Melatonin enhances the hypoxic response of rat carotid body chemoreceptor

Melatonin attenuates carotid chemoreceptor response to hypercapnic acidosis and may contribute to the effect of circadian rhythms on the chemoreflex. The purpose of this study was to test the hypothesis that melatonin modulates rat carotid chemoreceptor response to hypoxia. To examine the effect of melatonin on the hypoxic response of the chemosensitive cells, cytosolic calcium ([Ca2+]i) was measured by spectrofluorometry in fura-2-loaded type-I (glomus) cells dissociated from rat carotid bodies. Melatonin (0.01-10 nm) did not change the resting Ca2+]i level of the glomus cells but it concentration-dependently increased peak Ca2+]i response to cyanide or deoxygenated buffer. An agonist of melatonin receptors, iodomelatonin also enhanced the Ca2+]i response to hypoxia. The melatonin-induced enhancement of the Ca2+]i response was abolished by pretreatment with nonselective mt1/MT2 antagonist, luzindole, and by MT2 antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest that melatonin receptors in the glomus cells mediate the effect of melatonin on the chemoreceptor response to hypoxia. In addition, melatonin increased the carotid afferent response to hypoxia in unitary activities recorded from the sinus nerve in isolated carotid bodies superfused with bicarbonate-buffer saline. Furthermore, plethysmographic measurement of ventilatory activities in unanesthetized rats revealed that melatonin (1 mg/kg, i.p.) increased the ventilatory response to hypoxia. Hence, the circadian rhythm of melatonin in arterial blood can modulate the carotid chemoreceptor response to hypoxia. This modulation may be a physiological mechanism involved in the day-light differences in ventilatory activities.     

Chronic hypoxia modulates the function and expression of melatonin receptors in the rat carotid body

Melatonin modulates the carotid chemoreceptor response to chemical stimuli, and chronic hypoxia changes circadian activities and carotid body function. The purpose of this study was to test the hypothesis that chronic hypoxia alters the function and expression of melatonin receptors in the rat carotid body. Effects of melatonin on the carotid responses to hypercapnic acidosis and to hypoxia were determined by spectrofluorometric measurement of cytosolic calcium ([Ca(2+)](i)) in fura-2-loaded type-I (glomus) cells dissociated from carotid bodies obtained from normoxic (Nx) or chronically hypoxic (CH) rats breathing 10% oxygen for 4 wk. In the Nx control, melatonin concentration dependently attenuated the peak [Ca(2+)](i) response to hypercapnic acidosis, whereas it augmented the [Ca(2+)](i) response to cyanide or deoxygenated buffer. Yet, melatonin enhanced the peak [Ca(2+)](i) responses to hypercapnic acidosis or hypoxia in the CH glomus cells. An agonist of melatonin receptors, iodomelatonin also elevated the hypercapnic or hypoxic responses in the CH groups. The melatonin-induced changes in the [Ca(2+)](i) responses were abolished by pretreatment with nonselective mt(1)/MT(2) antagonist, luzindole, and by MT(2) antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest a functional modulation of melatonin receptors in the glomus cells in chronic hypoxia. To evaluate the level of expression of the melatonin receptors, in situ hybridization study with antisense mt(1) and MT(2) receptor mRNA oligonucleotide probes was performed on the Nx and CH carotid bodies. There were significant increases in the expression of mt(1) and MT(2) receptors in the CH comparing with the Nx group. Taken together, our results suggest an upregulation of the carotid expression of melatonin receptors by chronic hypoxia, which modulates the carotid response to melatonin for the circadian influence on breathing.     

Vardenafil increases coronary flow response to hypercapnic acidosis in isolated guinea pig heart

The hypothesis was tested that vardenafil, a PDE5 inhibitor, specifically enhances coronary vasodilation during acidosis. In isolated constant pressure perfused guinea pig hearts, infusion of vardenafil (≤ 1 μM) increased coronary flow concentration-dependently 34 % above baseline. In parallel, cGMP release increased (0.44 ± 0.094 vs. 0.14 ± 0.017 pmol/min·g at 0.5 μM vardenafil vs. baseline). Flow increases occurred in the absence of changes in heart function (LVP, heart rate, dP/dt_max, heart rate – pressure product). Infusion of the NO synthase blocker L-NMMA (100 μM) caused a rightward shift of the dose-response curve of vardenafil. To test whether vardenafil treatment may enhance metabolic coronary vasodilation, arterial pCO₂ was raised from 38 to 61mmHg, which resulted in a steady state flow increase of 18.8 ± 4.5%. Infusion of vardenafil, given at a threshold flow enhancing concentration, doubled the coronary flow response during hypercapnic acidosis to 38.4 ± 4.2 % (p = 0.004). This flow amplification during acidosis was not shared by the K_ATP channel opener cromakalim, indicating a specific effect of vardenafil on flow control during myocardial acidosis. We conclude that vardenafil specifically relaxes coronary resistance vessels through NO/cGMP-dependent pathways and increases the coronary flow response toward hypercapnic acidosis. This finding further supports the importance of the NO-cGMP axis in mediation of this flow response.     

