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Plasma immunoreactive endothelin in essential hypertension
Plasma Immunoreactive Endothelin in Essential Hypertension MASAKAZUKOHNO,MD., KENICHIYASUNARI,M.D., KOH-ICHIMURAKAWA,M.D., KOJIYOKOKAWA,M.D., TAKESHIHORIO,M.D.,TOSHIKIFUKUI,M.D.,TADANAOTAKEDA,M.D, Osaka,Japan
PURPOSE: Endothelin plays a role in the regulation of vascular tonus. Therefore, it has been hypothesized that increased production or release of endothelin or both may contribute to the pathogenesis of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, plasma immunoreactive endothelin concentrations were measured in patients with essential hypertension. PATIENTS AND METHODS: W e measured plasma immunoreactive endothelin concentrations in 42 subjects with essential hypertension, 12 subjects with borderline hypertension, and 25 normotensive control subjects. RESULTS:The concentrations were higher in hypertensive patients than in borderline hypertensive patients and normotensive subjects (beth p <0.05), although values in normotensives and hypertensives overlapped. Reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassay showed two components of plasma endothelin, one corresponding to synthetic endothelin-I (121) and the other corresponding to synthetic big endothelin (human, 1-38). The HPLC prof'fle of plasma endothelin of hypertensive patients was the same as that of normotensive subjects. Hypertensives with reduced glomerular f'fltration rates or increased serum creatinine levels had higher plasma endothelin concentrations than hypertensive patients as a whole (p <0.05). Mean blood pressure and serum creatinine levels were correlated to plasma endothelin in the hypertensives. Correlation was negative between glomerular f'fltration rate and the endothelin level in the hypertensives. CONCLUSION:Plasma endothelin was elevated in m~ny hypertensive patients with severe hypertension or renal involvement. Its major components were endothelin-1 and big endothelin.
he role of the vascular endothelium in the regulaT tion of vascular tonus has been the focus of much interest. It has been reported that endothelial cells release a short-lived endothelium-derived relaxing factor (EDRF) in response to vasoactive agents such as bradykinin and acetylcholine [1-3], and that one EDRF has been identified to be nitric oxide or a related substance [4]. On the other hand, several recent reports have described a protease-sensitive vasoconstrictor activity in supernatants of cultured endothelial cells [5-7]. Yanagisawa et al [8] isolated an endothelium-derived 21-residue vasoconstrictive peptide, endothelin, from the culture supernatant of porcine aortic endothelial cells, determined its amino acid sequence, and cloned the peptide precursor. Recently, porcine endothelin was found to be identical with human endothelin [9]. We have reported that immunoreactive endothelin is released from cultured porcine endothelial cells [10] and from porcine aortic strips with an intact endothelium [11] in a time-dependent way. This peptide has potent, long-lasting vasoconstrictor activity and is closely associated with calcium influx, probably through voltage-dependent calcium channels [8,12]. The intravenous infusion of synthetic endothelin-1 causes a prolonged elevation in blood pressure and a marked reduction in renal blood flow in anesthetized rats [13], and this peptide causes a lasting elevation in intracellular calcium in cultured vascular smooth muscle cells [14,15]. These observations suggest that endothelin plays a role in the regulation of vascular tonus. Therefore, increased production or release of endothelin or both may contribute to the pathogenesis Of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, we measured plasma immunoreactive endothelin concentrations in patients with essential hypertension. The results were compared with those of patients with borderline hypertension and normotensive control subjects. PATIENTS AND METHODS
From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. This study was supported by a Grant-in-aid for Scientific Research(6148210) from the Ministry of Education, Science, and Culture, Tokyo, Japan. Requests for reprints should be addressed to Masakazu Kohno, M.D., The First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Manuscript submitted November 15, 1989, and accepted in revised form February 23, 1990.
