Indole based antioxidants for the treatment of ischemia reperfusion injury

With the number of ischemia reperfusion (I/R) injuries on the rise, and a lack of pharmacological intervention aimed at reducing free radical damage associated with I/R, we have developed 30 indole phenolic antioxidants that were synthesized by click chemistry to couple our indole with a phenolic or anisole derivative. The total antioxidant activity of the analogues was tested in vitro using the ferric thiocyanate lipid emulsion method. Compounds containing hydroxyl or methoxy aromatics at the 3 or 4 position on the aromatic coupled to the indole exhibited increased antioxidant scavenging. 4-methoxyindole derivatives (8a-e) exhibited increased scavenging (p \u3c 0.05) compared to the known antioxidant butylated hydroxyanisole (BHA)

Long-term high salt intake involves reduced SK Currents and Increased Excitability of PVN Neurons with projections to the rostral ventrolateral medulla in rats

Evidence indicates that high salt (HS) intake activates presympathetic paraventricular nucleus (PVN) neurons, which contributes to sympathoexcitation of salt-sensitive hypertension. The present study determined whether 5 weeks of HS (2% NaCl) intake alters the small conductance Ca2+-activated potassium channel (SK) current in presympathetic PVN neurons and whether this change affects the neuronal excitability. In whole-cell voltage-clamp recordings, HS-treated rats had significantly decreased SK currents compared to rats with normal salt (NS, 0.4% NaCl) intake in PVN neurons. The sensitivity of PVN neuronal excitability in response to current injections was greater in HS group compared to NS controls. The SK channel blocker apamin augmented the neuronal excitability in both groups but had less effect on the sensitivity of the neuronal excitability in HS group compared to NS controls. In the HS group, the interspike interval (ISI) was significantly shorter than that in NS controls. Apamin significantly shortened the ISI in NS controls but had less effect in the HS group. This data suggests that HS intake reduces SK currents, which contributes to increased PVN neuronal excitability at least in part through a decrease in spike frequency adaptation and may be a precursor to the development of salt-sensitive hypertension

High salt intake augments excitability of PVN neurons in rats: Role of the endoplasmic reticulum Ca\u3csup\u3e2+\u3c/sup\u3e store

High salt (HS) intake sensitizes central autonomic circuitry leading to sympathoexcitation. However, its underlying mechanisms are not fully understood. We hypothesized that inhibition of PVN endoplasmic reticulum (ER) Ca2+ store function would augment PVN neuronal excitability and sympathetic nerve activity (SNA). We further hypothesized that a 2% (NaCl) HS diet for 5 weeks would reduce ER Ca2+ store function and increase excitability of PVN neurons with axon projections to the rostral ventrolateral medulla (PVN-RVLM) identified by retrograde label. PVN microinjection of the ER Ca2+ ATPase inhibitor thapsigargin (TG) increased SNA and mean arterial pressure (MAP) in a dose-dependent manner in rats with a normal salt (NS) diet (0.4%NaCl). In contrast, sympathoexcitatory responses to PVN TG were significantly (p \u3c 0.05) blunted in HS treated rats compared to NS treatment. In whole cell current-clamp recordings from PVN-RVLM neurons, graded current injections evoked graded increases in spike frequency. Maximum discharge was significantly augmented (p \u3c 0.05) by HS diet compared to NS group. Bath application of TG (0.5 μM) increased excitability of PVN-RVLM neurons in NS (p \u3c 0.05), yet had no significant effect in HS rats. Our data indicate that HS intake augments excitability of PVN-RVLM neurons. Inhibition of the ER Ca2+ ATPase and depletion of Ca2+ store likely plays a role in increasing PVN neuronal excitability, which may underlie the mechanisms of sympathoexcitation in rats with chronic HS intake

