PDE4A (Cherry, 1995) identifying a new olfactory signal transduction pathway in these neurons. Their axons project to. 'necklace' glomeruli found in the caudal ...
624s
Biochemical SocietyTransactions ( 1 996) 24
15
IDENTIFICATION OF A NEW PDE2A ISOFORM AND EXPRESSION OF PDE2 IN A SUBSET OF OLFACTORY NEURONS & GASTROINTESTINAL CELLS. D.M. Jyilfs. W. K. SS. S d . H-I. Fulle. D. L. Garbers. M. D. fIQuslav & J. A. Beavo. Depts. of Pharmacology, U. of Washington, Seattle, WA 98195 & U. of Texas, Dallas, TX 75235, Dept of Biochemistry U. of Glasgow, Scotland G12 8QQ. The cGMP-stimulated phosphodiesterase (PDE2) is one member of the seven cyclic nucleotide phosphodiesterase (PDE) families responsible for the hydrolysis of cAMP and cGMP. The PDE2 family presently consists of two members, BTPDE2A1 & RNPDE2A2 (Yang1994). We have cloned a new member, designated PDE2A3, from mouse and bovine brain cDNA libraries and also the mouse isoform of PDE2A2. These isoforms vary in only the extreme amino terminal. When expressed in the neuronal cell line NIE-115, PDE2A3 is clearly associated with cell membranes based on activity, Western analysis and immunocytochemistry as compared to PDE2Al. RNase protection analysis shows that PDE2A2 & PDE2A3 are both expressed in mouse brain. As previously reported (Juilfs, FASEB 9:(6)) PDE2 is expressed in a subset of olfactory neurons in the nasal epithelium. This same subset is now shown to also contain the membrane receptor guanylyl cyclase-D but does not express other components of the olfactory cAMP/calcium signal transduction pathway such as adenylyl cyclase 111, PDElC2 (Yan, 1993, or PDE4A (Cherry, 1995) identifying a new olfactory signal transduction pathway in these neurons. Their axons project to 'necklace' glomeruli found in the caudal region of the olfactory bulb. This observation is different from other classes of odorant receptor containing neurons which project to single glomeruli. PDE2 is also found in a subset of cells in the mucosal layer of the gastrointestinal tract. Expression in the stomach is restricted to a few cells and increases in concentration along the intestine to reach high concentrations in the colon. PDE2 is also present in myenteric neurons and the Bowman's glands in the proximal duodenum. These observations suggest the presence of a cAMP pathway in these cells which is attenuated by cGMP.
16
SHINGOSINE INHIBITS STORE-DEPENDENT CALCIUM INFLUX IN CFPAC-I CELLS. *M.Cavazzoni, and Soriati. Dip. di Biologia E.S. Via lrnerio 42, and 'Dip. di Biochimica, Via lrnerio 48, UniversitL di Bologna, 40126 Bologna, Italy.
m,
Activation of the 'sphingomyelin cycle" by various cytokines results in the hydrolysis of membrane sphingomyelin and generation of ceramide and sphingosine in target cells. Many biological functions of sphingosine have been described so far, however its mechanism of action remains poorly understood. In the present study we report that in a cell line derived from an adenocaranoma of pancreatic duct (CFPAC1), sphingosine slightly reduced basal Ca2* influx, whereas dramatically inhibited capaatative Ca2' influx triggered by depletion of intracellular Ca2*stores. It is widely accepted that in non-excitable cells Ca2* entry across the plasma membrane is regulated by the state of filling of the intracellular Ca2* stores in the endo lasmic reticulum through a process called 'capaatative CaE entry". This Ca2* entry can be switched on by activation of PLC-linked receptors and by inhibitors of the Ca2+-ATPase of the endoplasmic reticulum, such as thapsigargin. Sphingosine (5pM) almost completely inhibited Ca2' influx triggered by both thapsigargin and UTP. At the same concentration, sphingosine reduced the uptake of rhodamine 123, a lipophilic fluorescent dye which is selectively accumulated by mitochondria in living cells, monitoring the energy-state of these organelles. It follows that inhibition of Ca2 entry by sphingosine might be due to mitochondria1 deenergization. This conclusion is in agreement with data indicating that Ca2* influx activated by store depletion, is modulated by a small reduction of ATP levels. Supported by Progetto Apoptosi, UniversitL di Bologna.
