Calmodulin-dependent Nitric Oxide Synthase Research Articles

Combination spinal analgesic chemotherapy: a systematic review.

Combination spinal analgesic chemotherapy: a systematic review.

Inhibitory effects of relaxin on human basophils activated by stimulation of the Fcε receptor. The role of nitric oxide

This study investigates whether relaxin (RLX), a hormone previously shown to inhibit mast cell function and to stimulate endogenous nitric oxide (NO) biosynthesis, counteracts the activation of isolated human basophils stimulated with anti-IgE or phorbol ester, and, if so, whether NO is involved. RLX reduced dose-dependently the expression of the activation marker CD63, the release of histamine and the rise of intracellular Ca 2+ levels which triggers granule release by stimulated basophils. RLX also blunts the ultrastructural signs of anaphylactic granule release. The effects of RLX appear to depend upon activation of Ca 2+/calmodulin-dependent NO synthase and endogenous NO production. They were reproduced by the NO donor sodium nitroprusside (SNP) and were reverted by the NO synthase inhibitor N ω-monomethyl- l-arginine, or by the NO scavenger oxyhemoglobin, or by blocking the NO physiological target guanylate cyclase with ODQ. In conclusion, RLX appears to play a role in down-regulating basophil function upon immunologic and nonimmunologic activation.

Effects of manganese on inositol polyphosphate receptors and nitric oxide synthase activity in rat brain.

The neurotoxic effects of excessive exposure to manganese (Mn) include degeneration of dopaminergic neurons, impairment of energy metabolism, and perturbations in phosphoinositide (PI) hydrolysis leading to altered calcium (Ca2+) homeostasis. This study is designed to assess the in vitro and in vivo effects of Mn on Ca2+/calmodulin-dependent neuronal nitric oxide synthase (nNOS) activity and on the regulation of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) receptors involved in intracellular and extracellular mobilization of Ca2+. In vivo Mn exposure significantly increased 3H-InsP3 and 3H-InsP4 binding in the cerebellum and the cerebral cortex in a dose-dependent manner. However, in vitro Mn decreased 3H-InsP3 binding and increased 3H-InsP4 binding. In vitro and in vivo exposure of Mn inhibited nNOS activity in the cerebellum and the cerebral cortex. Immunohistochemical studies also showed a notable decrease in nNOS immunoreactivity in the granule cell layer of the cerebellum, whereas no significant changes were observed in the cerebral cortex. These data suggest that Mn neurotoxicity may be due to altered calcium homeostasis by its modulation of inositol polyphosphate receptors. Further, the inhibition of nNOS by Mn is of considerable importance because NO regulates a number of neurotransmitter functions.

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells.

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat.

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells.

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.

Attenuated Angiotensin Ii-Response in Transgenic Mice Expressing the At1 Receptor in the Vascular Endothelial Cells.

P186 The angiotensin II AT1 receptor mediated vasoconstriction is believed to be due to direct stimulation of vascular smooth muscle cells by the hormone angiotensin II (AngII). Recent evidence suggests that AT1 receptors, also expressed on endothelial cells (EC), on stimulation couple to activation of Ca+2 calmodulin-dependent nitric oxide synthase and NO release i n vitro . These AT1 receptors may mediate a vasodilatory function in the endothelium in vivo , contributing to regulatory mechanisms in blood pressure control. To investigate the physiological role of AT1 receptors on ECs, we generated transgenic mice expressing a constitutively active AT1[N111G] receptor specifically in vascular ECs using the mouse tie gene promoter. Of the three tie-AT1 founder lines with varying copies of transgene, two (TG23 and TG26) showed EC-specific transgene RNA transcript, and receptor protein expression when stained with an epitope-specific antibody. Increased mortality complemented with lack of homozygosity was observed in heterozygous TG23 mice. Tail-cuff systolic blood pressures measured were significantly reduced in TG23 tie-AT1 mice (116±2 TG versus 142±2 control) suggesting a vasodilatory effect mediated by the transgenic receptor. Change in arterial pressure to infused AngII (1 μg/kg body wt.) measured was depressed by approx. 40 % in TG23 tie-AT1 mice compared to controls, and a lower dose (0.2 μg/kg body wt.) evoked no response. These results suggest that the AngII-mediated response in vivo seems to depend on the cell types of the vascular beds and their downstream signaling events which may have important regulatory functional consequences.

