Karen Helle

: In our recent paper on sympto-matic carbohydrate malabsorption, we searched forpotential regulatory gastrointestinal peptides in-volved in the response to a fructose-sorbitol load,observing, however, normal plasma values of gluca-gon-like peptide 1 (GLP-1) and peptide YY (PYY)[1]. Taking into account that enterochromaffin (EC)cells, a major source of circulating chromogranin A(CgA) [2], are implicated in the pathophysiology offunctional gastrointestinal disorders [3], we havenow assessed serum CgA in patients with self-reported food hypersensitivity, most of them (89%)having irritable bowel syndrome according to theRome II criteria. Using the same clinical settingas before [1], CgA was measured using an enzymeimmunoassay, following the manufacturer’s instruc-tions (ALPCO Diagnostics, Salem, N.H., USA;cat. #43-CHRHU-EO1.2), prior to, 60 min, and180 min after oral intake of 25 g fructose and 5 gsorbitol in 250 ml tap-water. The results arepresented in Figure 1.CgA belongs to a family of highly conserved acidicproteins that are produced, stored, and secretedby the diffuse neuroendocrine system, polymorpho-nuclear neutrophils, and the myocardium [4]. CgAserves as a prohormone for a range of peptides withpotential regulatory functions, but is best known toclinicians as a marker for neuroendocrine tumors. Anearly 40% lower than normal serum CgA inpatients with self-reported food hypersensitivity isin marked contrast to the elevated serum CgAcharacteristics of hypertension, inflammatory condi-tions, and a range of neuroendocrine tumors [4].The cause of this reduction could include bothintestinal (e.g. loss of EC cell functions or entericneuropathy) and extra-intestinal (e.g. effects of stressor altered sympathoadrenal activity) abnormalities.Intriguingly, a non-specific, 48% decline in plasmaCgA has been reported in response to infusionof somatostatin [5], and somatostatin analoguescan be used in the treatment of patients withcarcinoid tumors [6]. Somatostatin is secreted fromwidely distributed D cells and acts as an inhibitor ofrelease of several regulatory peptides. In the pan-creas, somatostatin is secreted in response to glucose[7] and blocks insulin and glucagon secretion [8]. Inthe small and large intestine, D cells have longslender apical extensions with luminal contact [9],and can respond to luminal factors, as well as toexogenous beta-3 adrenergic receptor agonists actingvia cholinergic neurons in the colonic myenteric andsubmucosal plexi [10]. Hypothetically, an above-normal release of somatostatin could account forthe reduced serum CgA in our patient group.Increased meal-stimulated plasma levels of somatos-tatin have indeed been reported in patients withirritable bowel syndrome [11].The low serum CgA presently observed in patientswith self-reported food hypersensitivity opens fora new approach to our understanding of this func-tional gastrointestinal disorder. Interestingly, frag-ments of CgA have antimicrobial effects and maymodulate gastrointestinal nociception and motility[4]. Our finding may therefore have importantclinical implications.

The concentrations of catecholamines in the heart chambers of elasmobranchs were measured by the fluorimetric method of Bertler et al. (1958). Noradrenaline (NA) can be detected in all the chambers, but the sinus venosus is by far the richest in NA. This can either be due to the presence of storage sites for this amine in the sinus wall, or to a transport of amine to the sinus venosus from the anterior chromaffin bodies. The sinus wall contains large numbers of "granule containing cells" and axon-like processes, both with numerous dense-core vesicles of about 1800 A diameter. The dense-core vesicles contain a uranophilic matrix indicating the presence of protein, phospholipids and/or nucleic acid. The reactions failed to demonstrate amine, which may be due to a loss of amine by diffusion, to a relatively low intravesicular amine concentration, or, to the absence of amines in these granule-containing cells and processes. Heavy accumulations of granule-containing processes occur in the subendothelial area. The endothelium contains fenestrae and pores through which granule-containing fibres protrude into the venous cavity. Granule-containing cells are innervated by presumed cholinergic nerve endings. It is suggested that the granule-containing cells and fibres belong to the neurosecretory system with a cholinergic input, releasing the contents of the dense-core vesicles into the blood stream at the level of the venous cavity.

