Cholecystokinin Immunoreactivity Research Articles

Co‐localization of nociceptive markers in the lumbar dorsal root ganglion and spinal cord of dromedary camel

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Immunohistochemical Study of Endocrine Cells in the Stomach and Small Intestine of the Lion (Panthera leo)

Abstract. The distribution and relative frequency of gastrointestinal endocrine cells were investigated in the stomach and small intestine (duodenum and jejunum) of a lion (Panthera leo). The present study was an immunohistochemical investigation of endocrine cells showing immunoreactivity for serotonin, gastrin, or cholecystokinin (CCK). Serotonin-immunoreactive (IR) cells were detected throughout the gastrointestinal tract with the highest frequency in the duodenum. Gastrin-IR cells were numerous in the pyloric region of the stomach and moderate numbers were also seen in the duodenum and jejunum. Moderate numbers of CCK-IR cells were only detected in the duodenum and jejunum.

Relationship of cannabinoid CB1 receptor and cholecystokinin immunoreactivity in monkey dorsolateral prefrontal cortex

Exposure to cannabis impairs cognitive functions reliant on the circuitry of the dorsolateral prefrontal cortex (DLPFC) and increases the risk of schizophrenia. The actions of cannabis are mediated via the brain cannabinoid 1 receptor (CB1R), which in rodents is heavily localized to the axon terminals of cortical GABA basket neurons that contain cholecystokinin (CCK). Differences in the laminar distribution of CB1R-immunoreactive (IR) axons have been reported between rodent and monkey neocortex, suggesting that the cell type(s) containing CB1Rs, and the synaptic targets of CB1R-IR axon terminals, may differ across species; however, neither the relationship of CB1Rs to CCK-containing interneurons, nor the postsynaptic targets of CB1R and CCK axon terminals, have been examined in primate DLPFC. Consequently, we compared the distribution patterns of CB1R- and CCK-IR structures, determined the proportions of CB1R and CCK neurons that were dual-labeled, and identified the synaptic types and postsynaptic targets of CB1R- and CCK-IR axon terminals in macaque monkey DLPFC. By light microscopy, CB1R- and CCK-IR axons exhibited a similar laminar distribution, with their greatest densities in layer 4. Dual-label fluorescence experiments demonstrated that 91% of CB1R-IR neurons were immunopositive for CCK, whereas only 51% of CCK-IR neurons were immunopositive for CB1R. By electron microscopy, all synapses formed by CB1R-IR axon terminals were symmetric, whereas CCK-IR axon terminals formed both symmetric (88%) and asymmetric (12%) synapses. The primary postsynaptic target of both CB1R- and CCK-IR axon terminals forming symmetric synapses was dendritic shafts (81–88%), with the remainder targeting cell bodies or dendritic spines. Thus, despite species differences in laminar distribution, CB1Rs are principally localized to CCK basket neuron axons in both rodent neocortex and monkey DLPFC. These axons target the perisomatic region of pyramidal neurons, providing a potential anatomical substrate for the impaired function of the DLPFC associated with cannabis use and schizophrenia.

Effect of intravenous infusion of proglumide on ruminal motility in conscious sheep (Ovis aries)

The effects of intravenous infusion of proglumide on regular ruminal contractions were examined in conscious sheep using doses that inhibit pancreatic exocrine secretion. After a control period of 20 min, proglumide was infused intravenously for 40 min at a dose of 15, 30 or 60 micromol/kg per min and venous blood was collected. The intravenous infusion of proglumide significantly increased the frequency of ruminal contractions at 15 micromol/kg per min without altering the amplitude, while it significantly decreased the frequency and amplitude of ruminal contractions at 30 and 60 micromol/kg per min in a dose-dependent manner. Proglumide did not increase contractile activity of the omasum, abomasum and duodenum or the plasma concentration of immunoreactive cholecystokinin (CCK). Application of proglumide at 1-30 mmol/L inhibited bethanechol-induced contraction in both longitudinal and circular muscle strips of the dorsal sac of the rumen. These results suggest that proglumide at a low dose acts indirectly on the rumen as a CCK receptor antagonist to increase the frequency of contractions, whereas at higher doses it inhibits cholinergic-induced contraction of the ruminal muscles or acts as an agonist to inhibit contractions in sheep. Hence, proglumide at high doses seems unsuitable for research or therapeutic use as a CCK receptor blockade in sheep.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.