Attenuated Angiotensin Ii-Response in Transgenic Mice Expressing the At1 Receptor in the Vascular Endothelial Cells.

P186 The angiotensin II AT1 receptor mediated vasoconstriction is believed to be due to direct stimulation of vascular smooth muscle cells by the hormone angiotensin II (AngII). Recent evidence suggests that AT1 receptors, also expressed on endothelial cells (EC), on stimulation couple to activation of Ca+2 calmodulin-dependent nitric oxide synthase and NO release i n vitro . These AT1 receptors may mediate a vasodilatory function in the endothelium in vivo , contributing to regulatory mechanisms in blood pressure control. To investigate the physiological role of AT1 receptors on ECs, we generated transgenic mice expressing a constitutively active AT1[N111G] receptor specifically in vascular ECs using the mouse tie gene promoter. Of the three tie-AT1 founder lines with varying copies of transgene, two (TG23 and TG26) showed EC-specific transgene RNA transcript, and receptor protein expression when stained with an epitope-specific antibody. Increased mortality complemented with lack of homozygosity was observed in heterozygous TG23 mice. Tail-cuff systolic blood pressures measured were significantly reduced in TG23 tie-AT1 mice (116±2 TG versus 142±2 control) suggesting a vasodilatory effect mediated by the transgenic receptor. Change in arterial pressure to infused AngII (1 μg/kg body wt.) measured was depressed by approx. 40 % in TG23 tie-AT1 mice compared to controls, and a lower dose (0.2 μg/kg body wt.) evoked no response. These results suggest that the AngII-mediated response in vivo seems to depend on the cell types of the vascular beds and their downstream signaling events which may have important regulatory functional consequences.

Serotonin 5HT1B/1D receptor agonists abolish NMDA receptor-evoked enhancement of nitric oxide synthase activity and cGMP concentration in brain cortex slices.

Our previous studies indicating that the function of excitatory amino acids, NMDA type receptor, is modulated by serotonin focused on the interaction between serotonin 5HT1B/1D and glutamate, NMDA receptor in brain cortex. The effect of agonists of 5HT1B/1D receptor, sumatriptan, and zolmitriptan on NMDA receptor-evoked activation of nitric oxide (NO) and cGMP synthesis in adult rat brain cortex slices was investigated. Two kinds of experiment were carried out using adult rats. In one of them, sumatriptan or zolmitriptan was administered in vivo subcutaneously (s.c.) in a dose of 0.1 mg per kg body weight. Brain slices were then prepared and used in the experiments or, in the other exclusively in vitro studies, both agonists at 10 microM concentration were added directly to the incubation medium containing adult rat brain cortex slices. The data obtained from these studies indicated that stimulation of NMDA receptor in brain cortex slices leads to a large increase in calcium, calmodulin-dependent NO synthase (NOS) activity and to significant enhancement of the cGMP level. This NMDA receptor-dependent NO and cGMP release was completely blocked by competitive and noncompetitive NMDA receptor antagonists APV (10 microM) or MK-801 (10 microM.), respectively. The specific inhibitor of Ca(2+)-dependent isoforms of NOS (N-nitro-1-arginine NNLA and 7-nitroindozole (7-N1)) eliminated the NMDA receptor-mediated enhancement of NO and cGMP release. Moreover, the serotonin 5HT1B/1D receptor agonists sumatriptan and zolmitriptan administrated in vivo (s.c.) or in vitro abolished NMDA receptor-evoked NO signalling in brain cortex. The potency of both agonists investigated directly in vitro was similar to their effect after in vivo administration. These results suggest that both serotonin 5HT1B/1D receptor agonists may play an important role in modulating the NO and cGMP-dependent signal transduction pathway in the brain. This effect of sumatriptan and zolmitriptan on NO signaling in the brain system should be taken into consideration when investigating their mechanism of action in the migraine attack.

