α1-Adrenoceptor Activation Increases Ecto-5′-Nucleotidase Activity and Adenosine Release in Rat Cardiomyocytes by Activating Protein Kinase C
Background Adenosine is an important regulator of many cardiac functions and is synthesized primarily by ecto- and cytosolic 5′-nucleotidase. We have previously reported that α1-adrenoceptor blockade attenuates adenosine release from ischemic myocardium, raising the possibility that α1-adrenoceptor activation activates 5′-nucleotidase. This study tested whether activation of protein kinase C by α1-adrenoceptor activation increases 5′-nucleotidase activity and augments adenosine release.
Methods and Results Cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode’s buffer solution. After stabilization, the cardiomyocytes were incubated with and without an exposure to norepinephrine (10−9 to 10−5 mol/L) while being treated with propranolol and yohimbine or with and without an exposure to methoxamine (10−9 to 10−5 mol/L). Ecto-5′-nucleotidase activity was increased by norepinephrine and methoxamine during 30 minutes in a dose-dependent manner, whereas cytosolic 5′-nucleotidase was not activated. These increases in ecto-5′-nucleotidase activity were inhibited by GF109203X, an inhibitor of protein kinase C, and mimicked by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. The increase in ecto-5′-nucleotidase was not prevented by cycloheximide. When ecto-5′-nucleotidase activity increased, adenosine release was augmented in methoxamine- and PMA-treated cardiomyocytes (1299±252% and 1372±149%, respectively) compared with the untreated group (578±26%). The increase in adenosine release was blunted by GF109203X and α,β-methyleneadenosine 5′-diphosphate, an inhibitor of ecto-5′-nucleotidase.
Conclusions Thus, we conclude that α1-adrenoceptor–mediated increases in ecto-5′-nucleotidase activity are attributed to activation of protein kinase C in rat cardiomyocytes.
Adenosine has been reported to modify several key cellular activities in the heart.1 2 3 Adenosine relaxes vascular smooth muscles,4 inhibits platelet aggregation5 6 and oxygen-derived free radical generation from polymorphonuclear leukocytes,7 and attenuates increases in myocardial contractility due to norepinephrine.8 9 In the heart, adenosine is released from cardiomyocytes, coronary endothelial and coronary smooth muscle cells, and leukocytes.2 3 During ischemia and hypoxia, adenosine is released from cardiomyocytes primarily through 5′-nucleotidase. This enzyme is known to be modulated by myocardial cellular metabolic factors, eg, ATP, ADP, and inorganic phosphate (Pi)10 11 12 : Ecto-5′-nucleotidase is inhibited by ATP and ADP,10 whereas cytosolic 5′-nucleotidase is activated by ATP and ADP and inhibited by Pi.11 12 On the other hand, norepinephrine is released from presynaptic vesicles in the ischemic heart,13 which may activate α1-adrenoceptors and protein kinase C through accumulation of diacylglycerol. Furthermore, myocardial ischemia translocates protein kinase C from the cytosol to the plasma membrane.14 These two activities may synergistically activate protein kinase C in the cardiomyocytes during ischemia. We previously reported that α1-adrenoceptor activity regulates release of adenosine,15 16 raising the possibility that α1-adrenoceptor activation may increase 5′-nucleotidase activity by activating protein kinase C.17
To test this hypothesis, we measured both ecto- and cytosolic 5′-nucleotidase activity and adenosine release with and without exposure to norepinephrine, methoxamine, and phorbol 12-myristate 13-acetate (PMA). We also tested whether the increase in 5′-nucleotidase activity is blocked by GF109203X and α,β-methyleneadenosine 5′-diphosphate (AOPCP). In addition, we examined whether augmentation of 5′-nucleotidase activity increases adenosine release from rat cardiomyocytes.
