T-cell-dependent antibody responses in the rat: forms and sources of keyhole limpet hemocyanin matter.

The T-cell-dependent antibody response (TDAR) is a functional assay used in immunopharmacology and immunotoxicology to assess ability to mount an antibody response to immunization. Keyhole limpet hemocyanin (KLH) is extensively used as the immunogen of choice in non-clinical and clinical settings. Native KLH is comprised of high molecular weight (HMW; 4-8 MDa) assemblies of KLH subunit dimers (> 600-800 kDa). It is not known how the different forms (HMW vs subunit) and manufacturing processes (commercial sources) may impact the nature of anti-KLH immune responses (e.g. magnitude and inter-animal variability). Anti-KLH IgM and IgG responses were studied in Sprague-Dawley rats immunized with different forms and commercial sources of KLH: 100 µg of HMW KLH from two different sources or subunit KLH from three different sources. Biophysical and biochemical analyses were conducted to characterize the KLH formulations. Anti-KLH IgM and IgG responses were measured using a proprietary indirect quantitative electrochemiluminescence immunoassay. The HMW KLH preparations showed a greater number of sub-visible particles (2-150 µm size range) than the subunit KLH preparations. All HMW KLH and all subunit KLH were equivalent on SEC (hydrodynamic volume), PAGE (size and charge), and SDS-PAGE (molecular radius). Robust primary and secondary anti-KLH responses were detected for both sources of HMW KLH. The subunit KLH immunizations resulted in lower IgG and IgM responses compared to the HMW KLH, with the exception of Stellar Biotechnologies subunit KLH that produced both robust primary and secondary responses, which approached the HMW KLH responses. Inter-animal variability for IgM and IgG responses was lower with HMW KLH than with subunit KLH. In conclusion, different forms and commercial sources of KLH were associated with different magnitudes and inter-animal variability in IgM and IgG responses, a critical finding to take into consideration when designing TDAR studies for robust immunotoxicology or immunopharmacology testing.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems.

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.

Regulating and assessing risks of cholinesterase-inhibiting pesticides: divergent approaches and interpretations.

This document presents a revised framework for conducting worker and dietary risk assessments for less-than-lifetime exposures to organophosphate or carbamate pesticides based on red blood cell (RBC) or brain acetylcholinesterase (AChE) inhibition or the presence of clinical signs and symptoms. The proposals for appropriate uncertainty factors are based on the biological significance of the cholinesterase (ChE) inhibition noted at the lowest-observed-effect level (LOEL) and the degree of uncertainty in the extrapolation between human and animal data. An extensive evaluation of industry data, not previously summarized, and the available literature indicate that the following risk assessment principles are supportable and protective of human health: Plasma ChE inhibition is not an adverse effect, and therefore should not be utilized in risk assessments. Red blood cell AChE is not associated with the nervous system and inhibition is not per se an adverse (neurotoxic) effect. When available, cholinergic effects or brain AChE inhibition data should take precedence over RBC AChE for determining no-observed-effect levels (NOELs). When available, human RBC AChE inhibition or cholinergic effects data should take precedence over animal data for determining NOELs. Due to the lack of adversity associated with inhibition of RBC AChE, the use of a 10-fold (10x) uncertainty factor from the NOEL is adequate when RBC AChE inhibition data from either animal or human studies are used to assess human risk. Due to greater potential for adversity, NOELs for brain AChE inhibition and cholinergic effects identified in animal studies should receive a default uncertainty factor of 100x; lower uncertainty factors may be used on a case-by-case basis. NOELs based on cholinergic effects noted in human studies should only require a 10x uncertainty factor, since an interspecies extrapolation factor from animals to humans is unnecessary. For RBC and brain AChE activity the threshold for defining a NOEL should be less than or equal to 20% difference from control activity in all species. For risk assessment purposes, duration and route of the study should reflect the expected duration and route of exposure for humans (i.e., a 21-d or 28-d dermal study for subchronic occupational dermal exposure assessment). When dermal data are not available, a subchronic oral toxicity study and an appropriate dermal penetration factor should be used. A general default of 10% absorption should be used, analogous to the United Kingdom and German exposure models that are widely used in Europe. The recommendations in this document are generally consistent with current risk assessment procedures used by Canada, the European Community (EC), and the United Kingdom (UK).

Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease.

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only approximately 65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only approximately 65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington's disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington's disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.

Identification and cloning of a brain autoantigen in neuro-behavioral SLE.