Evidence for a contribution of store-operated Ca2+ channels to NO-mediated endothelium-dependent relaxation of guinea-pig aorta in response to a Ca2+ ionophore, A23187.

A23187 (6S-[6alpha,8beta,9beta,11alpha]-5-(methylamino) -2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7- dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid, calcimycin), an antibiotic Ca2+ ionophore, produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features were functionally characterized of endothelium-dependent relaxant response of guinea-pig aorta to A23187, especially focusing on the possible Ca2+ source and Ca2+ mobilization mechanisms in endothelial cells responsible for the vasorelaxant response to the Ca2+ ionophore. A23187-induced endothelium-dependent relaxation was suppressed profoundly by N(G)-nitro-L-arginine (L-NNA; 3 x 10(-4) M) or calmidazolium (3 x 10(-5) M), suggesting that nitric oxide (NO) produced by the enhanced activation of Ca2+/calmodulin-dependent endothelial NO synthase (eNOS) is largely responsible for the relaxant response of this artery to A23187. In the Ca2+-free solution without EGTA, NO-mediated endothelium-dependent relaxation induced by A23187 was almost abolished, which suggests that Ca2+ entry from extracellular space into endothelial cells plays the key role in the A23187-induced functional vasorelaxation. On the other hand, SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole; 5 x 10(-5) M) and Ni2+ (3 x 10(-4) M), both of which inhibit capacitative Ca2+ influx through store-operated Ca2+ channels (SOCCs), attenuated significantly NO-mediated endothelium-dependent relaxation by A23187. Furthermore, A23187-induced endothelium-dependent relaxation was suppressed more strongly than endothelium-independent relaxation induced by SIN-1 (3-morpholino-sydnonimine), an NO donor, when aortic preparation was preconstricted with high KCl instead of agonistic stimulation (prostaglandin F2alpha). These findings suggest that NO-mediated endothelium-dependent relaxant response of guinea-pig aorta to A23187 is preceded by the increase in endothelial cytosolic free Ca2+ concentration ([Ca2+]cyt) due to the enhanced Ca2+ influx from extracellular space. In the enhanced Ca2+ entry leading to the stimulation of eNOS and NO-mediated functional relaxant response of guinea-pig aorta to A23187, activation of SOCCs but not the Ca2+ entry through plasma membrane Ca2+-specific routes made by A23187 seems to play the predominant role. It is most likely that A23187 acts primarily at the Ca2+ store sites in endothelial cells, which subsequently depletes stored Ca2+ to activate SOCCs via unidentified mechanisms.

Possible involvement of Ca2+ entry and its pharmacological characteristics responsible for endothelium-dependent, NO-mediated relaxation induced by thapsigargin in guinea-pig aorta.

Thapsigargin, a specific inhibitor of Ca(2+)-pump Ca(2+)-ATPase in the sarcoplasmic/endoplasmic reticulum (SR/ER), produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features of thapsigargin-induced endothelium-dependent relaxation were functionally characterized in the isolated guinea-pig aorta especially focusing on the Ca2+ mobilization mechanisms in endothelial cells. Thapsigargin-induced endothelium-dependent vascular relaxation was markedly suppressed by N(G)-nitro-L-arginine (L-NNA) and calmidazolium, suggesting that the vascular relaxation to thapsigargin is largely attributable to endothelium-derived nitric oxide (NO) produced as a result of the activation of Ca2+, calmodulin-dependent NO synthase (NOS). Removal of Ca2+ from the external solution abolished the endothelium-dependent relaxation of guinea-pig aorta in response to thapsigargin. Thapsigargin-induced endothelium-dependent relaxation was inhibited more strongly compared with the endothelium-independent relaxation to an NO donor, SIN-1 (3-(4-morpholinyl)-sydnonimine), when the artery preparation was preconstricted with a high concentration (80 mM) of KCl instead of agonistic stimulation. Endothelium-dependent relaxation induced by thapsigargin was not affected by diltiazem, a blocker of L-type voltage-gated Ca2+ channels. SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole) and Ni2+, both of which block capacitative Ca(2+) entry, did not show any appreciable inhibitory effects on the endothelium-dependent relaxation to thapsigargin. These findings suggest that in guinea-pig aorta, endothelium-dependent NO-mediated relaxation induced by thapsigargin is preceded by the increase in the cytosolic free Ca2+ concentrations ([Ca2+]cyt) following the depletion of stored Ca2+ in thapsigargin-sensitive store sites in endothelial cells. Although the increase in [Ca2+]cyt responsible for the activation of endothelium NOS leading to thapsigargin-induced vascular relaxation may be ascribed to the capacitative Ca2+ entry from extracellular space, the Ca2+ entry mechanism stimulated with thapsigargin is deficient in sensitivity to SK&F96365 and Ni2+ in the endothelium of guinea-pig aorta.

Endogenous nitric oxide synthesis: Biological functions and pathophysiology

Modern molecular biology has revealed vast numbers of large and complex proteins and genes that regulate body function. By contrast, discoveries over the past ten years indicate that crucial features of neuronal communication, blood vessel modulation and immune response are mediated by a remarkably simple chemical, nitric oxide (NO). Endogenous NO is generated from arginine by a family of three distinct calmodulin-dependent NO synthase (NOS) enzymes. NOS from endothelial cells (eNOS) and neurons (nNOS) are both constitutively expressed enzymes, whose activities are stimulated by increases in intracellular calcium. Immune functions for NO are mediated by a calcium-independent inducible NOS (iNOS). Expression of iNOS protein requires transcriptional activation, which is mediated by specific combinations of cytokines. All three NOS use NADPH as an electron donor and employ five enzyme cofactors to catalyze a five-electron oxidation of arginine to NO with stoichiometric formation of citrulline. The highest levels of NO throughout the body are found in neurons, where NO functions as a unique messenger molecule. In the autonomic nervous system NO functions as a major non-adrenergic non-cholinergic (NANC) neurotransmitter. This NANC pathway plays a particularly important role in producing relaxation of smooth muscle in the cerebral circulation and the gastrointestinal, urogenital and respiratory tracts. Dysregulation of NOS activity in autonomic nerves plays a major role in diverse pathophysiological conditions including migraine headache, hypertrophic pyloric stenosis and male impotence. In the brain, NO functions as a neuromodulator and appears to mediate aspects of learning and memory.Although endogenous NO was originally appreciated as a mediator of smooth muscle relaxation, NO also plays a major role in skeletal muscle. Physiologically, muscle-derived NO regulates skeletal muscle contractility and exercise-induced glucose uptake. nNOS occurs at the plasma membrane of skeletal muscle which facilitates diffusion of NO to the vasculature to regulate muscle perfusion. nNOS protein occurs in the dystrophin complex in skeletal muscle and NO may therefore participate in the pathophysiology of muscular dystrophy.NO signalling in excitable tissues requires rapid and controlled delivery of NO to specific cellular targets. This tight control of NO signalling is largely regulated at the level of NO biosynthesis. Acute control of nNOS activity is mediated by allosteric enzyme regulation, by posttranslational modification and by subcellular targeting of the enzyme. nNOS protein levels are also dynamically regulated by changes in gene transcription, and this affords long-lasting changes in tissue NO levels. While NO normally functions as a physiological neuronal mediator, excess production of NO mediates brain injury. Overactivation of glutamate receptors associated with cerebral ischemia and other excitotoxic processes results in massive release of NO. As a free radical, NO is inherently reactive and mediates cellular toxicity by damaging critical metabolic enzymes and by reacting with superoxide to form an even more potent oxidant, peroxynitrite. Through these mechanisms, NO appears to play a major role in the pathophysiology of stroke, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.

