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Am J Physiol Heart Circ Physiol 284: H2083-H2090, 2003. First published January 30, 2003; doi:10.1152/ajpheart.01007.2002
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Vol. 284, Issue 6, H2083-H2090, June 2003

Cholinergic regulation of pericyte-containing retinal microvessels

David M. Wu1,2, Hajime Kawamura2, Kenji Sakagami2, Masato Kobayashi2, and Donald G. Puro1,2,3

1 Neuroscience Graduate Program, 2 Department of Ophthalmology and Visual Sciences and 3 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48105


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca2+ levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca2+-activated chloride currents and caused pericytes to contract in a Ca2+-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.

calcium; capillary; chloride current; muscarinic; vasoactive


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RETINAL VASCULATURE is anatomically and physiologically specialized to distribute oxygen and nutrients to areas of metabolic need within a tissue that must be translucent. One adaptation that minimizes interference with passing light is the low density of retinal capillaries (6). However, the relative paucity of retinal microvessels leaves little functional reserve. As a result, perfusion of a capillary must be tightly matched to the needs of nearby retinal neurons. Local control of retinal blood flow is facilitated by the absence of autonomic innervation (35), which limits extrinsic oversight by the central nervous system, and the presence of an endothelial barrier, which restricts the effects of circulating vasoactive molecules. In addition, the lack of precapillary smooth muscle sphincters (20), which control local perfusion in most other tissues, suggests that the regulation of retinal blood flow may occur in part within capillaries, rather than exclusively at precapillary sites.

Candidates for regulating blood flow at the capillary level are the contractile pericytes, which are located on the abluminal wall of microvessels. By contracting or relaxing, these cells may control capillary perfusion (10, 23, 26, 30). It has been suggested that pericytes have a particularly important function in the retina because the retinal microvasculature has a higher density of these cells than any other vascular bed (1, 27).

At present, the mechanisms by which pericyte function is coupled with the metabolic demands of retinal neurons remain uncertain. One possibility is that certain retinal neurotransmitters, in addition to mediating neuron-to-neuron communication, also serve as neuron-to-vascular signals that link blood flow to retinal function. Consistent with this idea, retinal microvessels respond to the neurotransmitter dopamine with the activation of a hyperpolarizing ATP-sensitive potassium current (34).

In this study, we consider the possibility that another retinal neurotransmitter, acetylcholine, regulates the pericyte-containing vessels in the retina. In agreement with acetylcholine serving as a neuron-to-capillary signal, binding sites for muscarinic receptors are found in cultured retinal pericytes (4), and biochemical studies (5) indicate that these pericyte receptors are functional. Because the release of acetylcholine reflects the activity of starburst amacrine cells (15), this molecule is a candidate for linking neuronal activity, and thereby metabolic demand, with capillary perfusion. However, although the role of this neurotransmitter in the nitric oxide (NO)-dependent relaxation of vascular smooth muscle is well characterized (7, 8, 19), little is known about the effects of acetylcholine on retinal capillaries. We report that activation of muscarinic receptors increases the intracellular Ca2+ concentration ([Ca2+]i), ionic conductances, and contractile tone of pericytes located on retinal microvessels freshly isolated from the adult rat retina.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microvessel isolation. Microvessels from 6- to 8-wk-old rat retinas were freshly isolated using a modified "tissue-print" method (24, 34). Animal use conformed to the guidelines of the Use of Animals in Ophthalmic and Vision Research and of the University of Michigan Committee on the Use and Care of Animals. Rats (Harlan Sprague Dawley; Indianapolis, IN) were euthanized with CO2, and their retinas were rapidly removed and incubated in 2.5 ml of Earle's balanced salt solution (Life Technologies; Grand Island, NY) supplemented with 0.5 mM EDTA, 20 mM glucose, 15 U papain (Worthington Biochemicals; Freehold, NJ), and 2 mM cysteine for 30 min at 30°C while being bubbled with 95% O2-5% CO2 to maintain pH and oxygenation. Each retina was then transferred to solution A, which was composed of (in mM) 140 NaCl, 3 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 Na-HEPES, 15 mannitol, and 5 glucose at pH 7.4 with osmolarity adjusted to 310 osmol/l with water, and gently sandwiched between two glass coverslips (15 mm diameter, Warner Instrument; Hamden, CT). Vessels adhered to the coverslip that was in contact with the vitreal side of the retina. By repeating this tissue-print step two or three times, several coverslips with pericyte-containing microvessels could be obtained from each retina.