Cholecystokinin-immunopositive basket and Schaffer collateral-associated interneurones target different domains of pyramidal cells in the CA1 area of the rat hippocampus

Two types of GABAergic interneurone are known to express cholecystokinin-related peptides in the isocortex: basket cells, which preferentially innervate the somata and proximal dendrites of pyramidal cells; and double bouquet cells, which innervate distal dendrites and dendritic spines. In the hippocampus, cholecystokinin immunoreactivity has only been reported in basket cells. However, at least eight distinct GABAergic interneurone types terminate in the dendritic domain of CA1 pyramidal cells, some of them with as yet undetermined neurochemical characteristics. In order to establish whether more than one population of cholecystokinin-expressing interneurone exist in the hippocampus, we have performed whole-cell current clamp recordings from interneurones located in the stratum radiatum of the hippocampal CA1 region of developing rats. Recorded neurones were filled with biocytin to reveal their axonal targets, and were tested for the presence of pro-cholecystokinin immunoreactivity. The results show that two populations of cholecystokinin-immunoreactive interneurones exist in the CA1 area ( n=15 positive cells). Cholecystokinin-positive basket cells (53%) preferentially innervate stratum pyramidale and adjacent strata oriens and radiatum. A second population of cholecystokinin-positive cells, previously described as Schaffer collateral-associated interneurones [Vida et al. (1998) J. Physiol. 506, 755–773], have axons that ramify almost exclusively in strata radiatum and oriens, overlapping with the Schaffer collateral/commissural pathway originating from CA3 pyramidal cells. Two of seven of the Schaffer collateral-associated cells were also immunopositive for calbindin. Soma position and orientation in stratum radiatum, the number and orientation of dendrites, and the passive and active membrane properties of the two cell populations are only slightly different. In addition, in stratum radiatum and its border with lacunosum of perfusion-fixed hippocampi, 31.6±3.8% (adult) or 26.8±2.9% (postnatal day 17–20) of cholecystokinin-positive cells were also immunoreactive for calbindin. Therefore, at least two populations of pro-cholecystokinin-immunopositive interneurones, basket and Schaffer collateral-associated cells, exist in the CA1 area of the hippocampus, and are probably homologous to cholecystokinin-immunopositive basket and double bouquet cells in the isocortex. It is not known if the GABAergic terminals of double bouquet cells are co-aligned with specific glutamatergic inputs. However, in the hippocampal CA1 area, it is clear that the terminals of Schaffer collateral-associated cells are co-stratified with the glutamatergic input from the CA3 area, with as yet unknown functional consequences. The division of the postsynaptic neuronal surface by two classes of GABAergic cell expressing cholecystokinin in both the hippocampus and isocortex provides further evidence for the uniform synaptic organisation of the cerebral cortex.

Peripheral axotomy increases cholecystokinin release in the rat anterior cingulate cortex

The anterior cingulate cortex (ACC) is a limbic region with a high density of cholecystokinin (CCK) immunoreactivity, that has been suggested to be of importance for the affective and emotional component of pain. In the present microdialysis study, performed in the awake rat, we demonstrate a bilateral 4- to 6-fold increase of the potassium-induced release of CCK-like immunoreactivity (CCK-LI) in the ACC 2-3 weeks after a unilateral transection of the sciatic nerve (axotomy), an animal model of phantom limb or deafferentiation pain. Considering the implication of CCK in pain modulation and anxiety, we suggest that an altered activity of CCK containing neurons in the ACC may be of importance for the affective component of certain pain conditions.

Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin

The neuroendocrine control of the gonad is exerted primarily by the gonadotropin-releasing hormone neurons located in the septum and the hypothalamus. Despite their sexually dimorphic activity, tonic in males and phasic in females, these neurons have not appeared qualitatively different between sexes in intrinsic organization or chemical phenotype. Here, by using multiple-label immunocytochemistry, it is demonstrated that the phenotype of gonadotropin-releasing hormone neurons is sex specific. In females only, 54.5% of them co-expressed cholecystokinin immunoreactivity and 29.4% additionally expressed neurotensin immunoreactivity. These multipeptidergic neurons were observed in the hypothalamus but not in the septum. During postnatal development, cholecystokinin and neurotensin immunoreactivities were first detected in gonadotropin-releasing hormone-containing axons of the median eminence at vaginal opening, suggesting an involvement of the neuropeptides in peri-ovulatory events. This peptidergic phenotype was not apparent in females ovariectomized as adults but was reinstated by estradiol treatment. In adult males, the testicle does not control this phenotype because orchidectomized adults did not display it, whatever the post-operative delay (one to five weeks) or substitutive chronic steroid treatment (testosterone or estradiol). The testicle may, however, masculinize the phenotype neonatally because estradiol or testosterone treatment in adulthood induced an expression of cholecystokinin immunoreactivity in gonadotropin-releasing hormone-containing axons of the median eminence in both males and females that were gonadectomized at birth. This procedure, however, failed to significantly induce an expression of neurotensin immunoreactivity, suggesting a role of the postnatal ovary on this element of the chemistry of gonadotropin-releasing hormone neurons. Thus, the gonad permanently organizes the gonadotropin-releasing hormone neuronal population, resulting, at least in females, in a mosaic of phenotypically distinct, functional subunits.

