Identification of preoptic sleep neurons using retrograde labelling and gene profiling.

In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs.

Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

Cardiomyocyte expression and cell-specific processing of procholecystokinin.

Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptides has only been suggested using transcriptional measures or methods, with the post-translational phase of gene expression unaddressed. In this study, we examined the cardiac expression of the CCK gene in adult mammals and its expression at the protein level. Using quantitative PCR, a library of sequence-specific pro-CCK assays, peptide purification, and mass spectrometry, we demonstrate that the mammalian heart expresses pro-CCK in amounts comparable to natriuretic prohormones and processes it to a unique, triple-sulfated, and N-terminally truncated product distinct from intestinal and cerebral CCK peptides. Isoprenaline rapidly stimulated cardiac CCK gene expression in vitro and in vivo, which suggests that the cardiac-specific truncated pro-CCK may have pathophysiological relevance as a new marker of heart failure. The suggestion is confirmed by measurement of plasma from heart failure patients.

Dysgenesis of enteroendocrine cells in Aristaless-Related Homeobox polyalanine expansion mutations.

OBJECTIVES: Severe congenital diarrhea occurs in approximately half of patients with Aristaless-Related Homeobox (ARX) null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Because polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX mutation and in a mouse model of the corresponding polyalanine expansion (Arx). METHODS: Immunohistochemistry and quantitative real-time polymerase chain reaction were the primary modalities used to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats. RESULTS: An expansion of 7 alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin, glucagon-like peptide 1, and somatostatin populations were reduced, whereas the chromogranin A population was unchanged. In the mouse model, cholecystokinin and glucagon-like peptide 1 populations were also lost, although the somatostatin-expressing population was increased. The ARX protein was present in human tissue, whereas the Arx protein was degraded in the mouse intestine. CONCLUSIONS: ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Owing to protein degradation, the Arx mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX protein on its transcriptional targets in the intestine.

High-throughput solvent assisted ionization inlet for use in mass spectrometry.

In this work we developed a multiplexed analysis platform providing a simple high-throughput means to characterize solutions. Automated analyses, requiring less than 5 s per sample without carryover and 1 s per sample, accepting minor cross contamination, was achieved using multiplexed solvent assisted ionization inlet (SAII) mass spectrometry (MS). The method involves sequentially moving rows of pipet tips containing sample solutions in close proximity to the inlet aperture of a heated mass spectrometer inlet tube. The solution is pulled from the container into the mass spectrometer inlet by the pressure differential at the mass spectrometer inlet aperture. This sample introduction method for direct injection of solutions is fast, easily implemented, and widely applicable, as is shown by applications ranging from small molecules to proteins as large as carbonic anhydrase (molecular weight ca. 29,000). MS/MS fragmentation is applicable for sample characterization. An x,y-stage and common imaging software are incorporated to map the location of components in the sample wells of a microtiter plate. Location within an x,y-array of different sample solutions and the relative concentration of the sample are displayed using ion intensity maps.

Expression of the type 1 cannabinoid receptor (CB1R) in CCK-immunoreactive axon terminals in the basolateral amygdala of the rhesus monkey (Macaca mulatta).

Studies in rodents have shown that interactions between cholecystokinin (CCK) and the endogenous cannabinoid system in the basolateral nuclear complex of the amygdala (BNC) modulate anxiety-like behavior and fear learning/expression. One of the main cell types implicated is a CCK-immunoreactive (CCK+) basket cell that innervates the somata of pyramidal projection neurons (PNs) and expresses the type 1 cannabinoid receptor (CB1R) in its axon terminals. Although numerous studies have elucidated the anatomy and physiology of these CCK+/CB1R + interneurons in rodents, it has not been determined if they exist in primates. The present investigation used immunohistochemical techniques in the monkey to answer this question. It was found that the monkey BNC, as in rodents, has a very high density of CB1R + axons, including CB1R + axon terminals that form basket-like plexuses contacting somata of PNs. These axons, as well as axons in the neuropil, exhibit extensive colocalization of CCK and CB1R. These findings suggest that the same synaptic mechanisms involved in CCK-CB1R interactions in rodents may also apply to primates, and that therapies that target the cannabinoid system in the BNC may be useful for treating fear and anxiety in human patients.

