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Transport Atpase Lsozymes in Pig Cerebellar Purkinje Neurons

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Transport Atpase Lsozymes in Pig Cerebellar Purkinje Neurons The Journal of Neuroscience, March 1991, 1 I(3): 650-656 A Study of the Organellar Ca2+-Transport ATPase lsozymes in Pig Cerebellar Purkinje Neurons Lutgarl Plessers, Jan A. Eggermont, Frank Wuytack, and Rik Casteels Laboratorium voor Fysiologie, Campus Gasthuisberg, Katholicke Universiteit Leuven, B-3000 Leuven, Belgium Pig cerebellar Purkinje neurons express a high level of Ca*+- In view of the apparent importance of Ca’+ for the Purkinje transport ATPases in their intracellular Ca2+ stores. This was neuron, several groups have recently tried to characterize the shown at the mRNA level by Northern blotting and in situ components involved in the accumulation of Ca” in its intra- hybridization and at the protein level by Western blotting cellular stores. It was concluded that the Ca’+ pump in the and immunocytochemistry. The majority of the Ca2+-trans- intracellular storesof cerebellar Purkinje neurons is of the “car- port ATPases in these neurons belongs to the SERCAPb type diac” type (Kaprielian et al., 1989; Shahin et al., 1989). Mo- (i.e., the Ca*+-pump isoform found in most nonmuscle cells). lecular analysis of the organellar Ca’+ pump in different tissues The SERCA2a (cardiac/slow-twitch skeletal/smooth muscle) has indeed revealed a multigene family consisting of at least 3 Ca*+-pump isoform is expressed only at very low levels. The different genes:sarcoplasmic/endoplasmic reticulum Ca’+ pump main Ca2+-pump messenger is 6.0 kilobases long and be- 1 (SERCAl), SERCA2, and SERCA3 (Gunteski-Hamblin et al., longs to a class 4-type processing of SERCAS, which is 1988; Burk et al., 1989). The SERCAl Ca” pump is only ex- exclusively confined to the cerebrum and cerebellum. Phos- pressedin fast-twitch skeletal muscle. In contrast, the SERCAZ pholamban, a regulator of the SERCAS Ca2+-transport ATP- gene is widely expressedboth in muscle and in nonmuscletis- ase in cardiac/slow-twitch skeletal/smooth muscle, could sues.The significanceof the SERCA3 Cal’ pump is lessclear. not be detected in Purkinje neurons. Tissue-dependentalternative processingof the SERCA2 pre- mRNA results in 2 distinct SERCA2 CaL+-pumpisoforms. The The cytosolic free Ca’+ concentration ([Ca*+],,,) is a key regulator SERCA2a isoform is confined to slow skeletal, cardiac, and of neuronal cell functioning; hence, the neuronal mechanisms smooth muscle,whereas the SERCA2b isoform is expressedin involved in the regulation of [Ca2+lcY,have been intensively smooth muscle and nonmuscle tissues(Burk et al., 1989; Eg- investigated during the past years (Kennedy, 1989). In partic- germont et al., 1989, 1990a). ular, the cerebellar Purkinje cell has attracted much interest in Using antibodies specific for the SERCA2a and -2b isoforms, this respect becauseof its high content of proteins and enzymes aswell as antisenseRNA probesspecific for the distinct SERCA2 involved in Ca” regulation (Rosset al., 1990). Indeed, Purkinje mRNAs, we have studied the organellarCa’+ pump in cerebellar neurons have been reported to contain large amounts of cal- Purkinje cells. We present evidence derived from immunolog- bindin and parvalbumin, 2 Ca’+-binding cytosolic proteins ical techniques [Western blotting and immunocytochemistry (Rogers, 1989; Celio, 1990). Purkinje cellsalso contain the high- (ICC)] and from RNA hybridization studies[Northern blotting est level of inositol 1.95-tris phosphate (IP,) receptors in the and in situ hybridization (ISH)] that the majority of the orga- brain (Worley et al., 1989). This finding points to an important nellar Ca*+pumps in Purkinje neuronsare of the “non-muscle,” role for IP,-induced Ca’+ releasein their cellular function. In that is, the SERCA2b type. We could not find any evidence for addition, the kinase C isoform (isoform I), which is present at the presencein Purkinje neurons of phospholamban,the reg- a high concentration in Purkinje neurons (Huang, 1989), is a ulatory protein of the SERCA2 Ca” pumps in cardiac, slow- Ca’+- and phospholipid-dependent protein kinase C. Further- twitch skeletal, and smooth musclecells. more, Purkinje neurons are particularly rich in enzymes in- volved in the cGMP-dependent regulatory pathway: soluble Materials and Methods guanylate cyclase (Nakane et al., 1983) and nitric oxide (NO) RNA probes. Transcriptionvectors were constructed by subcloningthe followingrestriction fragments in pGEM-7Z(f)+ (Promega,Madison, synthetase(Bredt and Snyder, 1990), with NO being a possible WI): pSERCA2,ClaI-XhoI [nucleotides(nt)15 19-20271 ofpig SERCAZ regulator of soluble guanylate cyclase (Lohmann et al., 1981). class1 cDNA (Eggermontet al., 1989);PER-1, DraI-SphI (nt 3 182- Interestingly, the relaxant effect of cGMP in smooth muscle has 3385)of pig SERCA2class 1 cDNA, PER-2,XbaI-ScaI (nt 3250-361I) been attributed to its ability to decreasethe [Ca:+],,, (reviewed of pig SERCAZclass 2 cDNA; PER-3,BamHI-DraIII (nt 1120-l 539) by Walter, 1989). of pig SERCA2class 3 cDNA (cloneER-15; Eggermontet al., I990a); and pPLB, DraI-EcoRI (nt 