Arterioles of the microcirculation respond to the application of muscarinic agonists with multiple effects. For example, in the hamster cheek pouch, a single brief arteriolar application of methacholine causes dilations that are composed of at least two components. One dilation is seen at the site of application, and another dilation is conducted to locations remote from the application site (1). Thus there are two dilatory components after the application of a single muscarinic agonist, and current evidence indicates that these two components are caused by independent intracellular pathways (2,3).
Two components for muscarinic receptor-induced vascular responses also were reported for large vessels (4,5). The muscarinic receptor subtypes mediating these responses were shown to be different and not necessarily even on the same cell type. This study was performed to determine whether the two modes of muscarinic-induced dilation in the microcirculation are due to the stimulation of different subtypes of muscarinic receptors.
Animal preparation. Hamsters were anesthetized with phenobarbital (70 mg/kg, intraperitoneal), the tracheas were cannulated, and an intraperitoneal catheter was placed for the continuous infusion of saline (0.008 ml/min) and phenobarbital (0.075 mg/min). All procedures were approved by the University Committee on Animal Resources.
The cheek pouch was exteriorized and pinned to a pedestal on the stage of a microscope for intravital microscopy (6). Bicarbonate-buffered solution (in mM: NaCl, 128; KCl, 4.7; CaCl2, 2.0; MgSO4, 1.2; H2PO4−, 1.2; glucose, 5.0; pyruvate, 2.0; EDTA, 0.02; NaHCO3, 17.9) was heated to 37°C, and equilibrated with gas containing 0-5% O2, 5% CO2, and the balance, N2, to achieve a pH of 7.4. This was continuously applied to the preparation.
Video microscopy. A Wild Kombistereo M3Z microscope (Heerbrugg, Switzerland) was used to visualize the cheek-pouch vasculature. Dissection of the tissue, placement of pipettes, and cannulation of arterioles took place by using the low-power, binocular view, whereas arteriole diameter was monitored at high power by using compound microscopy with Koeller transillumination. During high-power viewing, a ×25 long-working-distance objective, 0.22 numeric aperture, was used, and the image was viewed with closed circuit video by using a Dage-MTI CCD72 camera and Dage-MTI HR1000 monitor (Michigan City, IN, U.S.A.).
Application of methacoline. Methacholine was topically applied onto the arterioles. Changes in diameter after the repeated application of methacholine were sequentially recorded locally (local response) at the site of application and at a locations along the arteriole 500 and 1,000 μm upstream (conducted or remote response) from the site of application.
The drug was applied for 5 s with pneumatic ejection from micropipettes (1-2 μm tip diameter) placed 10-15 μm from the arteriole. The minimal ejection pressure was used that would create the maximal response from whatever concentration was in the pipette. To determine the minimal pressure, the arteriole response was first tested by using the maximal available ejection pressure (40 p.s.i.), and then the pressure was slowly increased from zero until the same response was obtained (typically 1-2 p.s.i.). This procedure was shown in previous experiments to maximize the response to any given concentration of drug with the minimal quantity of drug, and thus assure that the concentration of drug reaching the arteriole was very close to the concentration within the pipette (7). Defining the ejection pressure in this way also assured that as the drug diffused away from the site of application, it quickly reached concentrations that were no longer vasoactive. Therefore, it was possible to obtain concentration-response relations. Applying control solutions by using this technique caused no change in vessel diameter.
Application of antagonists and blockers. Muscarinic antagonists were applied via the superfusion solution. Concentrations were cumulatively applied, and ≥20 min was allowed to pass between concentrations before the micropipette application of methacholine was repeated in the presence of the antagonist.
Methacholine, methscopolamine, and scopolamine were purchased from Sigma (St. Louis, MO, U.S.A.). AFDX-116 [(11-2[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepin-6-one)) was a gift from Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT, U.S.A.) and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) was purchased from Research Biochemicals International. All drugs except AFDX-116 were prepared as 0.1 mM stock solutions in distilled water. AFDX-116 was dissolved in water that had been acidified to pH 2.3 with HCl; it was then returned to pH 7 with NaOH. The quantity of salt added by this procedure was compensated for in the final solution. The in situ arterioles developed spontaneous tone, so no vasoconstrictor drugs were necessary to maintain vascular tone during the experiments.
Data acquisition and statistics
Arteriole diameter was measured with a video micrometer (Microcirculation Research Institute, College Station, TX, U.S.A.). The video micrometer output was continuously recorded to a computerized data-acquisition system (Strawberry Tree, Workbench). The transient changes in arteriolar diameter induced by drug application were sampled at 10 Hz for ≤1 min from the start of drug application. The collected data were smoothed with a running average of four points. The starting diameter (baseline), onset time of the response after injection, and the peak dilation during the first minute after the drug application were determined.
Data are presented as absolute diameters or changes in diameter from baseline. Even in experiments in which the baseline diameters of the arterioles changed, the data were ultimately analyzed for changes in diameter because normalizing the data had no effect on the final results. To test the effect of normalization, the arteriolar dilation was divided by the maximal possible dilation, which could have occurred; that was, the difference between the resting baseline diameter and the "maximal" arteriolar diameter (as determined by applying drops of 10−4M adenosine or 10−2M methacholine onto the preparation).