Electrophysiology. A coverslip containing microvessels was placed in a recording chamber, which was perfused with solution A plus additives as noted. Vessels were examined at ×400 magnification with an inverted Leitz Diavert equipped with phase-contrast optics. Pericytes could be identified by their characteristic "bump on a log" location on the abluminal wall of microvessels that had outer diameters of <7 µm (13, 24).

As detailed previously, the perforated-patch configuration of the patch-clamp technique was used to monitor the ionic currents and the membrane potentials of pericytes located on microvessels that had been isolated from a retina within 3 h (24). The pipette solution consisted of 50 mM KCl, 64 mM K2SO4, 6 mM MgCl2, 10 mM K-HEPES, 240 µg/ml amphotericin, and 240 µg/ml nystatin at pH 7.4 with the osmolarity adjusted to 280 mosmol/l. The pipettes, which had resistances of ~5 MOmega , were mounted in the holder of a patch-clamp amplifier (Axon Instruments; Union City, CA) and sealed to the cell bodies of pericytes. While amphotericin/nystatin perforated the patch, the access resistance to the pericytes studied decreased to <25 MOmega within 5 min.

To assess steady-state current-voltage relations, we evoked currents by voltage steps from a holding potential of -58 mV. There was a 9-s interval between each step of a voltage protocol, which was controlled by pCLAMP 8 software (Axon Instruments). Currents were filtered at 1 kHz with a four-pole Bessel filter, digitally sampled at 50-µs intervals with the use of an acquisition system (DigiData 1200B, Axon Instruments) and stored on a Pentium computer equipped with pCLAMP 8 and Origin (version 6.1, OriginLab; Northampton, MA) software for data analysis and graphics display. Adjustment for the calculated liquid junction potential (2) was made after data collection. In voltage-clamp experiments, the zero-current potential was defined as the membrane potential of the recorded pericyte. Voltage-step protocols were completed ~2 min after the onset of exposure to a cholinergic agonist. For continuous recordings of currents, pericytes were voltage clamped at -58 mV, and the currents were sampled at 10- or 100-ms intervals. In current-clamp experiments (Fig. 2), the holding current was zero and the sampling rate was 100 Hz.

The mean amplitude of the transient inward currents was calculated at 5 s for each interval by subtracting the amplitude of the current during the absence of any transient events from the average current amplitude, which was determined with pCLAMP and Origin software (11). The absolute values of this inward current are given in the text. As detailed previously (11, 24), the nonspecific cation (NSC) current was measured at -103 mV, which is the equilibrium potential for potassium (EK); the potassium current was measured at 0 mV, which is close to ENSC.

Because there are gap junction pathways within retinal microvessels (18), currents detected in a pericyte include not only those generated by the ion channels of the sampled cell, but also currents transmitted electronically from neighboring vascular cells (11, 18). This coupling of cells is likely to limit the spatial control of voltage during our electrophysiological experiments. Consistent with this, we detected a 39 ± 8% (n = 6) decrement in voltage between recording pipettes located at sites 400-450 µm (424 ± 12 µm) apart on freshly isolated microvessels. Clearly, a space clamp would be more controlled in short, rather than long, microvessels. However, the frequent occurrence of low membrane potentials and unstable recordings in microvessels shorter than ~300 µm indicated that cells in short capillary fragments are often damaged. For this reason, we recorded from pericytes in microvessels of >300 µm although the voltage clamp of distantly coupled cells would be less than that of the sampled pericyte. Yet, despite this limitation of the space clamp, we found previously in studies (14, 24) of isolated retinal microvessels that the reversal potentials for potassium, chloride, and NSC currents closely matched the calculated equilibrium potentials for these ions. Thus, for identification of ionic conductances, it appears that the voltage within a microvessel can be clamped reasonably well at the sites containing the bulk of the ion channels that contribute to the current which is detected in a sampled pericyte.

