The endocrine role for chromogranin A: a prohormone for peptides with regulatory properties.

Chromogranin A (CgA) belongs to the granin family of uniquely acidic secretory proteins co-stored and co-secreted with other hormones and peptides in elements of the diffuse neuroendocrine system. The granins arise from different genes and are characterized by numerous sites for post-translational cleavage into shorter peptides with postulated regulatory properties. This review is directed towards endocrine aspects of CgA and its biologically active peptides. There is ample evidence from in vitro studies of distinct effects and targets for three CgA-derived peptides, vasostatin-I, pancreastatin and catestatin. Endocrine regulations are indicated from in vivo studies, consistent with the postulated prohormone function of CgA for peptides with regulatory properties. Most of the effects fit into patterns of direct or indirect, inhibitory modulations of major functions, implicating CgA peptides in regulation of calcium and glucose metabolism, cardiovascular functions, gastrointestinal motility and nociception, tissue repair, inflammatory responses and as host defense peptides in the first phase of microbial invasions.

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A.

Catestatin (bCGA(344-364)), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA(344-358)) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA(344-358) in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA(344-358)) (named cateslytin) as a novel component of innate immunity.

Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods.

Chromogranin A (CgA) and chromogranin B (CgB) are acidic proteins stored in and released from hormone granules in endocrine and neuroendocrine tissue. The chromogranins are postulated to serve as pro-hormones to generate biologically active peptides, which may influence hormonal release and vascular functions or have antibacterial functions. Although N-terminal and C-terminal regions show some species amino acid homology, the chromogranins as a whole display considerable interspecies differences, which prevents their use in comparative studies of biological functions. We present four new radioimmunoassays for the measurement of defined N-terminal regions of CgA and CgB. A new radioimmunoassay for measurement of intact bovine CgA has also been developed. With these assays and two previously published ones, we have compared the cross-reactivity of chromogranins from man, cattle, sheep, goat, pig and horse and compared adrenomedullar content and serum levels of CgA from these species. We have also studied the influence of peptide concentrations and the ionic strength of the mobile phase on molecular weight estimations. Assays with antibodies directed against the N-terminal parts of CgA and CgB showed sufficient interspecies cross-reactivity to allow comparative quantification of the circulating levels in man, cattle, sheep, goat, pig and horse. Assays measuring the intact human or bovine CgA were not suitable for comparative purposes in samples from sheep, goat, pig and horse. Molecular interactions between vasostatin immunoreactive material and intact bovine CgA were demonstrated in gel permeation studies, suggesting that conclusions about the degree of N-terminal processing from elution profiles should be made with caution. Reliable interspecies comparison of chromogranins is difficult, but measurements with region-specific assays may be helpful to study concentrations of chromogranins and chromogranin-related peptides.

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.

The adrenomedullary venous vasculature as a target for endothelins: comparison of the porcine and bovine central adrenomedullary veins.

The functional properties of the isolated porcine and bovine central adrenomedullary veins were compared, with emphasis on the active tension responses to high K+, endothelin-1 (ET-1) and neuropeptide Y (NPY). In the porcine vein, the contraction evoked by ET-1 was 4--5-fold higher than with high K+, as in the bovine vein. The potencies for ET-1 were similar in ring and strip preparations of the porcine vein, with EC50 values 5--7-fold higher than in the bovine vein. In preparations previously exposed to ET-1 the contractions evoked by high K+ and NPY were potentiated and facilitated, respectively,. However, only in the porcine vein was the ET-1 contraction sustained. This contraction was effectively relaxed by milrinone, indicating a role for cGMP inhibited cyclic nucleotide phosphodiesterase in the sustained contraction. Caffeine and forskolin were also effective relaxants of contractions evoked by ET-1 in both veins, suggesting relaxation by elevated levels of cAMP. The K(+)-contracted porcine, but not bovine, vein was relaxed by acetylcholine (ACh) and vasointestinal polypeptide in a concentration-dependent manner, indicating species differences with respect to signal transduction leading to increases in cyclic nucleotides. In conclusion, these results demonstrate that ET-1 is the main constrictor of the porcine central adrenomedullary vein, with significant species differences in mode of contraction and relaxation. These findings suggest roles for the endogeneously released ET-1 and NPY in regulation of venous contractility within the adrenal gland of mammals.

Relaxing effects of cyclic GMP and cyclic AMP-enhancing agents on the long-lasting contraction to endothelin-1 in the porcine coronary artery.

