Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.     

Some Characteristics of a Peptidyl Dipeptidase (Kininase II) from Rat CSF: Differential Effects of NaC1 on the Sequential Degradation Steps of Bradykinin

Incubation of various authentic peptides with rat CSF in vitro and analysis of their products by HPLC demonstrated the presence in CSF of a peptidyl dipeptidase [peptidyl dipeptide hydrolase; angiotensin I converting enzyme (ACE); kininase II; EC 3.4.15.1] which sequentially degraded bradykinin (BK) by liberating the carboxy-terminal dipeptides and converted angiotensin I to angiotensin II. This CSF enzyme was gel-chromatographed by means of HPLC, and the molecular weight was estimated. The susceptibility to various peptidase inhibitors of the rat CSF enzyme, as well as the effect of NaCl on the degradation of BK and Hip-His-Leu catalyzed by it, was also determined. These properties were compared with those of ACE or kininase II from brain or other tissues, as described in the literature. NaCl was shown to exert specific and concentration-dependent effects on each step of the sequential degradation of BK, via BK(1-7) to BK(1-5), catalyzed by the enzyme. In addition, the enzyme system for metabolism of BK appears to differ between rat CSF and blood, the former containing exclusively kininase II, whereas the latter contains both kininase I (carboxypeptidase N; EC 3.4.12.7) and kininase II.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Activation of Amiloride-Sensitive Sodium Transport in C6 Glioma Cells

We have characterized, in C6 cells, an amiloride-sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation-exchange system can be induced within 24-36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium-dependent step with full activation obtained in the presence of the calcium ionophore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocytochalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride-sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal-dependent step. Since diacylglycerol is the presumed endogenous activator of protein kinase C, we studied the effects of dioleylglycerol. This intermediate of phospholipid turnover was found to increase specifically the amiloride-sensitive sodium pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

A modular map of Bradykinin-mediated inflammatory signaling network

Bradykinin, a member of the kallikrein-kinin system (KKS), is associated with an inflammatory response pathway with diverse vascular permeability functions, including thrombosis and blood coagulation. In majority, bradykinin signals through Bradykinin Receptor B2 (B2R). B2R is a G protein-coupled receptor (GPCR) coupled to G protein family such as Gα_qs, Gα_q/Gα_11, Gα_i1, and Gβ1_γ2_. B2R stimulation leads to the activation of a signaling cascade of downstream molecules such as phospholipases, protein kinase C, Ras/Raf-1/MAPK, and PI3K/AKT and secondary messengers such as inositol-1,4,5-trisphosphate, diacylglycerol and Ca²⁺ ions. These secondary messengers modulate the production of nitric oxide or prostaglandins. Bradykinin-mediated signaling is implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. Despite the biomedical importance of bradykinin, a resource of bradykinin-mediated signaling pathway is currently not available. Here, we developed a pathway resource of signaling events mediated by bradykinin. By employing data mining strategies in the published literature, we describe an integrated pathway reaction map of bradykinin consisting of 233 reactions. Bradykinin signaling pathway events included 25 enzyme catalysis reactions, 12 translocations, 83 activation/inhibition reactions, 11 molecular associations, 45 protein expression and 57 gene regulation events. The pathway map is made publicly available on the WikiPathways Database with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP5132. The bradykinin-mediated signaling pathway map will facilitate the identification of novel candidates as therapeutic targets for diseases associated with dysregulated bradykinin signaling.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00652-0.     

Ca2+ signalling underlying pancreatitis

In spite of significant scientific progress in recent years, acute pancreatitis (AP) is still a dangerous and in up to 5% of cases deadly disease with no specific cure. It is self-resolved in the majority of cases, but could result in chronic pancreatitis (CP) and increased risk of pancreatic cancer (PC). One of the early events in AP is premature activation of digestive pro-enzymes, including trypsinogen, inside pancreatic acinar cells (PACs) due to an excessive rise in the cytosolic Ca²⁺ concentration, which is the result of Ca²⁺ release from internal stores followed by Ca²⁺ entry through the store operated Ca²⁺ channels in the plasma membrane. The leading causes of AP are high alcohol intake and biliary disease with gallstones obstruction leading to bile reflux into the pancreatic duct. Recently attention in this area of research turned to another cause of AP – Asparaginase based drugs – which have been used quite successfully in treatments of childhood acute lymphoblastic leukaemia (ALL). Unfortunately, Asparaginase is implicated in triggering AP in 5–10% of cases as a side effect of the anti-cancer therapy. The main features of Asparaginase-elicited AP (AAP) were found to be remarkably similar to AP induced by alcohol metabolites and bile acids. Several potential therapeutic avenues in counteracting AAP have been suggested and could also be useful for dealing with AP induced by other causes. Another interesting development in this field includes recent research related to pancreatic stellate cells (PSCs) that are much less studied in their natural environment but nevertheless critically involved in AP, CP and PC. This review will attempt to evaluate developments, approaches and potential therapies for AP and discuss links to other relevant diseases.     

Histamine Evokes Greater Increases in Phosphatidylinositol Metabolism and Catecholamine Secretion in Epinephrine-Containing than in Norepinephrine-Containing Chromaffin Cells

Abstract: Chromaffin cells have H₁ histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from Chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP₁) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine Chromaffin cells. Histamine-stimulated InsP, accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP¹ accumulation in the two Chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K⁺, bradykinin, phorbol 12, 13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H₁ receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H₁ receptors on epinephrine cells may account for the greater effects of histamine on InsP₁ accumulation and catecholamine secretion in these cells.     

The kinin–kallikrein system: physiological roles,pathophysiology and its relationship to cancer biomarkers

Abstract

The kinin–kallikrein system (KKS) is an endogenous multiprotein cascade, the activation of which leads to triggering of the intrinsic coagulation pathway and enzymatic hydrolysis of kininogens with the consequent release of bradykinin-related peptides. This system plays a crucial role in inflammation, vasodilation, smooth muscle contraction, cardioprotection, vascular permeability, blood pressure control, coagulation and pain. In this review, we will outline the physiology and pathophysiology of the KKS and also highlight the association of this system with carcinogenesis and cancer progression.     

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism

Kinin B₁ and B₂ receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B₁ (B₁^−/−) and B₂ receptor (B₂^−/−) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B₁^−/− and B₂^−/− in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B₁^−/− and B₂^−/−. In contrast, in the presence of l-arginine, Ca²⁺ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B₁^−/− and B₂^−/−. The present study demonstrated that targeted deletion of B₁ or B₂ receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B₁^−/− and B₂^−/− vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.     

Characterization of the kallikrein-kinin system during the bovine ovulation process

The kallikrein–kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B₁R and the B₂R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B₁R and B₂R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24 h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B₂R expression in theca cells and B₁R expression in theca and granulosa cells showed different profiles during the periovulatory period (P < 0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P < 0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P > 0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation.