The sources and sequestration of Ca(2+) contributing to neuroeffector Ca(2+) transients in the mouse vas deferens

Abstract

The detection of focal Ca(2+) transients (called neuroeffector Ca(2+) transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca(2+) in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.

Figure 1. Purinergic neuroeffector Ca²⁺ transients (NCTs)

A small region close to the surface of the mouse vas deferens is shown during field stimulation (#) at 1 Hz (while recording images at 2 frames s⁻¹). Smooth muscle cells are heterogeneously loaded with the Ca²⁺ indicator Oregon-BAPTA. A, a set of selected frames to demonstrate that NCTs (arrows) are intermittently evoked at several sites within smooth muscle cells. The time at which the image was acquired is marked on each frame. B, another selection of images, from the same recording, showing all NCTs arising at a given site. Each frame should be compared with the control frame (A, frame 1). B, frame 5, shows synchronous events at two sites within the same smooth muscle cell.

Figure 2. Intracellular Ca²⁺ stores and NCTs

The amplitudes of NCTs under control conditions and in the presence of ryanodine (10 µm) are shown. Each point represents the amplitude of NCTs occurring at a single neuroeffector junction. The thin line is a line of equivalence (where the amplitudes are unchanged), while the thick line is a linear curve fit (assuming that the curve passes through 0,0). F is the fluorescent intensity, in arbitrary units; ΔF is the change in F from its resting level.

Figure 3. The rate of recovery of NCTs is slowed by CPA

A, a control recording from a smooth muscle cell during field stimulation (#). In this cell, NCTs arose at the location marked with an arrow with a probability of 0.06 per field stimulus. B, the same region in the presence of CPA (10 µm). Field stimuli still evoked NCTs with a similar probability (0.05 at this site), but the rate of the recovery of Ca²⁺ to its resting concentration is significantly slower. This is quantified for this junction in C. Points plotted are means ± s.e.m.

Figure 4. Caffeine evokes focal Ca²⁺ transients

A, images of several smooth muscle cells loaded with the Ca²⁺ indicator Oregon-BAPTA. B-F show maps (drawn to the same scale and position as the image in A) of the location of focal Ca²⁺ transients occurring immediately after field stimuli (dots; B, D and F), or without stimuli (crosses; frames C, E and F), at some time over 20 min of recording in control (B, C and F) or in the presence of caffeine (3mM; D, E and F). Note that the evoked focal Ca²⁺ transients (NCTs) cluster in characteristic locations within parts of each cell (B). Caffeine induces spontaneous focal Ca²⁺ transients at additional locations (E; F, a composite of B and E).

Figure 5. The effects of T-type Ca²⁺ channel blockers on EJPs and NCTs

A and B show the effect of Ni²⁺ or mibefradil (respectively) on EJPs during low frequency (0.33 Hz) stimulation. The recordings were zeroed with respect to the potential recorded by the microelectrode outside the cell at the end of each experiment. The sampling frequency in A is 100 Hz, while that in B is 4 kHz. The amplitudes of NCTs under control conditions and in the presence of either NiCl₂ (100 µm; C) or mibefradil (10 µm; D) are also shown. Each point represents the amplitude of NCTs occurring at a single neuroeffector junction. A line of equivalence (where the amplitudes are unchanged) is marked on each graph. At concentrations specific for T-type Ca²⁺ channel block, neither of these drugs significantly affects the amplitude of EJPs or NCTs.

Figure 6. Spontaneous Ca²⁺ waves in a smooth muscle cell

A, an example of a cell in which Ca²⁺ waves arose spontaneously and propagated bidirectionally. The locations of the wave fronts are plotted in B. In this cell the average speeds of the waves were 4.4 µm s⁻¹ (•) and 7.4 µm s⁻¹ (^). A and B have the same time scale.

Figure 7. A model for the generation and sequestration of NCTs

Following a nerve terminal action potential there is only a small probability (about 0.02) that a packet of ATP will be released. This ATP acts on postjunctional P2X₁ receptors. The influx of Ca²⁺ through the P2X receptors is sufficient to cause a significant change in the local Ca²⁺ concentration, which is amplified by CICR from the RYR receptor. At least some of this Ca²⁺ is sequestered by the sarcoplasmic reticulum (SR) Ca²⁺-ATPase.