Mechanical allodynia is a serious complication of painful diabetic neuropathy (PDN) with limited treatment options. The transient receptor potential canonical 5 (TRPC5) channel is a promising target in pain; however, its role in painful diabetic neuropathy has not yet been elucidated. In this study, we have investigated the role of TRPC5 channels using BTD [N-{3-(adamantan-2-yloxy)-propyl}-3-(6-methyl-1,1-dioxo-2H-1λ⁶,2,4-benzothiadiazin-3-yl)-propanamide)],a potent TRPC5 activator and HC070, as TRPC5 channel inhibitor in rat model of PDN. In this study, streptozotocin was used to induce diabetes in male Sprague-Dawley rats. The alterations in mechanical and thermal pain thresholds, nerve functional deficits in diabetic animals were assessed by various behavioral and functional parameters.TRPC5 involvement was investigated by treating neuropathic rats with BTD, TRPC5 channel activator (1 and 3 mg/kg, i.p. for 14 days) and HC070, a TRPC5 channel inhibitor (1 and 3 mg/kg). BTD and HC070 effects in pain reduction were assessed by western blotting, estimating oxidative stress and inflammatory markers in the lumbar spinal cord. BTD treatment (3 mg/kg, i.p.) once daily for 14 days ameliorated mechanical allodynia but not thermal hyposensation or nerve functional deficit in diabetic neuropathic rats. BTD treatment down-regulated TRPC5 expression by increasing the activity of protein kinase C. It also subsequently down-regulated the downstream pain markers (CAMKII, ERK) in the spinal cord. Additionally, a decrease in inflammatory cytokines (TNF-α, IL-6) also demonstrated BTD's potent anti-inflammatory properties in reducing mechanical allodynia. On the other hand, HC070 did not exert any beneficial effects on behavioural and nerve functional parameters. The study concludes that BTD ameliorated mechanical allodynia in a rat model of painful diabetic neuropathy not only through modulation of the TRPC5-CAMKII-ERK pathway but also through its anti-inflammatory and anti-apoptotic properties. Overall, BTD is a promising therapeutic molecule in the treatment of mechanical allodynia in painful diabetic neuropathy.

2023-01-01·Biological & pharmaceutical bulletin

Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca²⁺ Influx in Lung Microvascular Endothelial Cells.

Article

作者: Dong, Mingjie ; Zhang, Rui ; Zhou, Yan

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca²⁺ ([Ca²⁺]_i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca²⁺-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca²⁺]_i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca²⁺ imaging. We found that histamine increased [Ca²⁺]_i was maintained at a static elevated level after a transient peak. The elevated Ca²⁺ plateau was vanished when extracellular Ca²⁺ was removed, indicating Ca²⁺ influx from extracellular mediated the histamine-induced Ca²⁺ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca²⁺ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca²⁺ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

2022-12-01·Molecular metabolism

CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity

Article

作者: Zweifel, Larry S ; Hunker, Avery C ; Bosch, Martha A ; Rønnekleiv, Oline K ; Qiu, Jian ; Kelly, Martin J ; Stincic, Todd L

OBJECTIVE:

Proopiomelanocortin (POMC) neurons are the key anorexigenic hypothalamic neuron for integrating metabolic cues to generate the appropriate output for maintaining energy homeostasis and express the requisite channels as a perfect synaptic integrator in this role. Similar to the metabolic hormones leptin and insulin, glutamate also excites POMC neurons via group I metabotropic glutamate receptors (mGluR1 and 5, mGluR1/5) that activate Transient Receptor Potential Canonical (TRPC 5) Channels to cause depolarization. A key modulator of TRPC 5 channel activity is stromal interaction molecule 1 (STIM1), which is involved in recruitment of TRPC 5 channels from receptor-operated to store-operated calcium entry following depletion of calcium from the endoplasmic reticulum.

METHODS:

We used a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas) and a single guide RNA (sgRNA) to mutate Stim1 in POMC^Cre neurons in male mice, verified by qPCR of Stim1 mRNA expression in single POMC neurons. Whole-cell patch clamp experiments were conducted to validate the effects of Stim1 mutagenesis. Body weight and food intake were measured in male mice to assess disruptions in energy balance.

RESULTS:

Reduced Stim1 expression augmented the efficacy of the mGluR1/5 agonist 3, 5-Dihydroxyphenylglycine (DHPG) to depolarize POMC neurons via a Gα_q-coupled signaling pathway, which is an essential part of excitatory glutamatergic input in regulating energy homeostasis. The TRPC 5 channel blockers HC070 and Pico145 antagonized the excitatory effects of DHPG. As proof of principle, mutagenesis of Stim1 in POMC neurons reduced food intake, attenuated weight gain, reduced body fat and fat pad mass in mice fed a high fat diet.

CONCLUSIONS:

Using CRISPR technology we have uncovered a critical role of STIM1 in modulating glutamatergic activation of TRPC 5 channels in POMC neurons, which ultimately is important for maintaining energy balance.

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适应症	最高研发状态	国家/地区	公司	日期
焦虑症	临床1期	-	C.H. Boehringer Sohn AG & Co. KG	-
焦虑症	临床1期	-	Hydra Biosciences, Inc.	-
抑郁症	临床1期	-	Hydra Biosciences, Inc.	-
抑郁症	临床1期	-	C.H. Boehringer Sohn AG & Co. KG	-