Walter R. Kukovetz

High D-Glucose–Induced Changes in Endothelial Ca2+/EDRF Signaling are Due to Generation of Superoxide Anions

Pretreatment of porcine aortic endothelial cells with high D-glucose results in enhanced endothelium-derived relaxing factor (EDRF) formation (39%) due to increased endothelial Ca2+ release (57%) and Ca2+ entry (97%) to bradykinin. This study was designed to investigate the intracellular mechanisms by which high D-glucose affects endothelial Ca2+/EDRF response. The aldose-reductase inhibitors, sorbinil and zopolrestat, failed to diminish high D-glucose-mediated alterations in Ca2+/EDRF response, suggesting that aldose-reductase does not contribute to high D-glucose-initiated changes in Ca2+/EDRF signaling. Pretreatment of cells with the nonmetabolizing D-glucose analog, 3-O-methylglucopyranose (3-OMG), mimicked the effect of high D-glucose on Ca2+ release (41%) and Ca2+ entry (114%) to bradykinin, associated with elevated EDRF formation (26%). High D-glucose and 3-OMG increased superoxide anion (O2−) formation (133 and 293%, respectively), which was insensitive to inhibitors of cyclooxygenase (5,8,11,14-eicosatetraynoic acid [ETYA], indomethacin), lipoxygenase (ETYA, gossypol, nordihydroguaiaretic acid [NDGA]), cytochrome P450 (NDGA, econazole, miconazole), and nitric oxide (NO) synthase (L-omega N-nitroarginine), while it was diminished by desferal, a metal chelator. The gamma-glutamyl-cysteine-synthase inhibitor, buthioninesulfoximine (BSO), also increased formation of O2− by 365% and mimicked the effect of high D-glucose on Ca2+/EDRF signaling. The effects of high D-glucose, 3-OMG, and BSO were abolished by co-incubation with superoxide dismutase. Like high D-glucose, pretreatment with the O2−-generating system, xanthine oxidase/hypoxanthine, elevated bradykinin-stimulated Ca2+ release (+10%), Ca2+ entry (+75%), and EDRF (+73%). We suggest that prolonged exposure to pathologically high D-glucose concentration results in enhanced formation of O2−, possibly due to metal-mediated oxidation of D-glucose within the cells. This overshoot of O2− enhances agonist-stimulated Ca2+/EDRF signaling via a yet unknown mechanism.

Release of nitric oxide from donors with known half-life: a mathematical model for calculating nitric oxide concentrations in aerobic solutions

The NO concentrations released from donor compounds are difficult to predict as they are determined by formation and inactivation reactions. To calculate the concentrations of NO over time, we have developed a mathematical model which is based on a system of two differential equations describing the first order decomposition of the NO donor in association with the third order reaction of NO with oxygen. Although there is no closed formula for the solution, it can be easily computed by any standard numerical differential solver or simulation software with the following input parameters: initial concentration and decomposition rate constant of the NO donor, O2 concentration, and rate constant for NO autoxidation. The model was validated by monitoring NO release from 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) with a Clark-type NO-sensitive electrode at two different temperatures (25 and 37°C) and DEA/NO concentrations ranging from 1 to 10μM. Under all conditions, there was an excellent agreement between experimental and calculated data. In addition to the computer modeling, we present graphical plots which allow a rough but very easy estimation of the actual NO concentrations if appropriate computer software should not be available.

Exposure To Elevated D-Glucose Concentrations Modulates Vascular Endothelial Cell Vasodilatory Response

The possible role of endothelial dysfunction in early stages of uncomplicated diabetes mellitus was investigated in porcine aortic endothelial cells. Prolonged exposure to various D-glucose concentrations resulted in concentration-dependent amplification of agonist-induced Ca2+ mobilization, whereas L-glucose and D-mannitol failed to mimic the effect of D-glucose. This stimulatory effect of high D-glucose on endothelial Ca2+ mobilization could be antagonized by coincubation with cytochalasin B, which prevented D-glucose uptake into the cells. In agreement with its effect on agonist-induced Ca2+ response, prolonged preincubation with pathological D-glucose concentrations amplified formation of endothelium-derived relaxing factor, which is well established to be strictly attributable to increases in endothelial free Ca2+. In contrast to endothelium-derived relaxing factor formation stimulated by receptor-interacting autacoids, preincubation with high D-glucose failed to modulate A 23,187-induced endothelium-derived relaxing factor formation, which is attributable to unphysiological increases in endothelial free Ca2+ by this ionophore. Similar to its effect on D-glucose-mediated amplification of agonist-stimulated Ca2+ mobilization, cytochalasin B abolished the stimulatory effect of high D-glucose on endothelium-derived relaxing factor formation. We therefore suggest that prolonged exposure to pathological high D-glucose concentrations results in an enhanced endothelium-derived relaxing factor formation caused by amplification of agonist-stimulated Ca2+ mobilization in endothelial cells. This mechanism may be of particular importance representing a possible basis of pathological vasodilation and reduced peripheral resistance in early stages of diabetes mellitus.

