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(Hypertension. 1996;28:209-212.)
© 1996 American Heart Association, Inc.

Articles

Effects of Insulin on Calcium Metabolism and Platelet Aggregation

Mari Ishida; Takafumi Ishida; Norihisa Ono; Hideo Matsuura; Mitsuaki Watanabe; Goro Kajiyama; Masayuki Kambe; Tetsuya Oshima

the First Department of Internal Medicine (M.I., T.I., N.O., H.M., M.W., G.K.) and Department of Clinical Laboratory Medicine (M.K., T.O.), Hiroshima (Japan) University School of Medicine.

Correspondence to Tetsuya Oshima, MD, Department of Clinical Laboratory Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan.

	Abstract

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The influence of insulin on platelets in vitro has not been exhaustively investigated. To clarify whether insulin affects Ca²⁺ metabolism in platelets directly or through alteration of other systems regulating intracellular Ca²⁺ homeostasis, we examined the effect of insulin both alone and in combination with prostaglandin E₁ on platelet aggregation and Ca²⁺ metabolism. Incubation of rat platelets with insulin reduced thrombin-induced Ca²⁺ influx but did not change thrombin-evoked release of Ca²⁺ from internal stores or the size of internal Ca²⁺ stores. The interactive effects of insulin with prostaglandin E₁ were only additive, and insulin did not augment the effects of prostaglandin E₁ on platelet Ca²⁺ metabolism. In contrast, insulin did not inhibit thrombin-induced platelet aggregation but did augment inhibition of platelet aggregation by prostaglandin E₁. Our results suggest that insulin inhibits platelet function by both prostaglandin E₁–dependent and -independent mechanisms.

Key Words: insulin • calcium • prostaglandins • platelet aggregation

	Introduction

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Vascular disease is the most common complication in hypertension. Platelets play a key role in the development of vascular disease and related thrombotic disorders,¹ and platelets from spontaneously hypertensive rats exhibit increased cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in resting and stimulated conditions^{2 3 4} as well as various functional abnormalities.⁵ Recently, it has been reported that insulin resistance and hyperinsulinemia are observed in hypertensive subjects.⁶ A small number of reports have shown an effect of insulin on platelet aggregation,^{7 8 9} and their results are controversial. Furthermore, information is limited regarding the effect of insulin on Ca²⁺ mobilization in platelets,¹⁰ although cytosolic free Ca²⁺ is an essential intracellular second messenger that determines cell function.

It has been suggested for various cell types that insulin itself affects intracellular Ca²⁺ metabolism. Ca²⁺-ATPase has been shown to be affected by insulin in renal basolateral membrane,^{11 12} adipocytes,¹³ and liver plasma membrane.¹⁴ Furthermore, in vascular smooth muscle cells, it has been demonstrated that insulin attenuates agonist-induced Ca²⁺ influx and a voltage-dependent Ca²⁺ response¹⁵ and accelerates Ca²⁺ extrusion from the cytosol after agonist stimulation.¹⁶ Kahn and coworkers^{7 17} have found that insulin augments increases in the cellular level of cAMP induced by prostaglandin E₁ (PGE₁) in platelets. These results raise the possibility that Ca²⁺ metabolism in platelets is affected by insulin either directly or through alteration of other systems regulating intracellular Ca²⁺ homeostasis. The aim of this study was to examine the effect of insulin, both alone and in combination with PGE₁, on Ca²⁺ metabolism and platelet aggregation. The results may be helpful in understanding the role of insulin in the development of vascular disease in hypertensive individuals.

	Methods

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Materials
Bovine insulin was obtained from Sigma Chemical Co and dissolved in 0.01N HCl. Fura 2-acetoxymethyl ester (fura 2-AM, Molecular Probes) was dissolved in dimethyl sulfoxide (DMSO). Thrombin was dissolved in deionized water, ionomycin in DMSO, and PGE₁ in ethanol (all from Sigma). The final concentrations of DMSO and ethanol in media never exceeded 0.1%.

