Jens F. Rehfeld

Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system

Immunocytochemistry and radioimmunochemistry demonstrate the occurrence of the gastrointestinal hormone cholecystokinin (CCK) in both the central and peripheral nervous system of the guinea pig. CCK nerves are particularly numerous in the neocortex, the hippocampus, the amygdaloid nuclei, the hypothalamus, the spinal cord and in the colon. The nerves contain 5 molecular components of CCK, with gel chromatographical elution constants (Kav) of 0.05, 0.50, 0.90, 1.10 and 1.30, respectively. The four latter correspond to triacontatriapeptide CCK and its COOH-terminal dodeca-, octa- and tetrapeptide portions, respectively. Cholecystokinins are hence widely distributed in the nervous system and occur in the substantial quantities (greater than or equal to 0.2 nmol CCK-8-equiv./g) in several distinct regions.

Increased cardiac BNP expression associated with myocardial ischemia

Congestive heart failure is accompanied by increased cardiac brain natriuretic peptide (BNP) gene expression with elevated plasma concentrations of BNP and its precursor, proBNP. We investigated if myocardial ischemia in the absence of overt heart failure may be another mechanism for increased myocardial BNP expression. The BNP expression was examined in hypoxic myocardium of patients undergoing coronary bypass grafting surgery, in patients with coronary artery disease and normal left ventricular function undergoing percutaneous transluminal intervention therapy, and in heart failure patients without coronary artery disease. BNP mRNA was quantified by real‐time PCR, and plasma BNP and proBNP concentrations were measured with radioimmunoassays. Quantitative analysis of BNP mRNA in atrial and ventricular biopsies from coronary bypass grafting patients revealed close associations of plasma BNP and proBNP concentrations to ventricular, but not atrial, BNP mRNA levels. Plasma BNP and proBNP concentrations were markedly increased in patients with coronary artery disease but without concomitant left ventricular dysfunction. These results are compatible with the notion that myocardial ischemia, even in the absence of left ventricular dysfunction, augments cardiac BNP gene expression and increases plasma BNP and proBNP concentrations. Thus, elevated BNP and proBNP concentrations do not necessarily reflect heart failure but may also result from cardiac ischemia.

Evidence for a common evolutionary origin of gastrin and cholecystokinin

THE gastrointestinal hormones gastrin and cholecystokinin (CCK) are structurally and functionally related in that they have identical COOH-terminal pentapeptide sequences, which represent the biologically active portions of the molecules. The rest of each molecule only modifies its activity quantitatively and determines relative potencies for various targets. This has suggested that the two hormones have evolved from a common ancestral molecule1,2. To test this hypothesis we have investigated the occurrence of gastrin and CCK in different vertebrate species by immunocytochemistry and radioimmunochemistry. We now present evidence that in amphibians and teleosts gastrin and CCK are no longer recognisable as separate hormones.

PANCREATIC-POLYPEPTIDE RESPONSE TO FOOD IN DUODENAL-ULCER PATIENTS BEFORE AND AFTER VAGOTOMY

In 21 normal fasting subjects basal serum-pancreatic-polypeptide (H.P.P.), as measured by a specific radioimmunoassay, was 43 +/- 4 pmol/l (mean +/- S.E.M). This basal concentration was of the same magnitude as that of other gastrointestinal peptide hormones. In normal subjects the H.P.P. response to food was bisphasic. A rapid eightfold increase, reaching a maximum 20--30 min after beginning of the meal, was followed by a secondary, prolonged increase. H.P.P. did not return to basal concentration within five hours. 14 duodenal-ulcer (D.U.) patients were studied before and after truncal vagotomy. Before operation the D.U. patients had higher basal H.P.P. concentrations than the normal group (110 +/- 40 pmol/l) whereas their H.P.P. response pattern to food was similar to that of the normal subjects. After vagotomy the primary rapid increase in H.P.P. concentration was eliminated and the secondary, prolonged response was significantly reduced (P less than 0-005). The results indicate that H.P.P. is a digestive hormone under vagal control.

Impaired gastric acid secretion in gastrin-deficient mice

To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H(+)-K(+)-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H+-K+-adenosinetriphosphatase (H+-K+-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H+-K+-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.

Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin receptor-deficient mice

BACKGROUND & AIMS The cholecystokinin (CCK)-B/gastrin receptor is one of several regulators of gastric acid secretion and mucosal growth. To elucidate the contribution of this receptor relative to other trophic and secretory factors, mice that lack the CCK-B/gastrin receptor have been generated and studied. METHODS Both alleles of the CCK-B/gastrin receptor were inactivated by targeted gene disruption. Analysis of the mice included measurement of basal gastric pH and plasma gastrin levels. In addition, multiple gastric mucosal cell types were identified by immunostaining and quantified. RESULTS Homozygous mutant mice were viable, fertile, and appeared grossly normal into adulthood. The receptor-deficient mice exhibited a marked increase in basal gastric pH (from 3.2 to 5.2) and an approximately 10-fold elevation in plasma gastrin concentration compared with wild-type controls. In the stomach of mutant animals, parietal and enterochromaffin-like cells were decreased, providing a likely explanation for the reduction in acid output. In the antrum, a decrease in somatostatin cell density and an increase in the gastrin cell number were observed, consistent with the concomitant elevation in circulating gastrin. CONCLUSIONS Together, these findings demonstrate the importance of the CCK-B/gastrin receptor in maintaining the normal cellular composition and function of the gastric mucosa.

