Ketamine exerts negative inotropic effects in isolated myocardial preparations (1,2). The negative inotropic effect with ketamine is considered to be caused by decreased intracellular Ca2+ concentrations through inhibition of transmembrane influx of Ca2+ through L-type voltage-dependent Ca2+ channels and release of Ca2+ from sarcoplasmic reticulum (SR) (1–3). Activation of α1-adrenergic receptors by norepinephrine (NE) in cardiomyocytes causes the release of inositol 1,4,5-trisphosphate (IP3), which plays an essential role in contraction through releasing Ca2+ from SR. However, the effect of ketamine on NE-induced IP3 production is not clear.
Phosphatidylinositide metabolism involves two pathways: the phosphatidylinositol (PI) turnover pathway and the 3-phosphatidylinositide pathway. In the PI turnover pathway, activation of phospholipase C results in the hydrolysis of PI biphosphate (PIP2) and generation of IP3 and diacylglycerol, which activates protein kinase C (PKC). The 3-phosphatidylinositide pathway is regulated by the PI3-kinase. PI3-kinase phosphorylates phosphoinositide, PI 4-phosphate, and PIP2 at the D-3 position of the inositol ring (4). Phospholipase C and PI3-kinase share PIP2 as their substrate. IP3 is controlled by several factors in the PI turnover pathway. For example, phospholipase A2 positively regulates phospholipase C (5). Activation of PKC decreases agonist-induced IP3 formation (6). IP3 production by agonists depends on the concentration of intracellular Ca2+(7), as shown in Figure 1. Activation of the α1-adrenergic receptor stimulates hydrolysis of PIP2, resulting in increases of IP3 production. IP3 stimulates the release of Ca2+ from SR and activates PKC through diacylglycerol. The increase of intracellular Ca2+ stimulates phospholipase C and PKC. Activation of phospholipase C further releases IP3, but activation of PKC serves as negative feedback regulation for phospholipase C. We formed a hypothesis about the mechanism of ketamine modulation of NE-induced IP3 formation, as shown in Figure 1. We investigated whether ketamine affects NE-induced IP3 production and whether phospholipase C, phospholipase A2, PIP2, PKC, and alteration of intracellular calcium are involved in ketamine modulation of IP3 formation in neonatal cardiomyocytes. Neonatal cardiomyocytes are suitable for the study of inositol phosphate turnover in poorly developed SR and are also more sensitive to the negative inotropic effects of ketamine than adult cardiomyocytes.
This study was approved by the Animal Research Committee of Hirosaki University. Neonatal rats of 3 days were anesthetized with sevoflurane and killed by cervical dislocation. By using an aseptic technique, we isolated myocytes according to the method of Sadoshima et al. (8). 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 mM glucose, and 25 mM HEPES. The hearts were washed with 4 L of PBS. The ventricles were divided and kept in the ice-cold PBS. 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), and 15 g/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO). The liberated cells were collected by centrifugation at 200 g and resuspended in PBS. The pooled and washed cells were plated in T-75 cell culture flasks in medium 199 (containing Earle’s balanced salts, l-glutamine)-supplemented media (containing 5% horse serum, 3 mM 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 medium 199-supplemented media containing 0.1 mM 5-bromo-2′-deoxyuridine to inhibit cell division and thereby control nonmyocyte cell growth. The suspension was then allocated on 0.1% gelatin-coated 25-cm2 flasks for measurement of IP3. The culture medium was changed with the above media after 48 h. The study was performed 4 days after isolation of cardiomyocytes.
The culture medium was changed to serum-free medium 24 h before the cells were studied. Cells in each study were treated with 1 μM TMB-8 (an intracellular calcium antagonist) for 30 min; 100 μM (p-amylcinnamoyl) anthranilic acid (ACA) (a phospholipase A2 inhibitor) for 30 min; 10 μM U 73122 (a phospholipase C inhibitor) for 30 min; 10 nM wortmannin (a PI3-kinase antagonist) for 30 min; 10 nM of staurosporine (a nonselective PKC antagonist) for 30 min; or 100 nM of bisindolylmaleimide (a highly selective PKC antagonist) for 15 min before exposure to ketamine. Culture with 1 μM of ketamine was then treated for 20 min before adding NE (1 μM). TMB-8 inhibits the release of Ca2+ from SR.