Adenosine, norepinephrine, propranolol, yohimbine, PMA, AOPCP, and cycloheximide were obtained from Sigma Biochemical Co. GF109203X was obtained from Calbiochem. 2′-Deoxycoformycin was provided by Yamasa Shoyu Research Laboratories. 5′-Iodotubercidin was obtained from Research Biochemicals Inc. All reagents and chemicals were the highest grade obtainable.
Preparation of Cardiomyocytes
Cardiomyocytes were isolated from 128 adult male Wistar rats (200 to 250 g) as described previously.18 Briefly, heparin (600 U IP) was injected 30 minutes before anesthesia with pentobarbital (0.2 mg/g body wt IP). The hearts were removed quickly and perfused in a Langendorff perfusion apparatus, initially with 25 mL of Ca2+-free modified HEPES-KOH/Tyrode’s buffer solution (in mmol/L: NaCl 120, KCl 5, MgSO4 1.2, NaHCO3 5, glucose 10, and HEPES 20; pH 7.3) equilibrated with 95% O2/5% CO2 at 37°C and then with digestion solution (Ca-free solution containing 0.06 wt% crude collagenase and 0.1% fatty acid–free bovine albumin) for 30 minutes. The ventricle was removed and finely minced with scissors in Ca2+-containing HEPES/Tyrode’s solution (1 mmol/L CaCl2). The cardiomyocytes were mechanically dispersed by gentle pipetting, filtered through gauze, and suspended in Ca2+-containing solution. Cells were washed twice with Ca2+-containing solution, collected by centrifugation (50g, 1 minute), and resuspended in Ca2+-containing solution. Microscopy demonstrated that almost all of the isolated cells were cardiomyocytes (2% of the cellular components resembled fibroblasts) and that 86±4% of the isolated cardiomyocytes were morphologically (rod shaped) and metabolically (trypan blue exclusion test) intact.
Protocol 1: Effects of Norepinephrine, Methoxamine, and PMA on Ecto- and Cytosolic 5′-Nucleotidase Activity
After cardiomyocytes were isolated from rats (n=72), 10−6 mol/L norepinephrine was added to the suspension of 1.0 to 2.0×107 cells. Cardiomyocytes obtained from different rats were used in each experiment. Propranolol 10−5 mol/L and yohimbine 10−5 mol/L were added to the suspension 10 minutes before its exposure to norepinephrine. In a preliminary study, we confirmed that neither propranolol nor yohimbine affects ecto- and cytosolic 5′-nucleotidase activity (data not shown). First, we recorded sequential changes in ecto- and cytosolic 5′-nucleotidase activity after exposure to 10−6 mol/L norepinephrine (n=5). Next, we varied the concentration of norepinephrine from 10−9 to 10−5 mol/L and measured ecto- and cytosolic 5′-nucleotidase activity at 15 minutes after exposure to norepinephrine (n=5 in each dose). This 15-minute interval was necessary to reach a steady state for ecto- and cytosolic 5′-nucleotidase activity (Fig 1⇓). To test whether activation of ecto-5′-nucleotidase is attributable to activation of protein kinase C, the effect of GF109203X (10−7 mol/L) on the dose-response curve between norepinephrine and 5′-nucleotidase activity (n=5 in each dose of norepinephrine) was observed. Furthermore, to test whether protein synthesis is involved in activation of ecto-5′-nucleotidase, the effect of cycloheximide (1.4×10−6 g/mL), a blocker of protein synthesis, on the dose-response curves between norepinephrine and 5′-nucleotidase activity (n=5 in each dose of norepinephrine) was observed.