Procedures
Male Wistar rats were anesthetized with thiamylal sodium, and blood was drawn from the inferior vena cava into a syringe containing 3.8% trisodium citrate. Platelets were prepared and [Ca²⁺]_i was measured as previously described.^{2 3 4} In brief, after platelet-rich plasma was centrifuged, platelets were separated from plasma by gel filtration with a Sepharose 2B-CL column (Pharmacia LKB Biotechnology AB) that had been equilibrated with medium containing (mmol/L) NaCl 145, KCl 5, MgSO₄ 1, HEPES 10, and glucose 5 (pH 7.4). The platelet suspension was incubated with 2 µmol/L fura 2-AM for 30 minutes and refiltered through the Sepharose column for removal of extracellular fura 2-AM. Platelets were adjusted to a concentration of 5x10⁷ cells per milliliter, and CaCl₂ was added to the cell suspension at a final concentration of 1 mmol/L. The platelets were preincubated with 10^-6 mol/L insulin for 30 minutes and/or 10^-8 mol/L PGE₁ for 2 minutes. We preincubated the platelet suspension with insulin at room temperature to prevent an increase in resting [Ca²⁺]_i and attenuation of the response due to prolonged incubation at 37°C.² We performed only a subset of experiments to determine whether we could detect the effect of insulin pretreatment at 37°C. Controls were incubated with the vehicle of insulin and PGE₁. Aliquots of the cell suspension were stirred continuously in a thermostat-controlled cuvette at 37°C; fluorescence was monitored with a spectrofluorophotometer (RF-5000, Shimadzu) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. We measured resting [Ca²⁺]_i, thrombin-evoked changes in [Ca²⁺]_i in the presence or absence of extracellular Ca²⁺, and the increase in [Ca²⁺]_i in response to 5 µmol/L ionomycin in Ca²⁺-free buffer as an index of the size of intracellular Ca²⁺ stores.^{3 18} Corrections were applied for extracellular fura 2-AM leakage with the use of EGTA² and for autofluorescence by subtraction of the fluorescence of unloaded platelets and test reagents. Extracellular fura 2-AM leakage was determined for each sample. [Ca²⁺]_i was calculated by the general formula described by Grynkiewicz et al.¹⁹

For aggregation studies, platelets were prepared by gel filtration and adjusted to 5x10⁸ cells per milliliter. Washed platelets were incubated with insulin, PGE₁, or both using the protocol described above. Platelet aggregation was monitored on a six-channel aggregometer (NBS Hematoracer 601, Niko Bioscience) equipped with a stirring apparatus and thermostat-controlled cuvette holder. Platelet aggregation was expressed as percentage of maximal aggregation.

Values are expressed as mean±SE. Statistical analysis was performed with paired Student's t test. A value of P<.05 was accepted as significant.

	Results

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Effect of Insulin Alone and With PGE₁ on [Ca²⁺]_i
Insulin at 10^-6 mol/L caused a slight but significant decrease in resting [Ca²⁺]_i (Fig 1). In the presence of 1 mmol/L extracellular Ca²⁺, the thrombin-evoked increase in [Ca²⁺]_i was significantly attenuated by 10^-6 mol/L insulin (Fig 2). Insulin did not affect the thrombin-induced increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which represents Ca²⁺ release from internal stores (Fig 3), nor did it affect the size of internal Ca²⁺ stores (Fig 4).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effects of insulin, prostaglandin E1 (PGE1), or both on resting [Ca2+]i in rat platelets. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before measurement. Results are shown as mean±SE of 11 different experiments. *P<.01, **P=.0001 vs control; P<.01 vs insulin.

View larger version (37K): [in this window] [in a new window]   Figure 2. Effects of insulin, prostaglandin E1 (PGE1), or both on thrombin-evoked increases in [Ca2+]i in rat platelets in the presence of extracellular Ca2+. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before stimulation with 0.1 U/mL thrombin. Results are shown as mean±SE of nine different experiments. *P=.0001 vs control; P=.0001 vs insulin; ¶P<.001 vs PGE1.

View larger version (39K): [in this window] [in a new window]   Figure 3. Effects of insulin, prostaglandin E1 (PGE1), or both on thrombin-evoked increases in [Ca2+]i in rat platelets in the absence of extracellular Ca2+. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before stimulation with 0.1 U/mL thrombin. Results are shown as mean±SE of nine different experiments. *P<.01 vs control; P<.001 vs insulin.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effects of insulin, prostaglandin E1 (PGE1), or both on ionomycin-evoked increases in [Ca2+]i in rat platelets in the absence of extracellular Ca2+. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before stimulation with 5 mmol/L ionomycin. Results are shown as mean±SE of nine different experiments.

