Toxicokinetics and toxicity of atorvastatin in dogs.

HMG-CoA reductase inhibitors (e.g., statins) are an important clinical option to lower cholesterol and treat co-morbidities. Atorvastatin is the most prescribed statin and has obtained generic status. We recently had a clinical development program evaluating a combination of atorvastatin with a GPR119 agonist as a treatment for dyslipidemia, where toxicological evaluations in dogs were completed. There were several challenges related to selecting doses for atorvastatin, including understanding the dose-exposure relationship from different drug forms used by the innovator in their general toxicology studies, bioanalytical assays that did not separate and quantify parent from metabolites, and high variability in the systemic exposures following oral dosing. The studies in this report characterized the toxicokinetics and toxicity of atorvastatin in the dog for up to 13-weeks. Overall, there were no notable differences in the toxicokinetics of atorvastatin or the two active hydroxylated metabolites between the sexes at Week 13. However, systemic exposures were markedly lower at Week 13 compared to that observed at Week 4, suggesting induction of metabolism or reduced absorption from the gastrointestinal tract following oral dosing. Changes in laboratory chemistries included increased liver enzyme levels and lower cholesterol levels. Histopathologic evaluation revealed multifocal minimal to slight hemorrhages in the submucosa of the gallbladder; all findings were reversible. The information from these studies along with the existing clinical experience with atorvastatin can be used to design robust toxicology studies in dogs and reduce animal use.

Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position.

Drug interactions due to efflux transport inhibition at the blood-brain barrier (BBB) have been receiving increasing scrutiny because of the theoretical possibility of adverse central nervous system (CNS) effects identified in preclinical studies. In this review, evidence from pharmacokinetic, pharmacodynamic, imaging, pharmacogenetic, and pharmacovigilance studies, along with drug safety reports, is presented supporting a low probability of modulating transporters at the human BBB by currently marketed drugs.

Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium.

The International Transporter Consortium (ITC) organized a second workshop in March 2012 to expand on the themes developed during the inaugural ITC workshop held in 2008. The final session of the workshop provided perspectives from regulatory and industry-based scientists, with input from academic scientists, and focused primarily on the decision trees published from the first workshop. These decision trees have become a central part of subsequent regulatory drug-drug interaction (DDI) guidances issued over the past few years.

Intracellular drug concentrations and transporters: measurement, modeling, and implications for the liver.

Intracellular concentrations of drugs and metabolites are often important determinants of efficacy, toxicity, and drug interactions. Hepatic drug distribution can be affected by many factors, including physicochemical properties, uptake/efflux transporters, protein binding, organelle sequestration, and metabolism. This white paper highlights determinants of hepatocyte drug/metabolite concentrations and provides an update on model systems, methods, and modeling/simulation approaches used to quantitatively assess hepatocellular concentrations of molecules. The critical scientific gaps and future research directions in this field are discussed.

Rational use of in vitro P-glycoprotein assays in drug discovery.

P-glycoprotein (Pgp) affects the absorption, distribution, and clearance of a variety of compounds. Thus, identification of compounds that are Pgp substrates can aid drug candidate selection and optimization. Our goal was to evaluate three assays used to determine whether compounds are Pgp substrates. Sixty-six compounds were tested in monolayer efflux, ATPase, and calcein-AM assays. Assay results yielded two categories of compounds. Category I (n = 35) exhibited concordance across the assays. Category II (n = 31) revealed differences among the assays that related to the apparent permeability (P(app)) of the compounds. Within category II, two groups were discerned based on the absence (group IIA, n = 10, nontransported substrates) or presence (group IIB, n = 21, transported substrates) of monolayer efflux. Detection of efflux (group IIB) was associated with compounds having low/moderate P(app) values (mean = 16.6 nm/s), whereas inability to detect efflux (group IIA) was associated with compounds having high P(app) values (mean = 535 nm/s). The calcein-AM and ATPase assays revealed Pgp interactions for highly permeable group IIA compounds but were less responsive than monolayer efflux for low/moderate P(app) compounds of group IIB. All assays detected substrates across a broad range of P(app), but the efflux assay was more prone to fail at high P(app), whereas the calcein-AM and ATPase assays were more prone to fail at low P(app). When P(app) is low, efflux is a greater factor in the disposition of Pgp substrates. The efflux assay is more reliable at low/moderate P(app) and is the method of choice for evaluating drug candidates despite low throughput and reliance on liquid chromatography with tandem mass spectrometry.

Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors.

P-Glycoprotein (Pgp) and cytochrome P450 3A (CYP3A) are important enzymes affecting the disposition of HIV protease inhibitors (HIV PIs). After multiple dosing experiments in rats, decreases in the plasma concentrations and area under plasma concentration-time curve (AUC) for HIV PIs have been observed. The purpose of these studies was to determine the changes in Pgp and CYP3A expression and HIV PI plasma exposure after multiple doses of HIV PIs. Male rats were orally dosed with an amprenavir prodrug (450 mg/kg/day amprenavir-equivalent) or nelfinavir (175 mg/kg/day) for 1 or 14 days. Relative to day 1, the C(max) and the AUC for amprenavir at day 14 were decreased by 33 and 51%, respectively. Similarly, the plasma concentration of nelfinavir at 1 h after the last dose (C(max)) was reduced by 52% after multiple doses. Compared with controls, dosing of amprenavir for 14 days increased intestinal Pgp and hepatic CYP3A protein levels by 59 and 151%, respectively, but did not alter intestinal CYP3A protein levels. In contrast, amprenavir treatment did not result in an increase in hepatic CYP3A activity. Nelfinavir treatment increased expression of intestinal Pgp and hepatic CYP3A levels by 83 and 85%, respectively, but not hepatic Pgp or intestinal CYP3A. HIV PIs also induced Pgp expression in the LS174T human intestinal cell line. These results indicate that HIV protease inhibitors induce both intestinal Pgp and hepatic CYP3A and suggest that induction of Pgp and CYP3A is a possible mechanism reducing drug exposure after multiple doses.

Prospective CYP2D6 genotyping as an exclusion criterion for enrollment of a phase III clinical trial.

A phase III study was performed to compare the efficacy and safety of lamotrigine (Lamictal), desipramine (Norpramin), and placebo in the treatment of unipolar depression. Desipramine is extensively metabolized by cytochrome P450 2D6 (CYP2D6), and kinetics of this compound are altered in poor metabolizers. Genotyping was utilized to exclude poor metabolizers in order to increase subject safety and to eliminate the need to continuously monitor plasma desipramine levels. As part of screening, subjects were genotyped for the *3(A), *4(B), and *5(D) alleles, which identify approximately 95% of poor metabolizers. Extensive metabolizers were eligible for randomization to the lamotrigine, desipramine, or placebo arm. Follow-up genotyping for the *6(T) and *7(E) alleles was performed after study enrollment and was used to identify poor metabolizers who may have been incorrectly identified as extensive metabolizers upon initial three-allele screening. Of 628 subjects screened for *3(A), *4(B), *5(D) alleles, 590 (93.9%) were classified as extensive metabolizers. The remaining 38 (6.1%) subjects were poor metabolizers and excluded. Subsequent *6(T) and *7(E) testing revealed that two poor metabolizers had been enrolled, and the follow-up genotyping provided an explanation for the high desipramine plasma concentrations in one subject. No differences in phenotypic or allelic frequencies were found between the study population and literature populations. However, the frequency of poor metabolizers varied among clinical sites (0-15%). For a compound that is extensively metabolized by CYP2D6, prescreening subjects for *3(A), *4(B), *5(D), *6(T) and *7(E) alleles can increase subject safety and eliminate the need to continuously monitor drug plasma concentrations.