The granin family comprises altogether 7 different proteins originating from the diffuse neuroendocrine system and elements of the central and peripheral nervous systems. The family is dominated by three uniquely acidic members, namely chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). Since the late 1980s it has become evident that these proteins are proteolytically processed, intragranularly and/or extracellularly into a range of biologically active peptides; a number of them with regulatory properties of physiological and/or pathophysiological significance. The aim of this comprehensive overview is to provide an up-to-date insight into the distribution and properties of the well established granin-derived peptides and their putative roles in homeostatic regulations. Hence, focus is directed to peptides derived from the three main granins, e.g. to the chromogranin A derived vasostatins, betagranins, pancreastatin and catestatins, the chromogranin B-derived sec...

The discovery in 1953 of the chromaffin granules as co-storage of catecholamines and ATP was soon followed by identification of a range of uniquely acidic proteins making up the isotonic vesicular storage complex within elements of the diffuse sympathoadrenal system. In the mid-1960s, the enzymatically inactive, major core protein, chromogranin A was shown to be exocytotically discharged from the stimulated adrenal gland in parallel with the co-stored catecholamines and ATP. A prohormone concept was introduced when one of the main storage proteins collectively named granins was identified as the insulin release inhibitory polypeptide pancreastatin. A wide range of granin-derived biologically active peptides have subsequently been identified. Both chromogranin A and chromogranin B give rise to antimicrobial peptides of relevance for combat of pathogens. While two of the chromogranin A-derived peptides, vasostatin-I and pancreastatin, are involved in modulation of calcium and glucose ...

Endothelium-independent vasoconstrictor responses in isolated segments of human internal thoracic artery (ITA) and saphenous vein (SV) were used as a bioassay system for the vasoinhibitory activity of bovine chromogranin A (CGA). Preincubation with vasostatin (0.8 micrograms/ml), containing the N-terminal domain of CGA, (CGA1-76, CGA1-113 and CGA1-143ff), inhibited the contractile responses evoked by 80 mM K+, 2.6 microM noradrenaline (NA), or 65 nM endothelin-1 (ET-1) in Ca(2+)-free solution in SV but not in ITA. The results demonstrate a vasoinhibitory activity in vasostatin and show that there is a marked difference between the arterial and venous segments in the Ca2+ independent component of the inhibitory response. A vascular role for the N-terminal domain of CGA is indicated, presumably by inhibiting Ca2+ release from intracellular stores in the human vein but not the artery.

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked beta-adrenoceptor, of high K+ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP (pD2 = 7.52 +/- 0.18, n = 6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a beta 2-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (alpha) = 0.27 +/- 0.01, n = 8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and beta-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the alpha-adrenoceptor mediated increase in contractility.

... Edited by Karen B. Helle and Dominique Aunis Volume 483 TAURINE 4: Taurine and Excitable Tissues Edited by Laura Della Corte ... Contributors xiii Guldborg Serck-Hanssen Department of Physiology, University of Bergen, 5009 Bergen, Norway Seung Ho So National Creative ...

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked beta-adrenoceptor, of high K+ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP (pD2 = 7.52 +/- 0.18, n = 6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a beta 2-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (alpha) = 0.27 +/- 0.01, n = 8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and beta-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the alpha-adrenoceptor mediated increase in contractility.

Fibroblast adhesion can be modulated by proteins released by neuroendocrine cells and neurons, such as chromogranin A (CgA) and its N-terminal fragment vasostatin-1 (VS-1, CgA(1-78)). We have investigated the mechanisms of the interaction of VS-1 with fibroblasts and of its pro-adhesive activity and have found that the proadhesive activity of VS-1 relies on its interaction with the fibroblast membrane via a phospholipid-binding amphipathic alpha-helix located within residues 47-66, as well as on the interaction of the adjacent C-terminal region 67-78, which is structurally similar to ezrin-radixin-moesin-binding phosphoprotein 50 (a membrane-cytoskeleton adapter protein), with other cellular components critical for the regulation of cell cytoskeleton.

