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Am J Physiol Heart Circ Physiol 290: H925-H934, 2006. First published November 18, 2005; doi:10.1152/ajpheart.01012.2005
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Extracellular lactate as a dynamic vasoactive signal in the rat retinal microvasculature

Shigeki Yamanishi,1 Kozo Katsumura,1 Takatoshi Kobayashi,1 and Donald G. Puro1,2

1Department of Ophthalmology and Visual Sciences and 2Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Submitted 23 September 2005 ; accepted in final form 9 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that extracellular lactate regulates the function of pericyte-containing retinal microvessels. Although abluminally positioned pericytes appear to adjust capillary perfusion by contracting and relaxing, knowledge of the molecular signals that regulate the contractility of these mural cells is limited. Here, we focused on lactate because this metabolic product is in the retinal extracellular space under both physiological and pathophysiological conditions. In microvessels freshly isolated from the adult rat retina, we used perforated-patch pipettes to monitor ionic currents, fura-2 to measure calcium levels, and time-lapse photography to visualize changes in mural cell contractility and lumen diameter. During lactate exposure, pericyte calcium rose; these cells contracted, and lumens constricted. This contractile response appears to involve a cascade of events resulting in the inhibition of Na+/Ca2+ exchangers (NCXs), the decreased of which function causes pericyte calcium to increase and contraction to be triggered. On the basis of our observation that gap junction uncouplers minimized the lactate-induced rise in pericyte calcium, we propose that the NCXs inhibited by lactate are predominately located in the endothelium. Indicative of the importance of endothelial/pericyte gap junctions, uncouplers of these junctions switched the pericyte response to lactate from contraction to relaxation. In addition, we observed that hypoxia, which closes microvascular gap junctions, also switched lactate’s effect from vasocontraction to vasorelaxation. Thus the response of pericyte-containing retinal microvessels to extracellular lactate is metabolically modulated. The ability of lactate to serve as a vasoconstrictor when energy supplies are ample and a vasodilator under hypoxic conditions may be an efficient mechanism to link capillary function with local metabolic need.

capillaries; retina

AN ESSENTIAL TASK of the circulatory system is to efficiently match blood flow to meet metabolic demand. In the retina, this is a major challenge because of its low capillary density (6). Although the paucity of capillaries minimizes interference with the passage of light, it also makes it especially critical to have a tight functional link between local perfusion and metabolic need. Hence, not unexpectedly, the vasculature of the retina is highly specialized for the local regulation of blood flow. For example, input from extrinsic sites is minimized by the lack of autonomic innervation (33) and the presence of endothelial tight junctions. In addition, the regulation of vascular resistance by contractile pericytes located on the abluminal wall of microvessels (≤7-µm diameter) appears to be particularly important in the retinal vasculature, which has the highest density of these mural cells (25). By contracting and relaxing, pericytes provide potentially potent, decentralized sites at which capillary perfusion can be regulated to meet metabolic demand (12, 24, 28). Because of these specializations, retinal microvessels are proving to be useful in the quest to elucidate the mechanisms that link capillary function with local metabolism (16, 19, 22, 24).

Almost certainly, key components in the regulation of blood flow are extracellular molecules that transmit information to the circulatory system about local metabolic needs. However, knowledge of the vasoactive signals that control capillary blood flow is limited. In this study, we began to test the hypothesis that extracellular lactate regulates the function of pericyte-containing retinal microvessels. Lactate is of interest because retinal cells produce this metabolic product even when their oxygen supply is adequate (4, 18, 29, 30). In fact, it is estimated that >80% of the glucose used aerobically in the retina is converted to lactate (29). Because retinal cells export lactate to prevent intracellular acidosis, this product of metabolism is normally in the retinal extracellular space (10). Even though the concentration of lactate at sites adjacent to the retinal vasculature is not known, under normoxic conditions the vitreous contains ~10 mM of this metabolic product (2). Under hypoxic conditions, lactate production and release are augmented at least severalfold (29, 30). Thus extracellular lactate is available to serve as a vasoactive signal under physiological and pathophysiological conditions.

The hypothesis that lactate plays a vasoregulatory role in the retina has recently received experimental support. For example, the intravenous administration of lactate increases retinal blood flow in humans and rats (7, 13). In addition, investigators have demonstrated that the microinjection of lactate into the vitreous of the porcine eye causes retinal vessels of >70-µm diameter to dilate (3). However, the effect of lactate on pericyte-containing retinal microvessels has not previously been studied. Here, we report on experiments using retinal vessels freshly isolated from the adult rat. We found that extracellular lactate caused mural cells to contract and lumens to constrict in pericyte-containing microvessels whose energy supply was ample. In contrast, pericytes located on hypoxic microvessels relaxed during exposure to lactate. This dual vasoactive capability may provide an efficient mechanism to link capillary perfusion with retinal metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Microvessel isolation. Animal use conformed to the guidelines of the Association for Research in Vision and Ophthalmology. The University of Michigan Committee on the Use and Care of Animals reviewed and approved the protocols for animal use. As detailed recently (16), 6- to 8-wk-old Long-Evans rats (Charles River, Cambridge, MA) were killed with a rising concentration of carbon dioxide, and their retinas were rapidly removed and incubated for 30 min at 30°C in 2.5 ml Earle’s balanced salt solution supplemented with 0.5 mM EDTA, 20 mM glucose, 15 U papain (Worthington Biochemicals, Freehold, NJ), and 2 mM cysteine; to maintain pH and oxygenation, 95% oxygen-5% carbon dioxide was bubbled into this solution. Subsequently, retinas were transferred to solution A, which consisted of 140 mM NaCl, 3 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM Na-HEPES, 15 mM mannitol, and 5 mM glucose at pH 7.4 with osmolarity adjusted to 310 mosmol/l. Each retina was then gently sandwiched between two glass coverslips (15-mm diameter, Warner Instrument, Hamden, CT). Retinal vessels adhered to the coverslip contacting the vitreal side of the retina. Repetition of this tissue print step resulted in several vessel-containing coverslips being obtained from each retina. Photomicrographs of microvessels freshly isolated by this technique are in a number of our previous publications (15, 20, 23, 27, 31, 32). Experiments were performed using vessels that had been isolated within 3 h.

