Fredrik Kamme

Johnson & Johnson, New Brunswick, New Jersey, United States

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Publications (17)69.52 Total impact

  • Journal of Biological Chemistry 08/2009; 284(31):le6. · 4.65 Impact Factor
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    ABSTRACT: Functional alterations in striatal projection neurons play a critical role in the development of motor symptoms in Parkinson's disease (PD), but their molecular adaptation to dopamine depletion remains poorly understood. In particular, type and extent of regulation in postsynaptic signal transduction pathways that determine the responsiveness of striatal projection neurons to incoming stimuli, are currently unknown. Using cell-type-specific transcriptome analyses in a rodent model of chronic dopamine depletion, we identified large-scale gene expression changes, including neurotransmitter receptors, signal transduction cascades, and target proteins of dopamine signaling in striatonigral and striatopallidal neurons. Within the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) cascade of enzymes that plays a central role in signal integration of dopaminoceptive neurons multiple catalytic and regulatory subunits change their mRNA expression levels. In addition to the number of genes the fact that the alterations occur at multiple levels stresses the biological relevance of transcriptional regulation for adaptations of postsynaptic signaling pathways. The overall pattern of changes in both striatonigral and striatopallidal neurons is compatible with homeostatic mechanisms. In accordance with the distinct biological effects of dopamine D(1) and D(2) receptor stimulation, the alterations of the transcriptional profiles most likely result in prodopaminergic phosphorylation patterns. Our data provide insight into the disease-related plasticity of functional genomic networks in vivo that might contribute to the protracted preclinical phase of PD. In addition, the data have potential implications for the symptomatic treatment of the disease.
    Journal of Neuroscience 06/2009; 29(21):6828-39. · 6.91 Impact Factor
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    ABSTRACT: Motor symptoms in Parkinson's disease (PD) are associated with complex changes of firing properties in basal ganglia output neurons (BGON). The abnormalities are generally attributed to altered synaptic input and potential post-synaptic mechanisms are currently unknown. Our cell-type selective transcriptome analyses of BGON in the rat 6-hydroxydopamine (6-OHDA) model of PD identified the ion channel HCN3 as a likely contributor to altered neuronal excitability. Quantitative PCR experiments confirmed the HCN3 upregulation in the rat and mouse 6-OHDA models and also demonstrated selectivity of the effect for HCN3. In accordance with the mRNA expression data, in vitro whole cell patch-clamp recordings in BGON showed increased HCN3 current amplitudes and increased rebound excitability in BGON of 6-OHDA treated rats. These data establish HCN3 up-regulation as a novel candidate mechanism that might contribute to the in vivo changes of electrical activity in basal ganglia output neurons of the parkinsonian brain.
    Neurobiology of Disease 05/2009; 34(1):178-88. · 5.62 Impact Factor
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    ABSTRACT: Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.
    Journal of Biological Chemistry 01/2009; 284(5):2811-22. · 4.65 Impact Factor
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    ABSTRACT: Relaxin-3 is a potent agonist for both G-protein coupled receptors (GPCR) RXFP3 (also known as GPCR135) and RXFP4 (also known as GPCR142) while insulin-like peptides 5 (INSL5) is a selective RXFP4 agonist. INSL5 is also a weak (low affinity) RXFP3 antagonist. RXFP3 and RXFP4 share about 50% homology. We have used gain-of-function (RXFP3 --> RXFP4) and loss-of-function (RXFP4 --> RXFP3) chimeras to identify the domains critical for the binding and activation induced by INSL5. Replacing extracellular loop (EL) 1 or EL3 of RXFP3 with the corresponding domains from RXFP4 does not change the RXFP3 pharmacological profile. Exchanging the N-terminus and EL2 of RXFP3 with these of RXFP4 results in a chimeric receptor (CR5) with a high affinity for INSL5. However, in contrast to native RXFP4, INSL5 does not elicit an agonist response from CR5. Conversely, replacing the N-terminus and EL2 of RXFP4 with counterparts from RXFP3 (CR15) results in a chimeric receptor for which relaxin-3 and INSL5 are high and low affinity agonists, respectively. Further mutagenesis studies indicate that transmembrane (TM) domains 2, 3 and 5 of RXFP4 are critical determinants of functional receptor activation by INSL5. Replacement of TM2, 3, and 5 of RXFP3 with equivalent domains from RXFP4 results in a chimeric receptor that can be activated by INSL5. These results suggest that the N-terminus and EL2 domains of RXFP3 and RXFP4 are involved in ligand binding while TM2, 3, and 5 are critical for receptor activation.