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Between January and September 1989, we recruited 79 patients for this study from a population of approximately 350 patients with various diseases seen in our department. Routine laboratory studies of all patients included assays of serum electrolytes, serum creatinine, blood urea nitrogen, and the fasting blood glucose level, liver function tests, urinalysis, a chest roentgenogram, and an electrocardiogram. Based on the results of the laboratory tests and the World Health Organization classification [16], subjects were placed into one of the following three diagnostic categories. Normal blood pressure was defined as a systolic pressure equal to or below 140 mm Hg and a diastolic pressure equal to or below 90 mm Hg. Hypertension was defined as a systolic pressure equal to or greater than 160 mm Hg or a diastolic pressure equal to or
ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
greater than 95 mm Hg, or both. The term "borderline hypertension" was used to denote blood pressure values between the normal and hypertensive ranges as described earlier. Secondary hypertension was excluded by the taking of a clinical history, physical examination, routine laboratory tests including measurem e n t s of p l a s m a r e n i n a c t i v i t y , a l d o s t e r o n e , catecholamine, and cortisol, and an excretory urogram or renal arteriogram. Five of the 42 hypertensive subjects were admitted to our department as hypertensive emergencies. None of these patients had evidence of cardiac, renal, or hepatic failure or of diabetes. In addition, none had clinical evidence of pulmonary disease, angina pectoris, or myocardial infarction. Only hypertensive subjects who had no previous antihypertensive drug therapy or whose antihypertensive drug therapy had been washed out for at least 2 weeks were included in the study. Arterial blood pressure was measured by mercury sphygmomanometer after the patient had rested sitting for 15 minutes in a quiet, warm room. The mean of three blood pressure measurements obtained at three different times was used to classify the subjects. A blood sample (10 mL) was drawn immediately into ice-chilled siliconized disposable glass tubes containing Trasylol (5 × 105 kallikrein inactivator units/ L) and ethyldiaminetetraacetic acid (1 g/L). Plasma was separated by centrifugation for 10 minutes at 4 ° C and immediately frozen and stored at - 8 0 ° C for several days. Immunoreactive endothelin was extracted from plasma as previously reported [17]. Briefly, 5 mL of plasma was diluted with 5 mL of 4% acetic acid. After centrifugation, the solution was pumped at the rate of i mL/minute through a Sep-Pack Cas cartridge (Water Associates, Milford, Massachusetts). After the cartridge was washed with distilled water, the adsorbed peptides were eluted with 86% ethanol in 4% acetic acid. After evaporation with a centrifugal evaporator (model RD-31, Yamato Scientific Co., Tokyo, Japan), the dry residue was dissolved in an assay buffer. The recovery rate was calculated by the addition of three amounts of cold endothelin-1 (0.5 pg/mL [0.20 pmol/L], 1.0 pg/mL [0.40 pmol/L], and 5.0 pg/mL [2.01 pmol/L]) to plasma with dextran-coated charcoal. The recovery rate was 63 4- 6%. The concentration of plasma immunoreactive endothelin was measured using antibody against synthetic endothelin-1 (Peninsula Laboratories Inc., Belmont, California) and 12~I-endothelin-1 (Amersham Japan, Tokyo, Japan) as a tracer. This antibody reacts 100% with endothelin-1 (121) and cross-reacts 84% with endothelin-2, 5% with endothelin-3, and 14% with big endothelin (human, 138). It did not show any cross-reactivity with somatostatin,/~-endorphin, human secretin, angiotensin II, vasopressin, or a-human atrial natriuretic peptide. Radioimmunoassay was done in the assay buffer of 0.01M sodium phosphate, pH 7.4, containing 0.05M sodium chloride, 0.1% bovine serum albumin, 0.1% Nonidet NP-40, and 0.01% sodium azide, by a method described elsewhere [17]. In brief, 100 pl of the sample or 100 ul of standard endothelin-1 was dissolved in the assay buffer and then incubated for 24 hours at 4°C. Approximately 15,000 counts/minute of 125I-endothelin-1 was added to each reaction and incubated for an additional 24 hours. After the second 24-hour incuba-
1 : 8
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endothelin- 1 (pg/tube) Figure 1. Standard curve of the endotheiin radioimmunoassay. The mean inhibition of binding of 1251-endothelin-1 in the presence of 0.5 to 100 pg of synthetic endothelin-1 was calculated from five consecutive assays. Curves constructed from the results of serial dilutions with assay buffer of an extract of plasma from a hypertensive subject ( 0 - 0 ) and normotensive control (z~-z~) with assay buffer were parallel to the standard curve (o-e). B / B o = percent of maximum 12~lendothelin-1 bound.
tion, 100 td of diluted normal rabbit serum and 100 #l of diluted goat anti-rabbit immunoglobulin G serum were added and the mixture was again incubated for 24 hours. After the third incubation, the precipitate was collected by centrifugation at 1,700 × g for 30 minutes. The supernatant was removed by aspiration and the pellet was counted for lesI with a gamma counter. The effective range of the standard curve was between 0.5 and 100 pg of endothelin-1 per assay tube. The 50% intercept was 21 pg of endothelin-1 (Figure 1). The interassay variation was 13% and the intraassay variation was 7%. Reverse-phase high-performance liquid chromagraphy (HPLC) was performed with an octadecylsilica column (0.46 X 25.0 cm, Gasukuro Kogyo Inc., Tokyo, Japan) eluted with a linear gradient of acetonitrile from 33% to 51% in 0.09% trifluoroacetic acid and 0.01M sodium chloride with a flow rate of 1 mL/minute; 0.5-mL fractions were collected and assayed for radioimmunoassay. For chromatographic analysis of immunoreactive endothelin, 30 mL of pooled plasma was separated and treated by reverse-phase HPLC. Serum sodium and potassium, serum and urine creatinine, and blood urea nitrogen were assayed by a routine automatic method. The glomerular filtration rate (GFR) was calculated by the endogenous creatinine clearance. Left ventricular hypertrophy was determined by electrocardiographic criteria. Statistical analysis was done by Student's t-test or Scheffe's test for multiple comparisons preceded by analysis of variance [18]. Linear regression analysis was used to study the relation of the plasma immunoreactive endothelin concentration to the mean blood pressure, GFR, or the serum creatinine level. Values were expressed as mean 4- SD. RESULTS
The characteristics of the normotensive, borderline hypertensive, and hypertensive groups are shown in
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ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
profile of the hypertensive subjects was not different from that of the normotensive control subjects. The mean blood pressure and the serum creatinine level were both correlated with the plasma immunoreactive endothelin concentration in the hypertensive group (Figures 4A and 4B). A negative correlation between GFR and plasma immunoreactive endothelin concentration was found in the hypertensive group (Figure 4C). Table II shows the plasma immunoreactive endothelin concentration for various complications of hypertension. The 11 hypertensive subjects with increased serum creatinine levels (greater than 1.2 mg/ dL [106.1 #mol/L]) and the 12 subjects with a decreased GFR (less than 70 mL/minute) had significantly higher concentrations than the hypertensive group as a whole (both p <0.05). The hypertensive subjects with left ventricular hypertrophy did not have higher concentrations of plasma immunoreactive endothelin than the entire hypertensive group.
