The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels.

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.     

N-terminal chromogranin-derived peptides as dilators of bovine coronary resistance arteries

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 vasostatin I are regulatory peptides of relevance for the coronary microcirculation and that a G(alphai) sub-unit and K(+) channel activation may be involved in the signal pathway.     

Chlorogenic acid: Potential source of natural drugs for the therapeutics of fibrosis and cancer

Fibrosis and cancer is described by some epidemiological studies as chronic stages of different disease conditions typically characterized by uncontrolled accumulation of extra-cellular matrix (ECM), thereby leading to inflammation of tissues and organ (lungs, heart, liver and kidney) dysfunction. It is highly prevalent, and contributes to increased mortality rate worldwide. Currently, the therapeutical approaches involving selected medications (bemcentinib, pirfenidone and nintedanib) obtained synthetically, and used in clinical practices for fibrosis and cancer management and treatment has shown to be unsatisfactorily, especially during progressive stages of the disease. With regards to finding a more potent, effective, and promising curative for fibrosis and cancer, there is need for continuous experimental studies universally. However, phytochemical constituents’ particularly phenolic compounds [Chlorogenic acid (CGA)] obtained from coffee, and coffee beans have been predominantly utilized in experimental studies, due to its multiple pharmacological properties against various disease forms. Considering its natural source alongside minimal toxicity level, CGA, a major precursor of coffee have gained considerable attention nowadays from researchers worldwide, owing to its wide, efficacious and beneficial action against fibrosis and cancer. Interestingly, the safety of CGA has been proven. Furthermore, numerous experimental studies have also deduced massive remarkable outcomes in the use of CGA clinically, as a potential drug candidate against treatment of fibrosis and cancer. In the course of this review article, we systematically discussed the beneficial contributions of CGA with regards to its source, absorption, metabolism, mechanistic effects, and molecular mechanisms against different fibrosis and cancer categorization, which might be a prospective remedy in the future. Moreover, we also highlighted CGA (in vitro and in vivo analytical studies) defensive effects against various disorders.     

Calorimetric,volumetric and structural studies of the interaction between chlorogenic acid and dipalmitoylphosphatidylcholine bilayers

Chlorogenic acid (CGA) is the main component of coffee and an antioxidant. CGA has been reported to bear various good health effects. At the same time, it has been found that the addition of CGA induces an undesirable deformation of red blood cells. This fact suggests that CGA may bind to the proteins or/and membrane lipids of red blood cells. This study aimed to examine how CGA binds the bilayers of phosphatidylcholine (PC), one of red blood cells' primary lipids. To this end, we investigated the effect of CGA on the phase behavior and the structure of dipalmitoyl-PC (DPPC) bilayers in the form of multi-lamellar vesicles. Calorimetry and dilatometry measurements showed that the DPPC chain melting transition cooperativity decreases as increasing CGA concentrations. In addition, X-ray diffraction results showed that the lamellar repeat periodicity becomes disordered, and the periodicity disappears completely at high CGA concentrations. Together with these findings, it can be inferred that the CGA molecules do not penetrate inside the DPPC bilayers but bind to their surface in a negatively charged form.     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

The metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil

We examined the involvement of chlorogenic acid (CGA) and salicylic acid (SA) in the stress-induced flowering of Pharbitis nil (synonym Ipomoea nil). The incorporation efficiency of exogenously applied CGA and the deactivation rate of incorporated CGA were determined in cotyledons by high-performance liquid chromatography. The assay plants could not incorporate a sufficient amount of CGA via roots. The perfusion technique by which the assay solution was forced into the plant from the cut end of the hypocotyl improved the efficiency of CGA incorporation. However, no flower-inducing activity was detected, indicating that CGA was not involved in flowering. It was concluded that the close correlation between CGA content and flowering response is merely coincidence or a parallelism. Flowering under long-day conditions induced by low-temperature stress was completely inhibited by aminooxyacetic acid (AOA), an inhibitor of phenylalanine ammonialyase. The flower-inhibiting effect of AOA was nullified by co-applied t-cinnamic acid and by benzoic acid. This indicates that the metabolic pathway from t-cinnamic acid to SA via benzoic acid is involved in the stress-induced flowering. The results indicate that the metabolic pathway of SA is involved in the stress-induced flowering of P. nil not the metabolic pathway of CGA.     

Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme.

Clostridium sp. G0005 produces a cell-bound glucoamylase (CGA). The gene encoding CGA has been sequenced. The deduced amino acid sequence begins with a putative 21-residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp. G0005, and that the modified residue is important in the cell-bound form of mature CGA. Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the overall primary structure. CGA is the first bacterial glucoamylase to be cloned. The CGA gene was expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5' non-coding region and the N-terminal coding region of the gene were replaced with the lac promoter. Kinetic studies of the cloned enzyme purified from E. coli were performed with a set of linear malto-oligosaccharides as substrates, and the subsite affinity was calculated from the kinetic parameters. CGA had typical kinetic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose-hydrolyzing activity than other eukaryotic glucoamylases.     

Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.

Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.     

Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma

The function of chromogranin A (CGA) is reviewed, and the radioimmunometric determination of plasma CGA was evaluated as a marker of pheochromocytoma using a comparison of pheochromocytoma patients immediately before surgery (group P, n=25, 635+/-451 ng/ml) with other groups of patients, i.e. pheochromocytoma patients approximately 1 year after removal of tumor (group PP, n=13, 69+/-33 ng/ml), medullary thyroid carcinoma patients (group M, n= 22, 106+/-59 ng/ml), congenital adrenal hyperplasy patients (n=33, 65+/-40 ng/ml), and controls (n=31, 66+/-29 ng/ml). A CGA level above cut off value 130 ng/ml was found in 24 of 25 patients in group P, 1 (relapse) of 13 patients in group PP, and 4 of 22 patients in group M. In the group P we found a significant association between the size of the tumors removed and plasma CGA concentrations (p=0.0016), and also a significant (p=0.0016) relationship between plasma CGA concentrations and PASS score rating the malignity of pheochromocytoma. We can conclude that plasma CGA concentration as determined by radioimmunometric assay (which is simple without the necessity of special laboratory equipment) is an effective marker of pheochromocytoma with association to malignity and tumor mass.     

Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.     

Chlorogenic acid participates in the regulation of shoot,root and root hair development in Hypericum perforatum

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l^?1 did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes.     

Engineering plants with increased levels of the antioxidant chlorogenic acid

The trend to view many foods not only as sustenance but also as medicine, so-called functional foods, is increasing. Phenolics are the most widespread dietary antioxidants, and among these, chlorogenic acid (CGA) accumulates to high levels in some crop plants. CGA acts as an antioxidant in plants and protects against degenerative, age-related diseases in animals when supplied in their diet. cDNA clones encoding the enzyme that synthesizes CGA, hydroxycinnamoyl-CoA quinate: hydroxycinnamoyl transferase (HQT), were characterized from tomato and tobacco. Gene silencing proved HQT to be the principal route for accumulation of CGA in solanaceous species. Overexpression of HQT in tomato caused plants to accumulate higher levels of CGA, with no side-effects on the levels of other soluble phenolics, and to show improved antioxidant capacity and resistance to infection by a bacterial pathogen. Tomatoes with elevated CGA levels could be used in foods with specific benefits for human health.     

Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.

Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.     

