This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Kudoh, A. Articles by Matsuki, A. Search for Related Content PubMed PubMed Citation Articles by Kudoh, A. Articles by Matsuki, A.

---

PEDIATRIC ANESTHESIA

Ketamine Inhibits Inositol 1,4,5-Trisphosphate Production Depending on the Extracellular Ca²⁺ Concentration in Neonatal Rat Cardiomyocytes

Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki, Aomori, Japan

Address correspondence and reprint requests to Akira Kudoh, MD, Department of Anesthesiology, University of Hirosaki School of Medicine, 5 Zaifucho, Hirosaki, 036-8562, Aomori, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
We investigated the effect of ketamine on inositol 1,4,5-trisphosphate (IP3) formation in rat cardiomyocytes. After the addition of 1 µmol/L ketamine, IP3 production in the presence of 0.5, 1, 5, 10, and 30 mmol/L Ca²⁺ significantly decreased from 537.1 ± 8.3, 590.7 ± 12.9, 690.6 ± 7.9, 754.8 ± 12.5, and 823.7 ± 15.2 pmol/mg protein to 467.0 ± 8.3, 483.8 ± 11.0, 512.6 ± 21.3, 612.1 ± 16.9, and 652.6 ± 17.3 pmol/mg protein, respectively. When exposed to TMB-8 (a intracellular calcium inhibitor), IP3 production decreased significantly from 347.2 ± 27.3 to 283.8 ± 20.4 pmol/mg protein in the presence of 1 µmol/L ketamine, but A23187, which increases intracellular calcium, did not affect the inhibition of IP3 production by ketamine. These results demonstrate that ketamine decreases IP3 formation through inhibition of the calcium ion-sensing receptor and that IP3 formation reduced by ketamine is not affected by the alteration of intracellular calcium.

Implications: Ketamine has a negative inotropic effect in isolated cardiomyocytes. The negative inotropic effect was associated with a decrease in inositol 1,4,5-trisphosphate production, and the inhibitory action was enhanced depending on the concentration of extracellular Ca²⁺.

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Myocardial contractility is clinically enhanced by ketamine as a result of sympathomimetic stimulation through the central nervous system and activation of the baroreceptor and the inhibition of neuronal uptake of catecholamine (1). However, ketamine is reported to have a negative inotropic effect in isolated cardiomyocytes (2). Waxman et al. (3) showed that ketamine has a direct depressing effect on myocardial contractility in the absence of sympathetic stimulation. The negative inotropic effect with ketamine could be caused by a decrease in Ca²⁺ release from intracellular stores and an interference with Ca²⁺ influx through a voltage-dependent Ca²⁺ channel (5).

Bradykinin (BK) is an important mediator of the physiological response to injury, pain, and trauma (6) and simultaneously has a positive inotropic effect on the heart. The hormone is a mediator of the protection of myocardium by angiotensin-converting enzyme (ACE) inhibitors (7). The ACE inhibitor inhibits the degradation of BK and potentiates the pharmacological action of BK. The stimulation of BK receptors results in increased inositol 1,4,5-trisphosphate (IP3) production. BK has been demonstrated to act on G-protein–coupled receptors to activate phospholipase C (PLC) and to stimulate the rapid formation of IP3, which leads to an increase in intracellular Ca²⁺ from sarcoplasmic reticulum (SR) and regulates myocardial contraction (8). G protein in isolated cardiac membrane preparations has been shown to increase IP3 production depending on the concentration of extracellular Ca²⁺ at physiological ranges (9) through the Ca²⁺-sensing receptor, which links the PLC and controls phosphoinositide turnover and intracellular calcium release (10). The extracellular Ca²⁺ concentration may thus play a regulatory role in receptor-mediated phosphoinositide turnover (11).

