Changes In Cyclic GMP Research Articles

Mitogen-activated Protein Kinase p38 Regulates the Wnt/Cyclic GMP/Ca2+ Non-canonical Pathway

The non-canonical Wnt/cyclic GMP/Ca(2+)/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/G alpha t2/PDE6 triad activated in response to Wnt5a, we observed a strong activation of the mitogen-activated protein kinase p38 in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation was rapid, sensitive to pertussis toxin, to siRNA against either G alpha t2 or p38 alpha, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca(2+). Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway.

Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation.

Natriuretic peptides (NP) were first identified in animals where they play a role in the regulation of salt and water balance. This regulation is partly mediated by intracellular changes in cyclic GMP (cGMP). NP immunoanalogues occur in many plants and have been isolated, with two NP encoding genes characterised in Arabidopsis thaliana L. (AtPNP-A and AtPNP-B). Part of AtPNP-A contains the region with homology to human atrial (A)NP. We report here on the effects of recombinant AtPNP-A and smaller synthetic peptides within the ANP-homologous region with a view to identifying the biologically active domain of the molecule. Furthermore, we investigated interactions between AtPNP-A and the hormone, abscisic acid (ABA). ABA does not significantly affect Arabidopsis mesophyll protoplast volume regulation, whereas AtPNP-A and synthetic peptides promote water uptake into the protoplasts causing swelling. This effect is promoted by the membrane permeable cGMP analogue, 8-Br-cGMP, and inhibited by guanylate cyclase inhibitors indicating that increases in cGMP are an essential component of the plant natriuretic peptides (PNP) signalling cascade. ABA does not induce cGMP transients and does not affect AtPNP-A dependent cGMP increases, hence the two regulators differ in their second messenger signatures. Interestingly, AtPNP-A significantly delays and reduces the extent of ABA stimulated stomatal closure that is also based on cell volume regulation. We conclude that a complex interplay between observed PNP effects (stomatal opening and protoplast swelling) and ABA is likely to be cell type specific.

Age-related changes in cyclic GMP and PKG-stimulated cerebellar Na,K-ATPase activity

Energy deficiency and dysfunction of the Na,K-ATPase are common consequences of many pathological insults. Glutamate through cyclic GMP and cyclic GMP-dependent protein kinase (PKG) has been shown to stimulate α 2/3-Na,K-ATPase activity in the central nervous system. Thus, a slight impairment of this pathway may amplify the disruption of ion homeostasis in the presence of a non-lethal insult. We investigate the effect of aging (4, 12 and 24 months) on the glutamate-cyclic GMP–PKG modulation of α 1, α 2/3-Na,K-ATPase activity in rat cerebellum and the stimulation of the glutamate-cyclic GMP–PKG pathway at different levels. Cyclic GMP levels and α 2/3-Na,K-ATPase activity were progressively decreased from 4 and 24 month-old animals. However, PKG basal activity was reduced between 4 and 12 months, and no additional change was observed at 24 months. The ability of 8-Br-cyclic GMP to stimulate PKG activity was only reduced between 12 and 24 months. Moreover, glutamate or 8-Br-cyclic GMP promoted a smaller increase of α 2/3-Na,K-ATPase activity at 24 months, when compared to 4 and 12 months. In spite of the age-related reduced basal levels of cyclic GMP, the production induced by CO or NO was not age-related. Finally, inhibition of PKG activation by KT5823 revealed a lower sensitivity of the enzyme at the older age. Taken together, these data show that basal age-related decline in sodium pump activity is a consequence of changes in different steps of the cyclic GMP–PKG pathway. On the other hand, age-related reduction in glutamate positive modulation of cerebellar α 2/3-Na,K-ATPase is linked to a defective PKG signaling pathway.