TABLE I Characteristics of the Normotensive, Borderline Hypertensive, and Hypertensive Subjects* Normotensive Borderline Hypertensive (n = 25) (n = 12) (n = 42)
Characteristic Mean age (years) Men (%) MAP (mm Hg) Serum creatinine (mg/dL) BUN (mg/dL) Serum sodium (mmol/L) Serum potassium (mmol/L)
50 =1=7 64 91 =1=9 0.9 4- 0.1 16=1=3 140 =1=1 3.9+0.1
49 4- 7 67 101 =t=3 0.9 =1=0.2 16=t=3 139 =t= 1 3.9=1=0.1
52 =1=7 67 132 -4- 20 1.0 =t=0.3 18+5 140 =1=1 4.0=t=0.1
MAP = meanblood pressure;BUN = bloodureanitrogen. * Valuesare mean:1: SD. Serumcreatinine, 1.0 mg/dL = 88.4 #mol/L; 13UN, l0 mg/dL = 3.6 mmol/L.
Table I. The three groups were similar except for the blood pressure levels. There were no significant differences in serum sodium, potassium, creatinine or blood urea nitrogen among the three groups. Plasma immunoreactive endothelin concentrations are shown in Figure 2. Mean plasma immunoreactive endothelin concentrations in the hypertensive, borderline hypertensive, and normotensive groups were 1.1 ± 0.7, 0.5 + 0.3, and 0.5 + 0.2 pg/mL, respectively (0.44 4- 0.28, 0.20 4- 0.12, and 0.20 4- 0.08 pmol/L, respectively). Plasma immunoreactive endothelin concentrations were increased significantly in the hypertensive subjects compared with values obtained for the borderline hypertensive subjects (p <0.05) and the normotensive control subjects (p <0.05). There was no significant difference in plasma immunoreactive endothelin concentrations between the borderline hypertensive subjects and the normotensive control subjects. Reverse-phase HPLC profiles of immunoreactive endothelin in extracts of pooled plasma from normotensive subjects and hypertensive subjects are shown in Figure 3. Two major components with immunoreactive endothelin were observed; one component was eluted in the position of standard endothelin-1 (1-21) and the other component was eluted in the POsition of standard big endothelin (human, 1-38), The elution
COMMENTS
Until recently, it was not even known whether endothelin was released into the circulation, but now data concerning human plasma endothelin concentrations are available. Cernacek and Stewart [19] and Ando et al [20] have demonstrated the presence of immunoreactive endothelin in the plasma of healthy subjects. The plasma immunoreactive endothelin concentration reported here for our normotensive control subjects was not very different from that reported by these other investigators. We found that plasma immunoreactive endothelin concentrations were elevated in many patients in whom blood pressure was particularly high or in whom renal function was impaired, although the values in normotensive and hypertensive subjects overlapped. Our results also showed that the major components of plasma endothelin immunoreactivity were endothelin-1 (1-21) and an intermediate form with 38 amino acid residues (so-called big endothelin). Elevated plasma endothelin concentrations may worsen renal function in hypertensive patients, especially in hypertensive patients with impaired renal
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June 1990 The American Journal of Medicine Volume 88
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Figure 2. Plasma i m m u n o r e a c t i v e e n d o t h e l i n c o n c e n t r a t i o n s in t h e normotensive, borderline hypertensive, and h y p e r t e n s i v e s u b j e c t s Plasma e n d o t h e l i n c o n c e n t r a t i o n , 1.O p g / m L = 0 . 4 0 p m o l / L .
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crine effects on adjacent vascular smooth muscle. Elevation in the plasma endothelin concentration may also induce proliferation in vascular smooth muscle cells, thereby contributing to the development of hypertension, because endothelin causes a rapid and transient increase in the c-los and c - m y c m R N A levels and stimulates D N A synthesis in such cells [23]. How-
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function. Animal experiments suggest that endothelin-1 infused intravenously acts on the renal circulation [13,21,22], reducing renal blood flow [13] and sodium excretion [21]. Furthermore, if the elevation of plasma immunoreactive endothelin reflects increased synthesis, and if the local concentration of endothelin at the site of production is greater than the plasma concentration, this peptide may contribute to the
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ever, the significance of the elevated plasma immunoreactive endothelin concentration in essential hypertension is not known. It remains to be clarified whether a low plasma concentration of endothelin would have a significant influence on vessel tone in the hypertensive state. Nevertheless, our results suggest an association between hypertension (especially severe hypertension with renal involvement) and plasma immunoreactive endothelin concentrations. There are several possible explanations for the elevated level of plasma immunoreactive endothelin in essential hypertension. One possibility is that an increase in the transmural pressure creates stress across the endothelial cell [24], activating the synthesis or release of endothelin. The sheer stress that would occur as the transmural pressure increases would stimu-
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ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
REFERENCES
TABLE II Mean Plasma Immunoreactive Endothelin Concentrations in Patients with Complications of Hypertension and in All Hypertensive Subjects
Number Left ventricular hypertrophy (by electrocardiogram) GFR (<70 mL/minute)
17 12
Serum creatinine (> 1.2 mg/dL)
11
All hypertensive subjects
42
Plasma Immunoreactive Endothelin (pg/mL [pmol/L]) 1.2 -4- 0.8 (0.48 =1=0.32) 2.0 -4- 0.5* (0.80 =1=0.20) 2.0 =1:0.5' (0.80 ± 0.20) 1.1 =1=0.7 (0.44 =1=0.28)
<0.05 comparedwith all hypertensivesubjects. Statisticalanalysiswas doneby Student's t-test. Serumcreatinine, 1.2 mg/dL = 106.1,~mol/L.