In murine models of SLE, particular patterns of abnormalities of social interaction and memory collectively known as neurobehavioral dysfunction (NBD) correlate with the occurrence of brain reactive autoantibodies. Study of the immunopathogenic effects of these antibodies has been limited by the absence of isolated autoantibodies and antigens. In order to identify the molecular targets, we isolated autoantibodies highly specific for brain plasma membranes from MRL/lpr mice. After immunoscreening a brain expression library with these brain specific autoantibodies, we identified a single cDNA clone of unique sequence and relevant anatomic distribution. Transcript for this cDNA is wide spread among mammalian species but appears to be present only in the brain. Addition features, suggesting this cDNA is pertinent for further study include (1) the expressed protein, called lupus brain antigen 1, reacts with the screening immunoglobulins as well as immunoglobulins from other strains of murine neuro-SLE not used to screen the library, but not with immunoglobulins from normal mice, (2) the transcript distribution within the brain is similar to immunochemical localization of binding of the spontaneous autoantibodies and (3) the localization of transcript within the brain, in the hippocampus, hypothalamus an cingulate gyrus, corresponds to anticipated anatomical regions of clinical dysfunction. Further, the transcript is a large, potentially structural molecule of unique sequence. Antibodies to this molecule may mediate changes in behavior either by direct interactions with the cognate antigen or by indirect influences through neuro-endocrine axes.

NMDA receptor overstimulation triggers a prolonged wave of immediate early gene expression: relationship to excitotoxicity.

Exposure of the rodent striatum to quinolinic acid (QA, N-methyl-D-aspartate receptor agonist) induces immediate early gene (IEG; c-fos, c-jun, jun-B, zif/268) expression that may extend 12-24 h after injection. In order to determine the specificity of the prolonged IEG response to the QA injection, the temporal pattern of c-fos mRNA expression was examined during the first 4 h after administration of saline or QA (40 micrograms). As early as 30 min after intrastriatal injection, both saline and QA increased c-fos mRNA levels. In the saline group, this increase in IEG expression was only transient and returned to baseline by 1 h. In contrast, c-fos mRNA levels within QA-injected animals continued to rise significantly at 1 and 4 h. In a second experiment, rats received 4 ng to 40-micrograms injections of QA followed by sacrifice at 6 h to determine if increasing QA doses caused the appearance of the prolonged IEG response phase. The prolonged IEG response was evident at 6 h only in animal groups that received higher dose ranges (4-40 micrograms) of QA. A final experiment was undertaken to determine if blockage of NMDA receptor stimulation would also inhibit the prolonged IEG response at 6 h in relationship to neuronal sparing evidenced at 24 h post-QA injection. The NMDA receptor antagonist, MK-801, blocked the prolonged IEG response at 6 h following QA (40 micrograms) injection while also preventing striatal neuropeptide mRNA decline by 24 h. Delaying the MK-801 administration for 1-2 h post-QA injection revealed that the intensity of the prolonged IEG mRNA response may be predictive of neuronal demise within the QA lesion site. These results suggest that prolonged IEG expression is associated with QA excitotoxicity of the rodent striatum and subsequent neuronal degeneration.

Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons.

The current report describes the molecular characterization of the human (the D4S234 locus) and mouse (the m234) homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene region. Sequence comparisons of full-length cDNA clones revealed that the mouse and human homologs encoded evolutionarily conserved 21-kDa proteins with greater than 90% amino acid sequence identity. Extensive sequence identity between the D4S234 gene and the rat p1A75 gene (a previously identified rat neuron-specific gene) showed that these genes are interspecies homologs. Furthermore, the D4S234 protein exhibited significant amino acid similarity to a 19-kDa mouse protein that localizes to the Golgi apparatus of embryonic neurons. However, nonconservative sequence differences suggested that these genes are independent members of a multigene family. Northern analyses revealed that rodent D4S234 expression occurred predominantly in the brain and included all brain regions. Neuron-specific expression was demonstrated using Northern analysis of cultured glial cells and quinolinic acid-treated rat brain samples. Minimal amounts of the rodent D4S234 mRNA were detected prenatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins identified an evolutionarily conserved hydrophobic sequence and a consensus nuclear localization signal in both genes. Immunofluorescence microscopy, using an antipeptide antibody, established that the human D4S234 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuronal cytoplasm by in situ hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation of the NTera 2 (NT2) cell line. Following differentiation into postmitotic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic staining and reduced or undetectable nuclear staining in many cells. The variation in the intracellular localization of the D4S234 protein in mitotic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein.

Preprotachykinin and preproenkephalin mRNA expression within striatal subregions in response to altered serotonin transmission.