Potentiation of tumor necrosis factor-α expression by YC-1 in alveolar macrophages through a cyclic GMP-independent pathway

Using cultured rat alveolar NR 8383 macrophages, this study investigated the effect of YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole], a soluble guanylyl cyclase (sGC) activator, on the production of tumor necrosis factor-α (TNFα). YC-1 enhanced lipopolysaccharide and interferon-γ (LPS/IFNγ)-induced TNFα formation in a concentration- and time-dependent fashion. YC-1 also caused an increasing effect on the TNFα mRNA level, suggesting that the transcriptional process was involved. However, further studies suggested that cyclic GMP did not mediate the potentiation of YC-1 on TNFα release, because (a) the sGC inhibitor and the protein kinase G inhibitor failed to block the effect; and (b) the cyclic GMP analogues, on the contrary, concentration-dependently diminished LPS/IFNγ-induced TNFα synthesis. In agreement with this finding, YC-1 produced changes in cell function but no changes in cyclic GMP and cyclic AMP levels or sGC activity. Pretreatment of the cells with cyclooxygenase inhibitors, a p38 mitogen-activated protein kinase inhibitor, a mitogen-activated protein kinase kinase (MEK) inhibitor, and a tyrosine kinase inhibitor did not attenuate the potentiation of TNFα release by YC-1. Cycloheximide prevented the YC-1-enhanced TNFα formation, implying that new protein synthesis was required. Interestingly, protein kinase C inhibitors enhanced the potentiation of YC-1 to a greater extent. Nevertheless, a protein kinase C activator, phorbol 12-myristate 13-acetate, failed to suppress the potentiation of TNFα production by YC-1. In summary, potentiation of TNFα release by YC-1 in LPS/IFNγ-activated alveolar macrophages is an additional mode of action of this compound that is independent of the elevation of cyclic GMP. Thus, caution needs to be used in attributing the YC-1-mediated response to the activation of sGC.

Relationship between the hemodynamic and platelet cyclic GMP changes induced by nitrates in essential hypertensives

Relationship between the hemodynamic and platelet cyclic GMP changes induced by nitrates in essential hypertensives

The subtype 2 of angiotensin II receptors and pressure-natriuresis in adult rat kidneys.

The present work examined the effects of the subtype 2 of angiotensin II (AT2) receptors on the pressure-natriuresis using a new peptide agonist, and the possible involvement of cyclic guanosine 3', 5' monophosphate (cyclic GMP) in these effects. In adult anaesthetized rats (Inactin, 100 mg kg(-1), i.p.) deprived of endogenous angiotensin II by angiotensin converting enzyme inhibition (quinapril, 10 mg kg(-1), i.v.), T2-(Ang II 4-8)2 (TA), a highly specific AT2 receptor agonist (5, 10 and 30 microg kg(-1) min(-1), i.v.) or its solvent was infused in four groups. Renal functions were studied at renal perfusion pressures (RPP) of 90, 110 and 130 mmHg and urinary cyclic GMP excretion when RPP was at 130 mmHg. The effects of TA (10 microg kg(-1) min(-1)) were reassessed in animals pretreated with PD 123319 (PD, 50 microg kg(-1) min(-1), i.v.), an AT2 receptor antagonist and the action of the same dose of PD alone was also determined. Increases in RPP from 90 to 130 mmHg did not change renal blood flow (RBF) but induced 8 and 15 fold increases in urinary flow and sodium excretion respectively. The 5 microg kg(-1) min(-1) dose of TA was devoid of action. The 10 and 30 microg kg(-1) min(-1) doses did not alter total RBF and glomerular filtration rate, but blunted pressure-diuresis and natriuresis relationships. These effects were abolished by PD. TA decreased urinary cyclic GMP excretion. After pretreatment with PD, this decrease was reversed to an increase which was also observed in animals receiving PD alone. In conclusion, renal AT2 receptors oppose the sodium and water excretion induced by acute increases in blood pressure and this action cannot be directly explained by changes in cyclic GMP.

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells.

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2. Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. N omega-nitro-L-arginine methyl ester (L-NAME; 20-300 microM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1beta and TNF-alpha was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 microM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 microM). 3. The inhibition elicited by L-NAME on PGE2-release by cytokine-treated astroglial cells was reversed by adding AA (40 microM), showing that the effect of NO on cytokine-dependent PGE2 release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine-treated astroglial cells was inhibited by indomethacin (10 microM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 microM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved. 4. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 microM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. 5. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL-1beta and TNF-alpha is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

Negative metabolic effects of cyclic GMP are altered in renal hypertension induced cardiac hypertrophy.