Influence of passive permeability on apparent P-glycoprotein kinetics.

PURPOSE: The objectives of this work were to evaluate the importance of moderate passive permeability on apparent P-glycoprotein (P-gp) kinetics, and demonstrate that inspection of basolateral to apical and apical to basolateral (BL-AP/AP-BL) permeability ratios may result in a compound being overlooked as a P-gp substrate and inhibitor of another drug's transport via P-gp inhibition. METHODS: The permeability ratios of nicardipine, vinblastine, cimetidine, and ranitidine were determined across Caco-2 monolayers that express P-gp, in the presence and absence of the specific P-gp inhibitor, GF120918. In addition, the permeability ratio of vinblastine was studied after pretreatment of Caco-2 monolayers with nicardipine, ranitidine, or cimetidine. Similar studies were repeated with hMDRI-MDCK monolayers. RESULTS: The permeability ratios for cimetidine and vinblastine were >2. The permeability ratios for nicardipine and ranitidine were close to unity, and were not affected by the addition of GF120918. Based solely on ratios, only compounds with moderate transcellular permeability (vinblastine and cimetidine) would be identified as P-gp substrates. Although the permeability ratios appeared to be unity for nicardipine and ranitidine, both compounds affected the permeability of vinblastine, and were identified as substrates and inhibitors of P-gp. Studies performed in hMDR1-MDCK cells confirmed these experimental results. Data were explained in the context of a kinetic model, where passive permeability and P-gp efflux contribute to overall drug transport. CONCLUSIONS: Moderate passive permeability was necessary for P-gp to reduce the AP-BL drug permeability. Inspection of the permeability ratio after directional transport studies did not effectively identify P-gp substrates that affected the P-gp kinetics of vinblastine. Because of the role of passive permeability, drug interaction studies with known P-gp substrates, rather than directional permeability studies, are needed to elucidate a more complete understanding of P-gp kinetics.

P-glycoprotein-mediated transport of morphine in brain capillary endothelial cells.

Cell accumulation, transendothelial permeability, and efflux studies were conducted in bovine brain capillary endothelial cells (BBCECs) to assess the role of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) transport of morphine in the presence and absence of P-gp inhibitors. Cellular accumulation of morphine and rhodamine 123 was enhanced by the addition of the P-gp inhibitors N-{4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquinolinyl)-ethyl]-phenyl}-9,10-dihydro-5-methoxy-9- carboxamide (GF120918), verapamil, and cyclosporin A. Positive (rhodamine 123) and negative (sucrose and propranolol) controls for P-gp transport also were assessed. Morphine glucuronidation was not detected, and no alterations in the accumulation of propranolol or sucrose were observed. Transendothelial permeability studies of morphine and rhodamine 123 demonstrated vectorial transport. The basolateral to apical (B:A) fluxes of morphine (50 microM) and rhodamine (1 microM) were approximately 50 and 100% higher than the fluxes from the apical to the basolateral direction (A:B), respectively. Decreasing the extracellular concentration of morphine to 0.1 microM resulted in a 120% difference between the B:A and A:B permeabilities. The addition of GF120918 abolished any significant directionality in transport rates across the endothelial cells. Efflux studies showed that the loss of morphine from BBCECs was temperature- and energy-dependent and was reduced in the presence of P-gp inhibitors. These observations indicate that morphine is transported by P-gp out of the brain capillary endothelium and that the BBB permeability of morphine may be altered in the presence of P-gp inhibitors.

Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor.

