Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes.

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca2+]i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca2+]i transient was reduced following treatment with 100 nM phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5 microM) and calphostin C (10 microM) increased [Ca2+]i transient amplitude, elevated basal [Ca2+]i and slowed the decay of the [Ca2+]i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca2+ channels was monitored indirectly as the rate of Mn2+ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn2+ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca2+ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca2+ content may be secondary to alterations of sarcolemmal Ca2+ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca2+ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca2+]i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca2+ influx, which may play a key role in setting the basal tone of cardiac muscle.

Inhibition of hepatic inositol 1,4,5-trisphosphate receptor function by ethanol and other short chain alcohols.

The ability of alcohols to regulate InsP3-receptor activity was examined in permeabilized hepatocytes. Incubation with 30-300 mM ethanol decreased the sensitivity to InsP3 for Ca2+ release, with little effect on the size of the Ca2+ store that could be released with maximal concentrations of InsP3. Ethanol (300 mM) increased the EC50 for InsP3 from a control value of 134.0 +/- 13.5 nM to 220.0 +/- 25.9 nM. Although ethanol also caused a partial depletion of the total pool of stored Ca2+, the ethanol-induced shift in InsP3 sensitivity was not secondary to this alteration in Ca2+ loading. Partial depletion of the Ca2+ stores with low doses of ionomycin and thapsigargin did not cause a shift in InsP3 sensitivity. Furthermore, measurements of InsP3 receptor channel activity using retrograde flux of Mn2+ to quench the fluorescence of Fura-2 within the Ca2+ stores demonstrated that ethanol inhibited InsP3-activated channel activity in the absence of stored Ca2+. Other short chain alcohols (methanol, 1-propanol and 1-butanol) also decreased the efficacy of InsP3 to release Ca2+. Measurements of [3H]-InsP3 binding demonstrated that ethanol decreased the total number of InsP3 binding sites without changing the KD. The effect of ethanol on InsP3 binding was apparent in the presence or absence of Ca2+ and was observed when the cells were pre-incubated with ethanol at either 37 degrees C or 4 degrees C. The initial rate of InsP3-induced Mn2+ quenching of compartmentalized Fura-2 was reduced by ethanol at all doses of InsP3. These data suggest that ethanol decreases the sensitivity of the intracellular Ca2+ store to release by InsP3, by reducing the number of channels that can be activated by InsP3.

Effect of oxidized glutathione and temperature on inositol 1,4,5-trisphosphate binding in permeabilized hepatocytes.

The effect of oxidized glutathione (GSSG) on inositol 1,4,5-trisphosphate (IP3) binding and the activity of IP3-gated Ca2+ channels was examined in permeabilized hepatocytes. The permeability properties of the channel were measured by using Mn2+ quenching of compartmentalized fura-2 at 37 degrees C and at 4 degrees C for comparison with IP3-binding measurements. GSSG (2 mM) increased the IP3-sensitivity of Mn2+ quenching, consistent with previous studies based on Ca(2+)-release measurements [Renard, Seitz and Thomas (1992) Biochem. J. 284, 507-512]. Measurements of [3H]IP3 binding were made at 4 degrees C after preincubation of permeabilized hepatocytes at 37 degrees C in the absence or presence of GSSG. Under these conditions GSSG stimulated IP3 binding by increasing the number of binding sites without changing the Kd. This effect was observed in the absence or presence of Ca2+, but was abolished when the preincubation with GSSG was carried out at 4 degrees C. Thimerosal also stimulated [3H]IP3 binding, but this effect was mediated both by an increase in the maximum number of binding sites and by a decrease in the Kd. The effects of thimerosal and GSSG were not additive. Further analysis of the effect of GSSG revealed that preincubation of permeabilized hepatocytes at 37 degrees C results in a progressive loss of [3H]IP3-binding sites that can be prevented and reversed by inclusion of GSSG. A parallel loss of IP3-sensitive Mn(2+)-quenchable stores was observed after incubation at 37 degrees C, and this could also be reversed by adding back GSSG. The loss of IP3 binding was not the result of IP3-receptor proteolysis, as judged by Western blotting of immunoreactive protein. The sensitivity of [3H]IP3 binding in permeabilized hepatocytes to varied ratios of GSSG and GSH suggests that the IP3 receptor responds to an oxidized redox environment such as that found in the lumen of the endoplasmic reticulum. GSSG had no direct effect on the ligand-binding activity of detergent-solubilized and partially purified IP3 receptors. We conclude that GSSG exerts an indirect effect on the IP3 receptors in permeabilized hepatocytes by preventing a temperature-dependent loss of IP3-binding sites. We suggest that the hepatic IP3 receptors may interact with a thiol-disulphide oxidoreductase that utilizes GSSG as a substrate and prevents inappropriate unfolding of the ligand-binding domain occurring after incubation of the receptor at 37 degrees C in vitro.

