PATHWAYS IN CALCIUM REGULATION

In smooth muscle, activation of receptors by agonists may induce production of second messengers such as IP3 and cyclic ADP ribose (cADPR) which induce Ca²⁺ release from the sarcoplasmic reticulum (SR) via receptor channels. Ca²⁺ itself may further trigger Ca²⁺ release from other stores (Ca²⁺-induced Ca²⁺ release). Ca²⁺ influx through membrane channels may increase intracellular Ca²⁺ levels, while Ca²⁺ reuptake into the SR and facilitated Ca²⁺ efflux through the membrane may decrease intracellular Ca²⁺. The relative roles of these different regulatory mechanisms differ between different smooth muscle types.

Given the rapid contraction of skeletal muscle, efficient Ca²⁺ release and reuptake is essential. In a skeletal muscle fiber, stimulation by an action potential triggers SR Ca²⁺ release via conformational changes in the SR membrane by charge coupling. Ca²⁺ ions diffuse rapidly down an extremely large electrochemical gradient over a very short distance. The SR then rapidly sequesters the released Ca²⁺ using an ATP-dependent pump. Agents such as beta-adrenoceptor agonists may enhance SR Ca²⁺ release, while agents such as thapsigargin (a tumor promotor), may decrease Ca²⁺ reuptake.Unlike skeletal muscle, Ca²⁺ influx is essential for cardiac contraction. Upon stimulation, Ca²⁺ influx triggers Ca²⁺ release from the SR (CICR). During relaxation, Ca²⁺ is actively sequestered by the SR and extruded across the membrane via a Na+-Ca²⁺ exchanger. Accordingly, agents such as nifedipine that inhibit Ca²⁺ influx decrease cardiac contractility.It is clear that the intracellular pathways involved in the elevation or reduction of Ca²⁺ in muscle under various conditions are complex, and only partially understood. The diverse research interests of different investigators, and the expertise of our collaborators, have led to a parallel focus of the laboratory on intracellular Ca²⁺ regulation in smooth, skeletal and cardiac muscles. Interactions between different laboratories have led to exciting experiments and results on the mechanisms underlying Ca²⁺ regulation. We have a number of ongoing projects that focus on:

Smooth Muscle

• Ca²⁺ sparks, waves and oscillations in airway smooth muscle.
• Role of cyclic ADP ribose in airway smooth muscle.
• Effect of volatile anesthetics on Ca²⁺ regulation in airway smooth muscle.
• Effect of nitric oxide (NO) on Ca²⁺ regulation in airway and coronary artery smooth muscles.
• Effect of beta-adrenoceptor agonists on Ca²⁺ regulation in airway smooth muscle.
• Effect of estrogens on Ca²⁺ regulation in coronary artery smooth muscle(collaboration).
• Comparison of cAMP versus cGMP effects on Ca²⁺ efflux in airway smooth muscle.

Cardiac Muscle

• Ca²⁺ sparks and waves in cardiac myocytes.
• Role of cyclic ADP ribose in cardiac Ca²⁺ regulation.
• Effect of volatile anesthetics on Ca²⁺ regulation in neonatal versus adult cardiac muscle.
• Effect of volatile anesthetics on Ca²⁺ regulation in presssure overload cardiac hypertrophy beta-adrenoceptor agonists on Ca²⁺ regulation in airway smooth muscle.
• Cytosolic versus mitochondrial Ca²⁺ regulation.

Skeletal Muscle

• Ca²⁺ sparks in skeletal myotubes.
• Effect of NO on Ca²⁺ regulation in single, type-identified diaphragm muscle fibers.
• Cytosolic versus mitochondrial Ca²⁺ regulation.

REAL-TIME CONFOCAL IMAGING OF INTRACELLULAR CA²⁺ EVENTS

Intracellular Ca²⁺ events may occur over a few milliseconds, as in skeletal muscle, or over hundreds of milliseconds, as in smooth muscle. Characterization of intracellular Ca²⁺ events requires adequate spatial and temporal sampling in order to properly interpret the relationships between Ca²⁺ and mechanical events in muscle cells. Real-time confocal imaging allows both spatial and temporal resolution of extremely rapid Ca²⁺ events in localized regions on single muscle cells. Using the Odyssey XL Confocal System (Noran Instruments), we can obtain Ca²⁺ measurements with a temporal resolution of 2 ms from a 0.5 µm3 volume within a cell.

We have used real-time confocal imaging to characterize Ca²⁺ sparks in airway and vascular smooth muscle cells, and in skeletal muscle fibers (see Samples of Recent Work). We believe that Ca²⁺ sparks represent unitary Ca²⁺ release events through ryanodine receptor channels in the SR. Ca²⁺ sparks may serve to "prime" the SR for agonist-induced Ca²⁺ release.
We have observed agonist-induced intracellular Ca²⁺ oscillations in airway smooth muscle cells, and dissected out the mechanisms underlying these oscillations (see Samples of Recent Work). Frequency and amplitude modulation of Ca²⁺ oscillations may be used by the cell to regulate the mean level of intracellular Ca²⁺, and thus modulate muscle contraction. In recent studies, using Ca²⁺ oscillations as a parameter, we have characterized the mechanisms by which NO modulates intracellular Ca²⁺.

In ongoing studies, we are dissecting out the mechanisms by which volatile anesthetics produce myocardial depression in neonatal versus adult hearts, and in cardiac hypertrophy. Furthermore, in recent studies, we have examined the spatial and temporal aspects of Ca²⁺ waves in cardiac myocytes (see Samples of Recent Work).