Brief, spatially localized Ca2+ transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these “junctional Ca2+ transients” (jCaTs) and postulated that they arose from Ca2+ entering smooth muscle cells through P2X1 receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X1-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca2+ entering the smooth muscle cell via P2X1 receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and α-latrotoxin (2.5 nM) markedly increased the frequency of “spontaneous” jCaTs. In the KO arteries, however, neither EFS nor α-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, α,β-methylene ATP (10.0 μM), elicited strong contractions and increased intracellular Ca2+ concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca2+ that enters the smooth muscle cells through P2X1 receptors activated by neurally released ATP and that this Ca2+ is involved in the initial rapid component of the sympathetic neurogenic contraction.
- arterial smooth muscle
- adrenergic and adenosine 5′-triphosphate
at the varicosities present along the terminals of sympathetic nerves in small arteries, three neurotransmitters are released: 1) norepinephrine (NE), 2) ATP, and 3) neuropeptide Y. It has been shown recently that “all three co-transmitters contribute significantly to vascular responses (contraction) and their contribution varies markedly with impulse numbers” (2). The relative contribution of the purinergic and NE components is dependent on the artery and the parameters of stimulation. NE is a powerful activator of neurogenic vasoconstriction, whereas neurally released neuropeptide Y may be modulatory or primarily trophic (7). Neurally released ATP activates an initial, rapid, and transient component of vascular contraction. Neurally released ATP also activates rapid, brief postjunctional membrane potential depolarization (excitatory junction potentials). Thus it has been postulated that sympathetically released ATP “may be important for redistribution of cardiac output during the” fight or flight “response, when blood flow is rapidly and preferentially directed to the heart and skeletal muscle as a result of vasoconstriction in kidneys and visceral organs” (24). This is thought to occur because neurally released ATP plays a greater role in activating contraction of proximal small arteries of cutaneous, mesenteric, and renal circulations than in small arteries from skeletal muscle and because, in the arteries in which ATP is important, sympathetic neurogenic contractions rise very much more rapidly than in those in which it is not (24).
In earlier studies, we showed novel spatially localized Ca2+ signals in the smooth muscle adjacent to perivascular nerves (14, 15). These were not attributable to NE, since they persisted in the presence of α1-adrenoceptor blockade (prazosin). They were abolished by the purinergic receptor blocker suramin. We called these Ca2+ transients “junctional Ca2+ transients” (jCaTs) and attributed them to neurally released ATP, but the particular purinergic receptor type involved was unknown. It is known that the P2X1 subtype of purinergic receptor is the predominant P2X receptor in vascular smooth muscle (6), and P2X1 is known to participate in mediating the neurotransmitter actions of ATP in vascular smooth muscle (17). Because P2X subtype-selective drugs are not available, P2X1-receptor-deficient mice have been developed (18). Using P2X1-receptor-deficient mice, Vial and Evans (27) demonstrated that homomeric P2X1 receptors underlie the artery smooth muscle phenotype and contribute ∼50% of the sympathetic neurogenic contraction (100 pulses at 10 Hz). In the present study, we used P2X1-receptor-deficient mice to test the hypothesis that P2X1 receptors are the integral inotropic purinergic receptors that underlie the jCaTs observed in arterial smooth muscle, and we examined some of the functions of these receptors in vascular contraction.
MATERIALS AND METHODS
Preparation of arteries and experimental solutions.
