cyclic-gmp and anantin

Atrial and brain natriuretic peptides (ANP and BNP) exist in the central nervous system and modulate neuronal function, although the locus of actions and physiological mechanisms are still unclear. In the present study we used rat spinal sacral dorsal commissural nucleus (SDCN) and hippocampal 'synaptic bouton' preparations, to record both spontaneous and evoked glycinergic inhibitory postsynaptic currents (sIPSCs and eIPSCs) in SDCN neurons, and the evoked excitatory postsynaptic currents (eEPSCs) in hippocampal CA3 neurons. ANP potently and significantly reduced the sIPSC frequency without affecting the amplitude. ANP also potently reduced the eIPSCs amplitude concurrently increasing the failure rate and the paired pulse ratio response. These ANP actions were blocked by anantin, a specific type A natriuretic peptide receptor (NPR-A) antagonist. The results clearly indicate that ANP acts directly on glycinergic presynaptic nerve terminals to inhibit glycine release via presynaptic NPR-A. The ANP effects were not blocked by the membrane permeable cGMP analog (8Br-cGMP) suggesting a transduction mechanisms not simply related to increasing cGMP levels in nerve terminals. BNP did not affect on glycinergic sIPSCs and eIPSCs. Moreover, both ANP and BNP had no effect on glutamatergic EPSCs in hippocampal CA3 neurons. The results indicate a potent and selective presynaptic inhibitory action of ANP on glycinergic transmission in spinal cord sensory circuits.

Topics: 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Anesthetics, Local; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cyclic GMP; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Hippocampus; Inhibitory Postsynaptic Potentials; Lidocaine; Neurons; Peptides, Cyclic; Potassium Channel Blockers; Presynaptic Terminals; Rats; Rats, Wistar; Spinal Cord; Thionucleotides

The retinal pigment epithelium (RPE) expresses aquaporin-1 (AQP1) and components of the natriuretic peptide signaling pathway. We hypothesized that stimulation of the natriuretic signaling pathway in RPE with atrial natriuretic peptide (ANP) and with membrane-permeable analogs of cGMP would induce a net apical-to-basal transport of fluid.. The hypothesis was tested using human RPE cultures that retain properties seen in vivo. Confluent monolayers were treated with ANP or membrane-permeable cGMP analogs in the presence of anantin, H-8, and an AQP1 inhibitor, AqB013. Fluid movement from the apical to basal chambers was measured by weight and used to calculate net fluid transport.. Our results demonstrated a 40% increase in net apical-to-basal fluid transport by ANP (5 μM) that was inhibited completely by the ANP receptor antagonist anantin and a 60% increase in net apical-to-basal fluid transport in response to the extracellularly applied membrane-permeable cGMP analog pCPT-cGMP (50 μM), which was not affected by the protein kinase G inhibitor H-8. The aquaporin antagonist AqB013 (20 μM) inhibited the cGMP-stimulated RPE fluid flux.. The effect of cGMP is consistent with an enhancement of the net fluid flux in RPE mediated by AQP1 channels. Pharmacologic activation of cGMP signaling and concomitant stimulation of fluid uptake from the subretinal space could offer insights into a new approach to treating or reducing the risk of retinal detachment.

Topics: Animals; Aquaporin 1; Atrial Natriuretic Factor; Biological Transport, Active; Blotting, Western; Cell Membrane Permeability; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Humans; Isoquinolines; Peptides, Cyclic; Retinal Pigment Epithelium; Water; Xenopus laevis

By the patch clamp technique, whole-cell currents induced by brain natriuretic peptide (BNP) from isolated bullfrog retinal Müller cells were studied. Application of 100 nM BNP induced a sustained inward current from these cells with a reversal potential of about 0 mV, and the current could be completely blocked by anantin, an antagonist of the A-type NP receptor (NPR-A) and CdCl(2), a blocker of cyclic nucleotide-gated (CNG) non-selective cation channels. Likewise, perfusion with the membrane-permeable cGMP analog 8-bromoguanosine-3',5'-cyclic monophosphate (8Br-cGMP) caused effects that are similar to those of BNP. Moreover, application of BNP failed to induce any current in the presence of 1 mM 8Br-cGMP. By calcium imaging, we further showed a significant increase in intracellular calcium levels ([Ca(2+)](i)) of all parts of Müller cells, including the endfoot, soma and processes following the perfusion of BNP, and the increase could be blocked by anantin. All these results suggest that NPR-A is expressed in bullfrog Müller cells, and activation of the receptor causes an increase of intracellular cGMP levels that activates CNG channels and thereby results in an increased calcium influx.

