Ontogeny of procholecystokinin processing in rat hypothalamus

The concentration of procholecystokinin (pro-CCK) in the fetal hypothalamus was 126 +/- 41 pmol/g (mean +/- SEM; n = 20), 22 +/- 9 pmol/g at day 7 postpartum and 3 +/- 2 pmol/g in the adult. In contrast, the concentration of bioactive carboxyamidated CCK rose from 6 +/- 2 pmol/g in the fetal hypothalamus to 52 +/- 10 pmol/g in the adult. The concentration of glycine-extended processing intermediates first decreased from 21 +/- 5 pmol/g in the fetus to 5 +/- 1 pmol/g at day 21 postpartum. Subsequently, the concentration rose to 21 +/- 4 pmol/g in the adult. The results show that the CCK gene is well expressed in the fetal hypothalamus. However, only a small fraction of pro-CCK reaches maturation before weaning. We conclude that expression of the CCK gene in the hypothalamus as bioactive peptide to a large degree is regulated at the posttranslational level.     

Progastrin and its products in the cerebellum

So far the only CNS neurons found to express gastrin have been hypothalamohypophyseal. Using a library of radioimmunoassays specific for different sequences of porcine progastrin in combination with chromatography and enzyme cleavages, 21 or 24 porcine cerebelli was now found to contain progastrin or its products. Sixteen cerebelli processed progastrin via glycine-extended intermediates to bioactive, carboxyamidated gastrin-17 and -34, half of the gastrins being tyrosine O-sulfated. The mean concentration of carboxyamidated gastrins was 0.8 pmol/g tissue (range less than 0.1-2.1 pmol/g), of glycine-extended intermediates 0.1 pmol/g (range less than 0.1-0.3 pmol/g) and of progastrin 0.4 pmol/g (range less than 0.1-1.0 pmol/g). The results show that the gastrin gene is expressed in more than a single region of the brain.     

Gastrin, cholecystokinin and their precursors in acoustic neuromas

Using sequence-specific radioimmunoassays the occurrence of 4 neuropeptides (cholecystokinin (CCK), gastrin, somatostatin, substance P and some of their precursors) was examined in 19 human acoustic nerve tumors and corresponding normal tissue. The normal acoustic nerve tissue obtained by autopsy contained traces of CCK, somatostatin, and substance P (5pmol/g), but neither proCCK, gastrin nor progastrin. In contrast 8 tumors expressed proCCK (range 0.2–4.5 pmol/g), 3 tumors gastrin (0.7–7.3 pmol/g) and 14 tumors progastrin (0.6–2.2 pmol/g). Traces of somatostatin were present in two and substance P in one tumor. The results show that acoustic neuromas often express the homologous CCK and gastrin genes, but process their propeptides poorly to transmitter-active peptides. The tumor synthesis of gastrin/progastrin contrasts to the rare, sporadic expression of the gastrin gene in normal nerve tissue.     

Measurement and characterization of neuronal cholecystokinin using a novel radioreceptor assay

This study describes a novel radioreceptor assay (RRA) for cholecystokinin (CCK) which is the first to measure and characterize brain CCK using a technique not dependent on the generation of peptide antibodies. The CCK RRA utilizes the mouse cerebral cortex CCK receptor as the binding source and [125I]BH-CCK-8 as the radiolabelled probe. [125I]BH-CCK-8 bound to the central CCK receptor with a Kd of 1.82 nM and a Bmax of 1.21 pmol/g tissue. Unlabelled CCK-8 displaced the specific binding of [125I]BH-CCK-8 with an inhibition constant of 3.84 nM. CCK was extracted (90% methanol) from discrete brain regions (mouse) and quantified using the CCK RRA. The amygdala contained the highest concentration of CCK (394 +/- 21 pmol/g tissue), followed by the olfactory bulbs (306 +/- 19 pmol/g tissue) and cerebral cortex (298 +/- 21 pmol/g tissue). Moderate levels of CCK were found in the hippocampus (212 +/- 18 pmol/g tissue), striatum (146 +/- 15 pmol/g tissue) and hypothalamus (129 +/- 9 pmol/g tissue). Low levels of CCK were recorded in the pons (45 +/- 5 pmol/g tissue), medulla (41 +/- 3 pmol/g tissue) and spinal cord (29 +/- 3 pmol/g tissue), whilst no CCK was detected in the cerebellum. The molecular forms of CCK in amygdala, cerebral cortex and hypothalamus were characterized using RRA in conjunction with HPLC. CCK-8 was identified as the major molecular form (88%, 94% and 91% of total CCK activity in amygdala, cortex and hypothalamus, respectively) with a smaller component attributable to CCK-4 (8%, 5% and 6% of the total CCK activity).     

