Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca2+ responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca2+]i within the SMCs, and the frequency of these Ca2+ oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca2+ oscillations that occurred with low frequencies and were preceded by small, localized transient Ca2+ events. The 5-HT–induced Ca2+ oscillations and contractions occurred in the absence of extracellular Ca2+ and were resistant to Ni2+ and nifedipine but were abolished by caffeine. KCl-induced Ca2+ oscillations and contractions were abolished by the absence of extracellular Ca2+ and the presence of Ni2+, nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT2–specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT2 receptors, induces arteriole contraction by initiating Ca2+ oscillations and that KCl induces contraction via Ca2+ transients resulting from the overfilling of internal Ca2+ stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca2+ oscillations and also by the relaxation rate of the SMC.
Pulmonary hypertension (PH) is characterized by a progressive elevation of pulmonary arterial pressure and pulmonary vascular resistance, which results in heart failure. The increase in pulmonary resistance results from vasconstriction and/or structural changes, including smooth muscle cell (SMC) hypertrophy and hyperplasia. The etiology of primary PH is poorly understood, whereas secondary PH usually results from hypoxia associated with respiratory disease or high altitude breathing (Farber and Loscalzo, 2004). The pulmonary artery distributes blood to the alveoli via intrapulmonary arteries and arterioles that follow the branching pattern of the airways. However, along the pulmonary artery, the phenotype and physiology of the SMCs can vary, and in different regions, SMCs may respond differently to vasoactive factors (Frid et al., 1997). Blood returns to the heart via larger veins that follow a route distinct from that of arterioles and airways. Consequently, an understanding of the physiological factors that regulate the resistance of the pulmonary circulation is necessary to identify the pathological basis of hypertension.
The contractile mechanisms of the pulmonary arteries have been generally investigated in SMCs isolated from large extrapulmonary arteries (Barnes and Liu, 1995). While these studies provided valuable information, it is difficult to know how well the responses of SMCs of the pulmonary artery reflect the physiology of small intrapulmonary arterioles. Therefore, we examined the responses of small intrapulmonary arterioles in lung slices to correlate the Ca2+ signaling of the SMCs with the contraction. These arterioles were studied by modifying our lung slice preparation used with confocal microscopy (Bergner and Sanderson, 2002a,b, 2003), by temporarily filling the arterioles with gelatin. While we simultaneously examined the contractile responses of both the bronchioles and arterioles, we report here the responses of the arterioles. An accompanying paper reports the airway responses (Perez and Sanderson, 2005).
5-HT is a potent vasoconstrictor of pulmonary blood vessels in animals and humans (MacLean et al., 2000) and is thought to contribute to the development of primary PH and secondary PH mediated by hypoxia. Secondary PH may also be induced by amphetamine-derived appetite suppressants that act as 5-HT agonists (Eddahibi et al., 2002). Consequently, we investigated the contractile and [Ca2+]i responses of arteriole SMCs to 5-HT. In addition, because membrane potential is important in contractility of other blood vessels, we examined the responses of intrapulmonary arterioles to KCl. We found that both 5-HT and KCl induced Ca2+ oscillations in the SMCs and that these Ca2+ oscillations were responsible for sustaining contraction. The frequencies of these Ca2+ oscillations were slower than those found in airway SMCs yet the contractile responses were larger. To resolve these data, we suggest that the contraction of the SMC is determined by the frequency of the Ca2+ oscillations as well as by the relaxation rate of the SMC; excessive contraction may therefore arise from a change in the Ca2+ signaling or relaxation rate of the SMCs.
Materials And Methods
All the materials and methods used have been previously described in detail (Perez and Sanderson, 2005); only a brief outline is given here. Reagents were obtained either from Invitrogen and GIBCO BRL or Sigma-Aldrich. Hanks' balanced salt solution was supplemented with 20 mM HEPES buffer (sHBSS). A KCl salt solution (K+-sHBSS) was prepared by substituting the sodium salts with potassium counterparts. High KCl isotonic solutions were prepared by mixing K+-sHBSS with normal sHBSS. Hanks' “0” Ca2+ solution was prepared by supplementing sHBSS without Ca2+ and Mg2+ with 0.9 mM MgSO4 and 1 mM Na2H2-EGTA.