The effects of lowered serotonin (5-hydroxytryptamine; 5-HT) neurotransmission on preprotachykinin (PPT) and preproenkephalin (PPE) mRNA levels were examined in subregions of the striatum. Adult male rats were treated systemically with para-chlorophenylalanine (pCPA; 350 mg/kg single i.p. injection) which reduced forebrain 5-HT amounts to approximately 20% of saline-injected controls at 24 and 48 h. As measured by Northern analysis, PPT and PPE mRNA levels were elevated 50% and 160% respectively in the anterior ventromedial striatum (region included nucleus accumbens). PPT mRNA levels were raised 90% in posterior striatum (at the level of the globus pallidus) by 48 h post-pCPA injection. To determine if increased PPT and PPE mRNA levels represented a transient response to brief 5-HT inhibition, additional experiments were performed to provide continual suppression of 5-HT within the striatum. First, rats received daily intraperitoneal injections of saline or the 5-HT1A receptor agonist, 8-OH-DPAT (1 mg/kg), for 7 days to reduce 5-HT release from raphestriatal terminals. In a parallel experiment, the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT, 5 micrograms), was stereotaxically injected into the striatum as a means to permanently remove 5-HT terminals. Although levels of each mRNA species were differentially sensitive to 5,7-DHT or 8-OH-DPAT, PPT and PPE mRNAs were lowered between 30-55% within the anterior dorsolateral and ventromedial striatum. Although these results support previous studies suggesting an overall positive regulatory role of serotonin on striatal tachykinin biosynthesis, PPT and PPE gene regulation in certain striatal subregions may by differentially sensitive to lowered 5-HT neurotransmission. This suggestion is supported by observations that acute systemic stimulation of 5-HT2A/C receptors with DOI (7 mg/kg single i.p. injection) raised PPT and PPE mRNA levels within anterior dorsolateral (30-60%) and posterior (100-200%) striata, but not within the anterior ventromedial striatum.

The identification of a functional nuclear localization signal in the Huntington disease protein.

Positional cloning has shown that the Huntington disease (HD) mutation is an expanded trinucleotide repeat in the IT15 gene. Although this mutation clearly produces the HD phenotype, the function of the Huntington disease protein remains undefined. One recent immunocytochemical study suggested that the IT15 protein preferentially localizes to the nucleus of affected neuronal cells. If this result is accurate, it could link the biochemical function of this protein to nuclear activities such as gene regulation. To examine the nuclear transport of the Huntington disease protein, we searched for basic peptide motifs that could produce nuclear localization. One peptide (RRKGKEK) was identified that is highly homologous to a consensus nuclear localization signal. When fused to the cytoplasmic reporter protein, beta-galactosidase, nuclear localization was observed in stably transformed human cell lines. In a complementary study, an anti-peptide polyclonal antibody, raised against a sequence adjacent to the putative nuclear localization sequence, detected the IT15 protein in the nucleus of human cells. These results extend and confirm the previous localization studies and identify an IT15 peptide motif that can function for nuclear localization.

Transcription of the Huntington disease gene during the quinolinic acid excitotoxic cascade.

Although Huntington disease (HD) is characterized by the selective neurodegeneration of the basal ganglia and cerebral cortex, efforts to define the disease pathology have been complicated by the widespread expression of the disease gene (IT15) throughout the body. In this study, we examined IT15 mRNA levels during the quinolinic acid (QA) excitotoxic cascade to determine whether neuronal and/or glial expression is regulated by neurodegeneration. Following an initial increase between 1 h and 6 h, IT15 mRNA levels declined in a pattern homologous to a group of neuron-specific genes. Decreased mRNA levels after 24 h demonstrated that glial transcription is not activated by neurodegeneration or gliosis. The 1 h and 24 h mRNA levels strongly suggest that IT15 transcription preferentially localizes to degenerating neurons.

The mouse plasma membrane Ca2+ pump isoform 1 promoter: cloning and characterization.

The expression of plasma membrane Ca2+ pump (PMCA) is regulated by various hormones or agonists via multiple second messenger pathways. Two different 5' segments of the PMCA1 gene (isoform 1) were cloned from a mouse genomic library. While one segment contained the 3' end of intron 1 and exon 2, the other segment was found to encompass the 5'-flanking region of the gene, exon 1, and the 5' portion of intron 1. Sequence analysis of the 5'-flanking region suggested the presence of the putative promoter. Four sites for initiation of transcription (spanning 64 bp) were identified by RNase protection assay and primer extension analysis. The promoter region was very GC-rich, contained no "TATA box," but had a "CAAT box" at -51. Comparison of sequence with known cis-regulatory motifs disclosed that the 5'-flanking region has a number of potential regulatory elements including an AP-1 site at -354, AP-2 binding sites at -267 and -123, Sp1 binding sites at -127, -111, and +3, and a cyclic AMP response element binding protein site at -67. To demonstrate promoter activity, a segment containing 611 bp of the promoter region (from -442 to +169) was subcloned in front of a promoterless chloramphenicol acetyltransferase (CAT) gene. This segment was able to drive the expression of chloramphenicol acetyltransferase in transient transfections of mouse (or human) neuroblastoma cells as well as rat aortic endothelial cells. Deletion analysis demonstrated that a fragment from -256 to +169 showed strong promoter activity, while a fragment from -117 to +169 had CAT activity that was not different from the vector control. The promoter was stimulated threefold by phorbol ester and twofold by cyclic AMP. These results provide further proof indicating up-regulation of the PMCA1 gene by multiple second messenger pathways.

