Two-pore K+ channels, NO and metabolic inhibition

Ischemic preconditioning is a potent endogenous mechanism protecting many organs from the devastating effects of prolonged ischemia. In the heart, NO is one mediator of this myoprotective response thought to involve activation of the K(ATP) channel. Ischemic preconditioning is known to be induced by metabolic inhibition using sodium cyanide (NaCN) in single cardiomyocytes. In the present study, we show for the first time that the end effector channel activated by NaCN has been incorrectly identified. The channel activated is not K(ATP) but instead belongs to the relatively new family of two-pore domain potassium channels (K2P). Further when activated by metabolic ischemia, the amplitude of K2P current is directly modulated by activators and inhibitors of the NO pathway.     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.     

Impact of combined NO and PG blockade on rapid vasodilation in a forearm mild-to-moderate exercise transition in humans

We tested the hypothesis that nitric oxide (NO) and prostaglandins (PGs) contribute to the rapid vasodilation that accompanies a transition from mild to moderate exercise. Nine healthy volunteers (2 women and 7 men) lay supine with forearm at heart level. Subjects were instrumented for continuous brachial artery infusion of saline (control condition) or combined infusion of N(G)-nitro-L-arginine methyl ester (L-NAME) and ketorolac (drug condition) to inhibit NO synthase and cyclooxygenase, respectively. A step increase from 5 min of steady-state mild (5.4 kg) rhythmic, dynamic forearm handgrip exercise (1 s of contraction followed by 2 s of relaxation) to moderate (10.9 kg) exercise for 30 s was performed. Steady-state forearm blood flow (FBF; Doppler ultrasound) and forearm vascular conductance (FVC) were attenuated in drug compared with saline (control) treatment: FBF = 196.8 +/- 30.8 vs. 281.4 +/- 34.3 ml/min and FVC = 179.3 +/- 29.4 vs. 277.8 +/- 34.8 ml.min(-1).100 mmHg(-1) (both P < 0.01). FBF and FVC increased from steady state after release of the initial contraction at the higher workload in saline and drug conditions: DeltaFBF = 72.4 +/- 8.7 and 52.9 +/- 7.8 ml/min, respectively, and DeltaFVC = 66.3 +/- 7.3 and 44.1 +/- 7.0 ml.min(-1).100 mmHg(-1), respectively (all P < 0.05). The percent DeltaFBF and DeltaFVC were not different during saline infusion or combined inhibition of NO and PGs: DeltaFBF = 27.2 +/- 3.1 and 28.1 +/- 3.8%, respectively (P = 0.78) and DeltaFVC = 25.7 +/- 3.2 and 26.0 +/- 4.0%, respectively (P = 0.94). The data suggest that NO and vasodilatory PGs are not obligatory for rapid vasodilation at the onset of a step increase from mild- to moderate-intensity forearm exercise. Additional vasodilatory mechanisms not dependent on NO and PG release contribute to the immediate and early increase in blood flow in an exercise-to-exercise transition.     

Chronic hypoxia increases the NO contribution of acetylcholine vasodilation of the fetal guinea pig heart

To investigate the effect of chronic hypoxia (HPX) on vasodilation of the fetal heart, we exposed pregnant guinea pigs to room air or 12% O(2) for 4, 7, or 10 days. We excised hearts from anesthetized fetuses (60 +/- 3 days; 65-day gestation = term) and measured changes in both the coronary artery pressure of the isolated constant-flow preparation and endothelial nitric oxide synthase (eNOS) mRNA of fetal ventricles. Dilator responses to cumulative addition (10(-9)-10(-5) M) of acetylcholine and sodium nitroprusside in prostaglandin F(2alpha) (5 x 10(-6) M)-constricted hearts were similar among normoxia (NMX), 4-, 7-, and 10-day HPX (control). Nitro-L-arginine (L-NA, 10(-4)M), a NOS inhibitor, inhibited maximal acetylcholine dilation of hearts exposed to 10-day HPX greater than NMX, 4-, and 7-day HPX. Hypoxia (after 7 and 10 days) increased eNOS mRNA of fetal ventricles compared with NMX and 4-day HPX. 4-Aminopyridine (3 mM), a voltage-dependent K(+)-channel inhibitor, inhibited acetylcholine- but not sodium nitroprusside-induced dilation of NMX and 10-day HPX hearts to a similar magnitude. Glibenclamide (10(-5) M), an ATP-sensitive K(+)-channel inhibitor, had no effect on vasodilation. We conclude that chronic HPX increases the contribution of NO but does not alter K(+)-channel activation in response to acetylcholine-stimulated coronary dilation. Thus increases in NO production via upregulation of eNOS gene expression may be an adaptive response to chronic HPX in the fetal coronary circulation.     