Secretagogues and Growth Factors in Fish and Crustacean Protein Hydrolysates.

: The search for new molecules in fish protein hydrolysates is of great interest in animal feeding as it is in aquaculture, fertilizer, cosmetic, and pharmacologic domains. Different sources of hydrolysates such as shrimp waste (Pandalus borealis), cod (Gadus morhua) head, and head and viscera of sardine (Sardina pilchardus), obtained after hydrolysis or autolysis, were tested on fibroblast cell cultures and by gastrin radioimmunoassay. The level of hydrolysis seems to play an important role in the presence of biological peptides. Elution profile on a gel filtration Sephadex G-50 column was used to estimate the degree of hydrolysis of the fractions studied. Growth-factor-like activities were found in less-hydrolyzed fractions. Conversely, the most-hydrolyzed fractions showed gastrin and cholecystokinin immunoreactivity.

The central 5-HT3 receptor in CNS disorders.

Among the characterized 5-HT receptors of the central nervous system, the type 3 receptor subtype (5-HT3R) is the only one known to be a ligand-gated ion channel. Its early pharmacological characterization and mapping by radioligand binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigro-striatal pathway and reduce alcohol consumption in experimental animals while antagonists of this receptor have been reported to treat anxiety disorders. Following the cloning of this receptor in 1991, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis. Here we summarize our recent efforts showing that 5-HT3R-expressing neurons are mainly GABA containing cells in the rat neocortex, olfactory cortex, hippocampus, and amygdala which also often contain cholecystokinin (CCK) immunoreactivity. These results provide a means to unify some of the initial pharmacological observations.

The potency of dietary amino acids in elevating plasma cholecystokinin immunoreactivity in cats is related to amino acid hydrophobicity

Incomplete agreement exists on the relative potency of amino acids in stimulating endocrine secretion of cholecystokinin (CCK). Species and methodological variations have been suggested to account for the apparent inconsistencies. In the present research, the CCK-releasing potency of dietary amino acids was evaluated in cats using plasma CCK-like immunoreactivity (CCK-LI) as an indicator of CCK secretion rather than pancreatic protein and enzyme secretion, as has been used in past research. Oral–gastric administrations of a casein-simulating amino acid mixture increased ( P<0.05) plasma CCK-LI but not to the extent of that observed for casein or sodium oleate. The response in plasma CCK-LI to administrations of 50 mM solutions of amino acids was significant ( P<0.05) for tryptophan, phenylalanine, leucine, and isoleucine and the response increased linearly ( P<0.01) with increasing amino side-chain hydrophobicity. Control administrations of water and saline also evoked elevation in plasma CCK-LI, but the responses were so transient that amino acid effects were not obscured. This was substantiated by the finding of a significant linear ( P<0.001) dose response to phenylalanine administration. Cholecystokinin-8, 33 and 58 were among the CCK molecular forms identified by HPLC in plasma after administrations of phenylalanine and water. The present findings indicate that lipophilic amino acids released during digestion account for at least part of the endocrine CCK response in cats to ingested protein. The greater CCK-releasing potency observed for intact protein relative to free amino acids may have been the result of a slow digestive release of amino acids, elaboration of peptide secretogogues or protection of protease-sensitive releasing factors.

