Release Of GMP Research Articles

ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis—A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing

The genome content of adeno-associated virus (AAV) vectors is critical to the safety and potency of AAV-based gene therapy products. Empty capsids are considered a product-related impurity and a critical quality attribute (CQA) of the drug product, thus requiring characterization throughout the production process to demonstrate they are controlled to acceptable levels in the final drug product. Anion exchange chromatography has been used to achieve separation between empty and full capsids, but requires method development and gradient optimization for different serotypes and formulations. Here, we describe an alternative approach to quantitation that does not rely on achieving separation between empty and full capsids, but instead uses the well-established relationship between absorbance at UV A260/A280 and relation to DNA/protein content, in combination with anion-exchange chromatography to allow one to calculate the relative proportion of empty and full capsids in AAV samples from a single peak. We call this approach ACUVRA: Anion-exchange Chromatography UV-Ratio Analysis, and show the applicability of the method through a case study with recombinant AAV2 (rAAV2) process intermediates and drug substance. Method qualification and GMP validation in a quality control (QC) laboratory results show that ACUVRA is a fit-for-purpose method for process development support and characterization, while also being a QC-friendly option for GMP release testing at all stages of clinical development.Graphical abstract

COVID-19 vaccine approvals: key lessons for mRNA therapeutics

The development of mRNA therapeutics is not a new phenomenon, but the key role played by mRNA-based vaccines in battling the COVID-19 pandemic has resulted a rapid acceleration in the development timeframe of this technology field. In a recent expert roundtable discussion, we spoke to a panel of experts within the field to better understand what lessons the vaccine and therapeutic fields can learn from each other. Here, we sum up some of their key thoughts. <div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/544f55bceea342b7b14d37488e0dc915.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#5D538A;">Venkata Indurthi</h3><h5 class="author-title" style="color:#5D538A;">Vice President Research and Development, Aldevron</h5><p class="focus-visible" data-focus-visible-added="">Venkata SK Indurthi, PhD is the Vice President of research and development at Aldevron (est. 1998, www.aldevron.com), a biologicals CDMO with sites in Fargo ND and Madison WI. He received his bachelor’s in engineering in biotechnology from SRM university (Chennai, India) and his Ph.D. in Pharmaceutical Sciences from North Dakota State University (Fargo, ND). After completing graduate school, he joined Aldevron as an assay development scientist and was focused developing assay for GMP release for biologics (DNA, RNA and protein). He then transitioned to a role of senior scientist, Product and Process Design (PPD) where he led the development of Aldervon’s mRNA process and platform and more recently as the Director of R&amp;D where he established all the R&amp;D efforts for Aldevron. Dr. Indurthi leads efforts that to develop new platforms and innovative manufacturing processes for the company including (not limiting to) the mRNA platform, plasmid DNA, gene synthesis and cell free synthetic DNA platform. His team also focuses on the development of new products for the company such as enzymes with improved attributes (increased yield, stability or activity). Dr. Indurthi’s research interests focus on the development of biologics. Particularly, new platform development from idea conception to commercialization. He has developed processes that are currently used for GMP manufacturing. Dr. Indurthi is also the operational head for the Aldevron RNA services. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/4d1901ea5f7e4906a920be4245fc9039.jpeg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#007589;">Joseph Barberio</h3><h5 class="author-title" style="color:#007589;">Director, Process Development, Strand Therapeutics Inc.</h5> Joseph Barberio is a biochemist and molecular biologist with a proven track record of solving complex problems with innovative solutions. He is the Director of mRNA Process Development at Strand Therapeutics and oversees Strand’s manufacturing strategy to support clinical development. With over fifteen years of industry experience, Joe specializes in process and analytical development and has extensive expertise in manufacturing of both viral and RNA based gene therapy medicinal products. The majority of Joseph’s career has been focused on building platforms for small biotech organizations. Most recently at bluebird bio, he constructed and led the mRNA process development team, a group designed to enable gene editing programs. Earlier in his career, Joe held process development roles at Moderna, Percivia, and Acceleron Pharma. In addition to his work at Strand, Joseph also serves on the Board of Directors of Sophie’s Hope Foundation, a non-profit charity supporting research for glycogen storage disease type 1b (GSD1b). </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/276f2895701f4c2e939bb8cc42991fec.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#00A795;">Scott Zobbi</h3><h5 class="author-title" style="color:#00A795;">Senior Business Development Manager, Custom POROS Chromatography Resins, Thermo Fisher Scientific</h5> Scott Zobbi is a Senior Manager of Business Development for Custom POROS Resins within the Bioproduction Division at Thermo Fisher Scientific. Scott has a B.S. in Biology from University of Connecticut and an MBA from the University of Massachusetts. Scott has worked on chromatography applications in the biotech industry for decades with 22 years spent at Thermo Fisher Scientific. His expertise is finding solutions to complex separation challenges draws on his experience in cGMP manufacturing, process development, customer training, sales and product management. In his current role, Scott is responsible for managing globally the Custom POROS Resin program, including working with customers to identify needs, in-house R&amp;D to develop solutions, and POROS manufacturing to commercialize bioprocess resins for GMP applications. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/7f8ed4c095f842d490465df72352b91a.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color: rgb(255, 127, 63);">Dr Christoph Kröner</h3><h5 class="author-title" style="color: rgb(255, 127, 63);">Director, DNA Process Development &amp; Cap Technology, BioNTech SE</h5> Christoph Kröner works as Director DNA Process Development &amp; Cap Technology at BioNTech RNA Pharmaceuticals GmbH with strong focus on developmental work for BioNTech’s various clinical projects using mRNA as drug substance. He has more than 10 years of experience working with nucleic acids like mRNAs. His work focused on mRNA-based therapeutics when he joined BioNTech in 2014 as a scientist. Christoph Kröner holds a diploma and PhD in chemistry from the University of Stuttgart, Germany. </div></div></div>

