Fernando Rodríguez-Pascual

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

Sodium nitroprusside, S‐nitroso‐N‐acetyl‐D,L‐penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38±0.02 μM. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half‐life (t1/2) of 3.9±0.2 min. The t1/2 for SPER/NO was 37±3 min. SNAP decomposed more slowly (t1/2=37±4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 μM and maximal effective concentration between 2.5 and 5 μM. For SNAP, threshold activation was seen at 1 μM, whereas full activation required a higher concentration (500–750 μM). The dose‐response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide‐deficient SNAP analogue or other NO donors. Although NO‐donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Functional coupling of nitric oxide synthase and soluble guanylyl cyclase in controlling catecholamine secretion from bovine chromaffin cells

This study was designed to evaluate whether the enzymes of the nitric oxide/cyclic-GMP pathway, nitric oxide synthase and soluble guanylyl cyclase, are functionally coupled in controlling catecholamine secretion in primary cultures of bovine chromaffin cells. In immunocytochemical studies, 80-85% of the tyrosine hydroxylase-positive chromaffin cells also possessed phenylethanolamine-N-methyltransferase, f1p4cating their capability to synthesize epinephrine. Immunoreactivity for neuronal-type nitric oxide synthase was found in over 90% of all chromaffin cells. Reverse transcription-polymerase chain reaction also demonstrated neuronal-type nitric oxide synthase messenger RNA. Immunoreactivity for soluble guanylyl cyclase was detectable in over 95% of chromaffin cells. Double-labeling immunofluorescence studies co-localized neuronal-type nitric oxide synthase and soluble guanylyl cyclase with tyrosine hydroxylase and phenylethanolamine-N-methyltransferase in the majority of chromaffin cells. Chromaffin cells possessed basal nitric oxide synthase activity which could be stimulated by acetylcholine and inhibited by NG-nitro-L-arginine methyl ester. Activation of soluble guanylyl cyclase by endogenously synthesized nitric oxide or the nitric oxide donor compound sodium nitroprusside was blocked by the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Catecholamine release and the increase in cytosolic Ca2+ concentration evoked by acetylcholine were enhanced by inhibitors of the endogenous nitric oxide/cyclic-GMP pathway such as NG-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor Rp-8-pCPT-cGMPS. These data indicate that chromaffin cells possess an autocrine nitric oxide/cyclic-GMP pathway tonically controlling the inhibition of catecholamine release.

Nitric Oxide-Sensitive Guanylyl Cyclase Activity Inhibition Through Cyclic GMP-Dependent Dephosphorylation

Abstract: The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter‐ and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine‐3′,5′‐monophosphate (cGMP)‐dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C‐type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP‐permeant analogue (which also activates PKG), in the presence of a broad‐spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)‐dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT‐5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the β subunit of sGC, and these changes correlated well with differences in SNP‐elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP‐stimulated cGMP rises without changing the SNP concentration required to produce half‐maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its β subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the β subunit.

Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture.

An ectoenzyme hydrolyzing diadenosine polyphosphates (ApnA) to AMP and Ap(n-1) has been studied in cultured chromaffin cells from bovine adrenal medulla. The KM value for extracellular Ap4A hydrolysis was 2.90 +/- 0.72 microM, the V(max) value obtained was 11.59 +/- 0.92 pmol/min x 10(6) cells (116 pmol/min.mg total protein). Ap3A, Ap5A, Ap6A, and Gp4G were competitive inhibitors of Ap4A hydrolysis with K(i) values of 3.65, 1.10, 1.20, and 2.65 microM, respectively. Phosphatidylinositol-specific phospholipase C removes the ApnA hydrolase activity from cultured chromaffin cells, suggesting an anchorage of this protein to the plasma membrane through the phosphatidylinositol. The turnover time for this enzyme calculated in the presence of cycloheximide was 38.94 +/- 1.53 hr for cultured chromaffin cells.

Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain.

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation. The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system.

