Purinergic and adrenergic Ca2+ transients during neurogenic contractions of rat mesenteric small arteries

Abstract

Contraction of small arteries is regulated by the sympathetic nervous system, but the Ca2+ transients during neurally stimulated contraction of intact small arteries have not yet been recorded. We loaded rat mesenteric small arteries with the fluorescent Ca2+ indicator fluo-4 and mounted them in a myograph that permitted simultaneous (i) high-speed confocal imaging of fluorescence from individual smooth muscle cells, (ii) electrical stimulation of perivascular nerves, and (iii) recording of isometric tension. Sympathetic neuromuscular transmission was achieved by electrical field stimulation (EFS) (frequency, 10 Hz; pulse voltage, 40 V; pulse duration, 0.2 ms) in the presence of capsaicin and scopolamine (to inhibit 'sensory' and cholinergic nerves, respectively). During the first 20 s of EFS, force rose to a small peak and then declined. During this time, junctional Ca2+ transients (jCaTs) were present at relatively high frequency. We have previously attributed jCaTs to influx of Ca2+ through post-junctional P2X receptors activated by ATP. Propagating asynchronous Ca2+ waves, previously associated with bath-applied alpha1-adrenoceptor agonists, were not initially present. During the next 2.5 min of EFS, force rose slowly, and asynchronous propagating Ca2+ waves appeared. The selective alpha1-adrenoceptor antagonist prazosin abolished both the slowly developing contraction and the Ca2+ waves, but reduced the initial transient contraction by only ~25 %. During 3 min of EFS in prazosin, the frequency of jCaTs declined markedly; at sites at which at least one jCaT occurred, the average probability of a jCaT was 0.008 +/- 0.002 pulse-1 in the first 20 s and 0.0007 +/- 0.0002 pulse-1 in the last 20 s. We suggest that (i) ATP released from sympathetic varicosities activates the initial, transient, contraction and the activator Ca2+ is derived largely from jCaTs, and (ii) sympathetically released noradrenaline (NA) activates the later, major contraction through mechanisms involving alpha1-adrenoceptors and which are associated with propagating Ca2+ waves.

Figure 4. Characteristics of Ca²⁺ waves during the predominantly adrenergic component of neurogenic contraction

A, recordings of force (upper trace) and fluorescence pseudo-ratio (lower trace) from a representative single cell during 3 min of EFS. B, frequency histogram of the times of occurrence of Ca²⁺ waves in 809 waves from 74 cells in one artery. C, scatter plot of peak pseudo-fluorescence ratio as a function of time of occurrence of the Ca²⁺ waves. Bar indicates the period of EFS. A video sequence, obtained at 2 images s⁻¹, during 3 min of EFS is available as Supplementary material (DOI:10.1113/jphysiol.2003. 043380).

Figure 2. Characteristics of junctional Ca²⁺ transients (jCaTs) during EFS in the presence of prazosin

Upper trace in A, force under control conditions. Lower trace in A, force in the presence of prazosin (10 μm). Bars below the force records indicate the times during which imaging was performed and during which the data illustrated in B and C were obtained. Ba, averaged image (30 frames) with representative AOIs shown from which fluorescence pseudo-ratios in were obtained. Bb, representative jCaTs recorded during the first 20 s of EFS from the three AOIs shown in Ba. Three jCaTs are marked (‘i’, ‘ii’ and ‘iii’). Ca–c, scatter plots of the peak fluorescence ratios of jCaTs, as a function of their time of occurrence, obtained during the three periods of time indicated by the bars in A; 0–20 s, 80–100 s and 160–180s. Different symbols represent data from different arteries. Cd–f, frequency histograms of jCaTs during the same periods. Bin size; 1 s. Data in C obtained from 5 arteries, consisting of 549 jCaTs. Mean values of peak fluorescence ratio (F/F₀) are: 2.033 ± 0.0261 (n = 408), 1.751 ± 0.040 (n = 90), and 1.782 ± 0.0648 (n = 51), in a, b, and c, respectively. Upper trace in D is force during 80 s EFS. Lower trace in D is average fluorescence from an entire image, such as that shown above, in Ba. Shaded bar indicates the period of EFS. Abrupt small increases in fluorescence are the upstrokes of individual jCaTs. A video sequence obtained at 30 images s⁻¹, and showing jCaTs during the initial 20 s of EFS in the presence of prazosin is available as Supplementary material (DOI:10.1113/ jphysiol.2003.043380).

Figure 1. Occurrence of junctional Ca²⁺ transients (jCaTs) during the predominantly purinergic component (A) and Ca²⁺ waves during the predominantly adrenergic component (B) of neurogenic contraction

Records at left of both A and B are the force produced by EFS for 3 min. The dotted boxes indicate the times during which images in a–c and traces in d were obtained. Aa and Ba, image obtained by averaging 30 frames (1 s) during the period indicated. Ab and Bb are virtual line-scan images derived from a selected single vertical line of pixels in images Aa and Ba, respectively (continuous white line). Ac and Bc are virtual line-scan images derived from a single horizontal line of pixels in images Aa and Ba, respectively (dotted white line). Traces in Ad are fluorescence pseudo-ratios (F/F₀) derived from the average fluorescence within selected areas of interest (AOIs) in the images in Aa (white boxes). Traces in Bd are derived from the line-scan image in Bb, at the lines indicated by the black arrows. All the data illustrated here were obtained at 30 images s⁻¹.

Figure 3. jCaTs originate near perivascular nerves

A, averaged image (n = 32) of perivascular nerves. Scale bar represents 25 μm. B, averaged image (n = 32) of smooth muscle cells below nerves with sites of origin of jCaTs marked (white boxes) and positions of nerve bundles from image A marked with white lines. The optical section in B is 1.2 μm deeper into the artery wall than that of A. C, frequency distribution histograms of the distances from sites of origin of jCaTs to the nearest nerve (black histogram) and distances of all pixels in the image from the nearest nerve (grey histogram).