(Hypertension. 1995;26:1070-1073.)
© 1995 American Heart Association, Inc.
Articles |
Calcium-ATPase and Insulin in Adolescent Offspring of Essential Hypertensive Parents
From the Hypertension Clinic, Children's Hospital "Ricardo Gutierrez," Buenos Aires, Argentina.
Correspondence to Beatriz Grunfeld, MD, Hipertensión Arterial, Hospital de Niños "R. Gutierrez," La Pampa 3635, 1430 Buenos Aires, Argentina.
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Abstract |
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Abstract A number of abnormalities in calcium homeostasis have been reported in patients with essential hypertension. In turn, insulin has been shown to influence the activity of the Ca2+-ATPase. We have previously shown that normotensive offspring of essential hypertensive individuals have an exaggerated insulin response to a glucose overload. Therefore, the aim of the present study was to evaluate basal and calmodulin-activated Ca2+-ATPase in red blood cells and its relationship to the insulin response during an intravenous glucose tolerance test in 27 normotensive adolescents with a family history of essential hypertension (F+) (mean age, 13.9±0.5 years) and in 10 control subjects matched for age and body mass index with no family history of hypertension (F-). The results (mean±SD) were as follows (µmol Pi/[mg protein/h]10-1): basal Ca2+-ATPase, 4.5±1.2 in F+ and 5.1±1.6 in F- (P=NS); calmodulin-activated Ca2+-ATPase, 13.6±3.9 in F+ and 16.2±1.7 in F- (P<.04). The insulin area under the curve after the glucose load was 3413±1674 µU/mL per hour in F+ and 2752±928 in F- (P=NS). Calmodulin-activated Ca2+-ATPase showed a negative correlation with the insulin area under the curve (r=-.59, P<.005) and cholesterol levels (r=-.38, P<.03). Urinary calcium excretion was 1.82±0.9 mmol/d in F+ and 2.47±0.9 mmol/d in F- (P=NS). Our findings indicate a diminished activity of calmodulin-stimulated Ca2+-ATPase despite increased levels of insulin, a known activator of this pump, further suggesting the presence of insulin resistance in normotensive offspring of essential hypertensive individuals. Since Ca2+-ATPase is an extrusion pump, a drop in its activity may lead to an increase in intracellular calcium accumulation and thus contribute to the development of hypertension.
Key Words: hypertension, essential • adolescent medicine • Ca2+-transporting ATPase • insulin
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Introduction |
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Anumber of disturbances in calcium metabolism have been reported in arterial hypertension.1 2 Lower serum ionized calcium levels, increased urinary excretion of calcium, raised intracellular calcium levels, and decreased Ca2+-ATPase or calmodulin-stimulated Ca2+-ATPase activities have been shown in platelets and erythrocytes obtained from primary hypertensive subjects.3 4 5 6 7
Abnormalities in plasma membranes of vascular smooth muscle, erythrocytes, and other cells have been reported in spontaneously hypertensive rats, indicating that the disturbance in cellular Ca2+ homeostasis seen in hypertension is not confined to one tissue but represents a more generalized defect.8 Studies on cellular calcium handling in erythrocytes and platelets from primary hypertensive subjects have suggested that the membrane defect relates to calcium efflux mechanisms.9 10 Since Ca2+-ATPase is an extrusion pump, a diminished activity would lead to intracellular calcium accumulation and thus contribute to increased peripheral vascular resistance and the development of hypertension.
Insulin is known to have a significant effect on several transmembrane ion-exchange systems, including Na+-K+-ATPase and Na+-H+ exchanger, and to directly stimulate Ca2+-ATPase activity in kidney, heart, liver, and adipocytes by increasing membrane calmodulin content and/or phosphorylation or by increasing the enzyme affinity for calcium.11 12 13 14
We and other researchers have previously shown that normotensive offspring of primary hypertensive individuals have an abnormal insulin response to a glucose challenge.15 Therefore, the aim of the present study was to evaluate Ca2+-ATPase and calmodulin-activated Ca2+-ATPase in red blood cell membranes and their relationship to the insulin response to an intravenous glucose load in normotensive adolescents with and without a family history of primary hypertension.
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Methods |
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Included in the study were 27 normotensive adolescents with a family history of primary hypertension (F+) (mean age, 13.9±0.5 years; range, 12 to 19 years; 10 females; at least one parent with long-standing hypertension as assessed by one of the authors) and 10 normotensive control subjects (mean age, 13.7±0.7 years; range, 12 to 19 years; 4 females) without a family history of hypertension (F-). All participants were healthy; were taking no medications; were white, from Spanish or Italian background; and were matched for age, body mass index, and pubertal stage (Tanner stages III and IV).
Procedures were carefully explained to both parents and children, and informed consent was obtained from parents.
All subjects came to the Hypertension Clinic after an overnight fast with a 24-hour urinary collection. A butterfly-like needle was placed in an antecubital vein, and a blood specimen was obtained for measurement of plasma glucose, insulin, cholesterol, high-density lipoprotein cholesterol, triglycerides, and Ca2+-ATPase and calmodulin-stimulated Ca2+-ATPase activities in red blood cell membranes. A glucose load (0.25 g/kg IV) as a 25% solution was then infused over approximately 2 minutes into a contralateral vein. Blood samples were obtained at 1, 3, 5, and 7 minutes after glucose injection for measurement of insulin and at 10, 20, 30, 40, 50, and 60 minutes for insulin and glucose.
Insulin was measured by radioimmunoassay.16 Calcium was measured in a 24-hour urinary collection by an atomic absorption spectrophotometer (Perkin-Elmer). Completeness of the collection was evaluated by measurement of urinary creatinine.
