Tissue printed cells from teleost electrosensory and cerebellar structures.

A modification of the tissue printing technique was used to acutely isolate and culture cells from the electrosensory lateral line lobe (ELL), corpus cerebelli (CCb), and eminentia granularis pars posterior (EGp) of the adult weakly electric fish, Apteronotus leptorhynchus. Cells were isolated without the use of proteolytic enzymes and tissue printed as a monolayer onto glass coverslips through centrifugation in the presence of a medium designed to preserve cell structure. Tissue printed cells were reliably distributed in an organotypic fashion that allowed for the identification of anatomical boundaries between the ELL and cerebellar regions, distinct sensory maps in the ELL, and specific cell laminae. Many cells were isolated with an excellent preservation of soma-dendritic structure, permitting direct identification of all electrosensory cell classes according to morphological or immunocytochemical criteria. Several classes of glial cells were isolated, including small diameter microglia and the complex arborizations of oligodendrocytes. A plexus of fine processes were often isolated in conjunction with cell somata and dendrites, potentially preserving synaptic contacts in vitro. In particular, immunolabel for gamma-aminobutyric acid (GABA) revealed a previously unrecognized network of GABAergic axonal processes in the CCb and EGp granule cell body and molecular layers. Tissue printed cells were readily maintained with an organotypic distribution of glial and neuronal elements for up to 27 days in culture. This procedure will allow for the isolation of electrosensory cells from adult central nervous system for electrophysiological analyses of membrane properties or synaptic interactions between identified cells.

Taurine depletion and excitation-contraction coupling in rat myocardium.

Although the sulfur-containing amino acid taurine is found in high concentrations in mammalian myocardium, its involvement in function of the cardiac myocyte remains unclear. To examine the effects of taurine depletion on cardiac mechanical function, rats were treated in vivo with the taurine transport antagonist guanidinoethane sulfonate (GES). After 6 weeks of treatment, myocardial taurine concentrations were decreased to < 40% of control, with no change in tissue DNA content. Right ventricular trabeculas from taurine-depleted rats exhibited significant reductions (P < .05) in isometric twitch force (Ft) at all [Ca2+]o levels and systolic sarcomere lengths examined. Taurine-depleted trabeculas also exhibited increased passive compliance. A slight (P < .05) rightward shift in the Ft-[Ca2+]o relation suggested a decrease in the sensitivity of the taurine-depleted muscles to [Ca2+]o. No changes were observed in the force-interval relation, suggesting that the transsarcolemmal Ca(2+)-handling mechanisms remained unchanged. The fraction of Ca2+ recirculated through the sarcoplasmic reticulum, inferred from the decay of postextrasystolic potentiation, was also not different in the taurine-depleted muscles. When force was expressed relative to the rate of stimulation, length of rest periods, or postextrasystolic potentiation, virtually all curves were super-imposable for control and taurine-depleted muscles, suggesting that the deficit was not dependent on Ca2+ handling. Thus, we conclude that in taurine-depleted muscles the force-generating processes showed the same regulation as in control muscle. Furthermore, the substantial deficit in force development is consistent with a reduced population of force generators on the basis of three pieces of evidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Impairment of cardiac contractility and sarcoplasmic reticulum Ca2+ ATPase activity by hypochlorous acid: reversal by dithiothreitol.

Isolated rat hearts perfused with 100 microM hypochlorous acid (HOCl), a powerful oxidant produced by activated neutrophils, exhibited progressive impairment of contractile performance suggestive of a cytosolic Ca2+ overload (increased left ventricular end-diastolic pressure, increased aortic root perfusion pressure, and depressed pulse pressure). Sarcoplasmic reticulum (SR) enriched microsomal preparations isolated from HOCl-perfused hearts showed a significant decline, when compared with control hearts, in both Ca2+ ATPase activity (123 +/- 40 vs. 473 +/- 46 nmol Pi.mg-1 protein.min-1) and Ca2+ uptake (12 +/- 5 vs. 46 +/- 4 nmol Ca2+.mg-1 protein.min-1). The sulfhydryl content in Ca2+ ATPase and other proteins, as determined by [14C]iodoacetamide binding, was also progressively depleted in HOCl-perfused hearts. Perfusion of the HOCl-treated hearts with dithiothreitol (DTT), a disulfide reducing agent, resulted in a time-dependent attenuation, and eventual partial reversal, of the dysfunction in both contractility and SR Ca2+ ATPase activity. Protein thiol levels were concomitantly restored to near control values. The data indicate that HOCl-induced contractile dysfunction in heart is related to the inactivation of the SR Ca2+ ATPase as a result of thiol oxidation and suggest that DTT is capable of reversing this dysfunction in situ by reducing the oxidized sulfhydryls in the Ca2+ ATPase.

Calcium homeostasis in rabbit ventricular myocytes. Disruption by hypochlorous acid and restoration by dithiothreitol.

Hypochlorous acid (HOCl) is a toxic oxidant produced by neutrophils at sites of cardiac inflammation. To examine the effect of this oxidant on Ca2+ homeostasis in the heart, isolated rabbit ventricular myocytes were iontophoretically loaded with the Ca2+ indicator fura 2 and superfused with 100 microM HOCl under voltage-clamp conditions. Ca2+ transients and the corresponding Ca2+ currents were elicited by 300-msec depolarizing pulses from -40 to 0 mV. Within 200 seconds after HOCl addition, the amplitude of the Ca2+ transients was reduced from 402 +/- 89 to 82 +/- 29 nM (p less than 0.01) while intracellular free ([Ca2+]i increased from 78 +/- 16 to 265 +/- 48 nM (p less than 0.01). During this time, the amplitude of the slow inward currents increased by 10%, while steady-state holding current remained stable. This sustained steady-state rise in [Ca2+]i occurred even in the absence of extracellular Ca2+ but was virtually abolished by a 20-second preexposure to 10 mM caffeine, suggesting that the major source of this Ca2+ was the sarcoplasmic reticulum. Although washout of HOCl failed to induce recovery, subsequent exposure to the dithiol reducing agent dithiothreitol caused a rapid restoration of both the steady-state [Ca2+]i and Ca2+ transient amplitude. We conclude that 1) HOCl caused a rise of [Ca2+]i by inducing the release of Ca2+ from internal stores and impairing cellular extrusion mechanisms and 2) these effects occur through alteration of protein thiol redox status.

Dithiothreitol restores contractile function to oxidant-injured cardiac muscle.

Reperfusion injury in ischemic myocardium is caused partially by polymorphonuclear leukocyte oxygen free radicals, the most toxic of which may be hypochlorous acid (HOCl). This study shows that dithiothreitol (DTT), a disulfide-reducing agent, can restore contractile function to cardiac muscles that had been exposed to physiological levels of HOCl. Isometrically contracting isolated rat papillary muscles which were exposed to HOCl (300 microM) showed a rapid and essentially complete loss of developed force, an increase in resting force, and a sharp decline in myocyte protein sulfhydryls (PSH). The addition of DTT (1 mM) after 40 min resulted in a significant (40%) restoration of contractile function. Earlier addition of DTT effected a more complete functional recovery. The DTT-induced recovery was accompanied by a matching increase in cellular PSH levels, suggesting that HOCl injury may be caused primarily by the oxidation of cysteine residues. These data suggest that DTT may prove to be useful in reversing oxidant injury in tissues exposed to oxygen free radicals.