Activation of no:cGMP pathway by acetylcholine in bovine chromaffin cells: Possible role of Ca2+ in the down-regulation of cGMP signaling

The production of cyclic GMP (cGMP) induced by acetylcholine and other stimuli was studied in bovine chromaffin cells. Acetylcholine increased intracellular cGMP in a transitory (peak at 2 min) and concentration-dependent manner (estimated half maximal increase, EC50 = 61 +/- 5 microM). NG-nitro-L-arginine methyl ester (NAME) inhibited such a rise in cGMP with a half maximal inhibitory concentration (IC50) of 231 +/- 55 microM. The acetylcholine-induced increase in cGMP was also inhibited by a calmodulin antagonist (calmidazolium, 30 microM) and by the absence of extracellular calcium. Other agents that strongly increased cytosolic calcium concentration ([Ca2+]i) as acetylcholine did, such as the nicotinic-agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), high-KCl (50 mM), and ionomycin, also caused a rise in cGMP in cultured bovine chromaffin cells. Veratridine, an activator of sodium channels, produced a slowly developing calcium increase and no significant cGMP production. The muscarinic-agonist, muscarine, failed to increase cytosolic calcium, and was the weakest stimulator of cGMP production. cGMP formation, induced by sodium nitroprusside (SNP, 100 microM) and by C-type natriuretic peptide (CNP, 100 nM), was inhibited by 30-40% by increasing [Ca2+]i with ionomycin. This inhibition was abolished by calmidazolium (30 microM) and by the absence of calcium in the extracellular medium. In conclusion, bovine chromaffin cells synthesize nitric oxide (NO) to activate guanylate cyclase in response to several stimuli, which increase [Ca2+]i. Moreover, the increase in [Ca2+]i also stimulates a Ca2+/calmodulin phosphodiesterase, which could down-regulate the levels of cGMP in these cells.

Modulation of the dihydropyridine-insensitive Ca2+ influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 microM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca2+]i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Combined incubations with nifedipine 10 microM and 8-Br-cGMP reduced KCl-evoked intracellular Ca2+ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) under conditions of low depolarization. Neomycin (0.2 mM) and omega-AgaToxin-IVA (AgTx) (1 microM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca2+ influx pathway in the chromaffin cell.

Diadenosine Polyphosphates from Neuro-Secretory Granules: The Search for Receptors, Signals and Function

The diadenosine polyphosphates -ApnA- are stored in secretory granules together with ATP and aminergic compounds: with serotonin in the dense granules of platelets, with acetylcholine in torpedo synaptic vesicles and with adrenaline and noradrenaline in chromaffin granules of adrenal medulla. The vesicular content in neural tissues is released to the extracellular media in a Ca2+ dependent way and in equimolecular ratios with respect to the other costored compounds. Cultured bovine neurochromaffin cells, torpedo synaptosomes and rat brain synaptic terminals, show the existence of specific binding sites for diadenosine polyphosphates. Two high affinity binding sites are found in these neural models with Kd values ranging from 0.1 to 0.7 nM for the first site and 5 to 6 nM for the second binding site. Displacement studies with P2-purinoceptor ligands present a particular potency order, suggesting the presence of a new receptor subtype in rat brain synaptic terminals, designated as P2d- The diadenosine polyphosphate receptors in neurochromaffin cells and vascular endothelial cells from adrenal medulla are coupled to Ca2+ release from internal stores and stimulation of Protein Kinase C. These intracellular signals cause the inhibition of catecholamine secretion from bovine chromaffin cells and induce a negative feedback for excitation in rat hippocampus. Both Ap4A and Ap5A evoke a fast rise in the [Ca2+]i in endothelial cells from bovine adrenal medulla, the effect is concentration dependent, and the EC50 is in the range 1–10 μM. The extracellular destruction of adenine dinucleotides is carried out by an ectodinucleoside polyphosphate hydrolase with high affinity, the Km values being close to 2 μM for the different diadenosine polyphosphates. The adenine mononucleotides produced are degraded to adenosine by the ecto-nucleotidases cascade. This nucleoside can be considered as the last extracellular product of purinergic transmission.