Perfusates. The perfusates containing 0–40 mM L-lactate (Table 1) were designed so that the sodium concentration, pH, and osmolarity were essentially the same. After adjustment of the pH to 7.4 with NaOH, the osmolarity of each perfusate was 309 ± 0.1 mosmol/l as measured by a vapor pressure osmometer (Wescor, Logan, UT). In addition, we designed the lactate-free perfusate so that its chloride concentration was the same, i.e., 124.2 mM, as that of the 20 mM lactate solution, which was the lactate-containing perfusate used most frequently in this study.


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  		Table 1. Composition of the perfusates

 
In experiments using BAPTA-AM, we exposed freshly isolated microvessels to this calcium chelator for 30 min before performing time-lapse photography or calcium imaging. In experiments using {alpha}-cyano-4-hydroxycinnamic acid (CHC), 5-(N,N-dimethyl) amiloride hydrochloride (DMA), KB-R7943, octanol, 18-{alpha}-glycyrrhetinic acid ({alpha}-GA), or ouabain, microvessels were initially exposed to these chemicals in the lactate-free perfusate for 5 min before their exposure to a lactate-containing perfusate that included one of these chemicals. In experiments using perforated-patch recordings from pericytes and the injection of Neurobiotin into these mural cells, we have confirmed that the gap junction uncouplers used in this study markedly increase the membrane resistance of pericytes and decrease the cell-to-cell spread of the gap junction permeant tracer (20). Because a 60- to 90-min exposure to 300 µM Gap 27 is required to close microvascular gap junctions, as detected by a >20-fold increase in membrane resistance in pericyte recordings using the perforated-patch technique (S. Yamanishi and D. Puro, unpublished observations), microvessels were exposed to this gap junction uncoupler in solution A for 2 h before time-lapse experiments. Because of the high cost of Gap 27 and our finding the effect of this gap junction uncoupler on membrane resistance persists for at least 30 min after its washout, Gap 27 was not included in the perfusates; experiments using Gap 27 were completed within 20 min after removal of microvessels from the Gap 27-containing solution. In experiments assessing the effect of hypoxia (see Figs. 2C and 7), perfusates were bubbled with 100% nitrogen gas for 15 min immediately before use. Microvessels were exposed to a lactate-free, nitrogen-treated bathing solution for 15 min before switching to a nitrogen-treated, lactate-containing perfusate.


Figure 2
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  		Fig. 2. Effect of extracellular lactate on pericyte calcium. A: relationship between the concentration of lactate and the maximum increase in pericyte calcium. The value at 0 mM lactate is the mean calcium concentration in 88 pericytes before their exposure to lactate. For the other data points, each is the mean of 22 ± 4 pericytes that had a significant (P < 0.05) lactate-induced increase in the intracellular calcium concentration; the calcium level rose in 50%, 53%, 100%, and 100% of the monitored pericytes during exposure to 0.2, 2, 20, and 40 mM lactate, respectively. At ≥0.2 mM lactate, the calcium concentration was significantly (P ≤ 0.017) greater than in the control group. B: plot of the intercellular calcium concentration vs. time for 6 pericytes located on a retinal microvessel. Bar indicates when the microvessel was exposed to a perfusate supplemented with 20 mM lactate. C: effect of lactate on pericyte calcium levels under various conditions. Five groups were exposed to 20 mM lactate without or with 1 mM CHC, 30 µM DMA, 30 µM KB-R7943, or 1 mM octanol; for the group labeled "lactate/hypoxia," microvessels were maintained in a nitrogen-treated, lactate-free perfusate for 15 min before exposure to the nitrogen-treated, 20 mM lactate perfusate (see MATERIALS AND METHODS). The various additives, as well as hypoxia, caused a significant (P < 0.001, Fisher’s exact test) decrease in the lactate-induced rise in pericyte calcium. For each group, 29 ± 2 pericytes were monitored.

 

Figure 7
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  		Fig. 7. Effect of conditions known to uncouple microvascular gap junctions. Labeled bars include groups of pericyte-containing microvessels that were exposed to 20 mM lactate without or with 1 mM octanol, 100 µM 18-{alpha}-glycyrrhetinic acid ({alpha}-GA), or 300 µM Gap 27. For the group labeled "lactate/hypoxia," microvessels were exposed to nitrogen-treated solution, as described in the MATERIALS AND METHODS. For the lactate, lactate/hypoxia, lactate + octanol, lactate + {alpha}-GA, and lactate + Gap 27 groups, time-lapse photography was used to monitor 274, 42, 36, 54, and 24 pericytes, respectively. Each of the additives, as well as hypoxia, caused a significant (P ≤ 0.003) change in the contractile response of pericytes to lactate.