Increased cerebrospinal fluid concentrations of C- but not N-terminal cholecystokinin fragments in multiple sclerosis

The sulfated N-terminus and the carboxyamidated C-terminus of cholecystokinin octapeptide (CCK-8) were radioimmunoassayed in cerebrospinal fluid from 19 patients with multiple sclerosis (MS) and 17 control subjects. While the N-terminal immunoreactivity was normal in all phases of MS, the concentration of C-terminal CCK immunoreactivity was significantly increased from 3.7 +/- 0.3 to 7.9 +/- 1.0 pmol/l (mean +/- S.E.M.) in both progressive and stable phases. The results indicate that C-terminal forms of CCK are released at an increased rate or that increased amounts of CCK-8 are released together with enhanced aminopeptidase activity in MS.     

Repeated administration of methamphetamine blocked cholecystokinin-octapeptide injection-induced c-fos mRNA expression without change in capsaicin-induced junD mRNA expression in rat cerebellum

In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.     

Strain differences in central nervous system concentrations of cholecystokinin between normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats

The regional brain and spinal cord concentrations of cholecystokinin-octapeptide (CCK-8) were measured in age-matched normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The relative order of distribution of CCK-8 in the WKY strain was hippocampus (20.5 +/- 1.3 pmol/g) greater than cortex greater than striatum = hypothalamus greater than midbrain = thalamus greater than spinal cord greater than medulla oblongata/pons (MO/P, 1.6 +/- 0.2 pmol/g) whereas in the SH strain this order was hippocampus (12.9 +/- 0.8 pmol/g) greater than cortex = striatum greater than hypothalamus greater than midbrain greater than thalamus = spinal cord greater than MO/P (1.4 +/- 0.2 pmol/g). The concentrations of CCK-8 in the cerebellum were at the level of assay sensitivity (0.5 pmol/g in both strains). In comparison to the WKY rats, the SH strain had significantly lower levels of CCK-8 in the hippocampus (-37%), cortex (-28%), spinal cord (-23%) and pituitary (-57%). The lowered levels of CCK-8 in the brain of the SH rat may be causally related to, or result from, the cardiovascular, behavioural or morphological abnormalities of this strain.     

Characteristics of 125I-Bolton-Hunter labelled cholecystokinin binding in human brain

The high affinity binding of 125I-Bolton-Hunter labelled CCK-8 (125I-BHCCK) was studied in human brain using ligand binding and autoradiographic techniques. High levels of 125I-BHCCK binding were observed in cortex, striatum and cerebellum. In cerebellar membranes 125I-BHCCK binding was inhibited by CCK analogues with an order of potency, caerulein greater than CCK-8 greater than CCK-33 greater than gastrin 1-17 = CCK-8 non-sulphated. CCK-4 was inactive. It is suggested that in human brain 125I-BHCCK labels a population of CCK receptors similar to those observed in guinea pig brain. The properties and distribution of these receptors differ from those of rat brain.     

Expression of the cholecystokinin gene in rat brain during development

The expression of the gene encoding for cholecystokinin (CCK) was studied pre- and postnatally in rat brain with a preproCCK-specific cDNA probe to quantitate mRNA. In addition, a C-terminal-specific radioimmunoassay was used to measure CCK-immunoreactive peptides. Poly(A)+-enriched RNA from rat brain was analyzed for the presence of CCK mRNA by agarose gel electrophoresis, followed by transfer to nitrocellulose and hybridization to a 32P-labeled CCK cDNA probe. An RNA of approximately 700 nucleotides was observed and there were no obvious CCK mRNA differences between samples taken at the different developmental stages. Brain preproCCK mRNA was detectable by embryonic day 14 and increased continuously until postnatal day 14 when it reached maximal concentrations. In young adult rats, levels were slightly decreased. CCK-immunoreactive peptides began to be detected in the samples from embryonic day 21, but marked development was seen only postnatally with a rapid increase occurring during the first 2 weeks after birth. The highest content of the peptide was present in young adult rats.     

