Abstract
By use of front-surface fluorometry and fura-2-loaded medial strips of the porcine coronary artery, cytosolic Ca++ concentration ([Ca++]i) and force development were monitored simultaneously to determine the mechanisms of vasorelaxation induced by the diadenosine polyphosphates (APnA) diadenosine 5′,5‴-P1,P4-tetraphosphate (AP4A) and diadenosine 5′,5‴-P1,P5-pentaphosphate (AP5A). APnA concentration-dependently inhibited the sustained elevations of [Ca++]iand force induced by U-46619, a thromboxane A2 analog, in the presence of extracellular Ca++. APnA shifted the [Ca++]i-force relation curves of contractions induced by various concentrations of high K+to the right. The AP4A-induced decreases in [Ca++]i and force were largely attenuated by tetrabutylammonium. The AP4A-induced decreases in force were attenuated by 4-aminopyridine and charybdotoxin. The AP5A-induced decreases in [Ca++]iand force were attenuated by tetrabutylammonium, 4-aminopyridine and charybdotoxin. In the absence of extracellular Ca++, APnA did not inhibit the transient elevations of [Ca++]i induced by histamine or caffeine. Both AP4A and AP5A increased intracellular cAMP content. We thus conclude that AP4A and AP5A relax the porcine coronary artery by decreasing [Ca++]i, possibly through the activation of K+ channels, but not through inhibition of intracellular Ca++ release and by decreasing the Ca++sensitivity of the contractile machinery. These effects were considered to be mediated by cAMP.
Diadenosine polyphosphates such as AP4A and AP5A have received considerable attention because of their multiple biological activities. These compounds are present in various types of cells, including platelets (Flodgaard and Klenow, 1982), chromaffin cells (Rodriguez del Castillo et al., 1988) and neural tissue (Pintor et al., 1992), and are implicated primarily in platelet functions (Flodgaard and Klenow, 1982;Luthje and Ogilvie, 1983; Zamecnik et al., 1992) and neurotransmission (Baxi and Vishwanatha, 1995).
In recent years, interest in APnA in vasomotor activity has been renewed. Busse et al. (1988) first described AP4A induced endothelium-independent vasoconstriction in rabbit mesenteric arteries. Subsequent studies also indicated that AP4A elicits increases in perfusion pressure of rat portal vein (Busshardt et al., 1989) and that AP4A also induces the vasoconstrictor responses mediated by P2X-purinergic receptors (Ralevic et al., 1995). AP5A was recently reported to be a novel vasoconstrictor agent isolated from human platelets (Schluteret al., 1994). These authors reported that AP5A induced increases in perfusion pressure of the vasculture in isolated perfused rat kidney and aorta. AP5A-induced vasoconstriction has also been documented in both the rat mesenteric artery (Ralevic et al., 1995) and human umbilical artery (Davies et al., 1995) .
AP4A also induces endothelium-dependent vasodilation in rabbit mesenteric arteries (Busse et al., 1988), and endothelium-dependent and -independent decreases in rabbit coronary perfusion pressure (Pohl et al., 1991). Intravenous administration of AP4A to a dog produced a dose-dependent decrease in mean arterial pressure (Kikuta et al., 1994), which thus indicates that AP4A may induce, either directly or indirectly, relaxation rather than constriction of vascular smooth muscle in vivo. However, the cellular mechanism for APnA-induced vasorelaxation has yet to be investigated extensively. In the present study, we determined the mechanism underlying the vasorelaxing effects of AP4A and AP5A on porcine coronary smooth muscle cells by use of the simultaneous measurements of [Ca++]i and force. We obtained evidence that AP4A and AP5A reduce [Ca++]i by modulating the function of K+ channels and decreasing the Ca++ sensitivity of the contractile machinery and, as a result, induce vasorelaxation. These effects were thought to be mediated by the increase in the cellular cAMP content.
Materials and Methods
Tissue preparation.