A Sau3A polymorphism in the 5' end of the IT15 gene that nonrandomly segregates with the Huntington disease trinucleotide expansion.

Genomic clones encompassing the Huntington disease (HD) mutation were used to isolate a probe that detects size changes in the restriction fragments that contain the HD trinucleotide repeat (TNR). This probe also detects a frequent Sau3A polymorphism (allele sizes 1.8-kb and 2.7kb), which maps approximately 950bp from the TNR. Examination of a number of HD families established that the frequency of the Sau3A alleles did not differ significantly between control and HD populations; however, the HD expansion was always present on a chromosome that contained the 1.8-kb Sau3A allele. This association between a specific allele and the HD TNR expansion was significant and could provide a clue to the chromosomal elements that produce the trinucleotide expansion on the Huntington disease chromosome.

Immediate early gene activation during the initial phases of the excitotoxic cascade.

Direct brain injections of the N-methyl-D-aspartate receptor agonist quinolinic acid (QA) trigger an excitotoxic cascade characterized by rapid neuronal death and glial/immune cell activation. The present study compared the timing of immediate early gene (IEG; c-fos, c-jun, jun-B, and zif/268) induction with the response of neuronal transcripts during the first 24 hr of a QA lesion within the rodent striatum. Following QA exposure, IEG mRNA induction periods extended from 30 min to 24 hr. Several characteristics of this prolonged transcriptional response suggest that separate cell populations (neuronal vs. glial) originate individual IEG phases during the first day of the lesion. The first IEG phase was rapid and peaked at 60 min. This initial IEG phase, likely neuronal in origin, was dominated by robust increases in the expression of c-fos, jun-B, and zif/268 mRNAs in contrast to small increases in c-jun expression. A second, delayed IEG phase was initiated after the first hour and extended to 24 hr. This IEG phase was more intense and continued beyond the period of neuronal survival as detected by the loss of neurotransmitter-specific mRNAs (preprotachykinin, preproenkephalin, and glutamic acid decarboxylase). During this phase, c-jun mRNA levels coordinately increased with c-fos. Interestingly, the transcriptional peak of the delayed IEG phase occurred between 4 and 12 hr, the time which corresponded to the rapid decline of neuronal transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Timing the excitotoxic induction of heat shock protein 70 transcription.

The time course of heat-shock protein 70 (HSP70) transcriptional induction was compared with neuronal- and non-neuronal-specific mRNAs following intrastriatal quinolinate (QA) injection. Within one hour of QA exposure, immediate-early gene (IEG; c-fos) activation preceded slight increases in glutamic acid decaroxylase (GAD) mRNA levels. However, glial fibrillary acid protein (GFAP) and HSP70 mRNA levels remained constant. After one hour, HSP70 mRNA levels surged 6 fold during a delayed transcriptional phase that was induced between 4-12 hours. This phase was characterized by massive increases in c-fos and GFAP mRNAs while GAD transcripts fell drastically suggestive of neuronal death. Therefore HSP70 genes may play an important role in glial/immune activation following rapid excitotoxic damage by direct injections of quinolinate.

An estimate of the number of genes in the Huntington disease gene region and the identification of 13 transcripts in the 4p16.3 segment.

Physical mapping and genetic linkage studies have positioned the Huntington disease (HD) gene to a relatively large genomic region in the distal portion of the short arm of human chromosome 4 (4p16.3). To estimate the number of genes present in this region and to identify candidate disease genes, several clones that map to the 4p16.3 segment have been examined for clusters of CpG-rich restriction sites and transcribed sequences. Thirteen expressed sequences were identified and were shown by pulsed-field gel electrophoresis not to cluster into a small segment of the 4p16.3 band. The frequency of transcripts in these clones suggests that the putative HD gene region contains about 100 genes.

Simultaneous reduction of the sarcolemmal and SR calcium ATPase activities and gene expression in cardiomyopathic hamster.

Altered calcium regulation is a prominent feature in the hereditary cardiomyopathy of the Syrian hamster. However, the activity of the two systems necessary for intracellular calcium homeostasis in the heart, the sarcolemmal and sarcoplasmic reticulum calcium ATPase pumps, have not been correlated. Using age- and pair-matched myopathic and control hamsters, a simultaneous reduction in gene expression and enzyme activity for these two pumps has been demonstrated. The concomitant alteration in gene expression as early as 1 month of age, preceding noticeable myocytolysis suggests that the depressed activity in these two calcium ATPase systems is not due to cell necrosis but at least in part due to reduction in their mRNA levels. Reduced capacity of the calcium pumps would result in calcium overload as well as impaired contractility that leads to the eventual heart failure in this animal model.

Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump.

Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.