Lung slices were prepared from BALB/C inbred mice between 7 and 9 wk old. The trachea was cannulated with an intravenous catheter, and after opening the chest cavity, the collapsed lungs were reinflated with ∼1.5 ml of air. A warm (37°C) solution of gelatin (type A, porcine skin, 300 bloom, 6% in sHBSS) was perfused through the intrapulmonary blood vessels, via the pulmonary artery. The lungs were deflated and refilled with a warm solution of 2% agarose (low gelling temperature) in sHBSS. Subsequently, a small volume of air was injected to flush the agarose-sHBSS out of the airways and into the distal alveolar space. The warm agar and the gelatin were gelled with cold sHBSS. A single lung lobe was removed and cut in serial sections of ∼130 μm thickness with a vibratome at ∼4°C starting at the lung periphery. Serum was not used because it contains 5-HT and growth factors (Abdullah et al., 1994). Slices were maintained in growth media at 37°C and 10% CO2 for up to 3 d. At 37°C, the gelatin in the blood vessel lumen dissolved, leaving the blood vessel lumen empty.
Measurement of the Contractile Response of Airways and Arterioles Induced by Agonists
Only arterioles which had not collapsed were used. Lung slices were mounted in a custom-made perfusion chamber and held in place with a small sheet of nylon mesh. A second coverglass edged with silicone grease was placed over the lung slice. Perfusion of the lung slice was performed by a gravity-fed perfusion system. The volume of the chamber was ∼100 μl with a perfusion rate of 800 μl/min. For phase-contrast microscopy, lung slice was observed with an inverted microscope with a 20× objective, and images were recorded using a CCD camera and image acquisition software (Video Savant). Digital images were recorded in time lapse (0.5 Hz) and stored on hard drive. The area of the airway and arteriole lumen was calculated, with respect to time, by pixel summing. Experiments were performed at room temperature.
Measurements of Intracellular Ca2+
Slices were incubated in 20 μM Oregon green 488 BAPTA-1, AM, 100 μM sulfobromophthalein (an inhibitor used to prevent dye extrusion through anion exchangers) and 0.2% pluronic F-127 for 30 min at 30°C followed by 30 min at room temperature and by an additional hour at room temperature in sHBSS containing 100 μM sulfobromophthalein. Lung slices were mounted in the perfusion chamber as described. Fluorescence imaging was performed using a video-rate confocal microscope (Sanderson and Parker, 2003; Sanderson, 2004). In brief, a 488-nm laser was used as the excitation wavelength. The resultant fluorescence (>510 nm) was detected by a photomultiplier tube and a frame capture board to form an image that was recorded to a hard disk. Most recordings were made in time lapse at 2 Hz. Changes in fluorescence intensity were analyzed by selecting regions of interest (ROIs) of ∼5 × 5 pixels. Average fluorescence intensities of an ROI were obtained, frame-by-frame, using the Scion Image software with custom written macros that allow tracking of the ROI within an SMC as it moved with contraction. Final fluorescence values were expressed as a fluorescence ratio (Ft/F0) normalized to the initial fluorescence (F0). For the higher speed recordings of Ca2+ waves and elemental Ca2+ events, images were acquired at 60 Hz (480 pixels × 170 lines). A line-scan analysis of these images was performed by extracting a row of pixels from each image and placing them sequentially, as a time sequence, in a single image. Paired Student's t tests were used to test for significant differences between means. All statistical values are expressed as mean ± SEM.
Online Supplemental Material
Videos (Videos 1–3) consisting of sequences of phase-contrast or fluorescence images were produced with Video Savant. Labels indicate the experimental conditions and the playback speed.
Characteristics and Morphology of Lung Slices
Because the intrapulmonary arteries are anatomically associated with the airways and follow a parallel course through the lungs, an airway and arteriole were readily identified in a lung slice (Fig. 1 A). A detailed description of the morphology of the slice preparation is given in a previous paper (Perez and Sanderson, 2005, Fig. 1). In lung slices, only the alveoli remain filled with agarose; the gelatin was removed from the arterioles by dissolving it at 37°C.
