High Glucose-enhanced Acetylcholine Stimulated CGMP Masks Impaired Vascular Reactivity in Tail Arteries from Short-Term Hyperglycemic Rats

Impaired vascular endothelium-dependent relaxation and augmented contractile responses have been reported in several models of long-term hyperglycemia. However, the effects of short-term ambient hyperglycemia are poorly understood. Since oxidative stress has been implicated as a contributor to impaired vascular function, we investigated the following:Aims: (1) the effects of high glucose exposure in vitro (7 – 10 days) on vascular relaxation to acetylcholine (Ach) and contractility to norepinephrine (NE) and KCl; (2) if NO-dependent cGMP generation is affected under these conditions; and (3) aortic redox status.Methods: Non-diabetic rat tail artery rings were incubated in normal (5mM) (control NG) or high (20mM) glucose buffer (control HG). Vascular responses to Ach, NE and KCl were compared to those of streptozotocin (SZ) diabetic animals in the same buffers (diabetic NG, diabetic HG). Ach stimulated cGMP levels were quantitated as an indirect assessment of endothelial nitric oxide (NO) production and oxidative stress evaluated by measuring vascular glutathione and oxidized glutathione.Results: Rings from diabetic rats in NG showed impaired relaxation to Ach (P = 0.002) but relaxed normally, when maintained in HG. Similarly, contractile responses to NE were attenuated in diabetic rings in NG but similar to controls in HG. HG markedly augmented maximal contraction to KCl compared to control and diabetic vessels in NG (P < 0.0001). Diabetic vessels in a hyperosmolar, but normoglycemic, milieu respond like those in HG. in vitro, HG for 2 hours changed neither relaxation nor contractile responses to NE and KCl in control rings. Basal cGMP levels were lower in aortae from diabetic animals pre-incubated in NG than in HG/LG or in control rings in NG (P < 0.05). cGMP responses to Ach were exaggerated in diabetic vessels in HG (P = 0.035 vs. control NG, P = 0.043 vs. diabetic NG) but not different between control and diabetic rings in NG. Vessels from diabetic animals had lower levels of GISH (P < 0.0001) and higher levels of GSSG (P < 0.0001) indicating oxidative stress.Conclusions: Our data indicate that endothelium dependent relaxation is altered early in the diabetic state and that increased NO responses may compensate for augmented oxidative stress but the lack of effect of short-term exposure of normal vessels to HG suggests that short-term hyperglycemia per se does not cause abnormal vascular responses.     

Effects of dopaminergic agonists and antagonists on cerebellar guanosine-3',5'-monophosphate (cGMP).

Apomorphine was found to cause an increase in cerebellar cGMP content. Bromocriptine, at a dose that caused stereotypies, neither elevated cGMP, nor blocked the apomorphine- induced rise in cGMP. The apomorphine-induced rise in cGMP was effectively blocked by haloperidol and some other neuroleptics, but not by sulpiride. These actions of the neuroleptics correlated with their ability to displace ³H-spiroperidol from striatal membranes, suggesting that dopamine receptor interactions were important in the cGMP changes noted. Based on the observation that haloperidol antagonized the increase induced by restraint, it is suggested that dopaminergic systems are involved in the reaction to stress.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide,adenosine, and prostacyclin in the pig

The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca²⁺ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca²⁺ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F_2α (PGF_2α). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5′-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone. J. Cell. Biochem. 70:49–59, 1998. © 1998 Wiley-Liss, Inc.     

Effects of ultraviolet radiation on the tension and the cyclic GMP level of bovine mesenteric arteries

Strips of bovine mesenteric arteries brought to sustained contraction by the addition of 3.0 microM phenylephrine relaxed when exposed to ultraviolet radiation (366 nm). The relaxation was reversible and associated with a rapid increase in the cGMP content. After termination of the radiation the cGMP level rapidly decreased below the basal level. The crude soluble guanylate cyclase from the artery was stimulated about 8-fold by ultraviolet radiation (366 nm). Neither the cGMP-phosphodiesterase activity nor the cAMP level were found to be changed during irradiation. The ultraviolet light-induced relaxation was not dependent on an intact intimal surface. Furthermore, the relaxing effect was found to be enhanced and accompanied by a larger increase of the cGMP level in nitroglycerin-tolerant arteries. The present results show that the ultraviolet light-induced relaxation in bovine mesenteric arteries is associated with a rapid increase in the cGMP content and that ultraviolet light and nitrocompounds may exert their relaxing actions through a common substance.     

Cyclic AMP-mediated increase in L-type calcium current (ICa,L) by nitroglycerin in guinea-pig ventricular myocytes.

The effects of nitroglycerin (NG) on the L-type Ca2+ current (ICa,L) were investigated in guinea-pig ventricular myocytes. NG did not affect the basal ICa,L significantly. The ICa,L was increased by NG when the ICa,L was augmented by the pre-treatment with isoproterenol (Iso), and this increase was abolished by the pretreatment with methylene blue (MB), a guanylate cyclase inhibitor. NG also increased ICa,L in the myocytes undergoing the pretreatment with isobutylmethylxanthine (IBMX). The NG-induced increase in ICa,L was also observed in the myocytes intracellularly dialyzed with cyclic adenosine monophosphate (cAMP). The order of the Iso-induced increase in ICa,L was Iso, IBMX and cAMP. Relatively a lower concentration of the extracellularly applied 8bromo-cyclic guanosine monophosphate (8Br-cGMP) increased ICa,L in the myocytes dialyzed with cAMP, whereas the higher concentration of 8Br-cGMP decreased ICa,L. NG produced a marked increase in cGMP levels, and a slight increase in cAMP levels in the ventricular tissues. These results suggest that the stimulatory effect of NG on the ICa,L is due to an inhibition of cGMP-inhibitable cAMP-phosphodiesterase (PDE) and a possible activation of cGMP-dependent protein kinase via the NG-induced increase in cGMP levels in guinea-pig ventricular myocytes.     

Increased Response to High KCl-Induced Elevation in the Intracellular-Ca2+ Concentration in Differentiated NG108-15 Cell and the Inhibitory Effect of the L-Type Ca2+ Channel Blocker, Calciseptine

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca²⁺]_i) by high-KCl application were investigated in the neuroblastoma×glioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca²⁺]_i by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt₂cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca²⁺ channel blocker, but not by N-, P- and R-type Ca²⁺ channel blockers. The IC₅₀ values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca²⁺]_i by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca²⁺]_i by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl₂). Together, these results suggest that the enhancement of the response effect of [Ca²⁺]_i by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.     

Reexamination of Opioid Stimulation of cGMP Formation in Cell Lines of Neuronal Origin

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings.2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma × glioma hybrid cells resembled the proportion of [Ca²⁺]_in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca²⁺ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines.3. The implication of the opioid-Ca²⁺-NO-cGMP cellular pathway for opioid activity in vivo is discussed.     

Activating Effect of NO on Ca2+-Dependent K+ Channels in Smooth Muscle Cells of the Main Pulmonary Artery of the Rabbit

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on outward transmembrane ion current in isolated smooth muscle cells (SMC) of the main pulmonary artery of the rabbit. We also studied the characteristics of unitary high-conductance Ca²⁺-dependent K⁺ channels (K_Ca channels) in the SMC membrane in the cell-attached and outside-out configurations. Nitroglycerin in a 10 M concentration increased the amplitude and intensified oscillations of outward transmembrane current induced by step depolarization. In this case, the threshold of activation of the current (–40 mV) did not change. If the potential was +70 mV, the transmembrane current in the presence of NG increased, as compared with the control, by 32.6 ± 19.4% (n = 6), on average. Simultaneous addition of 10 M NG and 1 mM tetraethylammonium chloride (TEA), a blocker of K_Ca channels, to the external solution at the potential of +70 mV decreased the amplitude of outward transmembrane current with respect to the control by 25.2 ± 11% (n = 6) and suppressed oscillations of this current. In the series of experiments carried out in the outside-out configuration (concentration of K⁺ ions in the external solution was 5.9 mM), we calculated the conductance of a single K_Ca channel, which was approximately 150 pS. In the case where the potential was equal to +40 mV, 1 mM TEA suppressed completely the current through unitary K_Ca channels. In the series of experiments performed in the cell-attached configuration, 100 M NG to a considerable extent intensified the activity of unitary high-conductance K_Ca channels by increasing the probability of the channel open state (P ₀), on average, by 80 ± 1%, as compared with the control. In this case, NG did not influence the conductance of single K_Ca channels. We concluded that the NO donor NG increases the amplitude of outward transmembrane current in SMC of the rabbit main pulmonary artery by stimulation of the activity of TEA-sensitive high-conductance K_Ca channels. Our experiments carried out on single K_Ca channels demonstrated that the activating effect of NG on K_Ca channels is realized at the expense of an increase in the P ₀ of these channels, but not of a change in the conductance of single channels.     

