Ronald R. Fiscus

Role of cyclic GMP in the regulation of neuronal calcium and survival by secreted forms of β-amyloid precursor

Abstract: The Alzheimers disease (AD) β‐amyloid precursor proteins (βAPPs) are large membrane‐spanning proteins that give rise to the βA4 peptide deposited in AD amyloid plaques. βAPPs can also yield soluble forms (APPss) that are potently neuroprotective against glucose deprivation and glutamate toxicity, perhaps through their ability to lower the intraneuronal calcium concentration ([Ca2+]i). We have investigated the mechanism through which APPss exert these effects on cultured hippocampal neurons. The ability of APPss to lower rapidly [Ca2+]i was mimicked by membrane‐permeable analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as agents that elevate endogenous levels of these cyclic nucleotides. However, only cGMP content was increased by APPs treatment, and specific inhibition of cGMP‐dependent protein kinase (but not cAMP‐dependent kinase) blocked the activity of APPss. A membrane‐permeable analogue of cGMP (8‐bromo‐cGMP) also mimicked the ability of APPss to attenuate the elevation of [Ca2+]i by glutamate, apparently through inhibition of NMDA receptor activity. In addition, 8‐bromo‐cGMP afforded protection against glucose deprivation and glutamate toxicity, and the protection by APPss against glucose deprivation was blocked by an inhibitor of cGMP‐dependent kinase. Together, these data suggest that APPss mediate their [Ca2+]i‐lowering and excitoprotective effects on target neurons through increases in cGMP levels.

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.

Role of cyclic-GMP in relaxations of vascular smooth muscle.

Relaxation of rat aorta segments with sodium litroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) ac-:cumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiogcaphy. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachi-donic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.

Atrial natriuretic factors stimulate accumulation and efflux of cyclic GMP in C6-2B rat glioma and PC12 rat pheochromocytoma cell cultures.

Abstract: Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6–2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates paniculate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6–2B and PC12 cells with the rank order of potency being atriopeptin HI = atriopeptin II ≥ human atrial natriuretic polypeptide ≥ atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6–2B cells fourfold and in PC12 cells 12‐fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6–2B cells (47‐fold) and PC12 cells (12‐fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (250 μM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells. Although the function of cyclic GMP in neural tissue is at present unclear, the cyclic GMP response observed in C6–2B and PC12 cells may provide important clues concerning the molecular mechanisms of action of ANF in the central and peripheral nervous systems. Since a significant amount of the generated cyclic GMP is released from these cells in response to ANF, extracellular as well as intracellular cyclic GMP may be important in mediating the actions of ANF in neural tissues.

Effects of atriopeptin on particulate guanylate cyclase from rat adrenal

Atriopeptin II activated particulate guanylate cyclase 5-10-fold in a concentration- and time-dependent fashion in crude membranes obtained from homogenates of rat adrenal cortex or medulla. Similar effects were observed with other atriopeptin analogs. Soluble guanylate cyclase and adenylate cyclase in these preparations were not activated. Accumulation of cyclic GMP in minces of adrenal cortex or medulla was increased 6-8-fold due to atriopeptin II activation of particulate guanylate cyclase. Several thiol-reactive agents blocked the activation of particulate guanylate cyclase, suggesting that free thiol groups on membrane proteins may be important in atriopeptin receptor-guanylate cyclase coupling.

[14] cGMP-dependent protein kinase activation in intact tissues

Publisher Summary Unlike cAMP-kinase that dissociates into regulatory and catalytic subunits upon activation with cAMP, cGMP-kinase remains as a holoenzyme upon binding of cGMP. An increase in the activity of purified cGMP-kinase is observed after adding cGMP in sufficient amounts to occupy the cGMP-binding sites. In intact cells, the enzyme is probably activated in a similar manner. Ideally, when measuring the activation of cyclic nucleotide-dependent protein kinases in intact tissues, the equilibrium should not be disturbed by the procedures of tissue extraction or assaying. In the case of cAMP-kinase, the fact that the regulatory and catalytic subunits dissociate upon activation helps to preserve the activation state of the enzyme when it is diluted into homogenizing buffer and further diluted into a kinase reaction solution for assaying. In contrast, activated cGMP-kinase, under similar conditions, begins to lose activity, 4 probably because of the rapid dissociation of cGMP from the enzyme as a result of dilution. Therefore, special procedures are needed to minimize cGMP dissociation.

Nω-nitro-L-arginine blocks the second phase but not the first phase of the endothelium-dependent relaxations induced by substance P in isolated rings of pig carotid artery

Summary: Endothelium‐dependent relaxations can be evoked by a variety of stimuli, among them substance P (SP), which is found in sensory nerve fibers supplying the adventitia‐media junction of most muscular arteries. This study determined the role of endothelium‐derived nitric oxide as a mediator of endothelium‐dependent relaxations to SP in isolated rings of the pig carotid artery suspended in organ chambers for isometric tension recording. SP (10−12‐10−7 M) caused concentration‐dependent relaxations of arteries precontracted with norepinephrine (10−7 M). The relaxations were characterized by a partially transient relaxation (phase 1) and a sustained relaxation of the artery (phase 2). The inhibitor of nitric oxide formation, N&ohgr;‐nitro‐L‐arginine (l‐NNA) methyl ester caused a gradual increase in tension, the phase I response at 3 × 10−10 to 3 × 10−7 M SP was shifted to the right, but the maximal relaxation was comparable in the presence of l‐NNA. However, the sustained relaxation after addition of substance P (phase II) was lost and tension in the presence of l‐NNA returned to a level above that induced by l‐NNA and norepinephrine (10−9 M). These results suggest that the endothelium‐dependent relaxations to SP, particularly the prolonged relaxation (phase II), are due to de novo synthesis of nitric oxide and hence fully abolished by a specific inhibitor.

Vasorelaxations induced by calcitonin gene-related peptide, vasoactive intestinal peptide, and acetylcholine in aortic rings of endothelial and inducible nitric oxide synthase-knockout mice.

The objective of this study was to determine if vasorelaxant responses to calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and acetylcholine are altered in aortic rings of mice lacking genetic expression of endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) genes (i.e., eNOS- and iNOS-knockout mice) as compared with control (wild-type) mice. Aortic rings from eNOS-knockout (eNOS (−/−)) mice did not relax in response to acetylcholine, thereby confirming previous reports. Aortic rings from iNOS-knockout (iNOS (−/−)) mice relaxed in response to acetylcholine in an endothelium-dependent manner. However, maximum relaxations in endothelium-intact rings were significantly (p < 0.05) larger than in control mice (85.3 ± 3.1% in iNOS (−/−) mice vs. 67.9 ± 5.6% in controls). CGRP caused concentration-dependent relaxations in aortas of all three types of mice: control mice, iNOS (−/−) mice, and eNOS (−/−) mice. Vasorelaxant responses to CGRP in control and iNOS (−/−) mice had identical relationships; both were partially dependent on endothelium. In eNOS (−/−) mice, dose-response curves of CGRP in endothelium-intact and endothelium-denuded rings were not significantly different, indicating loss of the partial dependence on endothelium. The vasorelaxant responses to VIP were completely dependent on endothelium in control and iNOS (−/−) mice. Maximum relaxations to VIP in iNOS (−/−) mice (77.4 ± 2.7%) were significantly greater than in control mice (64.0 ± 5.5%). Vasorelaxant responses to VIP in eNOS (−/−) aortic rings were also endothelium-dependent, but responses were greatly attenuated compared with wild-type mice. Relaxations induced by VIP (1 × 10−7M) in endothelium-intact aortic rings of eNOS (−/−) mice and control mice were 18.3 ± 5.4% and 64.0 ± 5.5%, respectively. These findings demonstrated that, in eNOS (−/−) mice, aortic vasorelaxant responses to CGRP were fully present but no longer dependent on the endothelium, and responses to VIP were greatly attenuated compared with control and responses to acetylcholine were abolished. In iNOS (−/−) mice, aortic vasorelaxant responses to VIP and acetylcholine were significantly greater than wild-type control, suggesting that induction of iNOS may have attenuated vascular responses to VIP and acetylcholine in wild-type controls.

Regulation of cyclic GMP synthesis and the interactions with calcium.

The formation of cyclic GMP (cGMP) by guahylate cyclase, and the properties of the soluble and particulate isoenzymes, are reviewed. Regulation of guanylate cyclase and cGMP accumulation in intestinal mucosa and vascular smooth muscle with and without, endothelium are summarized. The effects of E. coli heatstable enterotoxin, nitrovasodilators, endothelium-dependent vasodilators, and atrial natriuretic factors in these systems are discussed. A potential role for free radicals. Ca, and unsaturated fatty acids in cGMP synthesis is reviewed. These tissue systems (intestinal mucosa and vascular smooth muscle with or without intact endothelium) are presented as model systems and as examples of the regulation of cGMP accumulation in a single cell type by peptides, drugs, and cell-cell interactions that are required to generate cGMP with a hormone or drug.

Regulation of particulate guanylate cyclase by atriopeptins: relation between peptide structure, receptor binding, and enzyme kinetics

Structural analogs of atriopeptins (APs) were compared for their ability to activate particulate guanylate cyclase and bind to specific receptors in rat adrenal membranes. All analogs tested increase Vmax without altering the concentration of substrate required for half-maximum activity or the positive coperativity exhibited by the enzyme. Maximum velocities (pmoles of cGMP produced per min per mg protein) achieved in the absence and presence of APs were 128.3 +/- 6.6 and 283.8 +/- 20.6 using Mn2+-GTP, and 53.7 +/- 3.7 and 149.9 +/- 7.6 using Mg2+-GTP as the substrate, respectively. Although all APs were equally efficacious in activating the enzyme, their rank potency was ANF (8-33) = AP III = AP II greater than AP I when either divalent cation was used as the cofactor. The EC50 for activation of guanylate cyclase by AP I was about 10(-7) M, while that for the other peptides was about 10(-8) M, using either divalent cation cofactor. 125I-labeled ANF bound to rat adrenal membranes with a KD of 5.10(-10) M. Although all APs were equally efficacious in competing with labeled ANF for receptor binding, their rank potency was identical to that for enzyme activation. The Ki for AP I was about 10(-8) M, while that for the other peptides was about 10(-10) M. These data suggest that the carboxy terminal Phe-Arg present in the AP analogs except AP I and critical for biological and receptor-binding activity are also important in coupling receptor-ligand interaction with guanylate cyclase activation. The correlation between the rank order potency for receptor binding, enzyme activation, and the reported physiological actions of APs support the suggestion of a functional coupling between these proteins.