CCK-4: Psychophysiological conditioning elicits features of spontaneous panic attacks.

INTRODUCTION: Cholecystokinin-tetrapeptide (CCK-4) is an established model to generate subjective panic anxiety. CCK-4 injection also results in consistent and dose-dependent rise of stress hormones. Effects other than upon subjective panic and stress hormone activity have barely been examined. The purpose of the study was to investigate CCK-4 effects on emotional facial expression and especially on fear relevant facial muscles establishing therewith a more objective method to measure subjective panic anxiety. METHODS: 20 healthy male subjects were randomly and double-blindedly assigned in two groups (dose groups), each of which was investigated three times once with placebo and twice with 25 µg or 50 µg CCK-4 respectively. Subjects of each group were randomly assigned in two different balanced orders of investigations: CCK-CCK-Placebo vs. Placebo-CCK-CCK. Facial muscle and hypothalamo-pituitary-adrenocortical (HPA)-axis activity were recorded. RESULTS: CCK-4 led dose-dependently to an increase of panic anxiety, an activation of fear relevant facial muscles and a rise of stress hormones. Whereas placebo administration before CCK-4 revealed no significant panic and stress response, during placebo following CCK-4 stimulations a psychophysiological conditioning effect could be observed without rise in HPA-axis activity. DISCUSSION: Our findings indicate the possibility to measure different intensities of panic anxiety and conditioning effects with a facial EMG method. Dissociation of HPA-activity and fear relevant facial muscle activity is in accordance with former results about spontaneous panic attacks.

Gastric distention activates satiety circuitry in the human brain.

Gastric distention during meal ingestion activates vagal afferents, which send signals from the stomach to the brain and result in the perception of fullness and satiety. Distention is one of the mechanisms that modulates food intake. We measured regional brain activation during dynamic gastric balloon distention in 18 health subjects using functional magnetic resonance imaging and the blood oxygenation level-dependent (BOLD) responses. The BOLD signal was significantly changed by both inflow and outflow changes in the balloon's volume. For lower balloon volumes, water inflow was associated with activation of sensorimotor cortices and right insula. The larger volume condition additionally activated left posterior amygdala, left posterior insula and the left precuneus. The response in the left amygdala and insula was negatively associated with changes in self-reports of fullness and positively with changes in plasma ghrelin concentration, whereas those in the right amygdala and insula were negatively associated with the subject's body mass index. The widespread activation induced by gastric distention corroborates the influence of vagal afferents on cortical and subcortical brain activity. These findings provide evidence that the left amygdala and insula process interoceptive signals of fullness produced by gastric distention involved in the controls of food intake.

Aging and panicogenic response to cholecystokinin tetrapeptide: an examination of the cholecystokinin system.

Older age is associated with diminished symptomatic and cardiovascular response to the panicogenic agent cholecystokinin tetrapeptide (CCK-4). We hypothesized that circulating concentrations of endogenous CCK-4 and/or CCK-8 are increased in later life, possibly due to decreased enzymatic degradation, and that this is associated with desensitization of CCK-B receptors. The study group consisted of 20 healthy subjects aged 18-30 years and 20 healthy subjects aged 65-85 years. The two groups were compared on fasting basal plasma concentrations of CCK-4, sulfated CCK-8 (CCK-8s) and nonsulfated CCK-8 (CCK-8 ns), and on binding capacity of lymphocyte CCK-B receptors. Under single-blind (to subject) conditions, subjects were then administered an intravenous bolus of placebo, followed 50 min later by an intravenous bolus of 50 micro g of CCK-4. Plasma concentrations of total CCK (CCK(T)) were measured 2 min before and 2, 5, 10, and 15 min after each injection. Compared with younger subjects, older subjects had a significantly higher basal plasma concentration of CCK-8s and significantly diminished binding capacity of CCK-B receptors. Following injection of placebo, plasma CCK(T) concentrations did not significantly change from baseline in either age group, but the elderly had significantly higher concentrations than the young at 2, 5, and 10 min. Following injection of CCK-4, the plasma concentration of CCK(T) was highest at 2 min and declined after that. The elderly had significantly higher CCK(T) concentrations (ie. a slower decline in CCK(T)) than the young at 5, 10, and 15 min. These findings are consistent with our hypothesis and suggest that age-related changes in the CCK system could contribute to the diminished panicogenic response to exogenous CCK-4 in older persons.

Development of a sensitive and specific assay system for cholecystokinin tetrapeptide.

Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step system involves i) extraction of CCK fragments from plasma using reverse phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of peptides with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and 125I Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, sulfated CCK-8 (CCK-8S) and nonsulfated CCK-8 (CCK-8ns) with equal affinity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 +/- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equivalents. Concentrations of each individual peptide in plasma were determined to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively.

Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins.

The hypothesis that cellular protein carboxyl-methylation reactions recognize altered aspartyl residues as part of a protein repair pathway has been tested in an in vitro system using tetragastrin (Trp-Met-Asp-Phe-NH2) as a model sequence. The L-isoaspartyl form of tetragastrin, where the phenylalanine residue is linked to the side-chain carboxyl group of the aspartate residue ([iso-Asp3]tetragastrin), is a substrate for the erythrocyte protein carboxyl methyltransferases, while the normal form is not. The enzymatically produced alpha-methyl ester of [iso-Asp3]tetragastrin, [iso-Asp(OMe)3]tetragastrin, is unstable at pH 7.4 and 37 degrees C and spontaneously demethylates with a half-time of 41 min to an intermediate L-succinimide form ([Asu3]tetragastrin) that, in turn, spontaneously hydrolyzes with a half time of 116 min to give a mixture of normal tetragastrin (20%) and [iso-Asp3]tetragastrin (80%). This sequence of enzymatic and nonenzymatic reactions can be coupled in a single reaction mixture; the [iso-Asp3]tetragastrin that is produced upon succinimide hydrolysis can reenter the reaction sequence by enzymatic methylation, and the net result of the process is the conversion of the isomerized peptide to the normal peptide. The efficiency of this "repair" reaction is limited by a side reaction of racemization at the alpha-carbon of the succinimide (half-time = 580 min). In a 24-hr time period, normal L-aspartyl-containing tetragastrin is obtained in about 50% yield from the coupled reaction mixture; other products include [D-iso-Asp3]tetragastrin and [D-Asp3]tetragastrin. The versatile chemistry of succinimide peptides suggests that methylated L-isoaspartyl sites (and possibly methylated D-aspartyl sites) in cellular polypeptides can eventually yield "repaired" normal L-aspartyl sites through succinimide intermediates.

'In vivo' amplification of biological activity of tetragastrin by amino acid hydroxamates.

Rat blood was shown to contain an aminopeptidase which rapidly hydrolyses short peptides containing an aromatic amino acid as N-terminal residue. Using tetragastrin (Trp-Met-Asp-PheNH2) as substrate, we showed that some amino acid hydroxamates inhibit rat aminopeptidase activity 'in vitro' in the following order: HTrpNHOH greater than HPheNHOH much greater than HAlaNHOH. The same hydroxamates markedly enhanced the biological activity of tetragastrin 'in vivo'. The amplification of the secretory effect, correlated with the amount of the hydroxamate used, strongly suggests that these compounds can stabilize a number of active peptides in vivo by inhibiting their proteolytic degradation.

Degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma in vitro.

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products. CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min). The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide. CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids. CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.