Left circumflex coronary arteries (2–3 cm from the origin) were isolated from fresh porcine hearts at a local slaughterhouse immediately after the animals had been sacrificed. The tissue specimens were placed in ice-cold normal PSS and brought to the laboratory. After the segments were cut open longitudinally, the adventitia was trimmed away. The inner surfaces of the arteries were rubbed with cotton swabs to remove the endothelium. Each specimen was cut into similar sized strips (1 × 5 × 0.1 mm). Complete removal of the endothelium was confirmed by the lack of any relaxing response of the strips to 1 μM bradykinin.
Fura-2 loading.
Coronary arterial media strips were loaded with Ca++ indicator dye, fura-2, by incubating them in oxygenated (a mixture of 95% O2 and 5% CO2) Dulbecco’s modified Eagle’s medium containing 25 μM fura-2/AM (an acetoxymethyl ester form of fura-2) and 5% fetal bovine serum for 4 hr at 37°C. After loading with fura-2, medial strips were incubated in normal PSS for at least 1 hr at 37°C before starting the measurement, to remove the dye in the extracellular space and for purposes of equilibration. Loading the medial strips with fura-2 per se did not affect the contractility, as described previously (Abe et al., 1990;Hirano et al., 1990).
Front-surface fluorometry.
Changes in the fluorescence intensity of the fura-2-Ca++ complex were monitored simultaneously with the force development, by a front-surface fluorometer specifically designed for fura-2 fluorometry (CAM-OF3, Japan Spectroscopic Co., Tokyo, Japan), as described previously (Ushio-Fukai et al., 1993). The ratio of the fluorescence (500 nm) intensities at alternating 340-nm and 380-nm excitation wavelengths was monitored. The fluorescence ratio was expressed as a percentage, by assigning the values at rest in normal (5.9 mM K+) and 118 mM K+ PSS to be 0% and 100%, respectively. The absolute values of [Ca++]i of vascular strips were calculated according to the method of Grynkiewicz et al. (1985) with the Kd (apparent dissociation constant) of the fura-2-Ca++ complex of 225 nM (at 37°C). The absolute values of [Ca++]i for 0% and 100% levels were determined in separate measurements and were 108 ± 27 nM (n = 10) and 715 ± 103 nM (n = 10), respectively. The obtained [Ca++]i values are considered to be an approximation to the true [Ca++]i value, and the calibration of the absolute levels of [Ca++]i at the end of experiments is likely to be uncertain (Miyagi et al., 1995). Therefore, a statistical analysis of the [Ca++]i signal was performed with use of the percent fluorescence ratio.
Measurement of force development.
Coronary arterial media strips were mounted vertically in a quartz organ bath, and the isometric tension was measured, as described previously (Ushio-Fukaiet al., 1993). The strips were stimulated with 118 mM K+ PSS every 15 min during the fura-2 equilibration period (1 hr), and then the resting tension was increased in a stepwise manner to obtain the maximal tension development. The appropriate resting tension level obtained by this procedure was about 300 mg. At the beginning of each protocol, the responsiveness of each strip to 118 mM K+ PSS was recorded. The developed force was expressed in a percentage, by assigning the values at rest in normal PSS (5.9 mM K+) to be 0%, and those at steady state of contraction in 118 mM K+PSS to be 100%.
Assay of cAMP and cGMP.
The content of cAMP and cGMP in porcine coronary artery were assayed as described previously (Abeet al., 1994). After incubation of vascular strips with oxygenated (95% O2 and 5% CO2) normal PSS containing 10 μM AP4A or AP5A for 15 min at 37°C, the reaction was stopped by replacing the solution with ice-cold perchloric acid (6%). The strips were then homogenized in perchloric acid. The homogenate was centrifuged at 1500 ×g for 15 min. The supernatant was used to measure the cAMP and cGMP content by using radioimmunoassay kits (Yamasa, Tokyo, Japan). cAMP and cGMP levels were expressed as nanomoles or picomoles per milligram of wet weight of tissue (nmol or pmol/mg tissue).
Drugs and solutions.