Mechanism of vasorelaxation caused by N-Benzylsecoboldine in rat thoracic aorta

The vasorelaxing effect of N-benzylsecoboldine on the rat thoracic aorta was investigated, and we also compare it with nifedipine and cromakalim. In high K⁺ (60 mM) medium, Ca²⁺ (0.03–3 mM)-induced vasoconstriction was inhibited concentration-dependently by N-benzylsecoboldine, whereas this contraction was not altered by cromakalim. Cromakalim relaxed aortic rings precontracted with 15 but not 60 mM of K⁺. N-benzylsecoboldine and nifedipine were more potent and effective in producing relaxation in 60 mM than in 15 mM K⁺-induced contraction. N-benzylsecoboldine was found to be an ₁-adrenoceptor-blocking agent in rat thoracic aorta as revealed by its competitive antagonism of phenylephrine (PE)-induced contraction (pA₂=6.31 ± 0.04, pA₁₀=5.41 ± 0.03). This relaxing effect of N-benzylsecoboldine was not antagonized by indomethacin or methylene blue, and still persisted in endothelium-denuded aorta or in the presence of nifedipine (1 µM). The increase of inositol monophosphate caused by PE in rat aorta was significantly suppressed by N-benzylsecoboldine, but not by nifedipine or cromakalim. High concentration of N-benzylsecoboldine (100 µM) did not affect the contraction induced by B-HT 920, serotonin or PGF₂. Glibenclamide and charybdotoxin did not affect the relaxation of N-benzylsecoboldine in aortic rings precontracted with PE. Neither cGMP nor cAMP levels were changed by N-benzylsecoboldine. We suggest that N-benzyl-secoboldine relaxes rat thoracic aorta by suppressing the Ca²⁺ influx and also has antagonistic effect on ₁-adrenoceptors.     

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats

Uridine 5′-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor N^G-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y₂ receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE₂, PGF_2α, and PGI₂ in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA₂ levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y₂ receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

     

Depressing action of nitroglycerin on voltage-activated calcium current in isolated smooth muscle cells of the guinea pig gut

The effect of nitroglycerin (NG) on inward voltage-activated calcium current (I _Ca) was studied in isolated smooth muscle cells (SMC) of the guinea pigtaenia coli with the voltage clamp technique in an intracellular dialysis mode. Addition of NG (10^–7 to 10^–4 M) to the extracellular solution reduced theI _Ca amplitude and increased theI _Ca inactivation rate. The maximum inhibition ofI _Ca (on the average, by 41.7 ± 4.8%,n=13) was produced by 10^–4 M NG; the inhibition was dose-dependent. No shift of theI _Ca current-voltage curve under the NG influence was observed. Application of dibutyril-cGMP (2·10^–4 M), a membrane-penetrating analog of cyclic 3,5-guanosine monophosphate (cGMP), likewise decreased theI _Ca amplitude and increased its inactivation rate. The results obtained suggest that the NG inhibitory effect onI _Ca is related to a cGMP-dependent modulation of the voltage-activated calcium channels of L-type in the SMC membrane in the guinea pigtaenia coli.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 218–222, May–June, 1994.     

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca²⁺ signaling, and Ca²⁺ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca²⁺]_i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca²⁺ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca²⁺ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca²⁺ oscillations. NOC-5 inhibited the increase of [Ca²⁺]_i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP₃) in airway SMCs. The effect of NO on the Ca²⁺ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca²⁺ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca²⁺-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca²⁺ via the IP₃ receptor to decrease the frequency of agonist-induced Ca²⁺ oscillations.     

慢性缺氧对大鼠肺动胍内皮依赖性舒张反应及CGMP含量的影响

This experiment was designed to investigate whether chronic hypoxia affect rat pulmonary artery (PA) endothelium-dependent relaxation and the content of cGMP in PA. Both ACh and ATP could induce endothelium-dependent relaxation of PA, not prevented by indomethacin, but completely abolished by methylene blue. These results indicated that vasodilatation of PA induced by both ACh and ATP is mediated by EDRF (endothelium-derived relaxing factor). Chronic hypoxia significantly depressed PA endothelium-dependent relaxation. The percent relaxation of IPPA and EPPA by 10(-6) mol/L ACh was 61.3% and 59.2% of those in control, and the percent relaxation of IPPA and EPPA by 1.8 x 10(-5) mol/L ATP was 64.9% and 55.3% respectively of the control. Chronic hypoxia also depressed SNP-induced endothelium-independent relaxation. Chronic hypoxia significantly decreased the content of cGMP in PA. The basic level of cGMP was 51.9 +/- 5.7 (n = 14) in hypoxia group and 84.9 +/- 9.7 (n = 14) pmol/g wet wt. in control group (P less than 0.01). After treatment of PA with ACh (10(-7) mol/L), the content of cGMP was 91.4 +/- 7.3 (n = 5) pmol/g wet wt. in hypoxic group and 240.8 +/- 30.6 (n = 5) pmol/g wet wt. in control group (P less than 0.01). Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of soluble guanylate cyclase in vascular smooth muscle cells.     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.     