Melatonin inhibits nicotinic currents in cultured rat cerebellar granule neurons

There is limited data regarding the effects of melatonin on the activity of neuronal acetylcholine receptors (nAChRs) themselves. This study analyzes the effects of low concentrations of melatonin on nicotine-evoked currents from cerebellar granule neurons (CGNs) in culture. Using electrophysiological and Ca(2+)-imaging techniques, it was found a subset of rat CGNs to which nicotine application elicited both intracellular Ca(2+) transients and inward whole-cell currents. These responses were mediated by heteromeric nAChRs, as assessed by their sensitivity to nicotine and time constant of current decay. Preincubating the cells with low melatonin concentrations (down to 1 pm) significantly reduced the current amplitude in a dose-dependent manner, without affecting the receptor's apparent affinity and voltage-dependency, nor the current's rise and decay time course. The inhibitory effect of melatonin was significantly reduced by luzindole, a competitive antagonist of both MT(1) and MT(2) melatonin receptors. In conclusion, melatonin inhibits nicotinic currents through non-alpha7 heteromeric nAChRs expressed by CGNs in culture, an effect that appears to be at least partially mediated by melatonin membrane receptors. Direct modulation of nicotinic receptors is accomplished at doses that are likely to be physiologically relevant, thus providing a mechanism through which melatonin circadian rhythmic levels could modulate cholinergic activity.     

Melatonin stimulates proliferation and type I collagen synthesis in human bone cells in vitro

The pineal secretory product melatonin reportedly regulates release of growth hormone in humans and prevents phototherapy-induced hypocalcemia in newborn rats, suggesting that melatonin affects bone metabolism. Little is known about the effects of melatonin on bone in vitro or in vivo. The present study was undertaken to examine whether melatonin acts directly on normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO cells) to affect osteogenic action in vitro. The effect of melatonin on bone cell proliferation was determined using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xanilide (XTT) assay after a 24 hr incubation with melatonin. Melatonin significantly and dose-dependently increased the proliferation in HOB-M cells and SV-HFO cells by 215 +/- 22.1%, and 193 +/- 6.4%), respectively, with a maximal effect at a concentration of 50 microM. To evaluate the effect of melatonin on bone cell differentiation, alkaline phosphatase (ALP) activity, osteocalcin secretion and procollagen type I c-peptide (PICP) production (a measure of type I collagen synthesis) were measured after a 48 hr treatment. While melatonin at micromolar concentrations did not significantly affect either the ALP activity or the osteocalcin secretion, it significantly and dose-dependently increased the PICP production in HOB-M cells and SV-HFO cells by 983 +/- 42.2%, and 139 +/- 4.2%, respectively, with the maximal stimulatory doses between 50 and 100 microM. These results provide new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells in vitro, suggesting that melatonin may act to stimulate bone formation.     

Melatonin attenuates diabetic cardiomyopathy and reduces myocardial vulnerability to ischemia‐reperfusion injury by improving mitochondrial quality control: Role of SIRT6

Targeting mitochondrial quality control with melatonin has been found promising for attenuating diabetic cardiomyopathy (DCM), although the underlying mechanisms remain largely undefined. Activation of SIRT6 and melatonin membrane receptors exerts cardioprotective effects while little is known about their roles during DCM. Using high‐fat diet‐streptozotocin‐induced diabetic rat model, we found that prolonged diabetes significantly decreased nocturnal circulatory melatonin and heart melatonin levels, reduced the expressions of cardiac melatonin membrane receptors, and decreased myocardial SIRT6 and AMPK‐PGC‐1α‐AKT signaling. 16 weeks of melatonin treatment inhibited the progression of DCM and the following myocardial ischemia‐reperfusion (MI/R) injury by reducing mitochondrial fission, enhancing mitochondrial biogenesis and mitophagy via re‐activating SIRT6 and AMPK‐PGC‐1α‐AKT signaling. After the induction of diabetes, adeno‐associated virus carrying SIRT6‐specific small hairpin RNA or luzindole was delivered to the animals. We showed that SIRT6 knockdown or antagonizing melatonin receptors abolished the protective effects of melatonin against mitochondrial dysfunction as evidenced by aggravated mitochondrial fission and reduced mitochondrial biogenesis and mitophagy. Additionally, SIRT6 shRNA or luzindole inhibited melatonin‐induced AMPK‐PGC‐1α‐AKT activation as well as its cardioprotective actions. Collectively, we demonstrated that long‐term melatonin treatment attenuated the progression of DCM and reduced myocardial vulnerability to MI/R injury through preserving mitochondrial quality control. Melatonin membrane receptor‐mediated SIRT6‐AMPK‐PGC‐1α‐AKT axis played a key role in this process. Targeting SIRT6 with melatonin treatment may be a promising strategy for attenuating DCM and reducing myocardial vulnerability to ischemia‐reperfusion injury in diabetic patients.     