late the expression of endothelin mRNA in polygonal endothelial cells and would also increase the release of immunoreactive endothelin [25], although there is no direct evidence of a relation between sheer stress and persistent systemic hypertension. Another explanation is that endothelin or its degradation products may circulate because of the impaired renal function associated with the development of hypertension. This explanation is supported by the positive correlation of the serum creatinine level and negative correlation of GFR with the plasma immunoreactive endothelin concentration in the hypertensive group. The plasma immunoreactive endothelin level is high in uremic patients [19,26]. Bilateral nephrectomy significantly delays the disappearance of endothelin from plasma in rats, and the kidney plays a role in the plasma clearance of endothelin given intravenously [17]. These observations may support the second possibility given here. However, the exact mechanism of the elevation of the concentration of circulating immunoreactive endothelin in essential hypertension is not known. There was considerable overlap between the hypertensive and normotensive endothelin levels, and no difference between borderline hypertensive and normotensive endothelin levels. These findings suggest that an elevated level of endothelin is more strongly associated with the progress than with the onset of essential hypertension. Further investigation is required to clarify whether endothelin-1 or big endothelin acts as a circulating hormone and contributes to the pathophysiology of essential hypertension.
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1. Furchgott RF, ZawadzkiJV: The obligatory role of endothelin cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373-376. 2. Furchgott RF: Roleof endothelium in responsesof vascularsmooth muscle. Circ Res 1983; 53: 557-573. 3. Furchgott RF: The role of endothelium in the responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol 1984; 24: 175-179. 4. Palmer RMJ, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxingfactor. Nature 1987; 327: 524526. 5. Hickey KA, Rubanyi G, Paul RJ, Highsmith RF: Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol 1985; 248: C550-C556. 6. GillespieMN, OwasoyoJO, McMurtry IF, et al: Sustained coronary vasoconstriction provoked by a peptidergicsubstance releasedfrom endothelial cells in culture. J Pharmacol Exp Ther 1986; 236: 339-343. 7. O'Brien RF, Robbins RJ, McMurtry IF: Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 1987; 132: 263-270. 8.Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411-415. 9. Itoh Y, YanagisawaM, Ohkubo S, et al: Cloning and sequenceanalysisof cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBSLett 1988; 231: 440. 10. Kohno M, Murakawa K, Yokokawa K, et al: Production of endothelin by cultured porcine endothelial cells--modulation by adrenaline. J Hypertens 1989; 7(suppl 6): $130-$131. 11. Kohno M, YasunariK, MurakawaK, etal: Releaseof immunoreactiveendothelin from porcine aortic strips (abstr). Hypertension 1989; 14: 34. 12. Lippton H, Goff J, Hyman A: Effects of endothelin in the systemic and renal vascular beds in vivo. Eur J Pharmacot 1988; 155: 197-199. 13. YokokawaK, Kohno M, MurakawaK, et al:Acute effects of endothelin on renal hemodynamics and blood pressurein anesthetizedrats. Am J Hypertens 1989; 2: 715-717. 14. Marsden PA, Danthuluri NR, Brenner BM, BallermannBJ, BrockTA: Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem Biophys Res Commun 1989; 158: 86-93. 15. Kai H, Kanaide H, Nakamura M: Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitiveone in rat aortic smooth muscle cells in primary culture. Biochem Biophys Res Commun 1989; 158: 235-243. 16. World Health Organization: Arterial hypertension: report of a WHO Expert Committee. WHO Technical Report Series, 628. Geneva:World Health Organization, 1978. 17. Kohno M, Murakawa K, Yasunari K, et ah Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metabolism 1989; 38: 712-713. 18. Wallenstein S, Zucker Cl, FleissJL: Some statistical methods useful in circulation research. Circ Res 1980; 47: 1-9. 19.Cernacek P, Stewart DJ: Immunoreactive endothelin in human plasma: marked elevations in patients in cardiogenic shock. Biochem Biophys Res Commun 1989; 161: 562-567. 20. Ando K, Hirata Y, Shichiri M, Emori T, Marumo F: Presenceof immunoreactive endothelin in human plasma. FEBS Lett 1989; 245: 164-166. 21. Goetz KL, Wang BC, MadwedJB, et al: Cardiovascular, renal, and endocrine responses to intravenous endothelin in conscious dogs. Am J Physiol 1988; 255: R1064-R1068. 22. Miller WL, RedfieldMM, Bumett JC Jr: Integrated cardiac, renal, and endocrine actions of endothelin. J Clin Invest 1989; 83: 317-320. 23. Komuro I, KuriharaH, SugiyamaT, Takaku F, YazakiY: Endothelinstimulates clos and c-myc expression and proliferation of vascular smooth cells. EEBS Lett 1988; 238: 249-252. 24. Harder DR: Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res 1987; 60: 102-107. 25. YoshizumiM, KuriharaH, SugiyamaT, etal: Hemodynamicshear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 1989; 161: 859-864. 26. Koyama H, Tabata T, Nishizawa Y, et al: Plasma endothelin level in patients with uraemia. Lancet 1989; 1: 991-992.