The composition of normal PSS for fura-2 studies was as follows (mM): NaCl, 123; KCl, 4.7; NaHCO3, 15.5; KH2PO4, 1.2; MgCl2, 1.2; CaCl2, 1.25;d-glucose, 11.5. The Ca++-free solution (Ca++-free PSS) contained 2 mM EGTA instead of 1.25 mM CaCl2. High K+ PSS was prepared by replacing NaCl with equimolar KCl. PSS was bubbled with a mixture of 95% O2 and 5% CO2, and the resulting pH was 7.4. Fura-2/AM was purchased from Dojindo Laboratories (Kumamoto, Japan). AP4A, AP5A and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO). Bradykinin, ChTX and apamin were purchased from the Peptide Institute, Inc. (Osaka, Japan). U46619 was purchased from Funakoshi (Tokyo, Japan). Caffeine was obtained from Katayama Chemical (Osaka, Japan). Kits for radioimmunoassay of cAMP and cGMP were purchased from Yamasa (Tokyo, Japan). All other chemicals were obtained from Wako (Osaka, Japan).
Data analysis.
The values are expressed as the mean ± S.E.M. Student’s t-test was used to determine statistical significance between two groups, and analysis of variance was used to determine the dose-dependent effect of APnA on [Ca++]i and force. The number of experiments corresponds to the number of the animals used. The IC50 values, the concentrations that decreased the fluorescence ratio and force to 50% of the maximum response, were determined based on the concentration-response curve fitted according to a four-parameter logistic model (DeLean et al., 1978). The significance of the shift of the [Ca++]i-force relation curve was determined by an analysis of covariance. A value of P < .05 was considered to have statistical significance. All data were collected by a computerized data acquisition system (MacLab: Analog Digital Instruments, Castle Hill, Australia; and Macintosh: Apple Computer, Cupertino, CA).
Results
Effects of AP4A and AP5A on the [Ca++]i and force induced by U46619.
Figure 1, a and b, shows representative recordings of the effects of cumulative application of AP4A (0.01–10 μM) and AP5A (0.01–10 μM), respectively, on the [Ca++]i and force induced by 60 nM U46619, a thromboxane A2 analog. When the bathing medium was changed from normal PSS (5.9 mM K+) to 118 mM K+ PSS, both [Ca++]i and force rapidly increased and reached the plateau phases within 10 to 15 min. The values at the resting and plateau phases were designated to be 0% and 100% for both [Ca++]iand force. The application of 60 nM U46619 induced a rapid increase in [Ca++]i, which reached a peak level in 5 min and thereafter decreased to a plateau level within 10 min (60.52 ± 0.97%, n = 20). The force also rapidly increased and reached a plateau level within 10 min (95.27 ± 1.01%, n = 20). The applications of AP4A and AP5A during the U46619-induced sustained contraction caused concentration-dependent decreases in [Ca++]i and force of the coronary strips. Figure 1, c and d, shows a summary of the results obtained from seven and nine independent experiments performed in a manner similar to that shown in fig. 1, a and b. The cumulative application of AP4A and AP5A caused decreases in [Ca++]i and force (P < .05 by an analysis of variance) in a concentration-dependent manner. The IC50 values for [Ca++]i and force were 1.14 ± 0.29 μM and 1.03 ± 0.16 μM for AP4A (n = 7) and 0.82 ± 0.09 μM and 1.74 ± 0.34 μM for AP5A (n = 9), respectively. There was no significant difference in these IC50 values between AP4A and AP5A.
Effects of AP4A and AP5A on the [Ca++]i and force and the [Ca++]i-force relation (the sensitivity of the contractile machinery) induced by high K+ depolarization.
Figure2 shows the representative recordings of the effects of 10 μM AP4A (fig. 2, a–e) and AP5A (fig. 2, f–j), respectively, on the [Ca++]i and force induced by high K+ depolarization (20, 30, 40, 60 and 118 mM). The extent of the AP4A- and AP5A-induced decrease in force during the high K+ depolarization was much greater than that expected from the extent of reduction of [Ca++]i. This effect can be typically seen in figure 2, c, d, e, h, i and j, where AP4A (panels c, d and e) or AP5A (panels h, i and j) induced relaxation with little or no reduction of [Ca++]i. These observations indicated that AP4A and AP5A might decrease the Ca++ sensitivity of the contractile machinery. To further analyze the AP4A- and AP5A-induced change in the Ca++ sensitivity, the [Ca++]i (abscissa)-force (ordinate) relation curves were constructed with the data points obtained from several experiments done in a manner similar to shown in figure 2. As shown in figure 3, AP4A (panel a) and AP5A (panel b) shifted the [Ca++]i-force relation curves to the right (P < .05 by an analysis of covariance), which indicated that the reduction of force was much greater than that expected from the given reduction in [Ca++]i levels (the decrease in Ca++ sensitivity). AP4A and AP5A increased the ratio (%) values at which force was reduced by 50%, from 62.4 ± 6.2% to 76.3 ± 1.7% (AP4A); from 59.9 ± 8.7% to 77.1 ± 1.2% (AP5A).