Intracellular localization and activation of endothelial nitric oxide synthase.

Nitric oxide production by the Ca2+/calmodulin-dependent enzyme endothelial nitric oxide synthase primarily reflects changes in intracellular [Ca2+], increasing as Ca2+ rises and decreasing as Ca2+ falls. This simplistic bimodal mechanism of regulation has recently been refined by the finding that the binding of Ca2+/calmodulin to endothelial nitric oxide synthase involves the disruption of the association of endothelial nitric oxide synthase from the scaffolding protein caveolin and the subsequent translocation of the enzyme from plasmalemmal caveolae. Moreover, other endothelial nitric oxide synthase-associated proteins could account for a delayed Ca(2+)-independent activation of nitric oxide production, and may be involved with caveolin in the reversible trafficking of a large endothelial nitric oxide synthase-containing heterocomplex between the caveolae and cytosolic cell structures.

Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells.

Angiotensin II (ANG II) produces vasoconstriction by a direct action on smooth muscle cells via AT1 receptors. These receptors are also present in the endothelium, but their function is poorly understood. This study was therefore undertaken to determine whether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by the accumulation of nitrite and nitrate, was enhanced by 10(-7) M ANG II. The biological activity of the NO released by ANG II action was evaluated by measuring its guanylate cyclase-stimulating activity in smooth muscle cells. The guanosine 3',5'-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased by exposure of supernatant from ANG II-stimulated endothelial cells. These effects resulted from the activation of NO synthase, as they were inhibited by the L-arginine analogs. These ANG II actions were mediated by the AT1 receptor, as shown by their inhibition by the AT1 antagonist losartan. The cGMP production by reporter cells was inhibited by the calmodulin antagonist W-7, suggesting that ANG II activates endothelial calmodulin-dependent NO synthase. This hypothesis is also supported by the increase of intracellular free calcium induced by ANG II in endothelial cells. ANG II also stimulated luminol-enhanced chemiluminescence in endothelial cells. This effect was inhibited by N omega-monomethyl-L-arginine and superoxide dismutase, suggesting that this luminol-enhanced chemiluminescence reflected an increase in peroxynitrite production. Thus ANG II stimulates NO release from macrovascular endothelium, which may modulate the direct vasoconstrictor effect of ANG II on smooth muscle cells. However, this beneficial effect may be counteracted by the simultaneous production of peroxynitrite, which could contribute to several pathological processes in the vascular wall.

Response to: Possible Contribution of Transmembrane Ca sup 2+ Influx to Adenylate Cyclase-mediated NO-cGMP Relaxation in Rat Aorta