PURPOSE: To determine the role of P-glycoprotein (Pgp) on the CNS penetration of the HIV protease inhibitor (PI) amprenavir (141W94) and to test the hypothesis that co-administration of a second HIV PI (ritonavir) could enhance amprenavir's brain penetration in vivo. METHODS: Pgp-mediated efflux was investigated in vitro with Caco-2 cells and in vivo by whole-body autoradiography (WBA). "Genetic" mdr1a/1b double knockout mice, "chemical" Pgp knockout mice generated by administration of the Pgp inhibitor GF120918, and mice pretreated with ritonavir were used in WBA studies to investigate the effects of Pgp modulation on the CNS penetration of amprenavir. RESULTS: Amprenavir, indinavir, ritonavir, and saquinavir had 2- to 23-fold higher transport rates from the basolateral to apical direction than from the apical to basolateral direction across Caco-2 monolayers. Incubation with GF120918 negated this difference, suggesting that the efflux was Pgp-mediated. WBA studies demonstrated a 13- and 27-fold increase in the brain and a 3.3-fold increase in the CSF concentrations of amprenavir in mice pretreated with GF120918 and in mdr1a/1b double knockout mice. In contrast, pretreatment with ritonavir did not alter the CNS exposure of amprenavir. CONCLUSIONS: These results provide evidence that amprenavir and other HIV PIs are Pgp substrates and that co-administration of a specific Pgp inhibitor will enhance amprenavir's CNS penetration in vivo. These results will have an important therapeutic impact in the treatment of AIDS dementia.

Expression of a calmodulin-dependent phosphodiesterase isoform (PDE1B1) correlates with brain regions having extensive dopaminergic innervation.

Cyclic nucleotide-dependent protein phosphorylation plays a central role in neuronal signal transduction. Neurotransmitter-elicited increases in cAMP/cGMP brought about by activation of adenylyl and guanylyl cyclases are downregulated by multiple phosphodiesterase (PDE) enzymes. In brain, the calmodulin (CaM)-dependent isozymes are the major degradative activities and represent a unique point of intersection between the cyclic nucleotide- and calcium (Ca2+)-mediated second messenger systems. Here we describe the distribution of the PDE1B1 (63 kDa) CaM-dependent PDE in mouse brain. An anti-peptide antiserum to this isoform immunoprecipitated approximately 30-40% of cytosolic PDE activity, whereas antiserum to PDE1A2 (61 kDa isoform) removed 60-70%, demonstrating that these isoforms are the major CaM-dependent PDEs in brain. Quantification of PDE1B1 immunoreactivity on immunoblots indicated that striatum contains 3-17-fold higher levels of PDE1B1 than other brain regions, with lowest immunoreactivity in cerebellum. In situ hybridization demonstrated high levels of PDE1B1 mRNA in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Moderate mRNA levels were observed in dentate gyrus, cerebral cortex, medial thalamic nuclei, and brainstem, whereas negligible mRNA was detectable in the globus pallidus, islands of Calleja, substantia nigra, and ventral tegmental area. Immunocytochemistry confirmed that the majority of PDE1B1 protein was localized to the caudate-putamen, nucleus accumbens, and olfactory tubercle. Within the caudate-putamen, PDE1B1 immunoreactivity was ubiquitous, while PDE1A2 immunostaining was restricted to a minor subset of striatal neurons. The expression of PDE1B1 protein and mRNA correlate strongly with areas of the brain that are richest in dopaminergic innervation; indeed, there are strikingly similar distributions for PDE1B1 and D1 dopamine receptor mRNAs. Since D1 receptor binding activates adenylyl cyclase, and striatal neurons lack CaM-sensitive forms of cyclase, the high amount of this PDE implies an important physiological role for Ca(2+)-regulated attenuation of cAMP-dependent signaling pathways following dopaminergic stimulation.

Molecular cloning of DNA encoding a calmodulin-dependent phosphodiesterase enriched in striatum.