Differential effects of AP4A and AP5A on the contractions induced by high K+ depolarization to those by U46619.
As shown in figure 1, 10 μM AP4A or AP5A almost completely inhibited the increases in [Ca++]i and force induced by U46619, whereas 10 μM AP4A or AP5A had little effect on the increases in [Ca++]i and force induced by high K+ depolarization (fig. 2, a–j) These observations indicated that the increase in [Ca++]i induced by high K+ depolarization is more resistant to inhibition by AP4A or AP5A. To further characterize this, we made a new figure (fig.4) using parts of the data points shown in figure 1, c and d, and figure 3, a and b. At comparable [Ca++]i (30 mM K+ depolarization vs. 60 nM U46619) and force (60 mM K+ depolarization vs. 60 nM U46619) levels, both AP4A and AP5A induced smaller decreases in the [Ca++]i and force in the contractions induced by K+ depolarization than those induced by U46619 (fig. 4). These results suggest that the AP4A- and AP5A-induced decrease in [Ca++]i is caused, at least in part, by membrane hyperpolarization.
Effects of K+ channel blockers on AP4A- and AP5A-induced decreases in the [Ca++]i and force.
To determine whether or not K+channels are involved in the relaxation induced by APnA, we examined the effect of various K+ channel blockers on AP4A- and AP5A-induced decreases in [Ca++]i and force. The following K+ channel blockers were examined: 1 mM TBA (nonspecific K+ channel blocker), 100 nM ChTX (large conductance Ca++-activated K+ channel blocker), 30 μM 4-AP (voltage-dependent K+channel blocker), 3 μM glibenclamide (ATP-sensitive K+ channel blocker) and 1 μM apamin (small conductance Ca++-activated K+ channel blocker). These K+ channel blockers were applied 10 min before and during the application of 60 nM U46619. We chose the highest concentrations of K+ channel blockers that do not directly affect U46619-induced increases in [Ca++]i and force (Kawasaki et al., 1997). As shown in figure5, a and b, TBA partially inhibited the AP4A-induced decrease in [Ca++]i (P < .05 by an analysis of covariance) and TBA, ChTX and 4-AP partially inhibited the AP4A-induced decrease in force (P < .05 by an analysis of covariance). As shown in figure 5, c and d, TBA, ChTX and 4-AP partially inhibited the AP5A-induced decrease in [Ca++]i and force (P < .05 by an analysis of covariance). Glibenclamide and apamin had no significant blocking effect on the decreases in [Ca++]i and force induced by AP4A or AP5A (data not shown).
Effects of AP4A and AP5A on the elevation of [Ca++]i and force induced by histamine and caffeine in the absence of extracellular Ca++.
Figure6a shows representative time courses of the changes in [Ca++]iand force induced by histamine in Ca++-free PSS containing 2 mM EGTA. When vascular strips were exposed to Ca++-free PSS, [Ca++]i gradually declined to reach a steady state (−21.60 ± 2.18%,n = 8), whereas the force remained unchanged. The application of 10 μM histamine after a 10-min incubation in Ca++-free PSS caused transient elevations of [Ca++]i (22.51 ± 2.76%, n = 8) and force (48.04 ± 1.75%,n = 8) with peaks at 15 s and 30 s, respectively. As shown in figure 6, b and c, pretreatment with AP4A or AP5A for 10 min did not affect the transient elevations of [Ca++]i induced by histamine. However, AP4A and AP5A did inhibit the transient elevations of force induced by histamine. Figure 6d summarizes the results obtained from eight independent experiments.
