SHORT REPORT

Nisoldipine Blocks the Increase ofIntracellular Free Calcium-ionConcentration Associated with ElevatedSodium–lithium CountertransportActivity in Erythrocytes in Patients withNIDDMJ. Fujitaa, K. Tsudab, H. Obayashic, I. Fukuic, H. Ishidaa, Y. Seinoa

aDepartment of Metabolism and Clinical Nutrition, Kyoto UniversityFaculty of MedicinebDepartment of Integrated Human Science, Kyoto UniversitycKyoto Microbiological Institute, Kyoto, Japan

To understand the mechanism by which elevated sodium–lithium countertransport activity(SLC) associates with increased intracellular free calcium-ion concentration ([Ca2+]i), weinvestigated the relationship between SLC and the effects of the extracellular Ca2+

concentration ([Ca2+]o) and a Ca2+-channel blocker, nisoldipine, on [Ca2+]i in erythrocytesfrom 48 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). Therewas a significant correlation between SLC and [Ca2+]i. Nisoldipine in the incubationmedium significantly decreased [Ca2+]i, and there was a significant positive correlationbetween SLC and the degree of [Ca2+]i decrease. When the [Ca2+]o was elevated, [Ca2+]i

was significantly increased, but nisoldipine almost completely suppressed this increase of[Ca2+]i. There was a significant positive correlation between SLC and the degree of thesuppression. These data suggest that elevated SLC correlates with increased [Ca2+]i, andthat the increased [Ca2+]i might be due to the increased Ca2+ influx through adihydropyridine-sensitive Ca2+ pathway. 1997 by John Wiley & Sons, Ltd.

No of Figures: 5. No of Tables: 0. No of Refs: 15.

KEY WORDS Sodium–lithium countertransport activity Intracellularcalcium Nisoldipine NIDDM

Received 10 September 1996; revised 14 January 1997; accepted 19 January 1997

in erythrocytes from NIDDM, and determined theirIntroductionrelationship with SLC.

We have reported that elevated erythrocyte sodium–lithium countertransport activity (SLC) strongly reflects a Patients and Methodsfamily history of hypertension and that it can be a useful

The same 48 patients with NIDDM (23 men and 25marker of nephropathy in patients with non-insulin-women, aged 25 to 64 years) as in our previous report2dependent (Type 2) diabetes mellitus (NIDDM).1 Inwere included in this study. Using fasting blood samples,addition, our recent study suggested that increasedthe maximal rate of SLC was measured according to theintracellular free calcium-ion concentration ([Ca2+]i)method described by Canessa et al.,3 with modificationsmight be the underlying mechanism for the closeas described in our previous study.1,2association of elevated SLC with both predisposition to

Measurement of [Ca2+]i was performed using fura-2hypertension and nephropathy.2according to a method described by David-Dufilho etTo explore this mechanism further, we have done aal.4 with modifications.2follow-on study investigating the effects of nisoldipine,

a 1,4-dihydropyridine Ca2+-channel blocker, on [Ca2+]iEffects of [Ca2+]o and Nisoldipine on[Ca2+]iAbbreviations: [Ca2+]i: intracellular free calcium-ion concentration,

[Ca2+]o: extracellular free calcium concentration, SLC: sodium lithiumcountertransport activity To investigate the effects of extracellular Ca2+ concen-* Correspondence to: Dr J. Fujita, Department of Metabolism and

tration ([Ca2+]o) and nisoldipine on [Ca2+]i in vitro, [Ca2+]iClinical Nutrition, Kyoto University Faculty of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-01, Japan was measured in the following conditions:

499CCC 0742–3071/97/060499–04$17.50 1997 by John Wiley & Sons, Ltd. DIABETIC MEDICINE, 1997; 14: 499–502

SHORT REPORT

Figure 1. Condition A: Correlation between erythrocytesodium–lithium countertransport activity (SLC) and erythrocyte

Figure 3. The effects of nisoldipine and the influence of [Ca21]ointracellular free calcium-ion concentration ([Ca21]i); n 5 48,on [Ca21]i. The heights of columns indicate mean values andr 5 0.46, p , 0.01 ([Ca21]o 5 1 mmol l21)vertical bars indicate SD values of each condition (***p , 0.001)

