Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I–MYPT1 signaling may be of therapeutic benefit for combating these events.     

An inhibitor of cAMP-dependent protein kinase induces behavioural and neurological antidepressant-like effects in rats

Although it is well established that cyclic adenosine monophosphate (cAMP) signalling via cAMP-dependent protein kinase (PKA)within neurons plays an important role in depression and antidepressant treatment, the importance of several newly discovered targets that function independently from PKA, such as exchange protein activated by cAMP (Epac), remains unexplored in this regard. In this study we used a cAMP analogue that inhibits PKA but not Epac (Rp-8-Br-cAMP), to explore the modifying actions of these two targets on immobility in the forced swim test (FST) and cerebellar cAMP response element binding protein (CREB) phosphorylation in rats. In addition, we assessed central cAMP and cGMP levels and investigated the involvement of cGMP-dependent protein kinase (PKG) on any observed effects by using a selective PKG inhibitor (Rp-8-Br-PET-cGMPS).Interestingly, Rp-8-Br-cAMPS strongly reduced immobility in the FST and induced an increase in the phosphorylation of CREB in the cerebellum, effects that were unaltered by the co-administration of Rp-8-Br-PET-cGMPS. Furthermore, Rp-8-Br-cAMPS increased the accumulation of cAMP and cGMP in the hippocampus, frontal cortex and cerebellum of these rats. Together, these results suggest that in addition to activating PKA, elevated cAMP may also stimulate other targets that mediate antidepressant activity. According to the pharmacodynamic profile of Rp-8-Br-cAMPS and taking into consideration what has recently been discovered regarding the cAMP signalling system, a likely candidate is the guanine nucleotide exchange factor, Epac.     

cAMP and cGMP contribute to sensory neuron hyperexcitability and hyperalgesia in rats with dorsal root ganglia compression

Numerous studies have implicated the cAMP-protein kinase A (PKA) pathway in producing hyperexcitability of dorsal root ganglia (DRG) sensory neurons under conditions associated with pain. Evidence is presented for roles of both the cAMP-PKA and cGMP-protein kinase G (PKG) pathways in maintaining neuronal hyperexcitability and behavioral hyperalgesia in a neuropathic pain model: chronic compression of the DRG (CCD treatment). Lumbar DRGs were compressed by a steel rod inserted into the intervertebral foramen. Thermal hyperalgesia was revealed by shortened latencies of foot withdrawal to radiant heat. Intracellular recordings were obtained in vitro from lumbar ganglia after in vivo DRG compression. Activators of the cAMP-PKA pathway, 8-Br-cAMP and Sp-cAMPS, and of the cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMPS, increased the hyperexcitability of DRG neurons already produced by CCD treatment, as shown by further decreases in action potential threshold and increased repetitive discharge during depolarization. The adenylate cyclase inhibitor, SQ22536, the PKA antagonist, Rp-cAMPS, the guanylate cyclase inhibitor, ODQ, and the PKG inhibitor, Rp-8-pCPT-cGMPS, reduced the hyperexcitability of CCD DRG neurons. In vivo application of PKA and PKG antagonists transiently depressed behavioral hyperalgesia induced by CCD treatment. Unexpectedly, application of these agonists and antagonists to ganglia of naïve, uninjured animals had little effect on electrophysiological properties of DRG neurons and no effect on foot withdrawal, suggesting that sensitizing actions of these pathways in the DRG are enabled by prior injury or stress. The only effect observed in uncompressed ganglia was modest depolarization of DRG neurons by PKA and PKG agonists. CCD treatment also depolarized DRG neurons, but CCD-induced depolarization was not affected by agonists or antagonists of these pathways.     

Effect of nitric oxide on the maximal velocity of shortening of a mouse skeletal muscle