Effect of calcium on endothelium-derived relaxing factor formation and cGMP levels in endothelial cells.

Various stimulants of the release of EDRF (endothelium-derived relaxing factor) increased intracellular cGMP levels in bovine aortic endothelial cells. ATP was the most effective compound tested, increasing cGMP 7-fold, followed by the calcium ionophore, A23187 (4.8-fold), and bradykinin (4.0-fold). The EC50 values were similar to those obtained when EDRF release was measured with the bioassay technique, which suggests a stimulation of endothelial guanylate cyclase by EDRF. The direct acting stimulants of soluble guanylate cyclase, sodium nitroprusside and SIN-1 (3-morpholino-sydnonimine), also increased the cGMP content of endothelial cells by 9.4 and 7.2 times, respectively. The effects of both groups of stimulants on cGMP levels were antagonized by the lipoxygenase inhibitor, nordihydroguaiaretic acid, and by the radical scavenger, phenylbutylnitrone, whereas gossypol or canavanine only antagonized the EDRF-induced effect on endothelial cGMP levels. Bradykinin, ATP and A23187 also increased the uptake of 45CaCl2 into endothelial cells but since the complete removal of extracellular Ca2+ or blockade of Ca2+ transport by LaCl3 did not affect the ability of these compounds to elevate cGMP levels, the formation of EDRF appears not to be triggered by an influx of extracellular calcium. This study provides evidence that EDRF stimulators enhance cGMP levels in endothelial cells, probably due to a direct activation of guanylate cyclase by EDRF.

Novel guanylyl cyclase inhibitor, ODQ reveals role of nitric oxide, but not of cyclic GMP in endothelin-1 secretion

The role of nitric oxide (NO) and guanosine 3′,5′‐cyclic monophosphate (cyclic GMP) in cellular regulation of endothelin‐1 (ET‐1) secretion was investigated in cultured porcine aortic endothelial cells. NO synthase was inhibited with (l‐NNA) and guanylyl cyclase with the novel selective inhibitor, ODQ (1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one) (3 μM). Basal and phorbol ester (PMA)‐stimulated ET‐1 secretion were unaffected by ODQ, but stimulated secretion was increased by l‐NNA. In the presence of the NO donors, spermine/ NO, S‐nitroso‐glutathione (GSNO), and nitroprusside (NP) ET‐1 secretion was reduced, but ODQ had no effect on this inhibition, although it effectively inhibited cyclic GMP production. NO release from donors, measured with a sensitive NO electrode, was greatest for spermine/NO, intermediate for GSNO, minimal for NP and paralleled inhibition of ET‐1 secretion. The data suggest that in cultured endothelial cells, curtailment of ET‐1 secretion is mediated by NO and independent of cyclic GMP.

Activation of a small-conductance Ca2+-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.

Pharmacologic differentiation between endothelium-dependent relaxations sensitive and resistant to nitro-L-arginine in coronary arteries.

Summary: We investigated whether formation of endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in porcine and bovine endothelial cells (PAECs) was stimulated by different kinin receptors and studied pharmacologic differences and similarities between the two types of bradykinin-induced relaxation of bovine or porcine coronary arteries. Cultured PAECs were used for [3H]bradykinin binding assay and for measurement of the endothelial free [Ca2+]i by the fura-2/AM method. In organ bath studies with strips of bovine and porcine coronary arteries (endothelium intact), changes in length were recorded and cyclic GMP was measured by radioimmunoassay (RIA). Two bradykinin binding sites were detected, suggesting the presence of two subtypes of B2 kinin receptors. Bradykinin increased [Ca2+]i, and this action was antagonized by the B2 kinin receptor antagonist Hoe 140 and the K channel inhibitor tetrabutylammonium (TBA). Hoe 140 competitively antagonized the relaxing effects of bradykinin, whereas a B1 antagonist was inactive. L-ωN-nitro-arginine (L-NNA) diminished one part of bradykinin-induced relaxation and abolished the increases in cyclic GMP; TBA inhibited another part of the relaxing effect and attenuated (but not significantly) increases in cyclic GMP, and Hoe 140 completely inhibited relaxation and increases in cyclic GMP. The results indicate that the bradykinin response is mediated by biosynthesis of EDRF, which is sensitive to L-NNA, and of EDHF, which is sensitive to TBA.

