Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum

Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron-doped diamond microelectrode held at 1 V versus a Ag|AgCl reference electrode. This potential was sufficient to oxidise NO. Longitudinal muscle myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 μM nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N-nitro-L-arginine (L-NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation stimulated release produced a maximum current that corresponded nominally to 46 nM of NO. The oxidation currents decreased to 10 and 2 nM, respectively, when the tissue was perfused with tetrodotoxin and L-NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were 3-fold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real-time electroanalytical techniques

High mucosal serotonin availability in neonatal guinea pig ileum is associated with a low serotonin transporter expression

BACKGROUND & AIMS: 5-hydroxytryptamine is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterchromaffin cells (EC) which release 5-HT has not been studied in neonatals. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs. METHODS: 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and HPLC with electrochemical detection. RESULTS: An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 μM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs. adult tissues. There was no difference in 5-HT content between neonates and adults but 5-HIAA/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number but EC cells were found in the crypts in neonatal and along the villi in adult tissues. CONCLUSIONS: SERT expression is low in neonates and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may important for development of neurohumoral control of intestinal motor reflexes

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs

BACKGROUND: Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig small intestine. METHODS: Inhibitory neuromuscular transmission was investigated using in vitro ileal longitudinal muscle myenteric plexus (LMMP) preparations made from neonatal (≤ 48 h postnatal) and adult (~ 4 weeks postnatal) guinea pigs. KEY RESULTS: Amperometric measurements of nicotine induced nitric oxide release (measured as an oxidation current) from myenteric ganglia revealed larger currents in neonatal (379 ± 24 pA) vs. adult (119 ± 39 pA, P < 0.05) tissues. Nicotine-induced oxidation currents were blocked by the nitric oxide synthase (NOS) inhibitor, nitro-L-arginine (NLA, 100 µM). Nicotine-induced, NLA-sensitive oxidation currents could be detected in the tertiary plexus of neonatal but not adult tissues. Immunohistochemistry demonstrated stronger NOS immunoreactivity in neonatal compared to adult myenteric ganglia. Western blot studies revealed higher levels of NOS in neonatal compared to adult LMMP. Cell counts revealed that the total number of myenteric neurons in the small intestine was greater in adults than in neonatal guinea pigs, however the ratio of NOS:Calbindin neurons was significantly higher in neonatal compared to adult tissues. CONCLUSIONS: NO signaling to the longitudinal muscle is stronger in neonatal compared to adult guinea pig ileum. NOS-containing neurons are diluted postnatally by cholinergic and other, as yet unidentified neuronal subtypes

Macrophage depletion lowers blood pressure and restores sympathetic nerve alpha(2)-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats

We tested the hypothesis that vascular macrophage infiltration and O(2)(−) release impairs sympathetic nerve α(2)-adrenergic autoreceptor (α(2)AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3–5, 10–13, and 18–21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O(2)(−) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18–21. O(2)(−) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α(2)AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O(2)(−) while restoring α(2)AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O(2)(−), which disrupts α(2)AR function, causing enhanced NE release from sympathetic nerves

Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei

Background: The “Assay for Transposase Accessible Chromatin sequencing” (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. Results: In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. Conclusions: We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens. © 2022, The Author(s).Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood–testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p =.0329) and by 53% in a human Sertoli cell line (p =.0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5′-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p =.1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered. © 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]