Subcellular organization of calcium signalling in hepatocytes and the intact liver.

Hepatocytes respond to inositol 1,4,5-trisphosphate (InsP3)-linked agonists with frequency-modulated oscillations in the intracellular free calcium concentration ([Ca2+]i), that occur as waves propagating from a specific origin within each cell. The subcellular distribution and functional organization of InsP3-sensitive Ca2+ pools has been investigated, in both intact and permeabilized cells, by fluorescence imaging of dyes which can be used to monitor luminal Ca2+ content and InsP3-activated ion permeability in a spatially resolved manner. The Ca2+ stores behave as a luminally continuous system distributed throughout the cytoplasm. The structure of the stores, an important determinant of their function, is controlled by the cytoskeleton and can be modulated in a guanine nucleotide-dependent manner. The nuclear matrix is devoid of Ca2+ stores, but Ca2+ waves in the intact cell propagate through this compartment. The organization of [Ca2+]i signals has also been investigated in the perfused liver. Frequency-modulated [Ca2+]i oscillations are still observed at the single cell level, with similar properties to those in the isolated hepatocyte. The [Ca2+]i oscillations propagate between cells in the intact liver, leading to the synchronization of [Ca2+]i signals across part or all of each hepatic lobule.

Imaging of inositol 1,4,5-trisphosphate-induced Ca2+ fluxes in single permeabilized hepatocytes. Demonstration of both quantal and nonquantal patterns of Ca2+ release.

The subcellular organization and function of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores has been investigated in permeabilized hepatocytes using fluorescent probe techniques that monitor InsP3 action at the level of the Ca2+ storage organelles. Chlortetracycline fluorescence was used to follow alterations in luminal Ca2+, and InsP3-activated Mn2+ quench of compartmentalized fura-2 was used as a measure of the distribution and permeability of the InsP3-sensitive channels. Fluorescence imaging of single permeabilized hepatocytes attached to coverslips demonstrated that InsP3-sensitive Ca2+ stores are distributed throughout the cytoplasm, but are not present within the nuclear matrix. When hepatocytes were permeabilized in suspension, InsP3 activation of channel opening and Ca2+ release occurred in a quantal manner, such that the incremental magnitude of the response was determined by the dose of InsP3. Under these conditions dose-dependent steps of InsP3-induced Mn2+ entry into the stores occurred in the absence of changes in cytosolic or luminal Ca2+, providing evidence for a series of separate compartments with different sensitivities to InsP3. Electron microscopy studies revealed that the endoplasmic reticulum was extensively vesicularized when hepatocytes were permeabilized in suspension, whereas essentially normal endoplasmic reticulum structure was retained in cells attached to coverslips. In these attached cells the Ca2+ release and channel opening responses to InsP3 occurred in a nonquantal manner at the single cell level. Submaximal doses of InsP3 gave the same magnitude of response as a maximal InsP3 dose, although the rates of Ca2+ release and Mn2+ permeation through the InsP3-activated channels increased in a dose-dependent manner. Thus, in each cell the entire Ca2+ store was accessible for mobilization by all effective InsP3 concentrations. We conclude that the quantal release properties of the InsP3 receptor are not expressed in attached permeabilized liver cells because there is extensive luminal continuity within the InsP3-sensitive Ca2+ stores. This continuity appears to be disrupted when hepatocytes are permeabilized in suspension.