Conducted Arteriolar Dilations Persist in the Presence of Nitroarginine

Abstract

At least two mechanisms of arterial dilations are induced by the application of muscarinic agonists. One is caused by nitric oxide, and the second is the result of the release of an endothelium-dependent hyperpolarizing factor (EDHF) that has yet to be clearly defined. This study was performed to determine whether two modes of dilation also are present in the microcirculation. Arterioles (38 μm maximal diameter) in the cheek pouch of anesthetized hamsters were stimulated with the micropipette application of a muscarinic-receptor agonist (methacholine 10⁻⁴M; MCh). A single microapplication of MCh (5 s) caused dilation at the tip of the pipette, as well as a dilation remote from the site of application. Two modes of muscarinic receptor-induced dilation were suggested within arterioles because nitroarginine (10⁻⁶−10⁻³M) significantly decreased the local dilation from control of 14 ± 1.8 μm to 5 ± 0.9 μm in the presence of 10⁻⁴M nitroarginine, without significantly affecting the conducted response (control, 4.7 ± 0.8 μm; with 10⁻⁴M nitroarginine, 3.1 ± 0.5 μm). Therefore, in hamster cheek-pouch arterioles there are two modes of dilation caused by muscarinic agonists, and they are mediated by different mechanisms: one is nitric oxide and the other is consistent with a hyperpolarizing factor.

Muscarinic receptor-mediated relaxation of blood vessels has been repeatedly shown to be an endothelium-dependent process that is partially mediated by nitric oxide [NO; or endothelium-dependent relaxing factor (EDRF)] ^(1,2) and partially by a second endothelium-dependent mechanism involving a substance that hyperpolarizes smooth muscle (EDHF) ^(3-5). As these studies show, the presence of EDHF is unveiled when, in the face of blockade of NO synthase, there is a persistent endothelium-dependent dilatory component that also hyperpolarizes the cells in the vessel wall. In the microcirculation, microapplication of muscarinic agonist also causes dilations that can sometimes be separated into two components. It causes dilations at the site of drug microapplication, as well as conducted dilations that are remote from the application site.

Conducted vasomotor responses appear to result when changes in membrane potential are communicated from cell to cell in the vessel wall ^(6,7). A role for hyperpolarization in conducted dilations is supported by the fact that clamping the arteriole with high potassium concentrations can decrease the response ⁽⁸⁾, pinacidil (opener of potassium channels) can initiate the response ⁽⁹⁾, and potassium channel blockers can decrease the response ⁽¹⁰⁾. Thus hyperpolarization appears essential for conducted vasodilation. Because vascular cells can be hyperpolarized by NO ⁽¹¹⁾, this study was performed to determine whether NO can mediate both the local and conducted responses, or if the two responses are mediated by independent pathways.

METHODS

General preparation

Male golden hamsters (100-120 g) were anesthetized with sodium pentobarbital (70 mg/kg, intraperitoneal). The cheek pouch was exteriorized for intravital microscopy ⁽¹²⁾ after tracheostomy and endotracheal tube insertion. Continuous intraperitoneal injection of saline (0.008 ml/min) and anesthetic (0.075 mg/min) maintained the appropriate levels of hydration and depth of anesthesia. The cheek pouch was continuously superfused with 37°C physiologic salt solution (in mM: NaCl, 132; KCl, 4.7; CaCl₂, 2.0; MgSO₄, 1.2; and NaHCO₃, 20) equilibrated with 0%, or 5% O₂, 5% CO₂, and the balance N₂. Deep esophageal temperature of the hamster was maintained at 37°C by a combination of conducted and radiated heat.

The pouch was prepared by using no oxygen in the superfusate. All data were collected by using 5% O₂ to increase vessel tone and to give a uniform baseline diameter that was consistently <60% of the maximum vessel diameter. The in situ arterioles developed spontaneous tone, so no vasoconstrictor drugs were necessary to maintain vascular tone during the experiments. The "maximal" diameter of the arterioles being studied was determined at the start of experiment by applying a few drops of 10⁻²M methacholine directly onto the preparation.

The transilluminated pouch vasculature was viewed with a microscope (Wild Kombistereo M3Z; Leica, Inc., Deerfield, IL, U.S.A.) with a 0.22 nA, ×25 LWD objective lens. The scope was coupled to a video camera (CCD72; Dage-MTI Inc., Michigan City, IN, U.S.A.) and a video monitor (HR1000; Dage-MTI Inc.). Transient responses were measured in real time by using a video micrometer (Microcirculation Research Institute, College Station, TX, U.S.A.). This meter placed two parallel lines on the video monitor that were separated according to a manual control. A DC voltage signal from the meter corresponded with size of the separation. The video micrometer was calibrated with a stage micrometer. Final magnification to the video monitor was ∼×1,100.

