Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

Heme induces Cl^– secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl^– secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents (I_sc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced I_sc was caused by Cl^– secretion because it was inhibited in Cl^–-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl^– secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced I_sc. SnPP-induced I_sc was inhibited by the antioxidant vitamins, -tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl^– secretion can be regulated by the redox environment of the cell. heme oxygenase; electrolyte transport; carbon monoxide; cGMP; reactive oxygen species     

Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy

Hoshi, Yoko, Osamu Hazeki, Yasuyuki Kakihana, and MamoruTamura. Redox behavior of cytochrome oxidase in the rat brain measured by near-infrared spectroscopy. J. Appl.Physiol. 83(6): 1842-1848, 1997.Usingnear-infrared spectroscopy, we developed a new approach for measuringthe redox state of cytochrome oxidase in the brain under normalblood-circulation conditions. Our algorithm does not require theabsorption coefficient of cytochrome oxidase, which differs from studyto study. We employed this method for evaluation of effects of changesin oxygen delivery on cerebral oxygenation in rats. When fractionalinspired oxygen was decreased in a stepwise manner from100 to <10%, at which point the concentration of oxygenatedhemoglobin([HbO₂])decreased by ~60%, cytochrome oxidase started to be reduced.Increases in arterial PO₂ underhyperoxic conditions caused an increase in[HbO₂], whereas further oxidation of cytochrome oxidase was not observed. The dissociation of the responses of hemogloblin and cytochrome oxidase wasalso clearly observed after the injection of epinephrine under severelyhypoxic conditions; that is, cytochrome oxidase was reoxidized withincreasing blood pressure, whereas hemoglobin oxygenation was notchanged. These data indicated that oxygen-dependent redox changes incytochrome oxidase occur only when oxygen delivery is extremelyimpaired. This is consistent with the in vitro data of our previousstudy.

     

Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives.

The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Evidence for oxygen as the master regulator of the responsiveness of soluble guanylate cyclase and cytochrome c oxidase to nitric oxide

Haem is used as a versatile receptor for redox active molecules; most notably NO (nitric oxide) and oxygen. Three haem-containing proteins, myoglobin, haemoglobin and cytochrome c oxidase, are now known to bind NO, and in all these cases competition with oxygen plays an important role in the biological outcome. NO also binds to the haem group of sGC (soluble guanylate cyclase) and initiates signal transduction through the formation of cGMP in a process that is oxygen-independent. From biochemical studies, it has been shown that sGC is substantially more sensitive to NO than is cytochrome c oxidase, but a direct comparison in a cellular setting under various oxygen levels has not been reported previously. In this issue of the Biochemical Journal, Cadenas and co-workers reveal how oxygen can act as the master regulator of the relative sensitivity of the cytochrome c oxidase and sGC signalling pathways to NO. These findings have important implications for our understanding of the interplay between NO and oxygen in both physiology and the pathology of diseases associated with hypoxia.     

Control of the nitric oxide-cytochrome c oxidase signaling pathway under pathological and physiological conditions

Prominent among the mechanisms of interaction of nitric oxide (NO) with intracellular targets are the reactions with heme proteins. For example, the mechanism through which NO induces synthesis of the second messenger cyclic GMP involves the binding of NO to the heme in soluble guanylate cyclase. It has only recently been appreciated that NO binding to the binuclear oxygen binding site in cytochrome c oxidase may also serve as a signal transduction pathway. We postulate that NO is uniquely positioned to control mitochondrial respiration and in doing so regulates oxygen gradients within the cell. In this short overview the mechanisms of NO-dependent regulation of mitochondrial function will be discussed in the context of some of the biological and physiological consequences.     

Cu2+-induced modification of the kinetics of A beta(1-42) channels

We found that the amyloid peptide A(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu²⁺ or biological redox agents that have been reported to mediate A(1-42) toxicity. The A(1-42)-formed cation channel was inhibited by Cu²⁺ in cis solution ([Cu²⁺]_cis) in a voltage- and concentration-dependent manner between 0 and 250 µM. The [Cu²⁺]_cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 µM [Cu²⁺]_cis and partially reversible at 250 µM [Cu²⁺]_cis. The inhibitory effects of [Cu²⁺]_cis between 50 and 250 µM on the channel could not be reversed with addition of Cu²⁺-chelating agent clioquinol (CQ) at concentrations between 64 and 384 µM applied to the cis chamber. The effects of 200-250 µM [Cu²⁺]_cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 µM [CQ]_cis. The kinetic analysis of the data indicate that Cu²⁺-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu²⁺ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu²⁺-binding site of the A(1-42)-formed channels is modulated with Cu²⁺ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu²⁺ modulation of A and PrP channel proteins linked to neurodegenerative diseases. neurodegenerative diseases; transitional metals; ion channel pathologies; membrane injuries; calcium homeostasis     

Purification and properties of heme-deficient hepatic soluble guanylate cyclase: effects of heme and other factors on enzyme activation by NO, NO-heme, and protoporphyrin IX