Topics: Animals; Calcium; Cell Separation; Cyclic AMP; Cyclic GMP; Diagnostic Imaging; Fluorescent Dyes; Fura-2; Image Processing, Computer-Assisted; In Vitro Techniques; Natriuretic Peptide, Brain; Patch-Clamp Techniques; Peptides, Cyclic; Rana catesbeiana; Receptors, Atrial Natriuretic Factor; Retina

Natriuretic peptides (NPs) may work as neuromodulators through their associated receptors [NP receptors (NPRs)]. By immunocytochemistry, we showed that NPR-A and NPR-B were expressed abundantly on both ON-type and OFF-type bipolar cells (BCs) in rat retina, including the dendrites, somata, and axon terminals. Whole-cell recordings made from isolated ON-type BCs further showed that brain natriuretic peptide (BNP) suppressed GABAA receptor-, but not GABAC receptor-, mediated currents of the BCs, which was blocked by the NPR-A antagonist anantin. The NPR-C agonist c-ANF [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF(4-23)-NH2] did not suppress GABAA currents. The BNP effect on GABAA currents was abolished with preincubation with the pGC-A/B antagonist HS-142-1 but mimicked by application of 8-bromoguanosine-3',5'-cyclomonophosphate. These results suggest that elevated levels of intracellular cGMP caused by activation of NPR-A may mediate the BNP effect. Internal infusion of the cGMP-dependent protein kinase G (PKG) inhibitor KT5823 essentially blocked the BNP-induced reduction of GABAA currents. Moreover, calcium imaging showed that BNP caused a significant elevation of intracellular calcium that could be caused by increased calcium release from intracellular stores by PKG. The BNP effect was blocked by the ryanodine receptor modulators caffeine, ryanodine, and ruthenium red but not by the IP3 receptor antagonists heparin and xestospongin-C. Furthermore, the BNP effect was abolished after application of the blocker of endoplasmic reticulum Ca2+-ATPase thapsigargin and greatly reduced by the calmodulin inhibitors W-7 and calmidazolium. We therefore conclude that the increased calcium release from ryanodine-sensitive calcium stores by BNP may be responsible for the BNP-caused GABAA response suppression in ON-type BCs through stimulating calmodulin.

Topics: Animals; Atrial Natriuretic Factor; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Calmodulin; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Guanylate Cyclase; Heparin; Imidazoles; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Membrane Potentials; Natriuretic Peptide, Brain; Oxazoles; Patch-Clamp Techniques; Peptide Fragments; Peptides, Cyclic; Polysaccharides; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor; Receptors, Cytoplasmic and Nuclear; Receptors, GABA; Receptors, GABA-A; Retinal Bipolar Cells; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P<0.01) diuresis in SHR (0.21+/-0.04 vs 1.16+/-0.06 ml h(-1) 100 g(-1)), SD (0.42+/-0.06 vs 1.56+/-0.19 ml h(-1) 100 g(-1)) and WKY (0.12+/-0.04 vs 1.44+/-0.21 ml h(-1) 100 g(-1)). Moxonidine-stimulated urine output was lower in SHR than in SD and WKY. Moxonidine-stimulated sodium and potassium excretions were lower in SHR than in SD, but not WKY, demonstrating an influence of strain but not of pressure. Pretreatment with the natriuretic peptide antagonist anantin (5 or 10 microg) resulted in dose-dependent inhibition of moxonidine-stimulated urinary actions. Anantin (10 microg) inhibited (P<0.01) urine output to 0.38+/-0.06, 0.12+/-0.01, and 0.16+/-0.04 ml h(-1) 100 g(-1) in SD, WKY, and SHR, respectively. Moxonidine increased (P<0.01) plasma ANP in SD (417+/-58 vs 1021+/-112 pg ml(-1)) and WKY (309+/-59 vs 1433+/-187 pg ml(-1)), and in SHR (853+/-96 vs 1879+/-229 pg ml(-1)). These results demonstrate that natriuretic peptides mediate the urinary actions of moxonidine through natriuretic peptide receptors.

Topics: Animals; Antihypertensive Agents; Benzofurans; Clonidine; Cyclic GMP; Diuresis; Dose-Response Relationship, Drug; Female; Imidazoles; Imidazoline Receptors; Natriuresis; Natriuretic Peptides; Peptides, Cyclic; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Receptors, Drug; Yohimbine

Intracerebroventricular (i.v.t.) administration of renin (R) decreases urinary volume and increases urinary sodium excretion. We investigated whether i.v.t.-R-induced natriuresis could be associated with the release of atrial natriuretic peptide (ANP), its interaction with renal ANP-A receptors (ANPR-A) and the subsequent increase of urinary cyclic 3-5 guanosine monophosphate (cGMP).. In i.v.t. cannulated rats, the left carotid artery was catheterised with PE-50 tubing for blood collection, renin was injected i.v.t. and arterial blood samples were collected at 0, 2, 5, 10 and 15 minutes of injection, and urinary sodium and cGMP excretion at 1-, 3- and 6-hour periods of urine collection. Plasma ANP levels and urinary cGMP were determined by radioimmunoassay, and each urine sample was analysed for sodium concentration using a flame photometer.. Our results demonstrate that i.v.t.-R administration increases plasma ANP levels after two minutes of injection and urinary cGMP concentration at 1-, 3- and 6 hour period of urine collection. The natriuretic action induced by i.v.t.-R was blunted by peripheral administration of anantin, an ANPR-A antagonist. We assessed the role of brain angiotensin II (Ang II) AT1-receptors on the i.v.t.-induced antidiuresis, natriuresis and cGMP urinary excretion, the last as an indirect index of ANP secretion. Blockade of brain Ang II AT1-receptors with losartan (LOS; 120 microg/3 microl, i.v.t.), inhibited the antidiuretic action and blocked the increased urinary sodium and cGMP excretion induced by i.v.t.-R (7.14 mGU/5 microl). The increase in urinary cGMP was independent of nitric oxide synthase stimulation, since L-NAME pre-treatment did not alter the renal actions induced by i.v.t.-R.. Our results suggest that there is a link between the brain and the kidney. The activation of brain angiotensinergic neurons and stimulation of AT1- receptors may stimulate the release of ANP to the circulation. The released ANP circulates to the kidneys where it acts through renal ANPR-As and the consequent increase in cGMP to produce natriuresis.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Atrial Natriuretic Factor; Brain; Cyclic GMP; Diuresis; Injections, Intraventricular; Losartan; Male; Natriuresis; Neurotransmitter Agents; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, Atrial Natriuretic Factor; Renin; Time Factors