Pseudopod-like basal cell processes in intestinal cholecystokinin cells.

Cholecystokinin (CCK) is secreted by neuroendocrine cells comprising 0.1%-0.5% of the mucosal cells in the upper small intestine. Using CCK promoter-driven green fluorescent protein (GFP) expression in transgenic mice, we have applied immunofluorescence techniques to analyze the morphology of CCK cells. GFP and CCK colocalize in neuroendocrine cells with little aberrant GFP expression. CCK-containing cells are either flask- or spindle-shaped, and in some cells, we have found dendritic processes similar to pseudopods demonstrated for gut somatostatin-containing D cells. Most pseudopods are short, the longest process visualized extending across three cells. Pseudopods usually extend to adjacent cells but some weave between neighboring cells. Dual processes have also been observed. Three-dimensional reconstructions suggest that processes are not unidirectional and thus are unlikely to be involved in migration of CCK cells from the crypt up the villus. Abundant CCK immunostaining is present in the pseudopods, suggesting that they release CCK onto the target cell. In order to identify the type of cells being targeted, we have co-stained sections with antibodies to chromogranin A, trefoil factor-3, and sucrase-isomaltase. CCK cell processes almost exclusively extend to sucrase-isomaltase-positive enterocytes. Thus, CCK cells have cellular processes possibly involved in paracrine secretion.

Stabilized (111)in-labeled sCCK8 analogues for targeting CCK2-receptor positive tumors: synthesis and evaluation.

Radiolabeled cholecystokinin-8 (CCK8) peptide analogues can be used for peptide receptor radionuclide imaging and therapy for tumors expressing CCK2/gastrin receptors. Earlier findings indicated that sulfated CCK8 (sCCK8, Asp-Tyr(OSO(3)H)-Met-Gly-Trp-Met-Asp-Phe-NH(2)) may have better characteristics for peptide receptor radionuclide therapy (PRRT) than gastrin analogues. However, sCCK8 contains an easily hydrolyzable sulfated tyrosine residue and two methionine residues which are prone to oxidation. Here, we describe the synthesis of stabilized sCCK8 analogues, resistant to hydrolysis and oxidation. Hydrolytic stability was achieved by replacement of the Tyr(OSO(3)H) moiety by a robust isosteric sulfonate, Phe(p-CH(2)SO(3)H). Replacement of methionine by norleucine (Nle) or homopropargylglycine (HPG) avoided undesired oxidation side-reactions. The phenylalanine analogue Phe(p-CH(2)SO(3)H) of l-tyrosine, synthesized by a modification of known synthetic routes, was incorporated in three peptides: sCCK8[Phe(2)(p-CH(2)SO(3)H),Met(3,6)], sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)], and sCCK8[Phe(2)(p-CH(2)SO(3)H),HPG(3,6)]. All peptides were N-terminally conjugated with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and radiolabeled with In-111. In vitro binding assays on CCK2R-expressing HEK293 cells revealed that all three peptides showed specific binding and receptor-mediated internalization, with binding affinity values (IC(50)) in the nanomolar range. In vitro oxidation studies demonstrated that peptides with Nle or HPG indeed were resistant to oxidation. In vivo targeting studies in mice with AR42J tumors showed that tumor uptake was highest for (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)] (4.78 +/- 0.64 and 4.54 +/- 1.15%ID/g, respectively, 2 h p.i.). The peptide with the methionine residues replaced by norleucine ((111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H), Nle(3,6)]) showed promising in vivo characteristics and will be further investigated for radionuclide imaging and therapy of CCK2R-expressing tumors.

Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F.

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000----110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl-D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK-binding proteins in rat pancreas are (sialo)glycoproteins.

Expression of PCSK1 (PC1/3), PCSK2 (PC2) and PCSK3 (furin) in mouse small intestine.

The family of serine proteases known as the proprotein convertases subtilisin/kexin type (PCSK) is responsible for the cleavage and maturation of many precursor hormones. Over its three successive regions, the duodenum, the jejunum and the ileum, the small intestine (SI) expresses over 40 peptide hormones necessary for normal intestinal physiology. Most of these hormones derive from proteolytic cleavage of their cognate inactive polypeptide precursors. Members of the PCSK family of proteases have been implicated in this process, although details of enzyme-substrate interactions are largely lacking. As a first step towards elucidating these interactions, we have analyzed by immunohistochemistry the regional distribution of PCSK1, PCSK2 and PCSK3 in mouse SI as well as their cellular co-localization with substance P (SP), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and somatostatin (SS), 4 peptide hormones known to result from PCSK-mediated processing. Results indicate that PCSK1 is found in all three regions of the SI while PCSK2 and PCSK3 are primarily expressed in the upper two, the duodenum and the jejunum. In these proximal regions, PCSK1 was detectable in 100% of SP-positive (+) cells, 85% of CCK+ cells and 50% of GIP+ cells; PCSK2 was detectable in 40% of SS+ cells and 35% of SP+ cells; PCSK3 was detectable in 75% of GIP+ cells and 60% of SP+ cells. These histological data suggest that the 3 PCSKs may play differential and overlapping roles in prohormone processing in the three regions of the SI.

Combined administration of the mixture of honokiol and magnolol and ginger oil evokes antidepressant-like synergism in rats.

Magnolia bark combined with ginger rhizome is a common drug pair in traditional Chinese prescriptions for the treatment of depression. In the present study, we examined antidepressant-like effects of the mixture of honokiol and magnolol (HMM) from magnolia bark and essential oil from ginger rhizome (OGR) alone and in combination in chronic unpredictable mild stress (CUMS) of rats. Behavioral (sucrose intake, immobility time of forced swimming test) and biochemical parameters [serotonin (5-HT) in prefrontal cortex, hippocampus, and striatum, gastric mucosa cholecystokinin (CCK) and serum gastrin (GAS) levels] were simultaneously examined in the CUMS rats. 20 mg/kg HMM alone, but not OGR, significantly increased sucrose intake and reduced immobility time in the CUMS rats. Moreover, 20 mg/kg HMM and 14 mg/kg OGR in combination exhibited significant synergistic effects on sucrose intake increase and immobility time reduction in the CUMS rats. HMM elevated 5-HT levels in various brain regions, and OGR reduced gastric mucosa CCK and serum GAS levels in the CUMS rats. These results suggested that the synergistic antidepressant-like effects of compatibility of HMM with OGR might be mediated simultaneously by regulation of the serotonergic and gastroenteric system functions. These findings also provided a pharmacological basis for the clinical application of this drug pair of magnolia bark and ginger rhizome in traditional Chinese medicine.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn.

Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non-overlapping populations in laminae I-III, based on expression of substance P, gastrin-releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I-II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin-releasing peptide populations, but show limited overlap with the substance P cells. Laminae II-III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ-immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin-releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.

Influence of subliminal intragastric fatty acid infusion on subjective and physiological responses to positive emotion induction in healthy women: A randomized trial.