 Maximum velocity of shortening, V _o, was measured by the method of Edman [J Physiol (Lond) 291:143–159, 1979] on extensor digitorum longus muscles of a mouse in vitro at 20°C. Blockers of nitric oxide synthase, 10 mM nitro-l-arginine or 1 mM 7-nitroindazole, reduced V _o by 18% and 22%, respectively. On removal of the inhibitor, V _o returned to the control value. It was found that 10 mM nitro-d-arginine, an enantiomer of nitro-l-arginine inactive against nitric oxide synthase, did not affect V _o. A donor of nitric oxide, 0.1 mM nitroprusside, increased V _o by 15%. It removed the inhibition caused by nitro-l-arginine. Another donor of nitric oxide, 1 μM (±)-S-nitroso-N-acetylpenicillamine (SNAP), increased V _o by 8%. An inhibitor of cGMP synthase, 0.01 mM Ly-83583, decreased V _o by 18%. An analogue of cGMP, 0.1 mM 8-bromo-cGMP, increased V _o by 17%. A general inhibitor of phosphodiesterases, 0.02 mM 3-isobutyl-1-methylxanthine (IBMX), increased V _o by 17%. An inhibitor specific of cGMP phosphodiesterase, 0.01 mM dipyridamole, increased V _o by 8%. The maximal isometric force (F ₀) was not modified by the drugs, except by 7-nitroindazole and Ly-83583, which depressed F ₀ by 12%. The cGMP level in tetanized muscles decreased by 12–27% in the presence of blockers of nitric oxide synthase. We conclude that the level of intracellular nitric oxide modulates V _o through thecGMP pathway. Received: 16 February 1998 / Received after revision: 18 May 1998 / Accepted: 18 June 1998     

Cyclic AMP-dependent protein kinase mediates ocular dominance shifts in cat visual cortex

Visual experience during a critical period early in postnatal development can change connections within mammalian visual cortex. In a kitten at the peak of the critical period (approximately P28-42), brief monocular deprivation can lead to complete dominance by the open eye, an ocular dominance shift. This process is driven by activity from the eyes, and depends on N-methyl-D-aspartate (NMDA) receptor activation. The components of the intracellular signaling cascade underlying these changes have not all been identified. Here we show that inhibition of protein kinase A (PKA) by Rp-8-Cl-cAMPS blocks ocular dominance shifts that occur following monocular deprivation early in the critical period. Inhibition of protein kinase G by Rp-8-Br-PET-cGMPS had no effect, indicating a specificity for the PKA pathway. Enhancement of PKA activity late in the critical period with Sp-8-Cl-cAMPS did not increase plasticity. PKA is a necessary component of the pathway leading to cortical plasticity during the critical period.     

Both cGMP and peroxynitrite mediate chronic interleukin-6-induced negative inotropy in adult rat ventricular myocytes

We previously showed that chronic exposure to interleukin (IL)-6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)-dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO-associated negative inotropic effects of IL-6 during acute exposure; however, its role in chronic cardiac effects of IL-6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO⁻) in chronic IL-6-induced negative inotropy in ARVM. After ARVM were exposed to IL-6 for 2–24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp-8-Br-cGMP, an inhibitor of cGMP-dependent protein kinase G (PKG). Meanwhile, the IL-6-induced decrease in PRP at 2 h was blocked by ODQ or Rp-8-Br-cGMP. By contrast, ODQ or Rp-8-Br-cGMP only attenuated the inhibition of PRP induced by IL-6 after 24 h exposure. Furthermore, IL-6 time dependently increased superoxide anion production and ONOO⁻ formation; the latter was abolished by 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron (III) (FeTPPS), an ONOO⁻ decomposition catalyst. Interestingly, FeTPPS had no effect on the IL-6-elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO⁻ formation, abolished the IL-6-induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL-6-induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO⁻, at least in part, mediate the IL-6-induced inhibition of SR function after 24 h exposure.     

The effect of nitric oxide generators on ischemia reperfusion injury and histamine release in isolated perfused guinea-pig heart

Experiments were carried out to provide evidence of the effect ofl-arginine (l-Arg), its analogue N^G-monomethyl-l-arginine (MeArg) and of some nitrovasodilators (sodium nitroprusside, NaNP; 3-morpholino-sydnonimine, SIN-1) which spontaneously release nitric oxide (NO) on ischemia-reperfusion injury, histamine release and mast cell degranulation, occurring after multiple ligature and release of the left anterior descending (LAD) coronary artery in isolated perfused guinea-pig hearts. The reopening of the LAD coronary artery leads to a release of histamine related to a decrease in microdensitometry of cardiac mast cells and to calcium overload. The perfusion of the heart with NO-donors significantly reduces either the release of histamine, the loss of mast cell metachromasia and the overload of calcium. These effects were potentiated by SOD. The results suggest that the endogenous formation of NO and molecules able to generate NO have a role in the prevention of post-ischemic tissue injury.     

