(Hypertension. 1997;29:1007-1013.)
© 1997 American Heart Association, Inc.
Articles |
4Intracellular Free Calcium Abnormalities in Fibroblasts From Non–Insulin-Dependent Diabetic Patients With and Without Arterial Hypertension
From Istituto di Medicina Interna, National Research Council (CNR) Center for the Study of Aging, Università di Padova, and Istituto di Patologia Generale, Università di Ferrara (F. Di V.) (Italy).
Correspondence to R. Nosadini, MD, Istituto di Medicina Interna, Patologia Medica I, Policlinico Universitario, Via Giustiniani 2, 35128 Padua, Italy.
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Abstract |
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Abstract As arterial hypertension is frequently associated with diabetes, it is possible that altered intracellular free calcium ([Ca2+]i) handling, as reported in non–insulin-dependent diabetic patients, is accounted for by abnormalities caused by hypertension rather than diabetes. Our aim was to investigate [Ca2+]i transients triggered by two extracellular agonists, bradykinin and angiotensin II, with or without chronic insulin exposure, in cultured skin fibroblasts from 10 normotensive and 10 hypertensive non–insulin-dependent patients, matched for age, body mass index, and metabolic control, with fibroblasts from 10 healthy control subjects. Long-term cultured fibroblasts were loaded with fura 2-AM for measurement of [Ca2+]i. Resting [Ca2+]i levels were similar in the three groups of subjects. [Ca2+]i spikes stimulated by angiotensin II (0.1 µmol/L) and bradykinin (1 µmol/L) were significantly greater in hypertensive non–insulin-dependent diabetic patients (216±43 and 374±39 nmol/L, respectively) than in normotensive patients (174±16 and 267±55 nmol/L) and control subjects (188±29 and 320±78 nmol/L). Also, ionomycin evoked a greater [Ca2+]i response in hypertensive than normotensive non–insulin-dependent diabetic patients and in control subjects. Chronic insulin exposure increased by 70% to 90% the [Ca2+]i response to both angiotensin II and bradykinin in control subjects and normotensive non–insulin-dependent diabetic patients but not in hypertensive patients. The presence of abnormalities in [Ca2+]i transients in fibroblasts from only hypertensive non–insulin-dependent diabetic patients supports the possibility that these defects are a feature of concomitant arterial hypertension rather than of diabetes or its disturbed metabolic milieu.
Key Words: calcium, intracellular • diabetes, non–insulin-dependent • angiotensin II • bradykinin
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Introduction |
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Disturbances in intracellular free calcium ([Ca2+]i) homeostasis have been reported in a variety of cell models of experimental and human arterial hypertension.1 2 Resting unstimulated [Ca2+]i in vascular smooth muscle cells was found to be higher in spontaneously hypertensive than normotensive Wistar-Kyoto rats.3 Furthermore, arginine vasopressin–induced increases in vascular smooth muscle cell [Ca2+]i were higher in spontaneously hypertensive than Wistar-Kyoto rats.4 In human essential hypertension, an increased basal platelet [Ca2+]i is a widely established finding.5 6 7 8 Platelets of patients with essential hypertension also exhibit a larger agonist-induced increase in [Ca2+]i.8 9
Increased [Ca2+]i is frequently found in insulin-dependent and non–insulin dependent diabetes mellitus (NIDDM) and obesity.10 Not only [Ca2+]i but also its hormonal regulation have been reported as abnormal in NIDDM.11 Whether disturbances in [Ca2+]i in diabetes are primary (possibly genetic) defects or acquired metabolic abnormalities remains to be established.