ACETATE AS AN ACTIVE METABOLITE OF ETHANOL: NEURAL AND CARDIOVASCULAR IMPLICATIONS

Alcohol use disorders (AUD) and alcohol associated central nervous system (CNS) pathologies, including hypertension, excitotoxicity and dependence remain a large component of excessive ethanol intake. While studies involving ethanol and the intermediate metabolite, acetaldehyde have been appreciated, very little research has been done to explore the effects of the end metabolite, acetic acid/acetate on alcohol associated CNS pathologies. As such, we explored the effects of ethanol and acetate on CNS excitatory drive through modulation of N-methyl-D-aspartate receptor (NMDAR). In study 1 we demonstrated that microinjection of ethanol and acetate in the central nucleus of amygdala (CeA) of anesthetized rats increased sympathetic nerve activity (SNA) and mean arterial pressure (MAP) which was attenuated by local NMDAR antagonists and rostral ventrolateral medulla (RVLM) knockout. In study 2 we further expanded our study, including CeA microinjected acetate, ethanol and ethanol with acetaldehyde dehydrogenase (ALDH) inhibitor on the ethanol and acetate sympathoexcitatory effect. We demonstrated that local metabolism of ethanol to acetate and NMDAR activation is the major contributor to the alcohol induced sympathoexcitatory effect. Next, we demonstrated that acetate increased CeA-RVLM neuronal excitability through activation of NMDAR and that intraneuronal acidification with acetic acid increased neuronal activity, at least in part through NMDAR modulation. In neuronal cultures incubated with low doses of acetate, we found increases in pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1-beta (IL-1β), suggesting that acetate produces a CNS inflammatory effect. Overall, study 2 demonstrated that in vivo and in vitro, acetate has an excitatory effect in the CNS which may underlie numerous alcohol CNS pathologies. In study 3 we explored whether acetate was cytotoxic to dopaminergic like pheochromocytoma (PC12) cells through activation of NMDAR. We demonstrated that acetate activates NMDAR, resulting in increased cytosolic calcium and TNFα. Furthermore, acetate increased cytosolic reactive oxygen species (ROS) and apoptosis which ethanol did not. NMDAR blocker was unable to reverse acetate induced apoptosis, however it was able to abolish the influx of calcium and increases in TNFα. This suggests that acetate induced apoptosis is a result or contribution of several cellular pathways not limited to NMDAR activation alone. In study 4 we developed a method for analyzing cations, anions and acetate from liquid and tissue samples using ion chromatography. Since many CNS and peripheral pathologies give rise to electrolyte imbalance, including alcohol consumption, having an accurate method for determining these concentrations was not only crucial for alcohol metabolism, but also for a whole host of other CNS and peripheral pathologies. This study demonstrated that acetate was elevated in brain tissue relative to peripheral liver tissue, cation and anion concentrations from cerebrospinal fluid (CSF) and serum can be accurately replicated compared to alternative methodologies and that tissue electrolyte measurements can also be included through the use of one machine. Together, these studies provide new and exciting information for AUD

Acetic Acid: An Underestimated Metabolite in Ethanol-Induced Changes in Regulating Cardiovascular Function

Acetic acid is a bioactive short-chain fatty acid produced in large quantities from ethanol metabolism. In this review, we describe how acetic acid/acetate generates oxidative stress, alters the function of pre-sympathetic neurons, and can potentially influence cardiovascular function in both humans and rodents after ethanol consumption. Our recent findings from in vivo and in vitro studies support the notion that administration of acetic acid/acetate generates oxidative stress and increases sympathetic outflow, leading to alterations in arterial blood pressure. Real-time investigation of how ethanol and acetic acid/acetate modulate neural control of cardiovascular function can be conducted by microinjecting compounds into autonomic control centers of the brain and measuring changes in peripheral sympathetic nerve activity and blood pressure in response to these compounds

Long-Term High Salt Intake Involves Reduced SK Currents and Increased Excitability of PVN Neurons with Projections to the Rostral Ventrolateral Medulla in Rats

Evidence indicates that high salt (HS) intake activates presympathetic paraventricular nucleus (PVN) neurons, which contributes to sympathoexcitation of salt-sensitive hypertension. The present study determined whether 5 weeks of HS (2% NaCl) intake alters the small conductance Ca2+-activated potassium channel (SK) current in presympathetic PVN neurons and whether this change affects the neuronal excitability. In whole-cell voltage-clamp recordings, HS-treated rats had significantly decreased SK currents compared to rats with normal salt (NS, 0.4% NaCl) intake in PVN neurons. The sensitivity of PVN neuronal excitability in response to current injections was greater in HS group compared to NS controls. The SK channel blocker apamin augmented the neuronal excitability in both groups but had less effect on the sensitivity of the neuronal excitability in HS group compared to NS controls. In the HS group, the interspike interval (ISI) was significantly shorter than that in NS controls. Apamin significantly shortened the ISI in NS controls but had less effect in the HS group. This data suggests that HS intake reduces SK currents, which contributes to increased PVN neuronal excitability at least in part through a decrease in spike frequency adaptation and may be a precursor to the development of salt-sensitive hypertension

Long-Term High Salt Intake Involves Reduced Sk Currents And Increased Excitability Of Pvn Neurons With Projections To The Rostral Ventrolateral Medulla In Rats

