Introduction

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Histamine induces catecholamine release in several organ systems (Flacke et al., 1967; Albinus and Sewing, 1973; Marco et al., 1980). Myocardial ischemia is associated with an enhanced release of norepinephrine (Imamura et al., 1994, 1996; Obata et al., 1994). Adenosine, an endogenous nucleoside, is an important biochemical intermediate in cellular metabolism and has cardioprotective effects in myocardial ischemia (Lasley et al., 1990; Ely and Berne, 1992; Lasley and Mentzer, 1992; Thornton et al., 1992). Although the interaction between histamine and adenosine in the rat heart is unclear, recent reports have demonstrated the interaction between central histaminergic and adrenergic systems in cardiovascular response (Bealer, 1993; Bealer and Abell, 1994). Some investigators (Kitakaze et al., 1995; Sato et al., 1997) reported that enhanced activation of protein kinase C (PKC) increased 5'-nucleotidase activity, leading to an increased release of adenosine, in isolated rat cardiomyocytes. Furthermore, in isolated rat cardiomyocytes, the activation of 5'-nucleotidase was shown to be mediated by the activation of PKC (Kitakaze et al., 1995). Adenosine exerts multiple actions throughout the body and modifies various cardiovascular functions (Berne, 1980). The formation and release of adenosine in the ischemic myocardium is enhanced, and the adenosine is derived from the enzymatic dephosphorylation of adenosine 5'-monophosphate (AMP) by 5'-nucleotidase (Frick and Lowenstein, 1976; Thornton et al., 1992). It is suggested that, in dog hearts, stimulation of ₁-adrenoceptor augments adenosine production during ischemia by enhancing 5'-nucleotidase activity, which can limit the size of the infarct (Kitakaze et al., 1994). The present study was undertaken to clarify whether histamine affects the norepinephrine-mediated interstitial adenosine production.

To achieve this goal, we measured the concentration of interstitial adenosine in in vivo hearts using a flexibly mounted microdialysis technique that we developed (Obata et al., 1994, 1998). The production of adenosine under normoxic conditions is attributed primarily to the transmethylation of S-adenosylhomocysteine (SAH) catalyzed by SAH hydrase; the hydrolysis of AMP by ecto-5'-nucleotidase, the main pathway for adenosine production under ischemic conditions, is considered to be minimal (Sparks and Bardenheuer, 1986; Liu et al., 1991). To mimic ischemic conditions, we measured the concentration of dialysate adenosine under continuous supply of AMP, the substrate for 5'-nucleotidase, through the microdialysis probe. Using this system, we have reported that the level of AMP-primed dialysate adenosine reflects the activity of ecto-5'-nucleotidase in the particular site of the interstitial space of the myocardium (Obata and Yamanaka, 2000). The results in the present study demonstrate that histamine increased the production of interstitial adenosine via norepinephrine-mediated activation of ecto-5'-nucleotidase.

	Materials and Methods

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Animal Preparation

The study was performed with Wistar rats of either sex, weighing 300 to 400 g, that were anesthetized by an intraperitoneal injection of chloral hydrate (400 mg/kg). After intubation, the rat was mechanically ventilated with room air supplemented with oxygen. The chest was opened at the left 5th intercostal space, and the pericardium was removed to expose the left ventricle. In the case of reserpinized rats, reserpine (5 mg/kg) was injected intravenously 24 h before the experiment. All procedures in dealing with the experimental animals met the guideline principles stipulated by the Physiological Society of Japan and the Animal Ethics Committee of the Oita Medical University.