Furthermore, we tested the effect of an α1-adrenoceptor agonist, methoxamine, and an activator of protein kinase C, PMA. First, we recorded sequential changes in ecto- and cytosolic 5′-nucleotidase activity after exposure to 10−6 mol/L methoxamine (n=5) and 10−7 mol/L PMA (n=5). Next, we varied the concentrations of methoxamine from 10−9 to 10−5 mol/L and PMA from 10−10 to 10−6 mol/L and measured ecto- and cytosolic 5′-nucleotidase activity at 15 minutes after an exposure to methoxamine and PMA (n=5 in each dose). This 15-minute interval was necessary to reach a steady state for ecto- and cytosolic 5′-nucleotidase activity (Figs 2-4). To test whether activation of ecto-5′-nucleotidase is attributable to the activation of protein kinase C, the effect of GF109203X (5×10−7 mol/L) on the dose-response curves between methoxamine and PMA and ecto-5′-nucleotidase activity (n=5 in each dose of methoxamine and PMA) was observed. GF109203X is a specific inhibitor of protein kinase C. To test whether protein synthesis is involved in increases in ecto-5′-nucleotidase activity, the effect of cycloheximide (1.4×10−6 g/mL) on the dose-response curves between methoxamine and PMA and ecto-5′-nucleotidase activity (n=5 in each dose of methoxamine and PMA) was observed.
To determine whether norepinephrine, methoxamine, and PMA activate protein kinase C of the membrane fraction, we examined the time courses of changes in protein kinase C activity in the membrane and cytosolic fractions in the groups of norepinephrine (10−6 mol/L, n=5), methoxamine (10−6 mol/L, n=5), and PMA (10−7 mol/L, n=5) treatments. Since we found that activation of protein kinase C reaches a steady state in 15 minutes, this 15-minute interval was necessary to reach a steady state for protein kinase C activity. Therefore, we examined the dose-response relation between norepinephrine (10−9 to 10−5 mol/L, n=5 in each dose), methoxamine (10−9 to 10−5 mol/L, n=5 in each dose), and PMA (10−10 to 10−6 mol/L, n=5 in each dose) and protein kinase C activity at 15 minutes after exposure to each chemical.
Protocol 2: Release of Adenosine and 5′-AMP From Rat Cardiomyocytes Pretreated With and Without Norepinephrine, PMA, and GF109203X
In this protocol, cardiomyocytes were isolated from 56 adult male Wistar rats. Cardiomyocytes obtained from the different rats were used in each experiment. To verify that increases in ecto-5′-nucleotidase activity are involved in augmented adenosine release under normoxic and hypoxic conditions, we measured adenosine in the medium of the suspension with 1.0 to 2.0×107 rat cardiomyocytes pretreated with 10−6 mol/L of methoxamine or 10−7 mol/L of PMA with and without exposure to 10−5 mol/L of GF109203X for 30 minutes. Furthermore, to examine whether 5′-AMP, a substrate for ecto-5′-nucleotidase to produce adenosine, is also released from cardiomyocytes, we measured 5′-AMP in the medium of the suspension of cardiomyocytes at 15 minutes in each treatment. The cells were gently washed twice and were incubated in normoxic or hypoxic solutions bubbled with 95% O2/5% CO2 and 95% N2/5% CO2, respectively. The Po2 was 478±8 and 32±6 mm Hg, respectively, in the two solutions. To test the cause-and-effect relation between the ecto-5′-nucleotidase activation and the augmented adenosine release, we examined the effect of AOPCP (50 μmol/L) in all the groups. AOPCP was added after the baseline collection of suspension for adenosine. We made the cardiomyocytes hypoxic after the baseline measurements and during addition of AOPCP. The groups consisted of (1) no treatment, (2) methoxamine pretreatment, (3) PMA pretreatment, (4) methoxamine and GF109203X pretreatment, (5) PMA and GF109203X pretreatment, (6) AOPCP treatment, (7) methoxamine pretreatment with AOPCP treatment, and (8) PMA pretreatment with AOPCP treatment groups (n=5 in each group). Sequential changes in adenosine concentration were recorded after exposures to 10 μmol/L 2′-deoxycoformycin to inhibit adenosine deaminase and 1 μmol/L 5′-iodotubercidin to inhibit adenosine kinase.
