Zhong Li, Gleason Evanna L
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, United States.
Front Cell Neurosci. 2021 Aug 11;15:726605. doi: 10.3389/fncel.2021.726605. eCollection 2021.
The strength and sign of synapses involving ionotropic GABA and glycine receptors are dependent upon the Cl gradient. We have shown that nitric oxide (NO) elicits the release of Cl from internal acidic stores in retinal amacrine cells (ACs); temporarily altering the Cl gradient and the strength or even sign of incoming GABAergic or glycinergic synapses. The underlying mechanism for this effect of NO requires the cystic fibrosis transmembrane regulator (CFTR) but the link between NO and CFTR activation has not been determined. Here, we test the hypothesis that NO-dependent Ca elevations activate the Ca-dependent adenylate cyclase 1 (AdC1) leading to activation of protein kinase A (PKA) whose activity is known to open the CFTR channel. Using the reversal potential of GABA-gated currents to monitor cytosolic Cl, we established the requirement for Ca elevations. Inhibitors of AdC1 suppressed the NO-dependent increases in cytosolic Cl whereas inhibitors of other AdC subtypes were ineffective suggesting that AdC1 is involved. Inhibition of PKA also suppressed the action of NO. To address the sufficiency of this pathway in linking NO to elevations in cytosolic Cl, GABA-gated currents were measured under internal and external zero Cl conditions to isolate the internal Cl store. Activators of the cAMP pathway were less effective than NO in producing GABA-gated currents. However, coupling the cAMP pathway activators with the release of Ca from stores produced GABA-gated currents indistinguishable from those stimulated with NO. Together, these results demonstrate that cytosolic Ca links NO to the activation of CFTR and the elevation of cytosolic Cl.
涉及离子型γ-氨基丁酸（GABA）和甘氨酸受体的突触强度及信号取决于氯离子梯度。我们已经表明，一氧化氮（NO）可促使视网膜无长突细胞（ACs）内酸性储存库释放氯离子；暂时改变氯离子梯度以及传入的GABA能或甘氨酸能突触的强度甚至信号。NO产生这种效应的潜在机制需要囊性纤维化跨膜调节因子（CFTR），但NO与CFTR激活之间的联系尚未确定。在此，我们检验这样一个假说：依赖NO的钙离子升高会激活钙依赖性腺苷酸环化酶1（AdC1），从而导致蛋白激酶A（PKA）激活，已知PKA的活性可打开CFTR通道。利用GABA门控电流的反转电位来监测胞质氯离子，我们确定了钙离子升高的必要性。AdC1抑制剂可抑制依赖NO的胞质氯离子增加，而其他AdC亚型的抑制剂则无效，这表明AdC1参与其中。抑制PKA也可抑制NO的作用。为了验证该信号通路在将NO与胞质氯离子升高联系起来方面是否充分，我们在细胞内和细胞外零氯离子条件下测量了GABA门控电流，以分离细胞内氯离子储存库。环磷酸腺苷（cAMP）信号通路激活剂在产生GABA门控电流方面不如NO有效。然而，将cAMP信号通路激活剂与储存库中钙离子的释放相结合所产生的GABA门控电流与用NO刺激产生的电流无法区分。总之，这些结果表明，胞质钙离子将NO与CFTR的激活以及胞质氯离子的升高联系起来。