Deliberate release of genetically modified plants into the environment in Slovenia

Deliberate release of genetically modified higher plants (GMHPs) into the environment in Slovenia is regulated by the Law on the Management of Genetically Modified Organisms (ZRGSO) Ur. l. RS 23/2005 and 21/2010, III chapter. For each deliberate release of GMPs into the environment a license issued by the Ministry of Environment and Spatial Planning (MESP) must be acquired. The application or notification should contain a very accurate and complex description of the GMP, of the field where it will be released and of wider surroundings or environment. The application consists of Annex 2 with accessories: 1. Part A (technical data for the authorization of deliberate GMP release into the environment); 2. Part B (environmental risk assessment); 3. Application summary in Slovenian and English language) for the release of GMP into environment, which is transmitted to Brussels by MESP; 4. Extract from the Land Cadastre of the field to which the GMP will be released. The release procedure runs (till here) under the above mentioned Law, which has been in place for several years and which clearly defines that it is possible to release GMP in Slovenia. In the case of GM rice in 2011, the law applied till the site selection of the experiment. Here, the law was not sufficiently taken into account. It was prevailed by the regulation of Farmland and Forest Fund of the Republic of Slovenia and municipal decision, which was stronger than the national law and prevented the cultivation of GM rice in an area that is legally suitable for release of GMO into the environment. Rice is not grown in Slovenia and does not have wild ancestors or close relatives with whom it might mate. Nearest area of cultivation is in neighboring Italy, which is from potentially selected location in Slovenia more than 70 km away.

Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3′-terminal phosphate and 5′-OH

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2',3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5′-OH and either 2′,3′-cyclic phosphate or 3′-phosphate ends. The 3′-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5′-OH end attacks the 3′-phosphorus of the N(3′)pp(5′)G end to form a 3′,5′-phosphodiester and release GMP. Scheme 2 posits that the N(3′)pp(5′)G end is converted to a 2′,3′-cyclic phosphodiester, which is then attacked directly by the 5′-OH RNA end to form a 3′,5′-phosphodiester. Here we show that the sealing of a 2′,3′-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB–GMP adduct, and involves a kinetically valid RNA(3′)pp(5′)G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2′,3′-cyclic phosphate to a 3′-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB–GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3′-phosphate ligase steps of the RNA splicing pathway.

Automated, serum-free production of CTX0E03: a therapeutic clinical grade human neural stem cell line

Automation of cell culture processes is necessary to achieve scalable and reproducible manufacture of cell-based therapies. CTX0E03 is a near-clinic neural stem cell line for stroke therapy. The transfer of the CTX0E03 manufacture process from a manual to a completely automated process is demonstrated by (a) adapting the manual process to conform to the automated platform and (b) conducted a large scale (20 x T175 flasks) automated CTX0E03 expansion in accordance with the working bank to drug substance lot manufacture schedule. The automated lot was within the GMP release specification for viability, growth rate, nestin immunoreactivity and transcriptome equivalence.

Interaction of Metallothionein-2 with Platinum-Modified 5′-Guanosine Monophosphate and DNA

Human metallothioneins (MTs), a family of cysteine- and metal-rich metalloproteins, play an important role in the acquired resistance to platinum drugs. MTs occur in the cytosol and the nucleus of the cells and sequester platinum drugs through interaction with their zinc-thiolate clusters. Herein, we investigate the ability of human Zn 7MT-2 to form DNA-Pt-MT cross-links using the cisplatin- and transplatin-modified plasmid DNA pSP73. Immunochemical analysis of MT-2 showed that the monofunctional platinum-DNA adducts formed DNA- cis/ trans-Pt-MT cross-links and that platinated MT-2 was released from the DNA- trans-Pt-MT cross-links with time. The DNA- cis/ trans-Pt-MT cross-links were also formed in the presence of 2 mM glutathione, a strong S-donor ligand. Independently, we used 5'-guanosine monophosphate (5'-GMP) platinated at the N7 position as a model of monofunctional platinum-DNA adducts. Comparison of reaction kinetics revealed that the formation of ternary complexes between Zn 7MT-2 and cis-Pt-GMP was faster than that of the trans isomer. The analysis of the reaction products with time showed that while the formation of ternary GMP- trans-Pt-MT complex(es) is accompanied by 5'-GMP release, a stable ternary GMP- cis-Pt-MT complex is formed. In the latter complex, a fast initial formation of two Pt-S bonds was followed by a slow formation of an additional Pt-S bond yielding an unusual Pt(II)S 3N coordination with N7-GMP as the only N-donor ligand. The ejection of negligible zinc from the zinc-thiolate clusters implies the initial formation of Zn-(mu-SCys)-Pt bridges involving the terminal thiolate ligands. The biological implications of these studies are discussed.

Improving sensitivity in chiral supercritical fluid chromatography for analysis of active pharmaceutical ingredients

Despite its status as the preferred method for routine enantiopurity analysis in pharmaceutical research, supercritical fluid chromatography (SFC) has historically been unsuited for the accurate and precise measurements required for release testing of active pharmaceutical ingredients (APIs) under current good manufacturing processes (cGMPs). Insufficient signal to noise, as compared to HPLC, has heretofore been the major limitation of the chiral SFC approach. We herein describe an investigation into the fundamental limitations and sources of noise in the SFC approach, identifying thermal, electronic, and mechanical sources of noise within the flow cell as key parameters contributing to reduced sensitivity. A variety of instrument modifications are explored, ultimately leading to the development of a new and improved flow cell and other instrument modifications that allow suitable sensitivity and accuracy to carry out GMP release testing for enantiopurity analysis using SFC.