The Contractile Response of Arteriole–Bronchiole pairs to 5-HT, KCl, and ACH
The contractile response of arterioles and bronchioles to 5-HT, high KCl, and ACH were initially compared. In response to 1 μM 5-HT, the arteriole quickly contracted (Fig. 1, A and B, red line; Video 1), and this was followed by a slower contraction that reduced the arteriole size by 75 ± 6% after 5 min (8 slices from 3 mice). In response to 1 μM 5-HT, the airway reduced its lumen area by 41 ± 5% after 5 min (Fig. 1, A and B, blue line). Although the initial contraction rates induced by 5-HT in the arteriole and airway were similar, it is important to note that upon 5-HT removal, the relaxation of the arteriole occurred more slowly than that of the airway (Fig. 1, B and C). This differential relaxation rate appears to be related to the SMC type because it was evident at different sizes of contraction and was not correlated with the agonist inducing contraction (5-HT and ACH for airways or 5-HT and KCl for arterioles, see below). However, these differences in relaxation were not observed in response to a transient contraction induced by caffeine (6 slices from 3 mice). In response to 100 mM KCl, the arteriole displayed a large contraction (64 ± 8%) while the airway displayed only a small contraction (14 ± 3%, after 5 min, n = 5 slices from 3 mice). In each case, these contractions were irregular or spasmodic (Video 1). This was especially evident in the airway where the SMCs displayed uncoordinated twitching (Fig. 1 B, blue line). Removal of the KCl, again, resulted in a slow rate of arteriole relaxation. Perfusion with 1 μM ACH had no effect on the arteriole but induced the airway to contract by 57 ± 7% within 5 min (n = 8 slices from 3 mice) (Fig. 1 B; Video 1). Although the arterioles could be deformed by the contraction of the neighboring airway, this stretching did not induce arteriole SMC contraction. Perfusion of lung slices that had been precontacted (with 1 μM 5-HT) with 1 μM ACH did not alter arteriole contraction, but did result in a further contraction of the airway.
From these results, it is clear that the SMCs of arterioles and airways respond differently to different agonists and that the arterioles are not responsive to stretch. Because we characterized the response of the airways in a preceding paper, we focus here on characterizing the contractile and underling Ca2+ responses of arteriole SMCs to 5-HT and KCl and compare them to the responses of the airways.
Concentration Dependence of Arteriole Contraction to 5-HT and KCl
Increasing concentrations of 5-HT (from 0.01 to 0.5 μM) induced an increasing reduction in luminal size (Fig. 2, A and C). 1 μM 5-HT induced a maximal contraction of ∼77 ± 15% (n = 5 from 3 mice). ACH did not induce contraction in arterioles at any concentration tested (Fig. 2 C, n = 6 from 3 mice), even though stimulation of the same arteriole with 5-HT induced ∼80% reduction in size (n = 4 from 3 mice). Exposure to increasing concentrations of KCl (from 25 to 100 mM) induced increasing reductions in arteriole area (Fig. 2, B and D). At 100 mM KCl, a maximal contraction of 48 ± 4% (n = 4 from 3 mice) was achieved.
Type of 5-HT Receptor Involved in Arteriole Contraction
Because 5-HT2 and 5-HT3 receptors are coupled to increases in [Ca2+]i, antagonists and agonists for these receptors were used to identify the receptor type mediating arteriole contraction. Ketanserin, a 5-HT2–specific antagonist, at 10−8 M completely blocked the contraction induced by 5-HT (3 arterioles from 2 mice). In addition, the sequential stimulation with 10−8 to 10−6 M DOI (2, 5-dimethoxy-4-iodoamphetamine hydrochloride), a 5-HT2 specific agonist, induced a concentration-dependent arteriole contraction (3 arterioles from 2 mice). However, SR 57227 (4-amino-1-(6-chloro-2-pyridyl)-piperidine hydrochloride), a 5-HT3 agonist, at concentrations ranging from 10−7 through 10−5 M, did not induce contraction, although control stimulations with 5-HT induced contraction of the same arterioles (3 arterioles from 2 mice). These results suggest that the 5-HT2 receptor is mediating 5-HT responses in intrapulmonary arterioles.
Direct Action of KCl on Arteriole SMCs
To determine if KCl-induced arteriole contraction acts directly or indirectly by stimulating the release of 5-HT or nor-epinephrine from nerve terminals, we studied the effects of ketanserin (a 5-HT2 antagonist) and prazosin (an α1-adrenoreceptor antagonist) on contraction induced by KCl. Neither ketanserin nor prazosin had any effect on the contraction induced by KCl (3 arterioles from 2 mice). Similarly, neither ACH nor phenylephrine (PE) induced arteriole contraction. These results suggest that KCl acts directly on SMCs and not via the release of ACH, 5-HT, or nor-epinephrine.
Ca2+ Signaling in Arteriole SMCs Induced by 5-HT
In response to 1 μM 5-HT, the arteriole SMCs responded with an increase in [Ca2+]i followed by the onset of oscillations in [Ca2+]i (Fig. 3, A and B). These Ca2+ oscillations occurred asynchronously, with each cell displaying repetitive increases in [Ca2+]i at different times with respect to neighboring SMCs (Fig. 3 C). Individual Ca2+ oscillations occurred as waves propagating throughout the length of the arteriole SMCs (Video 2; Fig. 9). The increase in [Ca2+]i in the SMCs was accompanied by the contraction of the arteriole (Fig. 3 A; Video 2).