We designed this study to investigate the effects of ketamine on phosphatidyl inositol turnover and the process of calcium metabolism in neonatal rat cardio- myocytes.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
This study was approved by our institutional animal committee. Neonatal rats were anesthetized with sevoflurane and killed by cervical dislocation. Using an aseptic technique, we isolated myocytes according to the method of Sadoshima et al. (12). The hearts were rapidly removed and placed in ice-cold Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) containing 40 U/mL sodium heparin, 4 mmol/L glucose, and 25 mmol/L HEPES. The hearts were washed with 4 L of PBS. The atria and ventricles were divided and kept in ice-cold modified Eagle’s medium and PBS, respectively. The ventricles were minced with scissors into 1- to 3-mm3 fragments, which were then washed with PBS by gently stirring in a 37°C water-jacketed Erlenmeyer flask for 10 min. The tissue was then enzymatically digested five times for 10 min each with 10 mL of PBS containing 0.1% trypsin, 0.1% collagenase (type 5), 15 g/mL deoxyribonuclease 1, and 1% chicken serum. The liberated cells were collected by centrifugation at 200g and resuspended in PBS containing calf serum. The pooled and washed cells were plated in T-75 cell culture flasks in medium 199 (M199)-supplemented media (containing Earle’s balanced salts, 5% horse serum, 3 mmol/L pyruvic acid, MEN vitamins, 1 g/mL insulin, 1 g/mL transferrin, 10 ng/mL selenium, and 50 g/mL gentamicin). The nonadherent cells were harvested after incubation at 37°C for 60 min in a humidified incubator with 5% CO₂ in air. The cells were counted and resuspended in M199-supplemented media containing 0.1 mmol/L 5-bromo-2'deopxyuridiner to inhibit cell division and thereby control nonmyocyte cell growth. Fibroblasts were divided from cardiomyocytes. The suspension was then allocated on 0.1% gelatin-coated 25-cm² flasks for measurement of IP3. The culture medium was changed after 48 h with the above media and finally to serum-free medium 24 h before the cells were studied.

The study was performed 4 days after isolation of cardiomyocytes. The cultures were incubated at extracellular Ca²⁺ concentrations of 0, 0.5, 1, 5, 10, and 30 nmol/L for 120 min before adding ketamine. Ketamine was then treated for 30 min. Cells were treated with TMB-8 (an intracellular calcium antagonist that lowers intracellular calcium by blocking the efflux of Ca²⁺ from intracellular stores without affecting the influx), A23187 (an intracellular calcium agonist), W-7 (a calmodulin antagonist), nicardipine, and verapamil (voltage-dependent Ca²⁺ channel blocker) at extracellular Ca²⁺ concentrations of 0.5 mmol/L for 30 min before exposure to BK. There were eight subjects in each study.

Twenty-four hours before the experiments, the culture medium was changed to serum-free M199. After BK was added for 20 s, the reaction was stopped by aspiration of the media and addition of 5 mL of ice-cold 1 mol/L trichloroacetic acid (TCA) for each 1 mg of cells. The acid extract was homogenized and centrifuged at 0–4°C for 10 min at 1000g. TCA was removed from the extracts by adding 2 mL of a mixture of 3 vol of 1,1,2-trichloro-1,2,2-trifluoroethane plus 1 vol of trioctylamine for each 1 mL of TCA extract. The IP3 content in the aqueous top layer was determined by using a radioreceptor assay kit (NEN Research Products-Du Pont, Boston, MA).

Protein concentrations were determined by using the method of Bradford (13) using bovine serum albumin as standard.

BK, TMB-8, A23187, nicardipine, verapamil, W7, cell culture medium, supplements, and all chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Data are expressed as mean + SEM. Repeated- measures analysis of variance, followed by Bonferroni’s correction, was performed. P values <0.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
BK stimulated IP3 formation on cardiomyocytes of the neonatal rat in a dose-dependent manner. IP3 increased from a basal level of 75.2 ± 7.6 to the maximum of 770.3 ± 22.7 pmol/mg protein at extracellular Ca²⁺ concentrations of 0.5 mmol/L (Figure 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Effect of bradykinin on dose-dependently stimulated inositol 1,4,5-trisphosphate (IP3) production at extracellular Ca2+ concentrations of 0.5 mmol/L when applied for 20 s to cultures of neonatal rat cardiomyocytes. n = 8 for each data point.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of ketamine on bradykinin (BK)-induced 1,4,5-trisphosphate (IP3) production under various concentrations of extracellular Ca2+. The cultures were incubated at extracellular Ca2+ concentrations of 0, 0.5, 1, 10, and 30 mmol/L for 120 min before adding ketamine. Culture with 1 µmol/L ketamine was treated for 30 min. IP3 production was measured after BK (1 µmol/L) stimulation for 20 s, as described in Methods. n = 8 for each bar. Data are mean ± SEM. *P < 0.05, **P < 0.01 for BK-induced IP3 production with and within 1 µmol/L ketamine.