All experiments were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Adult wild-type (WT, +/+) and P2X1-receptor-deficient (KO, −/−) mice (18) were killed by lethal dose of CO2 and cervical dislocation. The mesenteric arcade was dissected from the abdominal cavity, rinsed free of blood, and placed in a temperature-controlled dissection chamber containing dissection solution (5°C) of the following composition (in mmol/l): 3.0 MOPS, 145.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 0.02 EDTA, 2.0 pyruvate, and 5.0 glucose (pH 7.4). Small arteries were dissected by methods described in detail previously for rat arteries (8). Segments of second-order arteries, 1–2 mm in length, were loaded with a calcium indicator, fluo-4. The average resting diameter for the WT arteries was 237.2 ± 8.8 μm (n = 19) and for the KO arteries was 243.2 ± 6.4 μm (n = 20). There was no statistically significant difference in the size of the two groups of arteries. The indicator in the “cell-permeant” acetoxymethyl ester form was dissolved in dissection solution: 5 μmol/l fluo-4/AM, 1.5% DMSO (vol/vol), and 0.03% Cremophor EL (vol/vol). Loading was allowed to proceed for 3 h at room temperature. The arteries loaded with fluo-4 were transferred to a recording chamber where the ends of the arteries were mounted on glass pipettes (tip diameter 60–100 μm) and secured by 10-0 sutures. One pipette was attached to a servo-controlled pressure-regulating device (Living Systems, Burlington, VT); the other was attached to a closed stopcock, to study the pressure-dependent effects in the absence of intraluminal flow. The intraluminal pressure was set to 30 mmHg. The arteries were equilibrated for over 1 h to initial experimental conditions (22–25°C). Physiological saline solution was a Krebs solution containing (contents in mmol/l) 112.0 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11.5 glucose, and 10.0 HEPES (pH 7.4), gassed with 5% CO2-5% O2-90% N2. Chamber Po2 was 90–100 mmHg, measured with an oxygen electrode (Microelectrodes; Londonderry, NH). Electrical field stimulation (EFS) was performed via two platinum wires placed in the bath parallel to the long axis of the artery and connected to a stimulator (model S48, Astro-Med). The intraluminal pressure was set to 30 mmHg; this pressure is below that at which myogenic tone develops in mice mesenteric arteries (11). Working at 30 mmHg allows us to make observations over a wider range of sympathetic nerve stimulation than would be possible without substantial movement at higher intraluminal pressures. However, even at 30 mmHg, the intensity of the EFS has to be kept at a low level to prevent movement. For the confocal experiments, the parameters of EFS were frequency 0.5–10 Hz, pulse duration 0.1 ms, and intensity 15–25 V; this was subthreshold for contraction. For the diameter experiments, the EFS frequency range was 2–16 Hz, duration was 0.1–0.2 ms, and intensity was 25–60 V. TTX (1.0 μM) was used to confirm that the responses produced were of neurogenic origin and not produced by direct electrical stimulation of smooth muscle cells. All drugs and chemicals were obtained from Sigma Chemical, except fluo-4/AM which was purchased from Molecular Probes and, α-latrotoxin and ryanodine from Calbiochem.
Image collection and data analysis.
For two-dimensional confocal imaging, we used a “real-time” confocal imaging system (Solamere Technology Group, Salt Lake City, UT) consisting of a Yokogawa confocal scanner (model CSU10) and an intensified charge-coupled device camera (model XR/Mega-10). This produced 30 images/s. Images of 75 × 50 μm were collected. The confocal imaging system utilized a Nikon inverted microscope equipped with a water objective lens (×60; numerical aperture 1.2).
Images were analyzed with the use of custom software written with Interactive Data Language (Research Systems). This software was used to obtain average fluorescence from areas of interest (AOIs) within the images. Fluorescence “pseudo-ratios” (F/Fo) were then constructed by dividing the average fluorescence (F) within the AOI, at each frame, by the average fluorescence during the first 2 s of the recording (Fo), before EFS began. Sites where Ca2+ transients arose within the smooth muscle cells were identified with the use of a computer program that allowed us to place an AOI in the region of a local change in fluorescence and to then interactively position the AOI such that the change was largest. The pseudo-ratio within the AOI was then calculated. The AOIs were 0.8 by 0.8 μm. The time of occurrence was defined as the first point at which the rate of change of the ratio exceeded 4.8 F/Fo s−1 (with an additional criterion for correction of noise). The peak value was obtained by fitting the next 10 points (330 ms) with a fourth-order polynomial and taking the maximum value. The half-time of the Ca2+ transients was obtained by fitting the data with a fourth-order polynomial and determining the time at which the F/Fo reached the value halfway between the peak value and the value before the transient. Transients where this value was not reached were discarded. Data are presented as means ± SE.