In the range of 0.01 to 1 μM, 5-HT induced a concentration-dependent increase in the frequency of the Ca2+ oscillations in arteriole SMCs (Fig. 4). At 0.01 μM, 5-HT induced a frequency of 0.6 ± 0.2 cycles min−1 (n = 7 cells from 3 slices from 2 mice) while at 1 μM, 5-HT induced a frequency of 6 ± 0.5 cycles min−1 (n = 13 cells from 6 slices from 3 mice) (Fig. 4 G).
Ca2+ Signaling in Arteriole SMCs Induced by KCl
High concentrations of KCl also induced increases in [Ca2+]i and Ca2+ oscillations in arteriole SMCs (Fig. 5; Video 3). However, the KCl-induced Ca2+ changes occurred more slowly than those induced by 5-HT, and an initial transient spike was often absent (Fig. 5 A). The frequency of the Ca2+ oscillations induced by 25–100 mM KCl was low (one to two per minute), and each Ca2+ transient had a long duration (19.1 ± 6.2 s). The KCl-induced Ca2+ oscillations of neighboring cells were asynchronous and could occur at different frequencies (Fig. 5 B). The sequential stimulation of the same slice with 1 μM 5-HT induced Ca2+ oscillations with a frequency (6.5 ± 0.3 cycles min−1, n = 4, 2 slices from 2 mice) that was significantly higher than those induced by 100 mM KCl in the same SMC (1.2 ± 0.2 cycles min−1; n = 5, 5 slices from 3 mice; P < 0.05, t test). However, there were no significant differences in the magnitude of the [Ca2+]i increases. Similar results were attained when the order of 5-HT and KCl application was reversed (P < 0.05, t test, n = 4 cells from 2 slices from 2 mice).
Role of Extracellular Ca2+ in SMC Contraction and Ca2+ Signaling
In the absence of extracellular Ca2+, 5-HT induced contraction of the arteriole to the same extent as in the presence of extracellular Ca2+ (Fig. 6 A; P < 0.01, calculated at 1 min after stimulation, n = 5 from 3 mice). In addition, the Ca2+ channel blockers NiCl2 or nifedipine were unable to inhibit the onset of the contraction induced by 5-HT (Fig. 6, B and C). By contrast, the absence of extracellular Ca2+ or the presence of NiCl2 or nifedipine completely abolished the contraction induced by KCl (Fig. 6, D–F). While effect of Ni2+ was reversible, the effect of nifedipine was not, a result consistent with a high affinity binding site for 1,4-dihydropyridines to transmembrane segments of the α1 subunits of L-type Ca2+ channels (Striessnig et al., 1998). These results indicate that extracellular Ca2+ or Ca2+ influx was not necessary to trigger the contraction induced by 5-HT, but was required to mediate contraction induced by KCl. However, after 3 min of 5-HT stimulation, in the absence of external Ca2+ or presence of NiCl2 or nifedipine, the normal secondary slow rate of contraction was arrested and, in some cases, the arteriole began to relax (Fig. 6, A–C).
In both the presence or absence of extracellular Ca2+, 5-HT induced an initial increase in [Ca2+]i followed by Ca2+ oscillations (Fig. 7). Although the Ca2+ oscillations persisted with an approximately constant frequency during the presence of the agonist and extracellular Ca2+ (Fig. 7, A and B), the oscillations slowed and ceased within 3 min in the absence of external Ca2+ (Fig. 7, C and D).
The ability of 5-HT to induce Ca2+ oscillations in the absence of external Ca2+ correlates with its capacity to initiate arteriole contraction in the absence of external Ca2+ (Fig. 6 A). Similarly, the inhibition of the Ca2+ oscillations after 3 min in the absence of Ca2+ correlates with the inhibition of the slow-rate contraction and the initiation of relaxation of the arteriole. In the absence of extracellular Ca2+, KCl did not stimulate any change in the [Ca2+]i (n = 3 arterioles from 2 mice), a result that correlates with the inability of KCl to induce arteriole contraction under these conditions (Fig. 6 D).