Resting [Ca²⁺]_i in platelets pretreated with both insulin and PGE₁ was similar to that in platelets treated with PGE₁ alone but lower than that in platelets treated with insulin alone (Fig 1). The thrombin-induced increase in [Ca²⁺]_i in the presence of extracellular Ca²⁺ was decreased by pretreatment with insulin or PGE₁ compared with controls and was further decreased by a combination of insulin and PGE₁ compared with insulin or PGE₁ alone (Fig 2). In platelets pretreated with both insulin and PGE₁, the thrombin-evoked increase in [Ca²⁺]_i in the absence of extracellular Ca²⁺ was suppressed compared with that in controls or with insulin alone and was similar to that in platelets pretreated with PGE₁ alone (Fig 3). These results indicate that the effects of insulin and PGE₁ together were only additive and that insulin did not affect the action of PGE₁ on Ca²⁺ metabolism. The size of intracellular Ca²⁺ stores was not altered by preincubation with insulin, PGE₁, or both (Fig 4).

In an additional experiment, we studied the effect of preincubation with insulin at 37°C (n=11). Incubation of washed rat platelets at 37°C for 30 minutes raised basal [Ca²⁺]_i (55.7±2.3 to 197.3±9.8 nmol/L) and attenuated the [Ca²⁺]_i response to 0.1 U/mL thrombin (401±18 to 129±10 nmol/L). In this situation, insulin alone, PGE₁ alone, and insulin and PGE₁ together decreased basal [Ca²⁺]_i (180.3±9.9, 178.5±10.0, and 170.5±7.6 nmol/L, respectively) and the thrombin-induced rise in [Ca²⁺]_i (110±7, 108±8, and 96±8 nmol/L, respectively). The ability to decrease basal [Ca²⁺]_i and the [Ca²⁺]_i response was greater in the incubation with insulin and PGE₁ than with insulin or PGE₁ alone. Thus, the effect of pretreatment with insulin and/or PGE₁ on platelet Ca²⁺ handling could be detected at 37°C.

Effect of Insulin Alone and With PGE₁ on Thrombin-Induced Platelet Aggregation
Insulin at 10^-6 mol/L did not change the maximal aggregation induced by thrombin (Figs 5 and 6), whereas 10^-8 mol/L PGE₁ reduced the maximal aggregation to 37.4±0.1% of controls. The maximal aggregation of platelets pretreated with both insulin and PGE₁ was inhibited to 20.4±0.1% of control values and was significantly depressed compared with that obtained with PGE₁ alone. These results suggest that insulin augments the inhibitory effects of PGE₁ on platelet aggregation.

View larger version (23K): [in this window] [in a new window]   Figure 5. Representative recording of the effect of insulin, prostaglandin E1 (PGE1), or both on thrombin-induced platelet aggregation. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before addition of 0.1 U/mL thrombin.

View larger version (27K): [in this window] [in a new window]   Figure 6. Effects of insulin, prostaglandin E1 (PGE1), or both on thrombin-induced platelet aggregation. Platelets were incubated with 10-6 mol/L insulin for 30 minutes and/or 10-8 mol/L PGE1 for 2 minutes before addition of 0.1 U/mL thrombin. Platelet aggregation is expressed as percentage of maximal aggregation. Results are shown as mean±SE of eight different experiments. *P<.01, **P=.0001 vs control; P=.0001 vs insulin; ¶P<.05 vs PGE1.

	Discussion

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This study showed that pretreatment of platelets with insulin reduced thrombin-induced Ca²⁺ influx, whereas it did not change thrombin-evoked release of Ca²⁺ from internal stores or the size of internal Ca²⁺ stores. The interactive effects of insulin with PGE₁ were only additive, and insulin did not augment the effects of PGE₁ on platelet [Ca²⁺]_i. In contrast, although insulin alone did not inhibit thrombin-induced platelet aggregation, it augmented inhibition of aggregation by PGE₁. This is the first report to show the effects of insulin and/or PGE₁ on both platelet aggregation and Ca²⁺ metabolism.

A previous report indicated a lack of effect of insulin on baseline or low-density lipoprotein cholesterol–stimulated [Ca²⁺]_i in human platelets.¹⁰ This discrepancy may result from the difference in cell metabolism between human and rat platelets, in agonists challenged (low-density lipoprotein cholesterol versus thrombin), or in the method of [Ca²⁺]_i determination (isolation of platelets and correction for extracellular dye).

Insulin attenuated the thrombin-induced increase in [Ca²⁺]_i in the presence of extracellular Ca²⁺, but it did not affect Ca²⁺ release from internal stores, indicating that this attenuation of the [Ca²⁺]_i response is due to blunting of Ca²⁺ influx. Our observation is similar to the study of Standley et al,¹⁵ who showed that insulin attenuated the arginine vasopressin–mediated increase in [Ca²⁺]_i by reducing Ca²⁺ influx in cultured vascular smooth muscle cells. Thus, the effects of insulin on Ca²⁺ metabolism in other cells are similar to those found for platelets in the present study. Several studies have shown that insulin has a stimulatory effect on the Ca²⁺-ATPase of various unstimulated cells, eg, renal basolateral membrane^{11 12} and liver plasma membrane.¹⁴ These observations are consistent with our finding that resting [Ca²⁺]_i was lower in insulin-treated platelets.