Microdialysis Technique

Details of the technique required for manipulation of the flexibly mounted microdialysis probe in in vivo rat hearts (to measure the interstitial adenosine) have been described previously (Obata et al., 1994). In brief, the tip of the microdialysis probe (3 mm in length and 220-µm o.d. with the distal end closed) was made of dialysis membrane (cellulose membrane 10 µm thick with a 50,000 molecular weight cut-off). Two fine silica tubes (75-µm i.d.) were inserted into the tip of a cylinder-shaped dialysis probe and served as an inlet for the perfusate and an outlet for the dialysate, respectively. The inlet tube was connected to a microinjection pump (CMA/100, Carnegie Medicine, Stockholm, Sweden), and the outlet tube led to the dialysate reservoir. These tubes were supported loosely at the midpoint on a semirotatable stainless steel wire so that their movement fully synchronized with the rapid up-and-down motion of the tip caused by the heart beats. The probe was implanted from the epicardial surface into the left ventricular myocardium and was perfused through the inlet tube with Tyrode's solution of the following composition (in mM): NaCl, l37; KCl, 5.4; CaC1₂, 1.8; MgC1₂, 0.5; NaH₂P0₄, 0.16; NaHCO₃, 3.0; glucose, 5.5; and HEPES, 5.0 (pH = 7.4 adjusted with NaOH). The Tyrode's solution that flowed out of the cut end of the inlet tube entered the extracellular space across the dialysate membrane by diffusion. The interstitial fluid diffused back into the cavity of the probe, and the dialysate left the probe through the orifice of the outlet tube. The perfusion rate was 1.0 µl/min. The relative recovery of adenosine measured using this flow rate (1.0 µl/min) was 18.0 ± 1.6% (n = 6).

Analytical Procedure

Measurements of Adenosine Concentration in Dialysate. The dialysate was collected (at the rate of 1.0 µl/min) into a series of wells for every 15 min consecutively (15 µl in each well). A 10-µl aliquot of the dialysate sample was used for the detection of adenosine, and we measured the concentration using reversed-phase high-performance liquid chromatography (HPLC). Separation of the compounds was achieved on Eicompak MA-5 ODS columns (5 µm, 4.6 × 150 mm; Eicom, Kyoto, Japan) with the mobile phase consisting of 200 mM KH₂PO₄ (pH = 3.8 adjusted with phosphoric acid) and 5% (v/v) acetonitrile. The flow rate was set at 1.0 µl/min using a pumping system (PU-980, JASCO Corp., Tokyo, Japan). The absorbance of the column eluate was monitored at 260 nm using an ultraviolet detector (UV-970, JASCO Corp.). The absorbance peak of adenosine was quantified by comparing the retention time and peak height with a known adenosine standard at concentrations of 1 and 10 µM. Concentrations of adenosine are expressed as a raw value unless otherwise indicated. The limit of the assay for adenosine is 0.42 µM.

Measurements of Norepinephrine Concentration in Dialysate. To determine the level of norepinephrine, the heart was perfused with Ringer's solution consisting of 147 mM NaCl, 2.3 mM CaCl₂, and 4 mM KCl (pH 7.4) (Obata et al., 1994; Yamazaki et al., 1997). Norepinephrine assay was performed using HPLC with an electrochemical procedure. To make the standard norepinephrine solution, norepinephrine was dissolved in the Ringer's solution. When the perfusion rate of 1.0 µl/min was used, the relative recovery, using the standard norepinephrine solution (1 µM), was l7.0 ± 0.7%. The dialysate samples were collected into a small collecting tube containing 15 µl of 0.1 N HClO₄ for every 15 min consecutively for the adenosine measurements. The samples were immediately injected into an HPLC-electrochemical system equipped with a glassy carbon working electrode (Eicom, Kyoto, Japan) and an analytic reverse-phase column on an Eicompak MA-5ODS column (5 µm, 4.6 × 150 mm; Eicom). The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g of 1-heptanesulfonic acid sodium salt, 0.1 g of Na₂EDTA, 3 ml of triethylamine, and 125 ml of acetonitrile. The pH of the solution was adjusted to 2.8 with 3 ml of phosphoric acid. When dialysate norepinephrine levels reached a steady state at 120 min after probe implantation, histamine was directly infused in rat heart through a microdialysis probe. The limit of the assay for norepinephrine is 0.005 µM.