It is likely that both hyperglycemia and insulin deficiency affect [Ca2+]i regulation.12 It has also been suggested that a primary abnormality in calcium homeostasis may be the basic defect initiating parallel impairments in insulin action and secretion10 and a common denominator for the association between NIDDM, hypertension, and obesity.13 The fact that an abnormality in calcium metabolism is a generalized disorder of the diabetic state is supported by the finding of an increased [Ca2+]i in various tissues in experimental diabetes, including vascular smooth muscle cells and adipocytes.12 14 A striking feature of the impaired [Ca2+]i metabolism of diabetes is the wide spectrum of abnormalities involved as well as the variability and specificity of derangements in different tissues. Insulin resistance in NIDDM may be at least partly explained by an increased [Ca2+]i that interferes with the action of insulin at a postreceptor step.15 As a consequence, high [Ca2+]i can cause hyperinsulinemia. Hyperinsulinemia and insulin resistance are not only common features of NIDDM and essential hypertension but might also be involved in their pathogenesis.16 On the other hand, insulin may play a role in blood pressure control by influencing [Ca2+]i. It has been suggested that insulin may alter vascular smooth muscle function by modulating agonist-induced calcium transients.17 These similarities between diabetes and essential hypertension suggest that alterations in [Ca2+]i handling might provide a common link for the frequent association of these two diseases.10
A central question is therefore whether disturbances in [Ca2+]i handling in diabetic patients are an inherent feature of this metabolic disease or are rather linked to hypertension. Cultured skin fibroblasts offer a useful in vitro model for investigation of intrinsic and possibly genetic defects in cell function independently of the environmental abnormality caused by diabetic disease in vivo.18 Therefore, we evaluated resting [Ca2+]i and the effect of direct acute stimulation with two extracellular agonists (bradykinin and angiotensin II [Ang II]) on [Ca2+]i in long-term cultured skin fibroblasts from NIDDM patients with and without hypertension and normal control subjects. We also assessed the effect of chronic exposure to insulin on resting [Ca2+]i and on [Ca2+]i transients triggered by extracellular agonists.
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Methods |
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Patients
Ten NIDDM patients with elevated blood pressure levels were recruited consecutively from the outpatient clinic of the Department of Internal Medicine at the University Hospital in Padua (Italy) and were matched for known diabetes duration, sex, and body mass index with 10 normotensive NIDDM patients. All patients were treated with sulfonylurea, metformin, or both. The diagnosis of NIDDM was assessed according to World Health Organization criteria.19 Arterial hypertension was newly diagnosed and defined according to the fifth report of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure (JNC-V) (systolic pressure
140 mm Hg and/or
diastolic pressure 90 mm Hg).20 All
subjects were of European origin and gave informed consent to the
study. The study protocol was approved by the Ethics Committee of the
Faculty of Medicine, University of Padua. All procedures were in
accordance with our institutional guidelines. Skin biopsy was
performed, and then hypertensive patients began antihypertensive
treatment. Family history for hypertension, NIDDM, and overt nephropathy were reported as positive when one or both parents had hypertension, NIDDM, or overt nephropathy. The diagnosis of hypertension was based on the use of antihypertensive treatment or documented elevated blood pressure levels. Patients with overt nephropathy had plasma creatinine levels higher than 150 µmol/L. The criteria for the diagnosis of NIDDM were the same for the two groups of normotensive and hypertensive NIDDM patients.
Ten healthy individuals without a family history of hypertension served
as control subjects. They were matched with the patients for age, sex,
and body mass index. The presence of diabetes or impaired glucose
tolerance was excluded by an abbreviated 75-g glucose tolerance test
(baseline and 2-hour blood glucose concentration) in nondiabetic
subjects. Their demographic, clinical, and biochemical features are
shown in Table 1
.
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Analytical Methods
On the morning of the skin biopsy, height and weight were
recorded with patients without shoes and in light indoor clothing;
blood was taken for determination of glycated hemoglobin
A1c21 and serum creatinine
(Jaffé reaction rate method) (range of normal values, 55 to
125 µmol/L).
Blood pressure (diastolic phase V) was measured to the nearest 2 mm Hg with subjects in the sitting position after 10 minutes of rest using a Hawksley random-zero sphygmomanometer (12x35-cm cuff) on at least 3 different days during 2 weeks. Causes of secondary hypertension were excluded by a complete medical examination, which included a 12-lead electrocardiogram.