Assistant Professor (Iranami).Professor and Chair (Hatano).Assistant Professor (Tsukiyama).Assistant Professor (Maeda).Assistant Professor; Department of Anesthesia; Wakayama Medical College; 7-Bancho-27; Wakayama City 640, Japan (Mizumoto).In Reply:-We appreciate the comments by Dr. Sheng-Nan Wu. He raises three important points; (1) the ability of isoproterenol to induce Ca2+ influx into endothelium, (2) inhibitory effect of halothane on Ca2+ ionophore (A23187)-induced endothelium dependent relaxation, and (3) the possibility of modification of the relaxation response to isoproterenol by Ca2+ channel blocking action of halothane. We discuss these points, respectively.From our findings and other reports (please see the article), it is likely that isoproterenol can not initiate nitric oxide (NO) production by vascular endothelium of other species except for rat and that adenylyl cyclase-mediated signal transduction within endothelium of other species differs from that of rat. From experiments using various inhibitors, we propose that cAMP-PKA play an important role in the activation of Ca2+ /calmodulin dependent NO synthase (cNOS) elicited by isoproterenol. The calcium ionophore, A23187, can increase intracellular Ca2+ in the endothelium not only via Ca2+ influx but also via release from intracellular store. The latter mechanism has been demonstrated to be essential for A23187-induced NO production by endothelium. [1,2]Thus, it is likely that halothane exerts the inhibitory action on NO-cGMP relaxation mechanism only when cNOS is activated by Ca2+ releasing mechanism.Halothane has been shown to modulate Ca2+ dynamics in vascular smooth muscle cells including the inhibition of Ca2+ influx and the facilitation of Ca2+ releasing from sarcoplasmic reticulum (caffeine-like action). [3]These dual actions of halothane on Ca2+ dynamics are likely to induce the net effects on vascular tone within a few minutes. Our experimental protocols basically comprised an initial equilibration period followed by elevation of vascular tone with noradrenaline and subsequent test of relaxation response of rings with sustained tone to isoproterenol in the absence or presence of various inhibitors or halothane. Aortic rings were exposed to halothane for more that 40 min, during which the modulation of halothane to Ca2+ dynamics might reach plateau. Therefore, isoproterenol was applied to the rings with sustained tone and Ca2+ dynamics in the presence of halothane. Further, our study demonstrated that isoproterenol produced endothelium-dependent and NO-mediated relaxation accompanying with the increases in cGMP contents of rat aortic rings and that halothane failed to affect not only the relaxation response but also the cGMP increase elicited by isoproterenol. It has been known that the post-cGMP cascade mediating relaxation of vascular smooth muscle cells includes the modulations of contractile proteins and of various ion currents (Na sup +, K sup + Cl sup - and Ca2+), however, the contribution of L-type Ca2+ inward current to it has not been well known. Even if halothane has an ability to inhibit the L-type of Ca2+ inward current through the membrane of vascular smooth muscle cells as shown in the short report by Dr. Wu et al., [4]in our opinion, it seems unlikely that this inhibitory action accounts for the failure of halothane to inhibit the isoproterenol-induced relaxation.Hiroshi Iranami, M.D.Assistant ProfessorYoshio Hatano, M.D.Professor and ChairYoshi Tsukiyama, M.D.Assistant ProfessorHiroshi Maeda, M.D.Assistant ProfessorKazuhiro Mizumoto, M.D.Assistant ProfessorDepartment of Anesthesia; Wakayama Medical College; 7-Bancho-27; Wakayama City 640, Japan(Accepted for publication March 18, 1997.)

Nitric oxide synthase activities in mammalian parotid and submandibular salivary glands

Nitric oxide is important as a physiological messenger molecule in various organs and cells. It is synthesized from the amino acid l-arginine by nitric oxide synthase. Here, the specific activities of nitric oxide synthase in the cytosolic fractions of rabbit, bovine, mice, rat, and guinea-pig parotid and submandibular glands were compared. Marked specific activities were detected in the rabbit and bovine parotid and submandibular glands and in the parotid of mice. The activity in rabbit parotid was highest and was similar to that in rabbit brain. The significant activities in the salivary glands were completely blocked in the absence of Ca 2+ or the presence of a calmodulin inhibitor. These findings suggest that the rabbit parotid glands are useful for studying the regulation of nitric oxide generation by Ca 2+/calmodulin-dependent nitric oxide synthase in salivary glands.

Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells.

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.