A murine cDNA for the 63-kDa calmodulin-dependent phosphodiesterase (CaM-PDE), PDE1B-1, was isolated by using polymerase chain reaction with degenerate primers followed by the cloning of a full-length cDNA from a whole-brain phage library. The nucleotide sequence of 2986 base pairs contains an open reading frame encoding a protein of 535 amino acids (M(r) = 61,231) with a predicted isoelectric point of 5.54. The deduced protein sequence shows approximately 60% identity with that of the 61-kDa isoform (PDE1A2), consistent with the proposal that these proteins arise from two separate genes [Novack, J. P., Charbonneau, H., Bentley, J. K., Walsh, K. A. & Beavo, J. A. (1991) Biochemistry 30, 7940-7947]. Southern blot analysis suggests high nucleotide-sequence conservation of the PDE1B1 gene among mammalian and avian species. A single approximately 3600-nucleotide mRNA transcript was seen in all brain regions, with striatum containing 4- to 30-fold higher levels than other areas. In nonneural tissues, low amounts of PDE1B1 mRNA were detected in lung, spleen, thymus, and testis; hybridization to several larger mRNA species was also seen in thymus and testis. By using nucleic acid probes for PDE1B1, the mechanisms that control its highly selective gene expression can now be studied at the molecular level.

Distribution and expression of SNAP-25 immunoreactivity in rat brain, rat PC-12 cells and human SMS-KCNR neuroblastoma cells.

Immunocytochemical, immunoblotting and in situ hybridization studies were used to map the distribution of SNAP-25 protein and mRNA in the rodent nervous system. These experiments demonstrated that subsets of neurons expressed SNAP-25, and that several patterns of expression emerged: SNAP-25 expression in caudate nucleus was initially concentrated in axons, which subsequently was localized in presynaptic regions of these axons. Other regions, typified by neocortex, showed developmental increases and persistent adult neuronal immunoreactivity for SNAP-25. Finally, olfactory bulb contained neurons which initially expressed SNAP-25, but lost expression during maturation. Additional studies in cultured human and rat cell lines derived from neural crest suggested that SNAP-25 is expressed in such lines, but not in glial or fibroblast lines. Differentiation of rat PC-12 cells with nerve growth factor failed to alter steady-state levels of SNAP-25 protein; similar responses were seen in human SMS-KCNR neuroblastoma cells differentiated using retinoic acid. The presence of SNAP-25 in presynaptic regions of numerous neuronal subsets and in neural crest cell lines suggests that this protein subserves an important function in neuronal tissues.

Expression of the calmodulin-dependent protein phosphatase, calcineurin, in rat brain: developmental patterns and the role of nigrostriatal innervation.

The distribution of neurons expressing the calmodulin-dependent protein phosphatase, calcineurin (CN) was characterized in developing and adult rat brain using a combination of immunocytochemical, immunoblot and in situ hybridization approaches. Immunoblot analysis revealed a strong increase postnatally in CN protein expression. Four differently-charged isoforms of CN were observed in adult brain with apparent regional differences in isoform expression. Immunocytochemistry showed highest levels of CN in hippocampus, striatum, substantia nigra, amygdala and septal nuclei with immunoreactivity first appearing in striatum and septal nuclei, followed by hippocampus, neocortex and limbic structures. In situ hybridization demonstrated that mRNA for the catalytic subunit of CN was seen as early as postnatal day (PND) 1 in striatum, cortex and hippocampus. Since immunoreactivity was not detectable until day 4, this suggests that mRNA expression may precede that of protein by several days in these regions. Lesioning of developing and adult nigrostriatal dopamine neurons either with 6-hydroxydopamine or by surgical hemitransection had little effect on expression of CN, suggesting that CN expression is not influenced transsynaptically by dopamine. Collectively, these findings demonstrate that CN protein and mRNA expression are subject to regional and temporal control during brain development suggesting that specific synaptic connections may influence CN gene expression. However, in striatum, dopaminergic innervation does not appear to affect CN levels.

Expression of calmodulin-dependent enzymes in developing rat striatum is not affected by perturbation of dopaminergic systems.