The 5-HT3Receptor Is Present in Different Subpopulations of GABAergic Neurons in the Rat Telencephalon

The type 3 serotonin receptor (5-HT3R) is a ligand-gated ion channel whose presence in the CNS has been established by radioligand binding, in situ hybridization, and immunohistochemical analysis. To analyze further the role of the 5-HT3R in the CNS, we used in situ hybridization and immunocytochemistry to determine that 5-HT3R-expressing neurons are mainly GABA-containing cells in the rat telencephalon. We determined that 5-HT3R/GABA-containing neurons do not exhibit somatostatin immunoreactivity but often contain cholecystokinin (CCK) immunoreactivity. 5-HT3R-expressing cells with CCK immunoreactivity were observed in the neocortex, olfactory cortex, hippocampus, and amygdala. The 5-HT3R/CCK interneurons represent between 35 and 66% of the total population of CCK-containing cells in the neocortex. Further characterization of the 5-HT3R/GABAergic neurons was based on their calcium-binding protein immunoreactivity and showed that these neurons lack parvalbumin (PV) and represent a subpopulation of calbindin (CB)-containing interneurons that were preferentially present in the CA1-CA3 subfield of the hippocampus. Although some 5-HT3R/GABAergic neurons with calretinin (CR) were found in the neocortex, olfactory cortex, hippocampus, and amygdala, these neurons were more often present in the agranular insular and piriform cortices. We conclude that the neuronal expression of the 5-HT3R is selective within the GABA neuron population in the rat telencephalon. These 5-HT3R-expressing interneurons might contain CCK, CB, and CR. We suggest that serotonin through the 5-HT3R may regulate GABA and CCK neurotransmission in the telencephalon.

Cortical peptide changes in Huntington's disease may be independent of striatal degeneration.

Patients with Huntington's disease (HD) develop pathological changes in cerebral cortex as well as in striatum. We studied levels of neuropeptide immunoreactivity in 13 areas of postmortem cerebral cortex dissected from 24 cases of HD and 12 controls. Concentrations of immunoreactive cholecystokinin (CCK-LI) were consistently elevated 57 to 153% in HD cortex. Levels of vasoactive intestinal polypeptide (VIP-LI) and neuropeptide Y (NPY-LI) were significantly increased in 10 and 8 of the 13 cortical regions, respectively. Concentrations of somatostatin (SRIF-LI) were increased in only 3 areas, while substance P (SP-LI) was, for the most part, unchanged. Detailed analyses of the CCK-LI and VIP-LI data showed there to be no relationship between the increased cortical peptide levels and the degree of striatal atrophy. We studied the same cortical peptides in rats with long-standing striatal lesions and found no significant changes of CCK-LI, NPY-LI, VIP-LI, or SRIF-LI in any of the 8 cortical regions that were examined. These results indicate that there are widespread and differential changes in cortical neuropeptide systems in HD and that these changes occur independently of the striatal pathology that characterizes the illness.

Distribution of cholecystokinin immunoreactivity in the BALB/c mouse forebrain: an immunocytochemical study

The present study describes cholecystokinin (CCK) immunoreactivity (CCK-IR) distribution in the brains of control and colchicine-treated mice. In the brains of control mice, the CCK-IR strongly revealed numerous axons and terminals. Perikarya exhibiting a faint to moderate immunoreactivity were also observed in areas such as cortices, hippocampus, amygdala, septum, and thalamus. The colchicine treatment did not seem to notably affect the brain CCK-IR innervation, but resulted in profound changes of the perikaryal staining. Indeed, the regions, which contained numerous moderately stained perikarya in the control animals, exhibited after colchicine treatment immunoreactive perikarya intensely stained but only in moderate number. This feature obviously appeared in the cortex in which, in addition to strongly stained perikarya, colchicine induced the appearance of numerous CCK-IR hillocks. In the lateral amygdala and thalamus of colchicine-treated animals, the somatic immunoreactivity was considerably decreased. The regions, such as paraventricular hypothalamic nucleus and bed nucleus of the stria terminalis, which in the control animals did not exhibit any stained perikaryon, showed a high number of strongly stained cell bodies after colchicine treatment. This study, mapping the mouse forebrain CCK-IR, demonstrated a wide distribution of this peptide. Moreover, CCK-IR is spontaneously visible in neurons of untreated mouse in some brain areas previously shown in the rat to exhibit CCK mRNA, but no clear perikaryal CCK-IR even after colchicine treatment.