 
Time-lapse photography. As illustrated previously (14, 16, 32), detection of contracting or relaxing pericytes is much more obvious and reliable when one views a time-lapse movie of a microvessel compared with looking at still pictures. For our time-lapse experiments, a coverslip containing freshly isolated retinal microvessels was positioned in a perfusion chamber (volume = 200 µl) on the stage of a Nikon Eclipse E800 equipped with differential interference contrast optics that permitted x1,000 magnification with the aid of a x100 oil objective. As detailed (14), images were recorded at 8-s intervals using a Nikon DCM1200 digital camera and ImagePro Plus software (Version 4.5, Media Cybernetics, Silver Spring, MD). Pericytes were identified by their characteristic "bump on a log" location on the abluminal wall of microvessels that had outer diameters of ≤7 µm. As in our previous studies (14, 16, 32), visual inspection of the time-lapse movies was used to detect the relaxation or contraction of pericytes during exposure to lactate. ImagePro software also was used to measure the diameters of microvascular lumens at sites in which a contracting pericyte and the adjacent lumen were simultaneously visible in time-lapse photographs; for Fig. 1B, lumen diameters near contracting pericytes were measured immediately before the onset of lactate exposure and at the time when the lactate-induced change in diameter was maximal.


Figure 1
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  		Fig. 1. Contractile effect of lactate. A: effect of lactate on pericyte contractility under various conditions. Labeled bars indicate groups in which the perfusate contained 20 mM lactate plus BAPTA-AM (10 µM), {alpha}-cyano-4-hydroxycinnamic acid (CHC, 1 mM), 5-(N,N-dimethyl) amiloride hydrochloride (DMA, 30 µM), KB-R7943 (KBR, 30 µM), or ouabain (1 mM). For each group, 92 ± 14 pericytes were monitored by time-lapse photography. Each of the various additives significantly (P ≤ 0.001, Fisher’s exact test) decreased lactate-induced pericyte contractions. B: lactate-induced vasoconstriction of pericyte-containing retinal microvessels. Lumen diameters were measured in 9 microvessels before and during exposure to 20 mM lactate. Error bars show SEs. *P = 0.014.

 
Calcium imaging. Freshly isolated microvessels were loaded with 5 µM fura-2 AM (Molecular Probes, Eugene, OR) at 37°C for 30 min. A coverslip containing fura-loaded microvessels was positioned in a chamber (volume = 1 ml), which was perfused at ~1.5 ml/min. To provide time for the AM ester to be cleaved, extracellular fura-2 AM was washed out with solution A for ~30 min. Digital imaging of fluorescence was performed at room temperature using an intensified CCD camera with a 12-bit dynamic range (Cool Snap HQ, Photometrics, Tucson, AZ); the light source was a high-intensity mercury lamp coupled to a monochromator (Cairn Research, Faversham, UK). MetaFluor software (Molecular Devices, Sunnyvale, CA) controlled the imaging equipment and collection of data. Microvessels were viewed with a Nikon Eclipse E600 FN microscope at x400 using a x40 water immersion objective. We measured fluorescent intensities at 340 nm and 380 nm from pericyte somas, which were defined as regions of interest using the imaging software. By measuring fluorescence from pericyte somas located at the edge of the endothelial wall, we could be assured that fluorescence changes in pericytes and not in endothelial cells were detected. In addition, because the endothelial cells of our isolated pericyte-containing microvessels were minimally loaded with fura-2, the contribution of endothelial fluorescence was minor. Autofluorescence was not detected in the microvessels. The fluorescence ratio (F340/380nm) was determined at 10-s intervals and subsequently converted to intracellular calcium concentration using a standard equation (8) in which Rmin and Rmax were determined by measuring the fluorescence ratio in fura-loaded pericytes located on freshly isolated microvessels exposed to the lactate-free perfusate (Table 1), which was supplemented with the calcium ionophore, ionomycin (5 µM), or which was modified by having 5 µM ionomycin, 3 mM EGTA, and no CaCl2.

The data points in Fig. 2A are the means of the difference between the maximal pericyte calcium concentrations measured during exposure to lactate and the average calcium concentration during the 24-s period immediately before lactate exposure. Because pericyte calcium levels tended to progressively increase when microvessels were exposed to perfusates that had been bubbled with nitrogen or that contained DMA, KB-R7943, or octanol, the lactate-induced changes in calcium concentration during exposure to these perfusates (Fig. 2C) were defined as the difference between the maximal calcium concentration detected during lactate exposure and the calculated basal calcium level, which was determined by interpolation of the calcium concentrations before and after lactate exposure; interpolated basal calcium concentrations were ≤150 nM.

Electrophysiology. A coverslip containing microvessels was placed in a recording chamber (volume = 1 ml), which was perfused (~1.5 ml/min) with solutions from a gravity-fed system using multiple reservoirs. Vessels were examined at x400 magnification with an inverted microscope equipped with phase-contrast optics. The perforated-patch configuration of the patch-clamp technique was used to monitor currents recorded from pericytes. The pipette solution consisted of 30 mM KCl, 20 mM NaCl, 65 mM K2SO4, 6 mM MgCl2, 10 mM Na-HEPES, 60 µg/ml amphotericin B, and 60 µg/ml nystatin at pH 7.4 with the osmolarity adjusted to 280 mosmol/l. The pipettes, which had resistances of ~5 M{Omega}, were mounted in the holder of a patch-clamp amplifier (model 3900A, Dagan, Minneapolis, MN); seals of ≥10 G{Omega} were made to the cell bodies of pericytes. As amphotericin/nystatin perforated the patch of membrane, the access resistance to the pericytes studied decreased to <25 M{Omega}. Currents were filtered at 1 kHz with a four-pole Bessel filter, digitally sampled at 250-µs intervals using a DigiData 1200B acquisition system (Axon Instruments), and stored by a computer equipped with pCLAMP and Origin (version 7, OriginLab, Northampton, MA) software for data analysis and graphics display. To determine the current-voltage relations, currents were evoked by voltage steps controlled with pCLAMP software (version 8.2, Axon Instruments) and measured as detailed (23). The zero-current potential was defined as the membrane potential. Adjustment for the calculated liquid junction potential (1) was made after data collection.

