cyclic-gmp and Bradycardia

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 μM. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

Topics: Binding Sites; Bradycardia; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Muscle Proteins; Nucleotides, Cyclic; Potassium Channels; Sinoatrial Node

Neural reflex mechanisms, such as the baroreflex, are involved in regulating cardiovascular system activity. Previous results showed that the ventral portion of the medial prefrontal cortex (vMPFC) is involved in modulation only of the cardiac baroreflex bradycardic component. Moreover, vMPFC N-methyl-D-aspartate (NMDA) receptors modulate the bradycardia baroreflex, but the baroreflex tachycardic component has not been investigated. Furthermore, glutamatergic neurotransmission into the vMPFC is involved in activation of the cardiac sympathetic and parasympathetic nervous system. Finally, it has been demonstrated that glutamatergic neurotransmission into the vMPFC can be modulated by the endocannabinoid system and that activation of the CB1 cannabinoid receptor by anandamide, an endocannabinoid, can decrease both cardiac baroreflex bradycardic and tachycardic responses. Thus, there is the possibility that glutamatergic neurotransmission into the vMPFC does not modulate only the cardiac bradycardic component of the baroreflex. Therefore, the present study investigated whether glutamatergic neurotransmission into the vMPFC modulates both cardiac baroreflex bradycardic and tachycardic responses. We found that vMPFC bilateral microinjection of the NMDA receptor antagonist AP7 (4 nmol/200 nl), of a selective inhibitor of neuronal nitric oxide (NO) synthase N-propyl (0.08 nmol/200 nl), of the NO scavenger carboxy-PTIO (2 nmol/200 nl), or of the NO-sensitive guanylate cyclase ODQ (2 nmol/200 nl) decreased the baroreflex activity in unanesthetized rats. Therefore, our results demonstrate the participation of NMDA receptors, production of NO, and activation of guanylate cyclase in the vMPFC in the modulation of both cardiac baroreflex bradycardic and tachycardic responses.

Topics: Animals; Autonomic Nervous System; Baroreflex; Bradycardia; Cyclic GMP; Guanylate Cyclase; Male; Nitric Oxide; Prefrontal Cortex; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Tachycardia

We previously reported that the leaf extract of Muntingia calabura L. (Tiliaceae) exerts a potent hypotensive effect in the normotensive rats. The antihypertensive activity of this plant extract, however, is currently unknown. In the present study, we investigated the antihypertensive effects of the n-butanol soluble fraction (BSF) from methanol leaf extract of M. calabura in spontaneously hypertensive rats (SHR), and delineated is underlying mechanisms. The intravenous bolus administration of the BSF (10-100 mg/kg) of M. calabura produced biphasic dose-related antihypertensive and bradycardiac effects in SHR. The BSF-induced initial cardiovascular depressive effects lasted for 10 min, and the delayed effects commenced 40 min and lasted for at least 120 min postinjection. These cardiovascular depressive effects of BSF treatments were greater in SHR than in normotensive Wistar-Kyoto (WKY) rats. Both the initial and delayed antihypertensive and bradycardiac effects of BSF (25 mg/kg, i.v.) in SHR, were significantly blocked by pretreatment with a nonselective nitric oxide (NO) synthase (NOS) inhibitor, a soluble guanylyl cyclase (sGC) inhibitor, or a protein kinase G (PKG) inhibitor. Moreover, the initial effects of BSF in SHR were inhibited by pretreatment with a selective endothelial NOS (eNOS) inhibitor; whereas the delayed effects were attenuated by a selective inducible NOS (iNOS) inhibitor. These results indicate that the BSF from the leaf of M. calabura elicited both transient and delayed antihypertensive and bradycardiac actions in SHR, which might be mediated through NO generated respectively by eNOS and iNOS. Furthermore, activation of sGC/cGMP/PKG signaling pathway may participate in the M. calabura-induced biphasic cardiovascular effects.