Experimental Protocol

We measured the time-dependent changes of the dialysate adenosine concentration in the presence of AMP (AMP-primed dialysate adenosine concentration) and evaluated the activity of ecto-5'-nucleotidase. Under a constant supply of AMP, the dialysate adenosine is considered to originate from enzymatic dephosphorylation of AMP by endogenous ecto-5'-nucleotidase since ,-methyleneadenosine 5'-diphosphate (,-meADP, 100 µM), an inhibitor of ecto-5'-nucleotidase, completely inhibited the AMP-primed dialysate adenosine (Obata and Yamanaka, 2000). Therefore, the level of dialysate adenosine measured in the presence of AMP is an appropriate measure of the activity of ecto-5'-nucleotidase in rat hearts in situ. In this series of experiments, AMP at a concentration of 100 µM was perfused throughout the experiment via the probe, and the dialysate sampling was started after a 30-min equilibration period.

Drugs Used

Histamine hydrochloride and AMP (Wako Pure Chemical Co., Osaka, Japan) and pargyline hydrochloride (Sigma, St. Louis, MO, and Osaka, Japan) were dissolved with the Tyrode's solution just before the start of experiments to acquire the desired final concentrations, as given in the text. ,-meADP (Sigma) and chelerythrine (Sigma) were dissolved in distilled water and kept as 10 mM stock solutions. Okadaic acid (a kind gift from Fugisawa Pharmaceutical Co., Osaka, Japan) was dissolved in dimethyl sulfoxide as a 10 mM stock solution. An appropriate volume of these stock solutions was added to Tyrode's solution just before use, as indicated under Results. Reserpine was purchased from Daiichi Pharmaceutical Co. (Tokyo, Japan).

Statistical Analysis

All values are expressed as means ± S.E.M. The significance of difference was determined by using ANOVA with Fisher's post hoc test. A P value of less than 0.05 was considered to be statistically significant.

	Results

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The Effect of Histamine on Adenosine Formation. We first examined the effect of histamine on the dialysate adenosine concentration in the presence of AMP and evaluated the activity of ecto-5'-nucleotidase in vivo. In this series of experiments, AMP was perfused throughout the experiments via a microdialysis probe. The dialysate sampling was started after a 30-min equilibration period, as described previously (Sato et al., 1997). After obtaining two control fractions (a dialysate of 30-45 and 45-60 min), histamine (100 µM) was introduced through the probe in the presence of AMP. The baseline level of dialysate adenosine measured in the absence of exogenous AMP was ~0.5 µM, which was ~18 times lower than the level of dialysate adenosine observed in the presence of 100 µM AMP (~9 µM). Histamine (100 µM) significantly increased the level of dialysate adenosine from 8.26 ± 0.66 to 11.68 ± 1.09 µM at 30 to 45 min after histamine was applied (n = 6, P < 0.05). After the removal of histamine from the perfusate, the adenosine concentration was decreased to 7.68 ± 0.95 µM in 30 min (Fig. 1A). In contrast, the introduction of ,-meADP (100 µM) significantly decreased the concentration of adenosine (0.51 ± 0.18 µM) within 45 min (a dialysate of 90-105 min) (n = 6, P < 0.05). After removal of these drugs from the perfusate, the concentration of dialysate adenosine was gradually restored and reached a level of 10.50 ± 1.85 µM (a dialysate of 135-150 min) (Fig. 1B). Similar experiments were repeated using various concentrations of histamine (10, 50, 100, and 500 µM), and the results are summarized in Fig. 2. Histamine had a tendency to increase the level of AMP-primed dialysate adenosine over the concentration range of 10 to 500 µM; the maximum effect, 143.8 ± 15.4% of control (n = 6, P < 0.05), was obtained at 100 µM histamine.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Effect of histamine on the production of interstitial adenosine in rat ventricular myocardium. A, sequential changes of the dialysate adenosine concentration measured in the presence of 100 µM AMP throughout. Histamine (100 µM) was added to the perfusate for 45 min, as indicated by a horizontal bar (n = 6). B, effect of ,-meADP (100 µM) on histamine-induced increases in dialysate adenosine (n = 6). The abscissa denotes the time in minutes before and after the introduction of histamine. Values are means ± S.E.M. *P < 0.05, **P < 0.001 versus predrug value.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effect of histamine on the AMP-primed (100 µM) dialysate adenosine. The ordinate scale indicates concentrations of dialysate adenosine, each measured 30 to 45 min after application of various concentrations of histamine (10, 50, 100, and 500 µM), and is shown as a percentage relative to the value measured just before histamine was applied (100%). Values are means ± S.E.M. (n = 6). ns, nonsignificant. *P < 0.05 versus predrug value (n = 6 in each column).