Calcium imaging. Freshly isolated pericytes were loaded with 1 µM fura 2-AM (Molecular Probes; Eugene, OR) at 37°C for 30 min. Afterward, the extracellular fura 2-AM was washed out with solution A for at least 30 min to give further time for the AM ester to be cleaved. A coverslip containing fura-loaded pericytes was positioned in a perfusion chamber, and digital imaging of fluorescence was performed at room temperature using an optical sensor (Sensicam, Cooke; Auburn Hills, MI). The light source was a high-intensity mercury lamp coupled to an Optoscan Monochromator (Cairn Research; Faversham, UK). Axon Imaging Workbench (Axon Instruments) was used to control the imaging equipment and collect data. Pericytes were identified with a Nikon Eclipse TE300 microscope at ×400 using a ×40 oil objective. We measured fluorescence from pericyte somas that were positioned like a bump on a log at the edge of the endothelial wall. In this way, fluorescence from pericytes, not endothelial cells, was measured. The fluorescence ratio (340:380) was converted to [Ca2+]i with the use of the equation in Ref. 9, as we have detailed previously (21). Autofluorescence, determined in unloaded cells, was not detected in the isolated microvessels.

Time-lapse photography. A glass coverslip containing freshly isolated microvessels was positioned in a specially built chamber (volume = 200 µl), which was perfused by a system of gravity-fed reservoirs. Pericyte-containing vessels were visualized by differential interference contrast optics at ×1,000 magnification with a Nikon Eclipse E800 equipped with a ×100 oil objective. To detect pericyte contractions, time-lapse images were recorded at 6-s intervals with a Nikon DCM1200 digital camera and ImagePro Plus Software (Media Cybernetics; Silver Spring, MD). Contractions were much more obvious in time-lapse movies (see http://ajpheart.physiology.org/cgi/content/full/284/6/H2088/DC1) than when viewed as single frames, which, by necessity, were used in Fig. 6. ImagePro Plus Software also facilitated the measurement of lumen diameters in the time-lapse photographs. Because, as noted by Sakagami et al. (23), contracting pericytes sometimes caused microvascular lumens to move out of the narrow depth of differential interference contrast focus, only those lumens that were in focus before and during exposure to oxotremorine-M had their diameters quantified.

Chemicals. Fura 2-AM was obtained from Molecular Probes (Eugene, OR). Other chemicals were obtained from Sigma/RBI (St. Louis, MO).

Statistics. Data are given as means ± SE. Unless otherwise noted, probability was evaluated by Student's t-test, paired or independent, as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cholinergic activation of ionic currents. To help test the hypothesis that cholinergic stimulation can influence the physiology of the pericyte-containing microvasculature, we monitored currents in pericytes located on microvessels freshly isolated from the adult rat retina. In a series of five recordings, we assessed the current-voltage relationship before and after supplementing the perfusate with the acetylcholine analog carbachol. As illustrated in Fig. 1, A and B, we did not detect a significant (P > 0.26) change in the steady-state currents that are due chiefly to the activity of voltage-dependent K+ (Kv) and NSC channels (24).


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  		Fig. 1.   Effect of carbachol on the currents monitored in a pericyte located on a freshly isolated microvessel. A: current traces from the pericyte before (control) and 2 min after the onset of exposure to 10 µM carbachol. The clamp protocol is shown below the current traces. B: current-voltage relations of the steady-state currents measured from the traces in A. C: continuous current record of the same pericyte voltage-clamped at -58 mV. The bar indicates when the perfusate (solution A) contained 10 µM carbachol. D: continuous current recording of a pericyte during exposure to 10 µM carbachol and 10 µM atropine as indicated by the labeled bars. The pericyte was voltage clamped at -58 mV. Carbachol induces an atropine-sensitive increase in the transient inward Ca2+-activated chloride (ClCa) currents recorded in pericyte-containing microvessels.

In contrast to the lack of a cholinergic effect on Kv and NSC currents, carbachol (10 or 20 µM) activated transient inward currents in 10 of 16 sampled pericytes (Fig. 1, C and D). Our previous demonstration (24) that, similar to the transient inward currents recorded in vascular smooth muscle cells (31), those of retinal pericytes have a reversal potential near the equilibrium potential for chloride, are blocked by a chloride channel blocker, and are activated by calcium, indicates that Ca2+-activated chloride (ClCa) channels generate these inward currents. The amplitude of the ClCa current monitored at a holding potential of -58 mV was 7.26 ± 1.75 pA (n = 16). During exposure of responsive microvessels to carbachol, the ClCa current increased significantly (P < 0.001, n = 10), from 7.26 to 33.8 ± 7.4 pA (holding potential = -58 mV). After 2 min of continued exposure to carbachol, the ClCa current remained significantly greater (P = 0.001) than the basal level. With a return to the carbachol-free perfusate (solution A), the amplitude of the ClCa current returned to the control level.