After NE was added for 20 s, the reaction was stopped by aspiration of the media and addition of 5 mL of ice-cold 1 M trichloroacetic acid (TCA) for each 1 mg of cells. The acid extract was homogenized and centrifuged at 0°C to 4°C for 10 min at 1000 g. 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. IP3 content in the aqueous top layer was determined by a radioreceptor assay kit from NEN Research Products (Boston, MA). Protein concentrations were determined by the method of Bradford (9) by using bovine serum albumin as standard.
NE, staurosporine, wortmannin, bisindolylmaleimide, TMB-8, PBS, trypsin, collagenase (type 5), deoxyribonuclease 1, chicken serum, cell culture medium, and supplements were purchased from Sigma. ACA and U 73122 were purchased from Biomol Research Laboratories (Hamburg, Germany). The radioreceptor assay kit for measurement of IP3 was purchased from NEN Research Products. Wortmannin, bisindolylmaleimide, ACA, and U 73122 were dissolved in 0.1% dimethyl sulfoxide.
The data were expressed as the mean ± sd. Each experiment was performed in duplicate in the same preparation, and then the experiments were repeated eight times with different cell preparations. Statistical comparisons were made by analysis of variance for repeated measures. When a significant F value was obtained, comparisons of means were performed with Student’s t-test for paired and unpaired samples. P values <0.05 were considered significant.
NE stimulated IP3 production in neonatal rat cardiomyocytes in a dose-dependent manner. Treatment of the cultures with 1 μM ketamine significantly decreased the IP3 response to 100 nM and to 1, 10, and 100 μM NE doses, from 396 ± 65, 542 ± 93, 717 ± 143, and 811 ± 152 pmol/mg protein to 227 ± 63, 398 ± 81, 569 ± 113, and 663 ± 109 pmol/mg protein, respectively. Ten micromolar ketamine further decreased IP3 production to 193 ± 74, 309 ± 89, 512 ± 93, and 559 ± 112 pmol/mg protein, respectively (Fig. 2).
We assessed the involvement of phospholipase C in NE-induced or ketamine-inhibited IP3 formation by using U 73122, a phospholipase C inhibitor. When the cultures were exposed to 10 μM U 73122, NE-induced IP3 production in the presence and absence of 1 μM ketamine changed from 309 ± 89 to 345 ± 52 pmol/mg protein and from 542 ± 93 to 526 ± 102 pmol/mg protein, respectively; 10 μM U 73122 did not significantly affect NE-induced IP3 in the presence or absence of ketamine.
Figure 3 shows the effects of ACA (a phospholipase A2 antagonist) on NE-induced IP3 production. Ten micromolar ACA was the maximal concentration for the inhibition of NE-induced IP3 production. We investigated whether phospholipase A2 is involved in ketamine modulation of NE-IP3. NE-induced IP3 production was significantly reduced by the phospholipase A2 antagonist with 100 μM ACA, from 398 ± 81 to 174 ± 46 pmol/mg protein. IP3 production was further significantly decreased by 1 μM ketamine to 126 ± 29 pmol/mg protein.
We studied whether a decrease in intracellular Ca2+ is associated with ketamine modulation of NE on IP3. IP3 production in the presence of 10 μM TMB-8 was 99 ±21 pmol/mg protein, which is not significantly different from buffer. Exposure to 10 μM of TMB-8 significantly decreased the ketamine modulation of NE (1 μM)-induced IP3 formation, from 398 ± 81 to 233 ±50 pmol/mg protein (Fig. 4).
We previously reported that the PI3-kinase antagonist stimulated hydrolysis of PIP2, resulting in an increase in IP3 formation (10). Thus, we studied whether PIP2 is involved in ketamine modulation of NE-IP3 by using wortmannin. Wortmannin (a PI3-kinase antagonist) significantly increased NE (1 μM)-induced IP3 production, from 542 ± 93 to 713 ± 101 pmol/mg protein, and the stimulation was blocked by 1 μM ketamine to 443 ± 87 pmol/mg protein (Fig. 5).
