Regulation of free Ca2+ concentration in hair-cell stereocilia.

By affecting the activity of the adaptation motor, Ca2+ entering a hair bundle through mechanoelectrical transduction channels regulates the sensitivity of the bundle to stimulation. For adaptation to set the position of mechanosensitivity of the bundle accurately, the free Ca2+ concentration in stereocilia must be tightly controlled. To define the roles of Ca2+-regulatory mechanisms and thus the factors influencing adaptation motor activity, we used confocal microscopy to detect Ca2+ entry into and clearance from individual stereocilia of hair cells dialyzed with the Ca2+ indicator fluo-3. We also developed a model of stereociliary Ca2+ homeostasis that incorporates four regulatory mechanisms: Ca2+ clearance from the bundle by free diffusion in one dimension, Ca2+ extrusion by pumps, Ca2+ binding to fixed stereociliary buffers, and Ca2+ binding to mobile buffers. To test the success of the model, we compared the predicted profiles of fluo-3 fluorescence during the response to mechanical stimulation with the fluorescence patterns measured in individual stereocilia. The results indicate that all four of the Ca2+ regulatory mechanisms must be included in the model to account for the observed rate of clearance of the ion from the hair bundle. The best fit of the model suggests that a free Ca2+ concentration of a few micromolar is attained near the adaptation motor after transduction-channel opening. The free Ca2+ concentration substantially rises only in the upper portion of the stereocilium and quickly falls toward the resting level as adaptation proceeds.

Plasma membrane Ca2+-ATPase extrudes Ca2+ from hair cell stereocilia.

Mechanically sensitive hair cells of the auditory and vestibular systems use Ca2+ to control adaptation of mechanical transduction, to effect frequency tuning, to trigger neurotransmitter release, and to mediate efferent synaptic signaling. To determine the role that pumps play in regulation of Ca2+ in the hair bundle, the organelle responsible for mechanoelectrical transduction, we localized and quantified the plasma membrane Ca2+-ATPase (PMCA) of the bundle. We found that each hair bundle contains approximately 10(6) PMCA molecules or approximately 2000 per square micrometer of bundle membrane and that PMCA is the principal calmodulin binding protein of the bundle. Consistent with biochemical estimates of PMCA density, we measured with extracellular Ca2+-selective electrodes a substantial Ca2+ efflux from bundles. The number of bundle Ca2+ pumps and magnitude of resting Ca2+ efflux suggested that PMCA should generate a substantial membrane current as bundles expel Ca2+. Measurement of whole-cell currents revealed a transduction-dependent outward current that was consistent with the activity of PMCA. Finally, dialysis of hair cells with PMCA inhibitors led to a large increase in the concentration of Ca2+ in bundles, which suggests that PMCA plays a major role in regulating bundle Ca2+ concentration. Our data further indicate that PMCA could elevate the extracellular Ca2+ concentration close to hair bundles above the low level found in bulk endolymph.

The selectivity of the hair cell's mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations.

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel's open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel's pore. These results demonstrate not only that the hair cell's transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.

ATPase activity of myosin in hair bundles of the bullfrog's sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.

Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.

A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.

Shipping stress and social status effects on pig performance, plasma cortisol, natural killer cell activity, and leukocyte numbers.

Crossbred pigs were used to evaluate the effects of shipping stress on natural killer (NK) cell activity, leukocyte numbers, plasma cortisol, and BW changes. In the first study, pigs were bled at a commercial farm and, after shipping, resident and shipped pigs were bled again. Plasma cortisol concentrations were not different (P > .10) because of large variation in cortisol concentrations. Furthermore, NK cytotoxicity was nondetectable among all pigs. A second study showed that plasma cortisol concentration rose by approximately 2.6 ng/mL (P = .018) for each minute after pigs were aroused. In the third, more controlled study, pigs were housed in pens of three pigs each. Video recordings were made during the first 24 h pigs were grouped to identify socially dominant, intermediate, and submissive pigs. At time zero (before shipping), resident pigs and those to be shipped had similar plasma cortisol concentrations. However, after the 4-h shipping experience, shipped pigs had elevated (P < .05) plasma cortisol compared with resident control pigs. Shipped pigs lost 5.1% of their BW (P < .05) compared with resident pigs, which gained .02% of their BW. Body weight change during shipping and plasma cortisol were negatively correlated (r = -.34, P = .04), indicating pigs that had greater adrenal response to shipping also lost more weight during shipping. Shipping reduced (P < .05) NK cytotoxicity among pigs of intermediate and submissive social status compared with shipped, dominant pigs. At the end of shipping or control treatments, the correlation between NK cytotoxicity and plasma cortisol was positive (r = .35, P = .036), indicating that pigs with greater cortisol response had greater NK cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropin stimulates natural killer cell activity.

Central release of CRH has recently been implicated in modulating natural killer cell (NK) activity independent of its role in activation of the hypothalamic-pituitary-adrenal axis. In the present study, NK- and interleukin-2 (IL-2)-activated NK cytotoxicity against K-562 target cells was examined in porcine lymphocytes cultured in vitro with ACTH or pig lymphocytes after iv or im ACTH administration. Physiological concentrations of porcine ACTH (10(-8)-10(-11) M) added to the culture medium had no direct influence on NK- or IL-2-stimulated NK cytotoxicity. In a second experiment four unrestrained pigs with indwelling catheters given an iv bolus of vehicle or ACTH (1 IU/kg BW) at 0800 h showed significantly elevated cortisol levels for 3 h after ACTH. Although serum cortisol had returned to baseline by 4 h after ACTH treatment, NK- and IL-2-stimulated NK cytotoxicity was dramatically elevated (P less than 0.01) compared to that in saline-injected controls. NK cytotoxicity in control pigs followed a diurnal pattern, with low morning and high evening cytotoxicity. Exogenous ACTH, given by bolus in the morning, prevented the normal morning decline in NK cytotoxicity. Because of this unexpected dramatic increase in NK- and IL-2-stimulated NK cytotoxicity in animals given ACTH, the experiment was replicated in two subsequent studies using 16 pigs (8 controls and 8 experimental) in each. Pigs were injected im with either ACTH (1 IU/kg BW) or an equivalent volume of saline at 0600 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and measure the cortisol response. As was observed in the previous study, NK- and IL-2-stimulated NK cytotoxicity was significantly greater (P less than 0.01) than that in saline-injected controls. In the final experiment pigs were given either ACTH (1 IU/kg BW) or an equivalent volume of saline at 1800 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and cortisol response. ACTH administered in the evening increased NK cytotoxicity, but the effect was only marginally significant and far less dramatic than in previous studies. Because ACTH had little effect on NK- and IL-2-stimulated NK activity in vitro, we hypothesize that the stimulatory effect of exogenous ACTH is mediated through an indirect mechanism, possibly through the suppression of central CRH as a result of elevated cortisol. This effect is more pronounced when the stimulatory dose of ACTH is given at a time in the circadian cycle when NK cytotoxicity is normally low.