BACKGROUND: Subliminal intragastric fatty acid infusion attenuates subjective and brain responses to negative emotion induction. However, the underlying gut-brain signaling mechanisms remain unclear, and it is unknown whether such effect equally applies to positive emotion. OBJECTIVE: We aimed to investigate the interaction between fatty acid-induced gut-brain signaling and subjective responses to positive emotion, and the potential mediational role of gastrointestinal (GI) hormones. DESIGN: Twelve fasting healthy women underwent intragastric infusion of 2.5 g lauric acid or saline, after which either positive or neutral emotion was induced for 30 min, in 4 separate visits. Appetite-related sensations, subjective emotional state, and GI hormones were measured at baseline and every 10 min after infusion. Heart rate variability was measured at baseline and at t = 20-30 min to quantify vagal tone (root mean square of successive differences, RMSSD), and sympathovagal balance (low frequency to high frequency ratio, LF/HF). RESULTS: Fatty acid infusion did not influence appetite-related sensations (as expected), nor emotional state ratings (contrary to expectations). As anticipated, fatty acid stimulated release of CCK at t = 20-40 min (p < 0.001), and GLP1 at t = 30-40 min (p < 0.001), but not PYY. Interestingly, positive emotion induction suppressed plasma octanoylated ghrelin at t = 20-40 min (p = 0.020). Further, both positive emotion and fatty acid attenuated RMSSD (p = 0.012 & 0.0073, respectively). Positive emotion attenuated LF/HF after fatty acid (p = 0.0006), but raised LF/HF after saline (p = 0.004). CONCLUSIONS: Subliminal fatty acid did not influence subjective responses to positive emotion induction. However, positive emotion induction suppressed octanoylated ghrelin release. Moreover, both positive emotion and subliminal fatty acid decreased cardiac vagal tone. Further, the fatty acid reversed the effect of positive emotion on sympathovagal balance.

Localization of cholecystokinin-like immunoreactivity in the central nervous system of Triatoma infestans (Insecta: Heteroptera).

The distribution of cholecystokinin-like immunoreactivity was studied in the central nervous system of the heteropteran insect Triatoma infestans using high-sensitivity immunocytochemistry. In the protocerebrum, CCK-IR somata were observed in the anteromedial, anterolateral and posterior cell-body layers. The neuropils displayed different densities of immunoreactive neurites. Few immunoreactive somata were found in the optic lobe in both the medial and lateral soma rinds, as well as in the proximal optic lobe. Immunoreactive fibers were present in the medulla and lobula neuropils. The sensory deutocerebrum contained a higher number of immunopositive perikarya than the antennal mechanosensory and motor center. The antennal lobe glomeruli displayed a moderate density of immunoreactive fibers. With regard to the subesophageal ganglion, numerous CCK-IR somata were found close to the root of the mandibular nerve; others were present in the soma rind of the remaining neuromeres. CCK-IR perikarya were present in both thoracic ganglia, with the abdominal neuromeres containing the highest number of positive somata. The neuropils of both ganglia showed moderate densities of immunopositive processes. The distribution of CCK-LI in somata and neuropils of central nervous system of T. infestans is widespread suggesting that a CCK-like peptide may act mainly as a neuromodulator in the integration of information from distinct sensory receptors.

Cardiac procholecystokinin expression during haemodynamic changes in the mammalian heart.

Cardiac myocytes express the cholecystokinin gene (CCK) at propeptide level. We recently reported that cardiac CCK expression is acutely regulated by isoprenaline in a porcine model. The regulation of CCK expression after myocardial infarction, in exercise, and in severe heart failure is, however, unknown. Cardiac tissue was obtained from healthy new-born and adolescent farm pigs. Myocardial infarction was induced by coronary artery occlusion in adult minipigs. Healthy male subjects performed a 3-hour exercise test, and patients with severe heart failure referred for right heart catheterization were included. Extracts of porcine cardiac tissue and human plasma were analysed with specific proCCK radioimmunoassays. Cardiac proCCK expression shifted from the right atrium in new-born piglets to include the left atrium in adolescent pigs. Regional proCCK expression in the adolescent pig heart was mainly confined to the atria without different expression in sinus node tissue. In adult minipigs with myocardial infarction, no changes in overall left ventricular function or proCCK expression were observed after 8 weeks. In healthy adults, proCCK in circulation increased markedly during exercise in parallel with pro-B-type natriuretic peptide. Finally, patients with severe heart failure displayed markedly increased proCCK - but not CCK - concentrations in plasma. Taken together, our data shows that regional proCCK expression reflects haemodynamic changes in the mammalian heart. The data supports the notion that cardiac CCK expression resembles that of cardiac natriuretic peptides in atria. The ventricular content of proCCK, however, differs from natriuretic peptides and suggests a distinct secretory pathway in ventricular cardiomyocytes.