Persistence of the releasable pool of CCK in the rat nucleus accumbens and caudate-putamen following lesions of the midbrain

Previous studies have identified populations of dopamine neurons in the midbrain that colocalize cholecystokinin some of which project to the nucleus accumbens and caudate-putamen. The contribution of dopamine-colocalized peptide to the total releasable pool of cholecystokinin in these brain regions was investigated using microdialysis. Dopamine, dihydroxyphenylacetic acid and cholecystokinin immunoreactive levels in dialysates of the posterior medial nucleus accumbens and medial caudate-putamen were determined following 6-hydroxydopamine lesions of the ventral tegmental area and substantia nigra or transection of the medial forebrain bundle. An 89–99% depletion in basal extracellular dihydroxyphenylacetic acid and an 87–99% decrease in veratridine-evoked extracellular dopamine levels was observed in the nucleus accumbens and caudate-putamen, 4 weeks after 6-hydroxydopamine lesion. No statistically significant difference was observed between lesioned and control animals in the basal or veratridine-evoked extracellular level of cholecystokinin immunoreactivity in either region. Similarly, transection of the medial forebrain bundle failed to significantly deplete the releasable pool of cholecystokinin immunoreactivity in the nucleus accumbens or caudate nucleus despite 89–99% depletions of dopamine and its metabolite. These data suggest that midbrain dopamine or non-dopaminergic cells are not the primary source of releasable cholecystokinin in the posterior medial nucleus accumbens and medial caudate-putamen measured by microdialysis.

Immunohistochemical studies of mesolimbic dopaminergic neurons in Fischer 344 and Lewis rats

Previous work has shown that the inbred Lewis and Fischer 344 rat strains differ in several behavioral measures related to mesolimbic dopamine function. Moreover, the ventral tegmental area (VTA) of the Lewis rat has been shown to contain higher levels of tyrosine hydroxylase compared to that of the Fischer rat by blot immunolabeling procedures. To investigate structural correlates of this biochemical difference, an immunohistochemical study of VTA dopaminergic neurons in these two strains was undertaken. Results show that the density and total number of tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat is about 50% of that found in the Fischer rat. In contrast, examination of the substantia nigra in the same sections revealed no differences in the density and number of tyrosine hydroxylase-positive cells between these strains. Fischer-Lewis strain differences were also evident for cholecystokinin immunoreactivity in the VTA, with much lower levels seen in the Lewis rat, consistent with the known colocalization of this neuropeptide in many VTA dopamine neurons. The finding of 50%v fewer tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat, along with our earlier results showing 45% higher levels of tyrosine hydroxylase by blot immunolabeling, would suggest much higher levels of tyrosine hydroxylase per VTA neuron in this strain. However, no obvious strain difference in the cellular intensity of tyrosine hydroxylase immunoreactivity could be detected by immunohistochemistry. Finally, the density of VTA dopamine neurons was assessed in 1-week-old Fischer and Lewis rats. In contrast to the results obtained for adult animals, no difference in the number of tyrosine hydroxylase-positive neurons was apparent in these young animals, indicating that the Fischer-Lewis strain difference in VTA dopamine neurons appears later in postnatal development. These anatomical findings shed new light on the differences in the mesolimbic dopamine system between Fischer and Lewis rats that may contribute to the behavioral differences exhibited by these animals.

Somatostatin, neuropeptide Y, neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy.

Somatostatin-, neuropeptide Y-, neurokinin B- and cholecystokinin-containing neurons were investigated in the rat hippocampus in two chronic models of temporal lobe epilepsy, i.e. 30 days after rapid kindling or electrically induced status epilepticus (post-status epilepticus). After rapid kindling, somatostatin immunoreactivity was strongly increased in interneurons and in the outer and middle molecular layer of the dentate gyrus. In four of six post-status epilepticus rats (status epilepticus I rats), somatostatin immunoreactivity was slightly increased in the dorsal but decreased in the ventral dentate gyrus and molecular layer. Somatostatin immunoreactivity decreased in neurons of the dorsal hilus in the two other post-status epilepticus rats investigated, while a complete loss was found in the respective ventral extension (status epilepticus-II rats). These changes were associated with a different extent of neurodegeneration as assessed by Nissl staining. Similarly, neuropeptide Y immunoreactivity was enhanced in neurons of the hilus and in the middle and outer molecular layer of the dentate gyrus in the dorsal hippocampus of rapidly kindled and status epilepticus-I rats. Neuropeptide Y and neurokinin B immunoreactivity was enhanced in the mossy fibers of all post-status epilepticus rats, but not in the rapidly kindled rats. In status epilepticus-II rats, neuropeptide Y-and neurokinin B-positive fibers were also detected in the infrapyramidal region of the stratum oriens of CA3 and in the inner molecular layer of the dentate gyrus in the dorsal and ventral hippocampus respectively, labeling presumably sprouted mossy fibers. Increased staining of neuropeptide Y and neurokinin B was found in the alveus after rapid kindling. Cholecystokinin immunoreactivity was markedly increased in the cerebral cortex, Ammon's horn and the molecular layer of the dentate gyrus in the ventral hippocampus of rapidly kindled and post-status epilepticus rats. The lasting changes in the immunoreactive pattern of various peptides in the hippocampus may reflect functional modifications in the corresponding peptide-containing neurons. These changes may be involved in chronic epileptogenesis, which evolves in response to limbic seizures.