In the coronary circulation, endothelin-1 (ET-1) evokes spasms which are difficult to treat when the endothelial integrity is compromised. This study compares several classes of relaxing agents on already established contractions to ET-1 in an in vitro model using ring segments of the porcine left descending coronary artery (pLAD). All segments were precontracted with 10 nmol/L ET-1. The calcium channel blocker isradipine was 300 times more potent than verapamil, but was only a partial relaxant; the maximal relaxation obtained was 52 +/- 2% (n = 6). Atrial natriuretic peptide (ANP) was an equally potent relaxant of the ET-1 contraction; however, it too was an incomplete relaxant, maximal relaxation being < 60%. A 50% relaxation of the ET-1 contraction was obtained with 0.28 +/- 0.24 mumol/L ANP, n = 4 (IC50). Comparison of cyclic nucleotide analogues revealed a 30 times higher potency for 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP)(IC50 44 +/- 11 mumol/L, n = 6) than for 8-bromo-cyclic adenosine monophosphate (8-Bi-cAMP) (IC50 1600 mumol/L, n = 6). The cyclic nucleotide phosphodiesterase (PDE) inhibitor milrinone, a PDE 3-inhibitor with an IC50 2.4 +/- 1.8 mumol/L, (n = 6) was 10 times more potent than rolipram (PDE 4-inhibitor), zaprinast (PDE 5-inhibitor) and vinpocentine (PDE 1-inhibitor). Withdrawal of these analogues and inhibitors from segments continuously exposed to 10 nmol/l ET-1 revealed that vinpocentine and 8-Br-cGMP were irreversible relaxants, in contrast to milrinone and 8-Br-cAMP. In conclusion, this study has demonstrated that cGMP-enhancing agents, such as the naturally occurring ANP, the calcium channel blocker isradipine, and the synthetic inhibitor of PDE 3, were the most effective relaxants of ET-1 evoked contractions in pLAD in vitro.

The adrenal medulla as a wet sponge: a role for the intramedullary venous vasculature?

Synchronous contractions in the intramedullary venous vasculature have been postulated to assist in the discharge of hormones from the stimulated adrenal medulla in a manner analogous to the squeezing of a wet sponge. This study reports on two experimental approaches to support the hypothesis that contractions in the venous vasculature may contribute to the hormonal efflux. Firstly, the bovine adrenal medulla was perfused retrogradely via the bovine central adrenomedullary vein end changes in the vascular volume were assessed as changes in wet weight of the perfused tissue. Stimulation with acetylcholine and carbachol resulted in repetitive, transient weight losses, suggesting cholinergically mediated reductions in the vascular volume. Secondly, the contractile properties of the longitudinal layers of smooth muscle cells in the intramedullary venous system were characterized, using the bovine central adrenomedullary vein as a model. The results showed that the longitudinal layers of this vein were, similarly to the circular layers, selectively contracted by endothelin-1 via an ETA-like receptor, by neuropeptide Y and by membrane depolarization (high K+). However, the vein was insensitive to electrical stimulation acetylcholine and carbacho, as well as to catecholamines. These results suggest neuropeptide Y, released from the cholinergically stimulated chromaffin cells, as the most likely mediator of stimulus-evoked synchronous contractions of the venous vasculature in the bovine adrenal medulla. Together, these experiments provide support for the 'wet sponge' hypothesis for hormonal discharge from the adrenal gland.

Human pheochromocytoma: different patterns of catecholamines and chromogranins in the intact tumour, urine and serum in clinically unsuspected cases.

Clinically unsuspected pheochromocytoma is usually discovered either at autopsy or during surgical intervention for unrelated conditions, despite often enormous neoplastic masses producing and storing catecholamine (CA). In order to assess whether these tumours share some common features we have compiled data for six patients admitted to hospital without previous diagnosis of their pheochromocytoma. The clinical variables and the morphological and immunohistochemical characteristics of the tumours revealed that these cases represented quite different expressions of adrenomedullary neoplasms. They differed not only with respect to nuclear ploidity and overall cytoplasmic morphology but also in catecholamine storage and expression of immunoreactive chromogranin A sequences in the intact tissue. In two of the patients hypertension had been overlooked as a diagnostic indicator of their CA-producing tumours. There was no clear relationship between the mean arterial pressure, the tumour content of CA and the serum levels of CA. Processed chromogranin A dominated in the serum of the two hypertensive cases. The 24-h urine values of CA and its main metabolite (vanillin mandelic acid) were, together with the serum values of chromogranin A and B, proportional to tumour mass and provided the most reliable diagnostic indicators for the non-hypertensive as well as the hypertensive cases.