Glomerular filtration rate was measured after a single intravenous injection of 51Cr-EDTA (Amersham International plc) by determining the radioactivity in venous blood samples taken from the other arm over 5 hours.22
Cell Culture
A skin biopsy was taken from the anterior surface of the left
forearm by excision under topical anesthetic (ethyl chloride).
Fibroblasts were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. After passage 4, cells were
harvested and stored in liquid nitrogen. For each experiment,
fibroblasts were then thawed and grown as described above. All
experiments were performed between passages 6 and 10. Cells were made
quiescent by 36 hours of serum
deprivation.18
Unless otherwise indicated, experiments were performed in saline solution (standard saline) containing (mmol/L) NaCl 125, KCl 5.0, MgSO4 1, NaHPO4 1, glucose 5.5, NaHCO3 5, CaCl2 1, and HEPES 20 (pH 7.4).
[Ca2+]i Measurement
[Ca2+]i was measured by a
microfluorimetric technique using the
[Ca2+]i-sensitive probe fura 2-AM.
Fluorescence measurement was performed in a
fluorescence spectrophotometer (Perkin-Elmer LS-50) equipped
with a thermostatically controlled cuvette holder and magnetic stirrer,
as previously described by Di Virgilio et al.23 Briefly,
fibroblasts were plated to confluence onto glass coverslips 36 hours
before the experiment in the absence of serum. Shortly before each
determination, the coverslips were rinsed and incubated for 30 minutes
at 37°C in standard saline containing 2 µmol/L fura 2-AM.
Coverslips were then rinsed to remove extracellular dye and were kept
in standard saline until used. Cells were excited at 340/380 nm, and
fluorescence was recorded at 500 nm.
Experimental Protocol
Resting and agonist-stimulated [Ca2+]i
values were measured in cells made quiescent by 36 hours of serum
deprivation. The effects on
[Ca2+]i of bradykinin (1 µmol/L), Ang
II (0.1, 1.0, and 10 µmol/L), ionomycin (100 µmol/L), and
thapsigargin (100 nmol/L) were determined by evaluating maximal
responses after addition of the agonist.
The effects of ionomycin and thapsigargin were evaluated in cells maintained quiescent in Ca2+-free saline containing EGTA. For evaluation of the effects of insulin treatment on [Ca2+]i responses to the previous agonists, cells were preincubated with insulin (1071 pmol/L), contained in the quiescent medium, for 36 hours.
Chemicals
All chemicals were of the purest grade available from Sigma
Chemical Co, except for fura 2-AM, which was from Boehringer
Mannheim Biochemica.
Statistical Analysis
Statistical analysis was performed by ANOVA; comparisons
between groups were conducted with the Newman-Keuls test or
2 analysis.24 Relationships
between variables were tested with linear regression
analysis. A two-tailed value of P<.05 was
considered significant. Data are given as mean±SD unless otherwise
stated.
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Results |
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Table 1
shows the clinical features of NIDDM patients with and
without arterial hypertension. The two groups were well
matched with control subjects with regard to age, body mass index, and
serum creatinine. No difference in diabetes duration and
glycated hemoglobin A1c was observed between normotensive
and hypertensive NIDDM patients. Family history of diabetes was similar in normotensive and hypertensive NIDDM patients. A positive family history of hypertension was more frequently found in hypertensive NIDDM than in normotensive NIDDM patients and control subjects (P<.01). A family history of overt nephropathy was reported in only 1 of 10 hypertensive NIDDM patients.
In the absence of insulin, resting [Ca2+]i
levels were similar in fibroblasts from NIDDM patients with and without
hypertension and control subjects and were within the range reported
for these or other cell types (Table 2). Addition of
1 µmol/L bradykinin evoked a fast and transient
[Ca2+]i rise followed by a smaller and
sustained shoulder (Fig 1).
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The kinetics (Fig 1) of the [Ca2+]i increase
after bradykinin addition were the same in the three groups
(time-to-peak, about 12 seconds), but the maximal increase above
resting level was higher in fibroblasts from hypertensive NIDDM
patients (374±39 nmol/L) than that observed in cells from normotensive
NIDDM patients and control subjects (267±55 and 320±78 nmol/L,
respectively), although the difference reached conventional
significance (P<.01) only between NIDDM patients.