To study the effect of PKC on ketamine modulation of NE-IP3, we studied the effects of staurosporine (a nonselective PKC antagonist) and bisindolylmaleimide (a highly selective PKC antagonist). Ten nanomolar staurosporine and 20 nM bisindolylmaleimide stimulated NE (1 μM)-induced IP3 production from 542 ± 93 to 668 ± 97 and 740 ± 43 pmol/mg protein. One micromolar ketamine significantly inhibited staurosporine- and bisindolylmaleimide-stimulated IP3 formation from 542 ± 93 to 483 ± 51 and 401 ± 43 pmol/mg protein, respectively (Fig. 6).
In cardiomyocytes, ketamine inhibited NE-induced IP3 production, and the inhibition was associated with PKC. Thus, the negative inotropic effect with ketamine seems to be partly caused by the inhibition of IP3 production. Kanmura et al. (11) demonstrated, in rabbit mesenteric artery, that the inhibitory action of ketamine on Ca2+ release from the intracellular stores may occur via the decreased IP3 formation, probably secondary to inhibition of phospholipase C. Rats et al. (12) also demonstrated that ketamine reduced phospholipase C activation in arterial smooth muscle. We investigated whether ketamine-inhibited IP3 is associated with phospholipase C. This study showed that NE-induced release of IP3 in the presence and absence of ketamine was insensitive to U 73122, which is a specific phospholipase C antagonist. U 73122 inhibits phospholipase C activation via phospholipase C β and C γ(13). The phospholipase C responsible for NE-induced IP3 production does not seem to belong to phospholipase C β and C γ. In addition, ketamine inhibition of NE-induced IP3 formation also seems to act through a different pathway from that of phospholipase C β and C γ. Phospholipase C is coupled to phospholipase A2, which positively regulates IP3 formation (5). This study demonstrated that phospholipase A2 antagonists inhibited NE-induced IP3 production. Denson et al. (14) suggested that ketamine is inhibitory at phospholipase A2 receptors and decreases arachidonic acid in pituitary cells. Therefore, we studied whether ketamine inhibition of NE-induced IP3 production is caused via phospholipase A2. However, the inhibition of NE-induced IP3 production by phospholipase A2 antagonists in maximal concentrations was further enhanced by ketamine. It is possible that the inhibition of NE-induced IP3 production by ketamine involves a different pathway than phospholipase A2.
Ketamine interferes with transmembrane influx of Ca2+ through L-type voltage-dependent Ca2+ channels and release of Ca2+ from intracellular stores, resulting in decreased intracellular Ca2+ concentrations (3). In this study, we found that TMB-8, an intracellular Ca2+ inhibitor, enhanced ketamine inhibition of IP3 production. A major effect of TMB-8 on cellular calcium metabolism is to prevent intracellular Ca2+ mobilization by inhibiting the IP3-induced Ca2+ release (6). As the increase in intracellular Ca2+ activates phospholipase C, which further increases intracellular Ca2+ through increases in IP3 production (1), a decrease in intracellular Ca2+ concentrations by ketamine contributes to a decrease in IP3 production.
Phosphatidylinositide metabolism involves two pathways: the PI turnover pathway and the 3- phosphatidylinositide pathway. In the PI turnover pathway, activation of phospholipase C results in the hydrolysis of PIP2 and generation of IP3. In the 3-phosphatidylinositide pathway, PI3-kinase plays an important regulatory role. The PI3-kinase phosphorylates phosphoinositide, PI 4-phosphate, and PIP2 at the D-3 position of the inositol ring (4). Our results indicate that the inhibition of the formation of phosphoinositides phosphorylated in position D-3 of the inositol ring by the PI3-kinase antagonist may lead to stimulation of the hydrolysis of PIP2, resulting in an increase in IP3 formation. Thus, ketamine decreases the conversion of PIP2 to IP3.
