, r⫽0.4) was also observed between MAP and fasting insulin levels both in EH and NC. Plasma insulin levels in EH and NC after a glucose oral load are depicted in Fig. 3A. Insulin levels in EH plasma were significantly higher (⬎40 U/mL) 120 min after the glucose load, compared to insulin levels in EH plasma at time 0, and to NC plasma insulin, both at 0 and 120 min. No significant difference was found between NC plasma insulin levels at 0 and 120 min. Plasma glucose levels in EH and NC after the glucose oral load are depicted in Fig.
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Table 1. Clinical and chemical characteristics of hypertensive patients (EH) and healthy subjects (NC)
Age (y) M/F BMI (kg/cm2) SBP (mm Hg) DBP (mm Hg) MAP (mm Hg) AST (U/L) ALT (U/L) Cholesterol (mg/dL) Triglyceride (mg/dL) Creatinine (mg/dL) Fasting glucose (mg/dL)
NC (n ⴝ 20)
EH (n ⴝ 20)
P
47 ⫾ 8.7 11/9 27 ⫾ 0.5 122.8 ⫾ 1.6 78.8 ⫾ 1.3 92.7 ⫾ 1.3 17.4 ⫾ 1.6 24.3 ⫾ 4.5 197 ⫾ 44 118.8 ⫾ 18.1 1.1 ⫾ 0.2 83.4 ⫾ 4
51.5 ⫾ 10 11/9 26.8 ⫾ 0.4 167.1 ⫾ 3.1 94.5 ⫾ 1.7 120.4 ⫾ 1.7 19 ⫾ 1.2 24.2 ⫾ 2 210 ⫾ 8.4 143 ⫾ 17.0 1.1 ⫾ 0.1 90.7 ⫾ 2
ns ns ⬍.0001 ⬍.004 ⬍.0001 ns ns ns ns ns ns
ns ⫽ not significant; M/F ⫽ male/female; BMI ⫽ body mass index; SBP ⫽ systolic blood pressure; DBP ⫽ diastolic blood pressure; MAP ⫽ mean arterial pressure; AST ⫽ aspartate aminotransferase; ALT ⫽ alanine aminotransferase. Values are mean ⫾ SEM.
3B. Fasting plasma glucose levels at time 0 were similar in NC and EH. After 120 min of the glucose load, both groups of patients showed a small nonsignificant increase in plasma glucose levels; nevertheless, within the normal range of plasma glucose. Fasting insulin levels and PMN [Ca2⫹]i showed a positive linear correlation (Fig. 4, P ⫽ .005; r ⫽ 0.7).
Discussion The ionic hypothesis, in which a steady-state elevation of cytosolic-free calcium occurs, plays an important role in hypertension.29 In animal models of hypertension and in EH patients, increased excretion of calcium in the urine was reported.22 In addition, increased [Ca2⫹]i was found in various cells of EH patients (ie, lymphocytes, erythrocytes, platelets, and vascular smooth muscle cells).24 All these cells were characterized by an impairment in their activity after the increase in [Ca2⫹]i. In hypertensive rats the increase in platelet [Ca2⫹]i was found to precede the development of high BP.17 In the present study, EH PMN
FIG. 1. Intracellular ionized calcium ([Ca2⫹]i) levels in healthy subjects (NC) and essential hypertension (EH) polymorphonuclear leukocytes, analyzed by flow cytometer as described in Methods. Data are presented as means ⫾ SEM. *P ⫽ .002.
showed increased levels of [Ca2⫹]i correlating positively with the individual’s BP and plasma insulin. We have recently reported that EH is accompanied by a primed state of PMN, causing OS and inflammation.10 Priming describes a state of PMN activation where a further stimulation causes the release of ROS and proteolytic mediators of tissue degradation.10,14 This contributes to OS, subsequent inflammation, and may trigger endothelial damage in an extent dependent on PMN priming. In addition, we have previously shown that in other clinical situations known to be associated with endothelial dysfunction and accelerated atherosclerosis such as uremia and type 2 diabetes, PMN are primed, contributing to OS and inflammation.10,14 In the above-mentioned EH study the rate of superoxide release from EH PMN was 1.8-fold faster than the rate from NC PMN reflecting PMN priming.10 The present study shows a similar increase (1.5fold) in [Ca2⫹]i of EH PMNs versus NC PMN, suggesting that the increase in [Ca2⫹]i may play a role in PMN priming. Alteration in calcium homeostasis facilitates stimulus-induced cellular activity by enhancing signal
FIG. 2. Linear correlation between mean arterial pressure (MAP) and [Ca2⫹]i (P ⫽ .006, r ⫽ 0.5) in EH patients (■) and NC subjects (Œ). Other abbreviations as in Fig. 1.
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FIG. 3. Plasma insulin (A) and glucose (B) levels in NC subjects and EH patients. Fasting plasma insulin and glucose levels were measured at time 0 and after 120 min of a glucose load. Data are presented as boxes and whiskers, showing the means and range of data. *P ⬍ .0005, EH patients 0 v 120 min; **P ⬍ .05, EH patients at 120 min v NC subjects both at 0 and 120 min. ns ⫽ not significant; other abbreviations as in Figs. 1 and 2.
transduction,30 and the role of [Ca2⫹]i as a second messenger in PMN priming was previously shown by Yee and Christou.14 They have shown an increased level of [Ca2⫹]i
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in primed PMN resulting in an enhanced superoxide release after stimulation, an observation that was also described in hemodialysis patients.13 To the best of our knowledge, the present report is the first to describe high levels of [Ca2⫹]i in EH PMN. One contradictory report exists where [Ca2⫹]i levels in EH patients did not differ from its levels in healthy subjects and no significant relationship was found between BP and [Ca2⫹]i.31 These conflicting data might arise from the usage of a different calcium-sensitive dye, quin 2, which was proven not as sensitive as Fluo-3 used in this study.25,26 Hence, we suggest that hypertension in EH patients is accompanied by an increase in PMN [Ca2⫹]i resulting in PMN priming. The PMN priming contributes to OS and inflammation, which may directly or indirectly harm the endothelium resulting in hypertension-related atherosclerosis in the long run. Although all the hypertensive patients in this study were nonobese, 80% were characterized by hyperinsulinemia after the oral glucose load, indicating the existence of insulin resistance in these patients. We also demonstrate a positive significant linear correlation of individual BP with both PMN [Ca2⫹]i and fasting plasma insulin levels, as well as a linear significant correlation between fasting insulin levels and MAP. Thus, these linked observations suggest a relation between plasma insulin, PMN [Ca2⫹]i, and BP. Insulin resistance causes hypertension by various mechanisms. Barbagallo et al32,33 showed that the property of cellular insulin resistance may not be related to the disease state per se as much as to the underlying abnormal cellular ion content (calcium and magnesium); these ions regulate the cellular responsiveness to insulin. Several in vitro reports have shown that insulin affects PMN directly by increasing myeloperoxidase activity34 and respiratory burst.35 One possible explanation can be that common channels exist both for [Ca2⫹]i and insulin in these cells, as found in smooth muscle cells.19 The effect of insulin on EH PMN remains to be further elucidated. In conclusion, PMN from EH patients show an increase in their [Ca2⫹]i. We propose this causes PMN priming, thereby contributing to OS and inflammation in EH. The correlation of individual BP with both PMN [Ca2⫹]i and plasma insulin levels can explain their involvement in the pathogenesis of hypertension-induced vascular injury, long before clinical evidence of target organ damage appears.
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