Adenosine and 5′-AMP Measurements
Details of the methods used to measure adenosine and 5′-AMP concentrations have been reported.15 16 19 20 To measure sequential changes in adenosine release from cardiomyocytes, we obtained the medium of the suspension of cardiomyocytes by centrifugation at 50g for 1 minute. We added 500 mmol/L EDTA and 10% trichloroacetic acid to the medium to inhibit degradation of adenosine and 5′-AMP. Trichloroacetic acid was removed by water-saturated ether. After centrifugation (1000g), the supernatant was collected and the adenosine and 5′-AMP concentrations were determined.
Adenosine in the solution (100 μL) was treated with succinic acid anhydride and trimethylamine in 100 μL of dioxane. After a 20-minute incubation at 37°C, the mixture was diluted with 100 μL of adenosine 2′,3′-O-disuccinyl-3-[125I]iodotyrosine methyl ester (0.5 pmol) and 100 μL of diluted antiadenosine serum. The mixture was kept in a cold-water (4°C) bath for 18 hours, and a second antibody solution (500 μL goat anti-rabbit immunoglobulin G antiserum) was added. After incubation for 1 hour at 4°C and centrifugation for 20 minutes at 2500g, the supernatant was removed and radioactivity remaining in the tube was countered in a gamma counter. We stored the samples obtained from the medium in which the cardiomyocytes were suspended.
The concentration of 5′-AMP was also determined by a UV method described previously.20 We used the same medium of cardiomyocytes for measurement of adenosine.
We stored the samples at −80°C until use for each assay.
Measurements of Activities of 5′-Nucleotidase and Protein Kinase C
On completion of the experiments, the rat cardiomyocytes were homogenized with a Potter-Elvehjem homogenizer (30 strokes) at 4°C for 5 minutes in 10 volumes of ice-cold 10 mmol/L HEPES-KOH buffer (pH 7.4) containing 0.25 mol/L sucrose, 1 mmol/L MgCl2, and 1 mmol/L mercaptoethanol. The crude homogenate was strained through a double-layer nylon sieve and homogenized again for 1 minute. For preparation of the crude membrane fraction, part of the homogenate was centrifuged at 1000g at 4°C for 10 minutes. The resultant pellet was washed three times and finally resuspended in the HEPES-KOH buffer. To prepare the cytosolic fraction, the remainder of the homogenate was centrifuged at 3000g at 4°C for 10 minutes, and the supernatant was centrifuged at 200 000g at 4°C for 1 hour. The membrane and cytosolic fraction were dialyzed at 4°C for 4 hours against HEPES-KOH containing 1 mmol/L MgCl2, 1 mmol/L mercaptoethanol, and 0.01% activated charcoal and divided into aliquots that were frozen immediately and stored at −80°C.
The activity of 5′-nucleotidase was measured by an enzymatic assay technique21 and is reported in units of nmol · mg protein−1 · min−1. 5′-Nucleotidase activity of membrane and cytosolic fractions were defined as ecto- and cytosolic 5′-nucleotidase activity, respectively. When cytosolic 5′-nucleotidase activity was measured, AOPCP (50 μmol/L) was added to inhibit contaminating ecto-5′-nucleotidase.
The activity of protein kinase C was measured by the enzyme assay using the RPN 77A kit (Amersham), which provides a simple and reliable method of estimating protein kinase C without extensive purification of the samples. Activity of protein kinase C was expressed as nmol · mg protein−1 · min−1. Protein concentrations were measured by the methods of Lowry et al22 using bovine serum albumin as a standard. Furthermore, to examine the Ca2+ and phospholipid dependencies of activity of protein kinase C, we measured protein kinase C activity adding 0.5 mmol/L in excess of EGTA to chylate Ca2+ and eliminating phosphatidyl-l-serine from the assay system.
Statistical analysis was performed with paired and unpaired t tests.23 Repeated-measures ANOVA followed by modified Bonferroni’s multiple comparison were also performed to evaluate intergroup differences in time courses and dose-response curves. All values were expressed as mean±SEM, and P<.05 was considered significant.