Because there are extensive gap junction pathways within retinal microvessels (20), currents monitored via a perforated-patch pipette sealed to a pericyte include not only the currents generated in the sampled pericyte but also currents transmitted electronically from neighboring vascular cells. Consistent with this, we previously reported that the amplitude of the current detected in a perforated-patch recording from a pericyte decreases by ~20-fold when gap junctions are uncoupled (14, 15, 20). Thus when gap junctions are open, it appears that ~95% of the current detected in a sampled pericyte is derived from its microvascular neighbors. As a result, pericyte recordings reflect the electrogenic activity of multiple microvascular cells.

Although the space clamp would be more controlled in short, rather than long, microvessels, 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 even though the voltage clamp of distantly coupled cells would be less than that of the sampled pericyte. However, despite space-clamp limitations, we previously found in studies of isolated retinal microvessels that the reversal potentials for ionic conductances closely matched the calculated equilibrium potentials (19, 23, 31, 32).

Chemicals. Unless otherwise noted, chemicals were obtained from Sigma (St. Louis, MO).

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


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lactate-induced pericyte contraction. To begin to test the hypothesis that lactate is a vasoactive signal in the retinal microvasculature, we used time-lapse photography to monitor the effect of this metabolic product on pericyte-containing microvessels freshly isolated from the adult rat retina. Consistent with lactate affecting pericyte function, an induced contraction of at least one pericyte was detected in 16 of the 31 microvessels monitored during exposure to 20 mM lactate; as detailed in MATERIALS AND METHODS, the sodium and chloride concentrations, as well as the osmolarity and pH, of the lactate-free and the lactate-containing perfusate were essentially the same. Even though vasoconstriction was induced by lactate in 52% of the microvessels, only a minority of the pericytes located on a responding microvessel contracted. Specifically, a contractile response was detected in 40 of the 274 (15%) pericytes monitored by time-lapse photography during exposure to 20 mM lactate (Fig. 1A); none of the observed pericytes relaxed.

In addition to facilitating the detection of changes in pericyte contractility, time-lapse photography also allowed us to quantify the effect of lactate on the diameter of microvascular lumens. A time-lapse movie of the effect of lactate on a pericyte-containing retinal microvessel is available online in the Supplemental Material to this study.1 In the microvessels in which lumens adjacent to contracting pericytes were in the focal plane of the differential interference contrast microscope, exposure to 20 mM lactate caused the lumen diameter to decrease significantly (P = 0.014), from 3.8 ± 0.6 to 2.1 ± 0.2 µm (Fig. 1B); vasoconstriction was initially detected 96 ± 7 s after the onset of lactate exposure. On the basis of our time-lapse studies, we concluded that extracellular lactate induces pericytes to contract and microvascular lumens to constrict.

Lactate-induced elevation of pericyte calcium. Because an increase in the intracellular calcium level can cause pericytes to contract (16, 32), we monitored the concentration of this divalent cation in pericytes located on retinal microvessels. As shown in Fig. 2A, extracellular lactate increased the pericyte calcium level in a dose-dependent manner. With exposure to 20 mM lactate, a rise in calcium level was detected in each of 28 monitored pericytes; an increase in pericyte calcium was initially detected 27 ± 1 s after the onset of lactate exposure. During sustained exposure to 20 mM lactate, the intracellular concentration of calcium initially increased to a peak and then declined toward the basal level (Fig. 2B). Consistent with the importance of calcium, lactate-induced contractions of pericytes were prevented (P = 0.007, Fisher’s exact test) by preincubating microvessels for 30 min in 10 µM BAPTA-AM, which is a cell-permeable calcium chelator; in fact, lactate caused pericytes to relax in the BAPTA-treated microvessels (Fig. 1A). From these experiments, we concluded that a rise in pericyte calcium is an essential step in the lactate-induced contraction of these mural cells. In addition, it appears that a decrease in pericyte calcium is not required for relaxation of these mural cells.

Lactate-induced hyperpolarization. Because many vasoactive molecules affect electrogenic processes in mural cells, we asked whether the membrane potential of pericytes was altered during exposure to extracellular lactate. In a series of 20 perforated-patch recordings, we found that 20 mM lactate induced a reversible increase in an outward current (Fig. 3, A and B) that significantly (P = 0.001) hyperpolarized pericytes from a resting membrane potential of –37 ± 1 to –46 ± 2 mV (Fig. 3C). Our finding that lactate has a hyperpolarizing effect strongly suggests that the voltage-dependent calcium channels expressed in pericyte-containing retinal microvessels (21, 23) do not play a significant role in mediating the lactate-induced rise in calcium and subsequent pericyte contraction.


Figure 3
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  		Fig. 3. Effect of lactate on currents and voltages monitored via the perforated-patch pipettes sealed to retinal pericytes. A: current traces before and during exposure to 20 mM lactate. The clamp protocol is shown at bottom. B: current-voltage (I-V) plots of the steady-state currents for the traces shown in A, as well as for the currents recorded from this same cell 5 min after cessation of lactate exposure (the darker line labeled "Recovery"). C: effect on the pericyte membrane potential of exposure to 20 mM lactate without or with 1 mM CHC, 30 µM DMA, 30 µM KB-R7943, and 1 mM ouabain. For each condition, 11 ± 5 pericytes were monitored. Before lactate exposure, membrane potentials were unaffected by CHC or DMA; KB-R7943 caused a 6.0 ± 0.6 mV (P = 0.006) hyperpolarization, and oubain caused a 5.2 ± 1.5 mV (P = 0.015) depolarization. Each additive significantly decreased the lactate-induced hyperpolarization; for KB-R7943, the P value was 0.05; for the other conditions, the P value was ≤0.001.