Histamine induces K+, Ca2+, and Cl- currents in human vascular endothelial cells. Role of ionic currents in stimulation of nitric oxide biosynthesis.

The nature of the membrane currents mediating agonist-induced Ca2+ entry and enhanced nitric oxide (NO) production in endothelial cells is still unclear. Using both perforated-patch and conventional whole-cell clamp technique, we have studied the membrane response associated with histamine stimulation of human vascular endothelial cells. In perforated-patch experiments, the initial histamine (10 mumol/L)-induced current reversed close to the K+ equilibrium potential and was blocked by tetrabutylammonium ions (TBA, 10 mmol/L). In addition, a TBA-insensitive current that developed slowly in the presence of histamine was recorded. This delayed histamine-induced current reversed close to neutral potential and was inhibited by SK&F 96365 (25 mumol/L), a putative blocker of receptor-operated Ca2+ channels. Similar histamine effects were observed in conventional whole-cell experiments using pipette solutions with low Ca(2+)-buffering capacity. Strong buffering of intracellular free Ca2+ suppressed the initial, but not the delayed, current response. The delayed component of histamine-induced current was substantially inhibited by the Cl- channel blocker N-phenylanthranilic acid (NPA, 100 mumol/L), and an eightfold change in the Cl- gradient shifted the reversal potential of this current by 30 mV. In Cl(-)-free solutions, histamine induced an SK&F 96365-sensitive NPA-resistant current, which, according to reversal potential measurements in 20 mmol/L extracellular Ca2+, corresponded to a cation conductance with 13- to 25-fold selectivity for Ca2+ over K+. Both SK&F 96365 and TBA strongly suppressed histamine-induced rises in intracellular free Ca2+ and cellular cGMP levels, whereas NPA did not. Our results provide the first demonstration that three distinct ionic conductances contribute to the histamine-induced membrane response of endothelial cells. It is suggested that histamine induces a Cl- conductance that is apparently not involved in Ca2+ homeostasis and regulation of NO biosynthesis, while, in parallel, joint activation of a rapidly induced K+ permeability and a slowly developing cation permeability mediate Ca2+ entry and stimulation of endothelial NO production.

Dual mechanism of the relaxing effect of nicorandil by stimulation of cyclic GMP formation and by hyperpolarization

In addition to previous results from our laboratory showing that micorandil relayed vascular smooth muscle by increasing evelic GMP levels it was shown to activate K-channel, as well. an effect that also leads to relavation. In the present study. we attempted to differentiate quantitatively between these two elieels in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclie GMP. When the strips were contracted by the thromhovane A analogue U 46619 (1 μM) with 10 μM methylene blue added. nieorandil produced 30–50 relavation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K to 80.4 mM (30-fold), nieorandil caused only 52 relaxation. whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechantsms f relaxation H nicorandil was further achicved through calculation of the cyclic GMP mediated component from a correlation between increases in cyclie GMP and percentage of relayation as, produced by mcorandil under condition, of inhibited hyperpolarization. i.e., in strips contracted with 1μM U 46619 or 26.8 mM K (10 fold) and exposed to either 30-fold K or 10 mM Ba Under both condition,. similar correlations between cylic GMP and relayation were ohtained. Because U 46619, in Addition to its contractile effect. partially antugonized the relaxation by nicorandil without changing cyclic GMP. the correlation was corrected for this effect and indicated a participation of cyclie GMP In the overall reluvunl response of 30–40 at low and 80–90 at high concentrations of nicorandil.

Mechanism of vasodilation by molsidomine

Molsidomine is enzymatically metabolized in the liver to SIN-1 and readily converted into the active metabolite SIN-1A, which carries a free nitroso group. Evidence obtained in isolated circular strips from bovine coronary arteries indicates that SIN-1 increases cyclic guanosine monophosphate in close association with its relaxant effects in coronary strips under various pharmacologic conditions, suggesting that cyclic guanosine monophosphate mediates relaxation. Various nitrovasodilators act by the same mechanism, which is stimulation of guanylate cyclase. In this study the effect of nitroglycerin depended on the presence of a special thiol, cysteine, whereas SIN-1 was active also in the absence of cysteine. Cysteine deficiency was found to be associated with tolerance. After prolonged exposure to the drug, tolerance toward nitroglycerin developed in coronary strips that was antagonized by cysteine. SIN-1 produced no significant tolerance and was also fully active in nitroglycerin-tolerant strips. We conclude that SIN-1 relaxes vascular smooth muscle by direct stimulation of guanylate cyclase, whereas nitroglycerin probably must be converted into a cyclase stimulator by a cysteine-dependent reaction.