Norepinephrine 10−6 mol/L increased ecto-5′-nucleotidase activity by 54.8±8.8% from 6.18±0.42 nmol · mg protein−1 · min−1 at 30 minutes (Fig 1⇑, top). Cytosolic 5′-nucleotidase activity (control value, 1.13±0.10 nmol · mg protein−1 · min−1) was not increased by exposure to 10−6 mol/L norepinephrine (Fig 1⇑, bottom). Methoxamine 10−6 mol/L also increased ecto-5′-nucleotidase activity by 53.0±10.3% from 5.95±0.85 nmol · mg protein−1 · min−1 at 30 minutes (Fig 2⇓, top); however, cytosolic 5′-nucleotidase activity (control value, 1.01±0.12 nmol · mg protein−1 · min−1) was not increased by exposure to 10−6 mol/L methoxamine (Fig 2⇓, bottom). The time courses of increases in ecto-5′-nucleotidase activity were similar between the norepinephrine- and methoxamine-treated groups, and increases in ecto-5′-nucleotidase activity reached a steady state at 15 minutes after exposure to each chemical. Ecto-5′-nucleotidase activity showed a positive dose-response relation to norepinephrine and methoxamine at concentrations between 10−9 and 10−5 mol/L (Fig 5⇓). GF109203X inhibited the dose-dependent increases in ecto-5′-nucleotidase activity due to norepinephrine and methoxamine (10−9 to 10−5 mol/L in each) (Fig 5⇓). However, cycloheximide did not inhibit the increases in ecto-5′-nucleotidase due to norepinephrine and methoxamine (Fig 5⇓). On the other hand, exposure to 10−7 mol/L PMA increased ecto-5′-nucleotidase activity by 56.2±9.2% from 6.63±1.69 nmol · mg protein−1 · min−1 at 30 minutes (Fig 3⇓, top); however, cytosolic 5′-nucleotidase activity (control value, 0.97±0.23 nmol · mg protein−1 · min−1) was not increased (Fig 3⇓, bottom). A positive dose-dependent relation between PMA and ecto-5′-nucleotidase was demonstrated (Fig 4⇓). This activation of ecto-5′-nucleotidase activity was inhibited by GF109203X and was not affected by cycloheximide. These results indicate that (1) α1-adrenoceptor activation increases activity of ecto-5′-nucleotidase, (2) the α1-adrenoceptor–mediated increases in ecto-5′-nucleotidase activity are attributable to activation of protein kinase C, and (3) activation of ecto-5′-nucleotidase is independent of protein synthesis.
To investigate whether norepinephrine, methoxamine, and PMA increase the protein kinase C activity of the cardiomyocytes, we measured protein kinase C activity of the membrane and cytosolic fraction exposures to norepinephrine, methoxamine, and PMA with and without concomitant exposures to GF109203X. Fig 6⇓ shows the time courses of the increases in the protein kinase C activity of the membrane and cytosolic fractions and the summation of both fractions after exposures to norepinephrine, methoxamine, and PMA. The protein kinase C activity in the membrane fraction was increased in 10 minutes after exposures to norepinephrine, methoxamine, and PMA, which preceded the activation of ecto-5′- nucleotidase. The extents of increases in protein kinase C activity 30 minutes after exposures to norepinephrine, methoxamine, and PMA were 88±16%, 99±19%, and 54±15%, respectively. In contrast, the protein kinase C activity of the cytosolic fraction did not increase after exposures to norepinephrine, methoxamine, and PMA. Fig 7⇓ shows the dose-response relations between the doses of norepinephrine, methoxamine, and PMA with and without concomitant exposures to GF109203X and the protein kinase C activity of the membrane and cytosolic fractions and summation of both fractions. Norepinephrine and methoxamine 10−9 to 10−5 mol/L and PMA 10−10 to 10−6 mol/L increased protein kinase C activity of the membrane fraction in a dose-dependent manner. Table 1⇓ shows the Ca2+- and phospholipid-dependent activation of protein kinase C. Depletion of Ca2+ and/or phospholipid decreased the basal activity of protein kinase C of the cytosolic fraction but not the basal activity of protein kinase C of the membrane fraction. On the other hand, depletion of Ca2+ and/or phospholipid blunted the activation of protein kinase C of the membrane but not of the cytosol fraction. Thus, the increase in protein kinase C of the membrane fraction after exposure to methoxamine and PMA was dependent on both Ca2+ and phospholipid.
