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Cartilage undergoes characteristic deformation following laser irradiation below the ablation threshold. Measurements of surface temperature and integrated scattered light intensity were performed during laser irradiation. Porcine auricular cartilage (1 - 2 mm thickness) was irradiated with an Nd:YAG laser (lambda equals 1.32 micrometer) with varying dose (J/cm2). Surface temperature was monitored using a single element HgCdTe infrared detector, responsive between 10 - 14 micrometer. A HeNe laser beam (lambda equals 632.8 nm) was incident on the back surface of the cartilage specimen and fractional integrated back scattered light intensity was measured using an integrating sphere and a silicon photodiode. Laser irradiation (2 W, 5.83 W/cm2, 50 Hz PRR) was allowed to proceed until surface temperature reached 70 degrees Celsius. Cartilage deformation was observed in each instance. Integrated scattered light intensity reached a plateau before the peak temperature (70 degrees) was reached. At increased laser power (10 W, 39.45 W/cm2, 50 Hz PRR), a feedback controlled cryogen spray was used to maintain surface temperature below 50 degrees Celsius. A similar plateau response was also noted in integrated scattered light intensity. This signal may be used to optimize the process of stress relaxation in laser cartilage reshaping. Several clinical applications are discussed. ©2005 Copyright SPIE - The International Society for Optical Engineering.

Members of the [Na.sup.2+]/[Ca.sup.2+] exchanger (NCX) family are important regulators of cytosolic [Ca.sup.2+] in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of [Na.sup.+]-dependent inactivation and requires much higher levels of regulatory [Ca.sup.2+] to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from [Na.sup.+]-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the [Na.sup.+]-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 [micro]M exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously. calcium homeostatis; [Na.sup.+]/[Ca.sup.2+] exchanger; development

The activity of the cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for [Na.sup.+] and [Ca.sup.2+]. In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by [Na.sup.+] and [Ca.sup.2+]. In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular [Na.sup.+] and [Ca.sup.+] ([Na.sup.x.sub.i] and [Ca.sup.2+.sub.i]). [Na.sup.+]/[Ca.sup.2+] exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of [Na.sup.+]- and [Ca.sup.2+]-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, [Na.sup.+]/[Ca.sup.2+] exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory [Ca.sup.2+], or exposure of patches to limited proteolysis by a-chymotrypsin, reveal that this 'coupling' is due to [Na.sup.+]-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by [Na.sup.+.sub.i]-dependent inactivation was not apparent. The current approach provides a novel means for assessment of [Na.sup.+]/[Ca.sup.2+] exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions. sodium/calcium exchange; coupled inactivation; ionic regulation; giant excised patch

The cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30[degrees]C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the N[H.sub.2]-terminal region of the molecule. Specifically, we found that ~75% of the [Na.sup.+]/[Ca.sup.2+] exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site. myocardial contractility; excitation-contraction coupling; salmonid; calcium handling

Ion transport and regulation of Na(super +)-Ca(super 2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(super +)-Ca(super 2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(super +)-Ca(super 2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(super +)(sub i)- (i.e., I(sub 1)) and Ca(super 2+)(sub i)-(i.e., I(sub 2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(sub 1) and I(sub 2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with (alpha)-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(sub 1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to 1(sub 2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(super 2+)(sub i), NCX1.3 showed a prominent decrease at higher concentrations (>1 (mu)M). This does not appear to be due solely to competition between Ca(super 2+) and Na(super +)(sub i) at the transport site, as the Ca(super 2+)(sub i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(super 2+)(sub i) had only modest effects on Na(super +)(sub i)dependent inactivation of NCX1.3, whereas I(sub 1) inactivation of NCX1.4 could be completely eliminated by Ca(super 2+)(sub i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(super +)-Ca(super 2+) exchange to fulfill tissue-specific requirements of Ca(super 2+) homeostasis. KEY WORDS: sodium-calcium exchange; regulation; alternative splicing