Uncovering the molecular basis of positive affect using rough-and-tumble play in rats: a role for insulin-like growth factor I.

Positive emotional states have been shown to confer resilience to depression and anxiety in humans, but the molecular mechanisms underlying these effects have not yet been elucidated. In laboratory rats, positive emotional states can be measured by 50-kHz ultrasonic vocalizations (hedonic USVs), which are maximally elicited by juvenile rough-and-tumble play behavior. Using a focused microarray platform, insulin-like growth factor I (IGFI) extracellular signaling genes were found to be upregulated by hedonic rough-and-tumble play but not depressogenic social defeat. Administration of IGFI into the lateral ventricle increased rates of hedonic USVs in an IGFI receptor (IGFIR)-dependent manner. Lateral ventricle infusions of an siRNA specific to the IGFIR decreased rates of hedonic 50-kHz USVs. These results show that IGFI plays a functional role in the generation of positive affective states and that IGFI-dependent signaling is a potential therapeutic target for the treatment of depression and anxiety.

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain.

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

The brain decade in debate: VIII. Peptide hormones and behavior: cholecystokinin and prolactin

This article is a transcription of an electronic symposium held on November 28, 2000 in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the peptide field with particular focus on central actions of prolactin and cholecystokinin. The comments in this symposium reflect the diversity of prolactin and cholecystokinin research and demonstrate how the field has matured. Since both peptides play a role in reproductive behaviors, particularly mother-infant interactions, this was the starting point of the discussion. Recent findings on the role of the receptor subtypes as well as interaction with other peptides in this context were also discussed. Another issue discussed was the possible role of these peptides in dopamine-mediated rewarding systems. Both prolactin and cholecystokinin are involved in mechanisms controlling food intake and somatic pain thresholds. The role of peripheral inputs through vagal afferents modulating behavior was stressed. The advent of knockout animals as potential generators of new knowledge in this field was also addressed. Finally, interactions with other neuropeptides and investigation of the role of these peptides in other fields such as immunology were mentioned. Knowledge about the central functions of prolactin and cholecystokinin has shown important advances. The role of these peptides in neurological and psychiatric syndromes such as anorexia, drug abuse and physiological disturbances that lead to a compromised maternal behavior seems relevant

The brain decade in debate: VIII. Peptide hormones and behavior: cholecystokinin and prolactin.

This article is a transcription of an electronic symposium held on November 28, 2000 in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the peptide field with particular focus on central actions of prolactin and cholecystokinin. The comments in this symposium reflect the diversity of prolactin and cholecystokinin research and demonstrate how the field has matured. Since both peptides play a role in reproductive behaviors, particularly mother-infant interactions, this was the starting point of the discussion. Recent findings on the role of the receptor subtypes as well as interaction with other peptides in this context were also discussed. Another issue discussed was the possible role of these peptides in dopamine-mediated rewarding systems. Both prolactin and cholecystokinin are involved in mechanisms controlling food intake and somatic pain thresholds. The role of peripheral inputs through vagal afferents modulating behavior was stressed. The advent of knockout animals as potential generators of new knowledge in this field was also addressed. Finally, interactions with other neuropeptides and investigation of the role of these peptides in other fields such as immunology were mentioned. Knowledge about the central functions of prolactin and cholecystokinin has shown important advances. The role of these peptides in neurological and psychiatric syndromes such as anorexia, drug abuse and physiological disturbances that lead to a compromised maternal behavior seems relevant.

Structural studies of a neuropeptide precursor protein with an RGD proteolytic site.

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.

An introduction to neuronal cholecystokinin.

This issue of Peptides was inspired by a gathering of CCK researchers at the first Neuronal Cholecsytokinin Gordon Conference. The papers in this issue reflect the diversity of CCK research and demonstrate how the field has matured. Reviews describe the regulation of CCK gene expression and CCK release, the nature of the hormone binding site of the CCK A receptor, interaction of CCK, dopamine and GABA, the role of CCK in thermoregulation, sexual behavior and satiety in rodents and humans. The research articles document features of cardiovascular regulation, reduced cocaine sensitization and decreased satiety in rats that lack the CCK A receptor. Pro CCK processing in neuroblastoma cells and the elevation of CCK levels in CSF in a model of chronic pain are detailed in other articles. Three articles using different behavioral paradigms in rat and sheep examine CCK in learning and memory. Two articles that examine CCK in different behaviors that have a dopaminergic component are included. Other articles describe the interaction between a 5HT(3) antagonist and CCK-induced satiety and c-fos activation and document secretion of oxytocin and vasopressin in female patients and controls in response to CCK 4 administration. There is good reason to believe that the future is bright for research on CCK. With the organization of national and international meetings, CCK researchers have a forum for communication. Opportunities for cooperation and collaboration have never been better. The easy integration of academic basic and clinical science with industrial science bodes very well for the advancement of our understanding of the multiple roles that CCK plays in the brain and for the future development of CCK-based therapies.

Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.

OLETF (Otsuka Long-Evans Tokushima Fatty) rats that lack the CCK 1 (A) receptor develop less behavioral sensitization to repeated cocaine treatment than wild type LETO (Long Evans Tokushima Otsuka) rats.

OLETF (Otsuka Long-Evans Tokushima Fatty) lacking the CCK 1 (A) receptor have similar spontaneous activity and locomotor response (horizontal and vertical activity) in response to a single injection of cocaine as the wild type LETO (Long Evans Tokushima Otsuka) rats. In contrast, the OLETF rats display more stereotypy in response to the first dose of cocaine than the LETO rats. Tested at 7 and 14 days after a one week daily treatment with cocaine, the LETO rats display robust behavioral sensitization to cocaine while the OLETF rats did not. These results support the hypothesis that endogenous CCK released by cocaine treatment and acting at CCK 1 receptors is required for the development and/or expression of this behavior.

Activation of CB1 cannabinoid receptors in rat hippocampal slices inhibits potassium-evoked cholecystokinin release, a possible mechanism contributing to the spatial memory defects produced by cannabinoids.

Cannabinoid use is known to disrupt learning and memory in a number of species. cholecystokinin (CCK) release and CCK receptors have been implicated in spatial memory processes in rodents. Rat hippocampal CCK interneurons express cannabinoid 1 receptors (CB1). The CB1 agonist R(+)WIN 55,212-2 (WIN+), at 1 and 10 micromol, strongly inhibited potassium-evoked CCK release from rat hippocampal slices, while the inactive isomer S(-)WIN,55,212-3 (WIN-) had no effect. CCK release from cerebral cortical slices was not altered by WIN+.

Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells.

Mammalian procholecystokinin (pro-CCK) is known to have three sulfated tyrosine residues, one of which is present in the CCK 8 moiety and two additional residues present in the carboxyl-terminal extension. In the present study, inhibition of tyrosine sulfation by sodium chlorate decreased the secretion of processed CCK 8 in CCK-expressing endocrine cells in culture. It was then demonstrated that when each of these tyrosines individually, as well as all three together, was mutated to phenylalanine and expressed in endocrine cells, CCK was still processed and secreted. However, the amount of CCK secreted varied with the type of mutation. Substitution of Phe to Tyr in CCK 8 reduced the quantity of secreted CCK 8 by 50%, and when all the sulfated Tyr were mutated to Phe the quantity of secreted CCK was reduced by about 70%, similar to what is observed with chlorate treatment. Changing of the putative phosphorylation site serine to alanine does not affect the processing. Serine phosphorylation at this site may play a functional role in regulatory events. Our results demonstrate that tyrosine sulfation alters the amount of secretion but is not an absolute requirement for the processing and secretion of CCK in this cell line. Tyrosine sulfation of CCK may still be important for its solubility, stabilization, and/or functional interaction.

Methodologies to study the induction of rat hepatic and intestinal cytochrome P450 3A at the mRNA, protein, and catalytic activity level.

Studies were conducted to characterize assays for the isolation and quantitation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline phosphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A in the in vitro hydroxylation of midazolam (MDZ) was also evaluated using isoform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration on in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigating the induction of CYP3A at the MRNA, protein, and catalytic activity within a single animal, a separate induction study was carried out with the CYP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were used to reliably detect differences in CYP 3A mRNA and immunoreactivity between DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A activity caused by DEX in comparison to VH. Collectively, these described techniques provide an experimental model to study xenobiotic induction of rat hepatic and intestinal CYP 3A from the molecular to the catalytic level in individual rats without the need for pooling of tissue.

PC2 and 7B2 null mice demonstrate that PC2 is essential for normal pro-CCK processing.

Analysis of CCK content in extracts of whole forebrain from PC2 and 7B2 null mouse brain showed a significant decrease relative to wild-type brains. More detailed analysis revealed that CCK 8 amide levels in cerebral cortex and forebrain regions were more decreased than in hypothalamus. CCK 8 content in PC2 null mouse intestines was identical to control. Null mutant brains contained less CCK 8 than wild type and no other forms were seen when analyzed by gel filtration chromatography. No brain area examined was completely devoid of CCK, suggesting that other enzymes can partially compensate for the loss of PC2. This is the first demonstration that any endoprotease is important for CCK processing but also suggest the presence of a redundant system to ensure production of active CCK in the brain.

Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture.

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.

Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation.