17
UNDEREXPRESSION OF THE 43 KDA INOSITOL POLYPHOSPHATE 5-PHOSPHATASE IS ASSOCIATED WITH CELLULAR TRANSFORMATION Caroline J. S d * ,Peter J. Little', John A. Hayman*,Christina A. Mitchell* *Department of Medicine, Monash M d c a l School, Box Hill Hospital, Box Hill 3 128, Melbourne, Austra1ia;'Baker Medical Research Institute, Alfred Hospital, Rahran 3181, Melbourne, Australia
In response to agonist stimulation the second messenger molecule inositol 1,4,5-trisphosphate (Ins(l,4,5)P1) mobilises calcium from intracellular stores. The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses h(1,4,5)P3 in a signal terminating reaction. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting NRK cells with the cDNA encoding the 43 kDa 5-phosphatase,cloned in the antisense orientation into the tetracycline-inducible expression vector pUHDIO-3. Antisense-transfectcd cells demonstrated a greater than 50% reduction in Ins(1,4,5)P3 5-phosphatase activity in both the total cell homogenate and the detergent-soluble membrane kaction of the cell, as compared to cells transfected with vector alone. Unstimulated antisensetransfected cells showed a concomitant two fold increase in Ins(l,4,5)P3 and four fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium Concentration of serum-deprived antisense-transfected cells (170 nM _+ 25) was increased 1.9 fold, compared to cells transfected with vector alone (90 nM 25). In addition, antisense-transfected cell lines with basal intracellular cell calcium concentrations greater than 150 nM, were noted to have a sigmhmt decrease (5 fold) in total intracellular calcium stores. Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7 fold faster rate, reached confluence at higher density, and demonstrated increased ['Hlthymidine incorporation, compared to cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in cellular Ins( 1,4,5)P35-phosphatase activity is.associated with cellular transformation and implicates the 43 kDa inositol polyphosphate 5-phosphatase as a potential tumour suppressor gene.
18
HYDROLYSIS OF PHOSPHATIDYLMOSITOL 3,4BISPHOSPHATE BY A 4-PHOSPHATASE ASSOCIATED \NTH THE P85/P110 PHOSPHOMOSITIDE 3-KINASE. A. Mundav. S. Brown, K.C.Caldwell I and C.A. Mitchell. Department of Medicine, Monash Medical School, Box RI1 Hospital, Vic, Australia 3128. 'Cell Pathology Laboratory, University of New Mexico, Albuquerque, New Mexico, USA, 87131 The phosphoinositide 3-kinase (PI 3-kinase) associates with activated tyrosine kinase receptors resulting in the production of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3.4bisphosphate (ptdIns(3,4)P2) and phosphatidylinositol 3,4,5trisphosphate (PtdIns(3,4,5)P~).The time course for formation of these 3phosphoinositides suggests PtdIns(3,4)P2 is dephosphoylated by a 4phosphatase forming F'tdIns(3)P. We report here the identification of a PtdIns(3,4)P2 4-phosphatase associated with the p85/110 form of the PI 3-kinase. N M 3T3 cells were stimulated with plateletderived growth hctor (PDGF) (30 ng/ml) for 030 minutes and the p85 subunit of the PI 3 - h e , or the PDGF receptor immunoprecipitated using spccific polyclonal antibodies. PI 3 - h e activity in the hunoprccipitates was determined using either PtdIns or PtdIns(4)P as the substrate. Within 10 minutes of PDGF-treatment, using PtdIns as a substrate, the formation of PtdIns(3)P was stimulated 1.6-fold in p85, and &fold in PDGF receptor immunoprecipitates. When PtdIns(4)P was used as a substrate in p85, or PDGF rcceptor immunoprecipitates 60% of the product the reaction aas PtdIns(3,4)P2 and the remainder PtdIns(3)P, suggesting the presence of PtdIns(3,4)P2 4-phosphatase. A 3.7-fold increase in total 3-phosphorylated phosphoinositides formed was observed in p85 immunoprecipitates following PDGF-treatment of N M 3T3 cells, however the hydrolysis of ptdlns(3,4)P2 to ptdIns(3)P was not enhanced. The immunoprecipitatcd 4-pho~phataS~ hydrolysed Ptdb~(~'P-3,4)P2forming Ptdh~(~*P-3)P, but did not hydrolyse inositol 1,3,4-trisphosphate. These studies indicate the presence of a PtdIns(3,4)P2 4-phosphatase associated with tbe p85 subunit of the PI 3-kinase.