Although we showed that ecto-5′-nucleotidase in rat cardiomyocytes is activated by α1-adrenoceptor activation through increased activity of protein kinase C in the membrane fraction, it is not clear whether the activation of ecto-5′-nucleotidase increases release of adenosine from cardiomyocytes. Therefore, we measured the adenosine concentration of suspension with isolated normoxic cardiomyocytes pretreated with and without a 30-minute exposure to 10−6 mol/L methoxamine and 10−7 mol/L PMA, with and without GF109203X or AOPCP. There were no differences in adenosine concentration in each group before additions of 2′-deoxycoformycin and 5′-iodotubercidin (basal adenosine concentration in the suspension [pmol/mg protein]: control group, 4.10±0.54; methoxamine-pretreated group, 4.55±0.52; PMA-pretreated group, 4.26±0.69; methoxamine and GF109203X group, 4.53±0.42; PMA and GF109203X group, 4.24±0.36; AOPCP group, 4.75±0.68; methoxamine and AOPCP group, 4.38±0.51; and PMA and AOPCP group, 4.49±0.49). Adenosine release was augmented in the methoxamine- and PMA-pretreated groups compared with the controls in the normoxic conditions (Fig 8⇓). Furthermore, this augmentation of adenosine release was blunted by concomitant exposures to GF109203X in the control levels. AOPCP decreased the accumulation of adenosine, indicating that ecto-5′-nucleotidase contributed to adenosine release. Interestingly, concomitant exposures to AOPCP in the methoxamine- and PMA-pretreated groups blunted the increases in adenosine release to the levels of the AOPCP-alone group, indicating that enhanced release of adenosine in the methoxamine- and PMA-pretreated groups is attributable to the activation of ecto-5′-nucleotidase. Additionally, adenosine release from hypoxic cardiomyocytes was higher after pretreatment with methoxamine and PMA than in controls (Fig 9⇓). The augmentation of adenosine release due to PMA was also blunted by concomitant exposures to GF109203X. There were no differences in adenosine concentration in each group before additions of 2′-deoxycoformycin and 5′-iodotubercidin and hypoxic procedure (basal adenosine concentration in the solution [pmol/mg protein]: control group, 3.43±0.37; methoxamine-pretreated group, 4.62±0.81; PMA-pretreated group, 4.44±0.64; methoxamine and GF109203X group, 4.52±0.34; PMA and GF109203X group, 4.23±0.25; AOPCP group, 4.78±0.65; methoxamine and AOPCP group, 4.62±0.76; and PMA and AOPCP group, 4.42±0.53). Table 2⇓ shows the 5′-AMP concentrations of the medium in which the cardiomyocytes were suspended. In the normoxic condition, we detected 5′-AMP in the medium, and the hypoxic condition increased the accumulation of 5′-AMP concentration in the medium. There were no significant differences in 5′-AMP concentration between all of the groups in normoxic and hypoxic cardiomyocytes.
