Inhibitors of the 5-lipoxygenase pathway activate pannexin1 channels in macrophages via the thromboxane receptor.

A multitude of environmental signaling molecules influence monocyte and macrophage innate and adaptive immune responses, including ATP and prostanoids. Interestingly, purinergic (P2) and eicosanoid receptor signaling interact such that the activation of P2 receptors leads to prostanoid production, which can then interfere with P2Y-mediated macrophage migration. Recent studies suggest that blockade of 5-lipoxygenase (5-LOX) in macrophages can activate a permeation pathway involved in the influx of dye and the release of ATP. Here, we provide evidence that pannexin1 (Panx1) is a component of this pathway and present the intracellular signaling molecules linking the thromboxane (TP) receptor to Panx1-mediated dye influx and ATP release. Using pharmacological tools and transgenic mice deficient in Panx1, we show that two 5-LOX pathway inhibitors induce ATP release and influx of dye in a Panx1-dependent manner. Electrophysiological recordings performed in wild-type and Panx1-deficient macrophages confirmed that these 5-LOX pathway inhibitors activate currents characteristic of Panx1 channels. We found that the mechanism by which Panx1 channels are activated under this condition involves activation of the TP receptor that is mediated by the cAMP/PKA pathway. This is to our knowledge the first evidence for the involvement of Panx1 in the TP receptor signaling pathway. Future studies aimed to clarify the contribution of this TP-Panx1 signaling network to macrophage immune responses are likely to be important for targeting inflammatory and autoimmune diseases.

Promises and pitfalls of a Pannexin1 transgenic mouse line.

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1(tm1a(KOMP)Wtsi). Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1(f/f) mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.

Contribution of pannexin1 to experimental autoimmune encephalomyelitis.

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.

Targeting pannexin1 improves seizure outcome.

Imbalance of the excitatory neurotransmitter glutamate and of the inhibitory neurotransmitter GABA is one of several causes of seizures. ATP has also been implicated in epilepsy. However, little is known about the mechanisms involved in the release of ATP from cells and the consequences of the altered ATP signaling during seizures. Pannexin1 (Panx1) is found in astrocytes and in neurons at high levels in the embryonic and young postnatal brain, declining in adulthood. Panx1 forms large-conductance voltage sensitive plasma membrane channels permeable to ATP that are also activated by elevated extracellular K(+) and following P2 receptor stimulation. Based on these properties, we hypothesized that Panx1 channels may contribute to seizures by increasing the levels of extracellular ATP. Using pharmacological tools and two transgenic mice deficient for Panx1 we show here that interference with Panx1 ameliorates the outcome and shortens the duration of kainic acid-induced status epilepticus. These data thus indicate that the activation of Panx1 in juvenile mouse hippocampi contributes to neuronal hyperactivity in seizures.

Diabetic foot resulting in amputation: our experience.

Background: The aim of our study was to early diagnosis of diabetic foot so that the complications can be prevented, to control the systemic infection and prevent the complications, to study the effectiveness of regular dressing in diabetic foot so as to prevent the local spread of infection and the ulcer and to conclude that early diagnosis, care and proper meticulous treatment of diabetic foot can prevent amputation. Methods: The present study was prospective, observational and longitudinal. Protocol of the procedure was formed along with Performa, Patient Information Sheet, Informed Consent Form and approval from Ethical Committee. The present study was carried out in surgery department of C.U Shah medical college, Surendranagar; Gujarat state. The study was carried out from 1st August 2011 to 30th September 2013. A total of one hundred patients admitted in surgery ward with diabetes type 1 or 2 with ulcer on foot having grade 1 or 2 of Wagner’s classification without any other co morbid condition. These patients undergo daily dressing with various dressing solutions according to their ulcer characteristics. All the patients given diet/oral hypoglycaemic drug/insulin for control of diabetes. Antibiotics given according to the infective status of the patients. Patients were either completely treated, went under skin grafting or ended up with amputation were recorded. Results: Of 100 cases studied, youngest patient was 32 years and oldest was 80 years of age. Highest number of cases was found in the age group 61-70 years (30%). Of the 100 cases studied in this series 36 (36%) patient were having Wagner’s class 1 ulcer and 64 (64%) patient having class 2 ulcers. Of 100 cases, various surgical treatment given to the patients according to the ulcer. In that 65(65%) debridement, 20 (20%) Incision & drainage, 10 (10%) STG, 5 (5%) fasciotomy. Most of the patients were undergone basic surgical procedure which is debridement on the 7th day follow up, out of 100 cases 70 patients came for follow up. Out of 70, all patients having healing ulcer. Out of 70 patients, 15(21.43%) patients were underwent STG on 15th day and other 55 (71.57%) patients having healing ulcer advised daily dressing with follow up after 1 week. Out of 30 patients, 3 (10%) patients underwent amputation on 7th day of follow up. On the 15th day new 5 (16.67%) patients underwent amputations, so total number of amputation done till date was 8 (26.67%). On 21st day, new 7 (23.34%) patients were underwent amputations and total number of amputations till date were 15 (50%). On 30th day, new 15 (50%) patients underwent amputations. Conclusions: Foot ulceration in diabetic patients is a resource consuming, disabling morbidity that often is the first step towards lower extremity amputation. Prevention is the best treatment.