Every living cell must detect, and respond appropriately to, external signals. The functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are thus intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. We have devised a method by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HT(Dro7) and 5HT(Dro1A)]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor was shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results uncovered a new role for cGMP signaling in tubule and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists.

Topics: Aequorin; Animals; Calcium; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Drosophila; Drosophila Proteins; Ligands; Malpighian Tubules; Membrane Glycoproteins; Peptides, Cyclic; Physiology; Radioimmunoassay; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Serotonin; Reverse Transcriptase Polymerase Chain Reaction; Second Messenger Systems; Signal Transduction; Transgenes

We tested both relaxation and cGMP generation by atrial (ANP), brain (BNP), and C-type natriuretic peptide (CNP) in oxytocin-stimulated myometrium from near-term pregnant guinea pigs to investigate the ability and mechanism of natriuretic peptides to inhibit myometrial contractility. Myometrial strips were contracted by 10(-8) M oxytocin, and relaxation to the cumulative addition (10(-9)-10(-6) M) of the natriuretic peptides measured. Maximal relaxation to BNP was significantly greater than to ANP (52 versus 32% respectively; p < 0.05), whereas CNP failed to produce relaxation. However, the increase in cGMP produced by BNP (10(-7) M) was significantly less than that produced by ANP (10(-7) M) (4.5 versus 7.0 times basal; p < 0.05); CNP did not increase myometrial cGMP. Anantin, a competitive blocker of the guanylate cyclase A receptor, significantly reduced the increase in cGMP produced by ANP and BNP, but had no effect on relaxation induced by either peptide. Rp-8-Br-cGMP, an inhibitor of the cGMP-dependent protein kinase, did not alter BNP-induced relaxation. The atrial natriuretic peptide-fragment 4-23 amide, a natriuretic peptide clearance receptor agonist, failed to inhibit oxytocin-stimulated myometrial contraction. We conclude that natriuretic peptide induced relaxation of oxytocin-stimulated myometrium from the pregnant guinea pig is not mediated by either guanylate cyclase A or B activation, is independent of the cGMP pathway, and does not involve clearance receptor activation. Our results suggest that natriuretic peptide-induced relaxation of pregnant myometrium is mediated via a novel mechanism.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Female; Guanylate Cyclase; Guinea Pigs; Muscle Relaxation; Myometrium; Natriuretic Peptide, Brain; Oxytocin; Peptides, Cyclic; Pregnancy; Pregnancy, Animal; Uterine Contraction

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.

Topics: Adjuvants, Immunologic; Adrenomedullin; Animals; Atrial Natriuretic Factor; Cats; Cell Line; Cell Line, Transformed; Cell Membrane; Cells, Cultured; Cholera Toxin; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; GTP-Binding Proteins; Guanylate Cyclase; Humans; Iris; Kinetics; Models, Biological; Muscle, Smooth; Peptides; Peptides, Cyclic; Pertussis Toxin; Time Factors; Vasodilator Agents; Virulence Factors, Bordetella

Anantin, a peptide binding to the receptor of the atrial natriuretic factor (ANF) was isolated from a strain of Streptomyces coerulescens. The molecule consists of 17 natural L-amino acids which form a peptidic ring system. It has a MW of 1,871.0. The chemical composition is C90H111N21O24. The compound was found to bind competitively to ANF-receptors from bovine adrenal cortex (Kd = 0.61 microM). Furthermore, it dose-dependently inhibited the ANF-induced intracellular cyclic guanosine monophosphate accumulation in bovine aorta smooth muscle cells. At the same concentration no agonistic effects were detectable in these cells. Thus, anantin is considered to be the first microbially produced antagonist of the cardiac hormone, ANF.

Topics: Adrenal Cortex; Amino Acids; Animals; Atrial Natriuretic Factor; Binding, Competitive; Cattle; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Esters; Fermentation; Mass Spectrometry; Muscle, Smooth, Vascular; Peptides, Cyclic; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Soil Microbiology; Spectrophotometry, Infrared; Streptomyces