Chlorogenic acid stimulates glucose transport in skeletal muscle via AMPK activation: a contributor to the beneficial effects of coffee on diabetes

Chlorogenic acid (CGA) has been shown to delay intestinal glucose absorption and inhibit gluconeogenesis. Our aim was to investigate the role of CGA in the regulation of glucose transport in skeletal muscle isolated from db/db mice and L6 skeletal muscle cells. Oral glucose tolerance test was performed on db/db mice treated with CGA and soleus muscle was isolated for 2-deoxyglucose transport study. 2DG transport was also examined in L6 myotubes with or without inhibitors such as wortmannin or compound c. AMPK was knocked down with AMPKα1/2 siRNA to study its effect on CGA-stimulated glucose transport. GLUT 4 translocation, phosphorylation of AMPK and Akt, AMPK activity, and association of IRS-1 and PI3K were investigated in the presence of CGA. In db/db mice, a significant decrease in fasting blood sugar was observed 10 minutes after the intraperitoneal administration of 250 mg/kg CGA and the effect persisted for another 30 minutes after the glucose challenge. Besides, CGA stimulated and enhanced both basal and insulin-mediated 2DG transports in soleus muscle. In L6 myotubes, CGA caused a dose- and time-dependent increase in glucose transport. Compound c and AMPKα1/2 siRNA abrogated the CGA-stimulated glucose transport. Consistent with these results, CGA was found to phosphorylate AMPK and ACC, consistent with the result of increased AMPK activities. CGA did not appear to enhance association of IRS-1 with p85. However, we observed activation of Akt by CGA. These parallel activations in turn increased translocation of GLUT 4 to plasma membrane. At 2 mmol/l, CGA did not cause any significant changes in viability or proliferation of L6 myotubes. Our data demonstrated for the first time that CGA stimulates glucose transport in skeletal muscle via the activation of AMPK. It appears that CGA may contribute to the beneficial effects of coffee on Type 2 diabetes mellitus.     

Calmodulin-dependent activation of calcineurin by chlorogenic acid

Chlorogenic acid (CGA) has been proved to be an activator of calcineurin (CN) in our previous research. In this study, the activation mechanism of CN by CGA was further explored. The results showed that although the purified CN was inactive in vitro if only Ca(2+)/calmodulin (CaM) existed without Mn(2+)/Ni(2+), CGA activated the inactive CN potently. It was found that CN's activity increased as the concentration of CGA increased and reached a plateau of 4- to 6-fold higher activity using p-nitrophenyl phosphate (pNPP) or phosphopeptide (32)P-RII as substrate. And the activation was CaM-dependent. Moreover, the fluorescent emission of CN had a 17 nm red shift in the presence of 128 muM CGA, and the quenching constant was 1.21x10(12) M(-1) . s(-1), which indicated that CGA bound to CN statically and changed its conformation. According to the kinetic analysis, CGA preferred to activate CN in a substrate noncompetitive manner. When Mn(2+) or Ni(2+) presented, CGA also activated CN with CaM-dependency by improving CN's affinity for Mn(2+) or Ni(2+). In addition, the inhibition of CN by Zn(2+) was partially eliminated by CGA chelation. Our findings suggested the activation of CN by CGA was in a CaM-dependent and substrate noncompetitive manner. This might provide the basis for the further study of CN-targeted activators.     

The primary structure of TE-6: a novel neuropeptide from the nematode Ascaris suum.

Extensive immunoreactivity (IR) towards a hexapeptide (sequence KGQELE), which flanks the C-terminus of the pancreastatin sequence in rat chromogranin A (CGA), is found throughout the nervous system of the nematode parasite Ascaris suum. The peptide IR was purified from the gonoduct of the parasite and found to have the sequence TKQELE. This peptide, designated TE-6, has some C-terminal homology with several regions of the CGA molecule. However, TE-6 was the only peptide isolated suggesting that either the nematode does not possess CGA, or that the -ELE regions of parasite CGA-like peptides which would be larger than TE-6 are not accessible to the antiserum in RIA, or are not being successfully extracted from the parasite. The N-terminus of TE-6 has little homology with any of the sequences preceding -ELE regions in CGA. This, and the fact that the tissue from which TE-6 was isolated does not contain IR towards another, highly conserved, region of the CGA molecule (WE-14) suggests that TE-6 may belong to a new class of regulatory peptide unrelated to CGA.