Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction

Nitric oxide (NO) diffuses as short‐lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO‐induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO‐sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO‐induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca²⁺‐free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca²⁺‐independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 337–346, 1999     

Effects of nitric oxide donors on Ca2+-dependent [14C]GABA release from brain synaptosomes: the role of SH-groups

Nitric oxide (NO) modulates processes of synaptic transmission at pre- and postsynaptic levels. In the present work we studied the mechanisms of action of NO on [gamma-14C]amino-n-butyric acid ([14C]GABA) release in rat cortical synaptosomes. NO donors--S-nitroso-L-cysteine and hydroxylamine (but not sodium nitroprusside)--inhibited the neurotransmitter efflux in a concentration range from 10 microM to 1 mM. Nitrosocysteine completely and selectively suppressed the Ca2+-dependent (vesicular) [14C]GABA release, while not affecting the Ca2+-independent component of the [14C]GABA transport. The influence of NO donors was not related to activation of guanylyl cyclase, since the membrane-permeable cGMP analog dibutyryl-cGMP did not mimic and the guanylyl cyclase inhibitor methylene blue did not change the NO effects. In contrast, the membrane-permeable SH-reagent N-ethylmaleimide (NEM) resembled the effects of NO donors on the Ca2+-dependent [14C]GABA release. The degree of inhibition of the release by nitrosocysteine, hydroxylamine, and NEM correlated with their ability to oxidize intra-synaptosomal SH-groups. These data suggest that synaptosomal sulfhydryl groups are the target for NO action at the presynaptic level. The NO-induced oxidation of thiols may be involved in physiological and, especially, pathological effects of nitric oxide in the central nervous system.     

Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO-evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics of cGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP-stimulated PDE (PDE 2). Analysis of NO-induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.     

Endogenous presynaptic nitric oxide supports an anterograde signaling in the central nervous system

The source size and density determine the extent of nitric oxide (NO) diffusion which critically influences NO signaling. In the brain, NO released from postsynaptic somas following NMDA-mediated activation of neuronal nitric oxide synthase (nNOS) retrogradely affects smaller presynaptic targets. By contrast, in guinea pig trigeminal motor nucleus (TMN), NO is produced presynaptically by tiny and disperse nNOS-containing terminals that innervate large nNOS-negative motoneurons expressing the soluble guanylyl-cyclase (sGC); consequently, it is uncertain whether endogenous NO supports an anterograde signaling between pre-motor terminals and postsynaptic trigeminal motoneurons. In retrogradely labeled motoneurons, we indirectly monitored NO using triazolofluorescein (DAF-2T) fluorescence, and evaluated sGC activity by confocal cGMP immunofluorescence. Multiple fibers stimulation enhanced NO content and cGMP immunofluorescence into numerous nNOS-negative motoneurons; NOS inhibitors prevented depolarization-induced effects, whereas NO donors mimicked them. Enhance of cGMP immunofluorescence required extracellular Ca(2+), a nNOS-physiological activator, and was prevented by inhibiting sGC, silencing neuronal activity or impeding NO diffusion. In conclusion, NO released presynaptically from multiple cooperative tiny fibers attains concentrations sufficient to activate sGC in many motoneurons despite of the low source/target size ratio and source dispersion; thus, endogenous NO is an effective anterograde neuromodulator. By adjusting nNOS activation, presynaptic Ca(2+) might modulate the NO diffusion field in the TMN.     

Modulation of neuronal nitric oxide release by soluble guanylyl cyclase in guinea pig colon

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Nitric oxide decreases pacemaker activity in lymphatic vessels of guinea pig mesentery

Intracellular microelectrode recordings were used to determine whether nitric oxide (NO), affects the pacemaker events that initiate vasomotion in lymphatic vessels of the guinea pig mesentery. This pacemaker activity is recorded as spontaneous transient depolarizations (STDs) and is likely to arise through synchronized Ca2+ release from intracellular stores. We show here that acetylcholine-induced endothelium-derived NO and exogenous NO released by sodium nitroprusside (SNP; 100 microM) and DEA-NONOate (500 microM) reduced the frequency and amplitude of STDs. This inhibition of STD frequency and amplitude was independent of the NO-induced hyperpolarization of the smooth muscle. The SNP-induced inhibition of STD frequency and amplitude was abolished during superfusion with the soluble guanylyl cyclase inhibitor ODQ (10 microM) and was diminished in the presence of cGMP and cAMP-dependent protein kinase inhibitors. The data are consistent with the hypothesis that NO inhibits vasomotion primarily by production of cGMP and activation of both cGMP- and cAMP-dependent protein kinases, which reduce the size and frequency of STDs, probably by acting on the underlying synchronized Ca2+ release from intracellular stores.     

Nitric oxide regulates the levels of cGMP accumulation in the cricket brain

Cricket brains were incubated in a saline containing nitric oxide (NO)-donor and phosphodiesterase inhibitor IBMX, which could activate soluble guanylate cyclase (sGC) to increase cGMP levels in the targets of NO. The increase of cGMP was detected by immunohistochemistry and enzyme linked immunosorbent assay. NO-induced cGMP immunohistochemistry revealed that many cell bodies of cricket brain showed cGMP immunoreactivity when preparations were treated with a saline containing 10 mM NO-donor SNP and phosphodiesterase inhibitor IBMX, but only a few cell bodies showed immunoreactivity when preparations were incubated without NO-donor. The concentration of cGMP in cricket brains were then measured by using cGMP-specific enzyme linked immunosorbent assay. Cricket brains were treated with a saline containing 1 microM of NO-donor NOR3 and 1 mM IBMX. The cGMP levels in the brain were increased about 75% compared to control preparations that was treated with a cricket saline containing IBMX. The level of cGMP decreased about 40% when preparations were incubated NOR3 saline containing sGC inhibitor ODQ. These results indicate that NO activates sGC and increases the levels of cGMP in particular neurons of the cricket brain and that the level of cGMP would be kept a particular level, which might regulate synaptic efficacy in the neurotransmission.     

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.     

STOP and GO with NO: nitric oxide as a regulator of cell motility in simple brains

During the formation of the brain, neuronal cell migration and neurite extension are controlled by extracellular guidance cues. Here, I discuss experiments showing that the messenger nitric oxide (NO) is an additional regulator of cell motility. NO is a membrane permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cells types in invertebrate models such as molluscs, insects and the medicinal leech provides insight how NO and cyclic nucleotides affect the wiring of nervous systems by regulating cell and growth-cone motility. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO signalling orchestrates neurite outgrowth and filopodial dynamics, cell migration of enteric neurons, glial migration and axonogenesis of pioneer fibers. Cultured insect embryos are accessible model systems in which cellular mechanisms of NO-induced cytoskeletal reorganizations can be analyzed in natural settings. Finally, I will outline some indications that NO may also regulate cell motility in the developing and regenerating vertebrate nervous system.     

cGMP-dependent and -independent angiogenesis-related properties of nitric oxide

Nitric oxide exerts a stimulatory role during postnatal angiogenesis. Although soluble guanylyl cyclase (sGC) mediates many of the effects of nitric oxide (NO) in the vascular system, the contribution of cGMP-dependent vs cGMP-independent pathways in NO-induced angiogenesis remains unclear. Herein, we determined the effects of a NO donor (sodium nitroprusside; SNP) and a NO-independent sGC activator (BAY 41-2272) in the growth and migration of rat aortic endothelial cells (RAEC). RAEC lack enzymatically active sGC as suggested by their inability to accumulate cGMP upon exposure to SNP. However, treatment of RAEC with SNP promoted a modest increase in their proliferation and migration that was dependent on extracellular signal regulated kinase1/2 activation. Moreover, when RAEC were exposed to vascular endothelial growth factor we observed an increase in migration that was inhibited by NO synthase, but not sGC, inhibition. Infection of cells with adenoviruses containing sGC greatly increased the efficacy of SNP as a mitogenic and migratory stimulus. We conclude that NO is capable of stimulating EC proliferation and mobility in the absence of sGC; however, increased intracellular levels of cGMP following sGC activation greatly amplify the angiogenic potential of NO.     

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.     

NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance Ca2+-activated K+ (BKCa) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884-1.3956 microl.min(-1).mmHg(-1)) over baseline (0.2373-0.5220 microl.min(-1).mmHg(-1)) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the BKCa channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and K+ eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility.     

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.     

Pharmacological approaches to nitric oxide signalling during neural development of locusts and other model insects

A novel aspect of cellular signalling during the formation of the nervous system is the involvement of the messenger molecule nitric oxide (NO), which has been discovered in the mammalian vascular system as mediator of smooth muscle relaxation. NO is a membrane-permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cell types in model insects such as Locusta, Schistocerca, Acheta, Manduca, and Drosophila shows that the NO/cGMP pathway is required for the stabilization of photoreceptor growth cones at the start of synaptic assembly in the optic lobe, for regulation of cell proliferation, and for correct outgrowth of pioneer neurons. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO, and potentially also another atypical messenger, carbon monoxide (CO), orchestrate cell migration of enteric neurons. Cultured insect embryos are accessible model systems in which the molecular pathways linking cytoskeletal rearrangement to directed cell movements can be analyzed in natural settings. Based on the results obtained from the insect models, I discuss current evidence for NO and cGMP as essential signalling molecules for the development of vertebrate brains.     

Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.     

Nitric oxide decreases endothelin-1 secretion through the activation of soluble guanylate cyclase

The use of exogenous nitric oxide (NO) has been shown to alter the regulation of other endothelially derived mediators of vascular tone, such as endothelin-1 (ET-1). However, the interaction between NO and ET-1 appears to be complex and remains incompletely understood. One of the major actions of NO is the activation of soluble guanylate cyclase (sGC) with the subsequent generation of cGMP. Therefore, we undertook this study to test the hypothesis that NO regulates ET-1 production via the activation of the sGC/cGMP pathway. The results obtained indicated that the exposure of primary cultures of 4-wk-old ovine pulmonary arterial endothelial cells (4-wk PAECs) to the long-acting NO donor DETA NONOate induced both a dose- and time-dependent decrease in secreted ET-1. This decrease in ET-1 secretion occurred in the absence of changes in endothelin-converting enzyme-1 or sGC expression but in conjunction with a decrease in prepro-ET-1 mRNA. The changes in ET-1 release were inversely proportional to the cellular cGMP content. Furthermore, the NO-independent activator of sGC, YC-1, or treatment with a cGMP analog also produced significant decreases in ET-1 secretion. Conversely, pretreatment with the sGC inhibitor ODQ blocked the NO-induced decrease in ET-1. Therefore, we conclude that exposure of 4-wk PAECs to exogenous NO decreases secreted ET-1 resulting from the activation of sGC and increased cGMP generation.     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。     

NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells.

Exposure of RINm5F cells to interleukin-1beta and to several chemical NO donors such as sodium nitroprusside (SNP), SIN-1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl-2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO-induced apoptosis in the pancreatic beta-cell line is independent of acute activation of the cGMP pathway.