Introduction

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It is now well established that a structurally diverse group of compounds that produce vasorelaxation via activation of vascular K_ATP exists (Triggle, 1990; Edwards and Weston, 1993). The more well known of these K_ATP opener vasodilators include clinically used antihypertensives such as minoxidil (via it's active metabolite, minoxidil sulfate) and pinacidil, as well as experimental drugs such as cromakalim (Edwards and Weston, 1993; Meisheri et al., 1993a). An extensive database is now available in vascular smooth muscle for these compounds with use of intact tissue pharmacology, intact tissue ⁴²K and ⁸⁶Rb fluxes, intact tissue membrane potential, single-cell membrane potential and whole-cell patch-clamp measurements, which collectively support the role of K_ATP activation as the primary mechanism for vasodilation produced by these agents (Edwards and Weston, 1993; Quast and Cook, 1989; Cook and Quast, 1990; Meisheri et al., 1993b; Xu and Lee, 1994). Although a consensus has emerged that the vascular K_ATP is the primary target for these vasodilators, the biochemical mechanism(s) by which these drugs activate the K_ATP has not been well understood. A breakthrough in this area was achieved in 1992 with the discovery of a specific binding site for P1075, a pinacidil-based potent K_ATP opener (Bray and Quast, 1992). In this and subsequent studies (Manely et al., 1993; Quast et al., 1993) with intact isolated rat aorta, a correlation between the functional effects of various K_ATP openers and blockers and their effects on specific binding of ³H-P1075 was established. These authors proposed that they have identified a functional receptor that mediates the vascular effects of various K_ATP openers as well as the K_ATP blocker, glyburide. This hypothesis was significant because previous attempts at identifying K_ATP opener receptor sites by use of radiolabeled cromakalim or minoxidil/minoxidil sulfate were unsuccessful (Coldwell and Howlett, 1987; Meisheri et al., 1991a, 1993a).

A large amount of in vitro as well as in vivo data are available which show that K_ATP openers produce vasodilation in a variety of vascular beds in various species (Cook and Quast, 1990; Shen and Venter, 1993). In contrast, as described above, characterization of the proposed vascular receptor site for P1075 is available to date only in rat aorta. We considered it important to develop a similar biochemical database in other vascular beds. Our working hypothesis was that there would be a tight coupling in a given vascular preparation between the presence of high-affinity ³H-P1075 receptor sites and the pharmacological effect of P1075. Therefore, the main objective of this study was to identify, characterize and compare specific binding of ³H-P1075 in vascular tissues from rat, rabbit and dog, with particular emphasis on correlating specific binding with functional vasorelaxation produced by P1075.

	Materials and Methods

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Five vascular preparations from three species were used in this study: rat aorta, rat superior mesenteric artery, rabbit superior mesenteric artery, dog coronary artery and dog superior mesenteric artery.

Preparation of Vascular Tissues

Rats and rabbits. Male Sprague-Dawley rats (250-300 g) and male New Zealand white rabbits (1.5-2.0 kg) were anesthetized with Metofane (methoxyflurane) and exsanguinated. The thoracic aorta (rat) and superior mesenteric artery (rat and rabbit) were carefully excised and placed in warm (37°C), oxygenated (100% O₂) PSS, pH 7.3.

Dogs. Male mongrel dogs (15-22 kg) were anesthetized with sodium brevital (approximately 100 mg/kg i.v.) and placed on a respirator while the superior mesenteric artery was isolated and removed. The heart was then quickly excised and the left circumflex coronary artery isolated. Both arteries were placed in warm, oxygenated PSS. Tissues from all three species were cleaned of adherent fat and connective tissue and cut into rings for use in either binding or vasorelaxation studies. Often binding experiments and vasorelaxation experiments were run on the same day so that rings from the same vascular preparation were used for both studies. This protocol allowed us to verify that the tissues were pharmacologically viable and responsive to P1075.