55l-737) of the pig phospholambancDNA clone8 (Verboomenet al., 1989).Antisense RNA probesfor Northern Received July 2, 1990; revised Sept. 26, 1990; accepted Oct. 10, 1990. blotting were synthesized by an in vitro transcription reaction as de- scribed previously (Eggermontet al., 1990a).RNA probesfor Northern We wish to thank Dr. K. P. Camobell (Howard Hushes Medical Institute. University of Iowa, Iowa City, IA) and Dr: L. R. Jones(Krannert Institute of blotting were labeled with [cGP]CTP up to 10y cpm per peg RNA. Cardiology, Indianapolis, IN) for the gift of the monoclonal antibodies and Dr. Antisense and sense RNA probes for in situ hybridization were labeled F. Vandesande (Neuroendocrinology and Immunological Biotechnology, K. U. following a standard protocol (Krieg and Melton, 1987) using 62.5 PCi Leuven, Belgium) for providing the PAP reagents. J.A.E. is a Research Assistant [c+S]CTP (1320 Ci/mmol; 12.5 mCi/ml) per transcription reaction. of the National Fund for Scientific Research (NWFO, Belgium). The total concentration ofCTPin the transcription mixture wasadjusted Correspondence should be addressed to Lutgart Plessers at the above address. to 14.5 @M with cold CTP. After DNase digestion of the transcription Copyright 8 1991 Society for Neuroscience 0270-6474/91/l 10650-07$03.00/O templates,the RNA probeswere purified on a SephadexG-50 spun The Journal of Neuroscience, March 1991, 1 I(3) 651 column. The RNA probes were subsequently fragmented to a length of synthetic peptide corresponds to the 9 C-terminal amino acids (aa) of approximately 100 nucleotides by alkaline hydrolysis in 40 mM Na- this cardiac muscle Ca2+-pump isoform (aa 989-997: -NYLEPAILE), HC0,/60 mM Na,CO, at 60°C for varying time intervals according to and for SERCA2b, to the 12 C-terminal amino acids of the organellar the in situ hybridization protocol of Amersham (Amersham Intema- Ca*+-pump isoform (aa 1031-1042: -STDTNFSDMFWS). For phos- tional plc, Amersham, UK). The probes were finally ethanol precipitated pholamban, a stretch of amino acids corresponding to a part of the and resuspended at a concentration of 5 ng RNA/fil. The specific activity putative cytosolic domain of the protein (aa 9-27: -RSAIRRASTI- of the j5S-labeled probes for in situ hybridization was 24 x lOa cpm/ EMPQQARQN-) was glutaraldehyde conjugated to BSA. For immu- fig RNA. _ nocytochemistry, antisera were diluted 24 hr prior to use in a 3% BSA- Preparation of vibratome and cryostat sections. Tissues were taken containing buffer in order to remove anti-BSA antibodies. from l-week-old piglets. For vibratome sections, the tissues were fixed Mouse monoclonal antibodies. A mouse monoclonal antibody (Mab) by overnight immersion in a solution of 4% paraformaldehyde in 0.15 IID8, against the canine cardiac sarcoplasmic/reticulum Ca2+ ATPase, M phosphate buffer (pH, 7.4), and 50-Nrn sections were cut on an Oxford was obtained from Dr. K. P. Camubell (Howard Hunhes Medical In- Vibratome (Oxford Instruments, UK). For cryomicrotomy, tissues were stitute, University of Iowa, Iowa ‘City, IA). This amibody does not quick frozen in isopentane precooled in liquid nitrogen and stored at discriminate between the 2 different SERCA2-type Cal’ pumps (Jor- -80°C until use. Sections (10 pm) were cut on a Jung microtome (W. gensen et al., 1988). A mouse monoclonal antibody IID (Adunyah et Dittes, Heidelberg, FRG). The sections were deposited on poly-L-lysine al., 1988) to canine cardiac phospholamban was obtained from Dr. L. coated slides, allowed to dry overnight at room temperature, and sub- R. Jones (Krannert Institute of Cardiology, Indianapolis, IN). sequently stored at -20°C. Slides destined for in situ hybridizations Preparation of membranefractions and immunoblot analysis. Cellular were made RNase free by overnight immersion in 2% (v/v) ABSOLVE membranes from tissues taken from a 20-kg pig were prepared by iso- detergent (DuPont/New England Nuclear, Boston, MA). pyknic equilibration in sucrose density gradients as described elsewhere Northern blotting. Pig cerebra and cerebella were removed immedi- (De Jaegere et al., 1990). SDS-PAGE, electroblotting, and immuno- ately after killing, frozen in liquid nitrogen, and stored at - 70°C. Total detection on Immobilon-P transfer membranes (Millipore Corporation, RNA was prepared according to the Chirgwin procedure (Chirgwin et Bedford, MA) by means of 4-chloro- 1 -naphthol were performed as de- al.. 1979). The isolation of polyA+-enriched RNA by means of an oligo- scribed earlier (Wuytack et al., 1989). (dT) spun column procedure (Pharmacia LKB Biotechnology, Uppsala, Indirect immunoperoxidase immunocytochemistry on cryostat sec- Sweden). electroohoresis of RNA. blotting. prehybridization, hybrid- tions. Sections were incubated at room temperature, unless indicated ization,‘and posthybridization washes were ail performed as described otherwise. Hydration and permeabilization was done by washing the previously (Eggermont et al., 1990a). Unless stated otherwise, solutions slides 3 x 5 min in 50 mM Tris/HCl (pH, 7.6) 0.9% NaCl, and 0.1% (SSPE and Denhardt’s solution) were of standard composition and ex- Triton X-100 (TBS-T).
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