Transsynaptic regulation is one mechanism that controls expression of several calmodulin (CaM)-dependent enzymes. This observation and the demonstration that expression of several CaM-dependent enzymes in developing striatum occurred with a spatial and temporal pattern similar to that seen for dopamine and tyrosine hydroxylase suggested that the nigrostriatal pathway may influence the expression of CaM-binding proteins (CaM-BPs) during striatal development. Therefore, the possible role of nigrostriatal dopamine systems regulating the expression of CaM-dependent enzymes was studied in Sprague-Dawley rats by using surgical hemitransections of brain, 6-hydroxydopamine lesions, and chronic haloperidol treatments. Alterations in CaM-BP expression following perturbation of the developing nigrostriatal tract were analyzed by using immunoblots, biotinylated CaM overlays, and enzyme assays. The extent of nigrostriatal lesions was assessed by using depletion of immunoreactive tyrosine hydroxylase levels in striatum. All three experimental paradigms failed to alter the normal developmental expression of CaM-dependent enzymes. From these results we conclude that the increased expression of CaM-dependent enzymes during striatal development is not directly dependent on synaptic input from the nigrostriatal dopamine system.

Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit.

A cDNA for the catalytic subunit of a calmodulin (CaM)-dependent protein phosphatase was cloned from Neurospora crassa. The open reading frame of 1557 base pairs encoded a protein of Mr approximately 59,580 and was followed by a 3'-untranslated region of 363 base pairs including the poly(A) tail. Based on primer extension analysis, the mRNA transcript in vivo was 2403 base pairs. Expression of this CaM-protein phosphatase mRNA was developmentally regulated, being highest during early mycelial growth; production of the corresponding protein followed mRNA with a time lag of 8-12 h. Polymerase chain reaction amplification of genomic DNA revealed three small introns, the positions of which coincided with those in the mouse gene, indicating evolutionary conservation of these structures. The deduced sequence showed approximately 75% identity with the mammalian homologue, calcineurin, in aligned regions. A region of 40 amino acids preceding the CaM-binding domain was essentially unchanged, suggesting conservation of a crucial interaction site. Three small segments in the carboxyl half of the protein were unrelated to the mammalian gene and may constitute "variable regions" that confer substrate specificity to the enzyme. An active recombinant catalytic subunit was expressed in bacteria and purified by CaM-Sepharose chromatography. This preparation was stimulated 2- 3-fold by CaM and showed a p-nitrophenol phosphatase activity equal to that of the bovine brain holoenzyme, although its dephosphorylation of phosphoprotein substrates was markedly different. These findings demonstrate that the catalytic subunit of this phosphatase can exhibit high activity in the absence of its intrinsic Ca(2+)-binding subunit.

Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) in rat brain.

The developmental expression and subcellular distribution of the neuron-specific 25-kDa synaptosomal protein (SNAP-25) were investigated by using Northern (RNA) blots, immunoblots, and immunocytochemistry. Both SNAP-25 protein and mRNA were present at low levels in embryonic day 15 rat brain, and levels of both increased during early postnatal maturation. Developmental immunoblots with antipeptide antisera demonstrated that a 25-kDa peptide was the major isoform in brain, and this form increased steadily from embryonic day 15 through adulthood. A second 27-kDa immunoreactive isoform was present in brain only during early development. Immunoblots of two-dimensional SDS/polyacrylamide gels revealed the presence of a predominant 25-kDa isoform of SNAP-25 in adult brain. Immunocytochemical studies indicated that as immunoreactivity for SNAP-25 increased during development, the cellular localization of SNAP-25 immunoreactivity concomitantly shifted from axons and cell bodies to presynaptic terminals. These data suggest that the SNAP-25 protein shifts in subcellular localization during development and may play a role in the establishment and stabilization of specific presynaptic terminals in brain.

Preparation, characterization and biological properties of biotinylated derivatives of calmodulin.