Microapplication of methacholine

Arterioles were stimulated to dilate with methacholine (10⁻⁴M) pneumatically ejected (Microinjector IM-200; Narishige Inc., Sea Cliff, NY, U.S.A.) from a micropipette, as shown in Fig. 1. Methacholine was applied for 5 s from 1- to 2-μm pipettes placed 10-15 μm from the arteriole.

FIG. 1: Technique used to study local and conducted dilations caused by the microapplication of methacholine. Methacholine was ejected from a small pipette placed near the arteriole, as described in Methods. Responses were measured at the site of application (A) and at a location 500 μm upstream from the application site (B). Sample changes in diameter are shown at the two locations. The dilations at both locations start ∼2.5 s after the beginning of the 5-s application. The solid bars at the beginning of each curve represent the time of application.

Ejection pressure was adjusted to apply the minimal amount of drug that would create the maximal response to the drug in the pipette. The maximal response of the arteriole to the drug in the pipette was determined by ejecting the drug with the maximal available pressure (45 p.s.i.). At this pressure, rapid bulk flow of drug created obvious movement of the arteriole and locations on the arteriole where pure pipette contents were being applied. The pressure was then returned to zero and slowly increased. The arteriolar response would increase with pressure until a point was reached (0.5-1.5 p.s.i.) at which the response was equal to that just defined by bulk flow of the drug. So although the actual concentrations at the arteriolar surface were not measured during the experiment, the response was the same as if bulk flow had created the response. Using the minimal possible pressure, the concentration of drug would quickly drop as it diffused away from the site of application. Applying drug with this technique, including dye to define the diffusion of drug, testing for the effect of duration of application, and defining the response profile along an arteriole have all diminished concerns about diffusion of drug to the remote sites where conducted responses were viewed ⁽¹³⁾. Injections of control solution, without drug, caused no arteriolar responses.

Changes in diameter after the application of agonists were sequentially recorded at the site of application (local) and at a location 500 μm upstream (conducted or remote) from the site of application. The response to methacholine was measured sequentially at each location because the locations could not be viewed simultaneously. An average response at each location was determined from one to three repeated trials, and a single arteriolar region was studied in each animal.

Application of nitric oxide synthase inhibitor

N^ω-nitro-L-arginine (10⁻⁶-10⁻³M; nitroarginine) was cumulatively applied to the pouch preparation via the superfusion solution. Each concentration of blocker was present ≥20 min before any responses to methacholine were repeated. The blocker had to be cumulatively applied because its effects were not reversible. Time controls by using this technique for stimulating the arteriole revealed that a consistent control dilatory response could be achieved for ≥5 h. This was most recently demonstrated in a study involving 13 animals in which the local dilation caused by the micropipette application of methacholine was 17.7 ± 1.6 μm at the onset of the experiments and 17.4 ± 1.3 μm 5 h later ⁽¹⁴⁾.

Drugs

Methacholine and nitroarginine were purchased from Sigma. All drugs were prepared as 10 mM stock solutions in distilled water, then diluted the day of the experiment with superfusion solution.

Data acquisition and statistics

The video micrometer output was continuously recorded to a computerized data-acquisition system (Workbench; Strawberry Tree Inc., Sunnyvale, CA, U.S.A.). A plot of vessel diameter versus time was regularly printed to keep a permanent record of the vessel diameter before and after the drug application. 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 4 points, and the starting diameter, onset time of the response after injection, and the peak dilation in the first minute after the drug application were determined.

The data are presented as absolute diameters or changes in diameter. Even in experiments in which the baseline diameters of the arterioles changed, the data are presented as absolute diameters, because normalizing the data to the new baseline diameter did not affect the final results. To test this, the arteriolar diameter response was normalized by dividing the change in diameter after a treatment by the maximal possible dilation that could have occurred; that was the difference between the resting baseline diameter and the maximal arteriolar diameter.

Repeated-measures analysis of variance (ANOVA) was used to determine whether differences caused by nitroarginine were significant (JMP statistical software; SAS Institute, Inc., Cary, NC, U.S.A.). Data are expressed as means ± SEM, unless otherwise noted. A value of p < 0.05 was considered significant.

RESULTS

Arteriolar regions were studied in eight animals. The maximal diameter of the arterioles in this study was 38 ± 1.9 μm. When methacholine was applied, the arteriole dilated briskly, both at the site of application (local) and at the remote site (conducted), as described in Fig. 1.

Blockade of responses with nitroarginine

Nitroarginine significantly decreased the resting baseline arteriolar diameters (Fig. 2) and significantly decreased the local response to methacholine microapplication (Fig. 3). The response was decreased by 65% when 10⁻⁴M nitroarginine was applied.