To assess whether the cholinergic activation of the microvascular ClCa currents was mediated via nicotinic or muscarinic receptors, we tested the effect of the selective muscarinic antagonist atropine (Fig. 1D). In five of five pericytes tested, exposure to atropine decreased the carbachol-induced ClCa current by >95%, resulting in a conductance amplitude that was not significantly (P = 0.42, n = 5) different than the control value. Similarly, exposure of microvessels to atropine inhibited the transiently occurring depolarizations caused by carbachol-induced ClCa channel activity (Fig. 2). Because atropine had no significant (P > 0.66) effect on the amplitude of the basal ClCa current, it does not appear that endogenous acetylcholine regulated ClCa channel activity in our isolated microvessels.


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  		Fig. 2.   Effect of carbachol on the pericyte membrane potential. A: continuous voltage record of a pericyte located on a freshly isolated microvessel. Throughout the recording period shown, the perfusate (solution A) was supplemented with 10 µM carbachol. The bar shows when 1 µM atropine was added to the carbachol-containing perfusate. B: voltage traces, which are from the same pericyte as in A, shown at a faster sweep speed than in A. The perfusate consisted of solution A (control) plus or minus carbachol without or with atropine. Atropine reversibly blocked the carbachol-induced transient depolarizations recorded in retinal pericytes.

To further establish a role for muscarinic receptors, we tested the effect of oxotremorine-M, which is a selective muscarinic receptor agonist. Similar to carbachol (Fig. 1D), this muscarinic agonist not uncommonly increased the frequency of the transient depolarizing events so dramatically during the initial minutes of exposure that there was a sustained inward conductance (Fig. 3A). In agreement with ClCa channels mediating the response to muscarinic receptor activation, the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 µM) significantly (P < 0.05, n = 3) inhibited the effect of oxotremorine-M (Fig. 3A). To obtain further evidence that muscarinic agonists activate chloride channels, we determined the reversal potential for the transient currents recorded in pericyte-containing microvessels during exposure to oxotremorine-M (Fig. 3B). Consistent with a muscarinic activation of chloride channels, the observed reversal potential of -23 ± 4 mV (n = 4) closely matched the calculated Nerstian value for the equilibrium potential for chloride, i.e., -22 mV. More evidence consistent with ClCa activation was that the oxotremorine-induced currents showed outward rectification (Fig. 3B), which is a characteristic of ClCa channels (17).


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  		Fig. 3.   Oxotremorine-induced currents monitored in pericytes located on freshly isolated microvessels. A, top: current trace monitored during exposure of a pericyte-containing microvessel to 10 µM oxotremorine-M. Bottom, current from same pericyte as in top during perfusion of 200 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) without and with 10 µM oxotremorine-M. B: currents recorded in a pericyte that was held at the various membrane potentials listed adjacent to each trace and was perfused with 10 µM oxotremorine-M. This muscarinic agonist activates chloride currents in retinal pericytes.

In a series of experiments, we quantified the effect of oxotremorine-M on the ClCa currents in pericytes. We found that oxotremorine-M (10 µM) increased the amplitude of the ClCa current in 22 of 29 sampled pericytes. Specifically, during oxotremorine exposure, the ClCa current of the responsive microvessels increased significantly (P < 0.001, n = 22), from 6.69 ± 0.88 to 42.74 ± 4.28 pA. After 2 min of continued exposure to this muscarinic agonist, the ClCa current remained significantly (P = 0.002) greater than the control level (Fig. 4). As with carbachol, oxotremorine-M did not significantly (P > 0.28, n = 16) affect the KV or NSC currents recorded in the pericyte-containing microvessels.


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  		Fig. 4.   Effect of oxotremorine-M on the pericyte ClCa current. Data points show the mean amplitude of the ClCa current in the nine sampled pericytes that were monitored for at least 2 min after the onset of exposure to oxotremorine-M. The bar shows when 10 µM oxotremorine-M was in the perfusate. This muscarinic agonist activates ClCa currents in pericyte-containing retinal microvessels.