View larger version (31K): [in this window] [in a new window]   Figure 3. Effect of ketamine on bradykinin (BK)-induced 1,4,5-trisphosphate (IP3) production, when exposed to TMB-8. Cardiomyocytes were incubated for 30 min at 37°C in the presence of 1 µmol/L TMB-8 (intracellular Ca2+ inhibitor) with or without 1 µmol/L ketamine for 30 min at 37°C. IP3 production was measured after BK (1 µmol/L) stimulation for 20 s, as described in Methods. n = 8 for each bar. Data are mean ± SEM. #P < 0.05, ###P < 0.001 versus BK-induced IP3 production in absence of ketamine and TMB-8. P < 0.01 versus BK-induced IP3 production in the presence of 1 µmol/L ketamine with and without TMB-8. P < 0.01 for BK-induced IP3 production in the presence of TMB-8 with and without 1 µmol/L ketamine.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of A23187 (an intracellular agonist) on inhibition of bradykinin (BK)-induced 1,4,5-trisphosphate (IP3) production by ketamine. Cardiomyocytes were incubated with 1 µmol/L A23187 for 30 min at 37°C. IP3 production was measured after BK (1 µmol/L) stimulation for 20 s, as described in Methods. n = 8 for each bar. Data are mean ± SEM.

View larger version (36K): [in this window] [in a new window]   Figure 5. Effect of ketamine on bradykinin (BK)-induced 1,4,5-trisphosphate (IP3) production during treatment with nicardipine and verapamil. Cardiomyocytes were incubated for 30 min at 37°C in the presence of 1 µmol/L nicardipine and verapamil without or with 1 µmol/L ketamine for 30 min. IP3 production was measured after BK (1 µmol/L) stimulation for 20 s, as described in Methods. n = 8 for each bar. Data are mean ± SEM. #P < 0.05 versus BK-induced IP3 production in the absence of ketamine, nicardipine, and verapamil.

View larger version (32K): [in this window] [in a new window]   Figure 6. Effect of ketamine on bradykinin (BK)-induced 1,4,5-trisphosphate (IP3) production during treatment with W-7 (a calmodulin antagonist). The cultures were incubated for 30 min at 37°C in the presence of W-7 (100 nmol and 1 µmol) without or with 1 µmol/L ketamine for 30 min at 37°C. IP3 production was measured after BK (1 µmol/L) stimulation for 20 s, as described in Methods. n = 8 for each bar. Data are mean ± SEM. *P < 0.05 versus BK-induced IP3 production in the absence of ketamine and W-7.

	Discussion

Top Abstract Introduction Methods Results Discussion References

G protein activated by extracellular Ca²⁺ stimulates PLC to activate mobilization of intracellular Ca²⁺ stores and stimulate Ca²⁺ entry across the plasma membrane depending on phosphoinositide turnover (16). The activation of the calcium ion-sensing receptor is also found with BK (17). Thus, our results suggest that ketamine attenuates IP3 production through the inhibition of the calcium ion-sensing receptor, G protein, or PLC. IP3 elicits a rapid release of calcium from intracellular stores and causes a positive inotropic effect in the heart. An increase in IP3 production correlates well with the increase in force of contraction.

In the present study, we showed that IP3 production is decreased by approximately 12% in the presence of 1 mmol/L ketamine. Schmitz et al. (4) demonstrated that an increase in IP3 by approximately 10% increases the force of contraction by approximately 10%–50%. Whether ketamine has negative inotropic effects in myocardium is controversial. Kanaya et al. (25) reported that ketamine inhibited contraction in ventricular myocytes, but the effect occurred at supraclinical concentrations. However, the inhibition by ketamine was prominent in a high concentration of extracellular Ca²⁺. IP3 production was decreased by approximately 19% in the presence of 10 mmol/L ketamine. The hypercalcemic state is often encountered in myocardial protection during cardiopulmonary bypass or in primary hyperparathyroidism. Therefore, in the hypercalcemic state, contractile activity inhibited by ketamine may be more prominent.