Evidence for a high-affinity uptake system for cholecystokinin octapeptide (CCK8) in rat cortical synaptosomes.

Given the high resistance of the cholecystokinin octapeptide (CCK8) to in vivo peptidase degradation, the possible existence of a reuptake system for this peptide was investigated. Efficient accumulation of intact, tritiated propionyl CCK8 ([3H]pCCK8) was observed following its incubation with rat cortical synaptosomes but not with cerebellar synaptosomes, where no cholecystokinin immunoreactivity was found. This uptake process appeared to be dependent on temperature, duration of incubation, concentration of radioligand, the presence of glucose and the integrity of the synaptosomes. A Lineweaver-Burk analysis indicated that the putative uptake process is characterized by a single Km value of 10.7 nM and a Vmax of 8.5 fmol/min/mg of protein. Carbonyl cyanide-m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked accumulation of [3H]pCCK8, whereas ouabain did not. The uptake was found to be highly specific since, among all the cholecystokinin analogues tested, only CCK8 and, to a lesser extent, CCK7, were able to inhibit [3H]pCCK8 uptake. The rate of [3H]pCCK8 uptake was not affected by CCK4, CCK5, D-Trp CCK8, BC 264, a potent and radioactivity was observed using [3H]pBC 264, a result which is not in favour of a cholecystokinin receptor-induced internalization mechanism. The potent and selective uptake mechanism characterized in this study could participate, in conjunction with extra and intracellular degradation of CCK8 by peptidases, in the interruption of cholecystokinin-conveyed messages in the brain.

Elevation of plasma cholecystokinin (CCK) immunoreactivity by fat, protein, and amino acids in the cat, a carnivore

The cat requires a diet high in protein and certain nutrients that are found only in animal tissue. It is possible that secretogogues of intestinal CCK in the cat may be different from those observed in non-carnivorous species. Plasma CCK concentrations were determined in cats ( n = 6) given by oral-gastric tube either casein, whey protein, corn oil, or corn starch suspended in water. CCK was measured by RIA with a tyrosine sulfate-specific, C-terminal antibody, DINO. HPLC of plasma revealed that most CCK-immunoreactivity (CCK-LI) was associated with CCK-33 and a late eluting peak, presumably CCK-58. Casein, whey protein, and corn oil increased ( P < 0.05) post-administration plasma CCK-LI, and at least for casein, the effect was dose related. An amino acid mixture approximating the residue composition of casein increased plasma CCK-LI ( P < 0.05), however, the increase tended to be less than that caused by casein. Evaluation of post-administration levels of plasma amino acids indicated that intact protein and amino acids in the intestinal lumen affect CCK release by different mechanisms. Collectively, the results indicated that although cats are carnivores cats and humans secrete CCK in response to the same nutrients.

Peptidergic innervation of the bovine vagina and uterus

The distribution of neuropeptide Y, substance P, vasoactive intestinal polypeptide, Leu5-enkephalin, bombesin/gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin, cholecystokinin and catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase was studied immunohistochemically in nerve fibres supplying the bovine vagina and uterus. The nerves containing tyrosine hydroxylase or dopamine-beta-hydroxylase and neuropeptide Y-immunoreactivity were particularly numerous in both organs. Substance P, vasoactive intestinal polypeptide and Leu5-enkephalin-containing nerves were less numerous whereas somatostatin and calcitonin gene-related peptide-immunoreactive nerves occurred occasionally. Bombesin/gastrin-releasing peptide and cholecystokinin immunoreactivities were not present in nervous fibers of the bovine uterus and vagina. Generally, the immunoreactive nerve terminals, fibers, networks or nerve bundles were present below the serous membrane, between smooth muscle cells of muscular layers, around blood vessels, in the submucosal layer and below the luminal epithelium of the uterus and cervix.