INTRODUCTION

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IN AN EARLIER STUDY (7), we found that N^G-nitro-L-arginine (L-NNA) and N^G-monomethyl-L-arginine (L-NMMA) inhibited the cerebral arteriolar dilation from cromakalim, minoxidil, and pinacidil, three agents that dilate vessels by opening ATP-sensitive potassium (K_ATP) channels. This effect of the arginine analogs was not dependent on blockade of nitric oxide synthase and was unrelated to nitric oxide, since the action of nitric oxide donors was not modified by blockade of K_ATP channels with glyburide (7). The inhibitory effect of arginine analogs was reversed by L-arginine, suggesting that the effect of the arginine analogs on potassium channels might involve competition with L-arginine. We suggested that K_ATP channels in cerebral arterioles require L-arginine for their function (7). In addition, we surmised that the requirement for L-arginine may be because of the presence of an arginine site on the channel that must be occupied for the ion channel to open (7). There is precedent for such an arrangement. A membrane calcium channel in neurons, the N-methyl-D-aspartate (NMDA) receptor, has a glycine site, which when occupied potentiates the action of agonists in opening this ion channel (6).

In the present study, we investigated further the role of arginine and other amino acids in the function of K_ATP channels in cerebral arterioles of anesthetized cats.

	METHODS

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Experiments were carried out in cats anesthetized with pentobarbital sodium (30 mg/kg iv). Additional doses of anesthetic were given as required to maintain surgical anesthesia, based on testing of corneal reflexes and on responses to tail pinch. The animals were subjected to tracheostomy and ventilated with a positive-pressure respirator. The end-expiratory CO₂ of the animals was continuously monitored with a Hewlett-Packard CO₂ analyzer and was maintained at a constant level of ~30 mmHg. Arterial blood pressure was measured with a pressure transducer connected to a cannula introduced into the aorta via the femoral artery. Arterial blood samples were collected for determination of arterial blood oxygen, CO₂ partial pressures, and pH at appropriate intervals during the experiment. Blood gas tensions and pH were measured with Corning electrodes. The rectal temperature of the animal was monitored continuously and was kept constant with the aid of a heating blanket.

The cerebral microcirculation of the parietal cortex was visualized through an acutely implanted cranial window as described in detail previously (8). The space under the cranial window was filled with artificial cerebrospinal fluid (CSF) identical in composition to that of cats. One port of the window was connected to a pressure transducer for continuous monitoring of intracranial pressure. The intracranial pressure was maintained at 5 mmHg by connecting another outlet of the window to a coiled plastic tube whose free end was placed at the appropriate height to give the desired pressure. Two ports of the cranial window were used as inlet and outlet, allowing topical application of various solutions either by filling the space under the window with stationary solution or by superfusion. Pial arteriolar diameter was measured with a Vickers image-splitting device attached to a Wild microscope. In each animal, several arterioles were observed covering a wide range of vessel caliber. The responses of small and large arterioles (smaller and larger than 100 µm in diameter, respectively) were analyzed separately to identify any size-dependent differences in responses.

Pinacidil, minoxidil, L-NNA, L-arginine, D-arginine, L-lysine, D-lysine, glycine, L-histidine, methyl-L-lysine, -hydroxylysine, dimethyl-L-arginine, dimethylarginine, and glyburide were obtained from Sigma. Hydroxylysine contained equal parts of D- and L-lysine. Pinacidil, minoxidil, and glyburide were first dissolved in ethyl alcohol to prepare a stock solution. The diluent had no significant effect on baseline vessel diameters. All other solutions were prepared directly in artificial CSF immediately before use and were equilibrated at 37°C in a water bath immediately before application.

The following series of experiments were done. 1) We tested whether L-arginine had a significant effect on the response to the known K_ATP channel opener pinacidil. Pinacidil was applied by filling the space under the window with the appropriate stationary solution for several minutes. Vessel diameters were measured between 2 and 4 min after the initiation of the application. This was sufficient for the vessels to reach a new steady state. The effect of pinacidil was evaluated in the presence of L-arginine in various concentrations from 0 to 500 µM.

2) The effects of various doses of L-arginine on the action of pinacidil or minoxidil were evaluated following pretreatment with L-NNA (250 µM) to block K_ATP channels. Responses to pinacidil or minoxidil in stationary solution were obtained before and after a 30-min topical application of 250 µM L-NNA. The responses to pinacidil or minoxidil were evaluated again in the presence of 5 µM D-arginine and 5 and 1,000 µM L-arginine. Similar experiments were conducted to evaluate whether other amino acids might have the same action as L-arginine. We tested the effects of D-lysine, L-lysine, glycine, L-histidine, methyl-L-lysine, dimethyl-L-arginine, dimethylarginine, and hydroxylysine on responses to pinacidil in several series of experiments.