Intracellular Ca²⁺ concentration can control IP3-mediated Ca²⁺ release (18). IP3 production by agonists are stimulated depending on the concentration of intracellular Ca²⁺ (18). Thus, it is possible that a concentration of intracellular Ca²⁺ diminished by ketamine leads to decreased IP3 production. Ketamine interferes with the trancemembrane influx of Ca²⁺ and release of Ca²⁺ from the intracellular store, resulting in decreased intracellular Ca²⁺ concentrations (19). Rusy et al. (2) showed that ketamine inhibits only the transmembrane influx of Ca²⁺ without affecting release from the intracellular store and contributes to decreasing intracellular Ca²⁺ concentrations. Ketamine inhibits transsarcolemmal Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels (9). However, we did not find that nicardipine and verapamil affect IP3 formation inhibited by ketamine. The link between IP3 and the voltage-dependent Ca²⁺ channel remains unclear. IP3 elicits a rapid release of calcium from the intracellular store. On the contrary, L-type voltage-gated channels are the main channel for slow and sustained calcium ion entry across the plasma membrane. These two phases of Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry across the plasma membrane are dependent on receptor-stimulated PIP2 as the same substrate as IP3 (20). Voltage-dependent Ca²⁺ channel is stimulated by IP3 (20); however, L-type Ca²⁺ channel blockers do not affect IP3 (5,15). These findings support the idea that IP3 formation suppressed by ketamine would not emanate from its inhibition of the voltage-gated Ca²⁺ channel. We demonstrated that TMB-8, an intracellular Ca²⁺ inhibitor, enhanced ketamine inhibition of IP3 production; however, increases of intracellular Ca²⁺ with A23187 could not block IP3 production inhibited by ketamine. This result suggests one possibility that inhibition of IP3 production by ketamine would not be associated with the concentration of intracellular Ca²⁺. Reduced release of Ca²⁺ from SR is believed to be essential in the negative inotropic action of ketamine (2). Riou et al. (19) reported that the function of SR is impaired by ketamine; however, the concentration of ketamine in their study was 100 µmol/L, which is 10–100 times higher than that used clinically. Connelly et al. (21) demonstrated that a clinical concentration of ketamine does not alter Ca²⁺ release from cardiac SR. In this study, we showed that a decrease in IP3 production by ketamine is mediated at the clinical concentration of ketamine. Therefore, the negative inotropic action of ketamine may be associated with the inhibition of the fast increase of Ca²⁺ from SR.

Calmodulin also has a regulatory role in Ca²⁺ release from SR. The protein could reduce rapid Ca²⁺ released by IP3 by decreasing the duration of Ca²⁺ channel open events in the rat heart (22). Therefore, we investigated whether calmodulin is associated with IP3 production or whether ketamine affects calmodulin. W-7 has been reported to increase Ca²⁺ release from the SR by interfering with the binding between the calmodulin receptor and the Ca²⁺-calmodulin complex (22). However, in the present study, we showed that W-7 does not affect IP3 production in the presence and absence of ketamine. Halothane and isoflurane have been reported to alter the Ca²⁺-binding affinity of calmodulin and to weakly reduce the activity of myosin light chain kinase (23). Our result indicates that calmodulin is not involved in the decrease in IP3 production by ketamine.

Neonatal myocardium is more sensitive than adult myocardium to extracellular calcium concentrations. This is associated with poorly developed sarcoplasmic reticulum of neonatal myocardium (24). Thus, contractile activity in neonatal myocardium seems to be dependent on the entry of extracellular Ca²⁺, rather than the release of Ca²⁺ from SR. IP3-induced Ca²⁺ release from SR may play a less important role in contractile activity compared with adult myocardium.

In conclusion, ketamine inhibits the BK-induced IP3 formation depending on the extracellular Ca²⁺ concentrations. The IP3 formation reduced by ketamine would be associated with the inhibition of the calcium ion sensing receptor, G protein, or PLC, rather than alteration of intracellular Ca²⁺ concentrations.