We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O2 consumption and that renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy would change this relationship. Four groups of anesthetized open-chest New Zealand white rabbits (N = 26) were utilized. Either vehicle or 3-morpholinosydnonimine (SIN-1) (10(-4) M, a guanylate cyclase activator) was topically applied to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. Guanylate cyclase activity was measured by conversion of GTP to cyclic GMP. 1K1C rabbits had a greater heart weight-to-body weight ratio (3.29 +/- 0.15) than controls (2.63 +/- 0.19). Systolic blood pressure was higher in 1K1C rabbits than in controls. In control rabbits, cyclic GMP levels (pmoles/g) were higher in SIN-1-treated (EPI: 7.5 +/- 1.6; ENDO: 8.1 +/- 1.5) than in vehicle-treated animals (EPI: 5.4 +/- 0.4; ENDO: 5.6 +/- 0.6). This effect was enhanced in 1K1C rabbits, with cyclic GMP levels in the SIN-1-treated group (EPI: 11.9 +/- 1.3; ENDO: 13.0 +/- 1.5) almost double those observed in the vehicle-treated group (EPI: 6.3 +/- 0.8; ENDO: 7.7 +/- 1.1). There were no significant differences in basal or maximally stimulated guanylate cyclase activity between controls and 1K1C rabbits. Myocardial O2 consumption (ml O2/min/100 g) was significantly less in the EPI region of control animals treated with SIN-1 (7.2 +/- 1.2) than in the same region of controls treated with vehicle (9.1 +/- 2.0). Myocardial O2 consumption was also significantly less in SIN-1-than vehicle-treated 1K1C animals (SIN-1-treated: EPI: 6.9 +/- 0.8; ENDO: 6.2 +/- 0.7; vehicle-treated: EPI: 10.0 +/- 0.8; ENDO: 12.5 +/- 3.0). There was no significant difference in O2 consumption between control and 1K1C animals after treatment with SIN-1. Thus, there was a greater elevation in cyclic GMP in 1K1C rabbits, but this did not result in a corresponding greater depression in O2 consumption. This suggests that cyclic GMP plays a role in the control of myocardial metabolism, and that the sensitivity of myocardial O2 consumption to changes in cyclic GMP is reduced by renal hypertension-induced cardiac hypertrophy.

Modulation of glutamate release by a nitric oxide/cyclic GMP-dependent pathway

The mechanism by which changes in cyclic GMP (cGMP) regulate glutamate release was investigated in rat cerebrocortical nerve terminals. The elevation of cGMP levels by inhibition of cGMP-phosphodiesterase with 2- o-propoxy-phenyl-8-azapurin-6-one (zaprinast) reduced the Ca 2+-dependent glutamate release evoked by depolarization with 30 mM KCl or 1 mM 4-aminopyridine. The nitric oxide (NO) donor S-nitroso- N-acetylpenicillamine also enhanced cGMP and reduced glutamate release. In addition, the membrane-permeable analogs 8-bromoguanosine 3′:5′-cyclic monophosphate (8-Br-cGMP) and N,2′- o-dibutyrylguanosine (dbcGMP) at 10 μM also mimic glutamate release inhibition. The reduction in glutamate release was observed with no modifications in the ATP/ADP ratio, and was reversed in the presence of the protein kinases inhibitor { N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide, HCl} (H-8). Interestingly, higher concentrations of dbcGMP (1 mM) abolished the inhibition observed with low concentrations although no facilitation was observed. This finding seems to indicate the existence of a dual role for cGMP in the control of glutamate exocytosis.

Glutamate toxicity in primary cerebellar cultures from mouse brain is unaffected by changes in cGMP levels.

During evaluation of potential end-points for in vitro neurotoxicity screening we investigated what influence changes in cyclic GMP (cGMP) levels might exert on the degree of glutamate (Glu)-induced neurotoxicity in primary cultures of mouse cerebellar granule cells. Depletion of Glu-stimulated cGMP levels by N-methyl-D-aspartate receptor antagonists fully protected against Glu-induced toxicity. However, when Glu-stimulated cGMP levels were either depleted or elevated by the use of a variety of pharmacological agents acting intracellularly at various points of the NO/cGMP signalling pathway the degree of cytotoxicity exerted by Glu was unaltered. These results imply that cGMP and NO do not modulate the toxic effects of Glu and are therefore unsuitable as biomarkers of excitotoxicity in these cells.

Effect of trilinolein on cyclic nucleotide formation in human platelets: relationship with its antiplatelet effect and nitric oxide synthesis

1. Trilinolein, a triacylglycerol with linoleic acid as the only fatty acid residue in all three esterified positions of glycerol, was recently reported to have an inhibitory effect on adrenaline-induced platelet aggregation. In the present study, we found that trilinolein at concentrations ranging from 0.01 to 1 microM increased cyclic GMP formation and decreased cyclic AMP formation in washed human platelets. Both NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue attenuated the trilinolein-induced increase in cyclic GMP. 2. Adrenaline decreased not only the production of cyclic AMP but also that of cyclic GMP. Trilinolein antagonized the inhibitory effect of adrenaline on cyclic GMP formation, but potentiated the inhibitory effect of adrenaline on cyclic AMP accumulation. 3. Both trilinolein and adrenaline enhanced intracellular calcium but the increment of intracellular calcium induced by them was much less than that produced by thrombin. 4. We propose that the anti-platelet effect of trilinolein is mediated through an increase in cyclic GMP, and that the change in cyclic GMP results from stimulation of nitric oxide synthesis in platelets. 5. We also propose that reduction of both cyclic AMP and cyclic GMP are involved in adrenaline-induced platelet aggregation.

The effect of endothelins on nitric oxide and prostacyclin production from human umbilical vein, porcine aorta and bovine carotid artery endothelial cells in culture.

1. This study has investigated the effects of the endothelin isopeptides, endothelin-1 (ET-1), ET-2 and ET-3 on the production of the endothelium-derived relaxing factors, nitric oxide (NO) and prostacyclin (PGI2) from primary cultures of endothelial cells obtained from human umbilical vein (HUVECS), porcine aorta (PAECS) and bovine carotid artery (BCAECS). 2. NO generation was assessed indirectly by measuring production of cyclic GMP and PGI2 formation was measured by radioimmunoassay of 6-keto PGF1 alpha. 3. In HUVECS, histamine (1 microM) increased cyclic GMP and 6-keto PGF1 alpha production by 12.6 +/- 2.0 and 4.9 +/- 0.7 fold respectively over the corresponding basal values. Haemoglobin (10 microM) and the NO synthase inhibitor NG-monomethyl-L-arginine (10 microM) significantly inhibited the increase in cyclic GMP formation in response to histamine but had no effect on 6-keto PGF1 alpha production. In contrast to histamine, the endothelin isopeptides (ET-1, ET-2 and ET-3; 0.01-1000 nM) produced no significant change in either cyclic GMP or 6-keto PGF1 alpha production in HUVECS. 4. In a separate series of experiments, ET-3 (0.01-1000 nM) also failed to produce any significant change in cyclic GMP or 6-keto PGF1 alpha production from primary cultures of PAECS and BCAECS. In contrast, bradykinin (0.1 microM) and sodium nitroprusside (1 mM) were used as positive control agents and increased cyclic GMP production in these cells. 5. In conclusion, the endothelin isopeptides do not release NO and PGI2 from primary cultures of HUVECS, PAECS and BCAECS. This suggests that endothelin receptors are either absent from these cells or are not coupled to NO or PGI2 production.

Dual Mechanism of the Relaxing Effect of Nicorandil by Stimulation of Cyclic GMP Formation and by Hyperpolarization

In addition to previous results from our laboratory showing that nicorandil relaxed vascular smooth muscle by increasing cyclic GMP levels, it was shown to activate K-channels as well, an effect that also leads to relaxation. In the present study, we attempted to differentiate quantitatively between these two effects in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclic GMP. When the strips were contracted by the thromboxane A2 analogue U 46619 (1 microM) with 10 microM methylene blue added, nicorandil produced 30-50% relaxation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K+ to 80.4 mM (30-fold), nicorandil caused only 52% relaxation, whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechanisms of relaxation by nicorandil was further achieved through calculation of the cyclic GMP-mediated component from a correlation between increases in cyclic GMP and percentage of relaxation as produced by nicorandil under conditions of inhibited hyperpolarization, i.e., in strips contracted with 1 microM U 46619 or 26.8 mM K+ (10-fold) and exposed to either 30-fold K+ or 10 mM Ba2+. Under both conditions, similar correlations between cyclic GMP and relaxation were obtained. Because U 46619, in addition to its contractile effect, partially antagonized the relaxation by nicorandil without changing cyclic GMP, the correlation was corrected for this effect and indicated a participation of cyclic GMP in the overall relaxant response of approximately 30-40% at low and less than or equal to 80-90% at high concentrations of nicorandil.

Nitroarginine does not Inhibit Lysophosphatidylcholine (LPC)-Induced Vascular Relaxation and Accumulation of Cyclic GMP

Lysophosphatidylcholine (LPC) is a potent endothelium-dependent vascular smooth muscle relaxant. The possibility that its action is mediated through endothelium-derived nitric oxide (EDNO), although suggestive, has not been proven. Both lysophosphatidylcholine and endothelium-derived nitric oxide relax by activating guanylate cyclase to form cyclic GMP. Based on the finding that EDNO formation is inhibited by NNA (N-omega-nitro-L-arginine), we followed cyclic GMP changes in bovine intrapulmonary arteries with LPC after incubation with NNA. Inhibition of cyclic GMP by LPC following NNA exposure would be suggestive of the production of EDNO by LPC. However, while NNA significantly inhibited accumulation of cyclic GMP after exposure to the calcium ionophore A23187 which releases EDNO, NNA failed to inhibit LPC-induced accumulation of cyclic GMP. The results indicate that LPC relaxes vascular smooth muscle through a non NO-mediated pathway.

Effects of saline loading during head down tilt on ANP and cyclic GMP levels and on urinary fluid excretion

In the present study the renal and humoral effects of acute saline infusions were investigated in six healthy male volunteers before, during and after a ten day period of −6° head-down-tilt (HDT). During the whole 23-day study period the subjects received a standardized diet including 40 ml water and 125 mg NaCl per kg body weight per day. After the infusion of 0.9% saline (22 ml/kg within 20 minutes) plasma atrial natriuretic peptide (ANP) levels were only slightly increased (not significant) at the end of the infusion, while plasma cyclic GMP levels were significantly increased by about 40% (p<0.05) one hour later. No difference was observed in the plasma ANP and cyclic GMP changes between the pre-HDT, the HDT and the post-HDT infusion experiment. Urine flow, sodium excretion and urinary cyclic GMP excretion were significantly increased (p<0.05 and below) by 100 to 300% during the second and third hour after each saline infusion. However, during these short-term periods only 20% of the infused water and less than 20% of the infused sodium were excreted. Furthermore, a significantly increased volume, sodium and cyclic GMP excretion was observed for over 48 hours after each fluid load experiment.These data suggest that HDT does not induce major alterations in the regulation of an acute saline infusion and plasma ANP does not play a major role in the diuretic/natriuretic effects of volume loading.

Cyclic Nucleotides Increase During Neuronally Induced Relaxation of Sphincteric and Nonsphincteric Gastrointestinal Smooth Muscle

Neuronal stimulation of most isolated precontracted gastrointestinal smooth muscle results in relaxation. This study examined the changes in cyclic nucleotide content associated with neuronally induced relaxation in sphincteric and nonsphincteric smooth muscle of the gut. Electrical field stimulation produced a frequency‐dependent relaxation of the guinea pig proximal colon (EF50= 1.6 Hz) and taenia coli (EF50= 2.4 HZ), and the canine proximal colon (EF50= 3.1 Hz) and internal anal sphincter (EF50= 3.0 Hz). Changes in cyclic nucleotide content occurred during EFS‐induced relaxation. Cyclic AMP levels were significantly increased in the guinea pig poximal colon and taenia coli and in the canine proximal colon. No change in cyclic GMP occurred in these smooth muscles. Conversely, in the canine internal anal sphincter the cyclic GMP content increased significantly with no change in cyclic AMP. These effects in this sphincter are similar to those identified previously in the lower esophageal sphincter of opossum, dog, and man. Tetrodotoxin blocked the EFS‐induced relaxation and the associated accumulation of cyclic nucleotides of all tissues. Therefore, EFS‐induced relaxation is mediated by the activation of neurons and the release of inhibitory neurotransmitter(s). Also, EFS‐induced relaxation is closely associated with increases in smooth muscle cyclic nucleotide content. Most importantly, the change in cyclic nucleotide accumulation that occurs during EFS‐induced relaxation is different for sphincteric and nonsphincteric smooth muscle, (i.e., different cyclic nucleotides are generated in sphincteric and nonsphincteric smooth muscle in response to stimulation of nonadrenergic, nonchohnergic, inhibitory enteric neurons).

Cyclic GMP release and vasodilatation induced by EDRF and atrial natriuretic factor in the isolated perfused kidney of the rat

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation. The renal vasodilator action of ANF however, is independent of EDRF, although the renal vascular endothelium cannot be discounted as a site at which ANF stimulates cyclic GMP production.

Effect of lysophosphatidylcholine on atherosclerotic rabbit arteries

We report here on the effect of an endothelium-dependent vascular smooth muscle relaxant, lysophosphatidylcholine (LPC) on rabbit aortic strips and on hemodynamic changes by LPC in atherosclerotic animals. Cyclic GMP changes induced by LPC in atherosclerotic vessels were also determined. Atherosclerosis was produced by feeding a high cholesterol and saturated fatty acid diet. LPC was injected into the left atrium and coronary flow was measured by radioactive microspheres; in vitro, relaxation of precontracted aortic strips by lysophosphatidylcholine was also recorded. LPC failed to increase coronary flow in the presence of atherosclerosis. In isolated aortic strips, dose-response curves with acetylcholine and LPC showed diminished relaxation in atherosclerotic preparations, and cyclic GMP production following LPC was reduced. The results demonstrate that vascular relaxation by LPC, together with its ability to activate guanylate cyclase is dependent on the functional and morphological integrity of the vascular wall.

Methylene blue but not changes in cyclic GMP inhibits resting and bradykinin‐stimulated production of prostacyclin by pig aortic endothelial cells

1. Primary cultures of pig aortic endothelial cells produced 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6-keto-PGF1 alpha production induced by bradykinin (1-100 nM) was concentration-dependent. 2. Treating endothelial cells with the inhibitor of soluble guanylate cyclase, methylene blue (0.1-20 microM) produced an irreversible reduction in resting and bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha with an IC50 of 0.5 +/- 0.1 microM. Treating endothelial cells with haemoglobin (10 microM) had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 3. Two stimuli that elevate the level of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in endothelial cells, 8-bromo cyclic GMP (30 microM) and atriopeptin II (0.1 microM), each had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. Furthermore, treating endothelial cells with either 8-bromo cyclic GMP (30 microM) or atriopeptin II (0.1 microM) had no effect on the ability of methylene blue (20 microM) to inhibit resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 4. Adding arachidonic acid (1 microM) to endothelial cells led to a marked stimulation of 6-keto-PGF1 alpha production. Treating cells with either methylene blue (20 microM) or the cyclo-oxygenase inhibitor, flurbiprofen (10 microM), inhibited both resting and arachidonic acid (1 microM)-induced production of 6-keto-PGF1 alpha. 5. In pig aortic endothelial cells methylene blue appears to block prostacyclin production by a mechanism independent of inhibition of soluble guanylate cyclase. Care should be exercised when using methylene blue as a selective inhibitor of endothelium-derived relaxing factor due to its additional ability to block production of the other endothelium-derived vasodilator, prostacyclin.

Correlation of lysophosphatidylcholine-induced vs spontaneous relaxation to cyclic GMP levels in rabbit thoracic aorta

We demonstrated previously that 10 −5M micellar solution of lysophosphatidylcholine (LPC) produced relaxation of rabbit thoracic aorta in a dose-dependent manner. It was also noticed that histamine (HA)-precontracted strips relaxed spontaneously in the absence of LPC. We now measured the cyclic GMP changes during these two types of relaxation. Results indicate that while LPC-induced relaxation was mediated through cyclic GMP, spontaneous relaxation was not. The vasodilating effect of LPC was not due to its interference with cellular viability by solubilizing the membrane, because repeated additions of LPC produced identical degrees of relaxation and after three consecutive additions of LPC, the strips continued to relax to the same degree with acetylcholine (Ach) demonstrating the functional integrity of the endothelium. LPC/Cholesterol dispersions (1:0.5 mole percent) relaxed the strips to the same extent as the micellar solution of LPC, while 1:1 mole percent did not. The results stress the role of cyclic GMP in LPC-induced relaxation. They further suggest that LPC/Cholesterol ratio may determine the availability of LPC and regulate LPC-mediated processes.