Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca(2+) transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small arteries during activation of alpha1-adrenoceptors. A method was developed to record the Ca(2+) transients within individual SMC during the changes in arterial diameter. Three distinct types of "Ca(2+) signals" were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca(2+) transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca(2+) transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca(2+) sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca(2+) sparks and an increase in the frequency of asynchronous wavelike Ca(2+) transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular [Ca(2+)] and may have a different mechanism than the asynchronous, propagating Ca(2+) transients.

Confocal microscopy of fluo-4 fluorescence in pressurized rat mesenteric small arteries subjected to low-frequency electrical field stimulation revealed Ca 2؉ transients in perivascular nerves and novel, spatially localized Ca 2؉ transients in adjacent smooth muscle cells. These muscle Ca 2؉ transients occur with a very brief latency to the stimulus pulse (most <3 ms). They are wider (Ϸ5 m) and last longer (t 1/2 , 145 ms) than Ca 2؉ sparks. They are abolished by the purinergic receptor (P2X) antagonist suramin, but they are totally unaffected by the ␣ 1 -adrenoceptor antagonist prazosin or by capsaicin (which inhibits the function of perivascular sensory nerves). We conclude that these novel Ca 2؉ transients represent Ca 2؉ entering smooth muscle cells through P2X receptors activated by ATP released from sympathetic nerves, and we therefore call them "junctional Ca 2؉ transients" or jCaTs. As expected from spontaneous neurotransmitter release, jCaTs also occur spontaneously, with characteristics identical to evoked jCaTs. Visualization of sympathetic neurotransmission shows that purinergic components dominate at low frequencies of sympathetic nerve fiber activation. S ympathetic perivascular nerves of small arteries release the triad of sympathetic cotransmitters, ATP, norepinephrine (NE), and neuropeptide Y (NPY). 1 On the postjunctional membrane, ATP activates (ionotropic) purinergic receptors (P2X) to allow influx of Na ϩ and Ca 2ϩ , 2 and NE activates (metabotropic) ␣ 1 -adrenoceptors to initiate phosphoinositide and other G protein-coupled signaling cascades 3 that result in Ca 2ϩ waves. 4,5 Surprisingly, recent studies indicate a predominant role for purinergic mechanisms (rather than adrenergic) in activating neurogenic contractions of small, third-to sixth-order arteries 6 ; the contractions of isolated rat mesenteric small arteries subjected to electrical field stimulation (EFS; 10 Hz, 1 second) are reduced to 65% of control values by the P2X receptor antagonist suramin. For the first time, we report novel Ca 2ϩ signals, generated by neurogenic activation of purinergic receptors in the smooth muscle cells of intact, pressurized small arteries. Materials and MethodsAll experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Male Sprague-Dawley rats, weighing 150 to 250 grams (Harlan, Indianapolis, Ind), were anesthetized with intramuscular ketamine (50 to 100 mg/kg) and killed by cervical dislocation. Mesenteric small arteries were dissected by methods described in detail previously. 5 Segments of third-or fourth-order arteries, 1 to 2 mm in length, were transferred to a recording chamber where their ends were mounted on glass pipettes (tip diameter 60 to 100 m) and secured by 10-0 sutures. One pipette was attached to a servo-controlled pressure-regulating device (Living Systems), while the other was attached to a closed stopcock, and the intraluminal pressure was set to 40 mm Hg. The vessel was then lo...

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 α1‐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 α1‐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 α1‐adrenoceptors and which are associated with propagating Ca2+ waves.

4. The extent of the hysteresis is sensitive to sarcomere length. The phenomenon is virtually absent above sarcomere lengths of about 2-2-2 3,um but becomes progressively greater at shorter sarcomere lengths. 5. The effect of sarcomere length on calcium sensitivity is restricted to the upward-going (increasing activation) part of the pCa-tension loop below 2 2 ,um. The downward-going (decreasing activation) part of the hysteretic relationship is virtually unaffected by sarcomere length up to 2 2 ,um. 6. Significant alterations in sarcomere length do not occur during tension development in the experiments described here: the phenomenon is not attributable to experimental artifacts of this kind.7. Hysteresis develops sufficiently rapidly to be consistent with a physiological relevance during the normal heart beat.8. The effects of sarcomere length show that the phenomenon is not due to force per se since, for example, greater peak force produces less hysteresis as sarcomere length is increased towards 2-2 ,um.9. Tonicity increase (by high-molecular-weight dextran), which shrinks the myofilament lattice, increases calcium sensitivity but reduces the effect of sarcomere length on calcium sensitivity.10. The results suggest that lattice shrinkage is the mechanism which accounts for hysteresis in, and the sarcomere length dependence of, calcium sensitivity in cardiac muscle.