 
Role of monocarboxylate transporters. We suspected that an essential step in linking exposure to lactate with the contractile response of pericytes is the transport of this metabolic product into microvascular cells. Consistent with this, both the lactate-induced rise in pericyte calcium (Fig. 2C) and the contractile response of these mural cells (Fig. 1A) were significantly (P < 0.001) diminished in the presence of CHC (1 mM), which is a blocker of the monocarboxylate transporters (MCTs) that move lactate along with protons across cell membranes (9). In addition, we observed that CHC prevented lactate from significantly (P = 0.5) affecting the membrane potential of pericytes (Fig. 3C).

Role of Na+/H+ exchangers. Because of the fact that MCTs transport protons along with lactate, we postulated that Na+/H+ exchangers (NHEs) could be activated in response to an influx of H+ during exposure to lactate. Suggestive of the importance of NHEs, we observed that DMA (30 µM), which inhibits NHEs (5), markedly (P < 0.001) diminished the lactate-induced rise in pericyte calcium (Fig. 2C). DMA also prevented (P < 0.001, Fisher’s exact test) the lactate-induced contraction of these mural cells (Fig. 1A); in fact, in the presence of DMA, lactate caused pericytes to relax. These experiments suggest that the contraction of pericytes in response to extracellular lactate is dependent on the activation of MCTs and NHEs. Also, consistent with NHEs being activated by lactate, inhibition of these exchangers significantly (P ≤ 0.001) diminished the lactate-induced hyperpolarization induced by lactate (Fig. 3C).

Role of Na+/Ca2+ exchangers. Because neither MCTs nor NHEs directly regulate intracellular calcium, the elevation of pericyte calcium during lactate exposure must involve additional events. As noted, it seems unlikely that voltage-dependent calcium channels are involved because lactate caused pericytes to hyperpolarize. An alternative mechanism that we considered involved microvascular Na+/Ca2+ exchangers (NCXs); we postulated that an influx of sodium via NHEs could secondarily affect the activity of NCXs, which in the forward mode exchange intracellular calcium for extracellular sodium and in the reverse mode import calcium and export sodium. To help assess the role of NCXs, we used an electrophysiological method to quantify the effect of lactate on the activity of NCXs, which are electrogenic because of their exchanging 3 Na+ for each Ca2+ (11). In a series of perforated-patch recordings, we measured the current that was sensitive to KB-R7943, which at 30 µM is reported to inhibit both forward and reverse NCX function (17). As illustrated in Fig. 4, the amplitude of the current inhibited by KB-R7943 diminished significantly (P = 0.009) during exposure of microvessels to 20 mM lactate. This observation led us to conclude that exposure of pericyte-containing retinal microvessels to extracellular lactate results in NCX inhibition. Of note, because retinal microvessels have extensive gap junction pathways (15, 20), our electrophysiological experiments could not establish whether the NCXs inhibited by lactate were located in the sampled pericytes and/or in their microvascular neighbors with which they were interconnected via gap junctions.


Figure 4
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  		Fig. 4. Effect of lactate on the KB-R7943-sensitive currents. A: I-V plots of steady-state currents before and during exposure to 30 µM KB-R7943, which is an inhibitor of Na+/Ca2+ exchangers (NCXs). A, inset: clamp protocol and the current traces whose I-V plots are shown. B: I-V plots of steady-state currents during exposure to 20 mM lactate without and with 30 µM KB-R7943. B, inset: current traces from which the I-V plots were generated; the clamp protocol was the same as in A. C: plots of the difference between the I-V curves in A and B. D: mean amplitude of the current inhibited by KB-R7943 in the absence and presence of 20 mM lactate. Currents were measured at –58 mV. For the lactate-free and lactate-containing groups, 9 and 5 pericytes were monitored, respectively. *P = 0.009.

 
To begin to assess the consequences of NCX inhibition, we determined the reversal potential for the current that was sensitive to 30 µM KB-R7943. In our experiments, the KB-R7943-sensitive current reversed at –27 ± 2 mV (n = 11) in the absence of extracellular lactate and at –29 ± 3 mV (n = 5) during exposure to 20 mM lactate; these potentials were not significantly different (P = 0.6). Because the reversal potential of the KB-R7943-sensitive current was significantly (P < 0.001) depolarized relative to the observed resting membrane potential of –37 ± 1 mV, we concluded that in our experimental preparation, the microvascular NCXs generate an inward current and thus function in the "forward mode," i.e., sodium is moved into and calcium out of the microvascular cells. On the basis of this finding, we predicted that lactate’s inhibition of NCXs would decrease the efflux of calcium via this exchanger and thereby result in a rise in intracellular calcium levels. Consistent with this scenario, KB-R7943 significantly (P < 0.001) inhibited the lactate-induced elevation of pericyte calcium (Fig. 2C). In addition, we found that exposure of microvessels to KB-R7943 significantly (P < 0.001) diminished the lactate-induced contraction of pericytes (Fig. 1A). Thus it appears that the inhibition of NCXs is a critical step in the sequence of events linking lactate exposure with the contraction of pericytes.

With evidence suggesting that NCX inhibition plays a key role in the response to lactate, we considered the possibility that inhibition of this electrogenic exchanger fully accounts for the hyperpolarization that occurs during lactate exposure. However, inconsistent with this, KB-R7943 decreased the hyperpolarization induced by 20 mM lactate by only 36 ± 11% (P = 0.05, n = 5, Fig. 3C). This observation suggests that in addition to NCXs, there must be at least one other electrogenic component whose activity is modified during exposure to lactate. In addition, because KB-R7943 only modestly diminished the lactate-induced hyperpolarization (Fig. 3C) but inhibited the lactate-induced rise in calcium by a robust 88 ± 1% (Fig. 2C), it appears that there is not a direct relationship between the rise in pericyte calcium during lactate exposure and the induced hyperpolarization of these mural cells. For this reason, it seem unlikely that voltage-independent calcium-permeable channels, which increase calcium influx as the membrane potential increases, account for the lactate-induced elevation of pericyte calcium. Rather, our assessment of the effects of lactate on retinal microvessels supports the idea that the inhibition NCXs is the critical event that results in the elevation of intracellular calcium levels and the triggering of pericyte contraction.

Role of Na+-K+ pumps. Because of our evidence that NHEs are involved in the lactate-induced contraction of pericytes (Fig. 1), we hypothesized that the NHE-mediated influx of sodium not only affects NCX function but also may activate Na+-K+ pumps. In agreement with electrogenic Na+-K+ pumps being activated, exposure of retinal microvessels to lactate significantly (P = 0.01) increased the amplitude of the current that was sensitive to ouabain, which inhibits this pump (Fig. 5). Furthermore, ouabain (1 mM) significantly (P = 0.001) reduced the lactate-induced hyperpolarization of pericytes (Fig. 3C). Consistent with the lactate-induced hyperpolarization being dependent on lactate’s effect on both Na+-K+ pumps and NCXs, the membrane potential of pericytes was not significantly (P = 0.2) altered when microvessels were exposed to lactate in the presence of both ouabain and KB-R7943 (Fig. 3C).


Figure 5
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  		Fig. 5. Effect of lactate on the ouabain-sensitive current. A: I-V plots of steady-state currents in the absence of extracellular lactate before and during exposure to 1 mM ouabain, which is an inhibitor of Na+-K+ pumps. B: I-V plots of steady-state currents during exposure to 20 mM lactate without and with 1 mM ouabain. C: plots of the difference between the I-V curves in A and B. D: mean amplitude measured at –58 mV of the current inhibited by ouabain in the absence and presence of lactate. For the lactate-free and lactate-containing groups, 13 and 7 pericytes were monitored, respectively. *P = 0.01.

 
We also asked whether the activation of Na+-K+ pumps regulates the contractile response of pericytes to lactate. Suggestive of this possibility, ouabain significantly (P = 0.001, Fisher’s exact test) decreased the percentage of pericytes that contracted during exposure to extracellular lactate (Fig. 1A). Taken together our experiments indicate that MCTs, NHEs, NCXs, and Na+-K+ pumps are involved in linking the exposure to lactate with the contraction of pericytes.

Differing contractile responses of arterioles and pericyte-containing vessels. In this first study of lactate’s effect on pericyte-containing microvessels, we were surprised to find that this molecule induced mural cell contraction and, thereby, vasoconstriction. These observations were unexpected because extracellular lactate is known to cause dilation of the smooth muscle-encircled vessels of the retina (3). Consistent with previous reports, our time-lapse studies of freshly isolated retinal arterioles also demonstrated that lactate caused the myocytes of these vessels to relax; lactate-induced contractions were never observed in arterioles (Fig. 6).


Figure 6
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  		Fig. 6. Effect of the lactate concentration on the percentage of microvessels with an induced contractile response. In the group with pericyte-containing microvessels, each data point consisted of 9.9 ± 3.7 vessels that were monitored by time-lapse photograph. For each data point in the arteriole group, 12.5 ± 1.8 vessels were monitored. Of note, lactate induced contractions in the pericyte-containing microvessels but relaxations in smooth muscle-encircled arterioles. *P ≤ 0.01 (Fisher’s exact test).

 
To better characterize the differing contractile responses of arterioles and pericyte-containing microvessels to lactate, we determined the dose dependence of the effect of lactate on contractility of mural cells. As shown in Fig. 6, pericyte contractions were elicited at lactate concentrations that had no detectable effect on the contractile tone of vascular smooth muscle cells. For example, 2 mM lactate induced contractions in 6 of the 11 pericyte-containing microvessels monitored by time-lapse photography but had no effect on the 15 observed arterioles (P = 0.001, Fisher’s exact test). We concluded from these experiments that under our standard assay conditions, smooth muscle-encircled arterioles and pericyte-containing retinal microvessels have markedly different sensitivities to extracellular lactate.

Contractile response of hypoxic retinal microvessels to lactate. Because retinal hypoxia markedly increases lactate production and release (29), we tested the contractile effect of lactate on isolated microvessels that were made hypoxic. As shown in Fig. 7, 20 mM lactate caused most of the responding pericytes in hypoxic microvessels to relax, rather than to contract; this was significantly different (P = 0.003) from our findings with microvessels that were maintained under nonhypoxic conditions. Consistent with this difference in contractile response, the lactate-induced rise in pericyte calcium was significantly (P < 0.001) diminished in hypoxic microvessels (Fig. 2C).

The question arose as to whether hypoxia alters the response of smooth muscle-encircled arterioles to extracellular lactate. However, unlike pericyte-containing vessels, arterioles responded to lactate in a similar manner under both control and hypoxic conditions. Namely, during exposure to 40 mM lactate, myocytes relaxed in 60% of the hypoxic arterioles (n = 5) monitored by time-lapse photography; no smooth muscle contractions were detected in response to lactate. This was identical to the effect of this metabolic product on retinal arterioles that were maintained under our control conditions (Fig. 6). Taken together, our observations indicate that under hypoxic conditions, lactate induces mural cell relaxation throughout the retinal vasculature, from the arterioles to the capillaries.

Role of gap junctions. By what mechanism does hypoxia switch the lactate-induced response of pericytes from contraction to relaxation? Because hypoxia closes gap junctions in retinal microvessels (19), we considered the hypothesis that inhibition of cell-to-cell communication via these junctions plays a role in switching the pericyte response from contraction to relaxation. Consistent with this hypothesis, treatment of microvessels with the gap junction uncouplers octanol, {alpha}-GA, or Gap 27 switched (P < 0.001) the lactate-induced contractile response of pericytes from contraction to relaxation (Fig. 7). In agreement with this observation, chemical uncoupling of gap junctions markedly (P < 0.001) diminished the effect of lactate on pericyte calcium levels (Fig. 2C). Thus it appears that the lactate-induced rise in pericyte calcium depends on cell-to-cell communication via gap junctions. To account for our findings with gap junction uncouplers and our evidence that a change in NCX activity during lactate exposure accounts for the rise in pericyte calcium (Fig. 2C), we propose that the NCXs affected by lactate are predominantly located in the vascular endothelium rather than in the pericytes.

We also assessed the effect of the gap junction uncoupler octanol on freshly isolated smooth muscle-encircled retinal arterioles. In contrast to its effect on pericyte-containing microvessels, octanol did not alter the effect of lactate on arteriolar tone (P = 1.0). As under control conditions, 60% of the retinal arterioles (n = 5) that were pretreated with 1 mM octanol relaxed during exposure to 40 mM lactate; no contractions were observed. Thus, in contrast to pericyte-containing microvessels, we did not detect a role for gap junctions in mediating the contractile response of retinal arterioles to extracellular lactate.


   DISCUSSION
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In this study, which is the first to assess the effects of extracellular lactate on pericyte-containing microvessels, we demonstrated that this metabolic product can serve as a vasoconstrictor. Using vessels freshly isolated from the rat retina, we observed in pharmacological experiments that the contractile effect of lactate appears to require MCTs, NHEs, and NCXs. A parsimonious explanation for our experimental findings is that a cascade of interrelated events links lactate exposure with vasoconstriction. Putative events include the MCT-mediated importation of protons along with lactate and the secondary NHE-mediated efflux of protons in exchange for sodium. We propose that an NHE-mediated influx of sodium accounts for the observed activation of Na+-K+ pumps and inhibition of NCXs. Although Na+-K+ pumps have a modulatory effect on the lactate-induced contractile response of pericytes, our experiments suggest that they do not play an essential role. On the other hand, it appears that the inhibition of calcium efflux via NCXs is the critical step in the series of events linking exposure to lactate with the elevation of intracellular calcium and the subsequent contraction of pericytes.

Role of microvascular gap junctions. Unexpectedly, we found that the vasoconstriction induced by lactate in pericyte-containing microvessels not only involves MCTs, NHEs, Na+-K+ pumps, and NCXs but also is dependent on gap junctions. To account for the importance of these intercellular pathways, we hypothesize that the presence of endothelial/pericyte gap junctions allows NCXs that are located in the endothelium to play a role in the regulation of the concentration of calcium within pericytes. As a consequence of this transcellular regulation, a decrease in calcium extrusion via endothelial NCXs causes pericyte calcium to rise and contraction to be triggered.

Another likely consequence of the role of gap junctions is that the contractile response of retinal microvessels to lactate is metabolically modulated. Indicative of this, we observed that lactate caused pericytes to contract in isolated microvessels maintained under control conditions, but under hypoxic conditions, which are known to close microvascular gap junctions (20), these mural cells relaxed during exposure to lactate. Our working hypothesis is that the hypoxia-induced closure of gap junctions prevents the NCXs located in the endothelium from regulating calcium levels in pericytes. Consistent with this, we observed that the effect of lactate on pericyte calcium was minimized in hypoxic microvessels. Although the mechanism by which this metabolic product causes pericytes to relax when microvascular gap junctions are closed is not known, our experiments indicate that, as with smooth muscle cells (26), the relaxation of pericytes does not necessarily require calcium levels to decrease. Even though more needs to be learned, our experiments strongly indicate that in pericyte-containing retinal microvessels, lactate serves as a vasoconstrictor when energy supplies are ample but is a vasodilator under hypoxic conditions.

Functional heterogeneity of retinal pericytes. Because only ~15% of the pericytes monitored by time-lapse photography had a detectable contractile response to ≥20 mM lactate, it appears that these mural cells are functionally heterogeneous. This conclusion is consistent with our previous observations using other putative vasoactive molecules (14, 16, 32). Although we cannot exclude that our isolation procedure caused some pericytes to become noncontractile, it appears that not all of the pericytes that are capable of contracting do so in response to lactate because significantly (P < 0.0001, Fisher’s exact test) more, i.e., 37%, are triggered to contract by extracellular ATP (16). However, the failure of most pericytes to contract in response to lactate does not appear to be due to the absence of an induced increase in intracellular calcium because the concentration of this divalent cation rose in all of the pericytes monitored during exposure to ≥20 mM lactate. Perhaps the threshold at which a rising intracellular calcium concentration causes contraction varies among the population of retinal pericytes. At present, the functional significance of retinal microvessels having a significant population of pericytes that do not contract in response to a vasoactive signal remains to be established. We postulate that because constriction at a single site along a tube is sufficient to limit flow, it is not necessary for all pericytes to contract in order for perfusion in a microvessel to be decreased. In fact, having only a minority of pericytes contract in response to lactate may be an energy-efficient mechanism to regulate capillary blood flow.