The Effect of Histamine on Norepinephrine Release. After obtaining two control fractions (a dialysate of 30-45 and 45-60 min), histamine (100 µM) was introduced through the probe in the presence of 100 µM AMP. As shown in Fig. 3A, histamine (100 µM) significantly increased the level of norepinephrine in the dialysate (n = 6, P < 0.05). In contrast, in rats treated with reserpine (see Materials and Methods), the levels of norepinephrine remained suppressed (n = 6) (Fig. 3B).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effects of histamine on the norepinephrine concentration in the dialysate. The histamine was introduced in the perfusate in intact (A) and reserpinized rat hearts (B), as indicated by horizontal bars. The abscissas show the time after implantation of the dialysis probe. Values are means ± S.E.M. (n = 6). *P < 0.05 versus predrug value (n = 6 in both A and B).

The Role of ₁-Adrenoceptors and PKC in the Histamine-Induced Adenosine Formation. We examined whether the histamine-induced increases of adenosine in dialysate were the result of increased PKC activity via ₁-adrenoceptor stimulation. To clarify this, we used prazosin, an ₁-adrenoceptor antagonist, or chelerythrine, a potent and selective PKC inhibitor, that interacts with the catalytic domain of this enzyme (Herbert et al., 1990). In the presence of prazosin (50 µM), histamine (100 µM) failed to increase the dialysate adenosine (Fig. 4A). In contrast, atenolol, a ₁-adrenoceptor antagonist, did not prevent the histamine-induced increase in dialysate adenosine even in the presence of a high concentration of atenolol (50 µM); histamine (100 µM) significantly increased the dialysate adenosine concentration (by 41.3 ± 13.8%, P < 0.05, not illustrated). On the other hand, in the presence of chelerythrine (10 µM), histamine did not increase the dialysate adenosine (Fig. 4B). In addition, in reserpine-treated animals, histamine did not significantly increase the level of adenosine in the dialysate (from 3.47 ± 0.63 to 3.41 ± 0.58 µM) (Fig. 4C). These results suggest that histamine-released endogenous norepinephrine increased the AMP-primed dialysate adenosine concentration (i.e., the activity of ecto-5'-nucleotidase) via activation of PKC. To further support this notion, we examined the effects of pargyline, a monoamine oxidase inhibitor, on the production of interstitial adenosine. In the presence of a high concentration of pargyline (100 µM), histamine (100 µM) significantly increased the dialysate adenosine from 11.34 ± 0.96 to 14.16 ± 0.70 µM at 30 to 45 min after pargyline was applied (n = 6, P < 0.05) (Fig. 5).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of prazosin and chelerythrine on the dialysate adenosine concentration. The effect of histamine (100 µM) on the adenosine concentration was studied in the presence of 50 µM prazosin (n = 5, A) or 10 µM chelerythrine (n = 6, B) in intact rat heart or in reserpinized rat heart (n = 5, C). Values are means ± S.E.M.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of high concentration of pargyline on the dialysate adenosine concentration under histamine in the perfusate. The abscissa denotes the time in minutes after the introduction of histamine. Pargyline (100 µM) was added to the perfusate for 45 min, as indicated by a horizontal bar (n = 6). Values are means ± S.E.M. *P < 0.05, versus predrug value.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of okadaic acid on the dialysate adenosine concentration under histamine in the perfusate. Okadaic acid (50 µM) was added to the perfusate for 45 min, as indicated by a horizontal bar (n = 6). Values are means ± S.E.M. *P < 0.05, versus predrug value.