PURPOSE: Endothelin plays a role in the regulation of vascular tonus. Therefore, it has been hypothesized that increased production or release of endothelin or both may contribute to the pathogenesis of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, plasma immunoreactive endothelin concentrations were measured in patients with essential hypertension. PATIENTS AND METHODS: W e measured plasma immunoreactive endothelin concentrations in 42 subjects with essential hypertension, 12 subjects with borderline hypertension, and 25 normotensive control subjects. RESULTS:The concentrations were higher in hypertensive patients than in borderline hypertensive patients and normotensive subjects (beth p <0.05), although values in normotensives and hypertensives overlapped. Reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassay showed two components of plasma endothelin, one corresponding to synthetic endothelin-I (121) and the other corresponding to synthetic big endothelin (human, 1-38). The HPLC prof'fle of plasma endothelin of hypertensive patients was the same as that of normotensive subjects. Hypertensives with reduced glomerular f'fltration rates or increased serum creatinine levels had higher plasma endothelin concentrations than hypertensive patients as a whole (p <0.05). Mean blood pressure and serum creatinine levels were correlated to plasma endothelin in the hypertensives. Correlation was negative between glomerular f'fltration rate and the endothelin level in the hypertensives. CONCLUSION:Plasma endothelin was elevated in m~ny hypertensive patients with severe hypertension or renal involvement. Its major components were endothelin-1 and big endothelin.
he role of the vascular endothelium in the regulaT tion of vascular tonus has been the focus of much interest. It has been reported that endothelial cells release a short-lived endothelium-derived relaxing factor (EDRF) in response to vasoactive agents such as bradykinin and acetylcholine [1-3], and that one EDRF has been identified to be nitric oxide or a related substance [4]. On the other hand, several recent reports have described a protease-sensitive vasoconstrictor activity in supernatants of cultured endothelial cells [5-7]. Yanagisawa et al [8] isolated an endothelium-derived 21-residue vasoconstrictive peptide, endothelin, from the culture supernatant of porcine aortic endothelial cells, determined its amino acid sequence, and cloned the peptide precursor. Recently, porcine endothelin was found to be identical with human endothelin [9]. We have reported that immunoreactive endothelin is released from cultured porcine endothelial cells [10] and from porcine aortic strips with an intact endothelium [11] in a time-dependent way. This peptide has potent, long-lasting vasoconstrictor activity and is closely associated with calcium influx, probably through voltage-dependent calcium channels [8,12]. The intravenous infusion of synthetic endothelin-1 causes a prolonged elevation in blood pressure and a marked reduction in renal blood flow in anesthetized rats [13], and this peptide causes a lasting elevation in intracellular calcium in cultured vascular smooth muscle cells [14,15]. These observations suggest that endothelin plays a role in the regulation of vascular tonus. Therefore, increased production or release of endothelin or both may contribute to the pathogenesis Of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, we measured plasma immunoreactive endothelin concentrations in patients with essential hypertension. The results were compared with those of patients with borderline hypertension and normotensive control subjects. PATIENTS AND METHODS
From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. This study was supported by a Grant-in-aid for Scientific Research(6148210) from the Ministry of Education, Science, and Culture, Tokyo, Japan. Requests for reprints should be addressed to Masakazu Kohno, M.D., The First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Manuscript submitted November 15, 1989, and accepted in revised form February 23, 1990.
