Increased intracellular Cl- Concentrations in Pulmonary Arterial Myocytes Associated with Chronic Hypoxic Pulmonary Hypertension

Chloride channels play an important role in regulating smooth muscle contraction and proliferation, and contribute to the enhanced constriction of pulmonary arteries (PAs) in pulmonary hypertension (PH). The intracellular Cl- concentration ([Cl-]i), tightly regulated by various Cl- transporters, determines the driving force for Cl- conductance, thereby the functional outcome of Cl- channel activation. This study characterizes for the first time the expression profile of Cl- transporters/exchangers in PA smooth muscle and provides the first evidence that the intracellular Cl- homeostasis is altered in PA smooth muscle cells (PASMCs) associated with chronic hypoxic PH (CHPH). Quantitative RT-PCR revealed that the endothelium-denuded intralobar PA of rats expressed Slc12a gene family-encoded Na-K-2Cl cotransporter 1 (NKCC1), K-Cl cotransporters (KCC) 1, 3 and 4, and Slc4a gene family-encoded Na+-independent and Na +-dependent Cl-/HCO3- exchangers. Exposure of rats to chronic hypoxia (10% O2, 3 weeks) caused CHPH and selectively increased the expression of Cl--accumulating NKCC1 and reduced the Cl--extruding KCC4. The intracellular Cl- concentration ([Cl-]i) averaged at 45 mM and 47 mM in normoxic PASMCs as determined by fluorescent indicator MEQ and by gramicidin perforated patch clamp technique, respectively, The ([Cl-]i was increased by ~ 10 mM in PASMCs of rats with CHPH. Future studies are warranted to further establish the hypothesis that the altered intracellular Cl- homeostasis contributes to the pathogenesis of CHPH.

BKCa Channel Inhibition by Peripheral Nerve Injury Is Restored by the Xanthine Derivative KMUP-1 in Dorsal Root Ganglia

This study explored whether KMUP-1 improved chronic constriction injury (CCI)-induced BKCa current inhibition in dorsal root ganglion (DRG) neurons. Rats were randomly assigned to four groups: sham, sham + KMUP-1, CCI, and CCI + KMUP-1 (5 mg/kg/day, i.p.). DRG neuronal cells (L4–L6) were isolated on day 7 after CCI surgery. Perforated patch-clamp and inside-out recordings were used to monitor BKCa currents and channel activities, respectively, in the DRG neurons. Additionally, DRG neurons were immunostained with anti-NeuN, anti-NF200 and anti-BKCa. Real-time PCR was used to measure BKCa mRNA levels. In perforated patch-clamp recordings, CCI-mediated nerve injury inhibited BKCa currents in DRG neurons compared with the sham group, whereas KMUP-1 prevented this effect. CCI also decreased BKCa channel activity, which was recovered by KMUP-1 administration. Immunofluorescent staining further demonstrated that CCI reduced BKCa-channel proteins, and KMUP-1 reversed this. KMUP-1 also changed CCI-reduced BKCa mRNA levels. KMUP-1 prevented CCI-induced neuropathic pain and BKCa current inhibition in a peripheral nerve injury model, suggesting that KMUP-1 could be a potential agent for controlling neuropathic pain.

A234 EVALUATING THE EFFECT OF STOOL SUPERNATANTS FROM IBS PATIENTS AND HEALTHY CONTROLS ON THE EXCITABILITY OF DRG NEURONS

Background Abdominal pain is commonly described in chronic disorders such as irritable bowel syndrome (IBS), but the underlying mechanisms are currently unclear. The stool metabolomic and microbiota profiles of IBS and healthy patients have shown distinct differences. Additionally, IBS stool supernatants have previously been demonstrated to induce hypersensitivity of nociceptive nerves in the ex vivo mouse colon, suggesting that mediators in the stool can sensitize nociceptors. However, the effects of healthy control (HC) or IBS patient stool supernatants on the excitability of DRG neurons have not been clarified. Aims To evaluate the effect of HC and IBS supernatant on DRG neurons. Methods HC (n=8 patients) or IBS (n=10 patients) stool was collected, dissolved and homogenized with bicarbonate-buffered Krebs solution at 37°C in a 1/10 dilution. DRG neurons from C57BL/6 mice were dissociated and incubated overnight with HC or IBS supernatant in a Krebs dissolution. Changes in DRG neuronal excitability were recorded using perforated patch-clamp techniques to measure the rheobase (amount of current needed to elicit an action potential). The effect of the IBS and HC stool supernatants on the resting membrane potential (RMP) was also recorded. Results Overnight incubations with supernatant of HC stool diluted in Krebs solution (n=28 neurons) did not significantly decrease the rheobase compared to control neurons (n=22) (62.7 ± 3.9 pA vs 64.2 ± 2.7 pA). In a parallel experiment, we evaluated the effect of IBS stool supernatants diluted in Krebs (n=52 neurons) and found that they significantly decreased the rheobase compared to the supernatant of HC diluted in Krebs and control neurons (52.3 ± 2.3; p<0.05). The data were analyzed with a one-way ANOVA and Tukey’s test. Incubations with IBS supernatant decreased the RMP compared to HC supernatant (-42.6 ± 0.6 mV vs. -46.0 ± 0.9 mV; p<0.01), which was calculated with an unpaired t-test. Conclusions These findings suggest that mediators in IBS stool increase the excitability of DRG neurons compared to HC stool supernatant, and thus may contribute to pain signaling in IBS patients. Funding Agencies CIHR

Downregulation of M Current Is Coupled to Membrane Excitability in Sympathetic Neurons Before the Onset of Hypertension

Neurohumoral activation is an early hallmark of cardiovascular disease and contributes to the etiology of the pathophysiology. Stellectomy has reemerged as a positive therapeutic intervention to modify the progression of dysautonomia, although the biophysical properties underpinning abnormal activity of this ganglia are not fully understood in the initial stages of the disease. We investigated whether stellate ganglia neurons from prehypertensive SHRs (spontaneously hypertensive rats) are hyperactive and describe their electrophysiological phenotype guided by single-cell RNA sequencing, molecular biology, and perforated patch clamp to uncover the mechanism of abnormal excitability. We demonstrate the contribution of a plethora of ion channels, in particular inhibition of M current to stellate ganglia neuronal firing, and confirm the conservation of expression of key ion channel transcripts in human stellate ganglia. We show that hyperexcitability was curbed by M-current activators, nonselective sodium current blockers, or inhibition of Na v 1.1-1.3, Na v 1.6, or I NaP . We conclude that reduced activity of M current contributes significantly to abnormal firing of stellate neurons, which, in part, contributes to the hyperexcitability from rats that have a predisposition to hypertension. Targeting these channels could provide a therapeutic opportunity to minimize the consequences of excessive sympathetic activation.

NKCC-1 mediated Cl− uptake in immature CA3 pyramidal neurons is sufficient to compensate phasic GABAergic inputs

Activation of GABAA receptors causes in immature neurons a functionally relevant decrease in the intracellular Cl− concentration ([Cl−]i), a process termed ionic plasticity. Amount and duration of ionic plasticity depends on kinetic properties of [Cl−]i homeostasis. In order to characterize the capacity of Cl− accumulation and to quantify the effect of persistent GABAergic activity on [Cl−]i, we performed gramicidin-perforated patch-clamp recordings from CA3 pyramidal neurons of immature (postnatal day 4–7) rat hippocampal slices. These experiments revealed that inhibition of NKCC1 decreased [Cl−]i toward passive distribution with a time constant of 381 s. In contrast, active Cl− accumulation occurred with a time constant of 155 s, corresponding to a rate of 15.4 µM/s. Inhibition of phasic GABAergic activity had no significant effect on steady state [Cl−]i. Inhibition of tonic GABAergic currents induced a significant [Cl−]i increase by 1.6 mM, while activation of tonic extrasynaptic GABAA receptors with THIP significantly reduced [Cl−]i.. Simulations of neuronal [Cl−]i homeostasis supported the observation, that basal levels of synaptic GABAergic activation do not affect [Cl−]i. In summary, these results indicate that active Cl−-uptake in immature hippocampal neurons is sufficient to maintain stable [Cl−]i at basal levels of phasic and to some extent also to compensate tonic GABAergic activity.

Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach

Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the ‘added buffer’ approach (Neher and Augustine, 1992) with perforated patch-clamp recordings (Horn and Marty, 1988). Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

Antagonistic modulation of NPY/AgRP and POMC neurons in the arcuate nucleus by noradrenalin

In the arcuate nucleus of the hypothalamus (ARH) satiety signaling (anorexigenic) pro-opiomelanocortin (POMC)-expressing and hunger signaling (orexigenic) agouti-related peptide (AgRP)-expressing neurons are key components of the neuronal circuits that control food intake and energy homeostasis. Here, we assessed whether the catecholamine noradrenalin directly modulates the activity of these neurons in mice. Perforated patch clamp recordings showed that noradrenalin changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the opposing effect on these neurons is mediated by the activation of excitatory α1A - and β- adrenergic receptors in NPY/AgRP neurons, while POMC neurons are inhibited via α2A – adrenergic receptors. Thus, the coordinated differential modulation of the key hypothalamic neurons in control of energy homeostasis assigns noradrenalin an important role to promote feeding.