Immunoreactive glycine-extended CCK peptides are found in normal mouse cerebral cortex and are very abundant in some CCK expressing endocrine tumor cells in culture. The glycine-extended forms in mouse cortex and in cell lines mirror their respective amidated forms. Mouse cerebral cortex, mouse AtT20 and rat WE cells produce mainly CCK 8 amide and CCK 8 Gly. In contrast, mouse intestinal STC-1 cells produce CCK 22 and CCK 8 amide along with forms of CCK Gly which are slightly larger than their respective amidated forms. The CCK 8 Gly-like peptide from AtT20 cells, after desulfation, co-eluted on HPLC with unsulfated CCK 8 Gly. Addition of copper and ascorbate to culture medium of WE cells caused a small increase in secretion of amidated CCK, without changing cellular levels of this peptide. Treatment with the amidation inhibitor diethyldithiocarbamate greatly decreased cellular content and secretion of CCK amide while it increased cellular content and secretion of CCK Gly. These results provide further evidence that glycine-extended CCK peptides are the immediate precursors of amidated CCK peptides.

Reduced levels of substance P in the brains of Cpe(fat)/Cpe(fat) mice.

This study examines the role of carboxypeptidase E (CPE) in processing pro tachykinin to form the final bioactive amidated undecapeptide, substance P (SP) in various rat brain regions. Cpe(fat)/Cpe(fat) mice brain tissue was analyzed for total SP forms (including intermediates), and final amidated SP was compared to Cpe+/Cpe+ and Cpe+/Cpe- controls. In all brain regions tested by radioimmunoassay, amidated fully processed SP was more than fivefold lower in Cpe(fat)/Cpe(fat) mice than in controls whereas total SP species levels were unchanged. This demonstrates that CPE is required for normal SP proteolytic processing. Substance P has numerous functions in the brain; therefore, SP deficiency due to the CPE mutation may contribute to the obese phenotype or even to other phenotypes not yet described in Cpe(fat)/Cpe(fat) mice.

Prohormone convertase 1 (PC1) when expressed with pro cholecystokinin (pro CCK) in L cells performs three endoproteolytic cleavages which are observed in rat brain and in CCK-expressing endocrine cells in culture, including the production of glycine and arginine extended CCK8.

Pro CCK was expressed in an L cell line engineered to express PC1 and the products secreted into the media were characterized by a combination of RIA, gel filtration and HPLC. PC1 released from L cells, cleaved pro CCK generating the amino terminal pro peptide. PC1 also generated a peptide which after carboxypeptidase B treatment, was detected with an antiserum specific for CCK Gly. Neither of these peptides was found in media from L cells expressing pro CCK alone. This CCK Gly immunoreactive peptide was similar in size to CCK 8, and after treatment with arylsulfatase and carboxypeptidase B, it co-eluted on HPLC with unsulfated CCK 8 Gly. These results agree with previous studies which support a role for PC1 in generation of CCK 8. This is the first demonstration that PC1 acting alone is able to cleave pro CCK liberating the amino terminal pro peptide and a glycine and arginine extended CCK 8 which is the immediate precursor of CCK 8 amide.

Prohormone and proneuropeptide processing. Recent progress and future challenges.

Our knowledge of prohormone and proneuropeptide processing and its relationship to the secretory pathway has advanced significantly in the last five years. The recent discovery of the prohormone convertase family of proteolytic enzymes has provided new candidates for the prohormone and proneuropeptide convertases. The increasing appreciation of the role of proteolysis in diverse cellular processes has also brought the prohormone processing field closer to the fields of growth factor processing, the role of host proteases in viral and bacterial pathogenesis and toxicity, control of the cell cycle, inflammation, and apoptosis. The last five years have been very productive, but the most interesting questions are still unanswered. Which enzymes are actually responsible for prohormone cleavages in specific tissues? What structural features of the prohormones determine where it will be processed or how it is recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? The availability of protease knockout mice and and a more detailed understanding of the complex biosynthetic activation of these enzymes will provide at least some of the answers.

Adult carboxypeptidase E-deficient fat/fat mice have a near-total depletion of brain CCK 8 accompanied by a massive accumulation of glycine and arginine extended CCK: identification of CCK 8 Gly as the immediate precursor of CCK 8 in rodent brain.

Cholecystokinin (CCK) amide concentrations were reduced over 85% in all the major brain regions of carboxypeptidase E (Cpe)(fat)/Cpe(fat) mice in comparison to control mice. Using an radioimmunoassay (RIA) specific for glycine-extended CCK (CCK Gly), low levels of CCK Gly were detected in control (0.65 ng/g tissue) and were even lower in Cpe(fat)/Cpe(fat) (0.246 ng/g) mice brain extracts. After treatment with carboxypeptidase B, the level of CCK Gly in Cpe(fat)/Cpe(fat) in these brain extracts was elevated to 33.5 ng/g, about 51-fold higher than in control. On gel-filtration chromatography and high-performance liquid chromatography (HPLC), this material coeluted with CCK 8 Gly. These results demonstrate that CPE is required for the correct processing of arginine- and glycine-extended CCK in all major regions of the mouse brain. These results support the hypothesis that CCK 8 Gly is the immediate precursor of CCK 8 amide in mouse brain, not larger amidated forms like CCK 22 or CCK 33.

Prohormone convertase 2 is necessary for the formation of cholecystokinin-22, but not cholecystokinin-8, in RIN5F and STC-1 cells.

Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing. Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.