614
June 1990 The American Journal of Medicine Volume88
Between January and September 1989, we recruited 79 patients for this study from a population of approximately 350 patients with various diseases seen in our department. Routine laboratory studies of all patients included assays of serum electrolytes, serum creatinine, blood urea nitrogen, and the fasting blood glucose level, liver function tests, urinalysis, a chest roentgenogram, and an electrocardiogram. Based on the results of the laboratory tests and the World Health Organization classification [16], subjects were placed into one of the following three diagnostic categories. Normal blood pressure was defined as a systolic pressure equal to or below 140 mm Hg and a diastolic pressure equal to or below 90 mm Hg. Hypertension was defined as a systolic pressure equal to or greater than 160 mm Hg or a diastolic pressure equal to or
ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
greater than 95 mm Hg, or both. The term "borderline hypertension" was used to denote blood pressure values between the normal and hypertensive ranges as described earlier. Secondary hypertension was excluded by the taking of a clinical history, physical examination, routine laboratory tests including measurem e n t s of p l a s m a r e n i n a c t i v i t y , a l d o s t e r o n e , catecholamine, and cortisol, and an excretory urogram or renal arteriogram. Five of the 42 hypertensive subjects were admitted to our department as hypertensive emergencies. None of these patients had evidence of cardiac, renal, or hepatic failure or of diabetes. In addition, none had clinical evidence of pulmonary disease, angina pectoris, or myocardial infarction. Only hypertensive subjects who had no previous antihypertensive drug therapy or whose antihypertensive drug therapy had been washed out for at least 2 weeks were included in the study. Arterial blood pressure was measured by mercury sphygmomanometer after the patient had rested sitting for 15 minutes in a quiet, warm room. The mean of three blood pressure measurements obtained at three different times was used to classify the subjects. A blood sample (10 mL) was drawn immediately into ice-chilled siliconized disposable glass tubes containing Trasylol (5 × 105 kallikrein inactivator units/ L) and ethyldiaminetetraacetic acid (1 g/L). Plasma was separated by centrifugation for 10 minutes at 4 ° C and immediately frozen and stored at - 8 0 ° C for several days. Immunoreactive endothelin was extracted from plasma as previously reported [17]. Briefly, 5 mL of plasma was diluted with 5 mL of 4% acetic acid. After centrifugation, the solution was pumped at the rate of i mL/minute through a Sep-Pack Cas cartridge (Water Associates, Milford, Massachusetts). After the cartridge was washed with distilled water, the adsorbed peptides were eluted with 86% ethanol in 4% acetic acid. After evaporation with a centrifugal evaporator (model RD-31, Yamato Scientific Co., Tokyo, Japan), the dry residue was dissolved in an assay buffer. The recovery rate was calculated by the addition of three amounts of cold endothelin-1 (0.5 pg/mL [0.20 pmol/L], 1.0 pg/mL [0.40 pmol/L], and 5.0 pg/mL [2.01 pmol/L]) to plasma with dextran-coated charcoal. The recovery rate was 63 4- 6%. The concentration of plasma immunoreactive endothelin was measured using antibody against synthetic endothelin-1 (Peninsula Laboratories Inc., Belmont, California) and 12~I-endothelin-1 (Amersham Japan, Tokyo, Japan) as a tracer. This antibody reacts 100% with endothelin-1 (121) and cross-reacts 84% with endothelin-2, 5% with endothelin-3, and 14% with big endothelin (human, 138). It did not show any cross-reactivity with somatostatin,/~-endorphin, human secretin, angiotensin II, vasopressin, or a-human atrial natriuretic peptide. Radioimmunoassay was done in the assay buffer of 0.01M sodium phosphate, pH 7.4, containing 0.05M sodium chloride, 0.1% bovine serum albumin, 0.1% Nonidet NP-40, and 0.01% sodium azide, by a method described elsewhere [17]. In brief, 100 pl of the sample or 100 ul of standard endothelin-1 was dissolved in the assay buffer and then incubated for 24 hours at 4°C. Approximately 15,000 counts/minute of 125I-endothelin-1 was added to each reaction and incubated for an additional 24 hours. After the second 24-hour incuba-
1 : 8
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Straight
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so
i 100
endothelin- 1 (pg/tube) Figure 1. Standard curve of the endotheiin radioimmunoassay. The mean inhibition of binding of 1251-endothelin-1 in the presence of 0.5 to 100 pg of synthetic endothelin-1 was calculated from five consecutive assays. Curves constructed from the results of serial dilutions with assay buffer of an extract of plasma from a hypertensive subject ( 0 - 0 ) and normotensive control (z~-z~) with assay buffer were parallel to the standard curve (o-e). B / B o = percent of maximum 12~lendothelin-1 bound.
tion, 100 td of diluted normal rabbit serum and 100 #l of diluted goat anti-rabbit immunoglobulin G serum were added and the mixture was again incubated for 24 hours. After the third incubation, the precipitate was collected by centrifugation at 1,700 × g for 30 minutes. The supernatant was removed by aspiration and the pellet was counted for lesI with a gamma counter. The effective range of the standard curve was between 0.5 and 100 pg of endothelin-1 per assay tube. The 50% intercept was 21 pg of endothelin-1 (Figure 1). The interassay variation was 13% and the intraassay variation was 7%. Reverse-phase high-performance liquid chromagraphy (HPLC) was performed with an octadecylsilica column (0.46 X 25.0 cm, Gasukuro Kogyo Inc., Tokyo, Japan) eluted with a linear gradient of acetonitrile from 33% to 51% in 0.09% trifluoroacetic acid and 0.01M sodium chloride with a flow rate of 1 mL/minute; 0.5-mL fractions were collected and assayed for radioimmunoassay. For chromatographic analysis of immunoreactive endothelin, 30 mL of pooled plasma was separated and treated by reverse-phase HPLC. Serum sodium and potassium, serum and urine creatinine, and blood urea nitrogen were assayed by a routine automatic method. The glomerular filtration rate (GFR) was calculated by the endogenous creatinine clearance. Left ventricular hypertrophy was determined by electrocardiographic criteria. Statistical analysis was done by Student's t-test or Scheffe's test for multiple comparisons preceded by analysis of variance [18]. Linear regression analysis was used to study the relation of the plasma immunoreactive endothelin concentration to the mean blood pressure, GFR, or the serum creatinine level. Values were expressed as mean 4- SD. RESULTS
The characteristics of the normotensive, borderline hypertensive, and hypertensive groups are shown in
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ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
profile of the hypertensive subjects was not different from that of the normotensive control subjects. The mean blood pressure and the serum creatinine level were both correlated with the plasma immunoreactive endothelin concentration in the hypertensive group (Figures 4A and 4B). A negative correlation between GFR and plasma immunoreactive endothelin concentration was found in the hypertensive group (Figure 4C). Table II shows the plasma immunoreactive endothelin concentration for various complications of hypertension. The 11 hypertensive subjects with increased serum creatinine levels (greater than 1.2 mg/ dL [106.1 #mol/L]) and the 12 subjects with a decreased GFR (less than 70 mL/minute) had significantly higher concentrations than the hypertensive group as a whole (both p <0.05). The hypertensive subjects with left ventricular hypertrophy did not have higher concentrations of plasma immunoreactive endothelin than the entire hypertensive group.