K(ATP)(+) channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation

The role of ATP-sensitive K(+) (K(ATP)(+)) channels, nitric oxide, and adenosine in coronary exercise hyperemia was investigated. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus and instrumented with a flow transducer on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Experiments were conducted at rest and during graded treadmill exercise with and without combined inhibition of K(ATP)(+) channels (glibenclamide, 1 mg/kg iv), nitric oxide synthesis (N(omega)-nitro-L-arginine, 35 mg/kg iv), and adenosine receptors (8-phenyltheophylline, 3 mg/kg iv). During control exercise, myocardial oxygen consumption increased ~2.9-fold, coronary blood flow increased ~2.6-fold, and coronary venous oxygen tension decreased from 19.9 +/- 0.4 to 13.7 +/- 0.6 mmHg. Triple blockade did not significantly change the myocardial oxygen consumption or coronary blood flow response during exercise but lowered the resting coronary venous oxygen tension to 10.0 +/- 0.4 mmHg and during exercise to 6.2 +/- 0.5 mmHg. Cardiac adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade. These results indicate that combined inhibition of K(ATP)(+) channels, nitric oxide synthesis, and adenosine receptors lowers the balance between total oxygen supply and consumption at rest but that these factors are not required for local metabolic coronary vasodilation during exercise.     

Ischemia reperfusion injury, KATP channels, and exercise-induced cardioprotection against apoptosis

Exercise is a potent stimulus against cardiac ischemia reperfusion (IR) injury, although the protective mechanisms are not completely understood. The study purpose was to examine whether the mitochondrial or sarcolemmal ATP-sensitive potassium channel (mito K(ATP) or sarc K(ATP), respectively) mediates exercise-induced cardioprotection against post-IR cell death and apoptosis. Eighty-six, 4-mo-old male Sprague Dawley rats were randomly assigned to treadmill exercise (Ex; 30 m/min, 3 days, 60 min, ～70 maximal oxygen uptake) and sedentary (Sed) treatments. Rats were exposed to regional cardiac ischemia (50 min) and reperfusion (120 min) or Sham (170 min; no ligation) surgeries. Exercise subgroups received placebo (saline), 5-hydroxydecanoate (5HD; 10 mg/kg ip), or HMR1098 (10 mg/kg ip) to inhibit mito K(ATP) or sarc K(ATP) channel. Comprehensive outcome assessments included post-IR ECG arrhythmias, cardiac tissue necrosis, redox perturbations, and autophagy biomarkers. No arrhythmia differences existed between exercised and sedentary hearts following extended-duration IR (P < 0.05). The sarc K(ATP) channel was confirmed essential (P = 0.002) for prevention of antinecrotic tissue death with exercise (percent infarct, Sed = 42%; Ex = 20%; Ex5HD = 16%; ExHMR = 42%), although neither the mito K(ATP) (P = 0.177) nor sarc K(ATP) (P = 0.274) channel provided post-IR protection against apoptosis (terminal deoxynucleotidyl transferase deoxy UTP-mediated nick-end labeling-positive nuclei/mm(2), Sham = 1.8 ± 0.5; Sed = 19.4 ± 6.7; Ex = 7.5 ± 4.6; Ex5HD = 14.0 ± 3.9; ExHMR = 11.1 ± 1.8). Exercise preconditioning also appears to preserve basal autophagy levels, as assessed by Beclin 1 (P ≤ 0.001), microtubule-associated protein-1 light-chain 3B ratios (P = 0.020), and P62 (P ≤ 0.001), in the hours immediately following IR. Further research is needed to better understand these findings and corresponding redox changes in exercised hearts.     

The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)‐injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT‐PCR/qRT‐PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down‐regulated in myometrium from LPS‐treated‐mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.     

ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (K(ATP)) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H(2)O(2), or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by K(ATP) channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the K(ATP) channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to K(ATP) channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N(omega)-nitro-l-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by K(ATP) channel activation. Coadministration of H(2)O(2) and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the K(ATP) channel. We conclude that ROS can trigger preconditioning by causing activation of the K(ATP) channel, which then induces generation of ROS and NO that are both required for preconditioning protection.     