Topics: Animals; Antihypertensive Agents; Bradycardia; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Plant Extracts; Plant Leaves; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction; Tiliaceae

The neuronal isoform of the enzyme nitric oxide synthase (nNOS) has been identified in the caudal ventrolateral medulla of the rat close to the location of cardiac vagal motoneurones. Therefore in this study we tested identified ventral medulla cardioinhibitory sites for the involvement of nitric oxide (NO) in the baroreceptor-heart rate reflex pathway. In rats anaesthetised with a mixture of urethane (650 mg kg(-1)) and chloralose (50 mg kg(-1)) i.v., blood pressure and heart rate were monitored continuously and using stereotaxic coordinates the ventrolateral caudal brainstem within and around the nucleus ambiguus was systematically explored for sites producing a bradycardia of >50 bpm, without a change in blood pressure, using D,L homocysteic acid (DLH, 0.2 M) microinjections (50 nl) from a glass micropipette. Identified sites were marked with pontamine sky blue. Microinjection of the NO donor sodium nitroprusside (SNP, 1 mM, 50 nl) at a cardioinhibitory site also produced a significant bradycardia (68+/-14 bpm) while the NOS inhibitor N(G)-nitro-l-arginine (l-NNA) (3 mM, 50 nl) caused a small significant increase in heart rate (5+/-1 bpm). Baroreceptor reflex gain measured by the response in heart rate to a change in blood pressure induced by phenylephrine i.v. was significantly increased (610+/-171%, p<0.05) during the steady state of the response to SNP, whereas it was significantly reduced (73+/-5%, p<0.01) by l-NNA injection at a medullary cardioinhibitory site. An inhibitor of soluble guanylyl cyclase, (1)H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 1 mM, 50 nl) also significantly reduced the baroreceptor reflex gain (63+/-8%, p<0.05). The results suggest that a NOS-cGMP signalling system in the baroreceptor reflex pathway distal to the NTS and closer to cardiac vagal motoneurones in the caudal ventral medulla contributes to enhancement of cardiac vagal tone.

Topics: Animals; Baroreflex; Bradycardia; Cyclic GMP; Heart Rate; Male; Medulla Oblongata; Neural Inhibition; Nitric Oxide; Rats; Rats, Wistar; Signal Transduction; Vagus Nerve

Endogenous opioids and nitric oxide (NO) are recognized modulators of cardiac function. Enkephalins and inhibitors of NO synthase (NOS) both produce similar interruptions in the vagal control of heart rate. This study was conducted to test the hypothesis that NO systems within the canine sinoatrial (SA) node facilitate local vagal transmission and that the endogenous enkephalin methionine-enkephalin-arginine-phenylalanine (MEAP) attenuates vagal bradycardia by interrupting the NOS-cGMP pathway. Microdialysis probes were inserted into the SA node, and they were perfused with nonselective (Nomega-nitro-l-arginine methyl ester) and neuronal (7-nitroindazole) NOS inhibitors. The right vagus nerve was stimulated and both inhibitors gradually attenuated the resulting vagal bradycardia. The specificity of these inhibitions was verified by an equally gradual reversal of the inhibition with an excess of the NOS substrate l-arginine. Introduction of MEAP into the nodal interstitium produced a quickly developing but quantitatively similar interruption of vagal bradycardia that was also slowly reversed by the addition of l-arginine and not by d-arginine. Additional support for convergence of opioid and NO pathways was provided when the vagolytic effects of MEAP were also reversed by the addition of the NO donor S-nitroso-N-acetyl-penicillamine, the protein kinase G activator 8-bromo-cGMP, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. MEAP and 7-nitroindazole were individually combined with the direct acting muscarinic agonist methacholine to evaluate potential interactions with muscarinic receptors within the SA node. MEAP and 7-nitroindazole were unable to overcome the bradycardia produced by methacholine. These data suggest that NO and enkephalins moderate the vagal control of heart rate via interaction with converging systems that involve the regulation of cAMP within nodal parasympathetic nerve terminals.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arginine; Bradycardia; Cyclic GMP; Dogs; Dose-Response Relationship, Drug; Enkephalin, Methionine; Enzyme Inhibitors; Indazoles; Methacholine Chloride; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Parasympathomimetics; Penicillamine; Phosphodiesterase Inhibitors; Sinoatrial Node; Vagus Nerve

Topics: Bradycardia; Calcium Channels; Cyclic GMP; Guanylate Cyclase; Humans; Receptors, Muscarinic

