András Kapus

Shrinkage-induced Protein Tyrosine Phosphorylation in Chinese Hamster Ovary Cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (≈42, ≈85, and ≈120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosm), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl− levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria.

The entrapment of the Ca2+ ‐sensitive fluorescence indicators fura‐2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ ([Ca2+]m) [(1987) Biochem. J. 248, 609–613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura‐2‐loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half‐maximal and maximal activation were attained at 0.8 and 1.6 μM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.

Is the mitochondrial Ca2+ uniporter a voltage-modulated transport pathway?

The influence of membrane potential (Δψ) on Ca2+ transport through the Ca2+ uniporter (UP) was investigated in fura‐2‐loaded rat heart mitochondria at physiologically relevant‐submicromolar‐external [Ca2+]. In the absence of Δψ the UP could not mediate Ca2+ uptake even when an 8‐fold external (∼500 nM) to internal (∼60 nM) [Ca2+] gradient was present and charge compensation was provided by acetate and the protonophore, CCCP. A small (∼−120 mV) and transient Δψ (generated by valinomycin) resulted in a rise in matrix [Ca2+] only when external [Ca2+] exceeded 150 nM. At physiologically high (∼−180 mV) and stable Δψ this threshold value for Ca2+ uptake dropped to 15 nM. The results indicate that (1) at physiological [Ca2+]m Δψ in addition to being a component of ΔμCa2+ seems to be necessary for providing a transport‐competent conformation for the UP; and (2) below a threshold [Ca2+]m the UP cannot operate even in the presence of a high electric driving force.

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F1-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca2+-uptake of mitochondria energized both by ATP or succinate without affecting the Ca2+-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the Pi and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.

Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+ 0/H+ i exchange process from the matrix side of the membrane. The Na+‐induced changes in the pH of the matrix (pHm) showed that: (i) the Na+ 0/H+ i exchange followed Michaelis‐Menten kinetics with respect to external Na+ with a K m of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+‐induced alkalinization occurred above a pHm of 7.45±0.09 (n=4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.

Ligands of purinergic receptors stimulate electrogenic H+-transport of neutrophils

The possible role of ATP, acting as a ligand on cell surface receptor was investigated in the activation of the electrogenic H+‐transporting pathway of porcine neutrophil granulocytes. (1) ATP brought about 2.1‐fold increase in the rate of H+‐efflux. (2) The order of potency of different nucleotides suggests, that ATP acts on P2 type purinoceptor. (3) The effect of the nucleotides was prevented by inhibition of phospholipase A2. (4) Inhibition of the metabolism of arachidonic acid (AA) via the cyclooxygenase pathway had no effect, whereas inhibition of the lipoxygenase pathway significantly enhanced H+‐release. This is the first report about activation of the H+‐transporter by physiological stimulator acting on the cell surface.