³H-P1075 Binding Studies

General. We followed the protocol developed by Quast et al. (1993) for determining ³H-P1075 binding. Vascular rings were freely suspended on hooks on metal rods and equilibrated for 90 min in 37°C PSS bubbled with 100% O₂, with PSS changed every 30 min. After equilibration, the rings were incubated for 90 min in warm, oxygenated PSS (2 ml) containing ³H-P1075 (1-5 nM; 1 nM = 260,000 dpm/ml). Nonspecific binding was defined by using 10 µM unlabeled P1075. After incubation, bound radioactivity was separated from the free by rinsing the tissues with an excess volume (200 ml) of ice-cold (4°C), vigorously bubbled PSS for 60 sec. The tissues were then gently blotted on filter paper, weighed and individually placed in scintillation vials containing 0.5 ml Solvable (0.5 M). After 30 min, solubilized tissues were supplemented with 0.5 ml of 1.0 N HCl and 15 ml of scintillation cocktail (Beckman Ready Safe) and counted for radioactivity with a Packard Tri-Carb 4640 liquid scintillation counter. Data were expressed as disintegrations per minute per milligram tissue wet weight, and specific binding was calculated as femtomoles per milligram tissue wet weight.

Effect of changing association/dissociation times on ³H-P1075 binding in intact rabbit mesenteric artery. This experiment was designed to study the effect of increasing association time and decreasing dissociation time on ³H-P1075-specific binding in rabbit mesenteric artery. The binding protocol remained the same as described above, except that tissues were incubated for 180 min (instead of the usual 90 min) in warm, oxygenated PSS containing ³H-P1075 (3 nM) and washed for 10 sec (instead of the usual 60 sec) in ice-cold PSS.

P1075 Vasorelaxation Studies

All experiments were conducted with a 20-ml isolated tissue bath system containing normal PSS buffer (pH 7.3) maintained at 37°C and bubbled with 100% O₂, as described previously (Meisheri et al., 1991b, 1993b). Each ring was suspended between two stainless steel hooks. One hook attached the ring to a force displacement transducer, the other to a fixed support rod. Isometric contractions were measured and recorded on a Grass model 7D polygraph linked to an MI² computerized data acquisition system. The resting tension and contractile agonist used for each preparation were: rat aorta (1 g, 0.1 µM NE); rat mesenteric artery (1 g, 10 µM NE); rabbit mesenteric artery (1 g, 3 µM NE); dog mesenteric artery (1 g, 3 µM NE); dog coronary artery (2 g, 500 nM U-46619, a stable thromboxane A₂ receptor agonist). The contraction produced in each preparation was close to maximal by the agonist used. At the plateau of each contraction, a cumulative relaxation response to P1075 was studied (1-100 nM). Based on the initial data, detailed dose-response curves for P1075 were generated in three tissues as follows: rat aorta (0.1 µM NE); rabbit mesenteric artery (3 µM NE); and dog coronary artery (20 nM and 500 nM U-46619). Cumulative relaxation dose-response curves were generated as described before (Meisheri et al., 1991b). Because P1075 relaxations are slow to plateau, a given tissue was exposed to only three to four concentrations of P1075. Thus, two rings from a given preparation were used to generate the full dose-response curve.

Solutions and Drugs

PSS contained (in mM): NaCl, 140; KCl, 4.6; CaCl₂, 1.5; MgCl₂, 1.0; glucose, 10.0; and HEPES, 5.0. The pH was adjusted to 7.3 with 1.0 N NaOH. NE (1-arterenol-HCl) was obtained from Sigma Chemical Co. (St. Louis, MO). U-46619 and P1075 were obtained from the Biological Screening Office at Pharmacia & Upjohn Inc. (Kalamazoo, MI). ³H-P1075 (specific activity, 118 Ci/mmol) was obtained from Amersham International (Amersham, UK). The radiolabel was stored in ethanol at 4°C.

Data Collection and Statistics

A computerized data acquisition system, MI² (Modular Instruments Inc., Malvern, PA), linked to a 16-tissue bath Grass polygraph system was used to record percent relaxations for the pharmacological studies. Graphs were generated with SLIDEWRITE. All data are expressed as mean ± S.E.M. (n). EC₅₀ values (effective concentration producing 50% of the maximum relaxation) were obtained with NLIN2, a SAS based computer program generated by Dr. M.N. Brunden (Pharmacia & Upjohn). Statistical significance was determined with the Student's t test with P  .05.