Acute hypoxia induces vasodilation and increases coronary blood flow by activating inward rectifier K+ channels

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 μm) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K_ATP channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A₃ receptor inhibitor); these increases were inhibited by the inward rectifier K⁺ (Kir) channel inhibitor, Ba²⁺. These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 μm) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.     

Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells

Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).     

Guanylyl cyclase inhibitors NS2028 and ODQ and protein kinase G (PKG) inhibitor KT5823 trigger apoptotic DNA fragmentation in immortalized uterine epithelial cells: anti-apoptotic effects of basal cGMP/PKG

The cGMP/protein kinase G (PKG) signalling pathway, at basal levels, has anti-apoptotic/pro-survival effects in certain neural cells. The present study determined apoptosis-regulating effects of basal cGMP/PKG in an immortalized uterine epithelial cell line, HRE-H9 cells, using two soluble guanylyl cyclase (sGC) inhibitors, NS2028 and ODQ, and a PKG inhibitor, KT5823. A new quantitative, ultrasensitive technique using capillary electrophoresis with laser-induced fluorescent detector (CE-LIF), recently developed in our laboratory, was used to quantify levels of apoptotic DNA fragmentation. NS2028 and ODQ increased apoptotic DNA fragmentation by 3.5- and 9-fold respectively, suggesting that lowering basal cGMP levels causes spontaneous apoptosis. 8-Br-cGMP, a cell-permeable cGMP analogue that directly activates PKG, reduced ODQ-induced apoptosis by 81%, indicating that replacement of lowered cGMP with a direct PKG activator prevents apoptosis. Western blot analysis, using PKG type I (PKG-I)-specific antibody, indicated that HRE-H9 cells express PKG-I at moderate levels. Inhibiting basal PKG activity with KT5823 increased apoptotic DNA fragmentation by 9.8-fold. Overall, the data show that inhibitors of basal sGC and PKG activities in immortalized uterine epithelial cells cause apoptosis, suggesting that normal basal levels of cGMP and PKG activity may be necessary to prevent a spontaneous development of apoptosis in these cells.     

Preservation of nitric oxide-induced relaxation of porcine coronary artery: roles of the dimers of soluble guanylyl cyclase,phosphodiesterase type 5, and cGMP-dependent protein kinase

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3′5′-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.     

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mm), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 m), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of l-arginine was reversed by administration of N -nitro-l-arginine methyl ester (l-NAME, 100 m), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 m), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3,5-cyclic monophosphate (8-B-cGMP) (1 mm), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETAINO) and endogenously generated (l-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.[/p]     

Both protein kinase G dependent and independent mechanisms are involved in the modulation of glutamate release by nitric oxide in rat hippocampal nerve terminals

We compared the effects of sodium nitroprusside (SNP), and of 8-bromo guanosine 3',5'-cyclic monophosphate (8-BrcGMP), on the 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate from hippocampal nerve terminals and further investigated the role of protein kinase G (PKG) in this mechanism. SNP and 8-BrcGMP dose-dependently inhibited glutamate release, however SNP concentrations ([SNP]) > 500 microM abolished the 4-AP evoked release, whereas 8-BrcGMP maximally inhibited the release by about 30%. The inhibition of glutamate release at low concentrations of SNP (< or = 5 microM) was of about 20%, and was reversed by Rp-8(4-chlorophenylthio)guanosine-3',5'-cyclic-monophosphorotioate ) (RpCPTcGMP, 50 nM), but the inhibition at higher concentrations (5 < SNP < or = 50 microM) was insensitive to the PKG inhibitor, but sensitive to [1 H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one] (ODQ), which partially prevented the inhibition. [SNP] > 50 microM strongly inhibited glutamate release, and this was not reversed by either inhibitor. Furthermore, [SNP] < or = 50 microM enhanced cGMP formation, and the observed effects were not related to either decreased Ca2+ entry or ATP/ADP levels. Our results indicate that NO/PKG is the signaling pathway underlying the inhibition of glutamate release at low concentrations of NO, and imply that other NO-dependent, but PKG-independent, mechanisms are activated and have complementary roles at higher NO concentrations.     