ABSTRACT The present study is the first to address the question of interaction between CGA and endothelial cells. It is evident that neither intact CGA nor N-terminal peptides activate endothelial cells by affecting the membrane potential. Nevertheless, bovine aorta endothelial cells (BAEC) bind and intemalise intact CGA in a temperature-dependent manner, and the binding occurs by low affinity and high capacity. BAEC do not express specific, high affinity membrane-associated binding sites for CGA. Cell surface charge appears, on the other hand, to be important for the low affinity binding, seemingly followed by non-specific adsorptive endocytosis involving plasmalemma1 vesicles. The different kinetics for CGA and inulin migration across the BAEC monolayer indicate that CGA is slowly transported by a transcellular route, consistent with transcytosis involving shuttle of vesicles between the luminal and abluminal side of the endothelial cell. Intemalised CGA may account for a pool of trapped CGA in equilibrium with circulating CGA. Taking into account the regional differences in endothelial properties and functions, the present approach to analyse CGA exchange in the macrovascular model should also be applied to endothelial cells derived from the microvasculature.

Vascular effects of atrial natriuretic polypeptide (APII), i.e. the peptide hormone released from the atrial myocardium, were investigated in segments of the human internal thoracic artery (ITA) and saphenous vein (SV) with intact (+E) or injured (-E) endothelium. All segments were subject to several cycles of agonists in order to detect tachyphylactic or facilitatory responses. Opposite, indirect effects on the noradrenaline contracted ITA and SV were obtained in response to APII at a supranormal concentration (50 nM) which had no direct relaxing action on the isolated segments in vitro. In ITA the noradrenaline contractures in subsequent cycles were reduced to 41 +/- 21% (+E) and 28 +/- 9% (-E), but in SV they were enhanced to 211 +/- 115% (+E) and 483 +/- 242% (-E) of those before APII exposure. Thus under in vitro conditions ITA could be indirectly relaxed by APII via tachyphylactic effect on the noradrenaline contracture. SV, on the other hand, was markedly potentiated by APII in its noradrenaline response. In injured endothelium these opposite effects were aggravated.

The relative importance of vascular relaxations induced by atriopeptins (AP), the beta-adrenoceptor agonist isoprenaline and of the neuropeptide VIP was studied in vitro on circular and longitudinal preparations of the rat portal vein. Two members of the rat atriopeptins (AP II and III) were equipotent with respect to relaxation of the spontaneously contracting outer, longitudinal layer and of the alpha 1-contracted inner, circular layer. The potency for AP II was about 13 times lower in the inner (pD2 = 7.48 +/- 0.73, n = 6) than in the outer layer (pD2 = 8.60 +/- 0.34, n = 6). No significant difference was apparent between the intrinsic activities for AP II in the two layers. The potencies for AP II were for both layers higher than those for VIP while the intrinsic activities for AP II were significantly lower than for VIP and for the reference agonist, isoprenaline in both layers. Atriopeptin II was equally efficient in relaxing the K+-depolarized and alpha 1-contracted longitudinal segments. Neither the beta-antagonist, propranolol nor the guanylate cyclase inhibitor, methylene blue, modified the potency or the intrinsic activity of AP II. These results suggest that concentrations of circulating atriopeptins above 10 nM may contribute to reduction of vascular tone by the methylene blue insensitive receptors for AP II and III in the portal-mesenteric vein region.

The relative importance of VIP in reduction of vascular tone was studied in circular and longitudinal preparations of the VIP-innervated rat portal vein. Exogenous VIP inhibited the methoxamine-evoked contractures in the atropine-blocked preparations with a lower potency in the inner, circular (pD2 = 6.4 +/- 0.5, n = 6) than in the outer, longitudinal layer (pD2 = 7.7 +/- 0.1, n = 6). VIP was also a less efficient relaxant (intrinsic activity (alpha) = 0.60 +/- 0.16, n = 6) of the inner than of the outer layer (alpha = 1.00). The selective (salbutamol) and the non-selective (isoproterenol) beta 2-agonists completely relaxed the methoxamine contractures in both layers and the potency (isoproterenol) was higher in the inner (pD2 = 6.39 +/- 0.32, n = 6) than in the outer layer (pD2 = 5.67 +/- 0.34, n = 6). Plasma from the portal-mesenteric vein of anaesthetized, fasting rats contained 0.036 nM VIP (median, n = 17), that is, several orders of magnitude lower than the range of VIP concentrations relaxing the methoxamine contracted vein preparations via VIP receptors of the apamin-blockable category. The results support the hypothesis that alpha 1-adrenoceptor-induced contractions in the circular layer are predominately relaxed via beta 2-adrenoceptors while relaxation of the outer layer may occur via VIP receptors, probably activated by local release of the neuropeptide.