The regulation of IP3-mediated Ca2+ release involves PKC (15–17). In coronary artery, phorbol 12 myristate 13 acetate (PMA), (a PKC agonist) decreased and staurosporine potentiated endothelin-stimulated inositol phosphates (15). In microsomes of neonatal rat cardiomyocytes, PMA downregulates α1-adrenoceptor-mediated PIP2 hydrolysis (16). Barr and Watson (17) reported that PKC inhibitors potentiated agonist-stimulated accumulation of inositol phosphates in astrocytoma cells. In this study, the observation that treatment with staurosporine and bisindolylmaleimide (PKC antagonists) stimulated IP3 formation indicates that there is feedback regulation of PKC to IP3. Ketamine inhibited the staurosporine- and bisindolylmaleimide-enhanced NE-induced IP3 production. Thus, ketamine seems to activate PKC in cardiomyocytes.
One of the difficulties in studying protein kinase mediation of drug actions is the limited availability of highly specific inhibitor compounds. The relative lack of selectivity of PKC inhibitors, such as staurosporine and polymyxin B, has been a limiting factor in evaluating the role of PKC in intracellular signaling, because these drugs also inhibit tyrosine kinase and protein kinase A (PKA). However, bisindolylmaleimide is a highly selective inhibitor of PKC (18). In this study, it is unlikely that the effects are due to the inhibition of PKA, because the concentration of bisindolylmaleimide used in this study was 40-fold less than the Ki for PKA (18). The bisindolylmaleimide may affect Ca2+ entry (19), MAPKAP kinase-1β (Rsk-2), or p70 S6 kinase (20). However, the effect of bisindolylmaleimide on Ca2+ entry occurred in a 250-fold larger concentration as compared with the concentration used in this study, and the relationship between MAPKAP kinase-1β (Rsk-2), p70 S6 kinase, and IP3 formation remains unclear. We used two PKC inhibitors, bisindolylmaleimide and staurosporine, and the same results were obtained in this study. Staurosporine is less specific than bisindolylmaleimide for PKC, but the identity of effects indicates that the drug acts by a common mechanism, i.e., PKC inhibition. ACA is a specific phospholipase A2 antagonist and does not affect phospholipase C (21). Wortmannin is a potent and selective inhibitor of PI 3-kinase (22). Because the concentration required for the half-maximal inhibition was as small as 10 nM or less when the inhibitor was added directly (22), the dose used in this study was adequate for the inhibition of PI3-kinase. The doses of TMB-8 were determined according to the study of Northover (23), who measured the concentration of intracellular calcium by using atrial muscle. Northover suggested that exposure of 2 μM TMB-8 for 15 minutes significantly decreased the concentration of intracellular calcium. Because a large concentration of TMB-8 has several actions, including depression of PKC and tyrosine kinase (24), the culture was exposed for 30 minutes at a concentration of 1 μM TMB-8 in this study.
The concentrations of ketamine used in this study were 1 and 10 μM. The plasma ketamine concentrations in humans are reported to be 4–12 μM for an IV dose of 2 mg/kg ketamine (10). The concentrations used in this study overlap the free plasma concentrations used clinically. Neonatal myocardium is more sensitive to extracellular calcium concentrations than is adult myocardium. This is associated with poorly developed SR of neonatal myocardium (25). The contractile activity in neonatal myocardium seems more dependent on the entry of extracellular Ca2+ rather than the release of Ca2+ from SR. Thus, IP3-induced Ca2+ release from SR may play a less important role in contractile activity as compared with adult myocardium; in addition, NE-induced IP3 formation in adult myocardium may have produced different results.
In conclusion, ketamine inhibits NE-induced IP3 formation in a dose-dependent manner. The inhibition is associated with PKC and a decrease in intracellular Ca2+ concentrations.
The authors thank Drs. S. F. Rabito and Egic (Department of Anesthesiology and Pain Management, Cook County Hospital) for their support of this research and their critical comments. The authors thank Dr. Paul Hollister for correction of English grammar and syntax.
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