	Results

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Rat aorta. Figure 1 shows ³H-P1075 binding and pharmacological data in intact rat aorta. As shown in figure 1A, ³H-P1075 at 1 and 3 nM produced high amounts of specific binding (total binding minus nonspecific binding, i.e., binding seen in the presence of 10 µM cold P1075). Specific binding as a percent of total binding in rat aorta was 68% at 1 nM and 58% at 3 nM ³H-P1075. Specific binding was calculated as 5.2 ± 0.4 (n = 6) and 9.6 ± 1.6 (n = 5) fmol/mg tissue wet weight at 1 and 3 nM ³H-P1075, respectively. At 5 nM ³H-P1075, specific binding was 9.9 fmol/mg tissue and was 48% of total binding (data not shown). These absolute numbers are very similar to those reported by Quast et al. (1993). Figure 1B shows the P1075 relaxation dose-response curve in rat aorta precontracted with 0.1 µM NE (roughly EC₉₀ concentration). The P1075 EC₅₀ for relaxation was 7.5 nM, which is similar to that reported by Bray and Quast (1992).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   (A) 3H-P1075 binding in intact rings of rat aorta. Nonspecific binding was determined by using 10 µM cold P1075. Total binding is significantly (P  .05) higher than nonspecific binding at each concentration of 3H-P1075. Each point is mean ± S.E.M. of four to six rings, and the figure was generated from 20 aortic rings from 7 rats. (B) P1075 vasorelaxation in NE (0.1 µM) precontracted rat aorta. Each point is mean ± S.E.M. of 6 to 13 rings, and the figure was generated from 14 rings from 2 rats. The P1075 EC50 = 7.5 nM.

Rabbit mesenteric artery. Figure 2 shows receptor binding and pharmacological data with P1075 in isolated rabbit mesenteric artery. In contrast to rat aorta, no significant specific binding of ³H-P1075 at 1 or 3 nM could be found in rabbit mesenteric artery (fig. 2A). It should be noted that the amount of nonspecific binding in rabbit mesenteric artery at 1 or 3 nM ³H-P1075 was statistically similar to rat aorta (compare figs. 1A and 2A). As shown in figure 2B, P1075 produced vasorelaxation in rabbit mesenteric artery precontracted with 3 µM NE (approximately EC₉₀ for contraction). The EC₅₀ for P1075 relaxation in rabbit mesenteric artery was 7.5 nM, like that in rat aorta. Thus, P1075 produced quantitatively the same pharmacological effect in rat aorta and rabbit mesenteric artery, but high amounts of specific binding could only be detected in rat aorta.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   (A) 3H-P1075 binding in intact rings of rabbit superior mesenteric artery. Nonspecific binding was determined by using 10 µM cold P1075. Total binding is not significantly (P  .05) different than nonspecific binding at either concentration of 3H-P1075. Each point is mean ± S.E.M. of seven to nine rings, and the figure was generated with use of 31 mesenteric rings from 6 rabbits. (B) P1075 vasorelaxation in NE (3 µM) precontracted rabbit mesenteric artery. Each point is mean ± S.E.M. of five rings, and the figure was generated from 10 rings from 2 rabbits. The P1075 EC50 = 7.5 nM.

Dog coronary artery. Figure 3 shows data in dog coronary artery for ³H-P1075 binding as well as P1075 vasorelaxation. Nonspecific binding was 40 to 45% less in the coronary artery than in rat aorta or rabbit mesenteric artery at a given ³H-P1075 concentration. ³H-P1075 produced small but statistically significant specific binding at 1 and 3 nM (fig. 3A). Specific binding in each case was 14% of the total binding. Specific binding was 0.24 ± 0.07 (n = 4) and 0.62 ± 0.04 (n = 4) fmol/mg tissue wet weight at 1 and 3 nM ³H-P1075, respectively. Thus, specific binding in the coronary artery was about 20-fold less than that found in rat aorta. When the radioligand concentration was increased to 5 nM in the coronary artery, specific binding did not increase (data not shown). The vasorelaxation data are shown in figure 3B. The P1075 EC₅₀ was 15 to 24 nM depending on the level of activation with U-46619. U-46619 at 20 nM produced 50% of the maximal contraction produced by 500 nM U-46619. It should be noted that 50 nM P1075 was still effective in producing maximal relaxation under both contraction conditions.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   (A) 3H-P1075 binding in intact rings of dog coronary artery. Nonspecific binding was determined by using 10 µM cold P1075. Total binding is significantly (P  .05) higher than nonspecific binding at each concentration of 3H-P1075. Each point is mean ± S.E.M. of four rings, and the figure was generated by using 16 coronary rings from 2 dogs. (B) P1075 vasorelaxation in U-46619 (20 or 500 nM) precontracted dog coronary artery. Each point is mean ± S.E.M. of 4 to 10 rings, and the figure was generated from 24 rings from 4 dogs. The P1075 EC50 = 15 and 24 nM at U-46619 concentration of 20 and 500 nM, respectively.