Effects of Activation of Protein Kinase C on Ecto-5′-Nucleotidase Activity and Adenosine Release
The present study demonstrates that activation of protein kinase C during α1-adrenoceptor stimulation increases ecto-5′-nucleotidase activity in rat cardiomyocytes and thereby contributes to increases in adenosine release from normoxic and hypoxic cardiomyocytes. Since increases in ecto-5′-nucleotidase activity due to the activation of protein kinase C do not necessarily result in increased adenosine release from rat cardiomyocytes, we had to consider the cause-and-effect relation between the increases in ecto-5′-nucleotidase activity and adenosine release. The major pathways of adenosine synthesis include enzymatic dephosphorylation of 5′-AMP by 5′-nucleotidase and the hydrolysis of S-adenosylhomocysteine (SAH) by SAH-hydrolase.24 5′-Nucleotidase exists in two forms in the myocardium: membrane-bound (ecto-5′-nucleotidase) and free in the cytoplasm (cytosolic 5′-nucleotidase), and both are capable of producing adenosine.25 26 27 Several lines of evidence have demonstrated that both ecto- and cytosolic 5′-nucleotidase are essential for the production of adenosine in the hypoxic cardiomyocytes.12 27 28 The present study reveals that ecto-5′-nucleotidase is more important for the pathway to increase adenosine release in rat cardiomyocytes, because (1) ecto-5′-nucleotidase activity was increased without altering cytosolic 5′-nucleotidase activity when release of adenosine was increased and (2) AOPCP, an inhibitor of ecto-5′-nucleotidase, blunted the increases in adenosine release from the rat cardiomyocytes (Figs 8⇑ and 9⇑). The present result is consonant with the work of Imai et al29 showing that ecto-5′-nucleotidase plays an essential role in releasing adenosine in hypoxic cardiomyocytes. In turn, Lloyd and Schrader30 showed that adenosine production by SAH-hydrolase is also essential in the normoxic condition, and we do not disagree with this. During ischemia and hypoxia, the contribution of SAH-hydrolase to adenosine release is believed to be relatively small, because increases in SAH, the substrate for SAH-hydrolase, are markedly less than the increases in 5′-AMP, the substrate for 5′-nucleotidase. In addition, we need to consider the activity of adenosine kinase and adenosine deaminase as potential determinants of adenosine production in rat cardiomyocytes.25 Schrader et al28 reported that adenosine kinase influences the net rate of adenosine production. Since we used 2′-deoxycoformysin and 5′-iodotubercidin to inhibit both enzymes and did not measure these two enzyme activities directly, the present study does not determine the role of activated protein kinase C in the modulation of the activity of either adenosine kinase or deaminase.
Because we found evidence that ecto-5′-nucleotidase plays an important role in adenosine production from cardiomyocytes, we needed to examine whether extracellular 5′-AMP is present around cardiomyocytes. We showed that 5′-AMP can be detected in the medium in which the cardiomyocytes are suspended and that hypoxia increases the amount of 5′-AMP. Even in the hypoxic condition, since most of the cardiomyocytes were intact, the detection of 5′-AMP may not be a reflection of cellular damages. Indeed, Bunger31 also reported that a compartment of 5′-AMP in the interstitial space exists in hearts. Furthermore, it is reported that 5′-AMP can be released from cells and is converted to adenosine outside the cells.32 33 Thus, we must consider the changes in the amount of 5′-AMP to be the cause of increased adenosine release in the norepinephrine-, methoxamine-, and PMA-treated conditions, because increases in adenosine release through ecto-5′-nucleotidase may simply be attributable to increased accumulation of the substrate, 5′-AMP. However, Table 2⇑ shows that 5′-AMP does not increase in these groups, indicating that changes in 5′-AMP are not the cause of increased adenosine release.