1. We tested the hypothesis that nitric oxide (NO) augments vagal neurotransmission and bradycardia via phosphorylation of presynaptic calcium channels to increase vesicular release of acetylcholine. 2. The effects of enzyme inhibitors and calcium channel blockers on the actions of the NO donor sodium nitroprusside (SNP) were evaluated in isolated guinea-pig atrial-right vagal nerve preparations. 3. SNP (10 microM) augmented the heart rate response to vagal nerve stimulation but not to the acetylcholine analogue carbamylcholine (100 nM). SNP also increased the release of [3H]acetylcholine in response to field stimulation. No effect of SNP was observed on either the release of [3H] acetylcholine or the HR response to vagal nerve stimulation in the presence of the guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM). 4. The phosphodiesterase 3 (PDE 3) inhibitor milrinone (1 microM) increased the release of [3H] acetylcholine and the vagal bradycardia and prevented any further increase by SNP. SNP was still able to augment the vagal bradycardia in the presence of the protein kinase G inhibitor KT5823 (1 microM) but not after protein kinase A (PKA) inhibition with H-89 (0.5 microM) or KT5720 (1 microM) had reduced the HR response to vagal nerve stimulation. Neither milrinone nor H-89 changed the HR response to carbamylcholine. 5. SNP had no effect on the magnitude of the vagal bradycardia after inhibition of N-type calcium channels with omega-conotoxin GVIA (100 nM). 6. These results suggests that NO acts presynaptically to facilitate vagal neurotransmission via a cGMP-PDE 3-dependent pathway leading to an increase in cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels. This pathway may augment the HR response to vagal nerve stimulation by increasing presynaptic calcium influx and vesicular release of acetylcholine.

Topics: Acetylcholine; Alkaloids; Animals; Bradycardia; Calcium Channel Blockers; Calcium Channels; Carbachol; Carbazoles; Cardiotonic Agents; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Female; Guanylate Cyclase; Guinea Pigs; Heart Rate; In Vitro Techniques; Indoles; Isoquinolines; Milrinone; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; omega-Conotoxin GVIA; Oxadiazoles; Phosphorylation; Presynaptic Terminals; Quinoxalines; Sinoatrial Node; Sulfonamides; Synaptic Transmission; Tritium; Vagus Nerve

Nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS). NO stimulates the soluble form of guanylyl cyclase (sGC) and induces accumulation of cyclic guanosine monophosphate (cGMP). The purpose of this study was to examine whether the cardiovascular responses induced by N-methyl-D-aspartate (NMDA) in the rostral ventrolateral medulla (RVLM) depend on the actions of NOS and sGC. In anesthetized cats, the extracellular NO level was measured by in vivo voltammetry using a nafion/porphyrine/o-phenylenediamine-coated carbon-fiber electrode. Microinjection of NMDA into the RVLM produced hypertension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a sGC inhibitor. These findings suggest that NO is involved in the NMDA-induced cardiovascular responses in the RVLM.

Topics: Animals; Blood Pressure; Bradycardia; Cats; Cyclic GMP; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Female; Guanylate Cyclase; Heart Rate; Hypertension; Indazoles; Male; Medulla Oblongata; N-Methylaspartate; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxadiazoles; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Signal Transduction

We tested the hypothesis that natriuretic peptide receptors (NPRs) that are coupled to cGMP production act in a similar way to nitric oxide (NO) by enhancing acetylcholine release and vagal-induced bradycardia. The effects of enzyme inhibitors and channel blockers on the action of atrial natriuretic peptide (ANP), brain-derived natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) were evaluated in isolated guinea pig atrial-right vagal nerve preparations. RT-PCR confirmed the presence NPR B and A receptor mRNA in guinea pig sinoatrial node tissue. BNP and CNP significantly (P < 0.05) enhanced the heart rate (HR) response to vagal nerve stimulation. CNP had no effect on the HR response to carbamylcholine and facilitated the release of [(3)H]acetylcholine during atrial field stimulation. The particulate guanylyl cyclase-coupled receptor antagonist HS-142-1, the phosphodiesterase 3 inhibitor milrinone, the protein kinase A inhibitor H89, and the N-type calcium channel blocker omega-conotoxin all blocked the effect of CNP on vagal-induced bradycardia. Like NO, BNP and CNP facilitate vagal neurotransmission and bradycardia. This may occur via a cGMP-PDE3-dependent pathway increasing cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels.