Different muscarinic-receptor subtypes were evaluated by using a variety of muscarinic antagonists. Differences in the KB for antagonism of the local and conducted responses would support the presence of selective muscarinic receptor subtypes for the different responses.
The KB for each antagonist was determined by using a null method of dose-ratio analysis (8). This technique has been shown to be effective for analyzing functional inhibition curves without requiring absolute values of the maximal response. The technique uses the concentrations of the agonist and antagonist shown to create half of the response caused by a given concentration of agonist. Thus functional inhibition curves were used to estimate the competitive antagonist affinities. The method was previously shown to be accurate and theoretically valid (9).
The following equation was used to determine the KB for each antagonist at each site of observation: Equation (1) where IC′50 was the concentration of antagonist required to decrease the response to 10−4M methacholine by half, Af was the concentration of methacholine used in the analysis (10−4M), and EC′50 was the concentration of methacholine required to reach half the response seen when 10−4M methacholine was applied.
According to the technique, a common, best-fit, slope was used in the fitting of logistic curves ("Solver" in Excel, Microsoft Inc., Redmond, WA, U.S.A.) to each individual arteriole's antagonist concentration-response relations. Dilations were normalized to the dilation obtained with 10−4M methacholine, so 1 was used as the maximal value in the logistic fit for determining the IC′50. The KB was then determined from individual IC′50 according the equation earlier.
Because it was not possible to perform both agonist and antagonist response relations on every arteriole, a constant value for EC′50 was used in the calculations. EC′50 was the concentration of methacholine required to reach half the response seen when 10−4M methacholine was applied. To verify that the mean EC′50 value was consistent between sets of experiments, a concentration-response relation for methacholine was determined on three different occasions, each with an n of at least five animals. The mean methacholine EC50 was not significantly different for any of the trials. The summary of these data has been previously reported (10) along with the actual EC50 values. The data were reanalyzed to obtain the average values for EC′50 by using the response to 10−4M methacholine as the maximal response in the logistic curve fits. The mean EC′50 was 1.4 × 10−6M for the local response and 5 × 10−5M for the conducted response. These values were used to calculate the antagonist KB and pKB.
Two-sided paired t tests for each antagonist were used to compare the pKB of the antagonist for the local and conducted responses (Excel; Microsoft). A value of p < 0.05 was considered significant. Data are expressed as means ± SEM unless otherwise noted.
A total of 45 hamsters was studied having an average weight of 127 ± 21 (SD) g. Average control baseline diameter of all the arterioles studied was 14 ± 3.7 μm, and average "maximal" diameter of 35 ± 3.5 μm.
The baseline diameter of the arterioles decreased dose dependently for all antagonists. The decrease in baseline diameter ranged from 70 to 90% of control and did not depend on any selective subtype of receptor (Fig. 1).
The concentration-response curves for the antagonists are shown in Fig. 2 The logistic curve fits displayed in Fig. 2 were created by using the averaged IC′50 values that were calculated for each antagonist from the normalized data. The values for IC′50 were determined for both remote locations (500 and 1,000 μm upstream) and were found to be the same. Therefore the data from the two locations were combined for the calculations of KB for conducted responses.
As shown in Table 1, pKB for scopolamine and was not different for the local and conducted responses. The pKB was significantly greater for the local response when pirenzepine or 4-DAMP was applied and was significantly greater for the conducted response when AFDX-116 was applied.
This study demonstrates that the sensitivity of antagonists for arteriolar responses to methacholine depends on the response being studied and the selectivity of the antagonist. The nonselective antagonist scopolamine showed no significant difference in the KB for the local and conducted responses, whereas subtype-selective antagonists demonstrated marked differences in KB for the receptors causing the two responses with antagonists selective for the m3 receptor showing the most potency and selectivity for the local response.
Selective antagonists are more sensitive pharmacologic tools for defining receptor subtypes than are selective agonists (11), so a series of selected antagonists were tested to determine whether any selectivity could be found for either the local or conducted response. Scopolamine, pirenzepine, 4-DAMP, and AFDX-116 were tested. Scopolamine was selected because it is not selective for any subtype and is reported to activate receptors that reside on the endothelium (12). Pirenzepine was selected because it is reportedly selective for m1 and m3 receptor subtypes; AFDX-116, because it is selective for m2 receptors; and 4-DAMP, because it is reportedly selective for m3-receptor subtypes (13-15).
The potency of 4-DAMP strongly suggests that m3-receptor subtype is involved in both responses, but the significant difference in sensitivity for the two responses also suggests that the receptors are not the same. If both the local and conducted responses were mediated by a common receptor, the KB would have been the same for the two responses, and the relative values for KB would not have changed with the different antagonists. Selectivity for the local response was only seen when a selective antagonist was used. The nonselective antagonist showed no selectivity for either response.
