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Canonical transient receptor potential (TRPC) channels control influxes of [Ca.sup.2+] and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the [Ca.sup.2+] influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced [Ca.sup.2+] response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a [Ca.sup.2+]-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy. [Ca.sup.2+] signaling | pyrazole compounds | TRPC channels | TRPC3

Caenorhabditis elegans navigates thermal gradients by using a behavioral strategy that is regulated by a memory of its cultivation temperature (Tc). At temperatures above or around the Tc, animals respond to temperature changes by modulating the rate of stochastic reorientation events. The bilateral AFD neurons have been implicated as thermosensory neurons, but additional thermosensory neurons are also predicted to play a role in regulating thermotactic behaviors. Here, we show that the AWC olfactory neurons respond to temperature. Unlike AFD neurons, which respond to thermal stimuli with continuous, graded calcium signals, AWC neurons exhibit stochastic calcium events whose frequency is stimulus-correlated in a To-dependent manner. Animals lacking the AWC neurons or with hyperactive AWC neurons exhibit defects in the regulation of reorientation rate in thermotactic behavior. Our observations suggest that the AFD and AWC neurons encode thermal stimuli via distinct strategies to regulate C. elegans thermotactic behavior. calcium imaging | G protein-coupled receptor AWC neuron | isothermal tracking

The objective of this trial was to determine how 25-hydroxyvitamin [D.sub.3] (25-OH [D.sub.3]) supplementation, altering supplemental dietary calcium, or their combination influence postmortem biochemical and tenderness changes in muscles from the round of mature cows. Twenty-seven Angus cows (3 to 7 yr old) were allotted randomly to 9 pens with 3 cows per pen. Treatments were arranged in a 3 x 3 factorial design with 3 dosages of 25-OH [D.sub.3] (0, 250, or 500 mg of 25-OH [D.sub.3] administered as a 1-time oral bolus 7 d before slaughter) and 3 percentages of supplemental limestone (0.5, 0.75, and 1.0%) replenished in the diet for 3 d before slaughter and after a 2-wk limestone withdrawal. Plasma samples were obtained during the feeding period. Upon slaughter, adductor, gracilus, pectineus, sartorius, semimembranosus, vastus intermedius, and vastus lateralis muscles were obtained and aged for 1, 3, or 7 d. Calcium concentrations were increased in plasma when 250 or 500 mg of 25-OH [D.sub.3] were administered (P [less than or equal to] 0.05). Calcium concentrations in muscle increased (P [less than or equal to] 0.001) when 500 mg of 25-OH [D.sub.3] were administered. Concentrations of 25-OH [D.sub.3] in meat and in plasma and 1,25-dihydroxyvitamin [D.sub.3] [1,25-[(OH).sub.2] [D.sub.3]] in plasma were increased when 25-OH [D.sub.3] was administered (P [less than or equal to] 0.05). The percentage of limestone replenished in the diet had no effect on 25-OH [D.sub.3] or 1,25-[(OH).sub.2] [D.sub.3] in meat or in plasma. Calpastatin activity was affected by treatments only in the gracilus and vastus intermedius muscles (P [less than or equal to] 0.05). Among all muscles and aging periods, calpastatin activity and intensity of troponin-T degradation product were related inversely. Results indicate that supplemental 25-OH [D.sub.3] has some influence on muscle characteristics known to improve tenderness, but improved tenderness was not observed. Key words: beef, calcium, cow, 25-hydroxyvitamin [D.sub.3], tenderness

Calcium signaling is critical for all cells. As a free ion ([Ca.sup.2+]), calcium links many physiological stimuli to their intracellular effectors by interacting with binding proteins whose occupancy determines the cellular effect of stimulation. Because binding site occupancy depends on the history of [Ca.sup.2+] concentration ([[Ca.sup.2+]]), [Ca.sup.2+] dynamics are critical. Calcium dynamics depend on the functional interplay between [Ca.sup.2+] transport and buffering systems whose activities depend nonlinearly on [[Ca.sup.2+]]. Thus, understanding [Ca.sup.2]. dynamics requires detailed information about these [Ca.sup.2+]. handling systems and their regulation in intact cells. However, effective methods for measuring and characterizing intracellular [Ca.sup.2+]. handling have not been available until recently. Using concepts relating voltage-gated ion-channel activity to membrane potential dynamics, we developed such methods to analyze [Ca.sup.2+] fluxes in intact cells. Here we describe this approach and applications to understanding depolarization-induced [Ca.sup.2+]. responses in sympathetic neurons.

The class I myosin My01c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by [Ca.sup.2+], permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a My01c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by My01c and modulated by the calcium transient. molecular motor | myosin