Background and purpose:Previous work has shown that N G -monomethyl-L-arginine (L-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N G , N G -dimethyl-L-arginine (ADMA), also exhibits this unusual selective blocking action. Experimental approach: The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels. Key results: ADMA (100 mM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300-1000 mM) had no greater effect. Although ADMA (100 mM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 mM had no effect on the maximal relaxation to ACh, although it produced a small (two-to threefold) reduction in sensitivity. ADMA and L-NMMA, like L-arginine (all at 1000 mM), protected ACh-induced relaxation against blockade by L-NAME (30 mM). Conclusions and implications:In the rat aorta, ADMA, like L-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and L-NMMA.

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BACKGROUND AND PURPOSEThe shearing forces generated by flow generally evoke dilatation in systemic vessels but constriction in the cerebral circulation. The aim of this study was to determine the effects of flow on the conduit artery delivering blood to the brain in the rat, that is, the carotid artery. EXPERIMENTAL APPROACHCarotid artery segments were mounted in a pressure myograph and pressurized to 100 mmHg. Changes in vessel diameter to flow (0.5-10 mL·min -1 for 2-10 min) at constant pressure were then measured using a video dimension analyser. KEY RESULTSFollowing the induction of tone, the onset of flow evoked a transient dilatation followed by a powerful constriction that was sustained until the termination of flow. Endothelial denudation or treatment with indomethacin, N G -nitro-L-arginine methyl ester, or the combination of apamin and TRAM-34 showed that the initial flow-mediated dilatation arose from the combined actions of endothelium-derived NO and endothelium-derived hyperpolarizing factor (EDHF). The flow-mediated constriction, which increased in magnitude with increasing flow rate and duration of flow, was also endothelium dependent, but was unaffected by treatment with superoxide dismutase, BQ-123, indomethacin, HET0016 or carbenoxolone. Flow-mediated constriction therefore appeared not to involve superoxide anion, endothelin-1, a COX product, 20-HETE or gap-junctional communication. CONCLUSIONS AND IMPLICATIONSAlthough a weak, transient flow-mediated dilatation is observed in the rat carotid artery, the dominant response to flow is a powerful and sustained constriction. Whether this flow-mediated constriction in the carotid artery serves as an extracranial mechanism to regulate cerebral blood flow remains to be determined.

Many cellular functions are regulated by agonist-induced InsP3-evoked Ca2+ release from the internal store. In non-excitable cells, predominantly, the initial Ca2+release from the store by InsP3 is followed by a more sustained elevation in [Ca2+]i via store-operated Ca2+ channels as a consequence of depletion of the store. Here, in smooth muscle, we report that the initial transient increase in Ca2+, from the internal store, is followed by a sustained response also as a consequence of depletion of the store (by InsP3), but, influx occurs via voltage-dependent Ca2+ channels. Contractions were measured in pieces of whole distal colon and membrane currents and [Ca2+]i in single colonic myocytes. Carbachol evoked phasic and tonic contractions; only the latter were abolished in Ca2+-free solution. The tonic component was blocked by the voltage-dependent Ca2+ channel blocker nimodipine but not by the store-operated channel blocker SKF 96365. InsP3 receptor inhibition, with 2-APB, attenuated both the phasic and tonic components. InsP3 may regulate tonic contractions via sarcolemma Ca2+ entry. In single cells,depolarisation (to ∼-20 mV) elevated [Ca2+]i and activated spontaneous transient outward currents (STOCs). CCh suppressed STOCs, as did caffeine and InsP3. InsP3 receptor blockade by 2-APB or heparin prevented CCh suppression of STOCs; protein kinase inhibition by H-7 or PKC19-36did not. InsP3 suppressed STOCs by depleting a Ca2+ store accessed separately by the ryanodine receptor (RyR). Thus depletion of the store by RyR activators abolished the InsP3-evoked Ca2+ transient. RyR inhibition (by tetracaine) reduced only STOCs but not the InsP3transient. InsP3 contributes to both phasic and tonic contractions. In the former, muscarinic receptor-evoked InsP3 releases Ca2+ from an internal store accessed by both InsP3 and RyR. Depletion of this store by InsP3 alone suppresses STOCs, depolarises the sarcolemma and permits entry of Ca2+ to generate the tonic component. Therefore, by lowering the internal store Ca2+ content,InsP3 may generate a sustained smooth muscle contraction. These results provide a mechanism to account for phasic and tonic smooth muscle contraction following receptor activation.

Ascorbate has both antioxidant and pro-oxidant activities. We have previously shown that plasma levels of ascorbate induce constriction and blockade of dilatation mediated by endothelium-derived hyperpolarizing factor (EDHF). In this study we sought to determine if these detrimental actions were mediated by a pro-oxidant action of ascorbate. Since trace levels of transition metal ions including, Cu2+ and Fe3+, promote oxidation of ascorbate, we examined the effects of the chelating agents, cuprizone and deferoxamine, and of CuSO4 and FeCl3 on ascorbate-induced constriction and blockade of EDHF in the perfused rat mesentery. Cuprizone abolished and Cu2+ but not Fe3+ ions enhanced both ascorbate (50 μM)-induced constriction and blockade of EDHF. The blockade of EDHF produced by ascorbate in the presence of CuSO4 (0.5 μM) was abolished by the hydrogen peroxide scavenger, catalase, but unaffected by the scavengers of hydroxyl radical or superoxide anion, mannitol and superoxide dismutase (SOD), respectively. Consistent with these observations, the oxidation of ascorbate by CuSO4 led to the rapid production of hydrogen peroxide. Catalase, mannitol and SOD had no effect on ascorbate-induced constriction. Thus, in the rat perfused mesentery, both the constrictor and EDHF-blocking actions of ascorbate arise from its oxidation by trace Cu2+ ions. The blockade of EDHF results from the consequent generation of hydrogen peroxide, but the factor producing constriction remains unidentified. These detrimental actions of ascorbate may help explain the disappointing outcome of clinical trials investigating dietary supplementation with the vitamin on cardiovascular health.