We also assessed the effect of muscarinic activation on ATP-sensitive K+ (KATP) currents. Although minimally active under control recording conditions, KATP currents are induced when microvessels are exposed to forskolin (34), an activator of adenylate cyclase. We found that 10 µM forskolin induced a 24 ± 6 mV hyperpolarization that was unaffected (P = 1.0, n = 4) by the addition of 10 µM oxotremorine-M to the perfusate. Taken together, our experiments demonstrate that activation of muscarinic receptors in retinal microvessels had no detectable effect on the currents generated by NSC, KV, and KATP channels, but markedly increased the activity of the depolarizing ClCa currents.

Cholinergic-induced increase in intracellular Ca2+ levels. The increase in ClCa currents recorded during exposure to oxotremorine-M likely reflected an increase in [Ca2+]i. To assess this possibility, we used the calcium indicator fura 2 to monitor the [Ca2+]i of pericytes. Figure 5 shows the effect of oxotremorine-M (10 µM) on the mean [Ca2+]i of 10 pericytes located on a freshly isolated microvessel. In a series of experiments, we found that this muscarinic agonist induced an increase in [Ca2+] in 31 of 37 sampled pericytes. In the responsive cells, [Ca2+]i rose from a basal level of 133 ± 3 nM to a peak of 190 ± 10 nM (P = 0.0001, n = 31). Thus activation of muscarinic receptors is linked with an increase in pericyte [Ca2+]i.


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  		Fig. 5.   Effect of oxotremorine-M on the pericyte intracellular Ca2+ concentration ([Ca2+]i). Data points show the mean [Ca2+]i of 10 pericytes located on a microvessel that was exposed to 10 µM oxotremorine-M during the period shown by the bar. Muscarinic stimulation increases the [Ca2+]i of retinal pericytes.

Cholinergic-induced contractions of pericytes. Because an increase in [Ca2+]i initiates contraction in other types of contractile cells (28), we asked whether pericytes contract during exposure to oxotremorine-M. This is of interest because others (23, 26, 30) have proposed that by contracting and relaxing, pericytes affect lumen size and thereby regulate capillary blood flow. To detect changes in pericyte contractility, freshly isolated retinal microvessels were viewed by differential interference contrast microscopy (Fig. 6A). During exposure to oxotremorine-M (10 µM), we observed at least one contracting pericyte in 13 of 17 (76%) microscopic fields monitored by time-lapse photography. Pericyte contractions continued throughout several minutes of oxotremorine exposure. Other than partial relaxations of pericytes that were phasically contracting during exposure to cholinergic agonists, we did not detect an induced decrease in contractility. These observations show that activation of muscarinic receptors induced contractile responses in most of the observed microvessels.


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  		Fig. 6.   Oxotremorine-induced contraction of a pericyte-containing microvessel. A: differential interference contrast photomicrograph of a bifurcating microvessel, which was freshly isolated from a rat retina. The arrowheads point to red blood cells located within the capillary lumen. The white box outlines the area shown at higher magnification in the bottom panels. Scale bar, 10 µm. B: data points show the lumen diameter, which was measured at the site indicated by the horizontal white line in the lower set of panels. The black bar indicates when 10 µM oxotremorine-M was in the perfusate. The colored symbols show the times at which the photographs in the bottom set of five panels were taken. The arrow in each of the five panels at bottom points to a perictye that contracted during exposure of the microvessel to oxotremorine-M. Associated with pericyte contraction, the adjacent capillary lumen was narrowed, and the squeezed erythrocyte was moved distally. The scale bar at bottom left shows 5 µm. Oxotremorine-M induces pericyte contraction and lumen constriction in retinal microvessels. Because the movement of a contracting pericyte is much more obvious than comparisons of pericyte shape in still pictures, a supplemental file containing a time-lapse movie of this microvessel is at http://ajpheart.physiology.org/cgi/content/full/284/6/H2088/DC1. Frames were acquired every 6 s for 5.75 min.