3) In another series of experiments, we tested whether binding of L-arginine on the channel is required for opening K_ATP channels by known agonists. We used an experimental design similar to what has been used to identify the glycine site in NMDA receptors (6). To this end, we first tested the effect of pinacidil (1 µM) by filling the window with stationary solution containing this agent. We then applied the same concentration of pinacidil by continuous superfusion at two different rates, 1 and 3 ml/min, in the absence and presence of L-arginine. Because the volume of the space under the cranial window is ~0.25 ml, these superfusion rates gave a turnover rate of 4-12 times/min. The rationale in using this procedure was that the continuous flow of fluid might wash arginine from binding on the channel, thereby allowing us to evaluate whether arginine bound to the channel is needed for the opening of the channel. In similar experiments, we used L-lysine and D-lysine instead of arginine.

In additional experiments, we alternated topical application of pinacidil (1 µM) by superfusion at 1 ml/min and in stationary solution to see how fast restoration of dilation occurred following application of the drug in flowing solution.

4) We investigated whether hydroxylysine, methyl-L-lysine, dimethyl-L-arginine, and dimethylarginine affected responses to pinacidil. To this end, responses to stationary pinacidil were tested before and after a 30-min topical application of each of these agents.

5) In another series of experiments, we tested whether L-arginine or L-lysine was capable of reversing the blockade of K_ATP channels induced by glyburide. To achieve this goal, we tested responses to stationary pinacidil before and after glyburide (1 µM) in the absence as well as in the presence of 1 and 5 µM L-arginine and 5 µM D-arginine. Glyburide was applied for 30 min. In preliminary experiments in two cats, every 15 min we tested responses to stationary pinacidil (1 µM) for 90 min after application of glyburide to ascertain the duration of the blockade. In all vessels tested, responses to pinacidil did not exceed 3% of the control response before glyburide application.

In two additional groups of cats, we tested responses to stationary pinacidil (0.5 and 1 µM) before and after glyburide in the presence of 0, 0.1, 0.25, 0.5, 0.75, and 1 µM L-arginine or L-lysine to determine the dose response of the effectiveness of these amino acids to block the inhibitory action of glyburide on K_ATP channels.

Statistical evaluation of the results was done with analysis of variance followed by t-tests modified for multiple comparisons.

	RESULTS

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Figure 1 shows that the responses to pinacidil were not modified in the presence of 5, 50, and 500 µM L-arginine.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Effect of L-arginine (L-Arg) on vasodilator responses to pinacidil in stationary solution in small (A) and large (B) cerebral arterioles. Values are means ± SE from 19 small and 15 large arterioles in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown. There was no significant change in responses to pinacidil from control in presence of L-arginine in any dose.

Figures 2 and 3 show that the responses to pinacidil or minoxidil were blocked after application of L-NNA and that this blockade was eliminated in the presence of 5 µM L-arginine but not in the presence of D-arginine. An increase in the concentration of L-arginine to 1 mM had no additional effect than that seen with the lower concentration. Figure 4 shows that L-lysine had effects similar to those of L-arginine but that D-lysine had no significant effect. Table 1 summarizes results of experiments in which several other amino acids were tested for their ability to reverse the blockade induced by L-NNA on responses to pinacidil. It is seen that glycine, L-histidine, methyl-L-lysine, -hydroxylysine, dimethyl-L-arginine, and dimethylarginine were incapable of reversing the blockade induced by L-NNA.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of L-arginine and D-arginine (D-Arg) on responses to stationary pinacidil after blockade with NG-nitro-L-arginine (L-NNA). Values are means ± SE from 19 small arterioles (A) and 15 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of values. Note that responses to pinacidil were significantly depressed after L-NNA in absence and presence of D-arginine but did not differ from control values in presence of L-arginine at either 5 µM or 1 mM.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of D-arginine and L-arginine on responses to topical application of minoxidil in stationary solution after blockade with L-NNA. Values are means ± SE from 18 small arterioles (A) and 12 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of data. Note that responses to minoxidil after L-NNA were significantly lower than control in absence as well as in presence of D-arginine but did not differ from control in presence of L-arginine at either concentration.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of D-lysine and L-lysine on responses to stationary pinacidil after blockade with L-NNA. Values are means ± SE from 16 small arterioles (A) and 13 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of data. Note that responses to pinacidil were significantly lower after L-NNA in absence as well as presence of D-lysine but did not differ from control in presence of L-lysine at either concentration.