TABLE I Characteristics of the Normotensive, Borderline Hypertensive, and Hypertensive Subjects* Normotensive Borderline Hypertensive (n = 25) (n = 12) (n = 42)
Characteristic Mean age (years) Men (%) MAP (mm Hg) Serum creatinine (mg/dL) BUN (mg/dL) Serum sodium (mmol/L) Serum potassium (mmol/L)
50 =1=7 64 91 =1=9 0.9 4- 0.1 16=1=3 140 =1=1 3.9+0.1
49 4- 7 67 101 =t=3 0.9 =1=0.2 16=t=3 139 =t= 1 3.9=1=0.1
52 =1=7 67 132 -4- 20 1.0 =t=0.3 18+5 140 =1=1 4.0=t=0.1
MAP = meanblood pressure;BUN = bloodureanitrogen. * Valuesare mean:1: SD. Serumcreatinine, 1.0 mg/dL = 88.4 #mol/L; 13UN, l0 mg/dL = 3.6 mmol/L.
Table I. The three groups were similar except for the blood pressure levels. There were no significant differences in serum sodium, potassium, creatinine or blood urea nitrogen among the three groups. Plasma immunoreactive endothelin concentrations are shown in Figure 2. Mean plasma immunoreactive endothelin concentrations in the hypertensive, borderline hypertensive, and normotensive groups were 1.1 ± 0.7, 0.5 + 0.3, and 0.5 + 0.2 pg/mL, respectively (0.44 4- 0.28, 0.20 4- 0.12, and 0.20 4- 0.08 pmol/L, respectively). Plasma immunoreactive endothelin concentrations were increased significantly in the hypertensive subjects compared with values obtained for the borderline hypertensive subjects (p <0.05) and the normotensive control subjects (p <0.05). There was no significant difference in plasma immunoreactive endothelin concentrations between the borderline hypertensive subjects and the normotensive control subjects. Reverse-phase HPLC profiles of immunoreactive endothelin in extracts of pooled plasma from normotensive subjects and hypertensive subjects are shown in Figure 3. Two major components with immunoreactive endothelin were observed; one component was eluted in the position of standard endothelin-1 (1-21) and the other component was eluted in the POsition of standard big endothelin (human, 1-38), The elution
COMMENTS
Until recently, it was not even known whether endothelin was released into the circulation, but now data concerning human plasma endothelin concentrations are available. Cernacek and Stewart [19] and Ando et al [20] have demonstrated the presence of immunoreactive endothelin in the plasma of healthy subjects. The plasma immunoreactive endothelin concentration reported here for our normotensive control subjects was not very different from that reported by these other investigators. We found that plasma immunoreactive endothelin concentrations were elevated in many patients in whom blood pressure was particularly high or in whom renal function was impaired, although the values in normotensive and hypertensive subjects overlapped. Our results also showed that the major components of plasma endothelin immunoreactivity were endothelin-1 (1-21) and an intermediate form with 38 amino acid residues (so-called big endothelin). Elevated plasma endothelin concentrations may worsen renal function in hypertensive patients, especially in hypertensive patients with impaired renal
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June 1990 The American Journal of Medicine Volume 88
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function. Animal experiments suggest that endothelin-1 infused intravenously acts on the renal circulation [13,21,22], reducing renal blood flow [13] and sodium excretion [21]. Furthermore, if the elevation of plasma immunoreactive endothelin reflects increased synthesis, and if the local concentration of endothelin at the site of production is greater than the plasma concentration, this peptide may contribute to the
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ever, the significance of the elevated plasma immunoreactive endothelin concentration in essential hypertension is not known. It remains to be clarified whether a low plasma concentration of endothelin would have a significant influence on vessel tone in the hypertensive state. Nevertheless, our results suggest an association between hypertension (especially severe hypertension with renal involvement) and plasma immunoreactive endothelin concentrations. There are several possible explanations for the elevated level of plasma immunoreactive endothelin in essential hypertension. One possibility is that an increase in the transmural pressure creates stress across the endothelial cell [24], activating the synthesis or release of endothelin. The sheer stress that would occur as the transmural pressure increases would stimu-
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ENDOTHELIN IN ESSENTIAL HYPERTENSION / KOHNO ET AL
REFERENCES
TABLE II Mean Plasma Immunoreactive Endothelin Concentrations in Patients with Complications of Hypertension and in All Hypertensive Subjects
Number Left ventricular hypertrophy (by electrocardiogram) GFR (<70 mL/minute)
17 12
Serum creatinine (> 1.2 mg/dL)
11
All hypertensive subjects
42
Plasma Immunoreactive Endothelin (pg/mL [pmol/L]) 1.2 -4- 0.8 (0.48 =1=0.32) 2.0 -4- 0.5* (0.80 =1=0.20) 2.0 =1:0.5' (0.80 ± 0.20) 1.1 =1=0.7 (0.44 =1=0.28)
<0.05 comparedwith all hypertensivesubjects. Statisticalanalysiswas doneby Student's t-test. Serumcreatinine, 1.2 mg/dL = 106.1,~mol/L.
late the expression of endothelin mRNA in polygonal endothelial cells and would also increase the release of immunoreactive endothelin [25], although there is no direct evidence of a relation between sheer stress and persistent systemic hypertension. Another explanation is that endothelin or its degradation products may circulate because of the impaired renal function associated with the development of hypertension. This explanation is supported by the positive correlation of the serum creatinine level and negative correlation of GFR with the plasma immunoreactive endothelin concentration in the hypertensive group. The plasma immunoreactive endothelin level is high in uremic patients [19,26]. Bilateral nephrectomy significantly delays the disappearance of endothelin from plasma in rats, and the kidney plays a role in the plasma clearance of endothelin given intravenously [17]. These observations may support the second possibility given here. However, the exact mechanism of the elevation of the concentration of circulating immunoreactive endothelin in essential hypertension is not known. There was considerable overlap between the hypertensive and normotensive endothelin levels, and no difference between borderline hypertensive and normotensive endothelin levels. These findings suggest that an elevated level of endothelin is more strongly associated with the progress than with the onset of essential hypertension. Further investigation is required to clarify whether endothelin-1 or big endothelin acts as a circulating hormone and contributes to the pathophysiology of essential hypertension.
618
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1. Furchgott RF, ZawadzkiJV: The obligatory role of endothelin cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373-376. 2. Furchgott RF: Roleof endothelium in responsesof vascularsmooth muscle. Circ Res 1983; 53: 557-573. 3. Furchgott RF: The role of endothelium in the responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol 1984; 24: 175-179. 4. Palmer RMJ, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxingfactor. Nature 1987; 327: 524526. 5. Hickey KA, Rubanyi G, Paul RJ, Highsmith RF: Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol 1985; 248: C550-C556. 6. GillespieMN, OwasoyoJO, McMurtry IF, et al: Sustained coronary vasoconstriction provoked by a peptidergicsubstance releasedfrom endothelial cells in culture. J Pharmacol Exp Ther 1986; 236: 339-343. 7. O'Brien RF, Robbins RJ, McMurtry IF: Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 1987; 132: 263-270. 8.Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411-415. 9. Itoh Y, YanagisawaM, Ohkubo S, et al: Cloning and sequenceanalysisof cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBSLett 1988; 231: 440. 10. Kohno M, Murakawa K, Yokokawa K, et al: Production of endothelin by cultured porcine endothelial cells--modulation by adrenaline. J Hypertens 1989; 7(suppl 6): $130-$131. 11. Kohno M, YasunariK, MurakawaK, etal: Releaseof immunoreactiveendothelin from porcine aortic strips (abstr). Hypertension 1989; 14: 34. 12. Lippton H, Goff J, Hyman A: Effects of endothelin in the systemic and renal vascular beds in vivo. Eur J Pharmacot 1988; 155: 197-199. 13. YokokawaK, Kohno M, MurakawaK, et al:Acute effects of endothelin on renal hemodynamics and blood pressurein anesthetizedrats. Am J Hypertens 1989; 2: 715-717. 14. Marsden PA, Danthuluri NR, Brenner BM, BallermannBJ, BrockTA: Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem Biophys Res Commun 1989; 158: 86-93. 15. Kai H, Kanaide H, Nakamura M: Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitiveone in rat aortic smooth muscle cells in primary culture. Biochem Biophys Res Commun 1989; 158: 235-243. 16. World Health Organization: Arterial hypertension: report of a WHO Expert Committee. WHO Technical Report Series, 628. Geneva:World Health Organization, 1978. 17. Kohno M, Murakawa K, Yasunari K, et ah Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metabolism 1989; 38: 712-713. 18. Wallenstein S, Zucker Cl, FleissJL: Some statistical methods useful in circulation research. Circ Res 1980; 47: 1-9. 19.Cernacek P, Stewart DJ: Immunoreactive endothelin in human plasma: marked elevations in patients in cardiogenic shock. Biochem Biophys Res Commun 1989; 161: 562-567. 20. Ando K, Hirata Y, Shichiri M, Emori T, Marumo F: Presenceof immunoreactive endothelin in human plasma. FEBS Lett 1989; 245: 164-166. 21. Goetz KL, Wang BC, MadwedJB, et al: Cardiovascular, renal, and endocrine responses to intravenous endothelin in conscious dogs. Am J Physiol 1988; 255: R1064-R1068. 22. Miller WL, RedfieldMM, Bumett JC Jr: Integrated cardiac, renal, and endocrine actions of endothelin. J Clin Invest 1989; 83: 317-320. 23. Komuro I, KuriharaH, SugiyamaT, Takaku F, YazakiY: Endothelinstimulates clos and c-myc expression and proliferation of vascular smooth cells. EEBS Lett 1988; 238: 249-252. 24. Harder DR: Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res 1987; 60: 102-107. 25. YoshizumiM, KuriharaH, SugiyamaT, etal: Hemodynamicshear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 1989; 161: 859-864. 26. Koyama H, Tabata T, Nishizawa Y, et al: Plasma endothelin level in patients with uraemia. Lancet 1989; 1: 991-992.