Effect of Caffeine on Arteriole Contraction Induced by 5-HT and KCl
To characterize the contribution of the intracellular Ca2+ stores and RyRs in the contraction of arterioles, we examined the effects of caffeine on the contractile response induced by 5-HT and KCl. In arteriole SMCs, caffeine induced a transient increase in [Ca2+]i that subsided to a sustained elevation in Ca2+, which itself returned to the prestimulatory level after the caffeine was removed (Fig. 8 A). The sustained elevation of Ca2+ was quickly abolished when extracellular Ca2+ was withdrawn (Fig. 8 B). In the absence of extracellular Ca2+, caffeine induced a transient Ca2+ increase without a sustained plateau (Fig. 8 C). However the readdition of external Ca2+ resulted in a sustained increase in Ca2+ (Fig. 8 C). In response to repeated exposures to caffeine, arterioles elicited single transient contractions that quickly relaxed to the prestimulatory state within a minute (Fig. 8 D). The response to both 5-HT and KCl was reversibly abolished by caffeine (Fig. 8, E and D). These results indicate that caffeine stimulates the release of Ca2+ from internal stores and stimulates a Ca2+ influx across the plasma membrane of arteriole SMCs. However, the sustained elevation of Ca2+ induced by caffeine does not appear to directly participate in the contractile response of arterioles. While these results are compatible with the expected result that a release of intracellular Ca2+ was necessary to trigger and sustain the contraction induced by 5-HT, they also highlight a significant dependence of the effects of KCl on internal stores.
Ca2+ Waves and Elemental Ca2+ Events in Arteriole SMCs Induced by 5-HT and KCl
To compare the spatio-temporal properties of the Ca2+ oscillations induced by 5-HT and KCl in arteriole SMCs, fast (60 fps) recordings of single SMCs were performed during continuous stimulation with each agonist. For analysis, line-scan plots from the longitudinal axes of single SMCs were made; these show approximately evenly spaced diagonal lines that represent the Ca2+ oscillations induced by 5-HT that consist of repetitive Ca2+ waves which propagated along the longitudinal axes of the cell (Fig. 9). While the wave velocity (slope of the lines) could change in magnitude and sometimes in direction, the average 5-HT–induced Ca2+ wave velocity was 21 ± 3 μm/s. No other changes in [Ca2+]i were observed in the period between two successive Ca2+ waves.
By contrast, KCl-induced Ca2+ oscillations appeared as longer lasting Ca2+ waves or tides. However, the mean wave propagation velocity (16 ± 3 μm/s) was similar to that observed with 5-HT. A unique feature of KCl-induced Ca2+ waves was that they were preceded by small, localized, and transient Ca2+ increases (elemental Ca2+ events) that did not spread throughout the whole cell (Fig. 9, arrows). These elemental Ca2+ events were initiated from one or more specific localizations in a cell with an average frequency of 8 ± 2 per min (5 cells from 3 slices from 2 mice). However the frequency of these events increased up to the time when the next Ca2+ wave was initiated. After each Ca2+ wave had subsided, a refractory period occurred before the Ca2+ events started to occur again. Ca2+ waves were usually initiated from one location showing the highest frequency of Ca2+ events. Each elemental Ca2+ event occurred with a diameter ∼4 μm, had a rise-to-peak time of 70 ± 30 ms, and a first order exponential decay rate of 385 ± 122 ms (Fig. 9, inset a, n = 4 cells from 3 slices from 2 mice). Ca2+ waves and elemental Ca2+ events were not observed during KCl stimulation in the presence of caffeine.
We used a lung slice preparation to study the contractile responses of intrapulmonary arterioles and the underlying changes in [Ca2+]i in their SMCs during stimulation with 5-HT and KCl. For the same reasons that apply to the study of bronchial airways (Bergner and Sanderson, 2002a), thin lung slices are well suited for the study of arterioles. The arterioles are easily identified, have reproducible contractile responses and the intracellular Ca2+ responses of their SMCs can be correlated with the contraction of the arteriole. In addition, the small intrapulmonary arterioles, at sites that are considered to be important in pulmonary hypertension, can be examined. A major advantage of the lung slice is the ability to simultaneously compare the responses of arteriole SMCs to those of airway SMCs. This allows for the instant collection of control data and facilitates an understanding of the specific physiological responses of each SMC type.
Vasoconstriction of pulmonary arteries to 5-HT has been observed in most animal species, including mice and humans (MacLean et al., 2000). In mouse lung slices, we found that 5-HT induced a concentration-dependent contraction of arterioles over the range of 0.01–1 μM with an EC50 of ∼50 nM. A similar sensitivity to 5-HT was observed in intrapulmonary artery rings from mice (EC50 = 80 nM) (Liu and Folz, 2004). In humans, the sensitivity of intrapulmonary arteries is reported to be slightly lower in intact (EC50 = 257 nM; MacLean et al., 1996) or endothelium-denuded intrapulmonary rings (390 nM; Cortijo et al., 1997). Differences in the sensitivity to 5-HT of intrapulmonary arteries between the mouse and human could be due to differences in the type(s) of 5-HT receptor expressed in each species.