In the absence of insulin, 0.1 µmol/L Ang II triggered a
[Ca2+]i rise similar to, albeit slower than
(time-to-peak, about 26 seconds), that evoked by bradykinin (Fig 1).
Peak increase was significantly higher (P<.01) in
fibroblasts from hypertensive NIDDM patients (216±43 nmol/L) than that
observed in normotensive NIDDM patients and control subjects (174±16
and 188±29 nmol/L, respectively) (Table 2).
We also studied the dose-response curve of
[Ca2+]i transients to 0.1, 1.0, and 10
µmol/L Ang II concentrations. Higher responses were observed in
hypertensive than in normotensive NIDDM patients and control subjects
at the lower Ang II concentrations. However, at maximal hormonal
stimulation, no further increase in [Ca2+]i
transients was seen in hypertensive NIDDM patients, whereas
normotensive NIDDM patients and control subjects exhibited a
significantly increased response compared with that in the previous two
steps (Fig 2). That the angiotensin effect
was receptor mediated was confirmed by the complete inhibition of the
[Ca2+]i spike by the specific Ang II receptor
antagonist saralasin (Fig 1d).
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) vs NIDDM patients without hypertension (
) and
normotensive control subjects (
). Mean±SD are shown.
To verify the possible influence of different variables on
[Ca2+]i transients, we performed multiple
linear regression analysis for the whole group of NIDDM
patients. In this model (with [Ca2+]i
transients triggered by Ang II or bradykinin as the dependent
variables), sex, known duration of diabetes, systolic and
diastolic blood pressures, and glycated hemoglobin were not
significantly related to [Ca2+]i changes
(Tables 3 and 4).
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We then tested the effect of chronic (36 hours) incubation with insulin
(1071 pmol/L) on resting [Ca2+]i levels and
on [Ca2+]i transients triggered by bradykinin
(1 µmol/L) and Ang II (0.1 µmol/L). Insulin treatment
significantly increased resting [Ca2+]i in
fibroblasts from all three groups (Table 2). Moreover, insulin
potentiated the agonist-induced [Ca2+]i rise
in control subjects and normotensive NIDDM patients but not
hypertensive NIDDM patients, thus bringing the
[Ca2+]i peak to about the same level in the
three cell populations.
To verify whether the differences in [Ca2+]i
handling after agonist addition were mediated by abnormalities in
receptor activities or in [Ca2+]i stores, we
also evaluated the effect of a Ca2+ ionophore, ionomycin,
in the absence of extracellular calcium. After a 30-minute incubation
period in Ca2+-free saline containing EGTA, the
[Ca2+]i response to ionomycin was higher in
NIDDM patients than in control subjects (Fig 3).
[Ca2+]i transients evoked by ionomycin in
fibroblasts from NIDDM patients with hypertension were significantly
greater than those of normotensive NIDDM patients. Chronic exposure to
insulin further emphasized ionomycin effects, although statistical
significance was reached only in normal control subjects and NIDDM
patients with hypertension (Fig 3).
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P<.05, 
P<.05, We also evaluated the effect of a specific endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, on [Ca2+]i in fibroblasts from NIDDM patients and control subjects. To prevent calcium influx, we incubated cells in Ca2+-free saline containing EGTA. Thapsigargin also caused a prolonged increase in [Ca2+]i, but this effect was similar in control subjects and NIDDM patients with and without hypertension and was not further enhanced by insulin pretreatment.
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Discussion |
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The prevalence of arterial hypertension is approximately twofold higher in NIDDM patients than in the normal population, and several studies have reported that more than 50% of NIDDM patients are hypertensive.25 Arterial hypertension may be a consequence of the metabolic abnormalities of diabetes (such as hyperinsulinemia, increased plasma volume, and hyperglycemia), or alternatively, an increase in blood pressure levels could be a simple clinical concomitant of NIDDM. The aim of the present study was to elucidate some putative pathophysiological mechanisms underlying the occurrence of hypertension in NIDDM patients.