ABSTRACT 1. DβH was released in parallel with CA and protein from acetylcholine-stimulated bovine adrenals perfused retrogradely with glucose-free tyrode.2. The low specific activity in freshly collected, unconcentrated perfusates indicated a considerable inactivation in the course of secretion.3. The extracellular DβH was immunologically identical to the intragranular enzyme, and also very similar in affinity to tyramine, inhibition by Cu2+ and resistance to trypsin. The active enzyme accounted, however, for only a small fraction of the total enzyme protein, obtained at a low recovery from the perfusate by affinity chromatography.

Synthetic neurotensin was shown to be a powerful contractant of the isolated rat portal vein. Concentration-dependent increases in basal tension and frequency of spontaneous contractions were obtained in response to single dose (10-1000 nM, final bath concentrations). The responses reached maxima within 2 min and differed in this respect from comparable responses to angiotensin II, substance P and bradykinin, which were maximal after 2-4 min. The "apparent affinity" for neurotensin (pD2 = 7.5 +/- 0.3, S.D., n = 10) was about 10 times less than for angiotensin II while higher than for both the other peptides and for norepinephrine. The "intrinsic activity" was 0.75 (+/- 0.13, S.D., n = 6) relative to angiotensin II during the first 2 min of the response. A pronounced degree of tachyphylaxis was observed; repetition of the neurotensin dose at the lower range of concentrations (1-10 nM) within 2-4 min was completely ineffective. The results suggest that there is a s...

1. The vasomotor responses to neuropeptides of the angular oculi and facial veins of reindeer were examined in vitro and correlated with the neuropeptide distribution in the perivascular nerves, as demonstrated by immunohistochemistry. 2. Nerves displaying calcitonin gene related peptide (CGRP)- or neuropeptide Y (NPY)-like immunoreactivity (-LI) were observed in the media of both veins, while very few fibers were immunoreactive to vasoactive intestinal polypeptide (VIP) or substance P (SP) in either vein. 3. The staining pattern for NPY-LI was largely identical to that of dopamine-beta-hydroxylase, a marker for noradrenaline (NA) producing fibers, indicating coexistence of NPY and NA. 4. Administration of NPY in vitro elicited contractions in both veins in the presence of propranolol, though more conspicuously in the angular oculi vein. 5. The peptide was without any modulating effect on NA-stimulated contractions in the angular oculi vein, whereas a small enhancement of the NA-ind...

By means of a monospecific antibody, dopamine beta-hydroxylase was monitored immunoelectrophoretically in various extracts of chromaffin granules. Approximately one-third of the dopamine beta-hydroxylase present was located in the membrane fraction and could only be liberated with detergent. The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. Furthermore, immunoelectrophoretic analysis in the presence of Triton X-100 plus the cationic detergent cetyltrimethylammonium bromide indicates additional heterogeneity of the membrane-bound dopamine-beta-hydroxylase. By limited proteolysis with chymotrypsin and thermolysin the amphiphilic form could be convered into its hydrophilic count...

Half a century after the discovery of chromogranin A as a secreted product of the catecholamine storage granules in the bovine adrenal medulla, the physiological role for the circulating pool of this protein has been recently coined, namely as an important player in vascular homeostasis. While the circulating chromogranin A since 1984 has proved to be a significant and useful marker of a wide range of pathophysiological and pathological conditions involving the diffuse neuroendocrine system, this protein has now been assigned a physiological "raison d'etre" as a regulator in vascular homeostasis. Moreover, chromogranin A processing in response to tissue damage and blood coagulation provides the first indication of a difference in time frame of the regulation of angiogenesis evoked by the intact chromogranin A and its two major peptide products, vasostatin-1 and catestatin. The impact of these discoveries on vascular homeostasis, angiogenesis, cancer, tissue repair and ...

Chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) belong to a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These "granins" are characterized by numerous pairs of basic amino acids as potential sites for intra- and extragranular processing. In response to adequate stimuli, the granins are coreleased with neurotransmitters and hormones and appear in the circulation as potential modulators of homeostatic processes. This review is directed towards functional aspects of the secreted CgA, CgB, and SgII and their biologically active sequences. Widely different effects and targets have been reported for granin-derived peptides. So far, the CgA peptides vasostatin-I, pancreastatin, and catestatin, the CgB peptides CgB(1-41) and secretolytin, and the SgII peptide secretoneurin are the most likely candidates for granin-derived regulatory peptides. Most of their effects fit into patterns of direct or indirect modu...

N-terminal peptides of chromogranin A and B (CGA and CGB) were compared for dilator responses in isolated bovine coronary arteries (bCoA), measuring diameter changes as a function of pressure. bCoA developed and maintained myogenic tone (MYT) at approximately 20% from 50 to 150 mm Hg. In contrast to CGB(1-40), CGA(1-40) and CGA(1-76) (VS-I) both displayed significant intrinsic vasodilator effects. CGA(1-40) reduced myogenic reactivity from 70 to 150 mm Hg (p<0.05, n=6). At 75 mm Hg, CGA(1-40) showed a concentration-dependent dilatation at 0.1 nM-10 microM. The dilator effect of CGA(1-40) persisted at moderately elevated [K(+)](e) (8.4-16 mM). However, this effect was diminished by pertussis toxin (PTX) and abolished by antagonists to several subtypes of K(+) channels (tetraethylammonium, Ba(2+) and glibenclamide). These results demonstrate that the N-terminal domain of CGA has dilator effect in the myogenically active bCoA. We propose that CGA(1-40) and the naturally occurring va...

Bovine adrenomedullary granules were separated into two subfractions by isopycnic density centrifugation. A small subfraction (approximately 10% of the total population) was sedimented into 2.2 M sucrose while the main population (80% of the total) was recovered at the interphase between 1.6 and 2.2 M sucrose. The concentrations of catecholamine (CA) and calcium showed marked seasonal variations for both subfractions, with lowest levels in the spring and highest levels in the winter. Throughout the year the concentrations of CA and calcium were 2-3 times higher in the minor subpopulation which also accounted for an abundance of noradrenaline (NA); on average 68% NA of total CA, 6.6 mumol CA and 225 nmol calcium/mg protein. The two subpopulations stored CA in similar ratios to ATP and calcium; i.e. 30 mol CA: 4 mol ATP: 1 mol Ca2+, indicating storage of CA largely independent of an equivalent amount of ATP, at least during winter when CA storage was 3.3 and 9.9 mumol/mg protein in th...

1. Right adrenal medullary and various cardiovascular responses to stimulation of the peripheral end of the right splanchnic nerve have been investigated in conscious calves, 2--5 weeks after birth. 2. The output of both adrenaline and noradrenaline was linearly related to stimulus frequency over the range 2.0--10.0 Hz, 2 1/2 min after stimulation was initiated. Peak outputs of both amines were obtained in response to stimulation at 15.0 Hz. 3. The output of adrenaline invariably exceeded that of noradrenaline, roughly in the proportion 3:2. At all frequencies tested between 7.0 and 40.0 Hz this difference was statistically significant (P less than 0.1). 4. Continuous stimulation at either 4.0 or 10.0 Hz produced a small but significant rise in the output of dopamine-beta-monooxygenase (DBH) activity from the right adrenal gland. Mean maximal outputs were obtained after 10 min; the levels were closely similar at both 4.0 and 10.0 Hz and could not be related to stimulus frequency or ...

Searching for endogenous proteolytic activities converting the membrane form of dopamine beta-hydroxylase (dopamine beta-monooxygenase, DBH) into the soluble and releasable form, DBH was monitored enzymatically and immunologically in aqueous and detergent-solubilized extracts of the adrenomedullary fractions. Degradation of the soluble DBH and acidic chromogranins by activation of endogenous proteases occurred during lysis in H2O. Shifts in the hydrophobicity of the membrane DBH were also apparent. Loss in enzyme protein or activity was, on the other hand, not observed for buffer-dialysed CG (pH 5-6). Limited proteolysis within the membrane phase was, however, indicated by the shift towards dominance of the intermediate hydrophobic DBH in the buffer-dialysed CG. By two-dimensional, crossed immunoelectrophoresis with cationic detergent the microsomal DBH was immunologically identical to the granule-bound enzyme but differed from the latter in molecular heterogeneity and in susceptibi...