Cholecystokinin peptides in the brain and pituitary of the bullfrog Rana catesbeiana: distribution and characterization

The distribution of CCK peptides in the bullfrog brain was determined with a CCK radioimmunoassay. Frog brain CCK distribution resembles rat porcine and human brain in that CCK concentration is moderate to high in hypothalamus, diencephalon, and medulla (3 18.4 ng/mg protein) and low in cerebellum (0.6 ng/mg protein). However, unlike all mammalian species examined, the CCK content of frog cerebral cortex, hippocampus and olfactory lobe is quite low (0.03 0.23 ng/mg protein). The elution of CCK-like peptides in frog brain extracts was determined on two HPLC systems. On both systems the bulk of the CCK-like material eluted with CCK 8 sulfate and separated from gastrin and other CCK peptides. These data suggest that though the chemical structure of CCK appears to be the same in the brains of frogs and mammals, the distribution of CCK in the brain appears to have shifted during the course of evolution, becoming a cortical, hippocampal, and olfactory system peptide only in more evolved organisms.     

Cholecystokinin peptides in the brain and pituitary of the bullfrog Rana catesbiana: distribution and characterization

The distribution of CCK peptides in the bullfrog brain was determined with a CCK radioimmunoassay. Frog brain CCK distribution resembles rat, porcine, and human brain in that CCK concentration is moderate to high in hypothalamus, diencephalon, and medulla (3–18.4 ng /protein) and low in cerebellum (0.6 ng/mg protein). However, unlike all mammalian species examined, the CCK content of frog cerebral cortex, hippocampus and olfactory lobe is quite low (0.03–0.23 ng/mg protein).The elution of CCK-like peptides in frog brain extracts was determined on two HPLC systems. On both systems the bulk of the CCK-like material eluted with CCK8 sulfate and separated from gastrin and other CCK peptides.These data suggest that though the chemical structure of CCK appears to be the same in the brains of frogs and mammals, the distribution of CCK in the brain appears to have shifted during the course of evolution, becoming a cortical, hippocampal, and olfactory system peptide only in more evolved organisms.     

A CHANGE IN SUSCEPTIBILITY OF RAT CEREBELLAR PURKINJE CELLS TO DAMAGE BY ACETALDEHYDE DURING FETAL, NEONATAL AND ADULT LIFE

Phillips S.C. & Cragg B.G. 1982 Neuropathology a id Applied Neurobiology 8, 455–463
A change in susceptibility of rat cerebellar Purkinje cells to damage by acetaldehyde during fetal neonatal and adult life
The sensitivity of rat cerebellar Purkinje cells to acetaldehyde during fetal, neonatal or adult life has been assessed by histological techniques. A pregnant female rat was exposed to ethanol vapour during the last 2 weeks of gestation while metabolism of acetaldehyde was blocked by injections of disulfiram (200mg/kg) on days E14, E16, E18 and E20. Purkinje cells were counted in the offspring 5 days after birth. The number of Purkinje cells in lobes I and VIII were 52% and 42% less than age matched controls. Smaller reductions were found in lobes V and X. The weight of the cerebellum was reduced by 42%. Neonatal rats received disulfiram (40mg) on the second day after birth and were exposed to ethanol vapour during daylight hours on the third and fourth days. Purkinje cells were counted at 5 days after birth, and losses similar to those described above were found. The weight of the cerebellum was reduced by 47%. Adult male rats were exposed to ethanol vapour for 2 weeks and given disulfiram (200mg/kg) on every second day. No reduction in cerebellar weight or loss of Purkinje cells was detected. When the dura overlying the cerebellum in adult rats was superfused with acetaldehyde solutions for 1 h, a 1–6 M solution caused degeneration whereas a 0–68 M solution did not. Thus the combination of high blood ethanol and acetaldehyde levels is damaging to perinatal Purkinje cells, whereas adult cells are resistant. We found the same ranking of susceptibility with ethanol alone (Philips & Cragg, 1982a and it was surprising that the cell losses with acetaldehyde were only marginally greater than those with ethanol alone, in view of the greater neurotoxicity of acetaldehyde during acute exposure (Phillips, 1981.     