Cyclic GMP Release by Acute Enhanced External Counterpulsation

Enhanced external counterpulsation (EECP) is a noninvasive, pneumatic technique that provides favorable effects in patients with coronary artery disease and heart failure. The mechanisms by which EECP exerts its beneficial effects remain poorly understood. Cyclic GMP (cGMP) regulates vascular smooth muscle tone that may improve arterial function. We investigated the effect of a single session of EECP on plasma and platelet cGMP in asymptomatic subjects with cardiovascular risk factors (HCVR) and in patients with coronary artery disease (CAD). Fifty-five subjects were included (25 HCVR and 30 CAD) and randomized into two groups to receive either sham (control) or active EECP during 1 h. Plasma and platelet cGMP were measured immediately before and after EECP by radioimmunoassay. One hour of EECP increased cGMP plasma concentration by 52% +/- 66% (SD) (P < .001) and platelet content by 19% +/- 28% (P < .01). The increase in plasma cGMP was particularly marked in CAD patients receiving active EECP (P < .01), mainly in those with low LDL-cholesterol. Platelets, inhibition of nitric oxide synthesis by N(G)-monomethyl-l-arginine (L-NMMA) reduced cGMP by 23% +/- 31% (P < .001), whereas presence of superoxide dismutase and inhibition of phosphodiesterase-5 increased cGMP by 46% +/- 49% and 70% +/- 77%, respectively (P < .001). In all of the cases EECP increased additional platelet cGMP content, which suggests nitric oxide synthase activation. Acute external counterpulsation showed that very early treatment increases the cGMP production that may participate in the mechanism by which EECP exerts its clinical benefit. Analysis of the modulation of platelet cGMP content suggests that EECP activated the nitric oxide-dependent pathways.

Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.

Atrial Natriuretic Factor and Angiotensin Ii Stimulate Nitric Oxide Release from Human Proximal Tubular Cells

1. It has been recently reported that angiotensin II can enhance atrial natriuretic factor-stimulated cyclic GMP release from brain capillary endothelial cells and stimulate directly the release of cyclic GMP by Neuro 2a cells. A possible mechanism mediating such cyclic GMP release could be via the production of nitric oxide and the resultant stimulation of soluble guanylate cyclase. 2. The ability of angiotensin II, atrial natriuretic factor and c(4-23) atrial natriuretic factor to stimulate nitric oxide production was investigated in primary cultures of human proximal tubular cells. 3. Freshly prepared human proximal tubular cells were seeded onto 6-well plates and allowed to reach confluence. Cells were then incubated with incremental concentrations of either angiotensin II, atrial natriuretic factor or c(4-23) atrial natriuretic factor alone for 1, 4, 12 or 24h or in the presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Angiotensin II was also incubated with human proximal tubular cells in the presence of the AT1 and AT2 receptor antagonists DuP 753 and PD 123319. 4. Incubation of human proximal tubular cells with angiotensin II, atrial natriuretic factor or c(4-23) atrial natriuretic factor produced a dose- and time-dependent increase in nitric oxide production, which was inhibited in the presence of NG-monomethyl-L-arginine. A similar increase in nitric oxide production was observed after incubation with atrial natriuretic factor or c(4-23) atrial natriuretic factor. 5. The angiotensin-induced increase in nitric oxide production was not inhibited in the presence of either the angiotensin AT1 or AT2 receptor antagonists DuP 753 or PD 123319. 6. This study demonstrates that primary cultures of human proximal tubular cells can be stimulated to produce nitric oxide by both atrial natriuretic factor and angiotensin II. Furthermore, the atrial natriuretic factor-induced response appears to be mediated via the atrial natriuretic factor-C receptor, while the angiotensin II-induced response appears to be mediated by a novel, as yet unidentified, angiotensin II receptor.