In WT mouse arteries, the potent P2X-receptor agonist α,β-methylene ATP (10 μM) evokes transient contractions as described previously in the rat (9) (Fig. 1A). At 10.0 μM, α,β-methylene ATP [a maximally effective dose (27)] evoked constriction to 0.578 ± 0.0164 of resting diameter (n = 6). These strong constrictions were associated with transient, spatially uniform increases in intracellular Ca2+ concentration ([Ca2+]i) in individual smooth muscle cells when viewed with the confocal fluorescence microscope (data not shown). In contrast, in the P2X1-receptor-deficient mice, contractile responses to P2X receptor agonists (10.0 μM α,β-methylene ATP) were not present (Fig. 1B). Similarly, there was no change in [Ca2+]i in the smooth muscle cells. This confirms previous work (27) that the P2X1-receptor subtype mediates responses to P2X-receptor agonists in arterial smooth muscle. The lack of response to α,β-methylene ATP in the P2X1-null arteries did not indicate impairment of their contractile function because they contracted in response to high-potassium solutions. The maximum constriction produced by potassium (100 mM) was 0.410 ± 0.0157 (n = 4) and 0.390 ± 0.0214 (n = 4) for WT and KO arteries, respectively. The potassium concentration at which half of this maximal constriction was reached was 25.57 ± 1.972 mM for WT and 26.71 ± 0.927 mM for KO arteries. There was no statistically significantly difference between the two groups in terms of either maximum constriction or half-maximal constriction to potassium and to the α1-adrenergic agonist phenylephrine (PE, 10.0 μM, Fig. 1D), similar to previous reports (27). The maximum constriction produced by PE (10 μM) was 0.307 ± 0.0111 (n = 6) and 0.288 ± 0.0078 (n = 6) for WT and KO arteries, respectively. There was no statistically significant difference between the two groups.
The sympathetic neurogenic contraction in response to trains of stimuli (EFS pulses) is normally biphasic (Fig. 2A) (5, 14, 19, 22, 24). In WT arteries, an initial, rapid, transient vasoconstriction, peaking and falling in the first 10 s, was followed by a slower further constriction during EFS at 2, 4, 8, and 16 Hz for 1 min (Fig. 2A). In the KO arteries, however (Fig. 2B), the initial rapid phase of constriction was absent. For the purpose of analysis of the differences between the neurogenic contractions of WT and KO arteries, the peak value reached in the first 10 s of stimulation and the average value for the last 10 s of a 1-min stimulation period were determined for constriction at each frequency of stimulation (Fig. 2, C–E; 6 WT and 6 KO arteries). The 1-min trains of EFS we used are not physiological but allowed time for the second phase of the neurogenic contraction to develop.
The data show that the rapid initial, transient neurogenic vasoconstriction is dependent on P2X1 receptors. We next used the selective α1-adrenoceptor antagonist prazosin to determine what component of the neurogenic contraction is dependent on α1-adrenoceptors (α1-AR). In WT arteries, the adrenergic antagonist prazosin (1 μM) had no significant effect on the first peak of the sympathetic neurogenic contraction at 16 Hz [100.29 ± 20.68% of control (n = 6)], whereas the amplitude of the steady-state neurogenic contraction was reduced significantly to 56.8 ± 5.63% (n = 6, P < 0.05) (see Fig. 3A). This steady-state constriction is similar to whose we have shown previously in rats (14). In P2X1-receptor-deficient arteries, however, α1-adrenoceptor blockade (0.1–1.0 μM prazosin) abolished the response to EFS (10 arteries) (Fig. 3B).
We conclude from these experiments, under the conditions used (pressurized arteries at 30 mmHg, 22–25°C, EFS up to 16 Hz), that the sympathetic neurogenic vasoconstriction is entirely dependent on functional P2X1 receptors and α1-AR. P2X1 underlies a rapid initial component, whereas α1-AR underlies a more slowly developing component. Although activation of Y1 receptors by neuropeptide Y (which is coreleased with ATP and NE) can cause strong vasoconstriction (4, 21), these receptors are evidently not involved in activating sympathetic neurogenic contractions.
The ability of sympathetic nerves to elicit Ca2+ transients in the arterial smooth muscle cells was investigated by using real-time confocal fluorescence microscopy. To examine specifically the effects of neurally released ATP and the role of P2X1 receptors, the experiments were performed in the presence of 1.0 μM prazosin to eliminate the contributions of α1-ARs. The focal plane was set on the superficial layer of smooth muscle cells adjacent to the perivascular nerves. We have previously shown in rat that the putative P2X1-receptor-dependent Ca2+ transients, jCaTs, arise in close proximity to perivascular nerves (14). In WT arteries, jCaTs similar to those described previously in rat were triggered by nerve stimulation (Fig. 4A; 40 × 20 μm section of a confocal image is shown). In the example, EFS (3 Hz) occurred between 10 and 20 s of the 30-s sample. Examples of data taken from AOIs within this image are shown in Fig. 4B. In this case, there were four sites (labeled 0-3) and seven events during EFS. The characteristics of the Ca2+ transients (time of onset, peak ratio, and Ca2+ transient half-life) were determined as outlined in Image collection and data analysis. The probability of occurrence of the evoked Ca2+ transients rose rapidly to a peak in the first 3 s, then declined to nearly baseline levels by 10 s (Fig. 4C; EFS, 3 Hz for 10 s, from 10 to 30 s of the recording; 6 WT arteries, images were 75 × 50 μm). No similar Ca2+ transients were detected in P2X1-receptor-deficient arteries (Fig. 4, D–F). The Ca2+ transients detectable in the P2X1-receptor-deficient arteries were Ca2+ sparks, as determined by analysis of their time course, and the effects of ryanodine (Fig. 5 and experiments described below).
The time course of the probability of occurrence of jCaTs during EFS appears to be appropriate to account for the initial rapid P2X1-receptor-dependent vasoconstriction identified above. Next we examined the effects of EFS frequency on jCaT frequency in WT and P2X1-receptor-deficient arteries. The intensity of the EFS was set to a level where there was a minimal amount of movement at the highest frequency examined (15–25 V). For all P2X1-receptor-deficient arteries, a voltage of 25 V was used. These data are shown in Fig. 5C. The diamond symbols represent the data from WT arteries and the stars represent the data from P2X1-receptor-deficient arteries. There is a clear increase in the number of events because the frequency of the EFS increases for WT arteries, whereas the Ca2+-sparks observed in KO mice were unaffected by the frequency of the EFS.
Data from four arteries (2 WT and 2 KO) exposed to TTX (1.0 μM) are also presented in Fig. 5C (× symbols). TTX eliminated the jCaTs elicited by EFS in WT arteries, leaving only a few Ca2+ sparks. For two WT arteries, the peak ratio of Ca2+ transients (F/Fo) was 2.214 ± 0.056 and the Ca2+ transient half-life was 0.204 ± 0.0150 s (86 transients, 2 arteries) before TTX application and after application of TTX, the peak ratio (F/Fo) was 1.760 ± 0.023 and the Ca2+ transient half-life was 0.091 ± 0.007 s (77 transients, 2 arteries). The differences in the Ca2+ transient half-life and peak ratio of Ca2+ transients were both statistically significantly different (P ≤ 0.001, Mann-Whitney rank sum test). For 2 KO arteries, the peak ratio of Ca2+ transients (F/Fo) was 2.074 ± 0.089 and the Ca2+ transient half-life was 0.113 ± 0.0182 s (23 transients, 2 arteries) before TTX application, and after application of TTX, the peak ratio (F/Fo) was 1.995 ± 0.071 and the Ca2+ transient half-life was 0.106 ± 0.006 s (36 transients, 2 arteries). The differences in the Ca2+ transient half-life and peak ratio of Ca2+ transients were not statistically significantly different.