Endogenous opioids modulate the effect of cholecystokinin on insulin release in dogs

Recently we have demonstrated in dogs and man that endogenous opioids participate in the regulation of pancreatic endocrine function following the ingestion of a meal. Since intestinal hormones such as cholecystokinin (CCK) are also released by the presence of nutrients in the gastrointestinal tract and participate in the postprandial stimulation of pancreatic endocrine function, an interaction between CCK and endogenous opioids seems possible. The present study was designed to examine this further. In a group of 8 conscious dogs the octapeptide of CCK was infused intravenously in its sulfated (CCK-8S) or nonsulfated (CCK-8NS) form and in addition the tetrapeptide of CCK (CCK-4) was given at increasing infusion rates of 50, 200 and 500 pmol/kg . h, respectively. The experiments were performed during a background infusion of saline to assess the effect on basal insulin and during a background infusion of glucose (0.2 g/min) to determine the effects on stimulated insulin release. The effect of endogenous opioids was examined by addition of the opiate-receptor antagonist naloxone. The studies demonstrate that in the basal state CCK-8S has no stimulatory effect on insulin secretion unless naloxone is added indicating that endogenous opioids help to prevent insulin secretion in the absence of elevated glucose levels. During i.v. glucose naloxone reduced the stimulatory effect of CCK-8S at 50 and 200 pmol/kg . h and that of CCK-4 at 50 pmol/kg . h. Infusion of CCK-8S and CCK-4 at 500 pmol/kg . h had no effect on glucose-stimulated insulin levels, however, the addition of naloxone elicited a significant stimulatory effect. These data demonstrate stimulatory as well as inhibitory effects of endogenous opioids depending on the dose of CCK-8 and -4. CCK-8NS reduced glucose-stimulated insulin release already at the lowest dose of 50 pmol/kg . h. This was reversed to a stimulatory effect with the addition of naloxone. These data demonstrate that the interaction between CCK-8 and -4 and endogenous opioids on prestimulated insulin secretion is much more dependent on the dose of CCK - low doses induce stimulatory and high doses inhibitory mechanisms via endogenous opioids. In view of previous in vitro and in vivo studies with exogenously infused opiate-active compounds it might be speculated that increasing doses of CCK elicit a parellel increase in the release of endogenous opioids which might be responsible for some but certainly not all of the effects observed recently for the action of naloxone in the post-prandial state.     

The regional and subcellular development of cholecystokinin immunoreactivity in vertebrate brain

We have previously demonstrated that the development of CCK in rat brain occurs during the first postnatal month. In order to determine whether the appearance of CCK is associated with specific aspects of brain histogenesis, we examined the development of brain CCK immunoreactivity in both precocial and altricial mammals and birds. The two precocial species (guinea pig and chicken) were found to achieve adult CCK concentrations prenatally, while the altricial species (zebra finch and rat) manifested adult brain CCK concentrations only after several weeks of postnatal development. In adulthood, both mammals showed relatively high forebrain CCK concentrations, while the two species of birds manifested much lower forebrain levels. Brainstem levels of CCK were similar in all species studied. In each species, the development of CCK followed a common time course across all major brain areas, although adult brainstem levels of CCK were generally attained shortly before adult forebrain levels. Correlation of our comparative ontogenetic data with known patterns of brain histogenesis indicated that CCK development follows regional neuroblastic proliferation, migration and differentiation, and occurs during or soon after local synaptogenesis. In the rapidly developing precocial chicken brain, CCK production precedes the postnatal gliogenic and myelinogenic increases in brain weight, suggesting that neurogenic production of CCK occurs independently of these non-neuronal maturation events. Subcellular fractionation of developing chicken brain revealed that a substantial fraction of brain CCK is localized in synaptosomes relatively early in embryogenesis; this synaptosomal localization becomes even more pronounced with further brain maturation. This early appearance of CCK in synaptic terminals indicates a correspondingly precocial maturation for the intraneuronal mechanisms subserving peptide cleavage, axonal transport and vesicular insertion, and suggests that CCK may be available for neurotransmission quite early in development. In an analysis of the molecular forms of CCK, gel filtration disclosed no differences between species or different brain areas in the form of CCK present. CCK-8 always predominated in brain, with smaller void volume (pro-CCK) peaks, and negligible amounts of CCK-33. Finally, duodenal CCK (largely CCK-33) appeared much earlier than brain CCK in all species examined, suggesting that the gut and brain CCK systems develop independently of one another.     