Figure 6d also summarizes the changes in [Ca++]i and force induced by caffeine in Ca++-free PSS containing 2 mM EGTA. The protocol for caffeine was similar to that for histamine (fig.6, a–c). The application of 20 mM caffeine after a 10-min incubation in Ca++-free PSS induced transient elevations of [Ca++]i (35.82 ± 1,95%, n = 5) and force (11.80 ± 0.83%,n = 5) with peaks at 30 s and 22 s, respectively. It is noteworthy that the [Ca++]i levels induced by 20 mM caffeine were significantly greater than those by 10 μM histamine (P < .01), whereas the forces induced by the former were smaller than those by the latter (P < .01) (fig. 6d). A pretreatment with AP4A and AP5A for 10 min did not affect the transient elevations of [Ca++]i and force induced by caffeine.
Effects of AP4A and AP5A on cellular cAMP and cGMP contents.
Figure 7 shows effects of AP4A and AP5A on the levels of cellular cAMP and cGMP contents of the porcine coronary artery in normal PSS. The cellular cAMP and cGMP contents were 0.21 ± 0.03 nmol/mg tissue (n = 14) and 2.04 ± 0.51 pmol/mg tissue (n = 14) at resting level (5.9 mM K+ PSS), respectively. When the vascular strips were exposed to 10 μM AP4A and AP5A for 15 min in normal PSS, the intracellular cAMP levels significantly increased to 0.31 ± 0.03 nmol/mg tissue (n = 14) and 0.32 ± 0.04 nmol/mg tissue (n = 14; vs. control, P < .05 for both, by student’s t test), respectively (fig. 7a). Neither AP4A nor AP5A induced any change in the intracellular cGMP levels (1.99 ± 0.34 and 2.38 ± 0.44 pmol/mg tissue) (fig. 7b).
Discussion
The present study investigated the mechanisms underlying the AP4A- and AP5A-induced direct relaxation of the porcine coronary artery without endothelium. The results obtained indicate that AP4A and AP5A induce relaxation of the porcine coronary artery by decreasing [Ca++]i (fig. 1), which may be partially caused by the opening of K+channels (fig. 5), and also by decreasing the Ca++ sensitivity of the contractile machinery (fig. 3). These effects were thought to be mediated by cAMP, because AP4A and AP5A increased the cellular cAMP content (fig. 7). In addition, AP4A and AP5A had no effect on the intracellular Ca++ release mechanism (fig. 6).
The relaxing effect of AP4A on the coronary artery has already been described previously. Pohl et al. (1991) first reported that AP4A induces endothelium-dependent and -independent decreases in rabbit coronary perfusion pressure. Nakae et al. (1996) also reported AP4A-induced coronary vasodilation in the porcine. However, the AP5A-induced vasorelaxation apparently has not been described previously. The results obtained in the present study indicate that AP4A- and AP5A-induced relaxation of the coronary artery is accompanied by a reduction of [Ca++]i. This mechanism could be clearly seen when precontraction was induced by U46619 (fig.1). However, when precontraction was induced by 60 mM or 118 mM K+ depolarization, the reduction of [Ca++]i was less obvious, although the reduction of force could still be detected (fig. 2). This result indicates that reduction of [Ca++]i is not the sole mechanism for the AP4A- or AP5A-induced coronary vasorelaxation.