High [Ca2+]i is potentially important in the pathogenesis of hypertension.13 It has been suggested that disorders in cellular calcium handling are the key link that explains the coexistence of hypertension and insulin resistance.15 Increased [Ca2+]i in vascular smooth muscle cells and increased reactivity to vasoactive calcium-mobilizing hormones, such as vasopressin4 26 and Ang II,26 may lead to increased vascular smooth muscle cell contractility and thus to elevated peripheral vascular resistance, decreased peripheral blood flow, and increased systemic blood pressure in NIDDM patients.27 28 29
The results of the present study show that stimulation of skin fibroblasts (cultured for several passages in identical media) using agonists such as bradykinin and Ang II evoked higher [Ca2+]i transients in NIDDM patients with arterial hypertension than in those without hypertension or in normal control subjects despite similarly higher blood glucose levels in both normotensive and hypertensive NIDDM patients in vivo. These findings suggest that arterial hypertension, rather than diabetes, may account for these abnormalities in cellular calcium handling. Although several studies using circulating blood cells showed that basal levels of [Ca2+]i are elevated in hypertensive individuals,5 6 7 8 we observed similar [Ca2+]i values in fibroblasts from the three groups. A possible explanation for this finding may be the experimental conditions of our study. In contrast with previous studies, fibroblasts were kept for several passages in well-defined media conditions and were made quiescent before agonist addition. Our data are in agreement with those reported recently by Siffert et al30 in immortalized lymphoblasts from normotensive subjects and essential hypertensive patients.
The differences observed in fibroblasts from NIDDM patients with hypertension could not be accounted for by a different pattern in other parameters of metabolic control—duration of diabetes or body mass index—which were similar to those of NIDDM patients without hypertension. Although NIDDM patients were not well matched for sex, it is unlikely that this may contribute to the differences observed because a multiple regression analysis performed for all groups of NIDDM patients did not show any significant relationship between sex and [Ca2+]i changes. It is also of note that blood pressure was not related to the [Ca2+]i response to Ang II and bradykinin, suggesting that the abnormalities were not simply secondary to actual blood pressure values.
Furthermore, skin fibroblasts were cultured in similar metabolic conditions in both control subjects and NIDDM patients for several passages. Hence, we suggest that abnormalities of the [Ca2+]i response to bradykinin and Ang II are an intrinsic feature of cells derived from hypertensive NIDDM patients rather than a simple consequence of the metabolic challenge of diabetes. This view is also supported by the finding of increased [Ca2+]i in normotensive offspring of parents with essential hypertension.31 It is of note that human skin fibroblasts have specific receptors for Ang II, which have been suggested to play an important role in the hypertrophy and proliferation of vascular smooth muscle cells.32 With regard to Ang II regulation of [Ca2+]i, the overall dose-response curve of [Ca2+]i was characterized by several and different abnormalities in NIDDM patients. At low, more physiological, Ang II concentrations, NIDDM patients with hypertension had higher [Ca2+]i surges in response to hormonal stimulation. However, when fibroblasts were exposed to pharmacological Ang II concentrations (10 µmol/L), [Ca2+]i transients were significantly higher in normal control subjects and normotensive NIDDM patients than in hypertensive NIDDM patients. These data suggest that hypertensive NIDDM patients had an increased sensitivity and lower maximal responsiveness to Ang II compared with normal control subjects and normotensive NIDDM patients. These abnormalities of [Ca2+]i response to Ang II could be accounted for by an increased number of Ang II receptors and by an impaired transduction of the signaling system at the postreceptor level.