Nitronyl nitroxides as probes to study the mechanism of vasodilatory action of nitrovasodilators, nitrone spin traps, and nitroxides: Role of nitric oxide

Nitronyl nitroxides have been used to trap nitric oxide ( ·NO) produced during visible irradiation of nitrovasodilators such as sodium nitroprusside (Joseph et al., Biochem. Biophys. Res. Commun. 192:926–934; 1993). We have also shown that nitrone and nitroso spin traps exert a potent vasodilatory effect in the isolated perfused rat heart (Konorev et al., Free Radic. Biol. Med. 14:127–137, 1993). The objective of this study was to investigate the effect of nitronyl nitroxides on the vasodilatory action of sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), α-(4-pyridyl-l-oxide)- N-tert-butyl nitrone (POBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy free radical (TEMPOL) in the isolated perfused rat heart model. In this study, we have used the following nitronyl nitroxides as nitric oxide traps: 2-(p-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-3-oxide 1-oxyl (SLI) and 2(1′,1′-dimethyl-2′-hydroxyethyl)-4,4,5,5-tetramethyl imidazoline-3-oxide 1-oxyl (SLII). Under in vitro conditions, both SLI and SLII trapped ·NO released from SNP/light treatment and from spontaneous decomposition of SNAP, forming the corresponding imino nitroxides, which were characterized by electron spin resonance (ESR) technique. In isolated hearts, SNP (2 μmol/1) and SNAP (20 μmol/l) increased coronary flow rate to a maximum of 185% and 190%, respectively. SNP-induced vasodilation was inhibited by SLI (0.05–3 mmol/l) from 162% to 131% of baseline, and SNAP-induced vasodilation was inhibited by SLII (0.05–1.2 mmol/1) from 190% to 136% of baseline. In contrast, neither SLI nor SLII inhibited the vasodilatory action elicited by POBN or TEMPOL. Only SNP and SNAP, but not POBN and TEMPOL, increased the cyclic GMP release into the coronary effluent. We conclude that coronary vascular relaxation induced by SNP and SNAP is mediated by ·NO and that the mechanism of the vasodilatory effect of POBN and TEMPOL does not involve ·NO release.

Endotoxin-induced nitric oxide synthesis in the perfused rat liver: Effects of l-arginine and ammonium chloride

We used the single-pass-perfused rat liver model to study short-term regulation of endotoxin-inducible nitric oxide synthesis by following the release of nitrite and nitrate, the oxidation products of nitric oxide, into the effluent perfusate. In endotoxin-pretreated livers, the basal nitrite+nitrate release was 5.3 +/- 1.2 nmol.gm liver-1.min-1. Nitrite and nitrate release was stimulated by L-arginine in a dose-dependent and saturable fashion. Maximal nitrite+nitrate release with 1 mmol/L L-arginine infused to the influent perfusate was 10.2 +/- 1.1 nmol.gm liver-1.min-1, with a half-maximal effect at 53 mumol/L L-arginine. In the absence of molecular oxygen, nitric oxide synthesis was inhibited. Ammonium chloride infusion also stimulated nitrite and nitrate release to a maximal rate of 9.2 +/- 0.8 nmol.gm liver-1.min-1 with half-maximal effects at 60 mumol/L ammonium chloride. Ammonium chloride-stimulated nitrite and nitrate release was abolished when urea synthesis was inhibited by bicarbonate-free liver perfusion. Citrulline and ornithine (200 mumol/L each) were without effect on nitrite and nitrate release. L-Nitroarginine methyl ester inhibited both, L-arginine-and ammonium chloride-induced nitrite and nitrate release. Stimulation of nitric oxide synthesis by L-arginine and ammonium chloride addition (1 mmol/L each) was accompanied by a threefold-to-fourfold increase of cyclic GMP release into the effluent perfusate. In livers of endotoxin-pretreated rats the urea production from L-arginine was higher than that in untreated livers, suggesting induction of an L-arginine transport system in hepatocytes by endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide participates in the regulation of pancreatic acinar cell secretion