Preparation of Isolated Red Blood Cell Membranes
One volume of red blood cells (washed three times with 150
mmol/L NaCl) was lysed in 8 vol lysing solution (1 mmol/L EGTA, 15
mmol/L Tris-HCl [pH 7.4], 1.4 mmol/L 2-mercaptoethanol) at 4°C.
Membranes were spun down at 10 000g during 20 minutes and
then washed twice with lysing solution. The membranes were then
suspended in 8 vol lysing solution, incubated 15 minutes at 37°C in
this solution, and spun down at 10 000g for 20 minutes.
This step was repeated once. Membranes were then washed with 8 vol of
15 mmol/L Tris-HCl (pH 7.4), resuspended in 1 vol of the same solution,
and stored at -20°C. This procedure yields membranes devoid of
endogenous calmodulin.
Ca2+-ATPase Activity
ATPase activity was measured at 37°C in a medium containing
(mmol/L) KCl 120, Tris-HCl (pH 7.4 at 37°C) 30, MgCl2 4,
EGTA 1, and ATP and CaCl2 2. Ca2+-ATPase
activity was taken as the difference between the activity measured in
the above medium and that measured in the same medium without
calcium.17
Statistical Analysis
Samples were analyzed with the unpaired t
test. Pearson correlation coefficients and stepwise multiple linear
regression analysis were used for assessment of relations
between variables. A value of P<.05 was accepted as
significant. Findings are expressed as mean±SD.
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Results |
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The clinical findings of the subjects are shown in the Table. There were no significant differences in body mass index, cholesterol levels, and systolic and diastolic blood pressures between adolescents with or without a family history of hypertension.
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Ca2+-ATPase was comparable in both groups (4.5±1.2 µmol Pi/[mg protein/h]10-1 in F+ and 5.1±1.6 in F-; P=NS) (Fig 1). Calmodulin-stimulated Ca2+-ATPase was significantly lower in adolescents with a family history of hypertension than in those without a family history of hypertension (13.6±3.9 µmol Pi/[mg protein/h]10-1 in F+ and 16.2±1.7 in F-; P<.04) (Fig 2). The insulin area under the curve after the intravenous glucose load was comparable in both groups (3413±1674 µU/mL per hour in F+ and 2752±928 in F-; P=NS).
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Calmodulin-stimulated Ca2+-ATPase was negatively correlated to both the insulin area under the curve (r=-.59, P<.005) (Fig 3) and serum cholesterol levels (r=-.38, P<.03) (Fig 4). Stepwise multiple linear regression analysis suggested that the insulin area under the curve may account for up to 36% of the variation in calmodulin-stimulated Ca2+-ATPase. When cholesterol was added to the model, the overall prediction equaled 42%. Urinary calcium excretion was comparable in both groups (1.82±0.9 mmol/d in F+ and 2.47±0.9 in F-).
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Discussion |
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Abnormalities in serum insulin levels and calcium metabolism have been reported in primary hypertensive subjects. However, it is not clear whether these changes precede the development of hypertension or are a consequence of high blood pressure.
Our findings indicate that normotensive adolescent offspring of primary hypertensive individuals have decreased calmodulin-stimulated Ca2+-ATPase activity, which is negatively correlated to both circulating insulin and cholesterol levels. A diminished Ca2+-ATPase or calmodulin-stimulated Ca2+-ATPase has been demonstrated in platelets and erythrocytes obtained from primary hypertensive subjects.6 10 18
Slightly reduced serum calcium and significantly raised plasma parathyroid hormone levels were also observed in prehypertensive young subjects genetically at risk for hypertension, supporting the view that disturbances in calcium metabolism are present in the early phase of primary hypertension and may precede the development of high blood pressure.19
Since Ca2+-ATPase is an extrusion pump, a diminished activity would lead to an intracellular calcium accumulation in vascular smooth muscle cells, and this may be of primary importance in the origin of increased peripheral vascular resistance, a characteristic feature of the hypertensive state. The reduced vasodilator response observed in obese adolescents that correlated with the degree of insulin resistance may be an expression of these findings.20
The abnormalities of intracellular calcium homeostasis described in this report may not be attributed directly to changes in calmodulin because calmodulin content and distribution in red blood cells from hypertensive subjects have been reported to be normal.10
Insulin is known to have significant effects on several transmembrane ion-exchange systems, including Na,K-ATPase and Na-H exchanger, and to directly stimulate Ca2+-ATPase activity in kidney, heart, liver, and adipocytes by increasing membrane calmodulin content and/or phosphorylation or by increasing the enzyme affinity for calcium.11 12 13 21 Since we found a decreased calmodulin-stimulated Ca2+-ATPase activity in the presence of high insulin levels, our data indicate the presence of insulin resistance and reveal still another link between abnormal insulin glucose metabolism and high blood pressure.
We have found decreased calmodulin-stimulated Ca2+-ATPase activity negatively correlated to increased serum cholesterol levels. It has been shown that the basal activity of Ca2+-ATPase as well as its response to calmodulin or to hormonal regulation depends in part on the lipid milieu.22 23 Abnormalities have also been previously described in human primary hypertension in other membrane transport systems, such as the Na,K-ATPase, Na-Li countertransport, and Na-H antiporter.24
Whatever the mechanisms involved, the above-described abnormalities in insulin, cholesterol, and Ca2+-ATPase activity as well as in sodium transport may be an expression of a widespread plasma membrane defect that precedes and may contribute to the development of high blood pressure.
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Acknowledgments |
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This work was supported by grant CONICET PID 3340/92. We are thankful to Fernando Rubinstein, MD, for his valuable advice in statistical matters.
Received June 19, 1995; first decision August 1, 1995; accepted August 18, 1995.
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