The mechanism other than the reduction of [Ca++]i for the AP4A- or AP5A-induced coronary vasorelaxation was explored in the experiments shown in figures 2 and 3. As shown in figure 2, c, d, e, h, i and j, where AP4A and AP5A induced relaxation with little or no reduction of [Ca++]i. AP4A and AP5A shifted the [Ca++]i-force curve to the right during stimulation with high-K+depolarization (fig. 3), which indicates that AP4A and AP5A decrease the Ca++ sensitivity of the contractile machinery. This mechanism also explains the observation that AP4A and AP5A inhibited the development of force without affecting the transient increase in [Ca++]i induced by histamine in Ca++-free medium (fig. 6). However, this is not case in the contractions induced by 20 mM caffeine in Ca++-free medium (fig. 6d). Neither AP4A nor AP5A were able to inhibit the caffeine-induced transient force development. In Ca++-free medium without APnA, 20 mM caffeine induced a much smaller force than 10 μM histamine, although the former induced a greater [Ca++]i elevation than the latter (fig. 6). We reported previously that caffeine increases cAMP, which markedly decreases Ca++ sensitivity of the contractile machinery (Watanabe et al., 1992). The inability for AP4A and AP5A to decrease the caffeine-induced contraction could be explained by the saturation of the effect of cAMP on the reduction of the Ca++ sensitivity, because the relaxing effect of AP4A and AP5A was also mediated by cAMP (fig. 7). We did not use permeabilized preparations, which could directly demonstrate the decrease in the Ca++ sensitivity, because the solution used for permeabilized cell preparations contains millimolar concentrations of ATP. These conditions may desensitize the purinergic and possibly adenosine receptors, that are proposed to be involved in AP4A- and AP5A-induced vasorelaxation (discussed later).
It is well known that contraction of vascular smooth muscle is regulated by agonist-mediated modulation of the [Ca++]i-force relation (= the Ca++ sensitivity of the contractile machinery) as well as the changes in [Ca++]i (Somlyo and Somlyo, 1994). As to the candidate mediator for the reduction of Ca++ sensitivity, cAMP and cGMP have been shown to decrease the Ca++ sensitivity of the smooth muscle contractile machinery (Nishimura and van Breemen, 1989). We have also shown in a previous study that nitroglycerine (Abe et al., 1990) and isoprenaline (Ushio-Fukai et al., 1993) shift the [Ca++]i-force relation to the right in the porcine coronary artery. It thus seems likely that the decreases in Ca++ sensitivity of the contractile machinery induced by AP4A or AP5A may be mediated by cAMP or cGMP. In the present study we determined the second messenger for AP4A- or AP5A-induced vasorelaxation. As shown in figure 7, both AP4A and AP5A increased the cellular contents of cAMP. These results also support the hypothesis that AP4A and AP5A decrease the Ca++ sensitivity of the contractile machinery.
The inhibitory effects of AP4A or AP5A on [Ca++]i and force were attenuated when the strips were precontracted by high K+ depolarization, as compared with those during U46619 stimulation (fig. 4). This observation evoked speculation that the reduction of [Ca++]imight involve membrane hyperpolarization mediated by the opening of K+ channels, because this mechanism is eliminated during high K+ depolarization. Recent evidence has suggested that vascular tone and membrane potential are regulated by several types of K+ channels, including Ca++-activated K+ channels, voltage-dependent K+ channels and ATP-sensitive K+ channels (Nelson and Quayle, 1995). Therefore, we investigated the effects of selective blockers for these K+ channels on the AP4A- and AP5A-induced decrease in [Ca++]i and force during the stimulation with U46619. Comparative studies with these selective K+ channel blockers, as shown in figure 5, suggest that the activation of large conductance Ca++-activated K+ channels and voltage-dependent K+ channels may contribute to the decrease in [Ca++]i and force. It has been hypothesized that cAMP may decrease [Ca++]i by a hyperpolarization (Somlyo et al., 1970) probably by stimulation of Ca++-activated K+ channel openings (Sadoshima et al., 1988). Thus, the present results that AP4A and AP5A significantly increased the intracellular cAMP levels (fig. 7) support the idea that AP4A and AP5A may induce membrane hyperpolarization mediated by the opening of K+ channels. Our results do not support the notion that ATP-sensitive K+ channels and small conductance Ca++-activated K+ channels play a major role in the AP4A- and AP5A-induced vasorelaxation and decrease in [Ca++]i.
Another potential mechanism for the reduction of [Ca++]i, namely the inhibition of Ca++ release from intracellular stores, was examined in the experiments shown in figure 6. AP4A and AP5A did not inhibit the histamine- and caffeine-induced release of intracellular Ca++ in the absence of extracellular Ca++. These observations suggested that AP4A and AP5A do not affect intracellular Ca++ release through inhibition of a receptor-coupled signal transduction pathway or through a direct effect on intracellular storage sites.
