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K B Helle

Publications and source records attributed to K B Helle.

At least 19 recordsLinked to original sources
PubMed2005

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.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Acetylcholine↗
PubMed1998

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

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.

Adrenal Medulla↗
PubMed1996

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.

Adrenal Gland Neoplasms↗
PubMed1995

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.

Adrenal Medulla↗
PubMed1995

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.

Adrenal Medulla↗
PubMed1994

Differential patterns of relaxation by atrial natriuretic peptide in major blood vessels of two distantly related teleosts.

In order to elucidate the role of the atrial hormone in the teleost circulation, the vascular effects of atrial natriuretic peptide (ANP) have been compared in major blood vessels of the cod (Gadus morhua) and of Atlantic salmon (Salmo salar). The relaxing effects of ANP from eel (eANP) have been examined in ventral aorta (VA) versus dorsal aorta (DA) in the cod and in VA versus the coeliaco mesenteric artery (CMA), a major branch of DA, in the salmon. The vessels were precontracted by acetylcholine (ACh) and adrenaline (A) and by the mammalian endothelium-derived vasoconstrictor peptide endothelin-1 (ET-1). The role of endothelial integrity for these responses has been assessed in vessels either mechanically probed or chemically impaired by indomethacin or the L-arginine analogue, NG-monomethyl-L-arginine (L-NMMA). Adrenaline and noradrenaline (NA) failed to contract the salmon VA. In the salmon vessels, eANP was without relaxing effects in ACh-contracted VA, while completely relaxing CMA when precontracted with ACh. The eANP was also a relaxant of A-contracted CMA and of ET-1-contracted VA. The cod vessels, which were insensitive to ACh were markedly relaxed by eANP when precontracted with either A (VA) or ET-1 (DA). In DA also the resting tension was reversed, an effect of eANP that was highly potentiated in mechanical probed vessels. Otherwise the relaxing effects of eANP in these vessels were seemingly independent of endothelial factors. In conclusion ANP is not a general relaxant of the precontracted VA which in teleosts is the first possible target vessel for the myocardial release of this hormone. On the arterial side ANP serves as a relaxant both in the salmon and the cod vessels, indicating that myocardial release of ANP in teleosts may have an important role in regulation of blood flow via diverse, species-specific effects on major blood vessels on both sides of the gills.

Acetylcholine↗
PubMed1994

Immunoreactive atrial natriuretic peptide and dopamine beta-hydroxylase in myocytes and chromaffin cells of the heart of the African lungfish, Protopterus aethiopicus.

The heart of the African lungfish, Protopterus aethiopicus, was examined for immunoreactive atrial natriuretic peptide (ANP) and dopamine beta-hydroxylase (D beta H) as markers for hormone secreting myocytes and chromaffin cells, respectively. Specific antibodies raised against rat alpha-ANP and rat D beta H were used for immunofluorescence microscopy and immunogold electron microscopy. D beta H-immunoreactive cells were restricted to subendocardial areas of the atrium whereas ANP immunoreactivity occurred throughout both the atrial and the ventricular myocardium, showing particularly strong staining intensity in the atrial myocytes. The granular ANP immunostaining in the atrial myocytes was frequently accumulated in the sarcoplasm. In the ventricular myocytes ANP immunoreactivity occurred as scattered granular staining throughout the sarcoplasm. ANP and D beta H immunofluorescence staining coincided with the presence of immunoreactive specific granules and secretory vesicles in the cardiac myocytes and chromaffin cells, respectively, as revealed by electron microscopy. The number of ANP-containing specific granules was generally high in the atrial myocytes, and they were frequently observed in clusters in subsarcolemmal areas. Granular frequency was considerably lower and the mean granular diameter was smaller (0.142 +/- 0.045 micron versus 0.213 +/- 0.049 micron) in the ventricular than in the atrial myocytes. The present results indicate that ANP and D beta H are phylogenetically highly conserved proteins from the dipnoi to the rat. The large amounts of ANP and of specific granules are consistent with an endocrine myocardium in the Protopterus heart. The presence of D beta H and secretory vesicles in the subendocardial chromaffin cells of the atrium suggests a local production of catecholamines from dopamine in the heart of this dipnoan.

Animals↗