Endothelial calcium-calmodulin dependent nitric oxide synthase in the in vitro vascular hyporeactivity of portal hypertensive rats

Background/Aims: Increased nitric oxide production has been implicated in impaired vascular responsiveness to vasoconstrictors in portal hypertension. However, there is no firm evidence concerning the involved nitric oxide synthase isoform. The present study investigated the possible contribution of one nitric oxide synthase isoform, the endothelial constitutive Ca 2+-calmodulin dependent, in the overproduction of nitric oxide in portal hypertension. Methods: Vascular responses to norepinephrine and acetylcholine were evaluated in isolated thoracic aortic rings from normal and portal vein stenosed rats. Results: An impaired concentration-dependent contraction to norepinephrine was observed in intact rings from portal hypertensive rats compared to controls. The hyporeactivity to norepinephrine was reversed after endothelium denudation, the inhibition of nitric oxide synthase with L-NOARG or the inhibition of calmodulin with W-7, but not after pre-incubation with indomethacin. Stimulation of intact rings with norepine phrine after the inhibition of calmodulin with calmidazolium was followed by a decreased vascular response in vessels from normal rats but not in those from portal hypertensive rats. Stimulation of intact rings with norepinephrine in a Ca 2+-free medium was followed by a decreased vascular response in vessels from both portal hypertensive and normal rats. No difference in vasoconstrictive responses was observed between the two groups after calmidoazolium or in a Ca 2+-free medium. Relaxation induced by acetylcholine in norepinephrine-precontracted rings was more marked in rings from portal hypertensive rats than in controls. No differences in the vasodilator responses were observed after relaxations had been inhibited by the removal of the endothelium, pre-incubation with L-NOARG, indomethacin, W-7 or calmidazolium and in a Ca 2+-free medium. Conclusions: This study demonstrates the involvement of the endothelial constitutive Ca 2+-calmodulin dependent nitric oxide synthase isoform in the overproduction of nitric oxide in portal hypertension.

Ca 2+/calmodulin-dependent cyclic GMP phosphodiesterase activity in granule neurons and astrocytes from rat cerebellum

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca 2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca 2+/calmodulin-dependent phosphodiesterase. At 10 μM cGMP, Ca 2+ (25 μM) stimulated basal (Ca 2+-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca 2+-dependent phosphodiesterase activity and exogenous calmodulin increased 3–4-fold the stimulatory potency of Ca 2+ in both cell types (EC 50 values 1.26±0.20 and 1.50±0.42 μM in the absence and 0.38±0.11 and 0.39±0.14 μM in the presence of 1 μM calmodulin, for neurons and astrocytes, respectively). In both cell types K m values for cGMP at 25 μM Ca 2+ were similar (1.72±0.20 and 1.92±0.09 μM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca 2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca 2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC 50 values at 1 μM cGMP: 31±10 μM and 46±6 μM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca 2+-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca 2+-dependent NO synthase type I is stimulated. © 1997 Elsevier Science B.V. All rights reserved.

Vasorelaxation, endothelium, and wine

The mechanism of the cardiovascular benefits of wine is not fully understood, and indeed, may be manifold [1–3]. We have evidence, at least in vitro, that there are compounds present in grapes and wines, particularly red wines, as well as many other food plants, which cause endotheliumdependent relaxation (EDR) of blood vessels via the arginine/nitric oxide/cyclic GMP pathway [4,5]. Nitric oxide (NO) was discovered in 1980 by Furchgott and Zawadzki [6], who coined the term endothelium-derived relaxing factor (EDRF). In 1987, Furchgott [7] and Ignarro et al. [8], working in separate laboratories, simultaneously reported that EDRF was indeed nitric oxide. Since that time, NO has been shown to play important roles in many physiological systems. NO is generated in endothelial cells by the Ca2+-calmodulin-dependent enzyme NO synthase (NOS), acting on its substrate, the amino acid L-arginine. NO is released from the endothelial cell (both luminal and abluminal sides) and easily traverses the distance to the vascular smooth muscle cell, where it diffuses in and acts on its target enzyme, guanylate cyclase. Guanylate cyclase, in turn, causes generation of cyclic GMP, which ultimately leads to vasorelaxation. NO donor drugs such as nitroglycerin deliver NO directly to the smooth muscle cell to cause vasodilation, thus bypassing the endothelial cell. NO exhibits several potentially significant cardiovascular protective effects: