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SUMMARY 1 ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2 Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3 Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4 The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I− > Cl− > Asp−) and inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5 Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6 In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.

4-AP Facilitates L-Type Ca Current. Background: Transient outward current (I to ) and L-type calcium current (I Ca ) are important repolarization currents in cardiac myocytes. These two currents often undergo disease-related remodeling while other currents are spared, suggesting a functional coupling between them. Here, we investigated the effects of I to channel blockers, 4-aminopyridine (4-AP) and heteropodatoxin-2 (HpTx2), on I Ca in cardiac ventricular myocytes. Methods and Results: I Ca was recorded in enzymatically dissociated mouse and guinea pig ventricular myocytes using the whole-cell voltage clamp method. In mouse ventricular myocytes, 4-AP (2 mM) significantly facilitated I Ca by increasing current amplitude and slowing inactivation. These effects were not voltage-dependent. Similar facilitating effects were seen when equimolar Ba 2+ was substituted for external Ca 2+ , indicating that Ca 2+ influx is not required. Measurements of Ca 2+ /calmodulin-dependent protein kinase (CaMKII) activity revealed significant increases in cells treated with 4-AP. Pretreatment of cells with 10μM KN93, a specific inhibitor of CaMKII, abolished the effects of 4-AP on I Ca . To test the requirement of I to , we studied guinea pig ventricular myocytes, which do not express I to channels. In these cells, 2 mM 4-AP had no effect on I Ca amplitude or kinetics. In both cell types, Ca 2+ -induced I Ca facilitation, a CaMKII-dependent process, was observed. However, 4-AP abolished Ca 2+ -induced I Ca facilitation exclusively in mouse ventricular myocytes. Conclusion: 4-AP, an I to blocker, facilitates L-type Ca 2+ current through a mechanism involving the I to channel and CaMKII activation. These data indicate a functional association of I Ca and I to in cardiac myocytes.

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174–179, 2000. © 2000 Wiley-Liss, Inc.

Quantitative structure-activity relationships have been used to predict the relative toxicity of organic compounds. Although not as common, ion characteristics have also proven useful for predicting the relative toxicity of metal ions. The purpose of this study was to determine if the relative toxicity of metal ions using the Microtox® bioassay was predictable using ion characteristics. Median effect concentrations (EC50s) were determined for 20 metals in a NaNO 3 medium, which reflected freshwater speciation conditions, using the Microtox bacterial assay. The log of EC50 values was modeled using several ion characteristics, and Akaike's Information Criterion was calculated to determine which ion characteristics provided the best fit. Whether modeling total ion (unspeciated) or free ion (speciated) EC50 values, the one variable which best modeled EC50s was the softness index (σ p , i.e., [coordinate bond energy of the metal fluoride - coordinate bond energy of the metal iodide]/[coordinate bond energy of the metal fluoride]), while a combination of χ m 2 r (χ m = electronegativity, r = Pauling ionic radius) and |log K OK | (absolute value of the log of the first hydrolysis constant, K OH for M n+ + H 2 O → MOH n-1 + H + ) was the best two-variable model. Other variables, including ΔE o and χ m 2 r (one-variable models) and (AN/ΔIP, ΔE o ) and (χ m 2 r, Z 2 /r) (two-variable models), also gave adequate fits. Modeling with speciated (free ion) versus unspeciated (total ion) EC50 values did not improve fits. Modeling mono-, di-, and trivalent metal ions separately improved the models. We conclude that ion characteristics can be used to predict the relative toxicity of metal ions whether in freshwater (NaNO 3 medium) or saltwater (NaCl medium) speciation conditions and that this approach can be applied to metal ions varying widely in both valence and binding tendencies.

Both relative toxicity and interactions between paired metal ions were predicted with least-squares linear regression and various ion characteristics. Microtox{reg_sign} 15 min EC50s (expressed as free ion) for Ca(II), Cd(II), Cu(II), Hg(II), Mg(II), Mn(II), Ni(II), Pb(II), and Zn(II) were most effectively modeled with the constant for the first hydrolysis (K{sub H} for M{sup n+} + H{sub 2}O {yields} MOH{sup a{minus}1} + H{sup +}) although other ion characteristics were also significant in regression models. The {vert_bar}log K{sub H}{vert_bar} is correlated with metal ion affinity to intermediate ligands such as many biochemical functional groups with O donor atoms. Further, ordination of metals according to ion characteristics, e.g., {vert_bar}log K{sub H}{vert_bar} facilitated prediction of paired metal interactions. Pairing metals with strong tendencies to complex with intermediate or soft ligands such as those with O or S donor atoms resulted in strong interactions.

Predominant methods for analyzing dose- or concentration-effect data (i.e., probit analysis) are based on the concept of individual tolerance or individual effective dose (IED, the smallest characteristic dose needed to kill an individual). An alternative explanation (stochasticity hypothesis) is that individuals do not have unique tolerances: death results from stochastic processes occurring similarly in all individuals. These opposing hypotheses were tested with two types of experiments. First, time to stupefaction (TTS) was measured for zebra fish (Brachydanio rerio) exposed to benzocaine. The same 40 fish were exposed during five trials to test if the same order for TTS was maintained among trials. The IED hypothesis was supported with a minor stochastic component being present. Second, eastern mosquitofish (Gambusia holbrooki) were exposed to sublethal or lethal NaCl concentrations until a large portion of the lethally exposed fish died. After sufficient time for recovery, fish sublethally exposed and fish surviving lethal exposure were exposed simultaneously to lethal NaCl concentrations. No statistically significant effect was found of previous exposure on survival time but a large stochastic component to the survival dynamics was obvious. Repetition of this second type of test with pentachlorophenol also provided no support for the IED hypothesis. We conclude that neither hypothesis alone was the sole or dominant explanation for the lognormal (probit) model. Determination of the correct explanation (IED or stochastic) or the relative contributions of each is crucial to predicting consequences to populations after repeated or chronic exposures to any particular toxicant.

In classical pharmacology, oral bioavailability of a toxicant is defined as that fraction of an orally administered dose reaching the systemic circulation of the animal. The present study estimates the bioavailability of methylmercury in channel catfish (Ictalurus punctatus) by comparing concentrations in the blood through time after oral and intra-arterial (IA) administration. Catfish were cannulated in the dorsal aorta and gavaged a pelleted feed that had been spiked with methylmercury. Each catfish was gavaged an increasing amount of spiked feed. Following oral dosing, serial blood samples were removed for more than 1,500 h. One month after removal of the last blood sample, the same fish were injected IA with methylmercury and serial blood samples were removed for more than 3,000 h. The area under the curve of the blood concentration-time curve extrapolated to infinity (AUC0→∞) was calculated from fish dosed orally and IA using both noncompartmental (trapezoidal) and compartmental methods. Bioavailability was estimated as the ratio of the dose-corrected oral AUC0→∞ to the IA AUC0→∞. Average bioavailability estimates from this approach were 33% using noncompartmental (range 14–55%) and 29% using compartmental (range 12–42%) methods and were correlated with the amount of food gavaged to the fish (r2 = 0.95, p = 0.026). Bioavailability estimates using the present methods were much lower than estimates using more conventional methods (i.e., assimilation efficiency estimates using mass balance), suggesting that conventional methods may overestimate the true bioavailability of toxicants in fish.