	Discussion

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The conversion of AMP to adenosine by 5'-nucleotidase may be a crucial step for cardioprotection during myocardial ischemia. We assessed the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) and examined the effects of histamine on the production of interstitial adenosine using a flexibly mounted microdialysis technique (Obata et al., 1994, 1998). We provided the evidence that the level of dialysate adenosine measured in the presence of AMP reflects the activity of ecto-5'-nucleotidase in the particular tissue (Obata and Yamanaka, 2000). In the present study, we have demonstrated that histamine enhanced the production of interstitial fluid adenosine produced via stimulation of ecto-5'-nucleotidase in rat hearts using microdialysis technique.

AMP-induced increases in the dialysate adenosine concentration was shown to be dependent on the AMP concentrations used, and the EC₅₀ of AMP was ~100 µM (Sato et al., 1997), a value close to the K_m (Michaelis constant) estimated for ecto- (rather than for cytosolic) 5'-nucleotidase in rat hearts (Sullivan and Alpers, 1971). The baseline level of dialysate adenosine was ~0.5 µM. Based on the recovery rate of tissue adenosine (18%), the concentration of adenosine in the interstitial fluid of the ventricular muscle located adjacent to the dialysis membrane was ~2.8 µM, a value comparable to that reported in other studies, i.e., 0.3 to 3.6 µM, using a conventional microdialysis technique (Van Wylen et al., 1990, 1992) or a porous nylon sampling disc technique (Zhu et al., 1991). Thus, the value of 2.8 µM observed in the present study is within the range obtained in previous studies. The AMP-induced adenosine was most probably generated by enzymatic dephosphorylation of AMP by ecto-5'-nucleotidase, and the baseline production of adenosine was probably derived from hydrolysis of SAH. In the presence of the selective inhibitor of ecto-5'-nucleotidase, ,-meADP (Hori and Kitakaze, 1991), at a concentration of 100 µM in the perfusate, AMP (100 µM)-induced increases of adenosine in the dialysate were completely inhibited and remained at ~0.51 µM, a level close to the baseline. Therefore, it is reasonably assumed that the level of dialysate adenosine is proportional to the adenosine concentration in the interstitial space of the myocardium and reflects the ecto-5'-nucleotidase activity in this tissue. The rationale and the relevance of this method were addressed in our previous reports (Sato et al., 1997; Obata and Yamanaka, 2000). In the present experiment, the administration of drugs was through the microdialysis probe. Although the exact mechanism by which histamine induced adenosine production is unclear, histamine clearly increased the level of adenosine in the rat heart (Fig. 1A). The introduction of ,-meADP in the presence of histamine significantly decreased the level of AMP-primed dialysate adenosine (Fig. 1B). Our preliminary observation showed that histamine not only increased the concentration of adenosine in the dialysate but also that of inosine, and in the presence of ,-meADP, histamine decreased the levels of both adenosine and inosine. Therefore, it is unlikely that the reduction of interstitial adenosine concentration by histamine in the presence of ,-meADP was due to increased activity of adenosine deaminase. Taken together, it is likely that the histamine-induced increase of adenosine was due to the activation of ecto-5'-nucleotidase. However, we cannot rule out other possibilities. For example, histamine attenuated the breakdown of adenosine by inhibiting adenosine deaminase, leading to the increase in dialysate adenosine. However, histamine (100 µM) did not affect the level of dialysate adenosine when measured in the absence of AMP (T. Obata, unpublished observation). Therefore, it is not likely that histamine attenuated the breakdown of adenosine. Namely, the effective concentrations outside the dialysis membrane are probably lower than the dialysate concentration. AMP supplied from an inlet tube diffused out into the interstitial fluid through the dialysis membrane and was converted to adenosine by endogenous 5'-nucleotidase. We examined the effect of ,-meADP, a selective inhibitor of ecto-5'-nucleotidase, which was unable to access to cytosolic 5'-nucleotidase because it cannot penetrate the sarcolemma of heart muscle cells (Headrick et al., 1992). Since ,-meADP completely inhibited histamine-induced increases in dialysate adenosine concentrations without affecting the baseline level of adenosine, the AMP-induced increase in the adenosine concentration was most probably derived from enzymatic dephosphorylation of AMP by ecto-5'-nucleotidase, and the baseline production of adenosine was probably derived from hydrolysis of SAH.