Comparison of mesenteric arteries from rat, rabbit and dog. Figure 4 provides data for ³H-P1075 binding in intact superior mesenteric arteries from rat, rabbit and dog. Like rat aorta, rat mesenteric artery showed very high specific binding to ³H-P1075 (fig. 4A). Specific binding was 79% and 64% of the total binding at 1 and 3 nM ³H-P1075. Specific binding in rat mesenteric artery was 5.8 ± 0.7 (n = 5) and 7.5 ± 1.1 (n = 5) fmol/mg tissue wet weight at 1 and 3 nM ³H-P1075. These numbers are similar to those found in rat aorta. In contrast, mesenteric artery from rabbit (fig. 4B) and dog (fig. 4C) failed to show any significant specific binding at 1, 3 or even 5 nM ³H-P1075.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   3H-P1075 binding in superior mesenteric artery from three species: rat (A), rabbit (B) and dog (C). Significant (P  .05) specific binding was only observed in rat mesenteric artery. Each data point is given as mean ± S.E.M. of four to nine mesenteric rings.

Detailed ³H-P1075 binding studies in rabbit mesenteric artery. Because rabbit mesenteric artery showed the same pharmacological sensitivity to P1075 as rat aorta (compare figs. 1B and 2B), further studies were conducted to detect specific binding of ³H-P1075 in rabbit mesenteric artery. Figure 5 shows the results of an experiment in which a 100-fold range of ³H-P1075 concentration was used, i.e., 0.1 to 13 nM (which is roughly 26,000 dpm/ml to 3.4 million dpm/ml incubation range). No significant specific binding could be detected at any of these concentrations.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   3H-P1075 binding in rabbit mesenteric artery. Binding was determined at 3H-P1075 concentration range of 0.1 to 13 nM. Nonspecific binding was determined with use of 10 µM cold P1075. Total and nonspecific binding are not significantly (P  .05) different from each other at any given 3H-P1075 concentration. Each data point is given as mean ± S.E.M. from four to nine rings. The S.E.M. are not shown when less than the size of the symbol.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Binding of 3 nM 3H-P1075 in rabbit mesenteric artery under two different conditions: one was 90-min incubation and 60-sec washout, and the other was 180-min incubation and 10-sec washout. In each case, total binding was not significantly (P  .05) higher than nonspecific binding. Each data point is given as mean ± S.E.M. from three mesenteric rings.

	Discussion

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This study describes the unexpected findings that not all vascular tissues show the presence of high-affinity receptor binding sites for the potent K_ATP opener, P1075. A comparison of ³H-P1075 binding in different vasculature from different species has not been reported. An important rationale for this study was to obtain a correlation between the pharmacological activity of P1075 (i.e., vasorelaxation) and the presence of specific receptor sites for P1075. Quast et al. (1993) have proposed that this receptor site not only mediates vasorelaxation by P1075 and related cyanoguanidines but also relaxation by all structurally different K_ATP openers. Our study, however, shows that there may not be a direct relationship between the presence of high-affinity P1075 receptor sites and vasorelaxation in a given vascular preparation. In fact, of the three species studied, only rat vasculature shows ³H-P1075-specific binding. In vasculature of rabbit and dog, more than 80 to 90% of the binding was determined to be nonspecific.

It is unlikely that our inability to detect specific binding of ³H-P1075 in some vasculature is caused by inappropriate experimental protocols or technical problems. With rat aorta, we have been able to duplicate qualitatively as well as quantitatively the results published by Bray and Quast (1992) and Quast et al. (1993), thus demonstrating that our experimental protocols can successfully detect and determine binding of ³H-P1075. Our ³H-P1075 binding data in rat aorta are in excellent agreement with those of Quast et al. (1993) both in terms of the absolute amount of specific binding (fmol/mg tissue wet weight) as well as in terms of specific binding as a percent of total binding. Furthermore, with this protocol, we were also able to demonstrate high amounts of ³H-P1075 specific binding in intact rat mesenteric artery. Additionally, our pharmacological studies in rat aorta have generated a P1075 EC₅₀ = 7.5 nM, which is very similar to the EC₅₀ = 8 nM reported by Quast et al. (1993). Thus, in both laboratories, use of the rat aorta preparation has yielded almost identical results in both biochemical and pharmacological studies. Another rat tissue, i.e., rat mesenteric artery, also shows a correlation between the presence of specific binding sites and sensitivity to vasorelaxation by P1075.