The role of nitric oxide (NO) in the regulation of exocrine secretion was investigated in isolated rat pancreatic acini. NO synthase activity was detected in the extract of acini and purified by ion-exchange and 2′, 5′-ADP agarose chromatographies. Enzyme activity was determined by conversion of 3H-arginine to 3H-citrulline, by measurement of nitrite (a breakdown product of NO) and by generation of cyclic GMP. Treatment of acini with L-arginine increased nitrite as well as cyclic GMP and amylase release, which were prevented by the nitric oxide synthase inhibitors N-monomethyl-arginine [NMMA] and N G-nitro-L-arginine [NNA]. These nitric oxide inhibitors also blocked carbachol-induced amylase release as well as elevation of acinar cell cyclic GMP. NNA was a potent inhibitor of carbamylcholine-induced amylase release (est. K i=2.2 uM). Nitric oxide apparently participates significantly in the overall control of pancreatic acinar cell secretory function.

Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [3H]Cyclic GMP was assayed in cells prelabeled with [3H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [3H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 microM (compared to 96 microM for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [3H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [3H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [3H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).

Cyclic GMP release and vasodilatation induced by EDRF and atrial natriuretic factor in the isolated perfused kidney of the rat

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation. The renal vasodilator action of ANF however, is independent of EDRF, although the renal vascular endothelium cannot be discounted as a site at which ANF stimulates cyclic GMP production.

On the presence of guanosine phosphate in the tail of bacteriophage T4

Treatment of gp18, a biologically active monomer of the structural protein of the bacteriophage T4 contractile sheath, with 0.6 m-HClO 4 leads to the release of GDP, GMP and inorganic phosphate. Each gp18 molecule is shown to carry three atoms of phosphorus. In the isolated protein preparation, gp18 and the nucleoside phosphate are in equimolar relation. It is suggested that in the native sheath-protein subunit, GDP and inorganic phosphate are united as GTP.

Catabolism of capped (3′-5′)- and (2′-5′)-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp 3A moiety was determined in the capped RNA model compounds GP 3A3′pA, Gp 3A3′pA-isoprop and Gp 3A2′pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp 3A cap moiety is hydrolyzed in (3′-5′) linked nucleotides only, whereas an extension of the Gp 3A in the 2′-direction prevents the nuclear triphosphatase to operate.

Interaction of substance P with dispersed pancreatic acinar cells from the guinea pig. Stimulation of calcium outflux, accumulation of cyclic GMP and amylase release.

In dispersed acinar cells from the guinea pig pancreas, substance P (SP) was found to stimulate outflux of 45Ca, cellular accumulation of cyclic GMP, and release of amylase. Maximal effects on accumulation of cyclic GMP and release of amylase were obtained with 3 x 10(-8) M of SP, 10(-7) M of Sp caused maximal outflux of 45Ca. These effects corresponded to 30-50% of the maximal effects obtained with caerulein, a cholecystokinin-like decapeptide. The concentrations of SP required for stimulation of 45Ca outflux, accumulation of cyclic GMP, and release of amylase correspond well with those which affect binding of 125I-tyr8-SP to pancreatic acinar cells.

Vagal stimulation and its role in eliciting gastrin but not glucagon release from the isolated perfused dog stomach.

Electrical stimulation (10 V, 10 Hz, 3 min) of both dorsal and ventral vagal trunks of the isolated canine stomach perfused with whole blood induced strong gastric contractions, transient release of cyclic GMP and marked release of gastrin. No gastric-glucagon release was elicited either at 'normal' (4.8 +/- 0.1 mmol/l) or at low (1.5 +/- 0.1 mmol/l) concentrations of blood glucose. It is concluded that, in conditions effective for the stimulation of gastrin release, electrical stimulation of the vagus nerves does not stimulate glucagon release from the isolated perfused dog stomach. Thus one of the well-accepted mechanisms controlling pancreatic-glucagon secretion, vagal stimulation, is ineffective on gastric-glucagon release.