We found here that arteriole contraction stimulated by 5-HT was associated with an initial increase in [Ca2+]i and fast Ca2+ oscillations followed by a period where the Ca2+ oscillations persisted with a slower but more constant frequency. Like the Ca2+ oscillations of airway SMCs, the oscillations spread along the cell as Ca2+ waves. The Ca2+ oscillations most probably result from repetitive cycles of Ca2+ release and reuptake by intracellular stores because Ca2+ oscillations could be initiated in absence of extracellular Ca2+ or in presence of Ca2+ channel blockers. However, the eventual cessation of the Ca2+ oscillations in the absence of extracellular Ca2+ suggests a need for some extracellular Ca2+ influx.
In view of the robust Ca2+ oscillation that we observed in intrapulmonary arterioles, it is surprising that Ca2+ oscillations were not observed by Guibert et al. (2004), who investigated 5-HT–induced Ca2+ signaling in segments of cannulated and pressurized rat intrapulmonary arteries. A reason for this difference, as the authors conceded, was probably the low spatio-temporal resolution of the Ca2+ measurements. Wide-field microscopy was used to observe a multicellular tissue with a sampling rate of one image/20 s. It is likely that the small increases in [Ca2+]i induced by increasing concentrations of 5-HT that correlated with contraction (Guibert et al., 2004) resulted from an averaging of increasing frequencies of asynchronous Ca2+ oscillation in multiple SMCs. Therefore, the interpretation that a sustained contraction of SMCs results from a sustained increase in [Ca2+]i induced by a constant influx of Ca2+ through receptor-operated channels should be reevaluated. Because pharmacological evidence supported the existence of receptor-operated channels in intrapulmonary arteries (Guibert et al., 2004), we suggest that these may be involved in refilling Ca2+ store to maintain the Ca2+ oscillations. The reliance on Ca2+ oscillations rather than sustained increases in [Ca2+]i to maintain contraction is also a way to avoid toxic effects of elevated [Ca2+]i (Savineau and Marthan, 2000; Lee et al., 2002).
The Ca2+ oscillations induced by 5-HT in lung arterioles were asynchronous between neighboring cells at all 5-HT concentrations. A similar finding is reported for rat mesenteric artery with PE-induced Ca2+ oscillations (Mauban et al., 2001). These observations are different to those reported for rat mesenteric arteries (Lamboley et al., 2003) where synchronization of the Ca2+ oscillations and recruitment of SMCs showing Ca2+ oscillations was observed with increasing concentrations of the PE. In contrast to our observations of both airway and arteriole SMCs, the sustained contraction of mesenteric arteries was observed only at high concentrations of PE and correlated with the recruitment and the synchronization of the Ca2+ oscillations (Lamboley et al., 2003). We have not observed any synchronization or substantial recruitment of cells with increasing agonist concentrations in either arteriole or airway SMCs. It is important to note that in lung arteriole and airway SMCs, as well as in rat mesenteric arteries (Mauban et al., 2001), each Ca2+ oscillation observed during 5-HT stimulation did not generate a transient contraction, but that the SMCs maintained a steady contractile state at all concentrations of 5-HT. This has the important implication that the contractile elements of the cell serve to integrate the stimuli encoded in the Ca2+ oscillations. Although, it has been reported that SMCs of a number of tissues, including airways (Kuo et al., 2003) and blood vessels (Christ et al., 1996), are electrically coupled, we have not observed Ca2+ waves spreading to adjacent cells in arterioles or airways. One explanation for this is that the concentration of inositol 1,4,5-trisphosphate (IP3) (a permeable messenger) would be expected to be similar in neighboring cells during global stimulation with the agonists.
In similarity to airway SMCs, the frequency of the Ca2+ oscillations and the extent of contraction induced by 5-HT in arterioles was concentration dependent. However, the frequency of the Ca2+ oscillations observed in airways was, in general, higher (∼20 or 30 cycles/min with maximal concentrations of 5-HT or ACH, respectively) than those observed in arterioles during 5-HT stimulation (approximately six cycles/min with maximal concentrations). Other systemic blood vessels have also been observed to have a concentration-dependent increase in contraction and Ca2+ oscillation frequency when stimulated with a variety of agonists (Lee et al., 2002). For example, in the rabbit inferior vena cava, PE ranging from 0.15 to 150 μM increased the frequency of Ca2+ oscillations from 3 to 30 cycles/min and the extent of contraction (Ruehlmann et al., 2000). PE was also found to stimulate a concentration-dependent increase in Ca2+ oscillations and contraction in rat mesenteric arteries (Mauban et al., 2001; Lamboley et al., 2003). This correlation between contraction and Ca2+ oscillation frequency suggests that the size of arteriole contraction is regulated by frequency modulation (FM) of the [Ca2+]i (Berridge et al., 2003) in a similar manner to that found for airway contraction.
The most likely mechanism for 5-HT–induced arteriole contraction and [Ca2+]i signaling is that 5-HT binds to a 5-HT2 receptor coupled through Gq protein to PLC (Hoyer et al., 2002). Activation of PLC generates IP3 to initiate the release of Ca2+ from the SR through IP3 receptors. Persistent oscillations in [Ca2+]i are produced by continuous cycles of release and reuptake of Ca2+ from internal stores. Support for this mechanism is provided by the fact that contraction was inhibited by ketanserin, a 5-HT2 receptor blocker and stimulated by DOI, a 5-HT2 receptor agonist. While 5-HT3 receptors can elevate [Ca2+]i, via a Ca2+ influx, 5-HT–induced increases in [Ca2+]i in the absence of extracellular Ca2+ and the 5-HT3 receptor agonist SR 57227 did not stimulate the arteriole contraction. It has also been suggested that the 5-HT1B/5-HT1D receptors participate in the contraction of intrapulmonary arteries in humans (MacLean et al., 1996; Cortijo et al., 1997; Morecroft et al., 1999), mouse (Keegan et al., 2001; Liu and Folz, 2004), and other species (Shaw et al., 2000; Murdoch et al., 2003). However, these receptors are coupled to Gi/o to produce a decrease in cAMP (Hoyer et al., 2002), and we believe that these receptors are not the major pathway for 5-HT–induced Ca2+ oscillations in arteriole SMCs.
While 5-HT appears to be a potent vasoconstrictor of pulmonary arterioles, the arterioles did not contract or relax in response to ACH at any concentration that produced contraction of airway SMCs. This suggests that the cholinergic pathway is not involved in the contractile response of the arterioles. In addition, arterioles precontracted with 5-HT did not relax in response to ACH. In other vascular SMCs, ACH induced relaxation via stimulation of the endothelial cells. However, the failure to observe this response in lung slices with constant perfusion could be explained by the fact that any factors released by endothelial cells would be washed away or by an inability of the cells to respond to ACH. To resolve these issues, further studies of the role of the endothelial cells in lung slices is required.
The mechanism by which KCl triggered Ca2+ oscillations in arteriole SMCs was dependent on extracellular Ca2+ influx, sensitive to NiCl2 and nifedipine, blocked by caffeine and characterized by Ca2+ waves occurring at very low frequencies. In contrast to 5-HT–induced Ca2+ waves, KCl-induced Ca2+ waves were preceded by small, localized Ca2+ increases or elemental Ca2+ events. As a result, we believe that KCl-induced membrane depolarization resulted in the activation of voltage-gated Ca2+ channels and that this, in turn, led to an influx of Ca2+. However, instead of directly inducing SMC contraction, this additional Ca2+ is loaded into the SR. As the Ca2+ load within the internal store increases, the chance that Ca2+ is released from the SR, through RyRs, is increased. After reaching a critical threshold, the elemental Ca2+ release is amplified by CICR to produce a long-lasting Ca2+ wave that results in contraction of the SMCs and the arteriole. Ca2+ waves induced by KCl did not occur synchronously in adjacent cells, which suggests that they do not result from cycles of depolarization and hyperpolarization in electrical coupled cells. This explanation for KCl-induced Ca2+ oscillations in arteriole SMCs is identical for airway SMCs. These responses to KCl also highlights the need for caution when assuming that sustained contraction results directly from increases in Ca2+ induced by depolarization.
The alternative hypothesis that KCl acts indirectly via the depolarization of neural terminals to locally release neurotransmitters (Lamont et al., 2003) does not appear to be applicable in lung slices. Most importantly, the arterioles did not respond to PE or ACH. Similarly, prazosin or ketanserin had no effect on the contraction induced by KCl. In addition, our experiments were performed with constant perfusion that would quickly wash away any endogenously released transmitters, yet the KCl effects persisted for longer periods and show similar responses during repetitive stimulations.
Probably, the most important implication of our results is that a sustained contraction of intrapulmonary arteriole SMCs is maintained and regulated by the frequency of Ca2+ oscillations rather than by a sustained Ca2+ elevation. We have reached an identical conclusion for the regulation of airway SMCs contraction. In addition, other recent studies using confocal Ca2+ imaging of nonintrapulmonary blood vessels or airways have observed Ca2+ oscillations rather than sustained elevations in Ca2+ during stimulation with agonists (Lee et al., 2002; Li et al., 2003; Wier and Morgan, 2003).