In contrast to rat, vascular tissues from rabbit or dog showed very little or no specific binding, in spite of the fact that pharmacological activity of P1075 could be readily demonstrated in these preparations. More detailed studies were carried out with use of rabbit mesenteric artery and dog coronary artery because of the extensive database available with K_ATP openers in these preparations (see Edwards and Weston, 1993; Meisheri et al., 1993b; Xu and Lee, 1994, for references). The data presented here show that these tissues are roughly as sensitive to P1075-induced vasorelaxation as rat aorta. The P1075 potency in these tissues was dependent on the preexisting contractile activation level. Thus, in rabbit mesenteric artery precontracted with 3 µM NE, the P1075 EC₅₀ was 7.5 nM, which is identical with that found in rat aorta. Dog coronary artery was slightly (2-3-fold) less sensitive, with P1075 EC₅₀ values of 15 and 24 nM depending on the activation level with U-46619. However, P1075 at 30 to 50 nM produced the same degree of maximal relaxation (>80%) in rat aorta, rabbit mesenteric artery and dog coronary artery regardless of the contractile activation level. Thus, similarity in the pharmacological sensitivity of rat aorta, rabbit mesenteric artery and dog coronary artery to P1075 would suggest that these tissues possess receptors with quite similar affinities and a very similar system for receptor-signal transduction coupling for producing vasorelaxation. However, there is a striking contrast in the detectability of specific receptor sites for P1075 in these tissues. Changes in the experimental protocol designed to enhance binding did not result in detection of putative P1075 binding sites. Thus, in rabbit mesenteric artery, even a 13-fold increase in ³H-P1075 concentration, i.e., from 1 nM (0.26 million dpm/ml) to 13 nM (3.4 million dpm/ml), failed to produce detectable specific binding. An increase in the association time (180 min) did not increase specific binding. Also, the possibility that the dissociation of the radiolabel from its receptor is faster in rabbit mesenteric artery was negated by the observation that decreasing washout time from 60 sec to 10 sec did not increase specific binding. Interestingly, at any given radioligand concentration, the nonspecific binding in rabbit mesenteric artery was very similar to that seen in rat aorta, and thus the key difference was the absence of specific binding in rabbit mesenteric artery when compared with rat aorta.

Thus, the data presented in this study show that the characteristics of ³H-P1075 binding in vasculature vary depending on the vascular preparation used or the species selected. The reasons for these differences remain to be investigated experimentally. The possibilities include differences in receptor affinity or maximum binding capacity or some combination of both. Alternatively, these data raise the question regarding the functional relevance of this binding site. It should be pointed out that, in general, it has been difficult to identify receptor sites for K_ATP openers as a class. Initial studies with ³H-labeled cromakalim failed to show specific binding to any site in any tissue studied, either in vitro or ex vivo (Coldwell and Howlett, 1987). Minoxidil sulfate has been suggested to produce vasorelaxation via a mechanism that involves covalent protein sulfation rather than a classical drug-receptor binding (Meisheri et al., 1991a, 1993a). It is interesting to note that even in rat aorta, ³H-P1075 binding does not behave like a classical receptor binding site because the specific binding of ³H-P1075 can only be detected with intact rat aorta and this specific binding is lost when membranes are prepared from tissue (Quast et al., 1993). There have been preliminary reports of identification of ³H-P1075 binding in smooth muscle cells isolated from rat aorta and calf coronary artery (Dickinson et al., 1993; Mannhold et al., 1996). There also has been a report describing a high-affinity specific binding site for another K_ATP opener with intact cultured rat insulinoma cells, but again the binding was lost when studied in membranes (Hoffman et al., 1993). Furthermore, the functional relevance of this binding in insulinoma cells is also unknown because K_ATP openers such as pinacidil, minoxidil sulfate and cromakalim are known to not inhibit insulin secretion at pharmacologically relevant concentrations (Garrino et al., 1989). More recently, a preliminary report identified ³H-P1075 binding in membrane preparations from various non-smooth muscle tissues (Dickinson et al., 1996). Further characterization of this binding and relevance of this binding site to that observed in intact cells or tissues would be of interest.

In summary, the present study shows that the proposed specific receptor site for ³H-P1075 varies in vasculature from different species and thus does not always provide direct correlation with the functional effects of P1075. The reasons for tissue and species dependency of this high-affinity receptor site remain to be established, but it becomes important to recognize these differences. Until such questions are resolved, it appears that rat is the most viable species for further biochemical characterization and investigation of the functional significance of this receptor site for P1075-induced, K_ATP-mediated vasodilation, as has been described previously (Bray and Quast, 1992; Quast et al., 1993).