	Acknowledgments

 
We thank Dr. S. F. Rabito, Department of Anesthesiology and Pain Management, Cook County Hospital, Chicago, IL, for her critical comments.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Lundy PM, Gverzdy S, Frew R. Ketamine: evidence of tissue-specific inhibition of neuronal and extraneuronal catecholamine uptake process. Can J Physiol Pharmacol 1985;63:298–303.[Medline]
2. Rusy BF, Amuzu JK, Bosscher HA, et al. Negative inotropic effect of ketamine in rabbit ventricular muscle. Anesth Analg 1990;71:275–8.[Abstract/Free Full Text]
3. Waxman K, Shoemaker WC, Lippmann M. Cardiovascular effects of anesthetic induction with ketamine. Analg 1980;59:355–8.[Abstract/Free Full Text]
4. Schmitz W, Jens Scholz HS, Steinfath M. Increase in IP3 precedes -adrenoceptor-induced increase in force of contraction in cardiac muscle. Eur J Pharmacol 1987;140:109–11.[Web of Science][Medline]
5. Yamazaki M, Ito Y, Kuse S, et al. Effects of ketamine on voltage-dependent Ca currents in single smooth muscle cells from rabbit portal vein. Pharmacology 1992;45:162–9.[Medline]
6. Proud D, Kaplan AP. Kinin formation: mechanisms and role in inflammatory disorders. Ann Rev Immunol 1988;6:49–93.[Web of Science][Medline]
7. Hartman JC, Hullinger TG, Wall TM, Shebuski RJ. Reduction of myocardial infarct size by ramiprilat is independent of angiotensin II synthesis inhibition. Eur J Pharmacol 1993;234:229–36.[Web of Science][Medline]
8. Callewaert G. Excitation-contraction coupling in mammalian cardiac cells. Res 1992;26:923–32.
9. Renard D, Poggioli J. Mediation by GTP S and Ca²⁺ of inositol triphosphate generation in rat heart membranes. J Mol Cell Cardiol 1990;22:13–22.[Web of Science][Medline]
10. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca²⁺-sensing receptor from bovine parathyroid. Nature 1993;366:575–80.[Medline]
11. De Jonge HW, Van Heugten HAA, Lamers JMJ. Signal transduction by the phosphatidylinositol cycle in myocardium. Mol Cell Cardiol 1995;27:93–106.[Web of Science][Medline]
12. Sadoshima J, Jahn L, Takahashi T, et al. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. J Biol Chem 1992;267:10551–60.[Abstract/Free Full Text]
13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.[Web of Science][Medline]
14. Bristow MR, Schwartz HD, Binetti G, et al. Ionized calcium and the heart: elucidation of in vivo concentration-response relationships in the open-chest dogs. Res 1977;41:565–74.
15. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 1984;312:315–21.[Medline]
16. Van Heughten HAA, De Jonge HW, Bezstarosti K, et al. Calcium and the endothelin-1 and 1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes. J Mol Cell Cardiol 1994;26:1081–93.[Web of Science][Medline]
17. Fasolato C, Pizzo P, Pozzan T. Receptor-mediated calcium influx in PC12 cells: ATP and bradykinin activate independent pathways. J Biol Chem 1990;265:20351–5.[Abstract/Free Full Text]
18. Zhang B-X, Zhao H, Muallem S. Ca²⁺-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca²⁺ release. Biol Chem 1993;268:10997–1001.[Abstract/Free Full Text]
19. Riou B, Lecarperntier Y, Viars P. Inotropic effect of ketamine on rat cardiac papillary muscle. Anesthesiology 1989;71:116–25.[Web of Science][Medline]
20. Kiselyov KI, Mamin AG, Semyonova SB, Mozhayeva GN. Low-conductance high selective inositol (1,4,5)-trisphosphate activated Ca²⁺ channels in plasma membrane of A431 carcinoma cells. FEBS Lett 1997;407:309–12.[Web of Science][Medline]
21. Connelly TJ, Ahern C, Coronado R. Ketamine, at clinical concentrations, does not alter the function of cardiac sarcoplasmic reticulum calcium release channels. Anesth Analg 1995;81:849–54.[Abstract]
22. Toyama J, Sugiura H, Kamiya K, et al. Ca²⁺-calmodulin mediated modulation of the electrical coupling of ventricular myocytes isolated from guinea pig heart. Cardiol 1994;26:1007–15.
23. Levin A, Blanck TJJ. Halothane and isoflurane alter the Ca²⁺ binding properties of calmodulin. Anesthesiology 1995;83:120–6.[Medline]
24. Klitzner T, Friedman WF. A diminished role for the sarcoplasmic reticulum in newborn myocardial contraction: effects of ryanodine. Pediatr Res 1989;26:98–101.[Web of Science][Medline]
25. Kanaya N, Murray PA, Damron DS. Propofol and ketamine only inhibit intracellular Ca²⁺ transients and contraction in rat ventricular myocytes at supraclinical concentration. Anesthesiology 1998;88:781–91.[Web of Science][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Kudoh, A. Articles by Matsuki, A. Search for Related Content PubMed PubMed Citation Articles by Kudoh, A. Articles by Matsuki, A.