Anant N. Malviya

“Tell Me Where Is Calcium Bred”: Clarifying the Roles of Nuclear Calcium

We are grateful to U. Aebi, E. Carafoli, L. Santella, O. H. Petersen, B. Himpens, A. K. Campbell, S. Bolsover, O. Oberleithner, F. McKeon, A. Rao, H. Bading, P. Nicotera, N. Divecha, J. Bustamante, and M. D. Bootman for discussions at the Conference and for providing the highlights of the topics discussed. S. Ott for secretarial assistance, G. Weil for computer graphics, and K. Langley for editing English usage are gratefully acknowledged.

UP-REGULATION OF DOPAMINE D2 RECEPTOR MRNA IN RAT STRIATUM BY CHRONIC NEUROLEPTIC TREATMENT

The effect of prolonged administration of antagonists on rat striatal dopamine D2 receptor binding and mRNA content was examined. Both haloperidol (2 and 4 mg/kg) and sulpiride (10 mg/kg) induced a significant rise in total D2 and D2(444) mRNA level and in Bmax. Regulation of messenger RNA accumulation is thus an important determinant of dopamine D2 receptor density.

Impaired gene transcription and nuclear protein kinase C activation in the brain and liver of aged rats

The expression of the hsp70 and c‐fos genes and the activation of nuclear protein kinase C (PKC) were studied in young and aged whole rats under heat‐shock conditions. The induction of hsp70 and c‐fos genes by heat shock were decreased several fold in the brain as well as in the liver of senescent animals. Nuclear run‐off transcription assay indicated that this age‐related impairment could be attributed to a block at the level of transcription. Nuclear PKC activation by heat shock was not apparent in old animals. Nuclear PKC involvement in the repression of transcription during senescence is postulated.

Regulation of signalling pathways to the nucleus by dopaminergic receptors

Dopamine regulates postsynaptic gene expression in the central nervous system. The pattern of gene expression is different from chronic vs acute stimulation of dopaminergic receptors. Signalling to the nucleus through dopamine receptors involves different second messenger systems, and each receptor subtype regulates multiple effectors. Long term adaptive changes in neuronal function following administration of dopaminergic drugs such as antipsychotic agent or drugs of abuse is one such example of molecular plasticity triggered by dopaminergic receptors. Role of dopaminergic receptors in the control of transcriptional events and immediate early gene regulation are reviewed.

Calcium signals in the cell nucleus Strasbourg, France, August 20-23, 1998

Just as the great explorations revealed a world far more diverse and rich than had been imagined before, a mass of fast accumulating data is disclosing a much more complex and sophisticated picture of the nucleus than hitherto assumed. The nucleus has long been thought of as some sort of enclosure circumscribed by a double membrane and containing the genetic material of the cell. The inner and outer nuclear membranes join periodically at the nuclear pores, forming complexes supposed to create large channels for the free diffusion of ions and small macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted, with nuclear Ca2+ signals originating from cytosolic Ca2+ waves merely by passive transmission through the nuclear pore complex (NPC). However, recent evidence has changed the mappa mundi of the cell. It has been shown that nuclear Ca2+ can be regulated independently of cytosolic Ca2+ changes. And, just as the Terra Incognita discovered beyond the Ocean Sea proved a fabulous New World, the nuclear envelope (NE) between the cytoplasm and nucleoplasm appears to play an essential role in the regulation of Ca2+ signals inside the nucleus. It contains proteins that regulate and respond to changes in nucleosolic Ca2+ concentration ([Ca2+]nucleosol). Furthermore, several specific Ca2+‐dependent nuclear functions have been described and the list is growing. The regulation of nuclear Ca2+ signals, and the role of nuclear Ca2+ as a specific regulator of nuclear events through Ca2+‐binding proteins, were the main themes of the recent EMBO workshop entitled ‘Calcium signals in the cell nucleus’, organized by A.N.Malviya and coworkers in Strasbourg, France. The existence of Ca2+ signals at the level of the nucleoplasm has never been a real issue, as the …

Pervanadate elicits proliferation and mediates activation of mitogen-activated protein (MAP) kinase in the nucleus.

There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen‐activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.

Separation from protein kinase C: a calcium-independent TPA-activated phosphorylating system

A calcium‐independent but 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐ or diacylglycerol‐activated phospholipid‐dependent phosphorylating activity has been separated from protein kinase C. This has been made possible by employing calcium‐dependent hydrophobic interaction chromatography. The material bound to phenyl‐Sepharose in the presence of calcium at low ionic strength was eluted with EGTA and was protein kinase C. While the unbound material passing through the phenyl‐Sepharose column showed no appreciable protein kinase C activity, instead it had a high phosphorylating activity manifested in the absence of calcium and in the presence of TPA plus phospholipid. The identification of this phosphorylating activity, distinct from protein kinase C, leads to important clues to cellular responses monitored by TPA in the absence of calcium.

Intracellular acidification mediates the proliferative response of PC12 cells induced by potassium ferricyanide and involves MAP kinase activation

Potassium ferricyanide is known to elicit cell growth and mitogenesis in various cells by stimulating a transplasma membrane electron‐transport system. When serum‐starved PC12 cells were treated with potassium ferricyanide, stimulation of mitogenesis was evidenced by enhanced DNA synthesis, as well as by increased cell numbers. Intracellular pH (pHi) of PC12 cells was measured at 37°C by microfluorimetric analysis of 2′,7′‐bis‐(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein (BCECF). The resting pHi of unstimulated cells was 7.52 (external pH 7.40). Addition of potassium ferricyanide (100 μM) decreased pHi by about 0.25 pH units. Lowering pHi to a similar extent, either by decreasing external pH (pHo) or by adding a weak acid, also elicited a mitogenic response, indicating that intracellular acidification by itself has growth factor‐mimicking, mitogenic effects. Nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) triggered proliferation without changes in pHi. The mitogenic treatments eliciting intracellular acidification did not activate protein kinase C (PKC) but stimulated the p42/p44 mitogen‐activated protein (MAP) kinase. Our results indicate that 2 distinct mitogenic pathways are active in PC12 cells: the first is independent of pHi and involves activation of the PKC pathway and the second requires a permissive pHi value around 7.25 and involves activation of the p42/p44 MAP kinase pathway.

Neuroleptics differentially induce jun family genes in the rat striatum

The effect of neuroleptics in single administration on the expression of genes of the jun family was studied in the rat striatum. jun B, but not jun D was dose-dependently and transiently induced. This effect was blocked by pretreatment with a specific dopamine D2 agonist. The expression of c-jun was also stimulated, although this did not reach statistical significance. Thus dopamine D2 receptors differentially regulate the expression of jun family members in the striatum.

Modulation of cytosolic protein kinase C activity by ferricyanide: priming event seems transmembrane redox signalling: A study on transformed C3H/10T1/2 cells in culture

Transformed 3T3/10T1/2 cultured cells incubated with ferricyanide caused a decrease of 2 mM EDTA extractable cytosolic protein kinase C activity in 2 min, whereas 5 or 20 min ferricyanide treatment reverted the enzyme activity to that observed without ferricyanide. The ferricyanide effect in 2 min was abolished by amiloride and sustained by ouabain. Thus, deactivation‐activation of cytosolic protein kinase C is attributed to an unknown signal generation during H+ accumulation coupled with the Na+/H+ exchange phase. In this mechanism the priming event concerns the transmembrane redox process shedding H+ into the cell interior while impermeant ferricyanide acts as a unique electron acceptor.