A. [Ca2+]o = 1 mmol l−1, nisoldipine (−)B. [Ca2+]o = 1 mmol l−1, nisoldipine (+) The effects of nisoldipine and the influences of [Ca2+]oC. [Ca2+]o = 2 mmol l−1, nisoldipine (−) on [Ca2+]i are shown in Figure 3. When the [Ca2+]o wasD. [Ca2+]o = 2 mmol l−1, nisoldipine (+). 1 mmol l−1, the addition of 10−6 mol l−1 of nisoldipine

significantly decreased the [Ca2+]i from 82.7 ± 2.2[Ca2+]i in each condition was expressed as [Ca2+]iA,nmol lRBC−1 ([Ca2+]iA) to 80.7 ± 1.9 nmol lRBC−1 ([Ca2+]iB)[Ca2+]iB, [Ca2+]iC, and [Ca2+]iD, respectively. Nisoldipine(p , 0.001). When [Ca2+]o was elevated from 1 totreatment was performed by incubating the fura-2 loaded2 mmol l−1, [Ca2+]i was significantly increased toerythrocytes in buffer A containing 10−6 mol l−1 of nisoldi-92.7 ± 2.5 nmol lRBC−1 ([Ca2+]ic) compared to [Ca2+]iApine (from 10−4 mol l−1 solution dissolved in ethanol) for(p , 0.001). The addition of 10−6 mol l−1 of nisoldipine10 min at 37°C. [Ca2+]o was then adjusted in the casesignificantly decreased [Ca2+]i to 83.7 ± 2.4 nmol lRBC−1of conditions C and D. All the procedures were performed([Ca2+]iD) compared to [Ca2+]iC (p , 0.001).in dark conditions.

The degree of decrease in [Ca2+]i from [Ca2+]iA to[Ca2+]iB (D[Ca2+]iA-B) was significantly correlated withStatistical AnalysesSLC (r = 0.49, p , 0.01; Figure 4). The degree of decrease

Statistical analyses were performed as described in our in [Ca2+]i from [Ca2+]iC to [Ca2+]iD (D[Ca2+] iC-D) alsoprevious report.2 significantly correlated with SLC (r = 0.47, p , 0.01;

Figure 5).Results

DiscussionIn the whole patient group, in the incubations withoutnisoldipine, [Ca2+]iA and [Ca2+]iC were significantly corre- The present study has demonstrated that erythrocyteslated with SLC (r = 0.46, p , 0.01 (Figure 1) and r = 0.44, from NIDDM patients with elevated SLC have increasedp , 0.01 (Figure 2), respectively).

Figure 4. Correlation between erythrocyte sodium–lithiumFigure 2. Condition C: Correlation between erythrocytesodium–lithium countertransport activity (SLC) and erythrocyte countertransport activity (SLC) and the degree of decrease in

[Ca21]i from [Ca21]iA to [Ca21]iB (D[Ca21]iA-B); n 5 48, r 5 0.49,intracellular free calcium-ion concentration ([Ca21]i); n 5 48,r 5 0.44, p , 0.01 ([Ca21]o 5 2 mmol l21) p , 0.01 ([Ca21]o 5 1 mmol l21)

500 J. FUJITA ET AL.

SHORT REPORTin vivo. If so, dihydropyridine Ca2+-channel blockersmight be useful in preventing the initiation and pro-gression of diabetic complications, such as nephropathyand macroangiopathy, through their anti-proliferativeeffects in addition to their anti-hypertensive effects.Further investigations are required to substantiate thispossibility. In addition, it is not certain if hypertensivesubjects behave in a similar manner to normotensivesubjects. Subjects with hypertension may have increasedSLC and higher intracellular calcium and differentresponses to nisoldipine.

In conclusion, our present study has shown thatnisoldipine can block the increase in [Ca2+]i associated

Figure 5. Correlation between erythrocyte sodium–lithium with elevated SLC. This suggests that the mechanism ofcountertransport activity (SLC) and the degree of decrease inthe close association of elevated SLC, with its known[Ca21]i from [Ca21]iC to [Ca21]iD (D[Ca21]iC-D); n 5 48, r 5 0.47,associations with predisposition to hypertension andp , 0.01 ([Ca21]o 5 2 mmol l21)diabetic nephropathy, includes increased Ca2+ influxthrough a dihydropyridine-sensitive Ca2+ pathway. Therelevance of these findings to diabetic nephropathy risk[Ca2+]i both in the basal state and in a high extracellular

Ca2+ concentration, and that nisoldipine can block this can be answered by prospective studies.increase in [Ca2+]i.

Dihydropyridine binding sites, although somewhatdifferent from those of excitable cells, have been found Acknowledgementson human erythrocyte membrane.5 The existence ofa voltage-dependent and/or dihydropyridine-sensitive This study was supported by Research Grants from thecation transport pathway has also been shown in human Ministry of Education, Science, Sports and Culture oferythrocyte membrane.6,7 In addition, an increase of Japan, and the Ministry of Health and Welfare of Japan,erythrocyte [Ca2+]i has been shown to be associated with and by a grant for diabetes research from Otsukaan increased dihydropyridine-sensitive calcium influx in Pharmaceutical Co., Ltd, Tokushima, Japan, and Takedaspontaneously hypertensive rats,8 and in patients with Medical Research Foundation, Osaka, Japan. We alsoessential hypertension.9,10 Therefore, the blocking effects thank Bayer Yakuhin, Ltd, Osaka, Japan for generouslyof nisoldipine on the increased [Ca2+]i associated with supplying nisoldipine.the elevated SLC found in our present study might beexerted through this VDCC-like channel.