Figure 5 shows that stationary pinacidil had a sustained vasodilator effect on cerebral arterioles for the 10-min period of observation. When, however, the same concentration of pinacidil was applied by continuous superfusion, the vasodilation was considerably less pronounced, progressively declined, and was completely eliminated within a few minutes. Pinacidil in the presence of 5 µM L-arginine applied by continuous superfusion had a sustained vasodilator effect for the full 10-min period of application, whereas pinacidil with D-arginine applied by superfusion had a transient effect. In 16 vessels in 3 cats, switching from CSF without additives to stationary pinacidil (1 µM) caused dilation that reached its maximum in 1.7 ± 0.12 min. In the same vessels, switching to stationary pinacidil (1 µM) from a 10-min superfusion at 1 ml/min with 1 µM pinacidil caused dilation that reached its maximum in 3.2 ± 0.47 min (P < 0.01). Figure 6 shows that continuous superfusion with a solution containing pinacidil and L-lysine caused sustained arteriolar dilation identical to that caused by stationary pinacidil, whereas pinacidil plus D-lysine by superfusion had virtually no effect.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Comparison of vasodilator responses to pinacidil applied topically as a stationary solution and by superfusion. Values are means ± SE derived from 21 small arterioles (A) and 17 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown in parentheses. Note that responses to pinacidil by superfusion in absence of L-arginine were significantly lower than responses to stationary pinacidil in both small and large arterioles. Responses to pinacidil by superfusion in presence of L-arginine were significantly greater than those seen during reperfusion in absence of amino acid but were significantly lower than with stationary pinacidil.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Comparison of response to pinacidil applied in stationary solution as well as by superfusion in absence and presence of D-lysine and L-lysine. Values are means ± SE from 20 small arterioles (A) and 12 large arterioles (B) from 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown in parentheses. Note that responses to pinacidil by superfusion in absence of additives as well as in presence of D-lysine were significantly less than those seen in response to stationary pinacidil, whereas responses to pinacidil by superfusion in presence of L-lysine did not differ from those seen with stationary pinacidil.

Figure 7 shows the effect of L-arginine in various concentrations on the action of pinacidil applied by superfusion at 1 ml/min. No dilation was seen in response to pinacidil in the presence of 0 and 0.25 µM L-arginine. Significant dilation was first seen at 0.5 µM, and at 1 µM L-arginine, the dilation was not different from that caused by stationary pinacidil.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of various concentrations of L-arginine on vasodilator effect of 1 µM pinacidil applied in flowing solution at 1 ml/min. Values are means ± SE of steady-state percent changes in diameter induced by pinacidil. Values were obtained from 17 small arterioles (A) and 15 large arterioles (B) in 5 cats. Note that no significant dilation was seen at 0 and 0.25 µM L-arginine in either small or large arterioles. Significant dilation was seen at 0.5-1 µM L-arginine. Dilation in presence of 1 µM L-arginine was not significantly different from that seen with stationary pinacidil.

Figure 8 shows that -hydroxylysine blocked completely responses to pinacidil. Methyl-L-lysine, dimethyl-L-arginine, and dimethylarginine did not affect responses to pinacidil in varying doses up to 1 mM (Table 2).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of -hydroxylysine on vasodilator responses to pinacidil in stationary solution. Values are means ± SE from 18 small arterioles (A) and 16 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown. Responses to pinacidil were significantly lower after hydroxylysine.

Figure 9 shows that the vasodilator response to pinacidil was blocked completely by pretreatment with glyburide and that in the presence of 1 µM L-arginine the blockade induced by glyburide was eliminated, thereby restoring responses to pinacidil to the control levels. L-Arginine (5 µM) had the same effect as the lower concentration, whereas D-arginine (5 µM) was ineffective.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Effect of D-arginine and L-arginine on responses to pinacidil in stationary solution after blockade with glyburide. Values are means ± SE from 20 small arterioles (A) and 14 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of data. Note that glyburide reduced responses to pinacidil significantly both in absence and presence of D-arginine, whereas in presence of L-arginine at either dose, responses did not differ from control.