The receptors responsible for the AP4A- and AP5A-induced coronary vasorelaxation were not explored in the present study. It has been reported that mammalian cells typically contain a specific AP4A hydrolase that hydrolyzes AP4A in an asymmetric fashion to yield AMP and ATP, which is converted to AMP and inorganic pyrophosphate; and dephosphorylation of AMP yields adenosine (Hankinet al., 1995; Luthje and Ogilvie, 1985, 1988; Ogilvieet al., 1989; Thorne et al., 1995). This enzyme also hydrolyzes higher homologs (e.g.,AP5A in an asymmetric fashion to yield ADP and ATP) and is presumed to be involved in the regulation of the intracellular level of such nucleotides (Guranowski and Sillero, 1992). Thus, it is possible that the application of AP4A and AP5A may produce adenosine, AMP, ADP and ATP, and stimulate such multiple receptors as nucleotide receptors and adenosine receptors. This may also partly explain why AP4A and AP5A have a different functional effect (constriction in some smooth muscle and relaxation in the other) at different potencies. For example, AP5A was approximately 10 to 1000 times more potent than AP4A for the contraction of guinea pig urinary bladder and vas deferens (Bo et al., 1994; Hoyleet al., 1995; Ralevic et al., 1995; Stone and Paton, 1989). For the vasoconstriction of rat and rabbit mesenteric vessels, AP5A was more potent than AP4A (Busse et al., 1988; Ralevicet al. 1995). On the other hand, there was no significant difference in the potency of AP4A and AP5A to induce relaxation of the guinea pig left atrium (Hoyle et al., 1996). We also observed almost equal potency for AP4A- and AP5A-induced coronary vasorelaxation. However, it is still possible that AP4A and AP5A might stimulate receptors as their own form without degradation because it has been reported that APnA can be considered as the long-lived substances (Busse et al., 1988; Hoyle et al., 1996; Pohl et al., 1991). In agreement with this speculation, the specific and saturable membrane receptors for AP4A have been reported to be present in brain, cardiac, liver, kidney, spleen and adipose tissue (Hilderman et al., 1991; Walker and Hilderman, 1993).
In summary, we obtained evidence that AP4A and AP5A induce relaxation of the porcine coronary artery by decreasing [Ca++]i, which may be partially caused by the opening of K+ channels, and decreasing the Ca++ sensitivity of the contractile machinery. These effects were thought to be mediated by cAMP. These substances may be naturally occurring coronary dilators.
Acknowledgments
We thank B. Quinn for comments on this manuscript. We also thank K. Kajishima for her excellent secretarial services.
Footnotes
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Send reprint requests to: Professor Hideo Kanaide, M.D., Ph.D.. Division of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3–1-1 Maidashi, Higashi-ku, Fukuoka 812–82, Japan.
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↵1 This study was supported in part by Grants-in-Aid for Developmental Scientific Research (no. 06557045), for General Scientific Research (nos. 07407022, 07833008) and for Creative Basic Research Studies of Intracellular Signaling Network from the Ministry of Education, Science, Sports and Culture, Japan, and also by Grants from Japan Research Foundation of Clinical Pharmacology and the Vehicle Racing Commemorative Foundation.
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↵2 Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812–82, Japan.
- Abbreviations:
- [Ca++]i
- cytosolic Ca++ concentration
- APnA
- diadenosine polyphosphates
- AP4A
- 5′,5‴-P1,P4-tetraphosphate
- AP5A
- diadenosine 5′,5‴-P1,P5-pentaphosphate
- TBA
- tetrabutylammonium
- 4-AP
- 4-aminopyridine
- ChTX
- charybdotoxin
- cAMP
- cyclic AMP (adenosine 3′,5′-cyclic monophosphate)
- cGMP
- cyclic GMP (guanosine 3′,5′-cyclic monophosphate)
- PSS
- physiological salt solution
- EGTA
- ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- Received March 7, 1997.
- Accepted July 16, 1997.
- The American Society for Pharmacology and Experimental Therapeutics