Purified hepatic soluble guanylate cyclase (EC 4.6.1.2) had maximal specific activities in the unactivated state of 0.4 and 1 μmol cyclic GMP min^?1 mg protein^?1, when MgGTP and MnGTP, respectively, were used as substrates. The apparent K_m for GTP was 85 or 10 μm in the presence of excess Mg²⁺ or Mn²⁺, respectively. Guanylate cyclase purified as described was deficient in heme but could be readily reconstituted with heme by reacting enzyme with hematin and excess dithiothreitol at 4 °C and pH 7.8. Unpurified guanylate cyclase was activated 20- to 84-fold by NO, nitroso compounds, NO-heme, and protoporphyrin IX. The purified enzyme was only slightly (2- to 3-fold) activated by NO and nitroso compounds but was markedly (50-fold) activated by NO-heme and protoporphyrin IX, achieving maximal specific activities of 10 μmol cyclic GMP min^?1 mg protein^?1. Enzyme activation by NO and nitroso compounds was restored by addition of hematin or by reconstitution of guanylate cyclase with heme. Excess hematin, however, inhibited enzyme activity. A partially purified heat-stable factor (activation-enhancing factor) was found to enhance (2- to 35-fold) enzyme activation without directly stimulating guanylate cyclase. In the presence of optimal concentrations of hematin, enzyme activation was still increased (2-fold) by the activation-enhancing factor but not by bovine serum albumin. Guanylate cyclase was markedly inhibited by SH reactive agents such as cystine, o-iodosobenzoic acid, periodate, and 5,5′-dithiobis (2-nitrobenzoic acid). In addition, CN^? and FMN inhibited enzyme activation by NO-heme, but not by protoporphyrin IX, and did not affect basal enzymatic activity. Hepatic soluble guanylate cyclase appears to possess SH groups required for catalysis and to require heme and/or other unknown factors for the full expression of enzyme activation by NO and nitroso compounds.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Endogenous nitric oxide is implicated in the regulation of lipolysis through antioxidant-related effect

We studied the influence ofnitric oxide (NO) endogenously produced by adipocytes in lipolysisregulation. Diphenyliodonium (DPI), a nitric oxide synthase (NOS)inhibitor, was found to completely suppress NO synthesis in intactadipocytes and was thus used in lipolysis experiments. DPI was found todecrease both basal and dibutyryl cAMP (DBcAMP)-stimulatedlipolysis. Inhibition of DBcAMP-stimulated lipolysis by DPI wasprevented by S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor. This antilipolytic effect of DPI was also preventedby two antioxidants, ascorbate or diethyldithiocarbamic acid (DDC).Preincubation of isolated adipocytes with DPI (30 min) before exposureto DBcAMP almost completely abolished the stimulated lipolysis.Addition of SNAP or antioxidant during DPI preincubation restored thelipolytic response to DBcAMP, whereas no preventive effects wereobserved when these compounds were added simultaneously to DBcAMP.Exposure of isolated adipocytes to an extracellular generating systemof oxygen species (xanthine/xanthine oxidase) or toH₂O₂ also resulted in an inhibition of thelipolytic response to DBcAMP. H₂O₂ or DPIdecreased cAMP-dependent protein kinase (PKA) activation. The DPIeffect on PKA activity was prevented by SNAP, ascorbate, or DDC. Theseresults provide clear evidence that 1) the DPI antilipolyticeffect is related to adipocyte NOS inhibition leading to PKAalterations, and 2) endogenous NO is required for the cAMPlipolytic process through antioxidant-related effect.

     

Carnosine as a regulator of soluble guanylate cyclase

The molecular mechanism of the participation of carnosine in the functioning of soluble guanylate cyclase is discussed. It is shown that carnosine inhibits the activation of soluble guanylate cyclase by sodium nitroprusside and a derivative of furoxan--1,2,5-oxadiazolo-trioxide (an NO donor). However, carnosine has no effect on stimulation of the enzyme by a structural analog of the latter compound, a furazan derivative (1,2,5-oxadiazolo-dioxide) that is not an NO donor; nor was carnosine involved in the enzyme activation by protoporphyrin IX, whose stimulatory effect is not associated with the guanylate cyclase heme. The inhibition by carnosine of guanylate cyclase activation by an NO donor is due to the interaction of carnosine with heme iron with subsequent formation of a chelate complex. It was first demonstrated that carnosine is a selective inhibitor of NO-dependent activation of guanylate cyclase and may be used for suppression of activity of the intracellular signaling system NO-soluble guanylate cyclase-cGMP, whose sharp increase is observed in malignant tumors, sepsis, septic shock, asthma, and migraine.     

Energy-dependent redox state of heme a+a3 and copper of cytochrome oxidase in perfused rat brain in situ

Using theblood-free perfused rat brain, we examined the redox behavior ofcytochrome oxidase of two chromophores, hemea + a₃ and copper.When perfusate inflow was stopped to induce global ischemia,the reduction of heme a + a₃ was triphasic,with a rapid phase, a slow phase, and a second rapid phase. Incontrast, the reduction of copper was monophasic after the rapid phaseof heme a + a₃. The triphasicreduction of heme a + a₃ was diminished by energy-depleting treatments, such as addition of an uncoupler. Thetime course of the reduction of copper was not affected by the energydepletion. During global ischemia the decrease in creatine phosphate nearly paralleled the reduction of hemea + a₃, whereas ATPremained at the control level until ~60% of hemea + a₃ was reduced inthe rapid phase. In the slow phase, ATP started to decrease with thereduction of copper. The redox behavior of copper was similar to theslow phase of the reduction of heme a + a₃ because ofthe higher oxygen affinity of copper than of hemea + a₃. Therefore,the rapid phase of the reduction of hemea + a₃ can be used asan alarm before a decrease in ATP, whereas the reduction of copperindicates a decrease in ATP under severe hypoxia. Thus the coppersignal in noninvasive near-infrared spectroscopy is a useful parameterfor the clinical setting.

     

Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.     

Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity

To clarify the contribution of intracellularCa²⁺ concentration([Ca²⁺]_i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca²⁺]_i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca²⁺]_i significantly increased andmyogenic tone developed in response to elevations of extracellularCa²⁺ concentration. The Ca²⁺ channelinhibitor nimodipine substantially decreased[Ca²⁺]_i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca²⁺]_i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca²⁺]_i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca²⁺ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca²⁺]_i.