Rat pial microvascular responses to melatonin during bilateral common carotid artery occlusion and reperfusion

The present study assessed the in vivo rat pial microvascular responses induced by melatonin during brain hypoperfusion and reperfusion (RE) injury. Pial microcirculation of male Wistar rats was visualized by fluorescence microscopy through a closed cranial window. Hypoperfusion was induced by bilateral common carotid artery occlusion (BCCAO, 30 min); thereafter, pial microcirculation was observed for 60 min. Arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (PCL), and capillary red blood cell velocity (V(RBC) ) were investigated by computerized methods. Melatonin (0.5, 1, 2 mg/kg b.w.) was intravenously administered 10 min before BCCAO and at the beginning of RE. Pial arterioles were classified in five orders according to diameter, length, and branchings. In control group, BCCAO caused decrease in order 2 arteriole diameter (by 17.5 ± 3.0% of baseline) that was reduced by 11.8 ± 1.2% of baseline at the end of RE, accompanied by marked leakage and leukocyte adhesion. PCL and capillary V(RBC) decreased. At the end of BCCAO, melatonin highest dosage caused order 2 arteriole diameter reduction by 4.6 ± 2.0% of baseline. At RE, melatonin at the lower dosages caused different arteriolar responses. The highest dosage caused dilation in order 2 arteriole by 8.0 ± 1.5% of baseline, preventing leakage and leukocyte adhesion, while PCL and V(RBC) increased. Luzindole (4 mg/kg b.w.) prior to melatonin caused order 2 arteriole constriction by 12.0 ± 1.5% of baseline at RE, while leakage, leukocyte adhesion, PCL and V(RBC) were not affected. Prazosin (1 mg/kg b.w.) prior to melatonin did not significantly change melatonin's effects. In conclusion, melatonin caused different responses during hypoperfusion and RE, modulating pial arteriolar tone likely by MT1 and MT2 melatonin receptors while preventing blood-brain barrier changes through its free radical scavenging action.     

Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor

Thirty percent of patients undergoing percutaneous transluminal coronary angioplasty develop recurrent disease within a year. This is usually due to the rapid accumulation of intimal smooth muscle cells and extracellular matrix, which causes luminal narrowing, and is probably orchestrated by several mitogenic and chemotactic factors, of which platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) appear to be particularly important. We have investigated the effects of administering a combination of neutralizing antibodies directed against PDGF-BB and bFGF on neo-intima development following balloon catheter injury in the rat carotid artery. Purified sheep anti-PDGF-BB and anti-bFGF immunoglobulins (IgGs) were administered singly and in combination prior to mechanical injury and daily until sacrifice, 8 days later. Plasma titres of exogenous anti-PDGF-BB and anti-bFGF were maintained at levels 10–20-fold higher than those required to neutralise the mitogenic and chemotactic effects of 20 ng/ml of PDGF-BB, or 10 ng/ml bFGF in vitro. Used singly, anti-PDGF IgG treatment was associated with a 47% reduction in intimal thickness and a 59% reduction in intimal:medial area ratio; anti-bFGF IgG administration caused a 53% reduction in intimal thickness, and a 50% reduction in intimal:medial area ratio. Treatment with a combination of these antibodies resulted in a 83.8% reduction in intimal thickness (P<0.05), and a 91% reduction in intimal:medial area ratio (P<0.01). The latter treatment was also associated with a significantly higher intimal cell density (14.2±1.6×10³ nuclei/mm²) compared to animals receiving non-immune IgG (7.8±0.8×10³ nuclei/mm²; P<0.025), although intimal and medial cell proliferation indices were not significantly different between the groups (P>0.05). Our results suggest that in this particular model, PDGF-BB and bFGF are the major factors controlling neointimal hyperplasia, and that these growth factors are operating principally via an effect on smooth muscle cell migration and extracellular matrix protein accumulation.