Functional differences between pericyte-containing and smooth muscle-encircled vessels. This study revealed substantial differences between the effect of lactate on pericyte-containing and smooth muscle-encircled retinal vessels. One difference was that pericyte-containing microvessels were markedly more sensitive than arterioles to extracellular lactate. For example, 0.2 mM lactate induced a vasoconstriction in 50% of the pericyte-containing microvessels but did not affect the contractile tone of myocytes in arterioles (P < 0.001). Although the basis for this difference remains to be established, one possibility is that capillaries express MCTs, the affinity of which for lactate is high, whereas retinal arterioles chiefly possess lower affinity MCTs.

In addition to there being a difference in lactate sensitivity, our experiments also revealed a difference in the vasomotor response of capillaries and arterioles to extracellular lactate. Under control conditions, lactate caused pericytes to contract but smooth muscle cells to relax. Because gap junctions appear to play a key role in the lactate-induced contraction of pericytes, the differing contractile responses of capillaries and arterioles may be a result of endothelial-to-mural cell communication being more effective in pericyte-containing microvessels than in smooth muscle-encircled vessels. Perhaps the relatively small size of pericytes compared with vascular smooth muscle cells accounts for the greater effectiveness of endothelial NCXs in the regulation of calcium levels in the mural cells of capillaries. Despite the need for more studies to clarify the underlying basis for these newly identified differences, our observations refute the traditional notion that pericytes and vascular smooth muscle cells respond identically to extracellular signals. Clearly, there is functional heterogeneity within the retinal vasculature.

What are the implications of capillaries and arterioles having different sensitivities and contractile responses to lactate? Our findings lead us to propose that lactate released from well-oxygenated retinal cells is a signal to selectively diminish local capillary perfusion. By this mechanism, blood flow may be shunted away from a locale that has a sufficient supply of energy. On the other hand, when hypoxia causes retinal cells to produce and release markedly greater amounts of lactate (29), the relaxation induced in both arterioles and capillaries by this metabolic product results in vasodilation throughout the vascular tree and, consequently, increased delivery of oxygen and nutrients. A more complete appreciation of the physiological differences between smooth muscle-encircled and pericyte-containing retinal vessels should result in a better understanding of how the vasculature of the retina functions to meet the metabolic needs of retinal neurons.

Caveats and conclusions. Our conclusions concerning the effects of lactate on the retinal microvasculature are based on experiments using freshly isolated vessels. One benefit of studying vessels in isolation is that confounding effects mediated by nonvascular retinal cells are eliminated. Also, in contrast to cultured mural cells, isolated vessels permit the study of pericytes as integral components of a multicellular microvascular unit. This advantage is important because our study indicates that the functional status of gap junctions is a critical factor in determining whether pericytes contract or relax in response to extracellular lactate. Yet, despite the advantages of our preparation, caution must be exercised. For example, it remains to be confirmed that the contractile effect of lactate in vivo involves MCTs, NHEs, NCXs, Na+-K+ pumps, and gap junctions, although at present an in vivo application of the imaging and electrophysiological techniques used in this study does not appear to be feasible. Another caveat is that our isolated microvessels were not internally perfused. As a consequence, possible effects of blood flow on vascular reactivity and intracellular signaling cascades remain unknown. In addition, a future challenge is to use nonpharmacological methods to more directly demonstrate that extracellular lactate affects the activities of various transporters, exchangers, and pumps, as well as gap junctions. Also, determination of the specific PO2 level at which gap junctions uncouple will be helpful to more fully assess the vasomotor response of the retinal microvasculature under hypoxic conditions that are less severe than those used in this first study of lactate’s effect on pericyte-containing microvessels. However, despite these caveats, freshly isolated vessels provide an experimental preparation to test new hypotheses concerning the mechanisms by which extracellular lactate regulates vascular function in the retina.

In summary, the results of this study indicate that the response of pericytes to extracellular lactate depends on a cascade of events involving MCTs, NHEs, Na+-K+ pumps, and NCXs. In addition, the functional status of endothelial/pericyte gap junctions appears to be critical in determining whether pericytes contract or relax during exposure to lactate. When gap junctions are opened, lactate causes pericytes to contract. Conversely, lactate induces these mural cells to relax when microvascular gap junctions are closed, as occurs with hypoxia (20). Thus it appears that lactate serves as a dual-action vasoactive signal. The dynamic nature of the contractile response of pericytes to this metabolic product is likely to provide an efficient mechanism to adjust microvascular function to match local metabolic needs.


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 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This project was supported by National Institutes of Health Grants EY-12505 and EY-07003 and a Senior Investigator Award from Research to Prevent Blindness.


   ACKNOWLEDGMENTS
 
We thank Bret Hughes for use of his calcium imaging equipment and Kenji Matsushita and Sophie Liao for helpful comments on earlier drafts of this manuscript.


   FOOTNOTES
 

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.

1 A time-lapse movie, included as Supplemental Material for this paper, is available online at http://ajpheart.physiology.org/cgi/content/full/01012.2005/DC1. The time-lapse movie shows a freshly isolated pericyte-containing retinal microvessel before, during, and after exposure to extracellular lactate. Differential interference contrast images were captured at 8-s intervals for a period of 30 min. This movie speeds up events by ~225-fold. Vasoconstriction occurred soon after the onset of exposure to lactate. The frames labeled "lactate" indicate when this pericyte-containing microvessel was exposed to 20 mM lactate. Arrows point to the region of maximal vasoconstriction. Back


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