The experiments described above showed that P2X1 receptors are essential for jCaTs but did not determine whether other molecules, such as ryanodine receptors, might be involved. We have previously shown that, to render ryanodine, which is consistently and quickly effective to deplete the sarcoplasmic reticulum (SR) of Ca2+ in rat mesenteric artery preparations, the use of caffeine is required (16). In the presence of ryanodine (40 μM), a brief exposure to caffeine (20 mM, 1 min) caused a transient contraction of the arteries and rendered them unaffected by caffeine for the remainder of the experiment; that is, no further contractions or Ca2+ transients could be evoked by caffeine, indicating depletion of the SR. In two ryanodine-treated WT arteries, jCaTs were still present, elicited by EFS as before ryanodine treatment. Their characteristics are shown graphically in Fig. 5, A and B. These Ca2+ transients were statistically significantly different (Mann-Whitney rank sum test) from those arteries that had not been treated with ryanodine. The peaks of the Ca2+ transients were significantly smaller (P < 0.05, F/Fo 1.970 ± 0.042, 42 events, 6 records, 2 arteries, relative to F/Fo 2.252 ± 0.038, 216 events, 12 records, 6 arteries, all with 10 s of 3-Hz EFS) and the Ca2+ transient half-lives were significantly slower (P < 0.01 0.234 ± 0.017 s, 42 events, 6 records, 2 arteries, relative to 0.195 ± 0.008 s, 216 events, 12 records, 6 arteries) than in control arteries. This reduction in size of the jCaTs presumably reflects a small contribution from SR Ca2+ release to the peak of the Ca2+ transient that is removed by the functional removal of the SR by ryanodine treatment. The slowing of the Ca2+ transients presumably reflects a SR contribution to Ca2+ removal from the cytoplasm. These transients were eliminated by the application of 1.0 μM TTX. In two P2X1-receptor-deficient arteries treated with ryanodine, no Ca2+ transients were detected. This suggests that the small Ca2+ transients in P2X1-receptor-deficient arteries were, in fact, Ca2+ sparks.
Finally, to confirm that the small Ca2+ transients remaining in P2X1-receptor-deficient arteries were indeed Ca2+ sparks, we examined the effects of α-latrotoxin, which evokes exocytotic release of neurotransmitters from a variety of nerve terminals but which should have no effect on Ca2+ sparks. The effect of 2.5 nM α-latrotoxin was examined in six arteries, three from WT mice and three from KO mice. These arteries were also treated with prazosin (1.0 μM), capsaicin (1.0 μM), and scopolamine (1.0 μM) to eliminate the effects of other neurotransmitters potentially released by α-latrotoxin. In the WT arteries, there were 1.143 ± 0.553 spontaneous events/30 s, which rose significantly to 9.909 ± 0.553 events/30 s in the presence of 2.5 nM α-latrotoxin (t-test, P < 0.05). There was no significant change in the frequency of Ca2+ sparks in the KO arteries (3.300 ± 1.012 events/30 s in control, 3.142 ± 1.933, n = 3 in the presence of 2.5 nM α-latrotoxin). In the KO arteries, there was no statistically significant difference between the transients observed before and after application of α-latrotoxin. Peak ratio (F/Fo) was 1.682 ± 0.0452 and Ca2+ transient half-life was 0.0951 ± 0.00788 s (n = 25) before α-latrotoxin application, and in the presence of α-latrotoxin, the peak ratio (F/Fo) was 1.574 ± 0.048 and Ca2+ transient half-life was 0.0935 ± 0.015 s (n = 21). The transients evoked by α-latrotoxin in the WT arteries had a peak ratio (F/Fo) of 1.914 ± 0.0628 and a Ca2+ transient half-life of 0.151 ± 0.0241 s (n = 95).