FIG. 2: The effect of nitroarginine on the baseline diameters of the arterioles. Two concentrations of nitroarginine tested had a significant effect on the baseline diameters of the arterioles. *p < 0.05 compared with the control value.

FIG. 3: The effect of nitroarginine on the local dilation (▪) and conducted dilation (•) caused by the microapplication of methacholine (n = 8). Two-way repeated-measures analysis of variance showed that nitroarginine had a significantly different effect on the local response compared with the conducted response (F = 8.7; p = 0.0024). Analysis of the responses at the individual locations revealed that there was a significant effect on the local dilation (*p < 0.05 compared with the control value; p = 0.012) and no significant effect on the conducted response (p = 0.23).

This is in sharp contrast to its effect on the conducted responses. According to the repeated-measures ANOVA, the nitroarginine did not significantly affect the conducted response.

In four of the eight experiments, an additional concentration of nitroarginine (10⁻³M) was tested to determine whether any further blockade could be obtained. When this was applied, no additional blockade of NO synthase was apparent. The baseline diameters were not significantly smaller, and the dilations caused by methacholine were not significantly changed by the increased amount of nitroarginine. Repeated-measures ANOVA on these four experiments gave the same results as the analysis of the original eight.

DISCUSSION

The data of the study show that the local and the conducted dilations caused by the single application of methacholine are mediated by different pathways. Nitroarginine was able to severely limit the local dilation while having a nonsignificant effect on the conducted response. This is consistent with previous studies on larger arteries showing that multiple mechanisms are involved in the dilations induced by muscarinic-receptor agonists ^(3-5). The report that muscarinic agonists can cause hyperpolarization of vessels and that changes in membrane potential can be communicated to remote sites to cause a change in arteriolar diameter ⁽⁶⁾ suggests that the response is mediated by hyperpolarization. Yet because nitroprusside does not cause a conducted response ⁽¹⁵⁾ and nitroarginine does not significantly decrease the conducted response, NO is not likely to play a major role in conducted vasodilations caused by methacholine. NO has been shown to affect membrane potential, but its effects are less consistent and depend on the conditions of the preparation ^(16-18). So although NO may affect membrane potential under special conditions, it was not apparent here.

This suggests that methacholine hyperpolarizes the cells in the vessel wall by a different pathway from that of NO. It could occur through direct receptor stimulation of a potassium channel ⁽¹⁹⁾, albeit this has never been demonstrated in vascular tissue, or through the release of a different substance that hyperpolarizes the cells. Possible substances that have been shown to be released after stimulation with methacholine and to cause hyperpolarization of cell membranes include prostanoids ⁽²⁰⁾, calcitonin gene-related peptide (CGRP) ⁽²¹⁾, and epoxyeicosatrienoic acids ⁽²²⁾. Thus the makeup of the substance called EDHF may well be a composite of a number of substances, depending on the tissue and the experimental conditions. The data presented herein simply showed that when the EDRF-dependent response was eliminated with nitroarginine, local and conducted responses persisted that could only be attributed to non-NO-dependent mechanisms causing conducted vasodilation.

The concentration of methacholine selected for these experiments was at the top of the concentration-response relation for methacholine on these arterioles ⁽¹³⁾. So the concentration decreased along the steep portion of the concentration-response relation as it diffused away. The concentration-response relation also demonstrated that the median effective concentration (EC₅₀) was significantly lower for the local response than for the conducted response. The relation between the two response was such that the conducted response was barely perceptible until concentrations of methacholine were used that caused the local response to be almost half the maximal response. Thus in my experiments, the conducted response did not persist simply because it was more sensitive to methacholine than was the local response.

The baseline diameter was significantly decreased by the presence of nitroarginine, suggesting that there was basal release of NO in these vessels. The effect of small decreases in diameter on the responses to methacholine was not likely to be significant in these experiments, however, because others have shown that smaller diameters and increased vessel wall tone would increase a vasomotor response, not decrease it ^{(14,23, 24)}. It addition, if the baseline diameter was affecting arteriolar responses, the effect should have been seen at both locations.

CONCLUSION

In summary, these studies showed that the microcirculatory responses to muscarinic agonists was composed of two components. One component mostly responsible for the local response to methacholine, was blocked by nitroarginine and was therefore assumed to be NO released from the endothelium. The other component mainly responsible for the conducted response was independent of nitroarginine. Its mechanism is unknown, but previous reports suggest a role for a substance that hyperpolarizes vascular cells ^(6-10).

Acknowledgment: I appreciate the excellent technical support offered by Judy Beckman.

This study was supported by NIH R29HL-01654. These data were first presented at the Annual Microcirculatory Society Meeting in April 1992.