Cholecystokinin-dopamine coexistence: electrophysiological actions corresponding to cholecystokinin receptor subtype

Cholecystokinin (CCK)-like peptides when administered intravenously produce 2 distinct actions on the single-unit activity of mesencephalic dopamine (DA) neurons in the rat: an excitatory action and a potentiation of the inhibitory effects of DA agonists. The ability of several CCK fragments that have been shown to bind selectively to the peripheral and/or the central CCK-binding sites were examined for their ability to induce either excitation or a potentiation of DA. Only sulfated CCK-8 was able to induce excitation of mesencephalic DA neurons, but both sulfated and unsulfated CCK-8, as well as CCK-4, potentiated the inhibitory effects of the DA agonist apomorphine (APO). CCK-3 failed to potentiate APO-induced inhibition. Both of these effects appeared to be confined to cell bodies in regions of the ventral tegmental area and substantia nigra, zona compacta that have been reported to contain both DA and CCK. Thus, CCK-like peptides that have been shown to bind to the high-affinity CCK binding site in brain potentiated the effects of DA. In contrast, the ability of CCK-like peptides to induce neuronal excitation corresponds with their affinity for the peripheral-type CCK binding site.     

Ontogeny of PHI in the rat brain

The regional distribution of immunoreactive PHI (IR-PHI) was investigated in rat brain between postcoitum (pc) and day 60 postpartum (pp). IR-PHI was undetectable in all regions of the foetal brain, and only very small amounts were found at day 7 pp. However, there was a dramatic increase thereafter reaching a peak at day 20 pp (e.g. in the hippocampus there was a 12-fold increase in the PHI concentration). Highest concentations were found in the cortex (40 ± 5 pmol/g) and the hippocampus (35 ± 8 pmol/g), with lower concentrations in the diencephalon (11 ± 4 pmol/g) and mesencephalon (10 ± 3 pmol/g). The brainstem and cerebellum contained very low amounts of IR-PHI. Permeation analysis of brain extracts, on Sephadex G50-superfine, indicated the presence of one major form of IR-PHI which eluted in a similar position to pure intestinal porcine PHI and human intestinal PHI.     

A CHANGE IN SUSCEPTIBILITY OF RAT CEREBELLAR PURKINJE CELLS TO DAMAGE BY ALCOHOL DURING FETAL, NEONATAL AND ADULT LIFE

Phillips S.C. & Cragg B.G.1982 Neuropathology and Applied Neurobiology 8, 441–454
A change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal and adult life
The sensitivity of rat cerebellar Purkinje cells to ethanol exposure during fetal, neonatal or adult life was assessed by histological techniques. Pregnant female rats were exposed to ethanol vapour during the last 2 weeks of gestation. Purkinje cells were counted 5 days after the pups were born. The number of Purkinje cells in lobe VIII was reduced by 45%, and the linear density of Purkinje cells in lobe I was 47% less than in controls not exposed to ethanol. Smaller reductions were found in other lobes. The weight of the cerebellum was reduced by 34%. Neonatal rats were exposed to ethanol vapour briefly during daylight hours on the third and fourth days after birth. Purkinje cells were counted on the fifth day after birth, and losses similar to those described above were found, with additional significant reductions of cell numbers in lobe I and of Purkinje cell density in lobe VIII. The weight of the cerebellum was reduced by only 4%. Adult male rats were exposed to ethanol vapour for 3 weeks and no Purkinje cell losses were subsequently found. The dura overlying the cerebellum of separate adult male rats was superfused with 100% ethanol for 1 h and no abnormalities were detected with electron microscopy in the exposed cortex 6 days later. It is remarkable that the brief neonatal treatment caused a more widespread loss of Purkinje cells than the 10 days of exposure to ethanol in utero , whereas the Purkinje cells present in adult animals show a great resistance to ethanol. The neonatal period seems to be a time of high susceptibility of Purkinje cells to ethanol.     

Cholecystokinin peptides and receptor binding in Alzheimer's disease

Summary Cholecystokinin (CCK) is a peptide that can be found in the cerebral cortex in high concentrations and is involved in learning and memory as well as neurodegenerative processes. Cortical brain samples from 9 patients with Alzheimer's disease and 9 matched control cases were studied with respect to the concentrations of various molecular forms of CCK and the CCK receptor binding characteristics. No differences were found between patients and controls in any of these measures. Significant correlations were found between the concentrations of CCK-8 sulphated and the three nonsulphated CCK peptides measured. In addition, the concentrations of CCK-4 and CCK-5 showed a highly significant and positive correlation.