Histamine is released during myocardial infarction and ischemic arrhythmias (Masini et al., 1987). We demonstrated that histamine increased the adenosine concentration measured in the presence of 100 µM AMP, which was inhibited by ₁-antagonist (prazosin) (Fig. 4A) or PKC inhibitor (chelerythrine, 10 µM) (Fig. 4B). Thus, we have shown a clear and important link between activation of PKC and the production of adenosine catalyzed by an enhanced activity of ecto-5'-nucleotidase in the rat heart in vivo. We previously reported that diacylglycerol, a potent PKC activator (Nishizuka, 1995), increased the AMP-primed dialysate adenosine (Sato et al., 1998). It is known that norepinephrine stimulates ₁-adrenoceptors and leads to activation of PKC (Fedida et al., 1993). During acute regional ischemia, the interstitial concentration of norepinephrine in the ischemia region is reported to be increased (Schömig, 1989). Taken together, the results suggest that histamine-released norepinephrine stimulated ₁-adrenoceptors and activated PKC, leading to activation of ecto-5'-nucleotidase and release of adenosine. We examined the effect of pargyline, a monoamine oxidase inhibitor, on the production of interstitial adenosine. In the presence of histamine, pargyline (100 µM) significantly increased the dialysate adenosine (Fig. 5). The results indicate that accumulation of norepinephrine in the extracellular fluid elicited by pargyline led to the production of adenosine. Our results indicate that histamine-released norepinephrine elevates adenosine by 5'-nucleotidase activation. Specifically, it has been shown that ₁-adrenoceptor stimulation and subsequent PKC activation is apparently one of the pathways that causes an adenosine rise through 5'-nucleotidase activation (Sato et al., 1997).

The steady-state production of adenosine, i.e., the steady-state concentration of dialysate adenosine, may depend on the equilibrium between phosphorylation and dephosphorylation of ecto-5'-nucleotidase. Okadaic acid enhances phosphorylation by inhibiting protein phosphatases (Bialojan and Takai, 1988). Our observation that okadaic acid enhanced the effect of histamine on the production of adenosine (Fig. 6) suggests that PKC phosphorylated ecto-5'-nucleotidase and increased its enzyme activity, leading to increased production of adenosine. As shown in Fig. 4, the adenosine level in reserpinized rat heart was about half that of nontreated control. Although the exact mechanism of reduced adenosine level in reserpinized rats is unclear, it may be explained as follows. Komachi et al. (1993, 1994) reported that membrane-associated immunoreactive protein kinase C was reduced in reserpinized rat brain. The change of PKC distribution may lead to decreased PKC activity and decreased phosphorylation of ecto-5'-nucleotidase, and as a consequence, decreased ecto-5'-nucleotidase activity would reduce the adenosine level. Ischemia activates PKC via an ₁-adrenoceptor-dependent and -independent mechanism. The latter mechanism of activation was secondary to translocation of PKC from the cytosol to the sarcolemma of cardiac muscles: the translocated PKC may then activate ecto-5'-nucleotidase, perhaps via modification of some of the latter enzyme from inside of the membrane, and as a consequence, interstitial adenosine would increase. Moreover, several lines of experimental evidence suggest that stimulation of a variety of G protein-coupled receptors (e.g., adenosine, A₁, ₁-adrenergic, muscarine, bradykinin, and endothelin-1 receptors) leads to the activation of PKC (Cohen and Downey, 1996). Although the contribution of histamine to this phenomenon is less known, it is possible that histamine, a catecholamine releaser, may contribute to ischemic preconditioning. However, further research is necessary to confirm the relation between histamine release and ischemic preconditioning. The present study provides in vivo evidence as follows: histamine-released norepinephrine stimulated an ₁-adrenoceptor-dependent and -independent mechanism. PKC phosphorylated ecto-5'-nucleotidase and enhanced its enzyme activity, leading to the increased production of adenosine in rat hearts. Estimation of ecto-5'-nucleotidase activity by using flexibly mounted microdialysis probes perfused with AMP may be useful in future studies to elucidate the regulatory influences of ecto-5'-nucleotidase on the production of adenosine.