An FM regulation hypothesis can explain the contraction of either the arteriole or airway SMCs, but the FM relationship of contraction to Ca2+ oscillation frequency is very different between airway and arteriole SMCs. For example, the size of arteriole contraction (∼70%) induced by slow Ca2+ oscillations (six per minute) stimulated by 5-HT was often greater than the contraction induced in airways (∼50%) by much faster oscillations (15–20 per minute) with the same agonist (Perez and Sanderson, 2005). To explain this difference, we hypothesize that the contractile state is determined, not only by the frequency of the oscillations but, also by the relaxation rate of the SMC. In this hypothesis, the rate at which SMCs develop Ca2+-dependent contraction is similar but the rate of relaxation from the contracted state serves to integrate the contractile response. A variation in this rate between SMC types will determine the frequency of the Ca2+ oscillations required to sustain contraction; a slow or fast relaxation rate will require low or high frequency Ca2+ oscillations, respectively.
The relaxation rate of airway SMCs appears to be faster than that of arteriole SMCs under all conditions. After the removal of 5-HT, the airways relax quicker than the arterioles. While SMC relaxation occurs following the cessation of the Ca2+ oscillations, the period between the stoppage of the Ca2+ oscillations and relaxation is shorter in airways. Under extracellular Ca2+-free conditions, the Ca2+ oscillations of both the airway and arteriole SMC rundown and the airway relax immediately but the arterioles only just began to relax after 5 min. Another major clue is presented by the twitching response of the SMCs in response to slow Ca2+ oscillations induced by KCl (Perez and Sanderson, 2005). The contractile state of the airway SMCs closely followed the changes in [Ca2+]i, indicating that the Ca2+-induced contraction can only be maintained for a short duration. By contrast, arteriole SMCs show modest twitching to the KCl-induced Ca2+ oscillations and maintained a substantial contraction. Thus, while both airway and arteriole SMCs quickly contract in response to Ca2+, arteriole SMCs sustain longer contractions to each Ca2+ pulse than the airway SMCs. Therefore, for airway SMCs to maintain contraction, the time interval between Ca2+ pulses must be short; a prediction that matches the fast frequency of Ca2+ oscillation observed in airway SMCs (Perez and Sanderson, 2005). Conversely, arterioles only require slow frequency Ca2+ oscillations to maintain contraction.
The immediate question raised by this differential relaxation hypothesis is the nature of the difference between the two SMC types. One possibility is that slower Ca2+ dynamics result in higher resting Ca2+ levels to delay relaxation. However, the base line of the Ca2+ oscillation does not seem to be correlated with the contractile state. Similarly, there is little difference between the duration of each Ca2+ oscillation or the basal Ca2+ level of the Ca2+ oscillations induced by KCl in airways and arterioles, yet the relaxation of the airways is faster. Thus, the relaxation time does not seem to be directly related to [Ca2+]i. To explain the different relaxation times, we speculate that the dissociation kinetics of dephosphorylated, but attached, actin–myosin crossbridges that are formed during actin–myosin cycling to produce contraction (Mijailovich et al., 2000) is slower in arteriole SMCs than in airway SMCs. This idea is supported by simulated contractions predicted by mathematical modeling of crossbridge formation in a Hai-Murphy four-state model (Hai and Murphy, 1989) in response to Ca2+ oscillations, but this idea will require further investigation.
In conclusion, intrapulmonary arterioles respond to 5-HT with a sustained contraction that is maintained by persistent and asynchronous Ca2+ oscillations of the associated SMCs. These Ca2+ oscillations are generated and propagated as waves by repetitive cycles of Ca2+ release and reuptake by the SR and require extracellular Ca2+ for store refilling. The size of the contraction of arterioles is regulated by the frequency of the Ca2+ oscillations in SMCs rather than by the amplitude of a sustained [Ca2+]i increase. We hypothesize that the relaxation kinetics of the SMC serves to integrate the Ca2+-dependent contractile response of the SMC. One implication of this hypothesis is that hyperactivity can result from a change in the relaxation kinetics of the SMC as well as from a change in the Ca2+ signaling of the SMC.
This work was supported by the National Institutes of Health grant HL71930 to M.J. Sanderson.
Lawrence G. Palmer served as editor.
Abbreviations used in this paper: FM, frequency modulation; 5-HT, 5-hydroxytrypamine; IP3, inositol 1,4,5-trisphosphate; PE, phenylephrine; PH, pulmonary hypertension; ROI, region of interest; SMC, smooth muscle cell.