Arterial size determines the enhancement of contractile responses after suppression of endothelium-derived relaxing factor formation

We studied the effect of endothelium-derived relaxing factor (EDRF) on norepinephrine-induced contractile responses and on the tissue guanosine-3,5-phosphate (cGMP) concentration of isolated rabbit arteries with an increasing endothelium to smooth muscle cell ratio (aorta, femoral and mesenteric arteries). After suppression of EDRF formation (either by N ^G-nitro-l-arginine or, in mesenteric arteries, by saponin), contractions elicited by cumulative doses of norepinephrine were unaltered in aorta but were enhanced by 22.5% in femoral arteries and by 44.3% in mesenteric arteries (at the highest norepinephrine concentration). The cGMP concentration (pmol/mg protein) of unstimulated, endotheliumintact vessels decreased after suppression of EDRF formation from 1.09±0.24 to 0.74±0.28 in aortic, from 2.86±0.4 to 0.61±0.19 in femoral and from 6.3±0.9 to 0.7±0.15 in mesenteric arterial segments. The basal cGMP concentration did not differ in endothelium-denuded segments of these arteries, suggesting a similar basal activity of soluble guanylate cyclase (sGC). A higher sensitivity of sGC may have contributed to the higher cGMP concentration observed in the smaller arteries, since in the presence of sodium nitroprusside the cGMP concentration of endothelium-denuded segments increased 1.8-fold in aortic, 2.9-fold in femoral and 2.4 fold in mesenteric arterial segments. However, these differences in sGC activation cannot be solely responsible for the high basal cGMP concentration in endotheliumintact mesenteric arteries. The greater ratio of endothelium to smooth muscle cell layers in the smaller arteries might result in a higher EDRF concentration in the vascular wall and subsequently in a higher cGMP concentration. In conclusion, these data support the view of a greater importance of EDRF-mediated vascular control in small arteries than in large conduit arteries.     

Hemodynamic Regulation of Inflammation at the Endothelial–Neutrophil Interface

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm² blocked by >80% the induction by 5 ng/mL TNFα of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-l-arginine methylester, l-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm². Also, neutrophil rolling lengths at 1 dyne/cm² were longer when TNFα-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.     

Inhibition of superoxide anion release from circulating neutrophils by l-arginine in man

Summary In animal studies of myocardial ischemia/reperfusion l-arginine reduces necrotic injury by preservation of endothelial function and attenuation of neutrophil accumulation in ischemic cardiac tissue. Because release of oxygen radical species by circulating neutrophils is important in endothelial function and ischemia-reperfusion injury, this study investigated the effect of intravenous administration of L-arginine on the in vitro release of superoxide anion of neutrophils in healthy young adults. Neutrophils were obtained at various time points before, during, and after infusion of l-arginine (17 mg kg^–1 min^–1 for 30 min) and analyzed for superoxide dismutase inhibitable reduction of ferricytochrome c. The spontaneously occurring respiratory burst of polymorphonuclear leukocytes at basal conditions was compared with that after triggering by 1 mol/l formylpeptide or 50 ng/ml phorbolester. Infusion of l-arginine inhibited both basal (P < 0.01) and formylpeptide-triggered (P < 0.05) release of superoxide anion did, but not affect release stimulated by phorbol 12-myristate 13-acetate. Pretreatment of neutrophils with 1 mmol/l l-arginine in vitro also significantly reduced formylpeptide-triggered (1 mol/l) superoxide anion release, suggesting that the affects observed after in vivo pretreatment may be due to direct action of l-arginine on neutrophils. These findings demonstrate the ability of L-arginine to reduce release of oxygen radical species by circulating neutrophils in man.Abbreviations HBSS Hank's balanced salt solution - FMLP formyl-Met-Leu-Phe - NO nitric oxide - PMA phorbol 12-myristate 13-acetate - PMNL polymorphonuclear leukocytes - SOD superoxide dismutase     

Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

The relaxant effect of hypercapnia (15% CO₂) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-l -arginine (l -NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of l -NOARG was completely reversed by l -arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO₂ elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. l -NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 m HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO₂. In vessel segments in which the endothelium had been removed beforehand 15% CO₂ induced relaxation that was not different from that seen in vessels with intact endothelium. l -NOARG had no affect in endothelium denuded vessels. The results suggest that high CO₂ elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by l -NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall.     