    European Journal of Pharmacology 05/2008; 590(1-3):43-52. · 2.59 Impact Factor
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    ABSTRACT: Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BDelta23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BDelta23-27)R/I5 binds to human GPCR135 (IC50=0.67 nM) and GPCR142 (IC50=2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2=9.15) but is not a human LGR7 ligand. Furthermore, R3(BDelta23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50=0.25 nM, pA2=9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BDelta23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BDelta23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.
    Journal of Biological Chemistry 09/2007; 282(35):25425-35. · 4.65 Impact Factor
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    ABSTRACT: Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC50 = 0.67 nm) and GPCR142 (IC50 = 2.29 nm) with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50 = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.
    Journal of Biological Chemistry 08/2007; 282(35):25425-25435. · 4.65 Impact Factor
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    ABSTRACT: The successful cloning and functional expression of the histamine H(3) receptor in the late 1990 s has greatly facilitated our efforts to identify small molecule, non-imidazole based compounds to permit the evaluation of H(3) antagonists in models of CNS disorders. High-throughput screening identified several series of lead compounds, including a series of imidazopyridines, which led to JNJ-6379490, a compound with high affinity for the human H(3) receptor. Analysis of structural features common to several series of non-imidazole H(3) receptor ligands resulted in a pharmacophore model. This model led to the design of JNJ-5207852, a diamine-based H(3) antagonist with good in vitro and in vivo efficacy but with an undesirable long half-life. However, further modifications of the template provided an understanding of the effect of structural modifications on pharmacokinetic properties, ultimately affording several additional series of compounds including JNJ-10181457, a compound with an improved pharmacokinetic profile. These compounds allowed in vivo pharmacological evaluation to show that H(3) antagonists promote wakefulness, but unlike modafinil and classical psychostimultants, they do not increase locomotor activity or produce any alteration of the EEG power spectral activity in rats. H(3) antagonists also increase extracellular acetylcholine and norepinephrine but not dopamine in rat frontal cortex and show efficacy in various models of learning-memory deficit. In addition, cFos immunoreactivity studies show H(3) antagonists activate neuronal cells in restricted rat brain regions in contrast to widespread activation after modafinil or amphetamine treatment. Therefore, H(3) antagonists are promising clinical candidates for the treatment of excessive day time sleepiness and/or cognitive disorders.
    Biochemical Pharmacology 05/2007; 73(8):1084-96. · 4.58 Impact Factor
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    ABSTRACT: A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the gamma-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.
    Journal of Biological Chemistry 11/2005; 280(42):35089-97. · 4.65 Impact Factor
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    ABSTRACT: Prokineticins 1 and 2 (PK1 and PK2) have been recently identified from humans and other mammals and play multiple functional roles. PK proteins are ligands for two G protein-coupled receptors, PK receptor 1 (PKR1) and PK receptor 2 (PKR2). Here, we report the molecular cloning and pharmacological characterization of an alternatively spliced product of the PK2 gene encoding 21 additional amino acids compared with PK2, designated PK2L (for PK2 long form). PK2L mRNA is broadly expressed, as is PK2. However, PK2L mRNA expression is lower in brain, undetectable in kidney, and much higher in lung and spleen than that of PK2. We expressed PK2L in mammalian cells and characterized the resulting peptide in comparison with PK1 and PK2. Biochemical characterization indicates that secreted PK2L protein is processed into a smaller peptide by proteolytic cleavage. We designate this smaller form of peptide as PK2beta. Coexpression of furin with PK2L significantly increased the PK2beta processing efficiency. Functional studies showed that PK1, PK2, and PK2beta stimulate intracellular Ca(2+) responses in PKR1-expressing cells with similar potencies. However, the PK2beta stimulus of Ca(2+) responses in PKR2-expressing cells is at least 10-fold less potent than that of PK1 or PK2. Differences in receptor selectivity combined with differential tissue expression patterns suggest PK2 and PK2beta might have different functions in vivo. PKRs have been reported to couple to G(q) and G(i) proteins. In this report, we show that PKs not only stimulate Ca(2+) mobilization but also induce cAMP accumulation in PKR-expressing cells.