A single application of methacholine to arterioles causes more than a single response. Dual responses after muscarinic-receptor activation have been shown in a number of tissues including myocardium, brain, parotid, blood vessels, stomach, and ileum (16-20). In the preparation used in this experiment, prior studies revealed that the local and conducted responses caused by the brief application of muscarinic agonist are mediated by separate intracellular mechanisms (2,3).
There are several possibilities for how multiple mechanisms could be initiated by the single application of a drug. There may be (a) differing subtypes of receptors that are selective for different responses, (b) a single receptor may have different affinities for the drug and different actions depending on the drug concentration or the cell type on which it resides, or (c) two processes within the cell may be stimulated by a single receptor either before or after activation of G protein. All possibilities have been described for muscarinic receptor-mediated responses (13,21-23). This study showed that the local and conducted responses caused by a single application of methacholine were due to the activation of different muscarinic receptors.
The antagonist affinity was the same for receptors causing both vasodilatory components when scopolamine was tested, indicating that it was possible to demonstrate a common affinity for both responses when a nonselective antagonist was used. On the other hand, when antagonists selective for a given subtype were tested, the apparent affinities for receptors causing the two dilatory components were significantly different. The apparent affinity of pirenzepine for receptors causing the local response was 2.4× greater than for the conducted response and was 6.6× greater for 4-DAMP.
In contrast, AFDX-116 (selective for the m2-receptor subtype) showed preference for the conducted response, albeit with only moderate sensitivity. The m2-receptor subtype was demonstrated to be important in the contraction of smooth muscle from artery that has been denuded of endothelium (24). How smooth-muscle constriction would contribute to endothelium-dependent conducted dilation is unknown. If the m2-specific antagonist was blocking receptors that caused constriction, then it seems the antagonist would cause dilation, not constriction of resting arterioles as demonstrated here. The fact that the current findings were made on arterioles may explain the difference because artery and arteriole receptor distribution are probably not the same. The low affinity for AFDX-116 suggests that the receptors that mediate the conducted response are unlikely to be m2 receptors. The fact that the drug is selective for the conducted response, however, implies that different receptors mediate the conducted response. The exact subtype and its specific cellular location remain to be discovered.
The high affinity of 4-DAMP for the receptors mediating both components indicates that m3 subtypes are important in dilation of arterioles but that it is more significant for the local response than for the conducted. Vasodilation mediated by the m3-receptor subtype was demonstrated in a number of studies (25-27). None of these studies tested for the conducted response, however. Receptors causing the conducted response have significantly less affinity for the antagonist that are selective for m1- and m3-receptor subtypes.
The KB reported here are consistent with antagonist affinities reported by others for vascular tissue. In vascular tissue, others have shown that the pA2 for 4-DAMP is 9.5, for AFDX-16 is 6.1, and for pirenzepine is 6.8 (28).
Numerous experimental and tissue variables limit the interpretation of these findings. Receptor distribution across the cell wall may be variable, and the penetration of the various antagonists will depend in the lipid solubility. Fat-soluble compounds readily affect receptors throughout the vessel wall, whereas water-soluble antagonists are limited to receptors on the external face of the endothelium or on the smooth muscle (29,30). Thus variations in the Hill coefficients could be partially attributed to variations in antagonist lipophilicity.
The interpretation is also complicated by the combined response that is present at the local site. Local responses are presumably a combination of the two components that cause local and conducted dilation. So activation of different receptors may cause cross-talk and a blended response that is not necessarily a simple summation of the two components. A third cause of variability may be inhomogeneous receptor distribution of subtypes. It may be that although the subtype controlling the two responses is the same, they are still not the same exact receptor. Receptors on one cell may cause the local response, whereas receptors on another cell may cause the remote response. Recent evidence indicates that methacholine can cause conducted signals to be generated in both the endothelium and the smooth muscle (31).
Baseline diameter was significantly decreased by all the antagonists. This suggests that some basal activation of muscarinic receptors is present in resting arterioles of the hamster cheek pouch. A couple of possible physiologic sources for acetylcholine have been suggested that could be important for controlling blood flow. Release of acetylcholine from the endothelium itself has been reported to occur in response to flow (32-34), and the neuromuscular junctions in the vicinity of the arterioles (35) could also be contributing.
In summary, these data are the first to suggest that different muscarinic receptors are selective for different modes of vasodilation in the microcirculation. Whether the subtype mediating the conducted response is different has yet to be conclusively demonstrated. Although antagonists selective for the m3 subtype appeared to be the most sensitive, the fact that there was a differential selectivity for the two responses indicates that the receptors mediating the two processes are unlikely to be the same. The receptors mediating the local response and the conducted response have different sensitivity for the muscarinic-receptor antagonists that are selective for the m3 receptor. Thus muscarinic receptors mediating the conducted response are either of a different subtype or are located on the vessel wall cells in positions that are unique from those causing the local response. These may be either on the same cell or on different cells within the vessel wall.
Acknowledgment: Much appreciation goes to Judy Beckman for her assistance in completing this study. This research was supported by NIH R29-HL49470.
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