Acute myocardial hypoxia increases BNP gene expression

It is well established that cardiac failure increases cardiac B‐type natriuretic peptide (BNP) expression due to myocardial stretching. However, patients with ischemic heart disease also display increased plasma BNP and proBNP concentrations despite preserved cardiac function. In this study, we examined whether acute myocardial hypoxia increases cardiac BNP expression. Surgical reduction of the blood flow to an area of the anterior ventricular wall in pigs reduced the myocardial oxygen tension from 46 ± 4 to 13 ± 5 mmHg. The tissue contents of VEGF and BNP mRNA increased 1.8‐fold and 3.5‐fold, respectively (n=10, P<0.005) in hypoxic compared with normoxic ventricular myocardium after 2.2 ± 0.2 h; the magnitude of the increase in BNP mRNA expression was positively correlated with that of VEGF in hypoxic myocardium (r=0.66, P<0.05). In support of a hypoxia‐induced increase of BNP gene transcription, the content of a premature BNP mRNA was increased in hypoxic myocardium (4.8‐fold, P<0.005) and in freshly harvested ventricular myocytes when kept in culture flasks and oxygen‐deprived for 3 h (2.2‐ fold, P=0.002). ProBNP peptide accumulated in the medium of freshly harvested ventricular myocyte cultures but was undetectable in ventricular myocardium, indicating rapid release of the newly synthesized proBNP peptide. Accordingly, the plasma proBNP concentration increased after 2 h of myocardial hypoxia (P=0.028). Cumulatively, the data suggest that acute hypoxia stimulates cardiac BNP expression.

Cholecystokinin octapeptide: Vesicular localization and calcium dependent release from rat brain in vitro

Abstract Sub-cellular fractionation tissue from rat hypothalamus and cerebral cortex in sucrose gradients indicated a concentration of cholecystokinin-like peptides in synaptosomal fractions. Lysis of synaptosomes yielded a vesicle rich fraction which was further enriched in cholecystokinin-like peptides, particularly the octapeptide (CCK-8). In vitro release experiments carried out using rat cerebral cortex tissue slices showed a calcium dependent release of cholecystokinin (primarily as CCK-8). The demonstration of a vesicular localization and calcium evoked release of cholecystokinin is consistent with a role for cholecystokinin as a neurotransmitter/neuromodulator in the CNS.

Expression but incomplete maturation of progastrin in colorectal carcinomas.

BACKGROUND To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue. METHODS Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods. RESULTS Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17-54 fmol/10(6) cells). Two cell lines secreted progastrin into the media (LoVo, 25 +/- 3 pmol/L; HCT116; 12 +/- 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4-2 pmol/g) than in normal tissue (range, 0.1-0.2 pmol/g). Only one tumor contained carboxyamidated gastrins. CONCLUSIONS Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

Neuronal Cholecystokinin: One or Multiple Transmitters?

The discovery of large amounts of regulatory peptides in the brain over the last decade is an important milestone in neurobiology. Neuropeptides are no longer considered a small group of hypothalamic substances regulating the secretion of pituitary hormones. Neuropeptides are potent transmitters in all parts of the central and peripheral nervous systems. Sometimes the peptides coexist and operate synergistically with the so-called classic transmitters (monoamines, acetylcholine, and amino acids) in acute synaptic transmissions. Possibly, neuropeptides are involved also in functions such as control of neuronal growth and metabolism (for recent general reviews, see Krieger, 1983; Krieger et al., 1984). Among the neuropeptide systems known today perhaps the largest group are hormones synthesized also in endocrine cells of the gastrointestinal tract and the pancreas. Thus, when they were found in the brain, methods for analysis were already at hand for some of these peptides-a fact that has advanced the study of the brain-gut peptides considerably. Although most of gut peptides occur only in discrete brain areas and/or in limited amounts (for instance, secretin, gastrin, insulin, glucagon, and motilin) others are distributed to most regions of the central and peripheral nervous systems [e.g., cholecystokinin (CCK), somatostatin, and vasoactive intestinal polypeptide (VIP)]. The CCK peptides, however, hold a unique position among brain peptides-including those originally isolated from brain tissue (substance P and other tachykinins, neurotensin, enkephalins, and others). First, the total amount of CCK in the mammalian CNS is higher than that of any other known regulatory peptide; and second, the distribution is unique, with the highest CCK concentrations in the cerebral cortex (Rehfeld, 197th; Larsson and Rehfeld, 1979a: Lamers et al., 1980; Beinfeld et al., 1981; Marley et al., 1984). The amounts and concentrations in the brain are significantly above also those of the recently discovered neuropeptide Y (Tatemot0 et al., 1982; Tatemoto, 1982), which by some has been claimed to be the most abundant peptide in mammalian brain (Allen et al., 1983; Adrian et al., 1983). In addition, CCK has aroused specific interest in schizophrenia research, because of its coexistence with 3,4-dihydroxyphenylethylarnine (dopamine) in the crucial mesolimbic neurons (Hokfelt et al., 1980a, 6) . The great interest in neuronal CCK has been emphasized by two recent international symposia devoted exclusively to brain CCK. Interested readers are referred to the proceedings from these meetings (de Belleroche and Dockray, 1984; Crawley and Vanderhaeghen, 1985). In spite of the substantial interest, fundamental questions about neuronal CCK are still unanswered, as reflected in the often confusing literature on CCK. The problem is not least attributable to ignorance of the extensive molecular heterogeneity of CCK. Therefore this review addresses the heterogeneity in terms of structure and biosynthesis of the different CCK molecules in the nervous system. In other words, is neuronal CCK a system comprising different neurotransmitters rather than one‘? As a starting point for neurochemists unfamiliar with gut endocrinology, a summary of the long history of intestinal CCK may give some perspective.