We (27) showed previously that P2X1 receptors underlie an important component of brief sympathetic neurogenic contractions of small arteries. Here we show directly that the P2X1 receptors are required to generate the Ca2+ transients (jCaTs) that activate this component of the neurogenic contraction; in P2X1-null mice, no jCaTs were ever observed.
The sympathetic neurogenic contraction in response to trains of stimuli is normally biphasic, and the initial rapid component is thought to be purinergic in origin, with the later maintained component thought to be primarily adrenergic in origin (5, 14, 19, 22, 24). In the present study, therefore, we used periods of EFS at a range of frequencies in WT and KO arteries to determine the influence of the P2X1-receptor component over a longer period of time. Physiologically sympathetic neuronal activity consists of brief bursts of action potentials separated by periods of quiescence, not the stochastic trains of impulses used in most physiological experiments (12). When physiological irregular patterns of stimulation are used, the contractile responses are significantly greater than when regular stimulation trains are used (20). For simplicity in comparing the responses of the P2X1-null mice with those of WT mice, we have used simple 1-min trains of regular pulses at a range of frequencies. The mouse WT arteries showed a biphasic constriction similar to that shown previously in arteries from other species and tissues (Fig. 2A). In WT arteries, the initial first peak of constriction was unaffected by prazosin, indicating no involvement of α1-adrenoceptors, whereas the maintained constriction was significantly reduced (Fig. 2A). In P2X1-null arteries, the initial first peak of vasoconstrictions was absent (Fig. 2B compared with Fig. 2A), whereas the later maintained component was abolished by prazosin, indicating the involvement of α1-adrenoceptors. Interestingly, adrenergic component in the P2X1-null arteries seemed to develop more slowly than in the WT arteries, perhaps reflecting the loss of some facilitatory effect (such as depolarization or Ca2+ entry) of the purinergic component.
As shown previously in rat arteries, jCaT frequency was greatest at the start of EFS, then fell to a lower level as the stimulation continued (Fig. 4, B and C). The decline in the frequency of jCaTs during maintained stimulation likely accounts for the transient nature of the purinergic component of the contraction and is most probably due to the decline in probability of release of ATP. The decline in ATP release could be either a decline in the number of ATP-containing synaptic vesicles (1, 23) or in the total number of vesicles released if the NE and ATP are coreleased (3). Overflow of ATP from the junction is transient (26), like the purinergic constriction and the time course of jCaT probability (Fig. 4C), suggesting that reduction in release rather than desensitization of the receptors is responsible for the transient nature of the contraction.
The mesenteric small arteries we studied here are part of the splanchnic circulation, which has a significant role in the regulation of systemic blood pressure (13). At rest, this circulation receives ∼60% of the cardiac output, contains about one-third of the total blood volume, and is the single largest reservoir of blood available to the circulatory system. Sympathetic nervous system activity, during the well-known fight or flight response, increases peripheral vascular resistance and mobilizes up to two-thirds of the reserve blood in the veins of the splanchnic circulation. Thus the purinergic component of this sympathetic vasoconstriction may be particularly important for initiating rapid redistribution of cardiac output to the heart and skeletal muscle, away from splanchnic, cutaneous, and renal circulations. Finally, pharmacological purinergic blockade in conscious rats did not change the mean arterial pressure but did markedly increase blood pressure variation (10, 25), suggesting a role for purinergic neuromuscular transmission in control of blood pressure also in the basal state.
The work was funded by grants from the National Heart, Lung, and Blood Institute (R01-HL-073094) and the Wellcome Trust.
We thank R. M. Saunders for assistance with the care and genotyping of the mice.
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.
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