Biotinylated derivatives of calmodulin (CaM) were prepared and their biological properties characterized by using enzyme assays, affinity and hydrophobic-interaction chromatography. Several N-hydroxysuccinimidobiotin derivatives [sulphosuccinimidobiotin (sulpho-NHS) and sulphosuccinimido-6-(biotinamido)hexanoate (BNHS-LC)] differing in spacer arm length were used to modify CaM. The shorter-spacer-arm CaM derivative (sulpho-CaM) activated CaM-dependent cyclic nucleotide phosphodiesterase and CaM-dependent protein kinase II; preincubation with avidin blocked its ability to activate these enzymes. The extended-spacer-arm derivative (BNHS-LC-CaM) activated CaM-dependent enzymes both in the presence and in the absence of avidin, suggesting that the longer spacer arm diminished steric effects from avidin preincubation. Other biotinylated CaM derivatives were prepared with biotinylated tyrosine and/or histidine residues (diazobenzoylbiocytin; DBB-CaM) or nucleophilic sites (photobiotin acetate; photo-CaM). These derivatives activated CaM-dependent enzymes in the presence and in the absence of avidin. Oriented affinity columns were constructed with covalently immobilized avidin complexed to each biotinylated CaM derivative. The chromatographic profiles obtained revealed that each column interacted with a specific subset of CaM-binding proteins. Elution profiles of biotinyl CaM derivatives on phenyl-Sepharose hydrophobic-interaction chromatography suggested that several derivatives displayed diminished binding to the matrix in the presence of Ca2+. Development and characterization of a series of biotinylated CaM molecules can be used to identify domains of CaM that interact with specific CaM-dependent enzymes.

Developmental expression of calmodulin-dependent cyclic nucleotide phosphodiesterase in rat brain.

The patterns of expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) have been studied in developing and adult rat brain using affinity-purified polyclonal antibodies against CaM-PDE. An immunocytochemical map of adult brain regions expressing CaM-PDE, constructed from serial coronal brain sections, illustrated that CaM-PDE was expressed in specific neuronal subpopulations throughout the adult rat brain. Immunoblot analysis coupled with subcellular fractionation indicated that CaM-PDE was primarily localized to cytoplasmic fractions, with a small amount associated with synaptosomal membranes. Immunoblots from developing brain indicated that CaM-PDE expression increased dramatically during postnatal days 7-20 (PND 7-20); parallel increases in CaM-PDE enzyme activity occurred during this same time. Immunocytochemical studies indicated that several distinct patterns of CaM-PDE expression occurred during development. Neocortex showed low levels of CaM-PDE immunoreactivity in neuronal somata of layers III, V and VI on PND 4 that increased by PND 11; the adult somatodendritic pattern of immunoreactivity was observed by PND 60. Similar patterns were observed in cerebellar Purkinje cells, with somatodendritic staining observed by PND 12. By contrast, caudate-putamen, the inferior olive and the hypoglossal nuclei expressed high levels of CaM-PDE on PND 4, with levels considerably lower in the adult animal. The different patterns of expression suggest that in neocortex and cerebellum, CaM-PDE increases during the period of neuronal differentiation and active synaptogenesis, while in the caudate-putamen, inferior olive and hypoglossal nucleus, high levels may be required early in development.

Developmental expression of neuronal calmodulin-binding proteins in rat brain.

The developmental patterns of calmodulin-binding proteins (CaM-BPS) in rat brain were examined using biotinylated calmodulin overlays of one- and two-dimensional gels. Hippocampus showed the earliest onset of CaM-BP expression (postnatal day 5; PND5), followed by cerebral cortex and striatum, both of which had detectable levels of CaM-BPs by PND7. Cerebellum had the latest onset of CaM-BP expression; CaM-BPs were not detectable until PND9. Very few CaM-BPs were present in brain before PND5 and all regions reached near adult levels by PND20. However, several unique CaM-BPs were seen in embryonic brain and these proteins may have an important role in developing neurons. These data suggest an orderly, complex expression of CaM-BPs which increases during times of synaptogenesis and synaptic maturation.