Mechanisms Underlying Nitroglycerin-Induced Suppression of Phenylephrine- and Caffeine-Evoked Contractions of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a strain measurement technique, we studied the mechanisms of the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on contractions of smooth muscles of the main pulmonary artery of the rabbit induced by phenylephrine and caffeine in normal Krebs solution (NKS) or in nominally calcium-free solution (NCFS). Phenylephrine applications caused contractions consisting of an initial fast phasic low-amplitude component followed by a tonic higher-amplitude component. After caffeine-induced monophasic low-amplitude contraction, tension of the smooth muscle strip shifted below the conventional zero. Addition of NG to NKS resulted in a decrease in the smooth muscle tension below the conventional zero. Under the influence of NG, the initial phasic component of phenylephrine-induced contraction was partially suppressed, whereas the next tonic component was suppressed to a greater extent. At the same time, NG exerted nearly no influence on the amplitude of caffeine-induced contractions. Washing out by NKS of phenylephrine dissolved in NCFS resulted in initiation of a fast phasic high-amplitude contraction. Such a contraction did not develop either in the presence of NG or phenylephrine in NCFS or in the case of washing out of caffeine dissolved in NCFS. Our findings allow us to conclude that phenylephrine or caffeine added to the superfusate induce contractions of the smooth muscle cells (SMC) of the main pulmonary artery of the rabbit due to activation of Ca²⁺ release from the respective intracellular calcium stores. In addition, calcium ions entering SMC through the calcium channels of the plasma membrane are also involved in activation of the phenylephrine-induced contraction. The inhibitory effect of NG on the phenylephrine-induced contraction is related to the influence of NO on the release of Ca²⁺ from the inositol trisphosphate-sensitive intracellular calcium store and receptor-operated inflow of Ca²⁺ to SMC. Nitroglycerin did not significantly influence the caffeine-induced contraction and, therefore, Ca²⁺ release from the caffeine-sensitive store.     

Evidence that lithium induces a glutamatergic: Nitric oxide-mediated response in rat brain

Studies have indicated the involvement of a glutamatergic mechanism in lithium (Li⁺) action. Glutamatergic agonists, such as kainic acid, are known to promote the synthesis of nitric oxide (NO) and to increase cGMP, while Li⁺ has displayed a similar, yet unexplained, ability to increase cGMP. NO synthesis is regarded as the principal prodromal event leading to the activation of the guanyl cyclase-cGMP transduction mechanism. In the present study, the involvement of the NO:cGMP pathway in the action of Li⁺ was examined, while the possibility of a glutamatergic mechanism in this response was also investigated. Parameters examined included cortical accumulation of cGMP and the stable oxidative metabolites of NO, viz. NO ₂ ^– and NO ₃ ^–, collectively expressed as NO ₂ ^–. A significant positive correlation was observed in the in vivo cGMP and NO ₂ ^– data throughout all the groups. Chronic treatment of rats with LiCl (0.3% m/m) engendered a significant increase in cGMP levels which was inhibited by the NO-synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester (L-NAME). Acute administration of kainic acid resulted in an increased accumulation of NO ₂ ^–, also prevented by concomitant L-NAME administration. In addition, a synergistic stimulatory response on cortical NO ₂ ^– was observed in the combination of LiCl and kainic acid. Collectively, these data implicate an involvement of a glutamatergic-mediated NO:cGMP transduction mechanism in the action of Li⁺.     

Impaired iNOS–sGC–cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS–sGC–cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague–Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight‐matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle + septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α₁ mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy

Increases inguanosine 3',5'-cyclic monophosphate (cGMP) induced bynitric oxide (NO), nitrovasodilators, and atrial peptides correlatewith relaxation of vascular smooth muscle. Relaxation of myometrialsmooth muscle by increases in cGMP, however, has required unusuallyhigh concentrations of the cyclic nucleotide. We tested the hypothesisthat the sensitivity of myometrium to relaxation by cGMP is increasedduring pregnancy. Aortic smooth muscle was more sensitive to relaxationby cGMP than myometrial tissues, and, contrary to our hypothesis,myometrium from pregnant rats was least sensitive. Although levels ofcGMP were elevated after treatment with the NO donor,S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels ofcGMP-dependent protein kinase (PKG) were significantly decreased inmyometrium from pregnant rats compared with myometrium from nonpregnantcycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, andprogesterone antagonized this response. We conclude that1) myometrial tissues from pregnantrats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP isaccompanied by progesterone-mediated decreases in the level of PKGexpression.