Evidence indicates that high salt (HS) intake activates presympathetic paraventricular nucleus (PVN) neurons, which contributes to sympathoexcitation of salt-sensitive hypertension. The present study determined whether 5 weeks of HS (2% NaCl) intake alters the small conductance Ca2+-activated potassium channel (SK) current in presympathetic PVN neurons and whether this change affects the neuronal excitability. In whole-cell voltage-clamp recordings, HS-treated rats had significantly decreased SK currents compared to rats with normal salt (NS, 0.4% NaCl) intake in PVN neurons. The sensitivity of PVN neuronal excitability in response to current injections was greater in HS group compared to NS controls. The SK channel blocker apamin augmented the neuronal excitability in both groups but had less effect on the sensitivity of the neuronal excitability in HS group compared to NS controls. In the HS group, the interspike interval (ISI) was significantly shorter than that in NS controls. Apamin significantly shortened the ISI in NS controls but had less effect in the HS group. This data suggests that HS intake reduces SK currents, which contributes to increased PVN neuronal excitability at least in part through a decrease in spike frequency adaptation and may be a precursor to the development of salt-sensitive hypertension

Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

Background Rice Callus Suspension Culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/Purpose The anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study Design Three cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10-18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. Results RCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10-18 by HPLC identified 8 peaks. Cell viability assay with fractions 10-18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS versus both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5’-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. Conclusion RCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets

High Salt Intake Augments Excitability Of Pvn Neurons In Rats: Role Of The Endoplasmic Reticulum Ca\u3csup\u3e2+\u3c/sup\u3e Store

High salt (HS) intake sensitizes central autonomic circuitry leading to sympathoexcitation. However, its underlying mechanisms are not fully understood. We hypothesized that inhibition of PVN endoplasmic reticulum (ER) Ca2+ store function would augment PVN neuronal excitability and sympathetic nerve activity (SNA). We further hypothesized that a 2% (NaCl) HS diet for 5 weeks would reduce ER Ca2+ store function and increase excitability of PVN neurons with axon projections to the rostral ventrolateral medulla (PVN-RVLM) identified by retrograde label. PVN microinjection of the ER Ca2+ ATPase inhibitor thapsigargin (TG) increased SNA and mean arterial pressure (MAP) in a dose-dependent manner in rats with a normal salt (NS) diet (0.4%NaCl). In contrast, sympathoexcitatory responses to PVN TG were significantly (p \u3c 0.05) blunted in HS treated rats compared to NS treatment. In whole cell current-clamp recordings from PVN-RVLM neurons, graded current injections evoked graded increases in spike frequency. Maximum discharge was significantly augmented (p \u3c 0.05) by HS diet compared to NS group. Bath application of TG (0.5 μM) increased excitability of PVN-RVLM neurons in NS (p \u3c 0.05), yet had no significant effect in HS rats. Our data indicate that HS intake augments excitability of PVN-RVLM neurons. Inhibition of the ER Ca2+-ATPase and depletion of Ca2+ store likely plays a role in increasing PVN neuronal excitability, which may underlie the mechanisms of sympathoexcitation in rats with chronic HS intake

Gut microbiota and short chain fatty acids: Influence on the autonomic nervous system

Reaching across multiple fields of focus, spanning from periodontistry to gastroenterology to neurobiology to behavior, interest in the influence of the microbiome in human physiology and pathology has risen over the past few decades. Microbiota co-exist in and on humans forming an evolutionarily symbiotic biological unit, a halobiont, in which disruptions in the relationship can occur through genomic alterations and mutations [1]. The human microbiome consists of bacteria, viruses, fungi, and protozoans that contribute 450 times more genes to this relationship and slightly outnumber human host cells [2, 3]. The bacteria in the gastrointestinal (GI) tract are of the most interest and exist within five phyla: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Verrucomicrobia. Within the Verrucomicrobia an interesting bacterium has emerged, Akkermansia muciniphila, a mucus-degrading bacterium that influences intestinal permeability [3, 4]. The composition of individual microbiota communities depends on host lifestyle and genetics [1]. Often the Firmicutes-to-Bacteroidetes ratio is considered a method for measuring the health of a community [2, 3, 4, 5, 6, 7] but has not been fully validated in other studies suggesting the measurement of phyla in feces as a diagnostic tool may not be practical. Malfunction in the GI tract impacts other systems and leads the loss of physiological function, which disrupts the relationship between microbes and host. Gut microbial community disturbances caused by the host lifestyle (antibiotic use, food consumption, and lack of exercise), results in a decrease in diversity and have been linked to cardiovascular diseases, such as hypertension [2, 3, 5, 8], neurodegenerative Parkinson’s and Alzheimer’s diseases [4, 6], and even obesity [7]