Both bradykinin and Ang II bind to specific receptors that use a GTP binding protein to activate phospholipase C, which in turn leads to an increase of [Ca2+]i due to the formation of inositol 1,4,5-triphosphate.33 Any alteration of this signaling pathway could determine the abnormalities observed in diabetic patients. Since our experiments with ionomycin demonstrated that the [Ca2+]i increase was greater in NIDDM patients with hypertension, it is unlikely that changes in agonist receptor status may explain our results. A greater rise in [Ca2+]i after ionomycin addition, in the absence of extracellular calcium, in NIDDM patients with hypertension suggests the presence of an increased [Ca2+]i pool in intracellular stores. On the contrary, no differences in [Ca2+]i response after the addition of thapsigargin, an inhibitor of the [Ca2+]-(Mg2+)-ATP of the endoplasmic reticulum,34 were observed between control subjects and NIDDM patients. These results suggest heterogeneity in the intracellular Ca2+ stores, some of which may be insensitive to thapsigargin but fully dischargeable by ionomycin.35
Chronic treatment with insulin clearly potentiated the [Ca2+]i spikes after agonist addition in normal control subjects and NIDDM patients without hypertension. The potentiation was particularly striking in the case of the [Ca2+]i rise triggered by Ang II, which was nearly doubled in insulin-treated fibroblasts. The molecular basis for insulin-dependent potentiation is not clear at present. It is known that the insulin receptor is not directly linked to generation of Ca2+-mobilizing second messengers, such as inositol 1,4,5-trisphosphate of cADP-ribose, but a cross talk exists between G protein–coupled Ca2+-mobilizing receptors and receptors with intrinsic tyrosine kinase activity such as the insulin receptor. Tyrosine kinase receptors can phosphorylate phospholipase C gamma and cause its translocation to the plasma membrane, where it can interact with its substrate, phosphatidylinositol 4,5-triphosphate.36 37 An additional explanation for the increased [Ca2+]i mobilization observed in insulin-treated cells could be an enhanced number of bradykinin and Ang II receptors. Ling et al38 have recently observed that chronic but not acute insulin exposure increases Ang II receptor density and channel sensitivity to [Ca2+]i in mesangial cells from rat glomeruli. We are currently performing experiments aimed at clarifying this issue. However, this latter finding could explain some of the abnormalities of the dose-response curve to Ang II stimulation we observed in hypertensive NIDDM patients.
Moreover, in contrast to the results obtained in cells from normal control subjects and NIDDM patients without hypertension, insulin did not potentiate the [Ca2+]i rise after agonist addition in fibroblasts from hypertensive NIDDM patients. Failure to respond to insulin could be due to a decrease of cell membrane insulin receptors or functional uncoupling of the receptors from the effector system. We have recently reported an impaired insulin action at the extrahepatic level on glucose uptake in hypertensive but not in normotensive NIDDM patients.39 40 Therefore, disturbances in insulin action related to hypertension rather than diabetic disease could account for the differences in [Ca2+]i homeostasis between hypertensive and normotensive NIDDM patients. It has been reported that the increase in [Ca2+]i reduces the ability of insulin to affect further increases in [Ca2+]i.41 Thus, it is possible that in hypertensive NIDDM patients, [Ca2+]i mobilization is already maximally stimulated. However, this latter hypothesis appears unlikely, as insulin further enhanced the effect of ionomycin on [Ca2+]i transients. Although the mechanism or mechanisms explaining the heterogeneity of [Ca2+]i responses to different agonists in fibroblasts from NIDDM patients remain unclear, cell overreactivity to Ca2+-mobilizing stimuli of NIDDM patients with hypertension could have important implications for the pathogenesis of blood pressure elevation, since Ca2+ is a pivotal mediator of the vasoconstrictive action of Ang II and alterations in resting or stimulated [Ca2+]i have been reported by many authors.8 9
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Acknowledgments |
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This work has been partially supported by National Research Council (CNR) grants, Progetto Finalizzato Invecchiamento No. 9300428.PF40, target projects BTBS, and ASCRO. We acknowledge the contribution of LADSEB-CNR (Prof Giovanni Pacini and Dr Karl Thomaseth) for supplying the method for mathematical analysis of glomerular filtration rate.
Received April 18, 1996; first decision June 17, 1996; accepted October 14, 1996.
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