Kahn et al^{7 17} have reported that insulin increases PGE₁ binding by increasing the number of PGE₁ receptors. On the basis of their study, insulin should amplify the effects of PGE₁ on [Ca²⁺]_i and aggregation. However, our results were partly the opposite of those expected; that is, insulin did not affect the action of PGE₁ on [Ca²⁺]_i, and the combined effects of PGE₁ and insulin on [Ca²⁺]_i were merely additive. Thus, insulin may modify Ca²⁺ metabolism in platelets through PGE₁-independent mechanisms. In contrast, insulin augmented the inhibition of thrombin-induced platelet aggregation by PGE₁, although insulin itself failed to inhibit platelet aggregation. Although we did not investigate other platelet functions, the present results suggest that the effects of insulin on platelet function may be mediated by PGE₁-dependent and -independent mechanisms.

The precise action of PGE₁ on platelet Ca²⁺ metabolism has not been fully clarified, although several studies have shown that the thrombin-induced increase in [Ca²⁺]_i in platelets is reduced by PGE₁ in the presence of extracellular Ca²⁺.^{20 21 22 23 24} Our results revealed that pretreatment with PGE₁ at a concentration of 10^-8 mol/L resulted in decreases in resting [Ca²⁺]_i, the thrombin-induced [Ca²⁺]_i response in the presence of extracellular Ca²⁺, and the release of Ca²⁺ from internal stores without altering the size of internal stores. Activation of platelets by thrombin is accompanied by phosphoinositide breakdown,²⁵ which results in the production of inositol trisphosphate (IP₃), inducing Ca²⁺ release from internal stores. Thus, our results suggest that PGE₁ modulates phosphoinositide breakdown and/or the action of IP₃ on Ca²⁺ stores. This hypothesis is supported by the observations that cAMP greatly inhibits the thrombin-induced formation of phosphatidic acid²⁶ and IP₃-induced Ca²⁺ release,²⁷ since PGE₁ is known to increase platelet cAMP concentration by stimulating adenyl cyclase.²⁸ The ability of PGE₁ to decrease resting [Ca²⁺]_i can be explained by recent studies which have shown that PGE₁ or cAMP stimulates Ca²⁺ extrusion or Ca²⁺ uptake in unstimulated platelets by dense tubules via Ca²⁺-ATPase.^{29 30}

In the present study, although the magnitude of inhibition of thrombin-induced Ca²⁺ increase by insulin in the presence of extracellular Ca²⁺ was similar to that caused by PGE₁, insulin did not affect platelet aggregation, and PGE₁ did. These results provide evidence that the inhibition of increase in [Ca²⁺]_i does not always result in inhibition of platelet aggregation and suggest that mechanisms other than alterations in cellular Ca²⁺ metabolism may contribute to platelet aggregation. The inhibitory effect of cAMP on platelet function may be mediated by inhibition of myosin light chain phosphorylation³¹ and protein tyrosine phosphorylation.²²

It has been reported that platelets from spontaneously hypertensive rats exhibit increased resting and agonist-stimulated [Ca²⁺]_i,^{2 3 4} although this was not the case in rats with acquired hypertension.^{32 33} Our results show that insulin reduces resting and thrombin-evoked increases in [Ca²⁺]_i and amplifies the antiaggregatory effect of PGE₁. Since there are several reports that hypertensive individuals exhibit insulin resistance and hyperinsulinemia, it could be predicted that platelets from hypertensive subjects would exhibit increased [Ca²⁺]_i and less sensitivity to the physiological inhibitory effect of PGE₁ in vivo, resulting in a greater incidence of vascular damage and related thrombotic disorders.

	Acknowledgments

 
This research was supported in part by a grant from Kurosumi Medical Foundation and Grants-in-Aid for Scientific Research (07407065 and 08457639) from the Ministry of Education, Science, and Culture, Japan. We thank Yuko Omura for her secretarial assistance.

Received April 10, 1995; first decision July 6, 1995; accepted April 8, 1996.

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ishida, M. Articles by Oshima, T. Search for Related Content PubMed PubMed Citation Articles by Ishida, M. Articles by Oshima, T.