Presynaptic, activity-dependent modulation of cannabinoid type 1 receptor-mediated inhibition of GABA release.

Endocannabinoid signaling couples activity-dependent rises in postsynaptic Ca2+ levels to decreased presynaptic GABA release. Here, we present evidence from paired recording experiments that cannabinoid-mediated inhibition of GABA release depends on the firing rates of the presynaptic interneurons. Low-frequency action potentials in post hoc identified cholecystokinin-positive CA1 basket cells elicited IPSCs in the postsynaptic pyramidal cells that, as expected, were fully abolished by the exogenous application of the cannabinoid receptor agonist WIN55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate] at 5 microM. However, the presynaptic basket cells recovered from the cannabinoid agonist-induced inhibition of GABA release when the presynaptic firing rate was increased to > or =20 Hz. Pharmacological experiments showed that the recovered transmission was exclusively dependent on presynaptic N-type Ca2+ channels. Furthermore, the increased presynaptic firing could also overcome even complete depolarization-induced suppression of inhibition, indicating that the magnitude of DSI markedly depends on the activity levels of basket cells. These results reveal a new locus of activity-dependent modulation for endocannabinoid signaling and suggest that endocannabinoid-mediated inhibition of GABA release may differ in distinct behavioral states.

Bacterial cells: The migrating kinase and the master regulator.

It is becoming clear that, as in eukaryotes, proteins in bacterial cells are targeted to specific cellular locations. The most recently discovered example is a remarkable histidine kinase that oscillates between polar and global distributions while temporally regulating transcription and DNA replication in Caulobacter.

Comparison of hepatic elimination of different forms of cholecystokinin in dogs. Bioassay and radioimmunoassay comparisons of cholecystokinin-8-sulfate and -33-sulfate.

The influence of hepatic transit on the ability of exogenous cholecystokinin-8-sulfate and -33-sulfate (CCK-8 and CCK-33, respectively) to stimulate gallbladder contraction and exocrine pancreatic secretion, as well as on the peripheral plasma concentration of each agent, was evaluated in five conscious dogs with pancreatic and gallbladder fistulas and complete portacaval transposition. The gallbladder pressure increments after portal administration of CCK-8 (0.125, 0.25, 0.50, and 1.0 microgram/kg per h for 5 min) were diminished by 36, 45, 39 and 25%, respectively, in comparison with those obtained with systemic administration of identical doses of CCK-8 (P less than 0.05). In a subsequent experiment, the integrated pancreatic juice volume, bicarbonate, and protein secretion were diminished by 22, 32, and 48%, respectively, during a 30-min infusion of CCK-8 (0.10 micrograms/kg per h) into the portal venous system, in comparison with the results obtained with systemic administration of CCK-8 (P less than 0.05). In contrast, the gallbladder pressure and pancreatic exocrine secretory responses to portal administration of CCK-33 did not differ significantly (P greater than 0.05) from the results obtained with systemic administration of CCK-33. Radioimmunoassay for CCK-8 in plasma showed that the integrated CCK-8 value during portal administration was significantly lower (P less than 0.05) than it was during systemic administration. The results for CCK-33, however, did not vary, whether it was given by a systemic or portal route (P greater than 0.05). Thus, the present study demonstrates that CCK-8 is partially inactivated by the liver whereas CCK-33 is not, which suggests that CCK-3 in the circulation may play a significant role in the physiologic regulation of the gallbladder and exocrine pancreas.