To help assess the role of intracellular calcium in linking the activation of muscarinic receptors with pericyte contraction, we tested the effect of a chelator of intracellular calcium, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). Time-lapse photography facilitated identification of pericyte-containing microvessels that contracted during oxotremorine exposure. Subsequently, four microvessels showing oxotremorine-induced contractions were exposed for 17 ± 1 min to a perfusate containing 10 µM BAPTA-AM. Four other contracting microvessels remained in solution A. When viewed at our standard ×1,000 magnification, the BAPTA-treated microvessels lacked detectable contractions during reexposure to oxotremorine-M. In contrast to the BAPTA-treated group, microvessels that were not exposed to BAPTA-AM contracted during a second exposure to oxotremorine-M. The inhibitory effect of this calcium chelator on oxotremorine-induced contractions in pericyte-containing microvessels was significant (P = 0.029, Fisher's test). These findings support the idea that a rise in [Ca2+]i links muscarinic receptor activation with pericyte contraction.

Although pericytes are located throughout the retinal microvasculature and, as noted above, a majority (76%) showed increased chloride currents during exposure to oxotremorine-M, this muscarinic agonist induced contractions in only ~10% of the pericytes. This contrasts with our recent observation that angiotensin II can evoke contractions in nearly all neighboring pericytes (M. Kobayashi and D. G. Puro, unpublished observations). Thus not all pericytes capable of contraction were induced to contract when oxotremorine-M was added to the perfusate. At present, the reason for this selectivity of responsiveness remains unclear.

Interestingly, most of the pericytes that were responsive to oxotremorine were observed chiefly at sites where the microvessels bifurcated. Specifically, of the 18 microvascular sites at which we detected oxotremorine-induced contractions, 13 (72%) were located at microvascular bifurcations. Because only ~20% of the length of the monitored microvessels was near (<= 30 µm) a bifurcation, the observed incidence of contractions at branching points was significantly (P = 0.007, Fisher's exact test) greater than would be predicted by a random distribution. Overall, we detected oxotremorine-induced contractions at 13 of 21 (62%) branch points monitored by time-lapse photography.

We also assessed whether pericyte contraction was associated with narrowing of the adjacent capillary lumen. Figure 6B shows an example of pericyte contraction and lumen constriction during exposure of an isolated microvessel to oxotremorine-M. In five microvessels, in which both a contracting pericyte and the adjacent endothelial lumen were in the focal plane of our differential interference contrast microscope, pericyte contraction was associated with a decrease in the diameter of the lumen from 3.32 ± 0.46 to 2.36 ± 0.56 µm (P = 0.011).

Taken together, our electrophysiological recordings, calcium imaging studies, and microscopic observations indicate that acetylcholine acts at muscarinic receptors to alter the physiology of pericyte-containing microvessels. By causing pericytes to contract and thereby capillary lumens to constrict, acetylcholine may serve to regulate blood flow in the microvasculature of the retina.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study support the hypothesis that acetylcholine serves as a vasoactive signal in the retina. In agreement with this possibility, the muscarinic agonist oxotremorine-M elicited an increase in [Ca2+]i in pericytes located on microvessels freshly isolated from the adult rat retina. Activation of muscarinic receptors also induced a marked increase in the activity of ClCa channels, which cause transient depolarizations of the pericytes. Furthermore, oxotremorine-M elicited calcium-dependent contractions of pericytes, chiefly near capillary bifurcations. Associated with pericyte contractions, the adjacent capillary lumens constricted. On the basis of these observations, it appears that acetylcholine may regulate capillary perfusion in the retina by modulating pericyte contractility.

The source of acetylcholine to activate muscarinic receptors of the retinal microvasculature is uncertain. Although acetylcholine is well established as a neurotransmitter that is released by the starburst amacrine cells (16, 29), there are no reports of retinal vessels being directly innervated by these neurons. Thus it appears that synaptically released acetylcholine would have to reach the capillaries by diffusion, or volume transmission. The possibility that acetylcholine may act via volume transmission, as well as at conventional synapses, is suggested by the observation that most of the neuronal M2 muscarinic receptors in the retina are located far from cholinergic synapses (32). On the other hand, the relatively high acetylcholinesterase activity in the retina must limit the distance over which acetylcholine diffuses before being hydrolyzed. However, volume transmission may be more extensive under pathophysiological conditions, such as diabetes, in which retinal acetylcholinesterase activity is substantially reduced (25).