Mechanism, temporal patterns, and magnitudes of the metabolic responses to the KATP channel agonist diazoxide

To assess the mechanism, temporal patterns, and magnitudes of the metabolic responses to the ATP-dependent potassium channel agonist diazoxide, neuroendocrine and metabolic responses to intravenous diazoxide (saline, 1.0 and 2.0 mg/kg) and oral diazoxide (placebo, 4.0 and 6.0 mg/kg) were assessed in healthy young adults. Intravenous diazoxide produced rapid, but transient, decrements (P = 0.0023) in plasma insulin (e.g., nadirs of 2.8 +/- 0.5 and 1.8 +/- 0.3 microU/ml compared with 7.0 +/- 1.0 microU/ml after saline at 4.0-7.5 min) and C-peptide (P = 0.0228) associated with dose-related increments in plasma glucose (P = 0.0044) and serum nonesterified fatty acids (P < 0.0001). After oral diazoxide, plasma insulin appeared to decline, as did C-peptide, again associated with dose-related increments in plasma glucose (P < 0.0001) and serum nonesterified fatty acids (P = 0.0141). Plasma glucagon, as well as cortisol and growth hormone, was not altered. Plasma epinephrine increased (P = 0.0215) slightly only after intravenous diazoxide. There were dose-related increments in plasma norepinephrine (P = 0.0038 and P = 0.0005, respectively), undoubtedly reflecting a compensatory sympathetic neural response to vasodilation produced by diazoxide, but these would not raise plasma glucose or serum nonesterified fatty acid levels. Thus selective suppression of insulin secretion, without stimulation of glucagon secretion, raised plasma glucose and serum nonesterified fatty acid concentrations. These findings define the temporal patterns and magnitudes of the metabolic responses to diazoxide and underscore the primacy of regulated insulin secretion in the physiological regulation of postabsorptive carbohydrate and lipid metabolism.     

Relative contribution of neurotransmission failure to diaphragm fatigue

Two procedures were used to estimate the relative contribution of neurotransmission failure (NF) to fatigue of the rat diaphragm at different rates of phrenic nerve stimulation. In one, direct muscle stimulation was intermittently superimposed on neural stimulation of the diaphragm, and the relative contribution of NF was estimated by the difference in generated tension. In a second procedure, diaphragm fatigue was induced by using either direct muscle stimulation (with complete blockade of the neuromuscular junction by d-tubocurare) or phrenic nerve stimulation. The relative contribution of NF to diaphragm fatigue was then estimated by comparing the force loss during these two modes of stimulation. With both procedures, it was observed that 1) the relative contribution of NF to diaphragm fatigue was less than 45% at each frequency of phrenic nerve stimulation; 2) the relative contribution of NF to diaphragm fatigue increased at higher rates of phrenic stimulation, reaching a maximum at 75 pulses/s; and 3) the relative contribution of NF to diaphragm fatigue reached a plateau after 2 min of repetitive stimulation.     

Relative contribution of agroforestry,rainforest and openland to local and regional bee diversity

Due to increasing human modification of tropical landscapes, the relative importance of natural habitats and agricultural systems has become a major conservation topic to counteract global species loss. We investigated the contribution of tropical primary forest, cacao agroforestry systems of varying management practices and openland to the temporal and spatial variation of diversity of native bee communities in the herb layer (Apidae, Hymenoptera) in Sulawesi (Indonesia). Local bee density and diversity were highest in openland, followed by agroforestry systems and were lowest in primary forests, revealing the importance of herbaceous food resources in the understorey. In contrast, highest regional bee richness was found in agroforestry systems, because of high community dissimilarity. Multidimensional scaling supported these findings with openland habitats showing more compactly clustered bee species communities than agroforestry habitats. In conclusion, the herb associated bee community profited from the opening of the landscape as a result of agricultural activities, while agroforestry systems increased bee species richness especially on a regional scale due to high management diversity.     

Relative contribution of abundant and rare species to species-energy relationships

A major goal of ecology is to understand spatial variation in species richness. The latter is markedly influenced by energy availability and appears to be influenced more by common species than rare ones; species-energy relationships should thus be stronger for common species. Species-energy relationships may arise because high-energy areas support more individuals, and these larger populations may buffer species from extinction. As extinction risk is a negative decelerating function of population size, this more-individuals hypothesis (MIH) predicts that rare species should respond more strongly to energy. We investigate these opposing predictions using British breeding bird data and find that, contrary to the MIH, common species contribute more to species-energy relationships than rare ones.     