N G-nitro-l-arginine antagonizes endothelium-dependent dilator responses by inhibiting endothelium-derived relaxing factor release in the isolated rabbit heart

The effects of a recently described inhibitor of endothelial NO synthesis, N ^G-nitro-l-arginine (l-NNA), on the vasomotor responses to endothelium-dependent and independent vasodilators, and on the release of endothelium-derived relaxing factor (EDRF), were studied in the isolated saline-perfused rabbit heart. Infusion of l-NNA (30 M) resulted in a 52±12% increase in basal coronary perfusion pressure. The vasomotor responses to 1 M acetylcholine (ACh) and serotonin after l-NNA became biphasic, showing a small transient dilation followed by a pronounced vasoconstriction. In contrast, the dilation observed with sodium nitroprusside was not affected by l-NNA. None of the above-mentioned effects was elicited by the Stereo-isomer d-NNA. Similarly, an increase in the basal coronary perfusion pressure by endothelin-1 (0.3 nM) to the same level as observed with l-NNA did not alter the vasomotor responses to ACh and sodium nitroprusside. The increase in cyclic GMP (cGMP) in platelets passing through the coronary vascular bed was used as an index of EDRF release. Platelet cGMP amounted to 0.50±0.10 pmol/mg protein after passage through the coronary bed of the unstimulated heart. When platelets were injected during an ACh infusion (1 M), a 2.7 fold increase in cGMP was observed (P<0.01). After a 30-min infusion with l-NNA, the cGMP content of platelets passing through the unstimulated heart was reduced by 62%. Likewise, the ACh-induced increase in platelet cGMP was totally blocked. These results show that l-NNA inhibits EDRF release, and is thus a potent and selective inhibitor of EDRF-mediated dilation in the isolated rabbit heart.     

Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries

Acetylcholine is believed to dilate normal blood vessels by promoting the release of a vasorelaxant substance from the endothelium (endothelium-derived relaxing factor). By contrast, if the endothelium is removed experimentally, acetylcholine constricts blood vessels. We tested the hypothesis that muscarinic cholinergic vasodilation is impaired in coronary atherosclerosis. Graded concentrations of acetylcholine and, for comparison, the nonendothelial-dependent vasodilator nitroglycerin were infused into the left anterior descending artery of eight patients with advanced coronary stenoses (greater than 50 percent narrowing), four subjects with angiographically normal coronary arteries, and six patients with mild coronary atherosclerosis (less than 20 percent narrowing). Vascular responses were evaluated by quantitative angiography. In several segments each of four normal coronary arteries, acetylcholine caused a dose-dependent dilation from a control diameter of 1.94 +/- 0.16 mm to 2.16 +/- 0.15 mm with the maximal acetylcholine dose (P less than 0.01). In contrast, all eight of the arteries with advanced stenoses showed dose-dependent constriction, from 1.05 +/- 0.05 to 0.32 +/- 0.16 mm at the highest concentration of acetylcholine (P less than 0.01), with temporary occlusion in five. Five of six vessels with minimal disease also constricted in response to acetylcholine. All vessels dilated in response to nitroglycerin, however. We conclude that paradoxical vasoconstriction induced by acetylcholine occurs early as well as late in the course of coronary atherosclerosis. Our preliminary findings suggest that the abnormal vascular response to acetylcholine may represent a defect in endothelial vasodilator function, and may be important in the pathogenesis of coronary vasospasm.     

Carbon dioxide induced vasorelaxation in rat mesenteric small arteries precontracted with noradrenaline is endothelium dependent and mediated by nitric oxide

Summary Hypercapnia induces initial constriction and prolonged relaxation of rat small mesenteric arteries. The mechanism of the relaxation is unknown, but has been attributed to lowering of pH_i in the vascular smooth muscle. In this study we have investigated the response to raised PCO₂ at constant pH_o, in mesenteric small arteries precontracted with noradrenaline. 10% CO₂ led to a fall in pH_i associated with acute potentiation of tension, and subsequent relaxation. The relaxation did not occur in arteries in which the endothelium had been removed, nor in arteries pretreated with the nitric oxide synthase inhibitor, L-NAME (10^–4M, N^G-nitro-L-arginine methyl ester). The D-enantiomer, D-NAME, was without effect. We conclude that hypercapnic-induced vasodilatation in this circulation occurs via endothelium derived nitric oxide production.