Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells.

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.

Identification of tyrosines in the putative regulatory site of the Ca2+ channel TRPV6.

In HEK293 cells, transfected with the Ca2+ channel protein TRPV6, Ca2+ influx is increased and TRPV6 is tyrosine phosphorylated following addition of the tyrosine phosphatase inhibitor N,N-dimethyl-hydroxamido hydroxovanadate to cells. This effect of DMHV is enhanced by co-transfection of cells with the tyrosine kinase Src and the tyrosine phosphatase 1B. It is abolished when cells had been treated with PP1, an inhibitor of Src family tyrosine kinases. PTP1B interacts with the N-terminal domain of TRPV6 within a region of amino acids 1-191 as shown by co-immunoprecipitation, bimolecular fluorescence complementation and the yeast 2-hybrid system. Point mutation of both tyrosines 161 and 162 in the TRPV6 protein abolishes the DMHV-effect on Ca2+ influx and tyrosine phosphorylation by Src. Single mutations of Y161 or Y162 shows that each of both tyrosines alone is sufficient for the DMHV-effect. We conclude that phosphorylation/dephosphorylation of tyrosines in position 161 and 162 is essential for regulation of Ca2+ influx through TRPV6 Ca2+ channels in HEK293 cells.

Inhibition of protein tyrosine phosphatase 1B by reactive oxygen species leads to maintenance of Ca2+ influx following store depletion in HEK 293 cells.

Depletion of inositol 1,4,5 trisphosphate-sensitive Ca2+ stores generates a yet unknown signal, which leads to increase in Ca2+ influx in different cell types [J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1-12]. Here, we describe a mechanism that modulates this store-operated Ca2+ entry (SOC). Ca2+ influx leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [L. Sternfeld, et al., Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells, Cell Signal 17 (2005) 951-960]. Since Ca2+ does not directly inhibit PTP1B, we assumed an intermediate signal, which links the rise in cytosolic Ca2+ concentration and PTP1B inhibition. We now show that Ca2+ influx is followed by generation of reactive oxygen species (ROS) and that it is reduced in cells preincubated with catalase. Furthermore, Ca2+-dependent inhibition of PTP1B can be abolished in the presence of catalase. H2O2 (100 microM) directly added to cells inhibits PTP1B and leads to increase in Ca2+ influx after store depletion. PP1, an inhibitor of the Src family tyrosine kinases, prevents H2O2-induced Ca2+ influx. Our results show that ROS act as fine tuning modulators of Ca2+ entry. We assume that the Ca2+ influx channel or a protein involved in its regulation remains tyrosine phosphorylated as a consequence of PTP1B inhibition by ROS. This leads to maintained Ca2+ influx in the manner of a positive feedback loop.

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells.

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.

Tyrosine phosphatase PTP1B modulates store-operated calcium influx.

We have studied modulation of "store-operated calcium influx" by tyrosine phosphatases in the pancreatic acinar cell line AR42J and in HEK 293 cells. We show that inhibition of tyrosine phosphatases by bis-(N,N-dimethyl-hydroxamido) hydrooxovanadate (DMHV) leads to an increase in Ca(2+) release-activated Ca(2+) (CRAC) entry. This effect can be blocked in the presence of 2-aminoethyldiphenyl borate (2-APB). Furthermore, transfection of HEK 293 cells with the human wild-type tyrosine phosphatase PTP1B leads to inhibition of CRAC influx, whereas transfection with the substrate-trapping mutant of PTP1B (D181A) slightly increases Ca(2+) influx. It also decreases enzymatic activity of PTP1B as compared to non-transfected cells. Our data suggest that CRAC influx is modulated by tyrosine phosphorylation and dephosphorylation which involves the tyrosine phosphatase PTP1B.

Hormonal control of ADP-ribosyl cyclase activity in pancreatic acinar cells from rats.

Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the beta-NAD+ analogs 1,N6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.