Relative contribution of gravity to pulmonary perfusion heterogeneity.

We designed a series of experiments and analyses to quantify the contribution of gravity to pulmonary perfusion heterogeneity. Regional pulmonary perfusion was measured in five anesthetized and ventilated dogs in both supine and prone positions by use of radiolabeled microspheres injected during apnea at functional residual capacity. Measurements of flow were repeated in each position, and the sequence of positions was prospectively designed to nullify any effect of order. The lungs of each animal were excised, perfused with saline until clear, dried at an inflation pressure of 25 cmH2O, and cut into 1.9-cm3 pieces. Each piece was weighed and the radioactivity determined in a scintillation counter. Measurement errors were minimized by excluding lung pieces that had greater than 25% airway and weighed less than 10 mg or greater than 60 mg. Weight-normalized flows in each position and repetition were determined for each lung piece. An analysis of variance model was used to identify the percentage of variation in regional flow that was due to position (supine vs. prone), to random error and time (measurement and repetition), and to structure, where structure was defined as the component of flow that remained constant across position and replication. The contributions of position, error/time, and structure to the total variability of flow across the five dogs were 7.8 +/- 0.6, 8.4 +/- 8.3, and 83.8 +/- 8.4%, (SD), respectively. Because the contribution of position represents the additive effect of gravity between two opposite positions, the contribution of gravity to perfusion heterogeneity in one position may be as little as 4%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.     

Purinergic contribution to circulatory, metabolic, and adrenergic responses to acute hypoxemia in fetal sheep

This study investigated the effects on femoral vascular resistance, blood glucose and lactate levels, and plasma catecholamine concentrations of fetal treatment with an adenosine receptor antagonist during acute hypoxemia in fetal sheep during late gestation. Under anesthesia, seven fetal sheep were instrumented between 117 and 118 days gestation (term is approximately 145 days) with vascular and amniotic catheters and an ultrasonic probe around a femoral artery. Six days after surgery, all fetuses were randomly subjected to a 3-h experiment consisting of 1 h of normoxia, 1 h of hypoxemia, and 1 h of recovery. This was done during either intravenous infusion of vehicle or the adenosine receptor antagonist [8-(p-sulfophenyl)-theophylline; 8-SPT] dissolved in vehicle. During vehicle infusion, all fetuses responded to hypoxemia with bradycardia, an increase in arterial blood pressure, and femoral vasoconstriction. Increases in blood glucose and lactate concentrations and in plasma epinephrine and norepinephrine concentrations also occurred in all fetuses during hypoxemia. Fetal treatment with 8-SPT markedly attenuated the bradycardic, hypertensive, vasoconstrictor, glycemic, and adrenergic responses to hypoxemia, but it did not affect the increase in blood lactate concentrations during hypoxemia. These data show that adenosine is involved in the mechanisms mediating fetal cardiovascular, metabolic, and adrenergic responses to hypoxemia in fetal sheep. Fetal treatment with 8-SPT mimics the effects of carotid sinus nerve section on fetal cardiovascular function during hypoxemia, suggesting a role for adenosine in mediating fetal cardiovascular chemoreflexes.     

Vascular and neural mechanisms of ACh-mediated vasodilation in the forearm cutaneous microcirculation.

The relative contribution of endothelial vasodilating factors to acetylcholine (ACh)-mediated vasodilation in the forearm cutaneous microcirculation is unclear. The aims of this study were to investigate the contributions of prostanoids and cutaneous C fibers to basal cutaneous blood flow (CuBF) and ACh-mediated vasodilation. ACh was iontophoresed into the forearm, and cutaneous perfusion was measured by laser-Doppler flowmetry. To inhibit the production of prostanoids, four doses of acetylsalicylic acid (ASA; 81, 648, 972, and 1,944 mg) were administered orally. Cutaneous nerve fibers were blocked with topical anesthesia. Cyclooxygenase inhibition did not change basal CuBF or endothelium-mediated vasodilation to ACh. In contrast, ASA (972 and 1,944 mg) significantly reduced the C-fiber-mediated axon reflex in a dose-dependent fashion. Blockade of C-fiber function significantly reduced axon reflex-mediated vasodilation but did not affect basal CuBF or endothelium-dependent vasodilation. The findings suggest that prostanoids do not contribute significantly to basal CuBF or endothelium-dependent vasodilation in the forearm microcirculation. In contrast, prostanoids are mediators of the ACh-provoked axon reflex.