The present results revealed that α1-adrenoceptor activation is an important regulator for activation of ecto-5′-nucleotidase. α1-Adrenoceptor activation is known to result in activation of protein kinase C and production of inositol 1,4,5-triphosphate (IP3), which may modulate the activity of ecto-5′-nucleotidase. The present study revealed that activation of protein kinase C is critically important, rather than the production of IP3, because (1) activation of ecto-5′-nucleotidase due to α1-adrenoceptor stimulation is prevented by GF109203X, an inhibitor of protein kinase C; (2) PMA activates ecto-5′-nucleotidase; (3) the time courses of protein kinase C activation precede the time courses of activation of ecto-5′-nucleotidase in the groups of norepinephrine, methoxamine, and PMA treatments; and (4) the time courses of the activation of ecto-5′-nucleotidase in the norepinephrine-, methoxamine-, and PMA-treated groups are comparable. As shown in Table 1⇑, protein kinase C in the membrane fraction was almost dependent both on Ca2+ and phospholipid, suggesting that protein kinase C (PKC) activated by norepinephrine, methoxamine, and PMA was conventional PKC (cPKC). The kinase activity independent of Ca2+ and phospholipid in Table 1⇑ may be attributable to protein kinase C other than cPKC, such as novel PKC (nPKC) or other type of protein kinase. Although we depleted phospholipid in the assay system to test the phospholipid dependency of kinase activity, the contamination of phospholipid of the membrane fraction cannot be excluded, which may partially account for the presence of phospholipid-independent kinase.
The subcellular mechanism by which protein kinase C activates ecto-5′-nucleotidase is an interesting issue, although we could not clarify the exact mechanisms to explain our observation. One possibility is that activation of protein kinase C changes the cellular membrane characteristics around ecto-5′-nucleotidase and inhibits the internalization of this enzyme. Another possibility is that activation of protein kinase C may phosphorylate and activate the proteins that regulate activation of 5′-nucleotidase. Allosteric factors related to 5′-nucleotidase may be partially responsible for the activation of this enzyme, and activation of protein kinase C may modify this allosteric effect. Although these hypotheses are plausible, no data to support or refute any one of these theories were generated from the present study.
Pathophysiological Relevance in the Heart
Myocardial ischemia has been reported to release norepinephrine from the presynaptic vesicles,13 which activates α1-adrenoceptors and thereby protein kinase C. Furthermore, ischemia translocates protein kinase C from the cytoplasm to the membrane,14 which means the activation of protein kinase C. In this situation, ATP and ADP are coreleased with norepinephrine from the presynaptic vesicles. Released ATP and ADP can be converted to 5′-AMP, which becomes the substrate for adenosine. Therefore, the phenomenon of this α1-adrenoceptor activation due to released norepinephrine increasing ecto-5′-nucleotidase activity may effectively convert 5′-AMP to adenosine. Importantly, α1-adrenoceptor–mediated increases in adenosine release may contribute to cardioprotection.3 15 16 Several lines of evidence support the proposition that adenosine is an important factor for cardioprotection against ischemic and reperfusion injury1 2 3 and that α1-adrenoceptor–mediated increases in ecto-5′-nucleotidase activity and release of adenosine improve reperfusion injury.3 15 16
The ultimate goal of cardioprotection is to limit infarct size in acute myocardial infarction. Administration of exogenous adenosine has been shown to limit infarct size, and adenosine mediates the infarct size–limiting effects of ischemic preconditioning.34 As a possible mechanism, we have shown that ischemic preconditioning is mediated by activation of ecto-5′-nucleotidase,35 36 37 which can be mediated by activation of protein kinase C during brief periods of ischemia.14 Thus, the present observation hints that the linkage between protein kinase C, ecto-5′-nucleotidase, and cardioprotection can be a clinical strategy to protect myocardium against ischemia and reperfusion injury.
This work was supported by a Grant-in-Aid for Scientific Research (03670449) from the Ministry of Education, Science, and Culture, Japan, and by a grant from the Uehara Memorial Foundation, Japan. The authors are grateful to Noriko Tamai, Yoshitomo Edahiro, and Shinya Suzuki for their technical assistance.
Reprint requests to Masafumi Kitakaze, MD, PhD, The First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Japan.
- Received August 22, 1994.
- Accepted November 26, 1994.
- Copyright © 1995 by American Heart Association
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