Topics: Acetylcholine; Animals; Atrial Natriuretic Factor; Bradycardia; Calcium Channel Blockers; Carbachol; Cardiotonic Agents; Cyclic GMP; Enzyme Inhibitors; Female; Gene Expression; Guanylate Cyclase; Guinea Pigs; Heart Rate; Isoquinolines; Milrinone; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Nitric Oxide; omega-Conotoxins; Phosphodiesterase Inhibitors; Polysaccharides; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Sulfonamides; Synaptic Transmission; Tritium; Vagus Nerve

The purpose of this study was to evaluate whether cordocentesis is associated with the release of vasoactive substances and whether there are differences between normally grown and growth-restricted fetuses.. 6-Keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin), endothelin-1, and cyclic guanosine monophosphate were measured in fetal blood at the beginning and closing of cordocentesis in 30 normally grown fetuses and 25 growth-restricted fetuses. This latter group was characterized by abnormal Doppler index values in umbilical artery and middle cerebral artery, suggestive of chronic hypoxemia as the causative factor of the impaired growth. In six growth-restricted fetuses bradycardia occurred at the end of the procedure. Umbilical artery pulsatility index was measured by Doppler ultrasonography immediately before and after the procedure.. The median interval between the two blood samples obtained by cordocentesis was 90 seconds (range 60 to 320 seconds). During this interval a significant rise of 6-keto-prostaglandin F1 alpha (p < or = 0.0001) and endothelin-1 (p = 0.03) was evidenced in normally grown fetuses. The increase in 6-keto-prostaglandin F1 alpha was significantly related (r = 0.52, p = 0.002) to the fall of umbilical artery pulsatility index occurring after the procedure. In growth-restricted fetuses cordocentesis induced a marked increase of endothelin-1 (p = 0.0002), which was significantly related to the severity of acidosis (r = 0.52, p = 0.018), whereas no modifications were evidenced for the other agents tested. The increase of endothelin-1 was higher in those growth-restricted fetuses showing bradycardia at the end of the procedure than in growth-restricted fetuses that did not (p = 0.04). The variations of the vasoactive substances assayed were not significantly related to the type of procedure (transamniotic or transplacental), the amount of blood aspirated during the procedure, the interval elapsing between the first and second samples, the gestational age at which the procedure was performed, and the degree of fetal smallness.. Cordocentesis induces the rapid release of vasoactive substances and the effect differs between normally grown and growth-restricted fetuses. This may explain the different hemodynamic response and the higher rate of complications occurring in the latter group after cordocentesis.

Topics: 6-Ketoprostaglandin F1 alpha; Bradycardia; Cordocentesis; Cyclic GMP; Endothelin-1; Female; Fetal Blood; Fetal Growth Retardation; Gestational Age; Humans; Pregnancy; Pulsatile Flow; Umbilical Arteries

The effects of a continuous i.v. infusion of urodilatin at a dose of 30 ng kg-1 min-1 were studied in a patient with congestive heart failure. After 30 min, urodilatin had induced a marked stimulation of plasma cyclic GMP concentrations. In parallel haematocrit increased. No significant diuresis and no change of invasive haemodynamics was observed. After 2 h the patient developed a profuse perspiration. Eighty minutes later he suffered from dizziness due to hypotension (blood pressure 80/40 mmHg) and a sudden bradycardia (50 bpm). Urodilatin was discontinued and symptoms were relieved by bed tilt and rapid infusion of isotonic saline solution. Mechanisms contributing to these adverse effects may be fluid extravasation to the third space and sympathoinhibitory effects known to occur with natriuretic peptide infusion.

Topics: Atrial Natriuretic Factor; Blood Pressure; Bradycardia; Cardiac Output; Cyclic GMP; Heart Failure; Heart Rate; Hematocrit; Humans; Hypotension; Infusions, Intravenous; Male; Middle Aged; Peptide Fragments