It has been reported that there is a significant correlation Referencesbetween SLC and total peripheral vascular resistance inpatients with essential hypertension.11 Elevated SLC also 1. Fujita J, Tsuda K, Seno M, Obayashi H, Fukui I, Seino Y.

Erythrocyte sodium–lithium countertransport activity as ahas been shown to be associated with renal hypertrophymarker of predisposition to hypertension and diabeticin IDDM,12 and with left ventricular hypertrophy innephropathy in NIDDM. Diabetes Care 1994; 17: 977–NIDDM.13 In addition, we have recently reported that982.

elevated SLC is significantly correlated with increased 2. Fujita J, Tsuda K, Seno M, Obayashi H, Fukui I, Seinointracellular sodium and [Ca2+]i concentration in NIDDM, Y. Elevated erythrocyte sodium–lithium countertransport

activity correlates with increased intracellular sodiumand suggested that this might explain the association ofand free calcium-ion concentration in Type 2 diabetes.elevated SLC with predisposition to hypertension andDiabetic Med 1996; 13: 53–58.nephropathy.2 These findings suggest that there are

3. Canessa M, Adragna N, Solomon HS, Connolly TM,common abnormalities of calcium handling in these Tosteson DC. Increased sodium–lithium countertransportcells, including erythrocytes. Although, the possible in red cells of patients with essential hypertension. N

Engl J Med 1980; 302: 772–776.link between SLC, predisposition to hypertension and4. David-Dufilho M, Montenay-Garestier T, Devynck MA.nephropathy, and changes in calcium influx remains

Fluorescence measurements of free Ca2+ concentration inspeculative and cannot be answered by this cross-human erythrocytes using the Ca2+-indicator fura-2. Cell

sectional study. It is possible that SLC and abnormal Calcium 1988; 9: 167–179.calcium transport across the cell membrane represent 5. Striessnig J, Zering G, Glossmann H. Ca2+ antagonist

receptor sites on red blood cell membranes. Eur Jtwo separate cell membrane defects.Pharmacol 1985; 108: 329–330.Dihydropyridine Ca2+-channel blockers have been

6. Halperin JA, Brugnara C, Tostesson MT, Ha TV, Tostessonreported to prevent proliferation of vascular smoothDC. Voltage-activated cation transport in human erythro-

muscle cells,14 and of renal mesangial cells.15 It is cytes. Am J Physiol 1989; 257: C986–C996.possible that nisoldipine in a therapeutic dosage could 7. David-Dufilho M, Astarie C, Pernollet MG, Bergougnan

L, Comte A, Dubray C, et al. Modulation by externalexert Ca2+ entry blocking effects on other types of cells

501NA+–LI+ COUNTERTRANSPORT AND INTRACELLULAR CALCIUM

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8. Morris MJ, David-Dufilho M, Devynck MA. In vivo and with insulin dependent diabetes mellitus (IDDM). Am Jin vitro effects of isradipine on cytosolic Ca2+ concentration Hypertens 1989; 2: 174–177.in erythrocytes from spontaneously hypertensive rats. Am 13. Sampson MJ, Denver E, Foyle WJ, Dawson D, PinkneyJ Hypertens 1992; 5: 887–891. J, Yudkin JS. Association between left ventricular hypertro-

9. Khalil-Manesh F, Venkataraman K, Samant D. Effects of phy and erythrocyte sodium–lithium exchange in normo-diltiazem on cation transport across erythrocyte mem- tensive subjects with and without NIDDM. Diabetologiabranes of hypertensive humans. Hypertension 1987; 9: 1995; 38: 454–46018–23. 14. Nilsson J, Sjolund M, Palmberg L, Von Euler AM, Jonzon

10. David-Dufilho M, Astarie C, Pernollet MG, Pino MD, B, Thyberg J. The calcium antagonist nifedipine inhibitsLevenson J, Simon A, et al. Control of the erythrocyte arterial smooth muscle cell proliferation. Atherosclerosisfree Ca2+ concentration in essential hypertension. Hyper- 1985; 58: 109–122.tension 1992; 19: 167–174. 15. Shulz PJ, Raij L. Inhibition of human mesangial cell

11. Weder AB, Fitzpatrick MA, Torretti BA, Hinderliter AL, proliferation by calcium channel blockers. HypertensionEgan BM, Julius S. Red blood cell Li+–Na+ countertransport, 1990; 15(suppl I): I76–I80.Na+–K+ cotransport, and the hemodynamics of hyperten-sion. Hypertension 1987; 9: 459–466.

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