    Molecular Pharmacology 07/2005; 67(6):2070-6. · 4.41 Impact Factor
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    ABSTRACT: We report the cloning, molecular characterization, and pharmacological characterization of the canine 5-HT2A and 5-HT2B receptors. The canine and human 5-HT2A receptors share 93% amino acid homology. The canine and human 5-HT2B receptors are also highly conserved (87% homology) with the exception of the carboxyl termini where the canine protein is 62 amino acids shorter. Both the canine 5-HT2A and 5-HT2B receptors have high affinity for [3H]5-HT (KD=4.50+/-0.89 nM and 3.10+/-0.82 nM, respectively) and, in general, the pharmacology of these two receptors matches closely the pharmacology of their human homologs for the 19 serotonergic ligands tested. However, the functional response (Ca2+ mobilization) of the canine 5-HT2B receptor to several agonists was weaker compared to the human 5-HT2B receptor. Using quantitative reverse transcriptase polymerase chain reaction, a high expression level of canine 5-HT2A receptor mRNA was detected in the brain and lower levels in peripheral tissues, whereas the highest levels of canine 5-HT2B receptor mRNA were observed in lungs and smooth muscles. A significant level of canine 5-HT2B receptor mRNA was detected in brain tissue. The availability of the full sequence and pharmacology of the canine 5-HT2A and canine 5-HT2B receptors provides useful information for the interpretation of previous and future pharmacological studies of 5-HT2A/2B ligands in dog.
    European Journal of Pharmacology 05/2005; 513(3):181-92. · 2.59 Impact Factor
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    ABSTRACT: Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.
    Journal of Biological Chemistry 02/2005; 280(1):292-300. · 4.65 Impact Factor
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    ABSTRACT: We describe a sensor capable of detecting single DNA molecules. The sensor is based on a single nanopore prepared in a polymer film by a latent ion track-etching technique. For this purpose, a polymer foil was penetrated by a single heavy ion of total kinetic energy of 2.2 GeV, followed by preferential etching of the ion track. DNA molecules were detected as they blocked current flow during translocation through the nanopore, driven by an electric field. The nanopores are highly stable and their dimensions are adjustable by controlling etching conditions. For detecting DNA, conical nanopores with opening diameters of 2 μm and 4 nm were used. The nanopore sensor was able to discriminate between DNA fragments of different lengths.
    Nano Letters - NANO LETT. 02/2004; 4(3).
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    ABSTRACT: Generating gene-expression profiles from laser-captured cells requires the successful combination of laser-capture microdissection, RNA extraction, RNA amplification, and microarray analysis. To permit single-cell gene-expression profiling, the RNA amplification method has to be sufficiently powerful to bridge the gap between the amount of RNA available from a single cell to what is required by the microarray, a gap that spans 5 to 6 orders of magnitude. This chapter focuses on the amplification of RNA using a two-round T7 RNA amplification method. The protocols described are adapted for laser-captured material and have been used to generate gene expression profiles from single laser-captured cells.
    Methods in molecular medicine 02/2004; 99:215-23.
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    ABSTRACT: Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.
    Journal of Neuroscience 06/2003; 23(9):3607-15. · 6.91 Impact Factor
  • Fredrik Kamme, Mark G Erlander
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    ABSTRACT: Gene expression profiling is increasingly being used to study complex disease processes, with a focus toward generating new hypotheses and identifying novel therapeutic approaches. This method requires not only the ability to assign expression data to the correct cell type, but also the aptitude to interpret the subsequent deluge of gene expression patterns. Single-cell gene expression analysis is currently used to generate data within the fundamental unit, the single cell, thereby freeing the analysis from assumptions or questions regarding cell population homogeneity, whether cell-type or temporal. Single-cell expression profiling also offers a highly parallel view of the workings of a gene regulatory network at one specific point in time, and will hopefully provide insights that could lead to an improved ability to interpret gene expression patterns.
    Current opinion in drug discovery & development 04/2003; 6(2):231-6. · 5.12 Impact Factor
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    ABSTRACT: Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of 'signature' genes (n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT(2B) receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.
    Brain Research 08/2002; 943(1):38-47. · 2.88 Impact Factor