The bovine central adrenomedullary vein: a target for endothelins.

This study reports on morphological and contractile properties of the bovine central adrenomedullary vein (bCAMV). Up to several layers of circularly orientated smooth muscle cells (SMC) were observed, however, without forming a continuous, closed sheath. Discrete bundles of eccentrically arranged, longitudinal SMC were also conspicuous. Chromaffin cells were in most cases located outside the SMC layers, while sometimes being in close apposition to the endothelium in areas without SMC. Circularly mounted preparations of the endothelium-denuded vessel responded selectively to high K+, endothelins (ETs) and neuropeptide Y (NPY). The threshold for ET-1 was 0.13 nM and the half maximally effective concentration (EC50) was 3 +/- 1 nM (n = 9). The order of potencies was ET-1 > or = ET-2 >> ET-3, suggesting a vascular receptor (ETA). Concentrations at and above EC50 frequently developed long-lasting oscillations during the spontaneous relaxation of the ET-1 evoked tension. This response was partly (21%) independent of extracellular Ca2+. A marked tachyphylaxis developed to ET-1 (3-30 nM), resulting, on the other hand, in facilitation of the subsequent constrictor responses to high K+ and NPY. Propranolol and phentolamine alone, or in combination, were without effects on the basal tension and on the above-mentioned responses to high K+, ET-1 or NPY, making a contribution from adrenoceptor activation unlikely. No response was obtained with exogenous catecholamines, acetylcholine or serotonin, nor with a series of peptides known to occur in the adrenal medulla. This study shows that bCAMV is not a passive capacitance vessel but appears unique among mammalian veins in being selectively regulated by ETs.

Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides.

Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system. Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity. The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein. The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis. Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence. This E. coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human. Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA. A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa. Four components (molecular mass range 12-18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA1-113) while the two others (molecular mass range 7-8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA1-76). From protein staining the ratio vasostatins II/I was 10:1. The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments. An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.

Role of the endothelium in regulation of vascular functions in two teleosts.

Functional and structural aspects of the vascular endothelium were studied in major blood vessels from two distantly related species, the Atlantic salmon (S. salar) and the cod (G. morhua). The ventral aorta (VA) of both teleosts and the dorsal aorta (DA) and the coeliaco mesenteric artery (CMA) of the cod and the salmon respectively were examined for endothelium dependent and independent responses to acetylcholine (ACh), adrenaline (A) and endothelin-1 (ET-1). In the salmon, endothelial probing resulted in reduced contractile responses to high K+ in both VA and CMA while the responses to ACh and A were reduced only in CMA. Indomethacin, but not L-NMMA, enhanced vasoconstrictions to high K+, ACh and A in the unprobed CMA. In the cod vessels the endothelial probing caused reduced contractile responses to the two effective vasoconstrictors in both vessels, to high K+ and A in VA and to high K+ and ET-1 in DA. Both indomethacin and L-NMMA enhanced contractile tension to A in VA, while indomethacin, but not L-NMMA, enhanced the constrictions by high K+ in VA and by ET-1 in DA. These experiments have revealed heterogeneous patterns of endothelial function in blood vessels of two teleosts, reflecting differences in endothelial morphology and in production of potent endothelial derived contracting factors as well as prostanoic and non-prostanoic endothelium-derived dilating factors.

Vasoinhibitory activity of synthetic peptides from the amino terminus of chromogranin A.

Naturally occurring amino terminal fragments of chromogranin A (CGA), the calcium-binding protein found in all endocrine secretory vesicles, have vasoinhibitory activity when tested in isolated segments of the endothelium-denuded human saphenous vein. Synthetic peptides corresponding to sequences within the first 76 residues of chromogranin A have been made and tested for biological activity. Full length vasostatin I (CGA1-76) (40 nM), but not the truncated vasostatin I, CGA1-40 (100 nM) mimics natural chromogranin A fragments in its inhibition of contractions induced by endothelin-1 (ET-1) in calcium containing medium. CGA1-40 (100 nM) mimics the inhibitory effect of the vasostatins on the contractions induced in the absence of extracellular calcium by high potassium and noradrenaline, but not by ET-1. The iodinated peptides both exhibit saturable binding in an aortic smooth muscle cell line, indicative of a single class of high affinity binding protein ('receptor' with an apparent KD of approximately 45 nM. This binding is not affected by endothelin-1. Iodinated peptides can be crosslinked to a single polypeptide in binding experiments performed on intact calf aortic smooth muscle cells.