Figures 10 and 11 show that ascending concentrations of L-arginine or L-lysine inhibited the glyburide-induced blockade of dilations by pinacidil. The effect of the amino acids was detectable at 0.5 µM, and inhibition was complete at 1 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Effectiveness of various concentrations of L-arginine in reversing blockade of vasodilator response to stationary pinacidil by glyburide. Values are means ± SE from 18 small arterioles (A) and 16 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of data. Note that reversal of blocking effect of glyburide is detectable at 0.5 µM and is complete at 1 µM.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Effectiveness of various concentrations of L-lysine (L-Lys) in reversing glyburide-induced blockade of vasodilator effect of stationary pinacidil. Values are means ± SE from 19 small arterioles (A) and 14 large arterioles (B) in 5 cats. Baseline diameters from which percent changes in diameter were calculated are shown above each set of data. Note that effect of L-lysine is detectable at 0.5 µM and becomes complete at 1 µM.

	DISCUSSION

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The findings reported above are consistent with the view that K_ATP channels in cerebral arterioles of cats have an amino acid site that must be occupied by either L-arginine or L-lysine for agonists, such as pinacidil, to open these channels. The amino acid site in these channels is evidently saturated at relatively low concentrations of L-arginine or L-lysine and is fully occupied under normal physiological conditions. This accounts for the absence of a potentiating effect of L-arginine, even in high concentrations, on the response to pinacidil. The blockade of K_ATP channels by arginine analogs and by hydroxylysine is attributable to the displacement of L-arginine or L-lysine from binding on the channel.

The superfusion experiments reported above show that the amino acid bound on the channel can be displaced by a flow of liquid but that upon cessation of the flow the amino acid is replenished within a few minutes from endogenous sources, thereby restoring responsiveness to potassium channel openers.

Understanding of the structure of K_ATP channels has improved considerably as a result of successful attempts at cloning these channels (1-5). As a result of these studies, it now appears that they consist of two components: an inward rectifying potassium channel and a sulfonylurea receptor. Coexpression of these two components reconstituted a potassium channel with all the properties expected of a K_ATP channel, including ATP sensitivity, responsiveness to potassium channel openers like diazoxide, and blockade by sulfonylureas (5).

Our finding that the blockade by glyburide was reversed by a low dose of L-arginine or L-lysine suggests strongly that these basic amino acids bind on the channel at a site situated on the sulfonylurea receptor. The findings are consistent with competition between these basic amino acids and glyburide for binding on this receptor. It is likely that the normally prevailing concentrations of these amino acids are sufficiently low for glyburide to exert its inhibitory effect. When the concentration of L-lysine or L-arginine exceeds 1 µM, the inhibitory effect of glyburide is eliminated completely.

The sulfonylurea receptor from pancreas from rat and hamster has recently been cloned (1). It consist of 1,582 amino acids, has 2 ATP binding sites, and belongs to the ATP-binding cassette family of proteins. It appears to function by modulating the activity of the potassium pore protein (2). Sulfonylurea receptor-like proteins have recently been identified in extrapancreatic tissues (3).

On the basis of the ability of various amino acids to block responses to pinacidil or their ability to reverse blockade of responses to pinacidil induced by L-NNA, we conclude that binding on the channel is through the free extra amino group of the basic amino acids, such as occurs in L-lysine and L-arginine. Clearly, there is also stereospecificity, since the D-isomers of these amino acids are ineffective. Methyl-L-lysine in which the amino group has a methyl substitution as well as dimethyl-L-arginine in which both guanidino amino groups are substituted are ineffective. The only exception is that dimethylarginine, which has a free extra amino group, was ineffective. This may be due to the fact that the presence of the two methyl groups on the companion guanidino amino group probably renders that portion of the molecule too bulky and prevents binding on the channel. It is also clear from the results that amino acids that have a substitution close to the amino group through which binding on the channel occurs block the channel. This is the case with L-NMMA, L-NNA, as well as hydroxylysine.

It is interesting to inquire whether or not our findings in the cerebral arterioles of cats apply to larger cerebral vessels, to vessels in other vascular beds, or to K_ATP channels in nonvascular tissues. McCarron et al. (10) found that isolated rat posterior cerebral arteries did not dilate in response to pinacidil or cromakalim applied by superfusion, a finding consistent with our results. However, mesenteric arteries in the same experiments dilated in response to cromakalim and pinacidil (10). Similarly, isolated porcine small coronary arteries dilated in response to K_ATP channel openers applied by superfusion (9). These findings suggest that the requirement for L-arginine or L-lysine for the opening of K_ATP channels in cerebral vessels may not be shared by such channels in other vascular beds. Such differences are not surprising. In a recent review, Quale et al. (11) pointed out a number of differences in K_ATP channels in different vascular beds as well as differences in the properties of these channels in vascular vs. nonvascular tissues. It should be noted, however, that final decision on these issues should be based on comparisons of the effect of K_ATP channel openers in stationary and flowing solution with and without L-arginine or L-lysine.