In addition to the starburst amacrine cells, another possible source of acetylcholine is the blood vessels themselves. Consistent with this possibility, isolated bovine retinal vessels were found to possess choline acetyltransferase (3), the rate-limiting enzyme for acetylcholine synthesis. However, it is not known whether capillaries synthesize acetylcholine, because the location of choline acetyltransferase within the retinal vasculature remains to be established. It appears that our isolated retinal microvessels lack sufficient endogenous acetylcholine to activate their muscarinic receptors, since atropine did not affect the basal ClCa currents monitored in perictyes. Although synthesis of acetylcholine in the pericyte-containing microvessels cannot be excluded, we postulate that the muscarinic receptors in retinal microvessels are activated by acetylcholine released from the starburst amacrine cells. Our recent finding that dopamine also alters the physiology of retinal pericytes (34) suggests that acetylcholine is not the only retinal neurotransmitter that serves as a vasoactive signal.

In freshly isolated retinal microvessels, we observed that activation of muscarinic receptors induced contractions of pericytes, chiefly at capillary bifurcations. At sites of pericyte contraction, the adjacent endothelial lumen narrowed. In contrast to this vasoconstrictive effect, the luminal diameter of capillaries at sites distant from bifurcations were reported to increase during exposure of isolated rat retinas to carbachol (26). Although the mechanism for a carbachol-induced vasodilation of retinal capillaries is uncertain, it is well known that the vascular endothelium of larger vessels synthesizes the vasodilator NO during exposure to acetylcholine (7, 19). This raises the question of whether the absence of relaxations in our isolated microvessels was due to endothelial damage and an inability to produce NO. Although we cannot exclude some injury, ~95% of the microvascular cells remain viable for at least 6 h after vessel isolation (12). In addition, endogenously synthesized NO tonically inhibits ClCa channels in most (~80%) of the isolated retinal microvessels (22). Thus in our experimental preparation, the direct contractile effect of cholinergic agonists on pericytes appears to overwhelm any indirect vasodilatory action of NO. Perhaps the absence of glial cells tightly ensheathing the isolated capillaries and our continuous superperfusion of microvessels with bathing solution limit the amount of NO that diffuses to the abluminal pericytes. In future studies, the use of NO synthesis inhibitors may better clarify the interactions of muscarinic receptors with the NO system in the retinal microvasculature. Also, more studies are needed to clarify the relative roles of the vasoconstrictive and vasodilatory responses of retinal capillaries to muscarinic receptor activation. However, despite remaining uncertainties, our experiments do demonstrate that one of the effects of activating muscarinic receptors in the pericyte-containing microvessels of the retina is a stimulation of vascular contractility and vasoconstriction, especially at capillary bifurcations.

Technical challenges preclude an in vivo application of the electrophysiological and imaging methods used in this study. Consequently, it remains to be demonstrated that the muscarinic effects observed in freshly isolated microvessels are induced by acetylcholine released within the retina in vivo. However, the use of isolated microvessels permitted patch-clamp recordings, calcium imaging, and time-lapse photography of fresh rather than cultured pericytes. Because retinal pericytes are coupled via gap junctions to dozens of neighboring vascular cells (11, 18), the ability to study a pericyte that is an integral component of a multicellular functional unit can reveal a more complete picture of how the retinal microvasculature responds to vasoactive signals. For example, although cholinergic effects on cultured retinal pericytes are minimal (33), we found that activation of muscarinic receptors markedly altered the ionic currents, the calcium concentration, and the contractility of pericytes located on retinal microvessels. A challenge for the future is to define the mechanisms by which the complexity of the cell-cell interactions contributes to the functional response of a retinal capillary.


    ACKNOWLEDGEMENTS

The authors thank Scott Salazay and Mitch Gillett for technical expertise and Bret Hughes for helpful discussions and generous use of equipment.


    FOOTNOTES

This work was supported by National Eye Institute Grants EY-12505 and EY-07003. D. M. Wu received a Physician-Scientist Training Award from the American Diabetes Association and a Research to Prevent Blindness Medical Student Research Fellowship. D. G. Puro is a Harrington Research to Prevent Blindness Senior Scientific Scholar.

Address for reprint requests and other correspondence: D. G. Puro, Dept. of Ophthalmology and Visual Sciences, Univ. of Michigan, 1000 Wall St., Ann Arbor, MI 48105 (E-mail: dgpuro{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 30, 2003;10.1152/ajpheart.01007.2002

Received 21 November 2002; accepted in final form 28 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(6):H2083-H2090
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